WO2022122833A1 - Physiological effects of activation of the alox15 pathway - Google Patents

Abstract

Systems and methods for treating an inflammatory condition in a subject are provided. One or more signal-conducting interfaces are placed in signaling contact with one or more splenic nerves of the subject. A connection is formed between a signal-generating source and the one or more signal-conducting interfaces. A stimulation is generated, by the signal-generating source, at the one or more signal-conducting interfaces, where the stimulation activates the ALOX15 pathway to achieve a change in a parameter.

Description

PHYSIOLOGICAL EFFECTS OF ACTIVATION OF THE ALOX15 PATHWAY

CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims priority to US Provisional Application 63/123,408, filed December 9, 2020, the contents of which are incorporated by reference herein in its entirety.

TECHNICAL FIELD

[0002] This specification describes technologies relating to activation of the ALOX15 pathway, such as by stimulation of neural activity in a nerve supplying the spleen.

BACKGROUND

[0003] Inflammation plays a fundamental role in host defenses and the progression of immune-mediated diseases [80], The inflammatory response is initiated in response to an injury and/or an infection by chemical mediators (e.g., cytokines and prostaglandins) and inflammatory cells (e.g., leukocytes). A controlled inflammatory response is beneficial, for example, in the elimination of harmful agents and the initiation of the repair of damaged tissue providing protection against infection. However, the inflammatory response can become detrimental if dysregulated, leading to a variety of inflammatory disorders such as rheumatoid arthritis, osteoarthritis, psoriatic arthritis, spondyloarthropathy, ankylosing spondylitis, psoriasis, asthma, allergies, septic shock syndrome, atherosclerosis, lupus, multiple sclerosis, inflammatory bowel disease, Crohn's disease, ulcerative colitis, and other clinical conditions mediated by chronic and/or acute inflammation.

[0004] Clinical trials have shown that electrostimulation of the vagus nerve can relieve symptoms associated with some inflammatory conditions (e.g, rheumatoid arthritis) [22], However, stimulation of the vagus nerve can produce undesired, non-specific side effects, due to the inadvertent modulation of the approximately 100,000 nerve fibers of which the vagus nerve is composed. Off-target effects that may occur due to such conventional methods include innervation of nerves leading to the brain (80%) and most of the organs of the body including the heart, liver and gastrointestinal tract (20%).

[0005] Advantageously, the spleen contains half of the body's monocyte population, making this organ the main contributor in inflammation, particularly in response to endotoxemic shock, and thus an attractive target for treatment of inflammatory conditions. The spleen is innervated by different nervous branches [47], and electrical stimulation of the splenic nerves is associated with vascular responses of the spleen [41], As such, electrical stimulation of the splenic nerves may be useful for treating conditions associated with inflammatory disorders [6], See, for example, United States Patent Application No. US 11/467,963, filed August 29, 2006; United States Patent Application No. US10/820,937, filed April 8, 2004; and United States Patent Application No. US10/820,677, filed April 8, 2004, each of which is hereby incorporated herein by reference in its entirety. However, electrical parameters used for stimulation of neural activity may lead to off-target effects such as a change in splenic arterial and venous blood flow, as well as changes in systemic arterial blood pressure and heart rate. Additional off-target effects induced via the autonomic nervous system may occur when using conventional techniques, due to the use of intervention sites that are positioned upstream of the target organ.

[0006] Thus, there is a need for improved systems and methods for stimulating neural activity in a nerve supplying the spleen for treating inflammatory disorders, including chronic and/or acute inflammatory disorders.

SUMMARY

[0007] Stimulation of neural activity in a nerve supplying the spleen (e.g., a splenic nerve), can be achieved using a variety of means, including but not limited to electrical signals, magnetic stimulation, infrared light stimulation, and/or ultrasound stimulation. For example, when using electrical signals for such purposes, stimulation of neural activity is caused by the influence of electrical currents of the electrical signal on the distribution of ions across the nerve membrane. The nature and amount of the electrical current utilized for stimulation of neural activity can be characterized by various parameters, including amplitude, pulse width, pulse height, total charge, waveform, frequency, and/or paradigm. Thus, in some embodiments, the stimulation that is supplied to the nerve by the electrical signal varies depending on chosen parameters of the electrical signal. In some instances, stimulation is induced by permanently or transiently-implanted electrodes, electrodes in direct contact with the nerve, externally-placed electrodes, and/or internally-implanted electrodes. In other instances, such as when using infrared light or ultrasound, stimulation is induced non-invasively and/or without direct contact of a signal-conducting interface and/or a signal-generating source with the nerve.

[0008] The present disclosure provides improved systems and methods that utilize stimulation of a splenic nerve for the modulation of the Lipoxygenase 15 (ALOX15) pathway, including neuromodulation of one or more inflammatory resolution pathways and one or more stimulation parameters for the same. For example, splenic nerve stimulation can induce changes in physiological levels of specialized pro-resolving lipid mediators (SPMs), in particular those regulated by the activity of the enzyme (ALOX15) on substrates (e.g., arachidonic acid) and thereby promoting attenuation of inflammation and resolution processes. SPMs, also referred to as “specialized pro-resolving mediators,” are lipid mediators that play a central role in reprogramming innate and adaptive immune responses via the regulation of immune cell recruitment and cytokine production.

[0009] In some instances, modulation of the ALOX15 pathway by splenic nerve stimulation is used to treat inflammatory conditions, such as chronic and/or acute inflammatory disorders.

Therapeutic effects of neuromodulation and regulation of the ALOX15 pathway can be assessed via measurements of one or more inflammatory cytokines or one or more SPMs, in accordance with some aspects of the present disclosure. For example, experimental results using validated large animal models with induced endotoxemia revealed that these immunomodulatory effects could be achieved in a manner that was well tolerated by the subject and did not affect nerve integrity, as evidenced by the lack of observable sensation in conscious animals undergoing stimulation (see, Examples 1 and 2, below). Splenic nerve stimulation can therefore be used to modulate cytokine production and promote resolution mechanisms via activation of at least the ALOX15 pathway, in a manner that minimizes undesirable off-target effects. Importantly, these mechanisms are associated with the action of the splenic nerve-released noradrenaline (NA) on spleen immune cells and are conserved across species. This is demonstrated by the observation that incubating human spleen-derived immune cells (splenocytes) with NA was able to increase levels of ALOX 15 -derived resolvin DI (RvDl) (see, Example 3 below).

[0010] Accordingly, technical solutions (e.g., computing systems, methods, and non- transitory computer readable storage mediums) for addressing the above-identified problems with treating inflammatory conditions are provided in the present disclosure.

[0011] The following presents a summary of the present disclosure in order to provide a basic understanding of some of the aspects of the present disclosure. This summary is not an extensive overview of the present disclosure. It is not intended to identify key/critical elements of the present disclosure or to delineate the scope of the present disclosure. Its sole purpose is to present some of the concepts of the present disclosure in a simplified form as a prelude to the more detailed description that is presented later.

[0012] Accordingly, the present disclosure provides a method of treating an inflammatory condition in a subject, the method comprising placing one or more signal-conducting interfaces in signaling contact with one or more splenic nerves of the subject, forming a connection between a signal-generating source and the one or more signal-conducting interfaces, and generating a stimulation at the one or more signal-conducting interfaces with the signal- generating source, where the stimulation activates the ALOX15 pathway.

[0013] In some embodiments, the one or more signal-conducting interfaces comprises an electrode (e.g., a cuff electrode, a circumferential cuff electrode, a catheter intravascular electrode, a stent, and/or a patch). In some embodiments, the one or more signal-conducting interfaces do not require an electrode. In some embodiments, the one or more signal- conducting interfaces is placed such that it is in direct physical contact with the one or more splenic nerves. In some embodiments, the one or more signal-conducting interfaces is placed externally (e.g, non-invasively applied).

[0014] In some embodiments, the connection between the signal-generating source and the one or more signal-conducting interfaces is a wireless connection. In some embodiments, the connection between the signal-generating source and the one or more signal-conducting interfaces is through a lead.

[0015] In some embodiments, the generating a stimulation at the one or more signal- conducting interfaces comprises generating a signal, at the signal-generating source, that is selected from the group consisting of: an electrical signal, an infrared signal, an ultrasound signal, a mechanical signal, and a magnetic signal.

[0016] In some embodiments, the generating the stimulation that activates the ALOX15 pathway produces an improvement in a physiological parameter in the subject, where the improvement in the physiological parameter is selected from the group consisting of: a reduction in one or more pro-inflammatory cytokines, an increase in one or more anti- inflammatory cytokines and/or one or more pro-resolving mediators, an increase in one or more catecholamines, changes in one or more immune cell population or one or more immune cell surface co-stimulatory molecules, a reduction in one or more factors involved in the inflammation cascade, and a reduction in one or more immune response mediators. In some embodiments, the generating the stimulation that activates the ALOX15 pathway produces an improvement in two or more physiological parameters in the subject, where the improvement in the physiological parameters is selected from the group consisting of: a reduction in one or more pro-inflammatory cytokines, an increase in one or more anti-inflammatory cytokines and/or one or more pro-resolving mediators, an increase in one or more catecholamines, changes in one or more immune cell populations or one or more immune cell surface co-stimulatory molecules, a reduction in one or more factors involved in the inflammation cascade, and a reduction in one or more immune response mediators. In some embodiments, the generating the stimulation that activates the ALOX15 pathway produces an improvement in three or more physiological parameters in the subject, where the improvement in the physiological parameters is selected from the group consisting of: a reduction in one or more pro-inflammatory cytokines, an increase in one or more anti-inflammatory cytokines and/or one or more pro-resolving mediators, an increase in one or more catecholamines, changes in one or more immune cell populations or one or more immune cell surface co-stimulatory molecules, a reduction in one or more factors involved in the inflammation cascade, and a reduction in one or more immune response mediators. In some embodiments, the generating the stimulation that activates the ALOX15 pathway produces an improvement in four or more physiological parameters in the subject, where the improvement in the physiological parameters is selected from the group consisting of: a reduction in one or more pro-inflammatory cytokines, an increase in one or more anti-inflammatory cytokines and/or one or more pro-resolving mediators, an increase in one or more catecholamines, changes in one or more immune cell populations or one or more immune cell surface co-stimulatory molecules, a reduction in one or more factors involved in the inflammation cascade, and a reduction in one or more immune response mediators. In some embodiments, the generating the stimulation that activates the ALOX15 pathway produces an improvement in five or more physiological parameters in the subject, where the improvement in the physiological parameters is selected from the group consisting of: a reduction in one or more pro-inflammatory cytokines, an increase in one or more anti-inflammatory cytokines and/or one or more pro-resolving mediators, an increase in one or more catecholamines, changes in one or more immune cell populations or one or more immune cell surface co-stimulatory molecules, a reduction in one or more factors involved in the inflammation cascade, and a reduction in one or more immune response mediators.

[0017] As disclosed herein, any embodiment disclosed herein when applicable can be applied to any aspect.

[0018] Various embodiments of systems, methods, and devices within the scope of the appended claims each have several aspects, no single one of which is solely responsible for the desirable attributes described herein. Without limiting the scope of the appended claims, some prominent features are described herein. After considering this discussion, and particularly after reading the section entitled “Detailed Description” one will understand how the features of various embodiments are used. INCORPORATION BY REFERENCE

[0019] All publications, patents, and patent applications mentioned in this specification are herein incorporated by reference in their entireties to the same extent as if each individual publication, patent, or patent application was specifically and individually indicated to be incorporated by reference. In the event of a conflict between a term herein and a term in an incorporated reference, the term herein controls.

BRIEF DESCRIPTION OF THE DRAWINGS

[0020] The implementations disclosed herein are illustrated by way of example, and not by way of limitation, in the figures of the accompanying drawings. Like reference numerals refer to corresponding parts throughout the several views of the drawings.

[0021] Figure 1 illustrates an example system for treating an inflammatory condition in a subject, in accordance with some embodiments of the present disclosure.

[0022] Figure 2 illustrates an example method for treating an inflammatory condition in a subject, in accordance with some embodiments of the present disclosure, where optional processes are indicated by dashed boxes.

[0023] Figures 3A, 3B, 3C, 3D, 3E, 3F, 3G, 3H, 31, 3J, 3K, 3L, and 3M show experimental results illustrating that splenic nerve stimulation (SpNS) releases NA that suppresses TNF-α in pig splenocytes. Fig. 3 A illustrates a pig splenic neurovascular bundle (NVB) section stained with antibodies against NF/βTub III (red) and MBP (green). Nuclei were counter stained with DAPI (blue). The insert shows a high magnification image of the fascicle indicated by the dashed white box; the arrowhead indicates an MBP positive axon. Figs. 3B, C show immunofluorescent images of pig SpN fascicles stained with antibodies against TH (red) and ChAT (green) in (B); TH (red) and CGRP (green) in (C). In both panels, cell nuclei were counterstained with DAPI (blue). Figs. 3D-F show immunofluorescent images of pig spleen stained with antibodies against CD1 lb (red) and TH (green) in (D); TH (red) and CD3 (green) in (E); CD1 lb (red) and CD3 (green) in (F). Fig. 3G is a schematic illustration of the experiments performed to quantify the amount of NA secreted during SpNS in pigs. Fig. 3H shows the concentration of NA (ng/mL) found within the SpV and the JV prior (baseline 1 and baseline 2) and during 2 stimulations (Stim 1 and Stim 2) of the pig splenic NVB. The graph on the right shows the data from both stimulations combined for all 4 pigs. Individual values (n=4), with mean + s.e.m. are shown. Fig. 31 is a schematic illustration of the in vitro splenocyte model used. Figs. 3 J, K illustrate quantification of TNF-α concentration in medium conditioned by splenocytes in CT (medium only), LPS, NA or NA + LPS conditions for 3 (J) and 24 (K) h expressed as % over LPS. Individual values (n=7), with mean + s.d. are shown. Fig. 3L illustrates quantification of TNF-a concentration (expressed as % over LPS) in medium conditioned by splenocytes in CT (medium only), LPS, NA or NA + LPS conditions 3 h after LPS exposure. In the latter group NA was added either 1 h before or 1 h after LPS exposure. Individual values (n=6), with mean + s.d. are shown. Fig. 3M: On the left, a schematic representation of the experiment in which the optimal time window between NA exposure and LPS challenge was investigated. On the right, the quantification of TNF-suppression (expressed as % over the maximum response) induced by NA at each time point tested. Data are shown as mean (n=3). ± s.e.m. *, P<0.05; **, P<0.01; ***, P<0.001.

[0024] Figures 4A, 4B, 4C, 4D, 4E, 4F, 4G, 4H, 41, and 4J show experimental results illustrating that SpNS and LVNS protect animals from cardiovascular collapse in a high dose endotoxemia model. Fig. 4A is a schematic illustration of the study design. The stimulation time point is marked with a lightning bolt sign. After a naive phase where ex vivo peripheral whole blood LPS assay was performed prior and after stimulation, a high dose (2.5 pg/kg, i.v.) of LPS was administered at the same time of a second stimulation. Peripheral blood was collected for routine hematology, biochemistry and cytokine analysis every 0.5 h. Fig. 4B illustrates quantification of the LPS-induced TNF-a from ex vivo cultured peripheral whole blood. The concentration of TNF-a expressed as % over the baseline value (defined as mean concentration of -2, - 1 and. 0 h time points), is shown as mean (n=6) ± s.d. Fig. 4C illustrates quantification of total white blood cell (WBC) count in peripheral blood samples during the naive phase and up to 0.5 h after in vivo LPS administration. Data are shown as mean (n=6) count ± s.e.m. Fig. 4D shows representative systemic mean arterial blood pressure (sMABP), HR and mCVP traces of a sham animal subjected to in vivo high dose LPS injection. Data are shown as % over baseline. The dashed line indicates the level at which the sMABP reaches 40 mmHg. Arrowheads indicate injections of vasopressin. Between 20 - 30 min after LPS injection the animal reached sMABP < 40 mmHg despite pharmacological treatment and had to be euthanized (humane endpoint). Fig. 4E illustrates the number of animals requiring vasopressin treatment after LPS injection. Single animals and mean (n=6) % ± s.e.m. are shown. Fig. 4F shows a box plot illustrating the lowest sMABP value (expressed as % over baseline) at 0.5 h post LPS injection. Single data points with median and min/max are shown. Baseline was defined as mean of 10 min prior to LPS injection. Fig. 4G illustrates the number of animals reaching humane endpoint (defined as sMABP < 40 mmHg, despite pharmacological treatment) within the 2 h window post in vivo LPS injection and therefore euthanized. Figs. 4H- J illustrate quantification of peripheral plasma levels of TNF-a and IL-6. Data are expressed as concentration (pg/mL) in (H), or as AUC (between 0 - 2 h post LPS) in (I) and (J). Data are shown as mean (n=6) ± s.e.m. *, P≤0.05; **, P≤0.01; ***, P≤0.001 or actual P values are shown.

[0025] Figures 5A, 5B, 5C, 5D, 5E, 5F, 5G, and 5H show experimental results illustrating that SpNS and eLVNS reduce TNF-a and IL-6 in a low dose endotoxemia model. Fig. 5A is a schematic illustration of the low dose (0.25 pg/Kg LPS, i.v.) endotoxemia model in pigs. Animals were divided into 5 groups (Sham, n = 7; Dexamethasone, n = 3; SpNS, n = 6; LVNS, n = 6; eLVNS, n=5). Dexamethasone (Dex) was administered immediately after induction of anesthesia (-2.5 h) and at the time of LPS injection (0 h). Electrical stimulation was delivered continuously from -2 to +1 h relative to LPS injection. Blood samples were collected every 0.5 h, between -2 and +4 h, from the JV for cytokine, as well as for hematology and biochemistry analysis. Fig. 5B illustrates concentration (pg/mL) of TNF-a (circles; left Y axis) and IL-6 (triangles; right Y axis) over time in all groups. Data are shown as mean (n=3-7) ± s.e.m. Figs. 5C, D illustrate quantification of total cytokine production as measured by AUC between 0.5 - 2 h post LPS for TNF-a (C) and between 1.5 - 4 h for IL-6 (D). Fig. 5E shows the concentration of TNF-a and IL-6 (expressed as AUC) for each individual animal within the 4 treatment groups. The solid lines indicate the mean TNF-a and IL-6 AUC values of the sham group. The bottom left quadrant in grey indicates the area in which therapeutic efficacy (concomitant reduction of TNF-a and IL-6, vs. mean sham values) is achieved. Figs. 5F-H illustrate quantification of peripheral blood white cell count in the different groups; specifically total white blood cells (F), monocytes (G) and lymphocytes (H) measured at time 0 h (prior to LPS injection). Figs. 5C-D and F-H: Individual values (n= 3-7) with mean + s.e.m. are shown. *, P<0.05; **, P<0.01; *** P<0.001; **** P<0.0001 or actual P values are shown.

[0026] Figures 6A, 6B, and 6C illustrate differential regulation of plasma lipid mediator profiles following acute SpN neuromodulation. Terminally anesthetized pigs received sham or SpN stimulation for 3 h (from -2 h to +1 h relative to LPS administration). Plasma was collected (A) at 0 h (immediately prior to LPS challenge), (B) 0.5 h, and (C) 2 h post LPS challenge and LM were investigated using LC-MS/MS-based lipid mediator profiling. Results were interrogated using Partial Least Square Discriminant Analysis. Left panels illustrate display score plots. Shaded area represents the 95% interval confidence. Right panels illustrate plots displaying the LM with the 10 highest VIP scores from component 1. n=6 for the sham group and n=6 for the SpNS group.

[0027] Figure 7 provides experimental results showing that acute SpN neuromodulation downregulates plasma prostaglandins and upregulates SPM. Terminally anesthetized pigs received sham or SpN stimulation for 2 h prior to LPS challenge, blood was collected just prior to LPS injection and LM were investigated using LC-MS/MS-based lipid mediator profiling. Flux down each of the bioactive metabolomes was assessed. The figure shows pathway analysis for the differential expression of mediators from the (left panel) EPA (EPA mediators indicated by dashed white lines) and AA, and (right panel) DHA and n-3 DPA (n-3 DPA mediators indicated by dashed white lines) bioactive metabolomes in the stimulated group when compared to the sham group. Results are expressed as the fold change. n=6 for the sham group and n=6 for the SpNS group.

[0028] Figure 8 provides experimental results showing that acute SpN neuromodulation shifts plasma lipid mediator profiles in response to LPS. Terminally anesthetized pigs received sham or SpN stimulation for 3 h (from -2 h to +1 h relative to LPS administration). Pigs were challenged with LPS at time 0 h, blood was collected after 0.5 h and LM were investigated using LC-MS/MS-based lipid mediator profiling. Flux down each of the bioactive metabolomes was assessed. The figure shows pathway analysis for the differential expression of mediators from the (left panel) EPA (EPA mediators indicated by dashed white lines) and AA, and (right panel) DHA and n-3 DPA (n-3 DPA mediators indicated by dashed white lines) bioactive metabolomes in the stimulated group when compared to the sham group. Results are expressed as the fold change. n=6 for the sham group and n=6 for the SpNS group.

[0029] Figures 9A, 9B, 9C, 9D, 9E, 9F, 9G, 9H, 91, 9J and 9K illustrate experimental results showing that chronic SpN neuromodulation evokes anti-inflammatory effects during LPS-induced endotoxemia. Fig. 9A illustrates a graph showing TNF-a concentration quantified in the peripheral plasma collected at different time points after intravenous (i.v.) injection of 0.025 pg/kg of LPS. The data are shown for non-surgical sham (grey circles), sham (black circles) and SpNS (grey triangles) animals. Fig. 9B illustrates a graph showing the quantification of the relevant TNF-a AUC (between 0.5-2 h post LPS) derived from Fig. 9A. Data are shown and were analyzed using the expressed as Logio normalized AUC values. The single data points with mean are shown. Figs. 9C-E illustrate graphs showing total white blood cell (C), neutrophil (D) or monocyte (E) cell counts from peripheral blood between 0 and 24 h post LPS injection. Figs. 9F and H illustrate dot plots (forward scatter vs. side scatter view) showing the changes over time (0, 3 and 24 hrs post LPS) of CD 16+ (F) and CD172a+ (H) gated monocyte populations in a sham and a SpNS representative pig. Figs. 9G and I illustrate quantification of peripheral blood monocytes stained with antibodies against CD 16 (G) or CD 172a (I) over time. Fig. 9 J illustrates representative histograms showing the changes over time of CD14 expression on CD16+ monocytes in a sham and a SpNS representative pig. Fig. 9K illustrates quantification of the median fluorescence intensity (MFI) of CD 14 expression on CD16+ monocytes over time. (A, C-E and G, I, K) Data are expressed as mean ± s.e.m. (G, I, K) Data are expressed as relative change over the baseline (value prior to LPS injection). (A-B) n=6 for SpNS; n=5 for sham. (C-E) n=6 for SpNS; n=5 for sham. (G, I, K) n=3 for SpNS; n=4 for sham. The sham group is shown in black and SpNS group in grey. * P<0.05; ** P <0.005.

[0030] Figures 10A, 10B, 10C, and 10D illustrate experimental results showing that chronic SpN neuromodulation reprograms peripheral blood lipid mediator profiles. Porcine splenic nerve was stimulated chronically for 8 days. Plasma was collected immediately prior to LPS challenge (A), 0.5 (B), 3 (C) and 24 (D) h post LPS challenge and lipid mediators were investigated using LC-MS/MS-based profiling. Results were interrogated using Partial Least Square Discriminant Analysis. Left panels illustrate display score plots. The shaded area represents the 95% interval confidence. Right panels illustrate plots displaying the lipid mediators with the 10 highest VIP scores from component 1. n=5 for the sham group and n=6 for the SpNS group.

[0031] Figure 11 illustrates experimental results showing that chronic SpN neuromodulation increases peripheral blood pro-resolving mediators prior to systemic LPS administration. A porcine splenic nerve was chronically-stimulated for 8 days and plasma was collected 24 h after LPS systemic administration. Lipid mediators were investigated using LC- MS/MS-based profiling. Flux down each of the bioactive metabolomes was assessed. The figure illustrates pathway analysis for the differential expression of mediators from the DHA and n-3 DPA (left panel; n-3 DPA mediators indicated by dashed white lines) and EPA and AA (right panel; EPA mediators indicated by dashed white lines) bioactive metabolomes in the SpNS group when compared to the sham group. Results are expressed as the fold change.

Solid line boxes indicate the SPMs upregulated in the SpNS group at 24h post LPS (vs. sham). n=5 for the sham group and n=6 for the SpNS group.

[0032] Figure 12A, 12B, 12C, 12D, 12E, 12F, 12G, 12H, 121, 12J, 12K, and 12L provide experimental results showing that chronic SpN neuromodulation does not cause systemic immune suppression in naive pigs. Figs. 12A-C are graphs showing the plasma TNF-a concentration following incubation of peripheral whole blood with either 0, 100 or 1000 ng/mL of LPS, performed at the different time points. Figs. 12D-F are graphs showing total white blood cell (D), neutrophil (E) or monocyte (F) cell counts from peripheral blood over time.

Figs. 12G and I are dot plots (forward scatter vs. side scatter view) showing the changes over time (0, 2 and 7 days) of CD 16+ (G) and CD172a+ (I) gated monocytes population in a sham (black) and a SpNS (grey) representative animals. Figs. 12H and J are graphs showing quantification of peripheral blood monocytes stained with antibodies against CD 16 (H) or CD172a (J) over time. Fig. 12K provides representative histograms showing the changes over time of CD14 expression on CD16+ monocytes in a sham and a SpNS representative animals. Fig. 12L illustrates quantification of the median fluorescence intensity (MFI) of CD 14 expression on CD 16+ monocytes. (H, J, L) Data are expressed as relative change over the average baseline value (average between -2, -1 and 0). (A-F and H, J, L) Data are expressed as mean ± s.e.m.. The sham group is shown in black and the SpNS group in grey. (A-F, H, J, and L) The black bar indicates the SpNS period. (A-F) n=6 for SpNS; n=6 for sham. (H, J, L) n=3 for SpNS; n=4 for sham.

[0033] Figures 13A, 13B, and 13C illustrates an example gating strategy for flow cytometric analysis. Fig. 13 A: Gating strategy for Panel 3: Initial gate on all cells in FSC vs. SSC view, then quadrant division of the CD8 vs. CD4 view, to identify CD4+, CD8+ and CD4+CD8+ (double positive) populations. Fig. 13B: Gating strategy for Panel 5: Initial gate on all cells in FSC vs. SSC view, then gate on CD14+ monocytes in SSC vs. CD14 view, or gate on CD16+ monocytes in SSC vs. CD16 view. CD16+ monocytes were further analyzed for their CD14 expression and split into CD141ow and CD14high populations. Fig. 13C: Gating strategy for Panel 7: Initial gate on all cells in FSC vs. SSC view, then gate on CD172a+ monocytes in SSC vs. CD172a view, or gate on CD163+ monocytes in SSC vs. CD163 view. FSC = Forward Scatter, SSC = Side Scatter.

[0034] Figures 14A, 14B, 14C, and 14D illustrate an exploration of marker expression on populations of peripheral blood monocytes. On the bottom set of plots (FSC vs. SSC view), blue cells mapped into the expected lymphocyte region, orange into monocyte region and green into granulocyte region (labelled circles). Fig. 14A: CD14+ cells were found in all three mononuclear cell subsets (low expression in a subset of lymphocytes, high expression in monocytes and granulocytes). Fig. 14B: CD16+ cells were found in all three mononuclear cell subsets (low expression in lymphocytes, high expression in monocytes and granulocytes). Fig. 14C: CD172a+ cells were found in monocyte and granulocyte populations (high expression in both). Fig. 14D: CD163+ cells were only found in the monocyte population. For all four markers, the monocyte population could be reliably separated on the SSC vs. marker view.

[0035] Figures 15A, 15B, 15C, 15D, and 15E collectively provide experimental results showing that chronic SpN neuromodulation does not affect peripheral leukocytes in naive pigs. Figs. 15A-E are graphs showing the quantification of peripheral leukocytes. In particular, the changes in peripheral CD4+ (A), CD8+ (B), CD4+CD8+ (C) lymphocytes, and CD14+ (D), or CD 163+ (E) monocytes are shown. Data were expressed as relative change of the proportion of positive cells over the baseline (average value of -2, -1 and 0 time point) at the different time points. Data are shown as mean ± s.e.m. Gating strategy is shown in Figs. 13A-C and 14A-D. (A-E) n=3 for SpNS; n=4 for sham. The sham group is shown in black and the SpNS group in grey.

[0036] Figures 16A, 16B, 16C, 16D, and 16E collectively provide experimental results showing that chronic SpN neuromodulation affects peripheral monocytes during endotoxemia. Figs. 16 A-E are graphs showing the quantification of peripheral leukocytes. In particular, the changes in peripheral CD4+ (A), CD8+ (B), CD4+CD8+ (C) lymphocytes, and CD 14+ (D), or CD 163+ (E) monocytes are shown. Data were expressed as relative change of the proportion of positive cells over the baseline (value at time 0 h, prior to LPS injection) at the different time points. Data are shown as mean ± s.e.m.. Gating strategy is shown in Figs. 13A-C and 14A-D. (A-E) n=3 for SpNS; n=4 for sham. The sham group is shown in black and the SpNS group in grey.

[0037] Figure 17 provides experimental results showing that chronic SpN neuromodulation leads to long term reprograming of peripheral blood lipid mediator profiles in response to LPS. A porcine splenic nerve was stimulated chronically for 8 days prior to systemic LPS administration and blood was collected just prior to LPS administration (0 h). Plasma lipid mediators were investigated using LC-MS/MS-based profiling. Flux down each of the bioactive metabolomes was assessed. The figure illustrates pathway analysis for the differential expression of mediators from the (top panel) EPA (EPA mediators indicated by dashed white lines) and AA, and (bottom panel) DHA and n-3 DPA (n-3 DPA mediators indicated by dashed white lines) bioactive metabolomes in the SpNS group when compared to the sham group. Results are expressed as the fold change. n=5 for the sham group and n=6 for the SpNS group.

[0038] Figure 18 illustrates an example assay showing the effect of NA on the production of ALOX 15 -derived Resolvin DI from human spleen-derived immune cells, in accordance with some embodiments of the present disclosure.

DETAILED DESCRIPTION

[0039] Introduction.

[0040] The inflammatory status of the body is monitored and regulated through the neuroimmune axis, connecting the brain to the immune system via both humoral and neural pathways [1-3], In particular, the “inflammatory reflex” [3] controls systemic immune responses; detection of inflammatory stimuli in the periphery is communicated to the brain that induces outflow of neural signals to promote peripheral immune responses proportionate to the threat. Studies in rodent models have identified the “cholinergic anti-inflammatory pathway” as the brain’s efferent response to infection and inflammation through peripheral neurotransmitters released in lymphoid organs, mainly the spleen [4, 5], Within this pathway the peripheral connection between the vagus nerve (VN), the splenic nerve (SpN) and its terminal release of noradrenaline (NA) into the spleen have been identified as crucial components of this neural circuit [6, 7],

[0041] Importantly, the anti-inflammatory pathway can be harnessed to promote immune control. For example, electrical stimulation of the cervical VN (vagus nerve stimulation; VNS) has been shown to be effective in reducing lipopolysaccharide (LPS)-induced systemic levels of tumor necrosis factor alpha (TNF-a) [4, 6, 7], as well as to be effective in pre-clinical rodent models of inflammatory diseases [4, 8-10], Additionally, early clinical feasibility studies have provided preliminary evidence of immunomodulatory effects of VNS in human patients [13, 14]

[0042] However, the functionally and anatomically complex composition of the VN limits its effectiveness as a medium for treatment of inflammatory conditions. In animals and humans, the VN contains both afferent and efferent axons, with motor and autonomic axons of varying size (large, medium and small) and degree of myelination (heavily myelinated, lightly myelinated and unmyelinated axons) innervating multiple organs and muscles [16], As a consequence, current VNS protocols result in activation of off-target circuits that can cause dysphonia, coughing, hoarseness, pain and dyspnea [14, 16, 17], although in most patients these can be managed and can also improve over time [18], The concomitant activation of both efferent and afferent circuits with currently available clinical systems can represent another challenge since it can potentially lead to opposing effects. Although the existence of opposing effects on inflammatory regulation has not yet been investigated, activation of efferent and afferent pathways within the VN can, for example, have contrasting effects on blood glucose regulation [18], Further, it remains unclear which axons (e.g., efferent vs. afferent, myelinated vs. unmyelinated) within the VN relay immunomodulatory signals to peripheral organs [19, 20], As a result, it is difficult to optimize stimulation parameters necessary to activate axons within the VN that carry signals to the spleen. Typically, clinical parameters have been selected based on the individual patient’s tolerance of off-target effects [13, 21] absent of evidence of direct activation of the anti-inflammatory pathway due to lack of an organ specific biomarker. Most of the evidence suggest that stimulation paradigms used in the clinic only activates large myelinated and some of the medium-sized myelinated axons [12, 21]; activation of small myelinated and unmyelinated axons, that are known to carry information to and from abdominal organs [16], is generally not achieved since they require much higher stimulation charges than those typically used in VNS [12, 21],

[0043] Accordingly, what is needed in the art are methods for modulating the immune response via the anti-inflammatory pathway that overcome the abovementioned disadvantages of VNS.

[0044] The spleen plays a major role as a reservoir of monocytes/macrophages and lymphocytes. These cells are activated during infection and inflammation, resulting in the production of cytokines and chemokines and in subsequent mobilization towards sites of inflammation/damage [52, 53], Since the SpN directly transmits neural signals to the spleen and is the fundamental nodal circuit in mediating the anti-inflammatory response [22], SpN stimulation (SpNS) may represent an alternative modality providing a more selective near organ modulation of the immune system.

[0045] The SpN consists of an abundant network of interconnecting fibers originating from abdominal ganglia [51, 29], This neuronal plexus runs along the splenic artery (SpA), together forming a neurovascular bundle (NVB), until it enters the splenic parenchyma where neurotransmitters, in particular catecholamines, are released, acting on immune cells as well as smooth muscle and endothelial cells. Proof of concept experiments in rodents have shown that immune responses can indeed be modulated by stimulation of the SpN with comparable cytokine suppressive effects to VNS [7, 23], For example, exogenous activation of neural pathways targeting the spleen via electrical stimulation of the cervical vagus nerve (VN) has been shown to induce cytokine modulation in small animals [4, 7], Using acute inflammatory models, this immunomodulatory effect has typically been demonstrated as a reduction in cytokine responses, particularly tumor necrosis factor alpha (TNF-a) [4, 7, 6], This effect has been shown to be dependent on the SpN, and on noradrenaline (NA) released from nerve terminals onto splenic leukocytes [6, 7], In addition, electrical stimulation of such neural pathways has been shown to reduce inflammation in animal models of arthritis [54, 10, 24] and inflammatory bowel disease (IBD) [55, 56], Importantly, clinical evidence suggests that neuromodulation of these pathways could be beneficial in patients with rheumatoid arthritis (RA) [13] and Crohn’s disease [14] via electrical stimulation of the VN.

[0046] However, the adaptation of stimulation parameters from rodent to human is hampered by anatomical (e.g, size of nerves), histological (e.g., number of axons, connective tissue thickness, proportion of adipose tissue) and immunological differences. These differences limit the usefulness of the rodent model for the development of clinical bioelectronic medicines, which typically involves the accurate estimation and validation of stimulation parameters in a surgically and anatomically correlated model to define device and therapy requirements for efficacious and successful treatment.

[0047] Accordingly, what is needed in the art are improved systems and methods for neuromodulation of the immune system via the splenic nerve. Examples 1 and 2 in the Examples section below illustrate systems and methods for immune system neuromodulation using splenic nerve stimulation that is validated in large animal models, human tissues and in silico modelling [11, 12], Specifically, neural pathways targeting the spleen also exist in the pig [57], and other large animals [56, 58] with gross anatomy, histology, physiology and immune functions more closely associated to humans. Compared to small animals, in some embodiments, the use of large animal models can be used to aid in human-relevant clinical device design and therapy translation.

[0048] Neuromodulation of the immune system by targeting the splenic circuits can further be used, in some embodiments, for treatment of one or more inflammatory conditions. The SpN offers a suitable target for such an approach, due to its major role in regulating immunological responses as well as its proximity to the target organ, potentially avoiding some off-target effects seen with more upstream intervention sites of the autonomic nervous system.

[0049] For example, it has been shown that the autonomic nervous system can regulate dynamic mechanisms associated with leukocyte recruitment and resolution processes during inflammation [60, 36], with neurotransmitters and the spleen playing a putative central role. Resolution of inflammation is an active process requiring fine regulation of biosynthetic pathways that lead to the production of lipid mediators, called specialized pro-resolving mediators (SPMs; [61]). SPMs play a central role in reprogramming both innate and adaptive immune responses to regulate immune cell recruitment and cytokine production, and loss of vagal signaling leads to a disruption in SPM biosynthesis and disrupted resolution mechanisms [60, 36],

[0050] As detailed herein (see the section entitled “Activation of ALOX15 Pathway,” below), SPMs include the resolvins, a class of metabolites derived from, for example, docosahexanoic acid (DHA) and eicosapentaenoic acid (EP A). DFIA and EPA can be metabolized to the D-series (e.g., RvDl, RvD2, RvD3, RvD4, RvD5, and/or RvD6) and E- series (e.g, RvEl, RvE2, RvE3, and/or RvE4) resolvins, respectively, via enzymatic activity of human lipoxygenase arachidonate 15-lipoxygenase (ALOX15). [0051] The present disclosure provides SpNS-induced activation of pathways associated with production of SPMs, particularly those derived from ALOX15 activity, for the treatment of one or more inflammatory conditions (e.g., chronic and/or acute inflammatory disorders). In some embodiments, the activation of the ALOX15 pathway comprises a change (e.g., an increase) in the physiological levels of SPMs (e.g., resolvins).

[0052] In some embodiments, the present disclosure provides stimulation parameters for acute and/or chronic stimulation of the SpN in a subject. In some embodiments, the presently disclosed systems and methods further comprise human-relevant stimulation parameters for therapeutic utility by acute and/or chronic stimulation of the SpN in the subject.

[0053] In some embodiments, the stimulating the SpN comprises neuromodulation of one or more inflammatory resolution pathways. For example, in some embodiments, the stimulating the SpN comprises reducing production of one or more inflammatory cytokines (e.g., TNF-a and/or IL-6). In some embodiments, the stimulating the SpN further comprises monitoring and/or determining the production or amount of one or more inflammatory cytokines (e.g., TNF-a and/or IL-6) to assess the efficacy of SpNS.

[0054] In some embodiments, the stimulating the SpN comprises increasing production of one or more inflammation-resolving mediators. In some embodiments, the stimulating the SpN further comprises monitoring and/or determining the production or amount of one or more inflammation-resolving mediators to assess the efficacy of SpNS.

[0055] In some embodiments, the stimulating the SpN comprises increasing production of SPMs, particularly ALOX 15 -derived SPMs. In some embodiments, the stimulating the SpN further comprises monitoring and/or determining the production or amount of SPMs, including ALOX 15 -derived SPMs.

[0056] In some embodiments, the activation of the ALOX15 pathway comprises increasing production of one or more ALOX 15 -derived resolvins, such as the D-series resolvins. In some embodiments, the activation of the ALOX15 pathway comprises increasing production of RvDl. In some embodiments, the systems and methods provided herein further comprise monitoring and/or determining the production or amount of one or more ALOX 15 -derived resolvins, such as the D-series resolvins (e.g., RvDl).

[0057] In an example method, a subject (e.g., a patient with a chronic and/or acute inflammatory condition) is implanted with a signal-conducting interface (e.g., a circumferential cuff electrode, using a minimally-invasive laparoscopic surgical procedure). The signal- conducting interface is placed in signaling contact with one or more splenic nerves in the subject (e.g., the circumferential cuff electrode interface is placed around the splenic nerve). A connection is formed between the signal-conducting interface and a signal-generating source (e.g., the circumferential cuff electrode is connected to an implantable pulse generator (IPG) to enable the delivery of neuromodulation). Neuromodulation of the immune system is performed via stimulation of the one or more splenic nerves (e.g., electrical stimulation using selected stimulation parameters such as amplitude, pulse width, pulse height, total charge, waveform, frequency, periodicity, duration, number of pulses, pulse sequence and/or paradigm).

Stimulation of the one or more splenic nerves activates the ALOX15 pathway, which can be determined, in some embodiments, by measuring and/or analyzing physiological responses such as cytokine production, cell phenotypic changes and SPMs present in peripheral blood prior to, during, and/or after SpN stimulation.

[0058] Reference will now be made in detail to embodiments, examples of which are illustrated in the accompanying drawings. In the following detailed description, numerous specific details are set forth in order to provide a thorough understanding of the present disclosure. However, it will be apparent to one of ordinary skill in the art that the present disclosure may be practiced without these specific details. In other instances, well-known methods, procedures, components, circuits, and networks have not been described in detail so as not to unnecessarily obscure aspects of the embodiments.

[0059] Definitions.

[0060] As used herein, the term “about” or “approximately” can mean within an acceptable error range for the particular value as determined by one of ordinary skill in the art, which can depend in part on how the value is measured or determined, e.g., the limitations of the measurement system. For example, “about” can mean within 1 or more than 1 standard deviation, per the practice in the art. “About” can mean a range of ±20%, ±10%, ±5%, or ±1% of a given value. The term “about” or “approximately” can mean within an order of magnitude, within 5-fold, or within 2-fold, of a value. Where particular values are described in the application and claims, unless otherwise stated the term “about” meaning within an acceptable error range for the particular value can be assumed. The term “about” can have the meaning as commonly understood by one of ordinary skill in the art. The term “about” can refer to ±10%. The term “about” can refer to ±5%.

[0061] As used herein, the term “subject” refers to any mammal.

[0062] As used herein, the term “treatment” refers to obtaining a desired physiologic effect.

The effect may be prophylactic in terms of completely or partially preventing a disease or condition or symptom thereof and/or may be therapeutic in terms of a partial or complete recovery from an injury, disease, or condition and/or amelioration of an adverse effect attributable to the injury, disease or condition and includes arresting the development or causing regression of a disease or condition. The effects may be a delay in onset, amelioration of symptoms, improvement in patient survival, increase in survival time or rate, improvement in cognitive function, etc. The effect may be improved health following eradication of the disease condition, e.g, a lessening of lasting effects caused by the disease or condition and/or long-term complications resulting from the disease or condition (e.g, during or after the partial or complete recovery from the disease or condition). The effect of treatment can be compared to an individual or pool of individuals not receiving the treatment.

[0063] In some embodiments, treatment refers to obtaining or inducing an improvement in one or more physiological parameters of a subject. Useful physiological parameters can include one or more of: the level of a pro-inflammatory cytokine, the level of an anti-inflammatory cytokine, the level of a pro-resolving mediator, the level of a catecholamine, the level of an immune cell population, the level of an immune cell surface costimulatory molecule, the level of a factor involved in the inflammation cascade, the level of an immune response mediator, and/or the rate of splenic blood flow.

[0064] In some embodiments, an “improvement in a physiological parameter” is taken to mean that, for any given physiological parameter, an improvement is a change in the value of that parameter in the subject towards the normal value or normal range for that value (e.g., towards the expected value in a healthy subject). In contrast, a “worsening of a physiological parameter” is taken to mean that, for any given physiological parameter, worsening is a change in the value of that parameter in the subject away from the normal value or normal range for that value (e.g, away from the expected value in a healthy subject).

[0065] As used herein, the term “acute inflammatory condition” refers to a rapid deterioration in a subject’s physiological status that may be life threatening if left untreated. Examples include trauma, sepsis, hemorrhage, severe hemophilia, severe episodes of lupus, episodes of severe Crohn’s, allograph/autograph rejection, anaphylaxis, endotoxic shock, acute respiratory distress syndrome (ARDS), severe respiratory distress syndrome (SARS), and coronavirus disease 19 (COVID-19). In some embodiments, subjects with an acute condition can require urgent medical care to relieve suffering and minimize morbidity and mortality risk. In some embodiments, treatments of acute medical conditions vary according to the disease, and, in some embodiments, success rates of treatments vary according to the severity of the condition. [0066] As used herein, the term “chronic inflammatory condition” refers to a condition that is characterized by prolonged clinical course during which there is little change or slow progression of underlying pathology. Examples of chronic inflammatory medical conditions include, but are not limited to, arthritis (e.g., rheumatoid arthritis), chronic pancreatitis, chronic obstructive pulmonary disease, or chronic heart failure. However, subjects with chronic conditions may suffer from acute exacerbations of the underlying disease process, and this is generally referred to as acute-on-chronic episodes. The distinction between acute and chronic medical conditions is well known in the art.

[0067] As used herein, the term “stimulation” refers to signaling activity for at least part of the nerve is increased compared to baseline neural activity in that part of the nerve, where baseline neural activity is the signaling activity of the nerve in the subject prior to any intervention. In some embodiments, stimulation results in the creation of neural activity which increases the total neural activity in that part of the nerve. As used interchangeably herein, the terms “neural activity” or “nerve activity” of a nerve refers to the signaling activity of the nerve, for example the amplitude, frequency and/or pattern of action potentials in the nerve. The term “pattern,” as used herein in the context of action potentials in the nerve, is intended to include one or more of: local field potential(s), compound action potential(s), aggregate action potential(s), magnitudes, frequencies, areas under the curve and other patterns of action potentials in the nerve or sub-groups (e.g., fascicules) of neurons therein.

[0068] As used herein, stimulation typically involves increasing neural activity, e.g., generating action potentials beyond the point of the stimulation in at least a part of the nerve. At any point along the axon, a functioning nerve will have a distribution of potassium and sodium ions across the nerve membrane. The distribution at one point along the axon determines the electrical membrane potential of the axon at that point, which influences the distribution of potassium and sodium ions at an adjacent point, further determining the electrical membrane potential of the axon at that point, etc. For example, in a nerve operating in its normal state, action potentials propagate between adjacent points along the axon, which can be observed using conventional experimentation. Stimulation of neural activity can be characterized as a distribution of potassium and sodium ions at one or more points in the axon, which is created by the application of a temporary external electrical field, rather than an electrical membrane potential at a point or adjacent points of the nerve as a result of a propagating action potential. The temporary external electrical field artificially modifies the distribution of potassium and sodium ions within a point in the nerve, inducing depolarization of the nerve membrane and generating a de novo action potential across that point. For example, a nerve operating in a disrupted state can be observed by a distribution of potassium and sodium ions at a point in the axon (e.g, the point which has been stimulated) that has an electrical membrane potential that is not influenced or determined by the electrical membrane potential of an adjacent point.

[0069] In some embodiments, stimulation of neural activity refers to increasing neural activity that continues past the point of signal application. In some such embodiments, the nerve at the point of signal application is modified such that the nerve membrane is reversibly depolarized by an electric field, generating a de novo action potential that propagates through the modified nerve. Hence, the nerve at the point of signal application is modified in that a de novo action potential is generated. When the signal is an electrical signal, the stimulation can be based on the influence of electrical currents (e.g, charged particles, which can be one or more electrons in an electrode in signaling contact with the nerve, or one or more ions inside and/or outside the nerve) on the distribution of ions across the nerve membrane.

[0070] As used herein, stimulation of neural activity includes full stimulation of neural activity in the nerve. In some embodiments, stimulation of neural activity refers to increasing total neural activity within the whole nerve. Stimulation of neural activity can be partial stimulation. Partial stimulation can be such that the total signaling activity of the whole nerve is partially increased, that the total signaling activity of a subset of nerve fibers of the nerve is fully increased (e.g, there is no neural activity in that subset of fibers of the nerve), or that the total signaling of a subset of nerve fibers of the nerve is partially increased compared to baseline neural activity in that subset of fibers of the nerve. For example, stimulation of neural activity can be an increase in neural activity of <5%, <10%, <15%, <20%, <25%, <30%, <35%, <40%, <45%, <50%, <60%, <70%, <80%, <90% or <95% in a nerve and/or a subset of nerve fibers of the nerve.

[0071] In some embodiments, neural activity is measured by methods known in the art, for example, by the number of action potentials which propagate through the axon and/or the amplitude of the local field potential reflecting the summed activity of the action potentials. Stimulation of neural activity can be an alteration in the pattern of action potentials. It will be appreciated that the pattern of action potentials can be modulated without necessarily changing the overall frequency or amplitude. For example, stimulation of neural activity can be corrective or partially corrective. As used herein, the term “corrective” is taken to mean that the modulated neural activity alters the neural activity towards the pattern of neural activity in a healthy subject (e.g, axonal modulation therapy). Thus, upon cessation of signal application, neural activity in the nerve closely or substantially resembles the pattern of action potentials in the nerve observed in a healthy subject than prior to signal application. In some embodiments, corrective stimulation is any stimulation as defined herein.

[0072] For example, in some embodiments, application of a signal results in an increase in neural activity, and upon cessation of signal application the pattern of action potentials in the nerve resembles the pattern of action potentials observed in a healthy subject. By way of further example, in some embodiments, application of the signal results in neural activity resembling the pattern of action potentials observed in a healthy subject and, upon cessation of the signal, the pattern of action potentials in the nerve remains the pattern of action potentials observed in a healthy subject.

[0073] As used herein, stimulation of neural activity further comprises altering the neural activity in various other ways, such as increasing a particular part of the baseline neural activity and/or stimulating new elements of activity (e.g., in particular intervals of time, in particular frequency bands, according to particular patterns, etc.).

[0074] In some embodiments, stimulation of neural activity is reversible. As used herein, “reversible” is taken to mean that the modulation of neural activity is not permanent. For example, in some embodiments, upon cessation of the application of a signal, neural activity in the nerve returns substantially towards baseline neural activity within 1-60 seconds, within 1-60 minutes, within 1-24 hours (e.g, within 1-12 hours, 1-6 hours, 1-4 hours, 1-2 hours), or within 1-7 days (e.g, 1-4 days, 1-2 days). In some instances of reversible stimulation, the neural activity returns substantially fully to baseline neural activity. That is, the neural activity following cessation of the application of a signal is substantially the same as the neural activity prior to a signal being applied. In some embodiments, cessation of a reversible stimulus returns the nerve or the portion of the nerve to its normal physiological capacity to propagate action potentials.

[0075] In some embodiments, stimulation of neural activity is persistent or substantially persistent. As used herein, “persistent” is taken to mean that the neural activity has a prolonged effect. For example, in some embodiments, upon cessation of the application of a signal, neural activity in the nerve remains substantially the same as when the signal was being applied, e.g., the neural activity during and following signal application is substantially the same.

[0076] Several aspects are described below with reference to example applications for illustration. It should be understood that numerous specific details, relationships, and methods are set forth to provide a full understanding of the features described herein. One having ordinary skill in the relevant art, however, will readily recognize that the features described herein can be practiced without one or more of the specific details or with other methods. The features described herein are not limited by the illustrated ordering of acts or events, as some acts can occur in different orders and/or concurrently with other acts or events. Furthermore, not all illustrated acts or events are used to implement a methodology in accordance with the features described herein.

[0077] Exemplary System Embodiments.

[0078] Details of an exemplary system are now described in conjunction with Fig. 1. Fig. 1 is a block diagram illustrating a system 100 in accordance with some implementations. The device 100 in some implementations includes at least one or more processing units CPU(s) 102 (also referred to as processors), one or more network interfaces 104 for connecting the device to a network, a user interface 106 having a display 108, an input device 110, a memory 111, and one or more communication buses 114 for interconnecting these components. The one or more communication buses 114 optionally include circuitry (sometimes called a chipset) that interconnects and controls communications between system components.

[0079] In some embodiments, each processing unit in the one or more processing units 102 is a single-core processor or a multi-core processor. In some embodiments, the one or more processing units 102 is a multi-core processor that enables parallel processing. In some embodiments, the one or more processing units 102 is a plurality of processors (single-core or multi-core) that enable parallel processing. In some embodiments, each of the one or more processing units 102 are configured to execute a sequence of machine-readable instructions, which can be embodied in a program or software. The instructions may be stored in a memory location, such as the memory 111. The instructions can be directed to the one or more processing units 102, which can subsequently program or otherwise configure the one or more processing units 102 to implement methods of the present disclosure. Examples of operations performed by the one or more processing units 102 can include fetch, decode, execute, and writeback. The one or more processing units 102 can be part of a circuit, such as an integrated circuit. One or more other components of the system 100 can be included in the circuit. In some embodiments, the circuit is an application specific integrated circuit (ASIC) or a field- programmable gate array (FPGA) architecture.

[0080] In some embodiments, the network is Internet, an internet and/or extranet, or an intranet and/or extranet that is in communication with the Internet. In some embodiments, the network is a telecommunication and/or data network. In some embodiments, the network comprises one or more computer servers that can enable distributed computing, such as cloud computing. In some embodiments, the network, with the aid of the system 100, can implement a peer-to-peer network, which may enable devices coupled to the system 100 to behave as a client or a server. Such systems can be connected through a communications network to the Internet. The communications network can be any available network that connects to the Internet. The communications network can utilize, for example, a high-speed transmission network including, without limitation, Digital Subscriber Line (DSL), Cable Modem, Fiber, Wireless, Satellite and, Broadband over Powerlines (BPL). Examples of networks accessed by network interface 104 include, but are not limited to, the World Wide Web (WWW), an intranet and/or a wireless network, such as a cellular telephone network, a wireless local area network (LAN) and/or a metropolitan area network (MAN), and other devices by wireless communication. The wireless communication optionally uses any of a plurality of communications standards, protocols and technologies, including but not limited to Global System for Mobile Communications (GSM), Enhanced Data GSM Environment (EDGE), high- speed downlink packet access (HSDPA), high-speed uplink packet access (HSUPA), Evolution, Data-Only (EV-DO), HSPA, HSPA+, Dual-Cell HSPA (DC-HSPDA), long term evolution (LTE), near field communication (NFC), wideband code division multiple access (W-CDMA), code division multiple access (CDMA), time division multiple access (TDMA), Bluetooth, Wireless Fidelity (Wi-Fi) (e.g., IEEE 802.11a, IEEE 802.1 lac, IEEE 802.1 lax, IEEE 802.11b, IEEE 802.11g and/or IEEE 802.1 In), voice over Internet Protocol (VoIP), Wi-MAX, a protocol for email (e.g., Internet message access protocol (IMAP) and/or post office protocol (POP)), instant messaging (e.g., extensible messaging and presence protocol (XMPP), Session Initiation Protocol for Instant Messaging and Presence Leveraging Extensions (SIMPLE), Instant Messaging and Presence Service (IMPS)), and/or Short Message Service (SMS), or any other suitable communication protocol, including communication protocols not yet developed as of the filing date of this document.

[0081] In some embodiments, the display 108 is a touch-sensitive display, such as a touch- sensitive surface. In some embodiments, the user interface 106 includes one or more soft keyboard embodiments. In some implementations, the soft keyboard embodiments include standard (QWERTY) and/or non-standard configurations of symbols on the displayed icons. The user interface 106 can be configured to provide a user (e.g., a health professional) with graphic showings of, for example, physiological data, signal feedback, disease conditions, and treatment suggestion or recommendation of preventive steps based on the disease conditions. The user interface may enable user interactions with particular tasks (e.g., reviewing the disease conditions and adjusting treatment plans). [0082] The memory 111 may be a non-persistent memory, a persistent memory, or any combination thereof. The non-persistent memory can include high-speed random access memory, such as DRAM, SRAM, DDR RAM, ROM, PROM, EEPROM, flash memory, whereas the persistent memory typically includes CD-ROM, digital versatile disks (DVD) or other optical storage, magnetic cassettes, magnetic tape, magnetic disk storage or other magnetic storage devices, magnetic disk storage devices, optical disk storage devices, flash memory devices, or other non-volatile solid state storage devices. Regardless of its specific implementation, the memory 111 comprises at least one non-transitory computer readable storage medium, and it stores thereon computer-executable executable instructions which can be in the form of programs, modules, and data structures.

[0083] In some embodiments, as shown in Fig. 1, the memory 111 stores the following:

• instructions, programs, data, or information associated with an operating system 116 (e.g., iOS, ANDROID, DARWIN, RTXC, LINUX, UNIX, OS X, WINDOWS, or an embedded operating system such as VxWorks), which includes various software components and/or drivers for controlling and managing general system tasks (e.g., memory management, storage device control, power management, etc.) and facilitates communication between various hardware and software components;

• instructions, programs, data, or information associated with an optional file system (which may be a component of operating system 116), for managing files stored or accessed by the system 100;

• instructions, programs, data, or information associated with an optional network communication module 118 for connecting the system 100 with other devices and/or to a communication network;

• a signal generation module 120 that generates a signal (e.g., an electrical, infrared, ultrasound, mechanical, and/or magnetic pulse) and comprising one or more signal parameters 122 (e.g., 122-1, 122-2,. . ,,122-P) including, for example, a periodicity, pulse sequence (e.g., pattern), number of pulses, pulse width, waveform, charge, amplitude (e.g., charge density per phase), frequency, and/or duration;

• an optional signal responsive module 124 that responds upon receipt of one or more inputs 126 (e.g., 126-1, 126-2,. . ,,126-Q) including, for example, a preconfigured and/or operator-selectable signal, an external trigger, and/or a signal indicative of a physiological parameter; • an optional physiological data store 128 comprising one or more physiological parameters (e.g., 130-1, 130-2,. . ,,130-R); and

• an optional data processing module 132 that processes signals indicative of one or more physiological parameters to determine one or more corresponding physiological parameters.

[0084] In some embodiments, the signal generation module 120 is connected to at least one current or voltage source. In some embodiments, the signal generation module is electrically coupled, via the system 100, to one or more electrodes. In some embodiments, the one or more electrodes are coupled to the system 100 via one or more electrical leads. In some embodiments, the system 100 is integrated with the one or more electrodes without leads. In other embodiments, the system 100 does not comprise a current or voltage source or one or more electrodes. In some embodiments, the signal generation module generates a signal comprising, for example, infrared light or ultrasound. In some embodiments, the signal generation module 120 generates the signal upon receipt of an input 126 to the optional signal responsive module 124. In some embodiments, the input is a signal generated by an operator, such as a physician and/or a subject undergoing treatment for an inflammatory condition. In some embodiments, the operator-generated signal is delivered upon operator interaction with an actuator (e.g., pressing or triggering the actuator).

[0085] In some embodiments, an input 126 is a signal indicative of a physiological parameter, such as a physiological parameter 130 stored in the optional physiological data store 128. For example, in some embodiments, a signal indicative of a physiological parameter for a respective subject is detected by an external physiological sensor (e.g., via electrical, radio frequency, and/or optical (visible or infrared) sensors), and the signal is processed by the data processing module 132 to determine a corresponding physiological parameter 130. In some embodiments, the data processing module 132 is configured for reducing the size of the data pertaining to the one or more physiological parameters for storing in memory 111 and/or for transmitting to an external system via the one or more communication buses 114 or the one or more network interfaces 104. Alternatively or additionally, in some embodiments, the data processing module 132 is configured to process the signals indicative of the one or more physiological parameters and/or process the determined one or more physiological parameters to determine a change in an inflammatory condition (e.g., a chronic and/or acute inflammatory condition) in the subject. [0086] In some embodiments, the physiological data store 128 includes physiological data pertaining to normal levels of the one or more physiological parameters. In some embodiments, the data is specific to a respective subject undergoing treatment for an inflammatory condition e.g., determined from various tests known in the art). In some embodiments, the data processing module 132 compares a physiological parameter determined from a signal detected by an external physiological sensor with the data pertaining to a normal level of the physiological parameter stored in the physiological data store 128, and determines whether the detected signals are indicative of an insufficient or excessive particular physiological parameter, and thus indicative of a change in the inflammatory condition in the subject.

[0087] Additional embodiments of systems for detecting, processing, and generating signals are described in International Patent Application PCT/GB2020/051451, entitled “Treatment of Acute Medical Conditions,” filed June 17, 2020, and International Patent Application PCT/GB2020/051458, entitled “Stimulation of a Nerve Supplying the Spleen,” filed June 17, 2020, each of which is hereby incorporated herein by reference in its entirety.

[0088] In various implementations, one or more of the above identified elements are stored in one or more of the previously mentioned memory devices and correspond to a set of instructions for performing various methods described herein. In some embodiments, the above identified modules, data, or programs (e.g., sets of instructions) are not implemented as separate software programs, procedures, datasets, or modules, and thus various subsets of these modules and data may be combined or otherwise re-arranged in various implementations. In some implementations, the memory 111 optionally stores a subset of the modules and data structures identified above. Furthermore, in some embodiments, the memory stores additional modules and data structures not described above. In some embodiments, one or more of the above- identified elements is stored in a computer system, other than that of the system 100, that is addressable by the system 100 so that the system 100 may retrieve all or a portion of such data.

[0089] Although Fig. 1 depicts a “system 100,” the figure is intended as a functional description of the various features that may be present in computer systems rather than as a structural schematic of the implementations described herein. In practice, items shown separately can be combined and some items can be separate. Moreover, although Fig. 1 depicts certain data and modules in the memory 111 (which can be non-persistent or persistent memory), these data and modules, or portion(s) thereof, may be stored in more than one memory. [0090] Methods as described herein can be implemented by way of machine (e.g., the one or more processing units 102) executable code stored on an electronic storage location of the computer system 100, such as, for example, on the memory 111. The machine executable or machine-readable code can be provided in the form of software. During use, the code can be executed by the one or more processing units 102. The code can be pre-compiled and configured for use with a machine having a processer adapted to execute the code, or can be compiled during runtime. The code can be supplied in a programming language that can be selected to enable the code to execute in a precompiled or as-compiled fashion.

[0091] Aspects of the systems and methods provided herein, such as the computer system 100, can be embodied in programming. Various aspects of the technology may be thought of as “products” or “articles of manufacture” typically in the form of machine (or processor) executable code and/or associated data that is carried on or embodied in a type of machine readable medium. Machine-executable code can be stored on an electronic storage unit, such as memory (e.g., read-only memory, random-access memory, flash memory) or a hard disk.

“Storage” type media can include any or all of the tangible memory of the computers, processors or the like, or associated modules thereof, such as various semiconductor memories, tape drives, disk drives and the like, which may provide non-transitory storage at any time for the software programming. All or portions of the software may at times be communicated through the Internet or various other telecommunication networks. Such communications, for example, may enable loading of the software from one computer or processor into another, for example, from a management server or host computer into the computer platform of an application server. Thus, another type of media that may bear the software elements includes optical, electrical and electromagnetic waves, such as used across physical interfaces between local devices, through wired and optical landline networks and over various air-links.

[0092] Systems and methods of the present disclosure can be implemented by way of one or more algorithms. An algorithm can be implemented by way of software upon execution by the one or more processing units 102.

[0093] While a system in accordance with the present disclosure has been disclosed with reference to Fig. 1, methods in accordance with the present disclosure are now detailed with reference to Fig. 2.

[0094] Exemplary Method Embodiments.

[0095] Referring to Block 200, the present disclosure provides a method of treating an inflammatory condition in a subject. [0096] Inflammatory conditions.

[0097] In some embodiments, a subject is any of the embodiments described herein (see, for example, the Definitions section above). In some embodiments, a subject is a human.

[0098] In some embodiments, the subject has been diagnosed with the inflammatory condition. For example, in some embodiments, a subject is a patient that has been diagnosed with a chronic or an acute inflammatory condition.

[0099] In some embodiments, the inflammatory condition is an increase in inflammation in a subject. In some embodiments, the inflammatory condition is any imbalance of pro- and/or anti-inflammatory markers and/or cytokines and/or mediators in a subject compared to a normal physiological homeostatic state, e.g, increased levels of one or more pro-inflammatory markers or cytokines and/or decreased levels of one or more anti-inflammatory metabolites (e.g, a specialized pro-resolving mediator,) compared to the normal physiological homeostatic state.

[00100] In some embodiments, the inflammatory condition is an acute or a chronic inflammatory condition. In some embodiments, the inflammatory condition is a disorder associated with inflammation (e.g., arthritis, inflammatory bowel disease, rheumatoid arthritis, or Crohn’s disease). For example, disorders associated with inflammation typically present with an imbalance of pro- and anti-inflammatory marker and/or cytokine and/or metabolite profiles compared to a physiological homeostatic state, e.g, increased levels of one or more pro-inflammatory markers and/or cytokines and/or decreased levels of one or more anti- inflammatory cytokines or metabolites (e.g, SPMs) compared to the normal physiological homeostatic state. In some embodiments, an inflammatory condition is associated with a medical or clinical condition, including chronic inflammatory disorders and/or acute inflammatory episodes associated with medical or clinical conditions.

[00101] In some embodiments, treatment of an inflammatory condition includes treatment of a subject suffering from, or at risk for developing, a disorder associated with inflammation, e.g, an inflammatory condition. In some embodiments, treatment of an inflammatory condition includes treating or ameliorating the effects of a disorder associated with inflammation, in a subject suffering from or at risk for developing the disorder, by reducing inflammation. In some embodiments, treatment of an inflammatory condition includes prophylactically treating a patient at risk for developing the inflammatory condition to prevent the onset of the condition and/or to ameliorate the effects of the condition after onset of the condition. For example, this can be achieved by decreasing the production and release of one or more pro-inflammatory markers and/or cytokines and/or increasing the production and release of one or more anti- inflammatory cytokines from the spleen (e.g., by reversibly electrically stimulating the splenic nerve). In some embodiments, treatment of an inflammatory condition includes a change (e.g., an increase) in a level of one or more SPMs. In some embodiments, treatment of an inflammatory condition includes a change (e.g., an increase) in a level of one or more ALOX15- derived SPMs. In some embodiments, treatment of an inflammatory condition includes a change (e.g., an increase) in a level of one or more resolvins (e.g., ALOX 15 -derived resolvins). In some embodiments, treatment of an inflammatory condition includes a change (e.g., an increase) in a level of one or more of the D-series resolvins, including RvDl, RvD2, RvD3, RvD4, RvD5, and/or RvD6.

[00102] In some embodiments, the inflammatory condition is an autoimmune disorder, such as arthritis (e.g., rheumatoid arthritis, osteoarthritis, psoriatic arthritis), Grave's disease, myasthenia gravis, thryoiditis, systemic lupus erythematosus, Goodpasture's syndrome, Behcet's syndrome, allograft rejection, graft-versus-host disease, ankylosing spondylitis, Berger's disease, diabetes including Type I diabetes, Reiter's syndrome, spondyloarthropathy psoriasis, multiple sclerosis, Inflammatory Bowel Disease, Crohn's disease, Addison's disease, autoimmune mediated hair loss (e.g., alopecia areata) and/or ulcerative colitis.

[00103] In some embodiments, the inflammatory condition is a disease involving the gastrointestinal tract and associated tissues, such as appendicitis, peptic, gastric and duodenal ulcers, peritonitis, pancreatitis, ulcerative, pseudomembranous, acute and ischemic colitis, inflammatory bowel disease, diverticulitis, cholangitis, cholecystitis, Crohn's disease, Whipple's disease, hepatitis, abdominal obstruction, volvulus, post-operative ileus, ileus, celiac disease, periodontal disease, pernicious anemia, amebiasis and/or enteritis.

[00104] In some embodiments, the inflammatory condition is a disease of the bones, joints, muscles and connective tissues, such as the various arthritides and arthralgias, osteomyelitis, gout, periodontal disease, rheumatoid arthritis, spondyloarthropathy, ankylosing spondylitis and/or synovitis.

[00105] In some embodiments, the inflammatory condition is a systemic or local inflammatory disease and/or condition, such as asthma, allergy, anaphylactic shock, immune complex disease, sepsis, septicemia, endotoxic shock, eosinophilic granuloma, granulomatosis, organ ischemia, reperfusion injury, organ necrosis, hay fever, cachexia, hyperpyrexia, septic abortion, HIV infection, herpes infection, severe acute respiratory syndrome (SARS), coronavirus infection (e.g., SARS-CoV infection or SARS-CoV-2 infection), organ transplant rejection, disseminated bacteremia, Dengue fever, malaria and/or sarcoidosis. [00106] In some embodiments, the inflammatory condition is a disease involving the urogenital system and associated tissues, including epididymitis, vaginitis, orchitis, urinary tract infection, kidney stone, prostatitis, urethritis, pelvic inflammatory bowel disease, contrast induced nephropathy, reperfusion kidney injury, acute kidney injury, infected kidney stone, herpes infection, and/or candidiasis.

[00107] In some embodiments, the inflammatory condition is a condition involving the respiratory system and associated tissues, such as bronchitis, asthma, hay fever, ventilator associated lung injury, cystic fibrosis, adult respiratory distress syndrome, acute respiratory distress syndrome, severe acute respiratory syndrome, pneumonitis, alveolitis, epiglottitis, rhinitis, achalasia, respiratory syncytial virus, pharyngitis, sinusitis, pneumonitis, influenza, pulmonary embolism, hyatid cysts and/or bronchiolitis.

[00108] In some embodiments, the inflammatory condition is a dermatological disease and/or condition of the skin (e.g., bums, dermatitis, dermatomyositis, burns, cellulitis, abscess, contact dermatitis, dermatomyositis, warts, wheal, sunburn, urticaria warts, and/or wheals), a disease involving the cardiovascular system and associated tissues (e.g., myocardial infarction, cardiac tamponade, vasculitis, aortic dissection, coronary artery disease, peripheral vascular disease, aortic abdominal aneurysm, angiitis, endocarditis, arteritis, atherosclerosis, thrombophlebitis, pericarditis, myocarditis, myocardial ischemia, congestive heart failure, periarteritis nodosa, and rheumatic fever, filariasis thrombophlebitis, and/or deep vein thrombosis), a cancer, tumor and/or proliferative disorder (e.g., Hodgkin's disease), and/or a nosocomial infection. In some embodiments, the inflammatory condition is an inflammatory or immune host response to any primary disease.

[00109] In some embodiments, the inflammatory condition is a disease involving the central or peripheral nervous system and associated tissues, such as Alzheimer's disease, depression, multiple sclerosis, cerebral infarction, cerebral embolism, carotid artery disease, concussion, subdural hematoma, epidural hematoma, transient ischemic attack, temporal arteritis, spinal cord injury without radiological finding (SCIWORA), cord compression, meningitis, encephalitis, cardiac arrest, Guillain-Barre, spinal cord injury, cerebral venous thrombosis and paralysis.

[00110] In some embodiments, the inflammatory condition is a disease associated with a particular organ (e.g., eye or ear). In some embodiments, the inflammatory condition includes an immune or inflammatory response such as conjunctivitis, iritis, glaucoma, episcleritis, acute retinal occlusion, rupture globe, otitis media, otitis externa, uveitis and Meniere's disease. In some embodiments, the inflammatory condition is post-operative ileus (POI). For example, POI is experienced by the vast majority of patients undergoing abdominal surgery and is characterized by transient impairment of gastro-intestinal (GI) function along the GI tract as well pain and discomfort to the patient and increased hospitalization costs. The impairment of GI function is not limited to the site of surgery, for example, patients undergoing laparotomy can experience colonic or ruminal dysfunction. POI is, at least in part, mediated by enhanced levels of pro-inflammatory cytokines and infiltration of leukocytes at the surgical site. Neural inhibitory pathways activated in response to inflammation contribute to the paralysis of secondary GI organs distal to the site of surgery. In some embodiments, the treatment of the inflammatory condition comprises treatment or prevention of POI (e.g, via stimulation of neural activity).

[00111] In some embodiments, the inflammatory condition is an autoimmune disorder (e.g, rheumatoid arthritis, osteoarthritis, psoriatic arthritis, spondyloarthropathy, ankylosing spondylitis, psoriasis, systemic, lupus erythematosus (SLE), multiple sclerosis, Inflammatory Bowel Disease, Crohn's disease, and/or ulcerative colitis) and/or sepsis.

[00112] In some embodiments, the inflammatory condition is a B cell mediated autoimmune disorders (e.g., systemic lupus erythematosus (SLE) and/or rheumatoid arthritis (RA)).

[00113] In some embodiments, the inflammatory condition is caused by or exacerbated by a bacterial infection. For example, the treatment of the inflammatory condition comprises treating inflammatory conditions caused or exacerbated by Escherichia coli, Staphylococcus aureus, Pneumococcus, Haemophilus influenza, Neisseria meningitides, Streptococcus pneumonia, Methicillin-resistant Staphylococcus aureus (MRSA), Klebsiella or Enterobacter infection.

[00114] In some embodiments, the inflammatory condition is caused by or exacerbated by a viral infection. For example, the treatment of the inflammatory condition comprises treating inflammatory conditions caused or exacerbated by coronaviruses, for example, severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) that causes coronavirus disease 19 (COVID- 19). For example, the invention is useful for treating multisystem inflammatory syndrome of children (MIS-C).

[00115] Splenic nerves.

[00116] Referring to Block 202, the method further comprises placing one or more signal- conducting interfaces in signaling contact with one or more splenic nerves of the subject. [00117] Innervation of the spleen is primarily sympathetic or noradrenergic, with peptide neurons likely representing the bulk of the remaining neurons. The human spleen is traditionally considered to be innervated by the splenic plexus surrounding the splenic artery only. The splenic artery is covered with nervous tissue, which is derived from the coeliac plexus and continues with the splenic artery to the spleen as the splenic plexus. The splenic plexus enters the spleen at the hilum where the splenic artery diverges in terminal branches and the splenic plexus continues with these branches into the parenchyma of the spleen. The splenic plexus includes several nerve fascicles which circumvent the main splenic artery from celiac artery to spleen, each nerve fascicle comprising a small bundle of nerve fibers. A nerve fascicle (or known as a peri-arterial nerve fascicle) that circumvents the splenic nerve is referred to herein as a splenic arterial nerve.

[00118] In some embodiments, the one or more splenic nerves is a single splenic nerve, such as a splenic arterial nerve. In some embodiments, the one or more splenic nerves comprises a sympathetic nerve. In some embodiments, the one or more splenic nerves is a plurality of splenic nerves. In some embodiments, the one or more splenic nerves comprises a splenic neurovascular bundle (NVB).

[00119] Nerve Stimulation

[00120] In some embodiments, a signal-conducting interface includes any device, medium, material, and/or component or part thereof capable of producing, transmitting, sending, carrying, and/or receiving a signal (e.g., an electrical signal, an infrared signal, an ultrasound signal, a mechanical signal, and/or a magnetic signal).

[00121] In some embodiments, a signal-conducting interface is an electrode that conducts (e.g, produces, transmits, sends, carries, and/or receives) electrical signals. In some embodiments, a signal-conducting interface is an interface (e.g, a device, medium, material, and/or component or part thereof) that conducts (e.g, produces, transmits, sends, carries, and/or receives) light (e.g., infrared, ultraviolet, and/or visible light signals). In some embodiments, a signal-conducting interface is an interface that conducts sound waves (e.g, ultrasound signals). In some embodiments, a signal-conducting interface is an interface that conducts a magnetic field (e.g., an electromagnetic pulse signal). In some embodiments, a signal-conducting interface is an interface that conducts a mechanical signal, such as a mechanical pulse. Methods for stimulating neural activity are described, for example, in Cotero et al., 2019, “Noninvasive sub-organ ultrasound stimulation for targeted neuromodulation,” Nat. Comm. 10:952; doi: 10.1038/s41467-019-08750-9; and Chang et al., 2020, “A Review of Different Stimulation Methods for Functional Reconstruction and Comparison of Respiratory Function after Cervical Spinal Cord Injury,” Appl. Bionics and Biomech., 8882430, 1-12; doi: 10.1155/2020/8882430, each of which is hereby incorporated herein by reference in its entirety.

[00122] As used herein, the phrase “signaling contact” refers to any placement or orientation where at least part of the signal applied via the one or more signal-conducting interfaces is received at the one or more splenic nerves.

[00123] In some embodiments, the one or more signal-conducting interfaces are placed in signaling contact with the one or more splenic nerves of the subject via an indirect contact (e.g., using a wireless contact). In some embodiments, the one or more signal -conducting interfaces are placed in proximity to but not directly contacting the one or more splenic nerves (e.g., internally or externally). In some embodiments, the placing the one or more signal-conducting interfaces in proximity to the one or more splenic nerves comprises placing the one or more signal-conducting interfaces on the skin of the subject and/or via a wearable device. In some embodiments, the placing the one or more signal-conducting interfaces in proximity to the one or more splenic nerves comprises placing the one or more signal-conducting interfaces at least 1 mm, at least 2 mm, at least 3 mm, at least 4 mm, at least 5 mm, at least 6 mm, at least 7 mm, at least 8 mm, at least 9 mm, at least 10, at least 11 mm, at least 12 mm, at least 13 mm, at least 14 mm, at least 15 mm, at least 16 mm, at least 17 mm, at least 18 mm, at least 19 mm, or at least 20 mm away from the subject.

[00124] In some embodiments, the method further comprises placing the one or more signal- conducting interfaces in physical contact with the one or more splenic nerves. For example, in some embodiments, the method further comprises isolating the one or more splenic nerves from connective tissue and the splenic vein of the subject. The one or more signal-conducting interfaces can then be placed (e.g., implanted) in direct physical contact with the isolated one or more splenic nerves.

[00125] In some embodiments, the placing the one or more signal-conducting interfaces in direct contact with the one or more splenic nerves of the subject comprises implanting the one or more electrodes internally. In some embodiments, the one or more splenic nerves are not isolated from the connective tissue and the splenic vein of the subject prior to placing the one or more signal-conducting interfaces in signaling contact.

[00126] In some embodiments, the implanting of the one or more signal-conducting interfaces comprises permanent or transient implantation. In some embodiments, the implanting of the one or more signal-conducting interfaces comprises implanting a signal-conducting interface internally in the subject.

[00127] Referring to Block 204, the method further comprises forming a connection between a signal -generating source and the one or more signal-conducting interfaces.

[00128] In some embodiments, the signal -conducting interface is physically connected to a signal-generating source. In some embodiments, the signal-conducting interface and the signal - generating source are components of a system (e.g., a device). For example, in some embodiments, the signal is produced and transmitted to the splenic nerve of the subject using a single device that is capable of producing and transmitting such signals. For example, the device can be an ultrasound machine or an infrared laser device, where the production and transmittal of the ultrasound signal is performed externally (e.g., noninvasively) by application of a device.

[00129] In some embodiments, the signal -conducting interface is connected to a signal- generating source using a connector, such as a lead. In some embodiments, the forming a connection comprises forming an electrical connection. In some embodiments, the signal- conducting interface is connected to a signal-generating source using a wireless connection.

[00130] In some embodiments, the one or more signal-conducting interfaces comprises one or more electrodes. In some embodiments, an electrode in the one or more electrodes is a cuff electrode, a circumferential cuff electrode, a catheter intravascular electrode, a stent, and/or a patch. In some embodiments, an electrode is a clip, a probe, or a pin type interface. In some embodiments, the one or more electrodes is a flat interface electrode which is flexible, particularly in embodiments where the one or more electrodes is configured for placement on or around the splenic nerve. In some embodiments, other electrode types are also suitable for use. Other electrode types suitable include cuff electrodes (e.g., spiral cuff, helical cuff or flat interface); hemi-cuff electrodes; a mesh, a linear rod-shaped lead, paddle-style lead or disc contact electrodes (including multi-disc contact electrodes); hook electrodes; sling electrodes; intrafascicular electrodes; glass suction electrodes; paddle electrode; and percutaneous cylindrical electrodes. In some embodiments, the one or more electrodes is a plurality of electrodes comprising at least a first electrode and a second electrode, referred to herein as a bipolar electrode configuration.

[00131] In some embodiments, the one or more signal-conducting interfaces is placed in physical contact with the one or more splenic nerves, where the physical contact comprises partial or full circumvention of the one or more splenic nerves. [00132] In some embodiments, the one or more electrodes is fabricated from, or is partially or entirely coated with, a high charge capacity material such as platinum black, iridium oxide, titanium nitride, tantalum, poly(elthylenedioxythiophene) and suitable combinations thereof. In some embodiments, the one or more electrodes are at least in part insulated from one another by a non-conductive biocompatible material. For example, in some embodiments the one or more electrodes are positioned on a non-conductive biocompatible material that is spaced transversely along the nerve when the device is in use. In some embodiments, the at least one electrode is modified with a coating and/or a surface treatment to modify the capacitance of the at least one electrode. In some embodiments, the coating and/or surface treatment comprises iridium oxide, titanium nitride, PEDOT/PEDOT-PSS, platinum black, laser roughened, electrical dissolution etching, chemical etching, and/or silicon carbide.

[00133] In some embodiments, the total surface area of an electrode in the one or more electrodes is between 0.01 to 0.9 cm², between 0.05 to 0.5 cm², or between 0.1 to 0.3 cm². In some embodiments, the total surface area of an electrode in the one or more electrodes is less than 0.2 cm². For example, in some embodiments, the total surface area of the electrode is about 0.05, about 0.06, about 0.07, about 0.08, about 0.09, about 0.10, about 0.11, about 0.12, about 0.13, about 0.14, about 0.15, about 0.16, about 0.17, about 0.18, or about 0.19 cm². In some embodiments, the width of an electrode in the one or more electrodes is between 1 and 4 mm, between 1 and 3 mm, between 2 and 4 mm, or between 2 and 3 mm.

[00134] See, for example, International Patent Application PCT/GB2020/051451, entitled “Treatment of Acute Medical Conditions,” filed June 17, 2020; International Patent Application PCT/GB2020/051458, entitled “Stimulation of a Nerve Supplying the Spleen,” filed June 17, 2020; and International Patent Application PCT/GB2018/052076, entitled “Electrode Devices for Neurostimulation,” filed July 23, 2018, each of which is hereby incorporated herein by reference in its entirety, for further details on signal-conducting interfaces, including electrodes and electrode devices, that can be used in accordance with the present disclosure.

[00135] In some embodiments, the signal -generating source generates an electrical signal, an infrared signal, an ultrasound signal, a mechanical signal, and/or a magnetic signal.

[00136] In some embodiments, the signal -generating source is an electrical source. In some embodiments, the electrical source is a pulse generator. In some embodiments, the pulse generator comprises a system 100 (see, for example, Fig. 1). For example, in some embodiments, electrical source is a pulse generator that applies a stimulation pulse sequence for splenic nerve stimulation. In some embodiments, the electrical connection between the electrical source and the one or more electrodes is a wireless connection. In some embodiments, the electrical connection between the electrical source and the one or more electrodes is through a lead. For example, in some embodiments, the method comprises forming an electrical connection between a pulse generator and a circumferential cuff electrode using a lead connector (e.g., a physical connection).

[00137] In some embodiments, the method further comprises implanting the signal- generating source (e.g., the pulse generator) within the subject. In some embodiments, the implanting the signal-generating source comprises permanently or transiently implanting the signal-generating source internally in the subject. In some embodiments, the signal -generating source is placed externally on the subject, such as on the skin of the subject and/or via a wearable device.

[00138] In some embodiments, the method further comprises forming an electrical connection between the one or more electrodes (e.g., a circumferential cuff electrode), the electrical source (e.g., a pulse generator), and at least one detector configured to detect one or more physiological parameters relating to the treatment of an inflammatory disorder. For example, in some embodiments, one or more physiological parameters includes a reduction in one or more pro-inflammatory mediators or cytokinesor chemokines, an increase in one or more anti-inflammatory cytokine (e.g., IL- 10) and/or one or more resolving mediator (such as resolvins, lipoxins, eicosanoids, maresins and protectins), an increase in one or more catecholamines or acetylcholine, changes in hematology or one or more cell counts (e.g., changes in immune cell population or one or more immune cell surface co-stimulatory molecules), a reduction in one or more factors involved in the inflammation cascade, and/or a reduction in one or more immune response mediators, as is further discussed below. For example, in some embodiments, the detector is configured for detecting biomolecule concentration using electrical, RF or optical (e.g., visible, infrared) biochemical sensors. In some embodiments, the at least one detector is configured to detect other physiological parameters such as blood flow rate in the spleen, blood flow rate in the splenic artery, blood flow rate in the splenic vein, spleen volume, neural activity in at least one splenic arterial nerve, and/or impedance of the at least one electrode.

[00139] For example, in some embodiments, the at least one detector is configured for detecting blood flow using intra- or peri-vascular flow tubes in or around the artery or vein. In some embodiments, the detector is configured to detect splenic artery contraction and blood flow changes using electrical impedance tomography, electrical impedance, stimulator voltage compliance, Doppler flow, splenic tissue perfusion, ultrasound, strain measurement, and/or pressure. In some embodiments, the at least one detector is configured to detect neural activity of at least one splenic nerve (e.g., a splenic arterial nerve) using an electrical sensor. In some embodiments, the detector is configured to detect neural activity of a single splenic nerve by detecting action potentials. In some embodiments, the detector is configured to detect neural activity of a plurality of splenic nerves (e.g., by detecting compound action potentials).

[00140] In some embodiments, the forming an electrical connection between the one or more electrodes, the electrical source, and the at least one detector comprises forming a wireless connection. In some embodiments, the forming an electrical connection between the one or more electrodes, the electrical source, and the at least one detector comprises forming a physical connection between any two or more of the one or more electrodes, the electrical source, and the at least one detector (e.g., via a lead).

[00141] Further details on signal-generating sources, including electrical signaling devices, that can be used in accordance with the present disclosure are described in, for example, International Patent Application PCT/GB2020/051451, entitled “Treatment of Acute Medical Conditions,” filed June 17, 2020; International Patent Application PCT/GB2020/051458, entitled “Stimulation of a Nerve Supplying the Spleen,” filed June 17, 2020; and International Patent Application PCT/GB2018/052076, entitled “Electrode Devices for Neurostimulation,” filed July 23, 2018, each of which is hereby incorporated herein by reference in its entirety.

[00142] Stimulation pulse sequences.

[00143] Referring to Block 206, the method further comprises generating a stimulation, at the one or more signal-conducting interfaces, with the signal -generating source. In some embodiments, the stimulation stimulates the one or more nerves of the subject. In some embodiments, stimulation of the respective one or more nerves comprises any of the embodiments described above (see, for example, the Definitions section above). In some embodiments, the generating a stimulation at the one or more signal-conducting interfaces comprises generating a signal, at the signal-generating source, that is selected from the group consisting of: an electrical signal, an infrared signal, an ultrasound signal, a mechanical signal, and a magnetic signal. In some embodiments, the stimulation, generated at the one or more signal-conducting interfaces with the signal-generating source, comprises a plurality of stimulation parameters. In some embodiments, the stimulation comprises a pulse (e.g., of electrical charge, light, heat, sound, pressure, magnetism, and/or other force or phenomenon). In some embodiments, the stimulation comprises a plurality of pulses (e.g., a pulse sequence). [00144] In some embodiments, the one or more signal-conducting interfaces comprises one or more electrodes, and the generating a stimulation comprises generating an electrical stimulation at the one or more electrodes (e.g, applying an electrical signal) to the one or more nerves of the subject. In some embodiments, the electrical signal is a non-destructive signal. As used herein, a “non-destructive signal” is a signal that, when applied, does not irreversibly damage the underlying neural signal conduction ability of the nerve. That is, application of a non-destructive signal maintains the ability of the nerve or fibers thereof, or other nerve tissue to which the signal is applied, to conduct action potentials when application of the signal ceases, even if that conduction is in practice artificially stimulated as a result of application of the non- destructive signal.

[00145] In some embodiments, the electrical signal is a voltage or a current waveform. In some embodiments, the electrical signal is characterized by one or more electrical signal parameters (e.g, amplitude, pulse width, pulse height, total charge, waveform, frequency, periodicity, duration, number of pulses, pulse sequence and/or paradigm). In some embodiments, the electrical signal is characterized by the pattern of application of the electrical signal to the nerve, referring to the timing of the application of the electrical signal to the nerve.

[00146] As an example, in some embodiments, the signal-generating source (e.g., electrical source) is a pulse generator, the stimulation (e.g, electrical stimulation) is a pulse sequence that comprises an active period followed by an inactive period, where the active period comprises a plurality of electrical pulses applied by the pulse generator to the one or more electrodes, and the inactive period comprises a predetermined amount of time in which no pulse is applied by the pulse generator to the one or more electrodes. In some embodiments, as used herein, a pulse sequence comprising an active period follow by an inactive period can also be referred to as an “on-off’ pattern, a “burst” pattern, and/or a “periodic” pattern. See, for example, further descriptions of periodic and/or burst patterns in the following sections of the present disclosure; “Paradigm.”

[00147] In some such embodiments, during the active period, the plurality of electrical pulses consists of between two and twenty electrical pulses. In some embodiments, the plurality of electrical pulses consists of between 2 and 10, between 10 and 20, between 20 and 30, between 30 and 40, between 40 and 50, between 50 and 100, between 100 and 200, between 200 and 300, between 300 and 400, between 400 and 500, between 500 and 600, between 600 and 700, between 700 and 800, between 800 and 900, between 900 and 1000, or between 1000 and 1500 electrical pulses. [00148] In some embodiments, a pulse in the plurality of pulses has pulse width of between 500 microseconds (ps) and 2000 ps. In some embodiments, each pulse in the plurality of pulses has a pulse width of between 500 microseconds (ps) and 2000 ps. In some embodiments, a pulse in the plurality of pulses has a total charge of between 5pC and 20 pC. In some embodiments, a pulse in the plurality of pulses has a bipolar waveform, a biphasic waveform or a symmetrical waveform. In some embodiments, the plurality of electrical pulses may have a frequency that is < 300Hz, preferably < 50 Hz, more preferably < 10 Hz. For example, the plurality of electrical pulses may be < 50 Hz, < 100 Hz, < 150 Hz, < 200 Hz, < 250 Hz or < 300 Hz. In other examples, the frequency of the electrical signal may be < 10 Hz, < 15 Hz, < 20 Hz, < 25 Hz, < 30 Hz, < 35 Hz, < 40 Hz, < 45 Hz, or < 50 Hz. In further examples, the plurality of electrical pulses may have a frequency that is < 1 Hz, < 2 Hz, < 5 Hz, or < 10 Hz. Additionally or alternatively, the frequency of the electrical signal may be > 10 Hz, > 15 Hz, > 20 Hz, > 25 Hz, > 30 Hz, > 35 Hz > 40 Hz, > 45 Hz, or > 50 Hz. In other examples, the plurality of electrical pulses may have a frequency that is > 0.1 Hz, > 0.2 Hz, > 0.5 Hz, > 1 Hz, > 2 Hz, or > 5 Hz. Any combination of the upper and lower limits above is also possible. In some embodiments, the plurality of electrical pulses have a frequency that is between 3 Hz and 25 Hz or between 6 Hz and 14 Hz.

[00149] In some embodiments, the active period may have a duration that is < 0.2 seconds, < 0.5 seconds, < 1 seconds, < 2 seconds, < 5 seconds, or < 10 seconds. Alternatively or additionally, the active period may have a duration that is > 0.1 seconds, > 0.2 seconds, > 0.3 seconds, > 0.5 seconds, > 1 seconds, > 2 seconds, or > 5 seconds. Any combination of the upper and lower limits above for the active period is also possible. In some embodiments, the active period has a duration of between 0.1 seconds and 30 seconds. In some embodiments, the active period has a duration of between 0.1 seconds and 10 seconds. In some embodiments, the active period has a duration of between 0.3 seconds and 1 second. In some embodiments, the active period has a duration of between 0.5 seconds and 30 seconds. In some embodiments, the predetermined amount of time of the inactive period is between 0.3 seconds and 1 second. In some embodiments, the active period has a duration of between 0.1 seconds and 5 seconds. In some embodiments, the predetermined amount of time of the inactive period may be < 1 seconds, < 3 seconds, < 5 seconds, < 10 seconds, < 15 seconds, < 20 seconds, < 25 seconds, or < 30 seconds. Alternatively or additionally, in some embodiments, the predetermined amount of time of the inactive period may be > 0.3 seconds, > 0.5 seconds, > 1 seconds, > 2 seconds, > 5 seconds, > 10 seconds, > 15 seconds, > 20 seconds, or > 25 seconds. Any combination of the upper and lower limits above for the predetermined amount of time of the inactive period is also possible. In some embodiments, the generating the pulse sequence is repeated a plurality of times. In some embodiments, the pulse sequence comprising an active period followed by an inactive period is used to treat a chronic and/or acute inflammatory condition.

[00150] As another example, in some embodiments, the signal -generation source (e.g., electrical source) is a pulse generator, the stimulation (e.g., electrical stimulation) at the one or more signal-conducting interfaces (e.g., electrodes) is a continuous stimulation pulse sequence applied by the pulse generator, the continuous stimulation pulse sequence has a frequency of < 50 Hz, preferably < 10 Hz, more preferably < 2 Hz, even more preferably < 1 Hz. For example, the frequency may be < 1 Hz, < 2 Hz, < 5 Hz, or < 10 Hz. In other examples the frequency may be < 0.1 Hz, < 0.2 Hz, < 0.3 Hz, < 0.4 Hz < 0.5 Hz, < 0.6 Hz < 0.7 Hz, < 0.8 Hz, or < 0.9 Hz. Additionally or alternatively, the frequency of the electrical signal may be > 0.1 Hz, > 0.2 Hz, > 0.5 Hz, > 1 Hz, > 2 Hz, or > 5 Hz. Any combination of the upper and lower limits above is also possible. As another example, the signal-generation source (e.g., electrical source) is a pulse generator, the stimulation (e.g, electrical stimulation) at the one or more signal-conducting interfaces (e.g., electrodes) is a continuous stimulation pulse sequence applied by the pulse generator, the continuous stimulation pulse sequence has a frequency of 15 Hz or less, and the continuous stimulation pulse sequence has a duration of greater than one minute.

[00151] In some such embodiments, a pulse in the continuous stimulation pulse sequence has a pulse width of between 200 ps and 1500 ps. In some embodiments, the continuous stimulation pulse sequence has a duration of between one minute and 15 minutes. In some embodiments, each pulse in the continuous stimulation pulse sequence has a pulse width of between 300 ps and 1200 ps. In some embodiments, a pulse in the continuous stimulation pulse sequence has a total charge of between 25pC and 45 pC. In some embodiments, a pulse in the continuous stimulation pulse sequence has a bipolar waveform, a biphasic waveform or a symmetrical waveform. In some embodiments, the frequency of the continuous stimulation pulse sequence is between 3 Hz and 14 Hz or between 6 Hz and 14 Hz. In some embodiments, the continuous stimulation pulse sequence is used to treat a chronic and/or acute inflammatory condition.

[00152] As another example, in some embodiments, the inflammatory condition is an acute inflammatory condition, the stimulation (e.g, electrical stimulation) is a continuous stimulation pulse sequence having a frequency of 15 Hz or less, and the continuous stimulation pulse sequence has a duration of greater than one minute. [00153] In some such embodiments, a pulse in the continuous stimulation pulse sequence has a pulse width of between 200 ps and 1500 ps. In some embodiments, the continuous stimulation pulse sequence has a duration of between one minute and 15 minutes. In some embodiments, each pulse in the continuous stimulation pulse sequence has a pulse width of between 300 ps and 1200 ps. In some embodiments, a pulse in the continuous stimulation pulse sequence has a total charge of between 25pC and 45 pC. In some embodiments, a pulse in the continuous stimulation pulse sequence has a bipolar waveform, a biphasic waveform or a symmetrical waveform. In some embodiments, the frequency is between 3 Hz and 14 Hz or between 6 Hz and 14 Hz.

[00154] In some embodiments, the plurality of stimulation parameters comprises one or more stimulation parameters selected based on a determination of stimulation tolerance and treatment efficacy for treating an inflammatory condition in a subject. For example, in some embodiments, the plurality of stimulation parameters can be used to evaluate the safety and long-term integration of the system and develop an understanding of the effects of acute or chronic neuromodulation on physiological functions. This is particularly important for minimizing off-target immunological effects, since general anesthesia is known to affect immunological functions [59], For example, validation of stimulation parameters using large animal models can reveal biological effects previously not studied in rodent models. Notably, as described in the Examples section below, SpN or VN neuromodulation promotes cardiovascular protection in endotoxemia models in pigs via a mechanism independent of cytokine modulation [51, 35], suggestive of additional pathways, beyond cytokine production, being regulated.

[00155] Additional details relating to stimulation parameters (e.g., amplitude, pulse width, pulse height, total charge, waveform, frequency, periodicity, duration, number of pulses, pulse sequence, paradigm, etc.) that are suitable in some embodiments for splenic nerve stimulation (including acute and chronic stimulation), activation of the ALOX15 pathway, and/or treatment of an inflammatory condition are provided below, in Example 2 (see Examples section below), and in International Patent Application PCT/GB2020/051451, entitled “Treatment of Acute Medical Conditions,” filed June 17, 2020; International Patent Application PCT/GB2020/051458, entitled “Stimulation of a Nerve Supplying the Spleen,” filed June 17, 2020; and International Patent Application PCT/GB2018/052076, entitled “Electrode Devices for Neurostimulation,” filed July 23, 2018, each of which is hereby incorporated herein by reference in its entirety.

[00156] Waveform [00157] In some embodiments, a pulse in the plurality of pulses is a square pulse. In some embodiments, a pulse in a plurality of pulses is a rectangular pulse. However, other pulse waveforms such as sawtooth, sinusoidal, triangular, trapezoidal, quasitrapezodial or complex waveforms can be used.

[00158] In some embodiments, a pulse in the plurality of pulses is biphasic. The term “biphasic” refers to a pulse that applies to the nerve over time both a positive and negative charge (e.g., anodic and cathodic phases). For biphasic pulses, the pulse width includes the time duration of a primary phase of the waveform, such as the anodic phase or the cathodic phase.

[00159] In some embodiments, a pulse in the plurality of pulses is charge-balanced. A charge-balanced pulse refers to a pulse that, over the period of the pulse, applies equal amounts (or thereabouts) of positive and negative charge to the nerve. In some embodiments, the biphasic pulses are charge-balanced. In some embodiments, the pulses are biphasic charge- balanced rectangular pulses.

[00160] In some embodiments, a pulse in the plurality of pulses is symmetric or asymmetric. A symmetric pulse is a pulse where the waveform when applying a positive charge to the nerve is symmetrical to the waveform when applying a negative charge to the nerve. An asymmetric pulse is a pulse where the waveform when applying a positive charge to the nerve is not symmetrical with the waveform when applying a negative charge to the nerve.

[00161] In some embodiments, a pulse in the plurality of pulses has a pulse width of between 200 and 4000 ps, between 400 and 3000 ps, or between 800 and 2000 ps. In some embodiments, a pulse in the plurality of pulses has a pulse width of between 100 and 3000 ps, between 200 and 2000 ps, or between 300 and 1500 ps.

[00162] Amplitude

[00163] In some embodiments, the amplitude of the electrical signal applied to the nerve is sufficient to evoke full or partial neural activity. In some such embodiments, the amplitude of the electrical signal is sufficient to evoke at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60% at least 70%, at least 80%, at least 90%, at least 95%, at least 98%, or at least 99% neural activity. For example, as described below in Example 2, in some embodiments, therapeutic immunomodulatory activity as measured by cytokine suppression can be achieved without requiring 100% nerve stimulation. In some embodiments, the amplitude of a stimulation applied to the one or more splenic nerves in the subject is selected based upon a determination of stimulation tolerance in the subject. In some such embodiments, the stimulation tolerance is determined based upon the type or severity of a respective inflammatory condition for the respective patient.

[00164] In some embodiments, a pulse in the plurality of pulses has an amplitude of between 0 and 40 mA. In some embodiments, a pulse in the plurality of pulses has an amplitude of between 0 and 5, between 5 and 10, between 10 and 15, between 15 and 20, between 20 and 25, between 25 and 30, between 30 and 35, or between 35 and 40 mA. In some embodiments, a pulse in the plurality of pulses has an amplitude of between 0 and 100 mA.

[00165] Frequency

[00166] In some embodiments, the frequency of a signal generated for splenic nerve stimulation can comprise a periodic and/or a continuous stimulation.

[00167] In some embodiments where the stimulation (e.g., electrical signal) is applied periodically, the stimulation can comprise a frequency of < 300 Hz, < 50 Hz, or < 10 Hz. For example, in some embodiments, the frequency of the stimulation (e.g, electrical signal) is < 50 Hz, < 100 Hz, < 150 Hz, < 200 Hz, < 250 Hz or < 300 Hz. In some embodiments, the frequency is < 10 Hz, < 15 Hz, < 20 Hz, < 25 Hz, < 30 Hz, < 35 Hz, < 40 Hz, < 45 Hz, or < 50 Hz. In some embodiments, the frequency is < 1 Hz, < 2 Hz, < 5 Hz, or < 10 Hz. In some embodiments, the frequency is > 10 Hz, > 15 Hz, > 20 Hz, > 25 Hz, > 30 Hz, > 35 Hz > 40 Hz, > 45 Hz, or > 50 Hz. In some embodiments, the frequency is > 0.1 Hz, > 0.2 Hz, > 0.5 Hz, > 1 Hz, > 2 Hz, or > 5 Hz. Any combination of the upper and lower limits above is also possible.

[00168] In some embodiments where the stimulation (e.g, electrical signal) is applied continuously, the stimulation can comprise a frequency of < 50 Hz, < 10 Hz, < 2 Hz, or < 1 Hz. For example, in some embodiments, the frequency of the stimulation (e.g., electrical signal) is < 1 Hz, < 2 Hz, < 5 Hz, or < 10 Hz. In some embodiments, the frequency is < 0.1 Hz, < 0.2 Hz, < 0.3 Hz, < 0.4 Hz < 0.5 Hz, < 0.6 Hz < 0.7 Hz, < 0.8 Hz, or < 0.9 Hz. In some embodiments, the frequency is > 0.1 Hz, > 0.2 Hz, > 0.5 Hz, > 1 Hz, > 10 2 Hz, or > 5 Hz. Any combination of the upper and lower limits above is also possible.

[00169] In some embodiments where the stimulation comprises a waveform comprising a plurality of pulses, the pulses are applied to the nerve at intervals according to the foregoing frequencies. For example, a frequency of 50 Hz results in 50 pulses being applied to the nerve per second.

[00170] In some embodiments, the plurality of electrical pulses is applied in a continuous or periodic (e.g, active/inactive or on-off) pattern according to any one or more of the foregoing frequencies, and/or any combination thereof as will be apparent to one skilled in the art. [00171] Paradigm

[00172] In some embodiments, the stimulation comprises one or more signals generated by the signal-generating source, where the one or more signals are characterized by an application pattern (e.g., a paradigm). For example, in some embodiments, the stimulation comprises a plurality of pulses (e.g., electrical, magnetic, sound, and/or light signals) that is generated, transmitted, and/or applied to the one or more splenic nerves of the subject according to a paradigm. In some embodiments, the pattern of application comprises continuous application, periodic application, and/or episodic application.

[00173] As used herein, “episodic” application refers to the application of the stimulation (e.g., electrical signal, magnetic signal, ultrasound signal, and/or infrared signal) to the nerve for a discrete number of episodes throughout a day. In some embodiments, each episode is defined by a set duration or a set number of iterations of the stimulation. For example, in some embodiments, the method further comprises repeating the generating the stimulation pulse sequence a plurality of times (e.g, according to a pattern or paradigm).

[00174] As used herein, “continuous” application refers to the application of the stimulation (e.g., electrical signal, magnetic signal, ultrasound signal, and/or infrared signal) to the nerve in a continuous manner. In some embodiments where the stimulation is applied continuously and episodically, the signal can be applied in a continuous manner for each episode of application. In some embodiments where the stimulation is a series of pulses, continuous application can comprise gaps between one or more pulses in the plurality of pulses (e.g, between the pulse width and the phase duration).

[00175] As used herein, “periodic” application refers to the application of the stimulation (e.g, electrical signal, magnetic signal, ultrasound signal, and/or infrared signal) to the nerve in a repeating pattern (e.g., an on-off pattern). In some embodiments where the stimulation is applied periodically and episodically, the signal can be applied in a periodic manner for each episode of application. In an example embodiment, a paradigm comprising an active period and an inactive period can comprise an active (e.g, “on”) period with a first duration, and an inactive (e.g, “off’) period with a second duration.

[00176] Episodic application

[00177] In some embodiments, the stimulation comprises at least 1, at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, at least 21, at least 22, at least 23, or at least 24 episodes per day. In some embodiments, the stimulation comprises no more than twenty-four episodes per day, no more than eighteen episodes per day, no more than twelve episodes per day or no more than six episodes per day. For example, in some embodiments, the number of episodes of signal application per day consists of one, two, three, four, five, six, seven, eight, nine, ten, eleven, twelve, thirteen, fourteen, fifteen, sixteen, seventeen, eighteen, nineteen, twenty, twenty-one, twenty-two, twenty-three or twenty-four. In some embodiments, the number of episodes per day comprises twenty-four or fewer times per day, thirty or fewer times per day, thirty-six or fewer times per day, forty-two or fewer times per day or forty-eight or fewer times per day.

[00178] In some embodiments, the stimulation is applied episodically every 2 to 3 hours. For example, in some embodiments, the stimulation is applied episodically once every 2 hours, 2 hour 15 min, 2 hour 30 min, 2 hour 45 min, or 3 hours. In some embodiments, the stimulation is applied episodically between one and five times per hour. In some embodiments, the stimulation is applied episodically up to a maximum of five times per hour, up to a maximum of ten times per hour, up to a maximum of fifteen times per hour or up to 20 times per hour.

[00179] In some embodiments, each episode is defined by a set duration or a set number of iterations of the stimulation. In some embodiments, each episode comprises applying to the nerve between 50 and 22000, between 50 and 10000, between 60 and 3000, between 100 and 2400 pulses, between 200 and 1200, or between 400 and 600 pulses of the stimulation. For example, in some embodiments, each episode comprises applying < 400, < 800, < 1200, < 1600, < 2000, < 2400, < 3000, < 10000, < 15000, < 18000, < 20000 or < 22000 pulses of the stimulation. In some embodiments, each episode comprises applying < 200, < 400, < 600, < 800, < 1000, or < 1200 pulses of the stimulation. In some embodiments, each episode comprises applying < 400, < 425, < 450, < 475, < 500, < 525, < 550, < 575, or < 600 pulses of the stimulation.

[00180] In some embodiments, each episode comprises between 20 and 450 iterations, between 20 and 400 iterations, between 20 and 200 iterations, between 20 and 100 iterations, between 20 and 80, or between 20 and 40 iterations of the periodic pattern. For example, in some embodiments, each episode comprises applying 20, 25, 30, 35, or 40 iterations of the periodic pattern, or any number therebetween. As another example, in some embodiments, each episode comprises applying 20, 25 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 90, 100, 150, 200, 250, 300, 350, 400 or 450 iterations of the periodic pattern, or any number therebetween.

[00181] In some embodiments, each episode comprises > 50 iterations, > 75 iterations, > 100 iterations or > 150 iterations. In some embodiments, each episode comprises < 200 iterations, < 150 iterations, < 75 iterations or < 50 iterations. Any combination of the upper and lower limits is also possible. For example, each episode can comprise > 50 iterations and < 200 iterations. In some embodiments, the number of iterations for a stimulation is reduced as the frequency is increased.

[00182] In some embodiments, the episodes are based on the subject’s sleep-wake cycle (e.g., episodes occur while the subject is asleep). In some such embodiments, the episodes can be applied whilst the subject is asleep (e.g., between 10 pm and 6 am). The sleep-wake cycle can be measured via known methods by detecting the subject’s circadian rhythm phase markers (e.g., cortisol level, melatonin level or core body temperature), and/or a detector for detecting the subject’s movements. In some embodiments, the episodes are applied whilst the subject is awake (e.g., between 6am and 10pm). In some embodiments, the episodes are applied over a sleep-wake cycle, over a 24-hour period.

[00183] Alternatively or additionally, in some embodiments the stimulation is applied episodically in regular intervals or in irregular intervals. For example, 6 episodes may be delivered per day, once every 2 hours, during the wake cycle of a patient. Different episodic intervals can be used between each episode, e.g., a first episodic interval can be used between first and second episodes and a second episodic interval different from the first episodic interval can be used between the second and third episodes. Different combinations of the upper and lower limits of the various parameters (e.g, number of episodes, episodic intervals, sleep-wake cycle and/or forms of stimulation) are possible and can be adjusted based on user or practitioner preferences to achieve a desired total charge delivered per day.

[00184] In some embodiments, the total charge delivered per day may be up to and including 900 mC per 30 minutes. For example, in some embodiments, the total charge delivered per day is less than or equal to 21,600 mC per day, less than or equal 25 to 600 mC per day, less than or equal to 500 mC per day, less than or equal to 400 mC per day, less than or equal to 300 mC per day, less than or equal to 200 mC per day, less than or equal to 100 mC per day, less than or equal to 75 mC per day, or less than 55 mC per day. In some embodiments, the total charge delivered per day is greater than or equal to 0.5 mC greater than or equal to 0.6 mC, greater than or equal to 0.7 mC, greater than or equal to 0.8 mC, greater than or equal to 1.0 mC, greater than or equal to 10 mC, greater than or equal to 20 mC, greater than or equal to 30 mC, greater than or equal to 40 mC, greater than or equal to 50 mC, greater than or equal to 60 mC, or greater than or equal to 70 mC per day. In some embodiments, the total charge is delivered episodically during different pulse burst paradigms. Any combination of the upper and lower limits for the total charge delivered per day is also possible. For example, the total charge delivered per day can be greater than or equal to 0.5 mC and less than or equal to 600 mC.

[00185] In some embodiments, a pulse in the plurality of pulses has a total charge of between 5pC and 20 pC. In some embodiments, a pulse in the plurality of pulses has a total charge of between 5 pC and 50 pC. In some embodiments, a pulse in the plurality of pulses has a total charge of between 5 and 10, between 10 and 15, between 15 and 20, between 20 and 25, between 25 and 30, between 35 and 40, or between 45 and 50 pC. In some embodiments, a pulse in the plurality of pulses has a total charge that is less than 50 pC, less than 40 pC, less than 30 pC, or less than 20 pC.

[00186] In some embodiments, each pulse in the plurality of pulses has a total charge of between 5pC and 20 pC. In some embodiments, each pulse in the plurality of pulses has a total charge of between 5 pC and 50 pC. In some embodiments, each pulse in the plurality of pulses has a total charge of between 5 and 10, between 10 and 15, between 15 and 20, between 20 and 25, between 25 and 30, between 35 and 40, or between 45 and 50 pC. In some embodiments, each pulse in the plurality of pulses has a total charge that is less than 50 pC, less than 40 pC, less than 30 pC, or less than 20 pC.

[00187] Periodic application

[00188] In some embodiments, a stimulation is applied in a repeating pattern (e.g., an on-off pattern), where the signal is applied is applied for a first duration, referred to herein as an “on” duration or “active period”, then stopped for a second duration, referred to herein as an “off’ duration or “inactive period.” The signal is subsequently applied again for the first duration, then stopped again for the second duration, etc.

[00189] In some embodiments, the active period (e.g, during which pulses at a certain frequency and amplitude are delivered to the nerve) has a duration of between 0.3 seconds and 1 second, and the predetermined amount of time of the inactive period (e.g, the time between on periods, during which no pulses are delivered to the nerve) is between 1 second and 10 seconds. In some embodiments, the active period has a duration of between 0.1 seconds and 5 seconds, and the predetermined amount of time of the inactive period is between 1 second and 1 minute.

[00190] In some embodiments, the active period has a duration of about 0.1, about 0.2, about 0.3, about 0.4, about 0.5, about 0.6, about 0.7, about 0.8, about 0.9, about 1, about 1.1, about 1.2, about 1.3, about 1.4, or about 1.5 seconds, and the predetermined amount of time of the inactive period is about 0.1, about 0.2, about 0.3, about 0.4, about 0.5, about 0.6, about 0.7, about 0.8, about 0.9, about 1, about 1.1, about 1.2, about 1.3, about 1.4, or about 1.5 seconds. [00191] In some embodiments, the active period has a duration of about 0.1, about 0.2, about 0.3, about 0.4, about 0.5, about 0.6, about 0.7, about 0.8, about 0.9, about 1, about 1.1, about 1.2, about 1.3, about 1.4, or about 1.5 seconds, and the predetermined amount of time of the inactive period is about 0.5, about 0.6, about 0.7, about 0.8, about 0.9, about 1, about 2, about 3, about 4, about 5 or about 6 seconds.

[00192] In some embodiments, the predetermined amount of time of the inactive period is between 1 second and 1 minute, between 1 minute and 2 minutes, between 2 minutes and 5 minutes, between 5 minutes and 10 minutes, between 10 minutes and 30 minutes, or more than 30 minutes.

[00193] In some embodiments, the active period has a duration of between 0.1 and 10 s and the inactive period has a duration of between 0.5 and 30 s. For example, the active period may be < 0.2 s, < 0.5 s, < 1 s, < 2 s, < 5 s, or < 10 s. Alternatively or additionally, in some embodiments, the active period has a duration of > 0.1 s, > 0.2 s, > 0.5 s, > 1 s, > 2 s, or > 5 s. Any combination of the upper and lower limits above for the on duration is also possible.

[00194] In some embodiments, the inactive period has a duration of < 1 s, < 3 s, < 5 s, < 10 s, < 15 s, < 20 s, < 25 s, or < 30 s. Alternatively or additionally, in some embodiments, the inactive period has a duration of > 0.5 s, > 1 s, > 2 s, > 5 s, > 10 s, > 15 s, > 20 s, or < 25 s. Any combination of the upper and lower limits above for the off duration is also possible.

[00195] In an exemplary embodiment, the periodic on-off pattern has an active period of 0.5 s and an inactive period of 4.5 sec. In another example, the periodic on-off pattern has an active period of 0.5s and an inactive period of 5 sec, for frequencies of up to 10 Hz. For a stimulation with a frequency higher than 10 Hz (e.g., 30 Hz) an example periodic on-off pattern has an active period of 0.1s and an inactive period of 3 s. In other words, a ratio of the active to inactive durations may be 1 :5. In some embodiments, the ratio of the active to inactive durations is 1 :6, 1;7, 1 :8, 1 :9, 1 : 10, 1 :20 or 1 :30. In some embodiments, the ratio of the active to inactive durations is 1 :10 for pulse frequencies up to 10Hz, and a ratio of the active to inactive durations is 1 :30 for pulse frequencies higher than 10Hz.

[00196] In some embodiments where the stimulation is applied periodically and episodically, the signal is applied in a periodic manner for each episode of application. In some embodiments, periodic application can also be referred to as a duty cycled application. A duty cycle represents the percentage of time that the signal is applied to the nerve for a cycle of the periodic pattern. For example, a duty cycle of 20% can represent a periodic pattern having an active period of 2 s, and an inactive period of 10 s. Alternatively, a duty cycle of 20% may represent a periodic pattern having an active period of 1 s, and an inactive period of 5 s. In other words, in some embodiments, periodic application can be referred to as on-off pattern stimulation, or burst stimulation.

[00197] In some embodiments, the duty cycle of the stimulation is between 0.1% and 100%. In some embodiments, the duty cycle of the stimulation is between 0.1% and 5%, between 5% and 10%, between 10% and 20%, between 20% and 30%, between 30% and 40%, between 40% and 50%, between 50% and 60%, between 60% and 70%, between 70% and 80%, between 80% and 90%, or between 90% and 100%.

[00198] In some embodiments, burst stimulation paradigms can be used for clinical and/or testing applications, such as long-term human use. For example, in some such embodiments, burst stimulation paradigms are used to minimize the impact of any neuromodulation therapy on the cardiovascular system. In some embodiments, such safeguards allow the clinical investigation of the efficacy of long-term SpN neuromodulation, in the presence of reduced effects on the cardiovascular system, while maintaining nerve stimulation and neurotransmitter release in the spleen.

[00199] Activation of ALOX 15 pathway.

[00200] As described above, the resolution of inflammation is an active process requiring fine regulation of biosynthetic pathways that lead to the production of lipid mediators, called specialized pro-resolving mediators (SPMs). SPMs include the resolvins, a group of oxidative metabolites of docosahexanoic acid (DHA) and eicosapentaenoic acid (EP A). For example, DHA and EPA can be metabolized to the D-series (e.g., RvDl, RvD2, RvD3, RvD4, RvD5, and/or RvD6) and E-series (e.g, RvEl, RvE2, RvE3, and/or RvE4) resolvins, via human lipoxygenase arachidonate 15 -lipoxygenase (ALOX15). Accordingly, referring to Block 208, the method further comprises generating a stimulation (e.g, an electrical signal, an infrared signal, an ultrasound signal, a mechanical signal, and/or a magnetic signal), where the stimulation treating the inflammatory condition activates the ALOX15 pathway.

[00201] Mammalian ALOX15 is an inducible and highly regulated enzyme that can counter regulate pro-inflammatory signaling through a variety of mechanisms. For instance, as described in Example 2 below, splenic nerve stimulation using acute and chronic stimulation in large animal models was shown to modulate the expression of specialized pro-resolving mediators in the systemic circulation prior to inflammatory challenge. Most of these changes were linked to the ALOX15 pathway, and these changes were also observed during an induced endotoxemia model following lipopolysaccharide (LPS) challenge, confirming the role of SpN neuromodulation in the activation of this pathway. In addition to the production of the D-series and E-series resolvins, ALOX15 is involved in the biosynthesis of lipoxins (e.g., LXA4, LXB4), hepoxilin isomers, and eoxins from arachidonic acid (AA), as well as other inflammatory regulators from linoleic acid, alpha-linolenic acid, and gamma-linolenic acid. Additional details relating to mammalian lipoxygenases, ALOX15, and intermediates and products of the ALOX15 pathway are found in Tian et al., 2017, “ALOX15 as a Suppressor of Inflammation and Cancer: Lost in the Link,” Prostaglandins Other Lipid Mediat; 132: 77-83, doi:

10.1016/j. prostaglandins.2017.01.002, and Kuhn et al., 2015, “Mammalian lipoxygenases and their biological relevance,” Biochim Biophys Acta; 1851(4): 308-330, doi:

10.1016/j .bbalip.2014.10.002, each of which is hereby incorporated herein by reference in its entirety.

[00202] Accordingly, in some embodiments, the activation of the ALOX15 pathway comprises a change (e.g., an increase and/or a decrease) in a physiological level of one or more substrates of a human lipoxygenase (e.g, ALOX15), including linoleic acid, alpha-linolenic acid, gamma-linolenic acid, arachidonic acid (AA), eicosapentaenoic acid (EP A), and/or docosahexaenoic acid (DHA).

[00203] In some embodiments, the activation of the ALOX15 pathway comprises an increase in enzymatic activity of arachidonate 15 -lipoxygenase (ALOX15).

[00204] In some embodiments, the activation of the ALOX15 pathway comprises a change (e.g., an increase and/or a decrease) in a physiological level of one or more metabolites in the arachidonic acid (AA) metabolism pathway, including 15S-HpETE, 15S-HETE, LTA4, 15S- Epoxytetraene, LXA4, LXB4, AT-LXA4, AT-LXB4, hepoxilin isomers, and/or eoxins.

[00205] In some embodiments, the activation of the ALOX15 pathway comprises a change (e.g., an increase and/or a decrease) in a physiological level of one or more metabolites in the eicosapentaenoic acid (EP A) metabolism pathway, including 18R-H(p)EPE, 5S-H(p)-18R- HEPE, 15S-HpEPA, and/or 15-S-HEPA.

[00206] In some embodiments, the activation of the ALOX15 pathway comprises a change (e.g., an increase and/or a decrease) in a physiological level of one or more metabolites in the docosahexaenoic acid (DHA) metabolism pathway, including 17S-H(p)DHA and/or 17S- HDHA.

[00207] In some embodiments, the activation of the ALOX15 pathway comprises a change (e.g., an increase and/or a decrease) in a physiological level of one or more specialized pro- resolving mediators (SPMs). In some embodiments, the SPM is a D-series resolving (e.g., RvDl, RvD2, RvD3, RvD4, RvD5, and/or RvD6), an E-series resolvin (e.g., RvEl, RvE2, RvE3, and/or RvE4), or a T-series resolving (e.g., RvTl, RvT2, RvT3, and/or RvT4). In some embodiments, the SPM is RvDl or RvD2. Figs. 11 and 17 include further examples of substrates (e.g., AA, EP A, and/or DHA), intermediates, metabolites, pro-resolving mediators, and/or enzymes for which levels can be modulated by splenic nerve stimulation for the treatment of inflammatory conditions, in accordance with some embodiments of the present disclosure. Changes in the physiological levels of additional members of the ALOX15 pathway (e.g., substrates, intermediates, metabolites, pro-resolving mediators, and/or enzymes) are possible, as will be apparent to one skilled in the art.

[00208] In some embodiments, the activation of the ALOX15 pathway comprises an increase in enzymatic activity of arachidonate 15 -lipoxygenase type II (ALOX15B).

[00209] Referring to Block 210, in some embodiments, the generating the stimulation that activates the ALOX15 pathway produces an improvement in a physiological parameter in the subject, where the improvement in the physiological parameter is one or more of the group consisting of: a reduction in one or more pro-inflammatory cytokines, an increase in one or more anti-inflammatory cytokines and/or pro-resolving mediators, an increase in one or more catecholamines, a change in an immune cell population or immune cell surface co-stimulatory molecules, a reduction in a factor involved in the inflammation cascade, and/or a reduction in one or more immune response mediators.

[00210] Assessment of effect of stimulation.

[00211] In some embodiments, treatment of an inflammatory condition in a subject comprises, for example, obtaining or inducing an improvement in one or more physiological parameters of the subject (see, Definitions). Useful physiological parameters can include one or more of: the level of a pro-inflammatory cytokine, the level of an anti-inflammatory cytokine, the level of a pro-resolving mediator (including but not limited to ALOX 15 -derived SPMs, ALOX 15 -derived resolvins, and/or D-series resolvins), the level of a catecholamine, the level of an immune cell population, the level of an immune cell surface costimulatory molecule, the level of a factor involved in the inflammation cascade, the level of an immune response mediator, and/or the rate of splenic blood flow.

[00212] In some embodiments, improvement in a physiological parameter is indicated by one or more of: a reduction in a pro-inflammatory cytokine, an increase in an anti-inflammatory cytokine and/or a pro-resolving mediator (including but not limited to ALOX 15 -derived SPMs, ALOX 15 -derived resolvins, and/or D-series resolvins), an increase in a catecholamine, a change in an immune cell population, a change in an immune cell surface co-stimulatory molecule, a reduction in a factor involved in the inflammation cascade, a change in the level of an immune response mediator and/or a decrease in splenic blood flow, any combination of one or more of these parameters, or any additions, modifications, or substitutions thereof, as will be apparent to one skilled in the art.

[00213] For example, in some embodiments, the administering the stimulation (e.g., an electrical signal, an infrared signal, an ultrasound signal, a mechanical signal, and/or a magnetic signal) that activates the ALOX15 pathway produces an improvement in a physiological parameter in the subject, where the improvement in the physiological parameter is one or more of the group consisting of: a reduction in one or more pro-inflammatory cytokines, an increase in one or more anti-inflammatory cytokines and/or one or more pro-resolving mediators (including but not limited to one or more ALOX 15 -derived SPMs, ALOX 15 -derived resol vins, and/or D-series resolvins), an increase in one or more catecholamines, changes in one or more immune cell population or immune cell surface co-stimulatory molecules, a reduction in one or more factor involved in the inflammation cascade, and/or a reduction in one or more immune response mediator.

[00214] As another example, in some embodiments, a continuous stimulation pulse sequence produces an improvement in a physiological parameter in the subject, where the improvement in the physiological parameter is one or more of the group consisting of: a reduction in one or more pro-inflammatory cytokines, an increase in one or more anti-inflammatory cytokines and/or one or more pro-resolving mediators (including but not limited to one or more ALOX15- derived SPMs, ALOX 15 -derived resolvins, and/or D-series resolvins), an increase in one or more catecholamines, a change in one or more immune cell population or immune cell surface co-stimulatory molecules, a reduction in one or more factors involved in the inflammation cascade, and/or a reduction in one or more immune response mediators.

[00215] As another example, in some embodiments, a stimulation pulse sequence comprising an active period and an inactive period produces an improvement in a physiological parameter in the subject, where the improvement in the physiological parameter is one or more of the group consisting of: a reduction in one or more pro-inflammatory cytokines, an increase in one or more anti-inflammatory cytokines and/or one or more pro-resolving mediators (including but not limited to one or more ALOX 15 -derived SPMs, ALOX 15 -derived resolvins, and/or D-series resolvins), an increase in one or more catecholamines, a change in one or more immune cell population or one or more immune cell surface co-stimulatory molecules, a reduction in one or more factors involved in the inflammation cascade, and/or a reduction in one or more immune response mediators.

[00216] Without being limited to any one theory of operation, upon stimulation of a splenic nerve, the spleen may: (a) decrease the secretion of a pro-inflammatory cytokine compared to baseline secretion; and/or (b) increase the secretion of an anti-inflammatory cytokines and/or a resolving mediator compared to baseline secretion. For example, in some embodiments, the decrease in a pro-inflammatory cytokine secretion is a decrease of < 5%, < 10%, < 15%, < 20%,

< 25%, < 30%, < 35%, < 40%, < 45%, < 50%, < 60%, < 70%, < 80%, < 90% or < 95%. In some embodiments, the increase in an anti-inflammatory cytokine and/or pro-resolving mediator secretion (e.g., ALOX 15 -derived SPMs, ALOX 15 -derived resolvins, and/or D-series resolvins) is an increase of < 5%, < 10%, < 15%, < 20%, < 25%, < 30%, < 35%, < 40%, < 45%,

< 50%, < 60%, < 70%, < 80%, < 90%, < 95%, < 100%, < 150%, < 200%, or < 500-1000%.

[00217] In some embodiments, measurement of a cytokine is performed using blood or serum samples (e.g., where cytokine concentrations are diluted after secretion into the circulation). For example, in some embodiments, the decrease in the level of a pro- inflammatory cytokine in the plasma or serum is a decrease of < 5%, < 10%, < 15%, < 20%, < 25%, < 30%, < 35%, < 40%, < 45%, < 50%, < 60%, < 70%, < 80%, < 90% or < 95%. In some embodiments, the increase in an anti-inflammatory cytokine secretion and/or pro-resolving mediator (e.g., ALOX 15 -derived SPMs, ALOX 15 -derived resolvins, and/or D-series resolvins) in the plasma or serum is an increase of < 5%, < 10%, < 15%, < 20%, < 25%, < 30%, < 35%, < 40%, < 45%, < 50%, < 60%, < 70%, < 80%, < 90%, < 95%, < 100%, < 150%, or < 200%.

[00218] In some embodiments, the level of catecholamine (e.g., norepinephrine or epinephrine), e.g., its level in the spleen or splenic vein, may increase, for example, by: < 5%, < 10%, < 15%, < 20%, < 25%, < 30%, < 35%, < 40%, < 45%, < 50%, < 60%, < 70%, < 80%, < 90%, < 95%, < 100%, < 150%, or < 200%. For example, stimulating a splenic nerve can decrease the level of a pro-inflammatory cytokine (e.g., TNFa) in the serum by 5%-99%, 10%- 90%, 20%-80%, 25%-70%, or 30%-60%.

[00219] Pro-inflammatory cytokines are known in the art. Examples of these include tumor necrosis factor (INF; also known as TNFa or cachectin), interleukin (IL)-la, IL-ip, IL-2; IL-5, IL-6, IL-8, IL- 15, IL- 18, interferon y (IFN-y); platelet-activating factor (PAF), thromboxane; soluble adhesion molecules; vasoactive neuropeptides; phospholipase A2; plasminogen activator inhibitor (PALI); free radical generation; neopterin; CD 14; prostacyclin; neutrophil elastase; protein kinase; monocyte chemotactic proteins 1 and 2 (MCP-1, MCP-2); macrophage migration inhibitory factor (MIF), high mobility group box protein 1 (HMGB-1), and other known factors.

[00220] Anti-inflammatory cytokines are also known in the art. Examples of these include IL-4, IL- 10, IL- 17, IL- 13, IL- la, and TNFa receptor.

[00221] Pro-resolving mediators are also known in the art. In particular, specialized pro- resolving mediators are a class of molecules produced during metabolism of polyunsaturated fatty acids via the action of several enzymes (e.g., lipoxygenase, cycl oxygenase, cytochrome P450 and others). They orchestrate the resolution of acute and chronic inflammation.

Examples include Lipoxins, Resolvins, Protectins and Maresins. In some embodiments, as described herein, physiological levels of specialized pro-resolving mediators are modulated via SpNS-induced activation of the ALOX 15 pathway.

[00222] ALOX 15 -derived SPMs include the D-series (e.g., RvDl, RvD2, RvD3, RvD4, RvD5, and/or RvD6), and E-series (e.g., RvEl, RvE2, RvE3, and/or RvE4) resolvins. Other ALOX 15 -derived metabolites include lipoxins (e.g., LXA4, LXB4), hepoxilin isomers, and eoxins, generated from arachidonic acid (AA), as well as other inflammatory regulators from linoleic acid, alpha-linolenic acid, and gamma-linolenic acid.

[00223] It will be recognized that some pro-inflammatory cytokines can act as anti- inflammatory cytokines in certain circumstances, and vice versa. Such cytokines are typically referred to as pleiotropic cytokines.

[00224] In some embodiments, factors involved in immune responses can be useful measurable parameters, for example, TGF, PDGF, VEGF, EGF, FGF, I-CAM, and/or nitric oxide.

[00225] In some embodiments, chemokines are also be useful measurable parameters of neuromodulation, such as 6cKine and MIP3beta, and chemokine receptors, including CCR7 receptor.

[00226] Changes in immune cell population (Langerhans cells, dendritic cells, lymphocytes, monocytes, macrophages), or immune cell surface co-stimulatory molecules (Major Histocompatibility, CD80, CD86, CD28, CD40) can also be useful measurable parameters. Applying a stimulation pulse sequence to a splenic nerve can cause reduction in total counts of circulating or tissue-specific (e.g., joint-specific in the case of rheumatoid arthritis) leukocytes (including monocytes and macrophages, lymphocytes, neutrophils, etc.). [00227] Factors involved in the inflammatory cascade can also be used as measurable parameters for effect of stimulation. For example, the signal transduction cascades include factors such as NFK-B, Egr-1, Smads, toll-like receptors, and MAP kinases.

[00228] In some embodiments, the physiological parameter is an action potential or pattern of action potentials in a nerve of the subject, where the action potential or pattern of action potentials is associated with the condition that is to be treated.

[00229] In some embodiments, the local physiological responses to splenic nerve stimulation (SpNS), such as change in splenic arterial blood flow (SpA BF), can be used as an indicative biomarker of SpN engagement to provide intraoperative confirmation for surgeons implanting a splenic nerve stimulation device for positioning and for parameter selection. For example, based on the correlation between SpN recruitment and SpA flow, SpA flow can be used as an indicator. In some embodiments, reduction in SpA BF can be used as a real-time dose-response biomarker for nerve engagement during surgical implantation of the bioelectronic device, and for patient-specific determination of the range of stimulation parameters.

[00230] In some embodiments, the efficacy of SpNS is assessed by measuring amplitude- and frequency-dependent changes in SpA BF and systemic mean arterial blood pressure (sMABP), which are directly correlated to nerve stimulation. These systemic physiological biomarkers can provide a surrogate of target engagement and can be used to determine nerve stimulation and facilitate the selection of neuromodulation parameters. Based on these findings, the feasibility and safety of chronic and/or acute SpN neuromodulation, e.g, during minimally- invasive surgery for esophageal cancer (esophagectomy), can be monitored and evaluated, e.g., to assess impact on inflammatory and physiological responses. See, for example, clinical trial NCT04171011, available online at clinicaltrials.gov. In some such embodiments, chronic and/or acute responses of SpN neuromodulation in humans can be assessed and used to provide information to support the use of intra-operative BF and blood pressure measurements for target engagement opportunities in future chronic implant clinical trials. In some embodiments, additional biological assays such as measurement of physiological parameters, contrast angiography, electrophysiology and histopathology can be performed to assess treatment efficacy, nerve integrity and/or subject tolerance and clinical status.

[00231] Methods of assessing these physiological parameters are known in the art. Detection of any of the measurable parameters can be performed before, during and/or after modulation of neural activity in the nerve. For example, a cytokine, chemokine, or a catecholamine (e.g., norepinephrine or epinephrine) can be directly detected, e.g., by ELISA. Alternatively, the presence or amount of a nucleic acid, such as a polyribonucleotide, encoding a polypeptide described herein may serve as a measure of the presence or amount of the polypeptide. Thus, it will be understood that detecting the presence or amount of a polypeptide will include detecting the presence or amount of a polynucleotide encoding the polypeptide. Specialized pro-resolving mediators can also be directly measured and quantified using methods described and available in the art. Such methods include liquid chromatography and tandem mass spectrometry.

[00232] Quantitative changes of the biological molecules (e.g., cytokines and pro-resolving mediators) can be measured in a living body sample such as urine or plasma. Detection of the biological molecules can be performed directly on a sample taken from a subject, or the sample can be treated between being taken from a subject and being analyzed. For example, a blood sample can be treated by adding anticoagulants (e.g., EDTA), followed by removing cells and cellular debris, leaving plasma containing the relevant molecules (e.g., cytokines and pro- resolving mediators) for analysis. Alternatively, a blood sample may be allowed to coagulate, followed by removing cells and various clotting factors, leaving serum containing the relevant molecules (e.g., cytokines and pro-resolving mediators) for analysis.

[00233] In some embodiments where the signal is applied while the subject is asleep, the method can further comprise determining the subject’s circadian rhythm phase markers, such as the level of cortisol (or its metabolites thereof), the level of melatonin (or its metabolites thereof) and/or core body temperature. Cortisol or melatonin levels can be measured in the blood (e.g., plasma or serum), saliva or urine. Methods of determining the levels of these markers are known in the art, e.g., by enzyme-linked immunosorbent assay (ELISA) or radioimmunoassay. If measurements of the subject’s circadian rhythm phase markers indicate circadian oscillations of inflammatory markers which may beneficially be regulated by application of a signal with a system in accordance with the present disclosure, then application of the stimulation pulse sequence at late night or early morning (when cortisol levels begin to rise) at a suitable periodicity according to the subject’s circadian rhythm may be appropriate.

[00234] As used herein, a physiological parameter is affected by the modulation (e.g., stimulation) of the splenic neural activity if the parameter changes (in response to nerve modulation) from the normal value or normal range for that value of that parameter exhibited by the subject or subject when no intervention has been performed, e.g., it departs from the baseline value for that parameter. Such a physiological parameter can include arterial pressure, heart rate or glucose metabolism. Suitable methods for determining changes in any these physiological parameters would be appreciated by one skilled in the art. One skilled in the art will appreciate that the baseline for any neural activity or physiological parameter in a subject need not be a fixed or specific value, but rather can fluctuate within a normal range or can be an average value with associated error and confidence intervals. Suitable methods for determining baseline values are known in the art.

[00235] As used herein, a physiological parameter is determined in a subject when the value for that parameter exhibited by the subject at the time of detection is determined. A detector (e.g., a physiological sensor subsystem, a physiological data processing module, a physiological sensor, etc.) is any element able to make such a determination. Thus, in some embodiments, the method further comprises a step of determining one or more physiological parameters of the subject, where the stimulation pulse sequence is applied when the determined physiological parameter meets or exceeds a predefined threshold value. In some such embodiments where more than one physiological parameter of the subject is determined, the stimulation pulse sequence is be applied when any one of the determined physiological parameters meets or exceeds its threshold value, or when all of the determined physiological parameters meet or exceed their threshold values. In some embodiments where the stimulation pulse sequence is applied by a system 100 e.g., a pulse generator), the system further comprises at least one detector configured to determine the one or more physiological parameters of the subject.

[00236] For any given parameter, the threshold value can be defined as a value indicative of a pathological state or a disease state. The threshold value can be defined as a value indicative of the onset of a pathological state or a disease state. Alternatively, the threshold value can be defined as a value indicative of a physiological state of the subject (that the subject is, for example, asleep, post-prandial, or exercising). Appropriate values for any given physiological parameter will be apparent to one skilled in the art (for example, with reference to medical standards of practice).

[00237] A threshold value for a given physiological parameter is exceeded if the value exhibited by the subject is beyond the threshold value (e.g., where the exhibited value is a departure from the normal or healthy value for that physiological parameter than the predefined threshold value). A departure from the normal or healthy value may be a value lower or higher than the predefined threshold value.

[00238] In some embodiments, the method further comprises administering to the subject a pharmaceutical composition for the inflammatory condition. For example, in some embodiments, the method further comprises administering an anti-inflammatory medication to the subject. In some embodiments, the administering is performed before the generating a stimulation (e.g., an electrical signal, an infrared signal, an ultrasound signal, a mechanical signal, and/or a magnetic signal) at the one or more signal-conducting interfaces (e.g, implanted or external electrodes) with the signal-generating source. In some embodiments, the administering is performed after the generating a stimulation at the one or more signal- conducting interfaces with the signal-generating source. In some embodiments, the administering is continued during, concurrently with and/or after the generating a stimulation at the one or more signal-conducting interfaces with the signal -generating source, for treatment of the inflammatory condition.

[00239] In some embodiments, the pharmaceutical composition is a nonsteroidal anti- inflammatory drug (NSAID), a steroid, a 5ASA, a disease modifying anti-inflammatory drug (DMARD) (e.g., azathioprine, methotrexate and/or cyclosporine), a biological drug (e.g., infliximab and adalimumab), and/or an oral DMARD (e.g., a Jak inhibitor).

[00240] Additional systems and methods for assessing efficacy of treatment of inflammatory conditions are described in International Patent Application PCT/GB2018/053731, entitled, “Treatment of Disorders Associated with Inflammation,” filed December 20, 2018, which is hereby incorporated herein by reference in its entirety.

[00241] Other Embodiments.

[00242] It will be apparent to one skilled in the art that the systems and methods for stimulating a splenic nerve in a subject provided herein can comprise any of the subjects, inflammatory conditions, implantation procedures, devices, stimulation parameters, stimulation pulse sequences, and/or methods for assessment of efficacy described in the foregoing sections and/or in the present disclosure. Further, it will be apparent to one skilled in the art that the method can comprise any embodiments described herein, or any substitutions, modifications, additions, deletions and/or combinations thereof.

[00243] It will be apparent to one skilled in the art that the systems and methods of treating an inflammatory condition (e.g., chronic and/or acute) in a subject by activating the ALOX15 pathway can comprise any of the subjects, inflammatory conditions, implantation procedures, devices, stimulation parameters, stimulation pulse sequences, and/or methods for assessment of efficacy described in the foregoing sections and/or in the present disclosure. Further, it will be apparent to one skilled in the art that the method can comprise any embodiments described herein, or any substitutions, modifications, additions, deletions and/or combinations thereof.

[00244] EXAMPLES.

[00245] EXAMPLE 1 - Validation of EIuman-Relevant Porcine Models for Near-Organ Neuromodulation of the Immune System via the Splenic Nerve. [00246] Materials and Methods

[00247] Acute terminal-anesthetized experiments

[00248] Sixty four farm pigs (female; Large white / British landrace cross) were sourced from a commercial pig farm and acclimatized at the research facility for a minimum of 7 days prior to the experiment; animals were used for electrophysiological studies (n=6, body weight 45 - 50 kg, age 10 - 12 weeks); high dose endotoxemia model (n=18; BW 45 - 50 kg, age 10 - 12 weeks); low dose endotoxemia model (n=27; BW 60 - 70 kg, age 12-14 weeks); and in vitro splenocytes experiments (n=7; BW 60 - 70 kg, age 12-14 weeks). Four Berkshire pigs (BW 60 - 70 kg, age 12-14 weeks) were used for in vivo NA measurement experiments. Animals were group housed on straw bedding and given food and water ad libitum until 12 hours prior to the experiment, at which point food was withheld. On the day of the experiment, animals were pre- medicated with ketamine (20 mg/Kg) and midazolam (0.5 mg/Kg) administered by intramuscular injection. Fifteen minutes after premedication, a 20 G intravenous catheter was placed in the auricular vein. General anesthesia was induced with propofol (2 mg/kg) administered intravenously. Animals were intubated with an endotracheal tube, and anesthesia was maintained with sevoflurane vaporized in a 50:50 mixture of oxygen and medical air. A continuous rate infusion of fentanyl (0.2 pg/kg/min) was started after induction and continued during the whole experimental procedure. For the low dose LPS endotoxemia model experiments fentanyl infusion was only used during the surgical preparation and then discontinued. Details of surgical procedure and monitoring are reported in the supplementary materials and methods below.

[00249] Pig splenocyte experiments (ex vivo, n=7)

[00250] General anesthesia, maintenance and surgical procedure were performed as described above. The major vessels (splenic, short gastric, and gastroepiploic arteries and veins) were sequentially ligated, and the vessels transected. The omentum was incised and the spleen removed. Animals were then euthanized by barbiturate overdose.

[00251] Spleens were cut into four sections, 5 g of tissue were sampled from the middle of each section, rinsed in cold PBS (10204733, Fisher Scientific) and passed through a metal strainer under gentle manual pressure using a 50 mL syringe plunger. Cell suspension was transferred to six 50 mL conical centrifuge tubes (E1450-0200, Starlab) and centrifuged for 10 minutes, 300 x g, at 4 °C. Supernatant was discarded and cells resuspended in 20 mL cold PBS, then mixed with 30 mL erythrocyte lysis buffer (8.29 g ammonium chloride (21236.267, VWR International Ltd.), 1 g potassium hydrogen carbonate (237205, Sigma-Aldrich), 37 mg EDTA disodium salt (ED2SS, Sigma-Aldrich) dissolved in 1 L DI water and filter sterilized) and left to stand for 10 minutes, mixing by inversion half way through. Centrifugation was repeated and supernatant discarded, and erythrocyte lysis was repeated. Leukocyte pellets were resuspended in 20 mL cold PBS and centrifuged for 5 minutes, 300 x g, at 4°C. Cells were resuspended in 20 mL cold PBS and passed through 70 pm cell strainers (352350, Scientific Laboratory Supplies Ltd., Nottingham, UK) into fresh tubes to remove debris, pooled, then centrifuged for 5 minutes as previously. Supernatant was discarded and cells resuspended in complete culture medium and counted by trypan blue exclusion (15250061, Fisher Scientific) using counting chambers (BVS100, Immune Systems Ltd.). Cells were then resuspended in medium (RPMI 1640 (72400-021, Fisher Scientific)), supplemented with 10% fetal bovine serum (FBS) (11550356, Fisher Scientific) and 1% Penicillin- Streptomycin (11548876, Fisher Scientific) and plated at 5xl0⁵/mL, in either 48- or 12-well flat bottomed culture plates as necessary (CC7682- 7548 & CC7682-7512 respectively, Starlab) and then used for experiments. For experimental details see the below supplementary materials and methods section.

[00252] NA in vivo measurements - Piss (in vivo, n=4)

[00253] Animal handling, management and anesthesia protocol on the day of the experiment was identical to that described above. Laparotomy was then performed to implant an electrode cuff around the splenic neurovascular bundle (NVB) (comprising the splenic artery and its nerve plexus). The leads were exteriorized and connected to an external stimulator. A catheter was inserted into the splenic vein (SpV) and routed towards the base of the spleen. Animals were allowed to stabilize for 30 minutes. Blood (5 mL) was collected at the same time from the SpV and the jugular vein (JV) over 60 seconds. Ten minutes later, splenic NVB stimulation (Stim 1) was performed for 1 minute at 10 Hz (bipolar, symmetrical biphasic rectangular pulses) and blood collected as before. After 30 minutes, blood sampling was performed again during another baseline (Baseline 2) and stimulation (Stim 2) procedure. A sham (no current applied) stimulation was used as control. Samples were transferred immediately to EDTA vacutainers, mixed by inversion and stored on ice. Plasma was isolated by centrifugation (2000 x g for 5 minutes), added to stabilizing solutions (as per ELISA instructions) and immediately frozen on dry ice. Frozen plasma aliquots were thawed and immediately analyzed by ELISA for quantification of Noradrenaline (NA) using the Noradrenaline Sensitive ELISA (DLD Diagnostika, cat. no. ea633/96), according to manufacturer’s instructions. Plates were analyzed using the Infinite® 200 PRO spectrophotometer and iControl software (Tecan Group Ltd.).

[00254] High dose LPS model - Pigs (in vivo, n=18) [00255] Animal handling, management and anesthesia protocol on the day of the experiment was identical to that described above, and the experiment set-up is illustrated in Fig. 4A.

Animals were randomly divided into 3 treatment groups with 6 animals per group, receiving either stimulation of the SpN (SpNS group; instrumented with an NVB cuff electrode and a SpA flow probe as described above), left vagus nerve stimulation (LVNS), or no stimulation (Sham group). Animals in the LVNS group were placed in dorsal recumbency, and the left ventral neck was clipped, aseptically prepared and draped in a routine fashion. Using aseptic technique, a 10 cm longitudinal skin incision was placed immediately to the left of the trachea from the larynx caudad. The incision was continued through the subcutaneous tissue and the sternohyoideus muscle until encountering the carotid sheath and left vagus nerve (LVN). A 1 cm segment of the LVN was circumferentially isolated from surrounding loose connective tissue by careful blunt dissection and was subsequently instrumented with a bipolar circumferential cuff electrode (2.0 mm diameter, 8 mm length; cathode surface area: 0.05 cm²; #1041.2179.01, CorTec GmbH). Sham group animals received the same surgical approach as the SpNS group and were instrumented with a SpA flow probe as described above; however, no NVB cuff electrode was implanted. In this group, the SpA was manually occluded for one minute achieving approximately 50 % flow reduction at the two stimulation time points, mimicking the SpA flow reduction evoked by SpNS. Three hours after initial stimulation animals were injected intravenously (i.v.) into the Jugular vein with 2.5 pg/kg body weight of LPS (purified lipopolysaccharides from the cell membrane of Escherichia coli (E. coli )O111 :B4; Sigma-Aldrich). Following LPS administration, animals were euthanized with an overdose of pentobarbital (administered i.v.) when the sMABP reached levels below 40 mmHg despite pharmacological treatment (defined as the humane endpoint), or when the animal completed the pre-determined study time window of 2 h post LPS injection. All of the details regarding stimulation parameters, blood sampling, LPS dosing and pharmacological treatment are reported in the supplementary materials and methods.

[00256] Low dose LPS model - Pigs (in vivo, n=27)

[00257] Animal handling, management and anesthesia protocol on the day of the experiment was identical to that described above, and the experiment set-up is illustrated in Fig. 5A.

Animals were randomly divided into 5 treatment groups: Sham (n=7), SpNS (n=6), LVNS (n=6), stimulation of the efferent trunk of the LVN (eLVNS) (n=5) and dexamethasone (Dex) (n=3). Animals within the Dex group received two i.v. boluses (0.5 mg/Kg each) after induction of anesthesia (2.5 h prior to LPS injection) and at the time of LPS injection. Animals within this group were maintained under general anesthesia for the same duration of the other animals, but no surgical procedure, a part for instrumentation (central vein and arterial catheters), was performed on these animals. The animals within the other groups were subjected to surgical procedures. The SpNS and Sham group received a laparotomy (as described above) and a transit time flow probe placed on the distal SpA. The SpNS group was also implanted on the proximal/middle NVB with a cuff electrode (5 mm diameter, 10 mm long spiral cuff electrode, CorTec GmbH). The LVN was accessed in animals belonging to LVNS and eLVNS groups. The VN was accessed as described above and implanted with a 2 mm cuff electrode (2.0 mm diameter, 8 mm length; cathode surface area: 0.05 cm²; #1041.2179.01, CorTec GmbH). After cuff implantation, the LVN was ligated just proximal to the cuff and then cut to eliminate central connections in the eLVNS group.

[00258] Electrical stimulation was delivered continuously for 3 h (-2 to +1 h relative to LPS injection) at 1 Hz; low frequency stimulation was chosen in order to prevent cardiovascular effects during the prolonged stimulation duration. The amplitude of the stimulation was such to recruit approximately 10 - 50% of the SpN axons and ca. 100% of the LVN axons. Two hours from initiation of the stimulation, LPS (0.25 pg/kg; E. coli O11 LB4; Sigma-Aldrich) was injected i.v. (via the Jugular vein). The LPS was prepared in sterile saline, then diluted in injectable saline (0.5 mL of LPS solution in 9.5 mL of saline) to achieve the right concentration and administered over a period of 5 min (2 mL/min). Peripheral blood samples were collected every 0.5 h from -1 h to +4 h relative to the LPS injection time point; blood was collected in plain and EDTA tubes for routine hematology and clinical chemistry. Additional EDTA samples were collected, immediately centrifuged at 2000 x g for 5 minutes at 4°C. Plasma was separated and immediately frozen on dry ice and stored at -80°C. Frozen plasma samples were used to measure TNF-a and IL-6 concentration using the commercially available ELISA kits (Porcine TNF-alpha; DY690B, and porcine IL-6, DY686; DuoSet Solid Phase Sandwich ELISA, R&D Systems).

[00259] Data analysis

[00260] For NA quantification, SpV and JV samples across time points were compared using repeated measures one-way ANOVA and Bonferroni correction for multiple comparisons. For splenocyte experiments, each group was compared to control (LPS group) using repeated measures one-way ANOVA and Bonferroni correction for multiple comparisons.

[00261] In the high dose endotoxemia model, ex vivo TNF-a levels were compared using two-way ANOVA and Bonferroni correction for multiple comparisons. For vasopressin use and changes in sMABP after in vivo LPS administration SpNS and LVNS groups were compared to the Sham control group using ordinary one-way ANOVA. For cytokine quantification the area under the curve (AUC) for both plasma TNF-a and IL-6 was calculated between 0 and 2.0 h post-LPS injection for SpNS and LVNS groups and compared using unpaired /-test.

[00262] In the low dose endotoxemia model, the AUC was calculated between 0.5 and 2.0 h post-LPS injection (AUC0.5- 2) for plasma TNF-a and between 1.5 and 4.0 h post-LPS injection (AUC1.5-4) for IL-6; these are the time periods during which the majority of the TNF-a and IL-6 increase, peak and subsequent decline occurred. Data were then compared using ordinary one- way ANOVA with each group compared to Sham controls. Hematology data were analyzed using two-way ANOVA and Dunnet correction for multiple comparisons.

[00263] Statistical significance was defined as P < 0.05, and analyses were performed with commercially available statistical software (JMP Pro 13.0.0 and GraphPad Prism 9.0).

[00264] Supplementary Materials and Methods

[00265] Immunohistochemistry and semi-thin sections of pig NVB

[00266] For immunohistochemistry, 4 pm paraffin embedded sections were dewaxed prior to heat antigen retrieval in citrate buffer pH 6.0. In brief, sections were incubated with tyrosine hydroxylase (TH) antibody (Mouse monoclonal anti TH, Abeam abl29991; dilution 1 :2000) and choline acetyltransferase antibody (Goat polyclonal anti ChAT, Millipore AB 144; dilution 1 :200); or TH and calcitonin gene-related peptide antibodies (Goat anti GCRP, Abeam AB36001; dilution 1 :3000); or neurofilament 200 and myelin basic protein (MBP) antibodies (Rabbit polyclonal anti NF200, Abeam ab8135; dilution 1 : 1000), (Rat monoclonal anti MBP; Abeam ab7349; dilution 1 :200). Fluorescent conjugated secondary antibodies were then incubated against the relevant host primary antibody. Alexafluor 488 and 594 nm secondary antibody (ThermoFisher) combinations were used to distinguish between the pairs of primary antibodies. Cell nuclei were counterstained with 4’,6-diamidino-2-phenylindole (DAPI). For each section, two different digital images were randomly captured at 20x magnification, and pseudocolored composites generated using appropriate software (AxioVision LE64). The proportion of positive fibers were quantified by manual counting in an area of 100 x 100 pm.

[00267] Pig surgery and monitoring

[00268] Following induction of stable anesthesia, bilateral indwelling jugular vein catheters and one femoral arterial catheter were placed under ultrasonographic guidance. Volume- controlled mechanical ventilation was maintained for surgery and implantation. Once instrumentation was complete animals were kept on spontaneous ventilation and data collection commenced when animals were in a steady-state as assessed by stable cardiovascular parameters. Routine anesthesia monitoring included vital parameters such as electrocardiogram and invasive arterial blood pressure (systolic, diastolic and mean), central venous pressure; end- tidal CO2 (ETCO2), fraction of inspired oxygen (FiO2), end-tidal sevoflurane (ETSev), pulse oximetry and core body temperature (via rectal probe). Some of these parameters (arterial blood pressure, central venous pressure, ECG, ETCO2, ETSev) were also digitally recorded using a 16 channel PowerLab acquisition system (AD Instruments) with LabChart 8 software at 2 kHz sampling frequency.

[00269] Animals were placed in right lateral recumbency, and the left lateral abdomen was clipped and aseptically prepared and draped in a routine fashion. Using aseptic technique, a 20 cm laparotomy incision was made in the second to last intercostal space, and access to the splenic base with associated neurovasculature was aided by the use of rib retractors. For the electrophysiological experiment, the splenic NVB was instrumented as detailed below. At the end of all experiments, animals were humanely euthanized with an overdose of pentobarbital administered intravenously, after which the splenic neurovasculature was rapidly harvested and fixed in 10% neutral buffered formalin (NBF; VWR) for histological analysis.

[00270] NA in vivo measurements - Pigs

[00271] Following plating, cells were placed in an incubator (37°C, 5% CO2) to acclimatize while treatments were prepared. Cell were treated with or without LPS and with or without NA as described below. 1 mL sterile saline (VETIVEX1, Dechra Veterinary Products Ltd.) was added to 1 mg LPS (L4391, Sigma-Aldrich). The vial was shaken vigorously, vortexed for 20 sec, and sonicated for 5 min. 30 pL aliquots of LPS were stored at -20°C. 10 mL sterile saline was added to 100 mg L-Norepinephrine hydrochloride (74480, Sigma-Aldrich Ltd.), mixed by pipette, and then vortexed for 20 sec. 100 pL aliquots NA were stored at -20°C. When needed, NA and LPS were thawed at room temperature, and LPS was sonicated for 5 min. Compounds were then diluted in culture medium as needed.

[00272] 3 h and 24 h conditions: plates were removed from the incubator and 41 pL of NA and 20 pL of LPS (control (CT) received medium only) were added to wells as appropriate, giving final concentrations of 8 pM NA and 100 ng/mL LPS. Plates were returned to the incubator for 3 or 24 h incubation. Time of NA addition was varied by +/-1 h to investigate impact of timing in relation to LPS exposure.

[00273] Time optimization: plates were removed from the incubator and NA-HC1 was added to the insert of duplicate wells, to give a final concentration of 8 pM NA. Plates were returned to the incubator for 1 h incubation. Medium was removed from the main wells and the plate centrifuged at 800 x g for 30 sec to empty transwell inserts of medium. Medium was again removed and 500 pL of fresh medium added to inserts to rinse cells. Plates were centrifuged again, and the transwell inserts containing cells were moved to clean 12-well plates containing 1.5 mL per well of fresh medium. At intervals of 1, 2, 4 and 25 h after initial NA exposure, LPS was added to the insert of duplicate wells to give a final concentration 100 ng/mL. Plates were returned to the incubator for 3 h incubation.

[00274] All experiments: following incubation, well contents were transferred by pipette to 1.5 mL microcentrifuge tubes (E1415-2231, Starlab) and centrifuged at 2000 x g for 5 min. The conditioned medium (CM) from each tube was collected, aliquoted, and immediately frozen on dry ice, before transfer to -80°C storage.

[00275] High dose LPS endotoxic shock model - Pigs

[00276] The experiment was divided in two parts.

[00277] In Part I of the experiment, stimulations were performed in naive animals and stimulation effects were evaluated by physiological responses and analyses of peripheral blood including an ex vivo LPS challenge with subsequent inflammatory cytokine assays. Triplicate samples of peripheral sodium heparinized venous blood (collected from the Jugular vein) were cultured for 4 h at 37°C in the presence of 0 pg/mL or 1 pg/mL of LPS (purified lipopolysaccharides from the cell membrane of Escherichia coli O111 :B4; Sigma-Aldrich). Samples were then centrifuged at 2000 x g and the supernatant stored at -80 °C until analysis for TNF-a by commercially available ELISA kits (Porcine TNF-alpha; DY690B; DuoSet Solid Phase Sandwich ELISA, R&D Systems), run as per the manufacturer's instruction. All samples were run as technical replicates (n=3) for each time-point and LPS concentration.

[00278] Part II of the experiment included evaluating stimulation effects in the same animals for up to 2 h after administering a high dose of LPS (2.5 pg/kg body weight; E. coli Ol l i :B4; Sigma Aldrich). The LPS was prepared in sterile saline, then diluted in injectable saline (0.5 mL of LPS solution in 9.5 mL of saline) to achieve the right concentration and administered i.v. over a period of 5 min; a second stimulation was delivered at the same time as the LPS injection. Again, stimulation effects were evaluated by physiological responses including sMABP and SpA BF. LPS injection caused an acute cardiovascular deterioration/decompensation, manifesting as unresponsive hypotension and/ or clinically significant changes in cardiac function. Standard clinical pharmacological therapies aimed at restoring homeostasis, such as vasopressin (2.5 IU bolus injections administered i.v. and repeated as needed) and anti-arrhythmic drugs (lidocaine; 2 mg/kg i.v. and/ or atropine; 40 pg/kg; i.v.) were given at the discretion of the anesthetists when animals were approaching the threshold of 40 mmHg of sMABP or they showed arrhythmias. The anesthetists were blind to the treatment groups (SpNS or Sham). Details of pharmacological treatment of individual animals are given in Table 3.

[00279] During Part I and II of the experiments blood samples were collected in plain and EDTA tubes for routine hematology and clinical chemistry. During Part II, prior to LPS injection and then every 0.5 h, additional samples were collected in EDTA tubes and immediately centrifuged at 2000 x g for 5 min at 4 °C. Plasma was then separated and immediately frozen on dry ice and subsequently stored at -80 °C. Frozen plasma samples were used to measure TNF-a and IL-6 concentration using the commercially available ELISA kits (Porcine TNF-alpha; DY690B, and porcine IL-6, DY686; DuoSet Solid Phase Sandwich ELISA, R&D Systems).

[00280] Identical stimulation parameters were used for stimulation in Part I and Part II of the experiment. Based on our electrophysiology data, the SpNS group received a stimulation (biphasic rectangular symmetric pulses delivered at 10 Hz and with 0.4 ms pulse width; and a fixed amplitude at 12 mA) to ensure target engagement as evaluated by reduction in SpA mean BF. The LVNS group received a 60 second stimulation with biphasic, symmetric, rectangular pulses delivered at 3.5 mA, 0.4 ms pulse width and 10 Hz. This resulted in suitable target engagement as defined by bradycardia, hypotension and bradypnea responses.

[00281] Results

[00282] Stimulation of SpN releases noradrenaline that is immunomodulatory in pigs

[00283] In line with electrophysiological experiments, histological examinations of splenic NVB demonstrated that both pig and human SpN are mainly composed of unmyelinated axons. Toluidine blue stained semi-thin sections did not show evidence of myelination in pig NVB sections. This was corroborated by immuno-histochemistry analysis where almost all (> 99%) of the neurofilament (NF)/piII-tubulin positive axons were found to be myelin basic protein (MBP)-negative, with very rare MBP-positive axons observed (Fig. 3A). Electron microscopic images of the human NVB showed a similar result: very rare myelinated axons were found interspersed among small unmyelinated axons. Sections of pig VN were used as a control for toluidine blue and MBP staining and showed the expected composition with myelinated and unmyelinated axons. [00284] It is known that the human SpN is composed of sympathetic neurons expressing tyrosine hydroxylase (TH) and lacking choline acetyltransferase (ChAT) [26, 29, 30], Here we observed that the pig SpN axons also stained positive for TH (Fig. 3B), and no ChAT -positive axons were identified. Sections of the VN, known to contain both fiber types, were used as a positive control and showed the expected mixed composition. Finally, a small proportion of pig SpN axons (< 5%) stained positive for calcitonin gene-related peptide (CGRP) (Fig. 3C), in line with recent human data [27], Within the pig spleen, TH-positive axons were again observed and found in close proximity to both CD1 lb myeloid cells (Fig. 3D) and CD3 lymphocytes (Fig. 3E) within the marginal zone, red pulp and periarteriolar lymphoid sheet (PALS) (Fig. 3E, F). Rarely nerve fibers could be observed within the B follicle areas (Fig. 3E).

[00285] Since NA is a key molecular component of the anti-inflammatory reflex in rodents, further studies were performed to verify whether SpNS induces release of NA in the pig spleen. Blood draining from the spleen within the SpV prior to and during 2 stimulations of the SpN (each delivered for 1 min at 10 Hz, 0.4 ms PW and 4.8 pC, 30 min apart) were collected from terminally-anesthetized pigs (Fig. 3G). As a control, blood from the jugular vein (JV) was also sampled. While baseline levels (prior to each of the stimulations) of NA were low in both SpV and JV, stimulation induced a significant increase in NA within the SpV only (Fig. 3H).

[00286] To investigate the immunomodulatory effects of SpNS, NA was utilized as a proxy of stimulation and its effect on isolated pig splenocytes was examined. Spleens were harvested from donor animals and processed to isolate total leukocytes. The culture was then challenged with LPS, with or without NA, and TNF-a accumulation in the conditioned medium (CM) was quantified (Fig. 31). NA was able to suppress LPS-induced TNF-a after both 3 h (Fig. 3J) and 24 h (Fig. 3K) of incubation. In addition, NA was able to reduce TNF-a (as measured 3 h post LPS exposure) when delivered 1 h before or 1 h after LPS exposure (Fig. 3L), with the former producing a stronger suppression (Fig. 3L). Prior to performing in vivo efficacy studies, a suitable time window was identified between stimulation (NA) and pro-inflammatory challenge (LPS) to achieve TNF-a suppression in pigs. Splenocytes were incubated with NA for 1 h prior to being washed and placed into fresh medium. Cells were then challenged with LPS at different times post NA removal and TNF-a accumulation was measured 3 h post LPS (Fig.

3M). The maximum NA-induced suppression of TNF-a (37.9 ± 8.3% reduction vs. LPS only) was observed when cells were challenged with LPS 2 h from initial NA exposure (Fig. 3M).

[00287] Taken together these data suggested that the pig SpN does indeed release NA which has a TNF-a suppressing effect in pig splenic cells similar to that demonstrated in rodents. [00288] Stimulation of splenic nerve or vagus nerve promotes immunomodulation in pigs

[00289] In order to assess the translatability of the anti-inflammatory pathway in vivo, two acute models of inflammation in pigs were used.

[00290] A high dose of LPS (2.5 pg/kg) was administered (i.v. into the JV) in terminally- anesthetized pigs (Fig. 4A). In this experiment, the protective effect of SpNS was tested in a model in which the complex LPS-induced inflammatory responses are known to cause acute cardiovascular compromise and death within a short time window [31, 32], Left VNS (LVNS) was used as a positive control, due to its reported protective effects in endotoxemia models, alongside a splenic nerve sham stimulation group. In order to select LVNS parameters, preliminary electrophysiology studies were performed to select the stimulation amplitude able to stimulate all fiber types contained within the cervical VN, including small and unmyelinated axons that are reported to be the main constituent of the abdominal portion of the VN in pigs and humans [16],

[00291] Stimulation of the SpN or LVN was delivered 3 h prior to LPS challenge and at the time of LPS injection (1 min stimulation duration both times). In both cases stimulation was delivered at 10 Hz as typically used in previous rodent efficacy studies and since it was able to produce a reliable release of NA in the experiments. Target engagement in the SpNS group was confirmed by stimulation responses resulting in a 37.41 ± 18.73% reduction in SpA mean BF, corresponding to approximately 25% eCAP activation (range 10 - 50%). Target engagement in the LVNS group was confirmed by presence of VNS-induced changes in physiology: bradycardia, hypotension and bradypnea. The sham animals received a mechanical partial occlusion of the SpA (causing about 50% SpA BF reduction) for 1 min at each stimulation time point to control for blood flow changes associated with SpNS.

[00292] Before and after the stimulation (or sham) period, peripheral whole blood was collected longitudinally to assess systemic immunomodulatory/ suppressive effects by using an ex vivo LPS-induced challenge. No differences between groups were observed in the ex vivo LPS-induced TNF-a levels up to 2 h post stimulation (Fig. 4B). Additionally, no difference in peripheral total WBC count (Fig. 4C) or other hematological parameters (Table 1) were observed between groups. Only time-dependent effects were observed in all groups and likely related to the procedure (surgery & anesthesia) per se. No significant changes over time were observed in biochemical parameters either (Table 2).

[00293] Upon LPS injection, leukopenia and hypotension were observed at 0.5 h post-LPS in the sham animals (Fig. 4C and D). Hypotension normally developed within 10 min following injection rapidly reaching sMABP values of 40 mmHg (Fig. 4D), accompanied by tachycardia and/or tachyarrhythmia (5 out of 6; Table 3), requiring the use of vasopressin (6 out of 6; Fig. 4D and E) and anti arrhythmic drugs (Table 3). Thirty minutes after LPS injection, the sMABP dropped to about 70% below the pre-LPS baseline in most sham animals (Fig. 4F). Stimulated animals, while still showing leukopenia (Fig. 4C), did not show extensive reduction in sMABP (Fig. 4E), and only a few animals displayed tachycardia and/or tachyarrhythmia (1 out of 6; Table 3), or required vasopressin (2 out of 6 SpNS, and 1 out of 6 LVNS; Fig. 4F). Animals that reached a sMABP of less than 40 mmHg, despite pharmacological treatment were euthanized. This humane endpoint was reached by 5 out of 6 sham animals within 2 h post-LPS and by only 1 out of 6 animals in both the SpNS and LVNS groups (Fig. 4G). Since sham animals received multiple treatments within the first 0.5 h and some animals reached the humane endpoint before this time point, comparison of cytokine levels was not possible in this model. However, TNF-a and Interleukin 6 (IL-6) were partially increased (vs. baseline) at 0.5 h in all groups, and both SpNS and LVNS animals showed similar trends in cytokine profiles over the course of 2 h post LPS (Fig. 4H). Quantification of the area under the curve (AUC) between 0 and 2 h post LPS showed no difference between SpNS and LVNS groups (Fig. 41, J).

[00294] These data indicated that SpNS and LVNS had similar protective effects in a model involving cardiovascular compromise caused by LPS.

[00295] Successively, in order to accurately assess the effect of SpNS on cytokine production and further assess translation of the anti-inflammatory pathway in pigs, a low dose endotoxemia model was used (0.25 pg/kg of LPS, i.v.) (Fig. 5A). This LPS dose caused peripheral cytokine and hematological changes, without severe cardiovascular effects, and therefore allowed serial peripheral blood sampling and cytokine analysis over several hours after LPS challenge in line with previous literature [34], The charge used for SpNS was in line with the previous experiment in order to produce a similar degree of nerve engagement (about 20-50% nerve stimulation). In addition to Sham (n=6) and LVNS (n=6), a group of animals (n=3) received i.v. injections of dexamethasone (0.5 mg/kg; 2.5 h before and at the same time of LPS injection), as a positive control for cytokine suppression; and another group (n=5) received stimulation of the efferent trunk of the LVN (eLVNS) in order to activate only peripheral, and not central (e.g., via the brain), connections to abdominal organs (Fig. 5A). In order to maximize effects over a long period of time, SpNS, LVNS and eLVNS were delivered for a longer time (from -2 h to + 1 h relative to LPS injection) at 1 Hz (instead of 10 Hz) to reduce cardiovascular changes associated with the stimulations. The charge used for LVNS and eLVNS groups was in line with the previous experiment.

[00296] Injection of this dose of LPS (2 h after stimulation initiation in accordance with the previous in vitro and in vivo results) also resulted in change in peripheral leukocyte counts. In all groups the typical peripheral white cell responses to LPS were observed. There was initial leukopenia with reduction in circulating neutrophils, monocytes and lymphocytes. This was followed by the leukocytosis with increase in circulating neutrophils while monocytes and lymphocytes remained low until the end of the observation period (4 h after LPS). A statistically significant (P<0.01) effect of time on total white blood cell (WBC) count, as well as lymphocyte, monocyte and neutrophil counts was observed in all groups. Dexamethasone showed the smallest changes in leukocyte counts vs. baseline (0 h) levels. In parallel to hematological changes, LPS caused a dynamic accumulation and clearance of peripheral TNF-a and IL-6 (Fig. 5B) during the course of the experimental window (4 h post LPS injection), reaching maximum at 1 h and 3 h post-LPS, respectively. Dexamethasone caused a strong suppression of LPS-induced TNF-a and IL-6 (Fig. 5B, C and D) compared to Sham.

Importantly, all electrically-stimulated groups displayed cytokine suppression, albeit only partially (Fig. 5B, C and D). Interestingly, SpNS and eLVNS both resulted in a similar reduction (vs. Sham) in TNF-a and IL-6 total concentrations, measured as AUC (Fig. 5C, D). Conversely, LVNS animals showed a highly variable response in TNF-a reduction (Fig. 5C), but a more reproducible reduction in IL-6 (Fig. 5D). When looking at each individual animal and the relative concentration of TNF-a and IL-6 (vs. Sham animals), SpNS had a global modulatory effect (grey box in Fig. 5E) in 4 out of 6 animals; for the VN groups, eLVNS had global modulatory effects on 5 out of 5, while LVNS in 3 out of 6 animals (Fig. 5E). Dexamethasone showed a modulatory effect in all 3 animals. Of note was the finding that the suppression of TNF-a and IL-6 induced by dexamethasone occurred in combination with a significant reduction of circulating leukocytes (Fig. 5F), mainly monocytes (Fig. 5G) and lymphocytes (Fig. 5H), within 2.5 h after administration of a single dose (0 h; prior to LPS administration). No significant changes in peripheral leukocyte counts (prior to LPS injection) were observed either in SpNS or eLVNS groups (Fig. 5F-H). A small but statistically significant reduction of circulating numbers of lymphocytes was observed instead in the LVNS group (Fig. 5H). [00297] Table 1. Peripheral blood hematology data from endotoxic shock model.

[00298] Table 1. The table shows the quantification of the hematology panels in the Sham, SpNS (referred to as SpN2S) and LVNS (referred to as LVN2S) groups. Mean values are averaged per time point within each treatment group. The first stimulation was given at time 0, whereas the second stimulation was administered immediately after taking the baseline sample. Abbreviations and units: WBC = total white blood cell count (xlO^A9/L); Neutroph = neutrophils (% of total WBC); Lymphoc = lymphocytes (% of total WBC); Monoc = monocytes (% of total WBC); Eosinoph = eosinophils (% of total WBC); Basoph = basophils (% of total WBC); RBC = red blood cell count (xlO^A9/L); HGB = hemoglobin (g/dL); HCT = hematocrit (%); PLT = platelet count (xlO^A9/L). [00299] Table 2. Peripheral blood biochemistry data from endotoxic shock model.

[00300] Table 2. Quantification of the biochemistry panel for each Sham, SpNS and LVNS.

Mean values are averaged per time point within each treatment group. The first stimulation was given at time 0 whereas the second stimulation was administered immediately after taking the baseline sample. Abbreviations and units: TP = total protein (g/L); Albumin (g/L), Globulin (g/L), Na = sodium (mmol/L); K = potassium (mmol/L); C1 = chloride (mmol/L); Ca = calcium (mmol/L); P = inorganic phosphorus (mmol/1); Urea (mmol/1); Creatinine (pmol/L); Cholesterol (mmol/L); T bilirubin = total bilirubin (gmol/1); Amylase (U/L); Lipase (U/L); ALT = alanine aminotransferase (U/L); CK = creatine kinase (U/L); ALP = alkaline phosphatase (U/L).

[00301] Table 3. Cardiovascular changes after LPS administration.

[00302] Table 3. Changes in systemic mean arterial blood pressure (referred to as MABP in this table) observed in the animals after LPS administration, and treatment administered to individual pigs. The time represents the time after LPS injection. MASS = external chest (cardiac) massage; VAS = administration of vasopressin (2.5 gg/kg i.v.); ATR = administration of atropine; LID = administration of lidocaine; Time Euth = time (min) from administration of the LPS to euthanasia; the pre-determined end-point was at 120 min.

[00303] Discussion

[00304] The foregoing studies demonstrate that immunomodulatory neural circuits previously described in rodents also exist in larger species such as the pig that represent, in some embodiments, a suitable correlate of the human anatomy and function. The foregoing studies show translation of the anti-inflammatory neuromodulation pathway in a clinically relevant species. This provides evidence that, in some embodiments, the pig is a suitable test-bed for refining clinical stimulation parameters in the context of neuro-immune modulation and testing interface designs for bioelectronic medicine development and future clinical use. Further, the present example illustrates the development of a novel surgical and anatomical approach to the anti-inflammatory pathway, which successfully targets the nodal point and distal nerve associated with the spleen and confirms target engagement through physiological, neurological, and immunological readouts.

[00305] The studies described herein show that, in some embodiments, immune responses in pigs can be regulated by modulation of vagus and splenic nerve activity, in line with previous rodent work [4, 7, 24], by using two different models of acute inflammation. For example, stimulation of splenic circuits via near organ stimulation of the SpN caused a 20-30% reduction in circulating levels of TNF-a and 40-50% reduction in IL-6 following a low dose LPS challenge. In support of this finding, similar immunomodulatory effects induced by cervical LVN stimulation were also observed. Interestingly, maximal stimulation (e.g., resulting in stimulation of all fiber types) of the efferent LVN led to a similar suppression profile, while the stimulation of the intact LVN did not result in a significant TNF-a reduction in this setting. Stimulation of the intact LVN in anesthetized rats resulted a highly variable response in TNF-a production, while eLVNS had a significantly more reproducible effect [20], Without being limited to any one theory of operation, the reason for this discrepancy in literature is not yet understood and may be related to different effects of afferent and efferent pathways [22] activated in the VN by electrical stimulation, especially under anesthesia where central mechanisms (e.g., via the brain) may be affected.

Together these data illustrate that neurotransmitter release induced by SpN or VN stimulation can regulate cytokine production following LPS. Importantly, the similar profile of SpNS and eLVNS supports the evidence that anti-inflammatory mechanisms can be regulated in abdominal organs, mainly the spleen. Earlier theories of the anti-inflammatory pathway, for example, posited a requirement in some instances for activation of efferent vagal circuits towards the spleen [3, 4, 6],

[00306] In some embodiments, NA may represent an important molecular mediator of the anti- inflammatory mechanisms also in the pig spleen. The studies described herein show that, in some embodiments, the SpN in pigs, in line with humans [26, 29], is catecholaminergic (TH-positive) in nature and releases NA upon stimulation. An in vitro splenocyte model demonstrated that NA is able to modulate TNF-a production, thus potentially linking in with the in vivo results. The importance of NA and its receptors expressed by immune cells within the spleen is also supported by previous rodent data. However, further studies may be required to better understand the molecular mechanisms and the molecular drivers of the immunomodulatory effects in pigs. For example, it is known that the SpN contains and releases other neurotransmitters, including adrenaline, CGRP, and neuropeptide-gamma [35], which could potentially drive additional/ differ ent immunomodulatory effects.

[00307] Novel effects associated with SpNS and LVNS, beyond modulation of cytokines, have been identified. For example, electrical stimulation prevented cardiovascular collapse in a pig acute high dose endotoxemia model, prior to cytokine flare. Since cardiovascular collapse in sham animals occurred earlier (within 0.5 h after LPS) than the time points at which cytokines reach significant up-regulation (1 h post LPS for TNF-a; 2 h post LPS for IL-6), the different cardiovascular response in stimulated animals could not be explained by cytokine modulation. Furthermore, while LVNS did not result in clear TNF-a modulation in the low dose LPS model, it did result in a similar (to SpNS) cardiovascular protective effect in the high dose model. This effect has also been observed previously in a different sepsis model in pigs, where VNS induced cardiovascular protection in the absence of cytokine modulation [35], Altogether this suggests that SpNS and LVNS may have broader immunological consequences and applicability in disease. The exact mechanism and mediators able to explain this substantial effect remains to be clarified.

Prevention of cardiovascular collapse in pigs has been previously shown in a very similar model where treatment of pigs (prior to high dose LPS injection) with diclofenac sodium promoted cardiovascular stability and improved survival without significant effects on cytokines [32], This effect was related instead to the suppression of lipid mediators, in particular inflammatory eicosanoids. Interestingly, vagotomy has been shown to delay infection resolution in mice by impacting lipid mediator production [36], This suggests a possible role of the same anti- inflammatory autonomic circuits in the regulation of mediators which may aid in resolution of inflammation, as well as cardiovascular protection. In line with this, it has been recently shown that the autonomic nervous system can regulate lipid mediators via NA action in the abdominal cavity [37], Thus, in some embodiments, stimulation of the SpN may result in regulation of lipid mediator production by immune cells and/or possibly by direct nerve release/production as recently observed for the VN [38], A broad spectrum of molecular mechanisms triggered by SpNS can, in some instances, support the development of neuro-immunomodulatory therapies. [00308] Importantly for clinical translation, hematological analysis showed that neither a short acute (1 min) or prolonged (2 hours) SpNS caused systemic immune-suppressive responses, as indicated by the numbers of circulating immune cells as well as by the ex vivo LPS-assay. This was also observed for eLVNS and partially with LVNS. However, SpNS and eLVNS had a very different profile in comparison to pharmacological treatment with dexamethasone, which promoted a substantial reduction in cytokine levels at the expense of systemic immune suppression (e.g., reduction of circulating levels of leukocytes). Thus, in some embodiments, neuromodulation can be used to provide beneficial therapeutic effect without causing significant suppression of systemic immune defenses.

[00309] Finally, biochemical analysis of pancreatic and kidney enzymes suggested that implantation and stimulation of the SpN (running close to the pancreas) did not cause acute adverse effects on organ function, when compared to sham and VN implanted pigs. Thus, chronic studies can also be used in some instances to further determine the effect of chronic implantation and daily stimulation of the SpN on hematological and biochemical parameters.

[00310] The data presented above provide a demonstration of the existence of anti-inflammatory circuits in a larger, clinically appropriate, translational species. Importantly, in some embodiments, the translational model described herein (low dose LPS challenge in adult pigs) can be leveraged for future neuro-immune modulation therapy development. Furthermore, shown herein is a nodal immunomodulatory therapy opportunity utilizing stimulation of the splenic nerve, which in some instances results in the reduction of inflammation and the prevention of cardiovascular collapse. Thus, in some embodiments, the use of human tissues and large animals provides a more suitable model for splenic nerve bioelectronic medicine development, as well as a dataset that can be used for the translation of near-organ neuromodulation of the immune system into first-in-human clinical trials.

[00311] EXAMPLE 2 - Splenic Nerve Neuromodulation Reduces Inflammation and Promotes Resolution in Chronically Implanted Pigs.

[00312] Materials and Methods

[00313] Stimulation parameters

[00314] Stimulation parameters and paradigms used are described in the relevant sections in the

Materials and Methods and the Results. [00315] Surgery. Bilateral indwelling jugular vein catheters and one femoral arterial catheter were placed under ultrasonographic guidance. Volume-controlled mechanical ventilation was maintained for surgery and implantation. Once instrumentation was complete animals were kept on spontaneous ventilation and data collection commenced when animals were in a steady-state. Routine anesthesia monitoring included vital parameters such as electrocardiogram and invasive arterial blood pressure (systolic, diastolic and mean). Arterial blood pressure was also digitally recorded using a 16 channel PowerLab acquisition system (AD Instruments) with LabChart 8 software at 2 kHz sampling frequency.

[00316] Animals were placed in right lateral recumbency, and the left lateral abdomen was aseptically prepared and draped in a routine fashion. Using aseptic technique, a 20 cm laparotomy incision was made in the second to last intercostal space, and access to the splenic base with associated neurovasculature was aided by the use of rib retractors.

[00317] Chronic conscious neuromodulation study methods

[00318] This study developed a minimally invasive, laparoscopic technique in a translational porcine model to implant a cuff electrode around the splenic NVB and an IPG to enable delivery of chronic neuromodulation. The tolerability of SpNS in conscious, freely behaving pigs was then evaluated, prior to quantification of multiple immunological parameters in both naive and endotoxin-challenged inflammatory conditions. These parameters included quantification of cytokines, flow cytometry and SPM analyses before, during and after LPS challenge. Under terminal anesthesia, contrast angiography, electrophysiology and histopathology demonstrated the integrity of the splenic NVB.

[00319] I. Animals

[00320] A total of 12 female Berkshire pigs were used for the chronic implant study. Due to the exploratory nature of the study and logistical difficulties associated with large-animal studies, animals progressed in 4 cohorts in weekly blocks. Each cohort contained animals receiving SpNS and non-stimulated sham animals to allow for any differences arising from different batches of LPS. Cohorts were used as a blocking factor in data analyses.

[00321] The pigs were sourced from a commercial pig farm, acclimatized at the research facility and underwent handling and socialization training for a minimum of 1 month prior to the experiment. Animals weighed 74-99 kg at start of study and were ~9 months old. They were individually housed in close apposition to allow visual and physical contact through slatted fencing, on straw bedding with environmental enrichment. Water was provided ad libitum and they were fed a commercial pelleted sow and weaner diet based on minimum basal and metabolic energy requirements.

[00322] II. The neuromodulation device

[00323] Stimulation lead. The stimulation lead consisted of a lead body with a distal end cuff electrode applied to the splenic NVB, and a proximal connector connected to the IPG manufactured from implant-grade silicone and metals. The design was able to support laparoscopic implantation for cuff placement around the splenic NVB. The cuff electrode was designed to interface with nerves located around the periphery of the splenic artery and contained two electrically active electrode arms and one inert middle arm for retention.

[00324] Implantable pulse generator. A commercially available implantable pulse generator

(IPG; 5cc mStim IPG, Integer CCC, Uruguay; customized by Galvani Bioelectronics) was connected to the stimulation lead to electrically stimulate the SpN. Two versions of the IPG were used on the current study. In cohorts 1 and 2, the IPG was capable of stimulation up to 15 μC (15.3 mA, 980 gs pulse width). In cohorts 3 and 4, the IPG was upgraded to provide output up to 40 μC (20.0 mA, 1980 μs). All other stimulation parameters remained identical and are detailed above. Differences in IPG output are noted in the relevant sections.

[00325] III. Surgery

[00326] Implantation of the neuromodulation device. Animals were anesthetized and underwent minimally invasive laparoscopic surgery via a left sided lateral approach. Following retraction of the stomach and then spleen, the splenic NVB was dissected free from the surrounding tissue and the circumferential cuff electrode was implanted around the splenic NVB. Stimulation applied with an external pulse generator (EPG (DS5, Digitimer, UK)) confirmed electrical integrity of the implanted stimulation lead and physiological functionality by a measured increase in sMABP. The IPG was implanted on the dorso-lateral thorax and the lead tunneled to connect to the IPG. A series of stimulations were then applied via the IPG to confirm functionality by comparison to changes evoked with the EPG, as well as IPG communication and charging before surgical closure of the trocar locations. [00327] Intra-operative neuromodulation of the SpN (at surgery and at termination). At implantation, neuromodulation was delivered at either 15 or 40 pC using a 10 Hz continuous paradigm. Intraoperative splenic nerve neuromodulation (10 Hz continuous; 60 s) consistently induces changes in physiological biomarkers [57] which enabled robust confirmation of nerve- target stimulation via the test system. Specifically, splenic blood flow is decreased, and systolic, diastolic and sMABP are increased during splenic nerve stimulation under anesthesia; these are caused by smooth muscle contraction within the artery and spleen. These changes are directly correlated with the amplitude and frequency of stimulation, and such changes are resolved upon the cessation of stimulation.

[00328] After completion of surgical implantation, a 3 min stable period of no stimulation was performed to obtain baseline values, including sMABP. Stimulation was applied via the IPG; the ability to evoke an increase in sMABP was used to demonstrate function of the implanted lead and effects on target physiology. A period of no stimulation (minimum 120 s) was performed to allow recovery of sMABP to ± 10% of pre-stimulation values between each stimulation. Impedance of the implanted stimulation lead electrodes (between the proximal and distal electrodes (cuff)) was measured prior to and following implantation and through the course of the study.

[00329] At termination, stimulation was also delivered using either the 10 Hz continuous or 10 Hz burst paradigm with an EPG up to 40 pC with additional measurement of SpA BF using an ultrasonic transit time probe (Transonics, USA) placed on the splenic artery (SpA), (immediately proximal to the branching of the gastroepiploic artery from the SpA). Flow changes were continuously monitored via a TS420 perivascular flow module (Transonics, USA), and measurements were digitally recorded using a 16 channel PowerLab acquisition system (AD Instruments) with LabChart 8 software at 2 kHz sampling frequency. Baseline values for sMABP and SpA BF, were generated by averaging the measurements obtained over the last 30 s prior to each stimulation. During stimulation, values were then expressed as percentage change from this baseline value to quantify the effect of stimulation. For each parameter, the maximum change occurring during the stimulation period was used for comparison.

[00330] Vascular access port implantation. After implantation and intraoperative stimulations, all animals were implanted with an intravenous catheter in the left external jugular vein, using a minimally-invasive ultrasound-guided approach, which was terminated with a subcutaneous vascular access port (VAP (Le Grand CompanionPort (CP305K) - Norfolk Vet Products, Skokie, IL, USA) to enable repeated, stress free, blood sampling in conscious animals.

[00331] Recovery. Animals were routinely recovered from anesthesia and returned to their pens. Recovery was assessed by return to normal behaviors, pain scoring, wound healing, and body weight.

[00332] IV. Determination of effects of chronic SpN neuromodulation

[00333] Tolerability to SpNS as determined by behavioral responses. Animals were given a 14 day recovery period following surgery after which those assigned to the neuromodulation group (n=6) were stimulated at either 15 or 40 μC using the 10 Hz burst paradigm (5 s duty cycle interval, 10% duty cycle). Stimulation was tested at ascending 1 mA intervals. For each stimulation current amplitude, stimulation was ramped up over 30 s and then held for a further 60 s (90 s total) under IPG control. Animals assigned to the non-stimulation group (n=6) were not stimulated.

[00334] During neuromodulation, animals were observed by two independent trained assessors for any responses indicative of perception of stimulation (scoring system and observed responses are described in supplementary methods). This assessment was performed at each incremental increase in current amplitude of stimulation; extreme responses or repeated abnormal behavior would have led to immediate cessation of stimulation. If this occurred a recovery period would be allowed and then, as appropriate, stimulation at either the next lowest or the same intensity level performed prior to increasing stimulation intensity again (see supplementary methods for detailed protocol).

[00335] Baseline blood testing. Animals were then maintained for a further 14 day period without stimulation, after which time all animals underwent baseline blood sampling taken from the VAP for hematology, clinical biochemistry and ex vivo LPS cytokine assays and flow cytometry (only performed in cohorts 3 and 4), nominally at day -2, -1 and 0, at the same time of day (around 10:00 h).

[00336] V. Biomarker assays in naive animals

[00337] To determine the effects of SpNS in naive animals, chronic neuromodulation therapy (10 Hz burst at IPG output of either 15 or 40 μC, 7 days a week) was initiated from Day 0, following the baseline testing. Cohorts 1 and 2 (n=6) received neuromodulation 6 times per day from 09:00 at 90 min intervals, while in cohorts 3 and 4 (n=6), neuromodulation was performed 12 times per day from 07:00 at 60 min intervals. Animals assigned to the non-stimulation group (n=6) were not stimulated. On Days 2 and 7, scheduled neuromodulation was stopped after the 09:00 stimulation and blood drawn from all animals around 10:00 for ex vivo LPS cytokine assays (plus, additional samples per baseline bloods as described above). Scheduled neuromodulation was then restarted so the animals received their 11 :00 therapy session.

[00338] Ex vivo LPS cytokine assays. Duplicate samples of peripheral sodium heparinized venous blood (collected via the VAP) were immediately incubated with either 0, 100 or 1000 ng/mL LPS for four hours (purified lipopolysaccharides from the cell membrane of E. coli O111 :B4; Sigma Aldrich (see supplementary materials and methods for further details). Samples were then centrifuged at 2000 xG and the supernatant stored at -80 °C until analysis for TNF-a by commercially available ELISA kits (Porcine TNF-a; DY690B; DuoSet Solid Phase Sandwich ELISA, R&D Systems), run as per the manufacturer's instruction. All samples were run as technical replicates (n=3) for each time-point and LPS concentration.

[00339] Flow cytometry methods. Sodium heparin vacutainers were filled with 10 mL blood collected from the VAP, mixed by inversion, and kept at 4°C until processing (which occurred within 2 h from collection). Blood was treated with red blood cell lysis buffer (ammonium chloride lysis buffer), then centrifuged at 2000 xG and washed twice in cold PBS. Cells were transferred to individual FACS tubes (352058, Scientific Laboratory Supplies) containing 900 pL FACSFlow™ buffer (342003, Becton, Dickinson UK Ltd., Wokingham, UK). Cells were then stained for CD4/CD8, CD16/CD14 and CD172a/CD163 using dye-conjugated antibodies. Isotype matched controls were used. All antibodies are listed in Table 4. The samples were analyzed on a BD FACSCalibur™ (Becton, Dickinson) and 10000 events acquired using BD CellQuest Pro™ software. Details of gating strategy are reported in the supplementary materials.

[00340] Elematology and biochemistry. Samples for hematology and biochemistry analyses were submitted to a commercial laboratory.

[00341] VI. Biomarker responses in animals in the endotoxemic phase

[00342] Neuromodulation was continued (12x/day) following the naive assessment described above and the next day in vivo LPS testing was performed. Sham animals were maintained for a matching time period. One sham animal was excluded from the in vivo LPS part of the study on veterinary recommendation for reasons not associated with the study. [00343] In vivo LPS and cytokines assay. Baseline blood was drawn in all animals at time 0 h and they were injected with LPS (0.025 pg/kg i.v. over 5 mins (E. coli O111 :B4; Sigma Aldrich); see supplementary materials and methods for LPS preparation. This dose of LPS was chosen based on previous work in this group [57] and from a pilot study as outlined below). Following LPS injection, blood samples were drawn at the following intervals: 0.5, 1, 1.5, 2, 3, 4, 6, 8 and 24 h. Samples were collected for cytokine analysis, hematology, biochemistry (at all timepoints), flow cytometry (at 0, 3 and 24 h) and Specialized Pro-Resolving Mediator analysis (at 0, 0.5, 3 and 24 h). Clinical reactions to LPS were carefully monitored to ensure (as predicted from the preliminary study) that only mild to moderate clinical signs occurred, such as shivering and lethargy; animals were predicted to remain responsive and appetent throughout. LPS injection was coordinated such that stimulation animals (n=6) received scheduled neuromodulation immediately before LPS injection, followed by an additional manual stimulation five minutes post-injection. For cytokine analysis, venous blood samples collected in EDTA tubes were immediately centrifuged at 2000 xG for 5 mins at 4 °C. Plasma was then separated and immediately frozen on dry ice and subsequently stored at -80 °C. These plasma samples were used to measure TNF-a concentration using the commercially available ELISA kits described above (R&D systems). All other blood analyses were run as described above.

[00344] Targeted, lipid mediator profiling. Specialized pro-resolving mediator analysis was performed on plasma samples taken as above during the endotoxemic phase in this conscious study. Additionally, similar analysis was performed on plasma samples collected during a low dose anesthetized endotoxemia study with acute SpNS [57] at 0, 0.5 and 2 h post LPS administration. This allowed for comparison between acute SpNS and chronic neuromodulation in conscious animals.

[00345] Plasma lipid mediators were extracted using solid-phase extraction columns as in [62], Briefly, prior to sample extraction, deuterated internal standards, representing each region in the chromatographic analysis were added to facilitate quantification. Samples were kept at -20° C for a minimum of 45 mins to allow protein precipitation. Supernatants were subjected to solid phase extraction, methyl formate and methanol fraction collected, dried and suspended in phase (methanol/water, 1 :1, vol/vol) for injection on a Shimadzu LC-20AD HPLC and a Shimadzu SIL- 20AC autoinjector, paired with a QTrap 6500+ (Sciex). An Agilent Poroshell 120 EC-C18 column (100 mm x 4.6 mm x 2.7 pm) was kept at 50° C and mediators eluted using a mobile phase consisting of methanol/water/acetic acid. QTrap 6500+ was operated using a multiple reaction monitoring method as in [62], Each lipid mediator was identified using established criteria including matching retention time to synthetic or authentic standards, an AUC >2000 counts and matching of at least 6 diagnostic ions in the MS/MS. Calibration curves were obtained for each mediator using lipid mediator mixtures at 0.78, 1.56, 3.12, 6.25, 12.5, 25, 50, 100, and 200 pg that gave linear calibration curves with an r² values of 0.98- 0.99.

[00346] VII. Data analysis

[00347] TNF-a, hematology bloodwork and flow cytometry data were analyzed in InVivoStat 4.0 (see Bate and Clarke, 2014) and visualized in Graphpad Prism 8.4.2.

[00348] Ex vivo LPS assay. The ex vivo TNF-a data were analyzed for each LPS concentration using a 2-way repeated measures mixed model approach, with neuromodulation as the treatment factor, timepoint (average of baseline, +2 and +7 days) as the repeated factor, and cohort as the blocking factor. The responses were logio transformed prior to analysis to stabilize the variance. Planned comparisons were then made at each timepoint, and the unadjusted p-values were corrected using Hochberg’s multiple comparison procedure.

[00349] In vivo LPS assay. The area under the curve (AUC) was calculated for plasma TNF-a between 0.5 and 2.0 h post-LPS injection (AUC0.5-2.0) for each animal individually; the time period during which the majority of the TNF-a increase, peak and subsequent decline occurred. Baseline for AUC for each animal was taken as the value of its baseline TNF-a prior to LPS injection. Data were analyzed using a 1-way ANCOVA approach, with neuromodulation or no neuromodulation as the treatment factor and baseline TNF-a (prior to LPS injection) as the covariate. The responses (AUC and baseline TNF-a) were logio transformed prior to analysis to stabilize the variance. The cohort was included as a blocking factor to account for day-to-day variability.

[00350] Hematology and biochemistry. The data were analyzed using a 2- way repeated measures mixed model approach, with neuromodulation as the treatment factor, timepoint (average of baseline, +2 and +7 days or 0-24 h for naive and endotoxemia phases respectively) as the repeated factor, cohort as the blocking factor and baseline cell count as the covariate. The responses and covariate were logio transformed prior to analysis to stabilize the variance. Planned comparisons were then made at each timepoint, and the unadjusted p-values were corrected using Hochberg’s multiple comparison procedure. [00351] Flow cytometry. The data were analyzed as per the hematology and biochemistry but without a covariate or transformation. Data were expressed as % change over the baseline value. Baseline value for the naive phase was defined as the average value across the 3 days (-2, -1, 0) prior to initiation of stimulation. For the endotoxemia phase the baseline was defined as time 0 h prior to LPS challenge.

[00352] Lipid mediators. Lipid mediators were analyzed by multivariate analysis performed using online open access metaboloanalyst (available online at metaboanalyst.ca/MetaboAnalyst/home.xhtml) using statistical analysis tool. Data were uploaded as concentration as .txt files. Features with a constant or single value across samples were deleted. Partial Least Square Discriminant Analysis was then performed following auto-scaling (mean- centered and divided by the standard deviation of each variable). Network analyses was performed on normalized concentrations (expressed as the fold change) of the lipid mediators from the Sham and SpNS groups and lipid mediator biosynthetic networks were constructed using Cytoscape 3.7.1.

[00353] VIII. Terminally-anesthetized procedures

[00354] Following in vivo LPS testing, 10 of 12 animals were terminally anesthetized for contrast angiography, SpNS and gross and histological pathology analysis. Two animals were excluded from terminal studies for use in further work (not described in this paper). Following anesthesia contrast angiography was used to show retention and alignment of the lead electrode around the splenic artery and 3D angiography to show the stimulation lead and IPG.

[00355] Open laparotomy surgery (as described above) was performed in all animals and splenic neuromodulation performed with the IPG and an EPG. Sham animals did not undergo terminal stimulation in order to preserve their status as sham animals, but were maintained under anesthesia for the same amount of time as those stimulated to control for any anesthesia-induced changes. Thresholds and stimulation response curves for changes in SpA blood flow (measured with an ultrasonic transit time probe (Transonics, USA) placed on the SpA) and sMABP were determined using methods as described above for intraoperative stimulation. The relationship between these thresholds and cytokine response in the in vivo TNF-a assay were determined within-animal.

[00356] Following measurement of blood pressure (BP) and blood flow (BF), a small SpN fascicle was dissected free from the NVB, more distal to the implanted lead cuff. Evoked compound action potential recordings were then performed in all animals to provide additional electrophysiological evidence of functional SpN fascicles. Five minutes prior to euthanasia 5000 IU heparin IV was administered to prevent post-mortem clotting. Immediately following euthanasia with pentobarbital, the chronic effects of the neuromodulation device on the splenic nerve and artery, and surrounding tissues were evaluated using gross and histopathologic evaluation. A full post mortem was performed and the following areas were grossly examined in detail: the abdominal wall and abdominal cavity immediately surrounding the implantation site (including the pancreas, spleen and liver); the IPG location and the neural interface (NI)/cuff.

[00357] Following gross pathology examination, the following tissues were harvested for histopathology: i) the entire segment of splenic artery including the cuff (left in situ) and surrounding muscle/fat tissue; ii) tissues from lead and IPG regions, including pancreas; iii) one section of spleen from an area close to the NI implantation site and one section lateral to the first section (by approx. 5cm); i.v.) one section of liver from the left lateral lobe, in the immediate vicinity of the implant site. All tissues were immersed in 10% neutral buffered formalin solution (minimum lOx volume of tissue sample) for 48 hours prior to immersion in 70% alcohol.

[00358] IX. Pilot study investigating LPS dose in non-surgical sham animals

[00359] A pilot study was performed in 4 Berkshire pigs (sex, age and size matched to above study) to inform the LPS dose for the in vivo challenge in this example. Additionally, they provided a group of non-surgical sham animals for comparison to animals implanted with electrodes and IPGs in the main study. Briefly, animals were anesthetized, a VAP implanted and then recovered (as described previously). In vivo LPS and cytokine assays were performed (as above) to assess the effect of two LPS dosages: 0.0025 gg/kg i.v. and 0.025 gg/kg i.v. The dose of 0.0025 gg/kg did not evoke TNF-a release (data not shown) unlike the 0.025 gg/kg dose which provided a robust, consistent cytokine response in the absence of any clinically adverse effects on the animals. On this basis, the 0.025 gg/kg dosage was selected for the main study.

[00360] Supplementary Materials and Methods

[00361] Chronic conscious neuromodulation study methods

[00362] I. Implantation of the neuromodulation device

[00363] Anesthesia. Animals were started on a course (9 days) of antibiotics (amoxicillin 15 mg/kg i.m.) and anti-ulcer medication (omeprazole 40 mg p.o.) 24 h before surgery and then continued as prescribed afterwards. Food, but not water, was withheld for 18 h prior to surgery and animals received a veterinary examination to ensure health status. After induction of anesthesia as below, analgesia and anti-inflammatory mel oxicam (0.4 mg/kg i.m.) was administered and continued in the recovery period as needed. Animals were pre-medicated with ketamine (20 mg/kg i.m.) and midazolam (0.5 mg/kg i.m.). Fifteen min after premedication general anesthesia was induced with propofol (2 mg/kg via an auricular vein catheter). Animals were intubated with an endotracheal tube, and anesthesia was maintained with sevoflurane vaporized in a mixture of oxygen and medical air. Volume-controlled mechanical ventilation was maintained throughout surgery, as were intravenous fluids (isotonic and colloidal fluids, glucose and electrolyte supplementation as needed). Intra-operative analgesia included buprenorphine (0.02-0.04 mg/kg i.v.) and fentanyl (2-5 gg/kg i.v.). Instrumentation and monitoring included electrocardiogram (ECG), heart rate (HR), invasive arterial blood pressure (systolic, diastolic and mean), respiratory rate (RR), pulse oximetry, capnography, spirometry, (including fraction of inspired oxygen (FiO₂), end-tidal sevoflurane (ETSev)), and core body temperature. Some of these parameters (arterial blood pressure, central venous pressure, ECG, ETCO₂, ETSev) were also digitally recorded using a 16 channel PowerLab acquisition system (AD Instruments) with LabChart 8 software at 2 kHz sampling frequency.

[00364] Laparoscopic surgery. A minimally-invasive laparoscopic surgical procedure was used. Animals were placed in right lateral recumbency, and the left thorax and lateral abdomen were aseptically prepared and draped in a routine fashion. A total of 7 trocars were used for each surgical procedure. The first trocar (15 mm diameter) was initially placed in the paralumbar region between the last rib and wing of the ilium using the Hasson technique. The abdomen was then insufflated with gas at a constant pressure ranging between 10-16 mmHg. At the same time the lead was prepared, inspected and tested in saline for electrical continuity (impedance). The laparoscopic camera was then inserted and used to select the ideal location for successive trocars. A second trocar (5 mm) was then placed along the cranio-ventral axis of the abdomen to provide an access port for stomach retraction tools. A third trocar (12 mm) was placed approximately 20 cm caudal from the second trocar for spleen retraction. Three additional trocars (12 mm) were placed approximately 15-20 cm dorsal from the retraction trocars to form a triangle just ventral to the last and second to last ribs. The two trocars at the base of the triangle (n.4 and 5) were used for dissection and cuff implantation, while the other (n.6) was used for the laparoscope.

[00365] The head of the spleen was retracted caudally by fixing a self-retaining laparoscopic

Overholt clamp at the base of the renosplenic ligament. The stomach was then retracted ventrally by using an articulating cobra liver retractor to provide access to the splenic NVB. Initial dissection of the peritoneum overlying the distal (closer to the spleen) splenic NVB was performed using a harmonic laparoscopic scalpel to prevent bleeding. The splenic NVB was then isolated by blunt dissection using a Maryland tool, 60 and 90-degree Overholt dissecting instruments and scissors. A region of approximately 2 cm of NVB was freed from the connective tissue and separated from the splenic vein. The stimulation lead was then prepared for deployment into the abdomen. A seventh trocar (5 mm) (n.7) was placed between the two access ports used for dissection. The lead was then introduced into the abdomen using atraumatic graspers to manipulate the lead. The lead was exteriorized by pulling the lead cap through trocar n.7. The circumferential cuff electrode was then implanted around the splenic NVB and impedance was checked using a Minirator (MR-PRO, NTI Audio, Switzerland) to confirm electrical continuity. Stimulation was then performed with an external pulse generator (EPG; DS5, Digitimer, UK) to confirm electrical integrity and physiological functionality by a measured increase in sMABP.

[00366] A pocket was created to accommodate the implantable pulse generator (IPG; Integer, CCC, Uruguay) dorsally, approximately above the third to last rib and in line with the position of the n.7 trocar. An incision of 5-8 cm was performed and pocket created by blunt dissection. The n.7 trocar was carefully removed, and the lead tunneled to the IPG pocket on the lateral thorax. Subsequently, the lead connector was attached to the IPG and the IPG implanted into the pocket on the lateral thorax. Instruments and trocars were removed. A series of stimulations were then applied via the IPG to confirm functionality by comparison to changes evoked with the EPG, and IPG communication and charging were confirmed. The trocar locations were then sutured closed. Stimulation was then delivered via the IPG up to either 15 or 40 pC using a 10 Hz continuous paradigm as detailed in main methods.

[00367] Vascular access port (VAP) implantation. Following implantation and stimulation, animals were placed in dorsal recumbency and the neck and scapula region prepared and draped for aseptic surgery. An intravenous catheter was placed, as below, in the left external jugular vein using a minimally invasive ultrasound-guided approach and terminated with a subcutaneous vascular access port (Le Grand CompanionPort (CP305K) - Norfolk Vet Products, Skokie, IL, USA). The VAP consisted of a titanium port with a silicone septum and an attachable rounded tip silicone catheter (7 French). Both the port and catheter were flushed with 0.9% saline prior to insertion. [00368] A 1 cm incision was made in the skin overlying the left external jugular vein in the mid- cervical region. An 8 Fr catheter introducer was placed into the jugular vein under ultrasound guidance and the catheter for the VAP passed through this introducer and advanced 9-10 cm distally. Tip localization was confirmed using fluoroscopy to ensure location was proximal to the heart. For placement of the port, a 5- to 6-cm curvilinear incision was made dorso-cranial to the scapula and 5 cm lateral to midline dorsal neck. Tissues overlying trapezius muscle were undermined to create a pocket for the port. The catheter was tunneled dorsally between the skin and subcutaneous tissues and attached to the port. The port was secured to the underlying musculature by using 3-0 polydioxanone suture at 2 anchor points on the port. Catheter patency was confirmed intraoperatively through withdrawal of a blood sample. The port and catheter were flushed with 5 to 6 mL 0.9% saline and locked with 5 mL heparinized saline (500 lU/mL, Hospira). VAPs were maintained as per below until blood sampling.

[00369] VAP maintenance and use. VAPs were maintained in all animals during the study period; they were accessed either for experimental procedures or minimally every 2 weeks for maintenance. Briefly, the animal was restrained in a crate to which it was habituated, and aseptic technique used throughout. Topical local anesthetic cream (lignocaine 2.5% and prilocaine 2.5%; EMLA 5% cream; Aspen medical) was rubbed in the skin over the access port, palpable under the skin, and left for a minimum of 1 h. After final skin preparation, the port was located and stabilized by holding the edge and a right angled Huber needle (22G 1”; Norfolk Vet Products, Skokie, IL, USA), attached to an extension set and syringe containing 0.9% saline flush solution, was inserted through the skin and into the dome of the port. Flush solution (5 mL) was introduced into the catheter, and then a syringe used to withdraw at least 3 times the volume of the VAP system, including the flush, which was discarded. Following this, either blood samples were taken or LPS injected. The catheter was then flushed again with 0.9% saline (3 times volume VAP system), followed by lock solution. If further samples were to be taken within 24 h, the needle remained indwelling in the port, attached to a sealed extension set and secured around the animal. When the system was accessed within 24 h and the needle left in place, it was flushed and locked with 5 mL heparinized saline (100 U/mL); when samples were not taken within that time prior to removing the needle, a lock solution of 5 mL heparinized saline (500 U/mL) was placed in the VAP.

[00370] II. Effects of chronic SpN neuromodulation in naive animals [00371] Tolerability to SpNS as determined by behavioral responses. After a 14-day recovery period from surgery, in those pigs assigned to the neuromodulation group, tolerability to SpNS (as per the conscious stimulation paradigm), was determined. Impedance was measured via the IPG before, during and after each stimulation session. Animals in the sham group did not receive any neuromodulation. Behavioral responses indicative of perception to SpNS were scored by two independent observers. Responses identified as most severe: abdominal wall contractions, distressed vocalization and excessive agitation/psychomotor activity, warranted immediate cessation of stimulation if seen individually and only on a single occasion. The animal was allowed to recover for five minutes, and following veterinary assessment, stimulated again at the next lowest intensity before attempt at step-up made again. Other less severe behaviors (startle response, scratching/rubbing, nose bumping against a solid object, kneeling, squatting, stomping and stretching) were scored as absent or present; and if present whether as a single occasion, intermittent or continuous. If a combination of two or more these behaviors was seen at greater than a single frequency, stimulation was ceased and after a five-minute recovery, retested at the same intensity. If the response was observed during the second stimulation at the same level, no further increase in stimulation would occur and effects of stimulation reassessed at the next lowest intensity to confirm absence of limiting behavioral changes. If no response was observed during the second stimulation at the same level, the stimulation would be increased in 1 mA levels until either an observed response or IPG maximum was reached.

[00372] Blood sampling at baseline in naive animals. Animals were then left for a further 14 days with no stimulation. Blood samples were then taken from all animals for baseline blood testing at Days -2, -1 and 0 and then following initiation of SpNS, on Days 2 and 7 from the VAP (as per above protocol). Blood samples for ex vivo LPS cytokine assay and flow cytometry were collected in sodium heparin-coated tubes. Bloods for clinical biochemistry were collected in lithium heparin- and non-coated tubes. Blood for hematology was collected in EDTA-coated tubes.

[00373] LPS preparation methods. A 1 mg vial of LPS (Purified lipopolysaccharides from the cell membrane of Escherichia coli O111 :B4; Sigma Aldrich) was reconstituted with 1 mL sterile saline to give 1 mg/mL solution. The vial was vortexed for 20 s and then sonicated for 5 min. Stock solution aliquots of 500 pg/mL were then made by adding 100 pL of 1 mg/mL LPS to 100 pL saline in individual tubes. These were again vortexed for 20 s and sonicated for 5 min and stored for up to 12 h at 4°C. Within 20 min of use, working solutions of LPS were made (50, 5 or 0 pg/mL): tubes were again vortexed and sonicated as above, before serial dilutions with sterile saline. Vortex and sonication were done between each dilution step. Working solutions were sonicated again immediately prior to incubation with blood samples. Within 15 min of blood collection, blood tubes were inverted to resuspend cells and 20 gL of working LPS dilutions (50, 5 or 0 gg/mL) were added to culture tubes, followed by addition of 980 pL of blood to each tube to achieve final concentrations of 1000, 100 or 0 ng/mL LPS. Two replicates were performed per final concentration. Samples were mixed by 3 inversions and transferred to a 37°C incubator, flat on a rocker for 4 h. Plasma was then separated by centrifugation for 10 min at 2000 xG to pellet cells and then removed by pipette and stored in cryovials at -80°C.

[00374] Flow cytometry methods. Data recorded by the flow cytometer was analyzed using FlowJo™ software (version 10.6.2). Each blood sample was divided in 7 aliquots to generate 7 different panels: 1) unstained; 2) isotype control for panel 3; 3) antibodies against CD4 and CD8; 4) isotype control for panel 5; 5) antibodies against CD14/CD16; 6) isotype control for panel 7; 7) antibodies against CD172a/CD163. Gates were applied according to the gating strategy (Fig. 13). Identical gates were applied for all samples (all experimental animals, days and cohorts). Panel 3 was used to distinguish T cell subsets based on their CD4 and CD8 expression; Panels 5 and 7 were used to analyze the monocyte population. Simple gating on all CD14⁺, CD16* or CD172⁺ cells resulted in inclusion of granulocyte and lymphocyte populations (Fig. 14). Because the primary interest in Panels 5 and 7 was in the monocyte population, gating was done in a view where the marker was plotted against Side Scatter. The position of monocyte population on the Side Scatter axis was, as expected, between lymphocytes and granulocytes. Although there is a slight overlap with these populations, using side scatter in combination with the marker resulted in a reliable separation of these three subsets. This strategy was therefore used as the final gating strategy.

Results are expressed as a percentage of its parent population. In the case of CD 14 expression on CD16⁺ monocytes, median fluorescence intensity was measured along with percentage of CD14^low and CD14^high cells.

[00375] III. Effects of chronic SpN neuromodulation during endotoxemia

[00376] LPS preparation for in vivo experiments. LPS stock solution of 1 mg/mL was prepared as above. A 110 dilution of this was made by diluting 100 gL of stock solution into 900 gL sterile saline, which was then vortexed for 20 s. The final concentration of LPS to be given based on the animal’s body weight (BW) was calculated (0.025 gg/kg of BW (kg)) and a 1 mL solution of 2x final LPS concentration solution made into a glass vial, vortexed for 20 s and kept on ice until use (within 30 min). Just prior to use, the vial was sonicated for 5 min, 500 pL were then diluted in 9.5 mL sterile saline and the solution injected IV over 2 min into each animal.

[00377] In vivo LPS and cytokines assays. Immediately prior to LPS, and following LPS injection, blood samples were drawn in the following tubes: flow cytometry in sodium heparin- coated; cytokine analysis, SPM analysis and hematology in EDTA-coated; clinical biochemistry in lithium heparin and non-coated. Blood samples for cytokine analysis, hematology and clinical biochemistry were drawn at: 0 (baseline), 0.5, 1, 1.5, 2, 3, 4, 6, 8 and 24 h. Blood samples for flow cytometry were drawn at: 0 (baseline), 3 and 24 h. Blood samples for SPM analysis were drawn at: 0 (baseline), 0.5 and 24 h.

[00378] Results

[00379] Chronic SpN neuromodulation - Implantation and intraoperative stimulation

[00380] A total of twelve female pigs underwent a novel minimally-invasive laparoscopic surgical procedure. This implantation procedure was refined through studies in both human and pig cadavers with input from human and veterinary surgeons. The splenic NVB was surgically-isolated from surrounding tissue, and a circumferential cuff electrode was implanted around the artery and nerve plexus. The electrode lead then exited the abdomen where it was connected to an implantable pulse generator (IPG). A catheter was placed in the jugular vein and connected to a subcutaneously- implanted vascular access port to enable longitudinal blood sampling.

[00381] During laparoscopic implantation of the stimulation lead and IPG, SpN neuromodulation with a 10 Hz continuous paradigm (60 s) was used to confirm both placement and function of the implanted lead, IPG and target engagement, as performed in the on-going clinical trial described previously. Animals were stimulated at their respective IPG charge outputs (see methods for details) of 15 pC or 40 pC. Stimulation evoked an increase in sMABP of 9.81±6.83 % (n=5) at 15 pC (15.3 mA; 980 ps pulse width) or 10.31±5.874 % (n=7) at 40 pC (20.0 mA; 1980 ps pulse width). In all animals the increase in sMABP was temporally-correlated to the onset and offset of stimulation, in line with the acute experiments described above, and was used as confirmation of target engagement and functionality of the implanted SpN neuromodulation system prior to animal recovery.

[00382] Following implantation surgery, all animals recovered well without complications. Electrodes were monitored throughout the course of the study to assess continued function of the IPG and stimulation lead integrity. Following implantation, impedance was 404.33±62.68 (n=10 of 12), and peaked around 11 days post-implantation (583.21±89.78 ) before plateauing around 540 for the remainder of the study.

[00383] Given the practical limitations of chronically-implanted large-animal studies and associated biomarker assays, animals proceeded through the study in four cohorts, with each cohort consisting of animals receiving chronic SpN neuromodulation, and sham animals implanted with an identical stimulation lead and IPG receiving no conscious neuromodulation. Evidence of mechanical failure of the electrode was noted in two animals prior to onset of chronic neuromodulation. Given that electrode failure was not caused by animal- or nerve-related issues, these animals were assigned to the sham group. Other animals were randomly assigned to either the SpNS or sham group.

[00384] Effects of chronic neuromodulation in naive animals

[00385] To assess behavioral tolerability of SpN neuromodulation, approximately 14 days following device implantation, SpNS pigs were stimulated using the 10 Hz burst paradigm using a step-up protocol between 1 and 15 or 40 μC (increments of 1-2 pC). The behavioral responses to stimulation were not assessed in sham animals. There was no evidence of behaviors or responses indicative of perception of neuromodulation of the SpN in any animal at any of the stimulation amplitudes used. Animals were then maintained without stimulation until approximately 28 days following device implantation.

[00386] The effects of chronic SpN neuromodulation were assessed to examine any potential immunosuppressive effects in animals naive to inflammatory challenge. This was performed by measuring ex vivo peripheral whole blood LPS-induced TNF-a production, hematological and clinical biochemistry measurements and flow cytometry. Baseline peripheral blood samples were collected from all the animals prior to initiation of chronic neuromodulation, nominally days -2, -1 and Day 0.

[00387] Chronic neuromodulation was initiated and maintained for 7 days at either 15 or 40 pC using the 10 Hz burst paradigm (5 mins / session; 12x / day; full parameters described in the methods) and further blood samples were collected on Days 2 and 7. There was a clear concentration-response relationship between LPS and the amount of TNF-a measured in the plasma isolated from whole blood incubated ex vivo with varying concentrations of endotoxin (Fig. 12A- C). Considering the neuromodulation period between day 0 and 7, there was no difference in TNF- a release between SpNS and sham animals. Additionally, there was no effect of SpNS on total white blood cell counts, neutrophils or monocytes (Fig. 12D, E and F respectively), or any other hematological or clinical biochemistry parameters, comparing either within the SpNS animal groups between baseline and Day 2 or Day 7, or between SpNS and sham animals.

[00388] To further evaluate effects on the immune system of naive animals, peripheral blood leukocytes were stained with antibody against the surface molecules CD 14 and CD 16, CD 172a and CD163, CD4 and CD8, to identify potential changes in major leukocyte subsets by flow cytometry. As expected CD 14, CD 16 and CD 172a markers were expressed not only by monocytes but also by lymphocytes and granulocytes. Plotting these markers against side scatter allowed these three populations to be distinguished and therefore monocytes to be specifically quantified (see Fig. 13 and 14). Subsequent analysis did not reveal any significant effect of SpNS within or between groups when quantifying the proportion of monocytes that were CD16⁺(Fig. 12G and H), CD172a⁺ (Fig. 121 and J), CD14⁺ (Fig. 15D) or CD163^T (Fig. 15E). No significant differences were observed when quantifying the expression (as median fluorescence intensity; MFI) of CD 14 on CD 16^T monocytes (Fig. 12Kand L). Furthermore, no changes were noted in the T cell compartment (Fig. 15A-C).

[00389] Effects of chronic neuromodulation during in vivo endotoxemia

[00390] Following evaluation of the effects of SpNS in naive animals, the effects of neuromodulation were assessed in an in vivo endotoxemia model. Neuromodulation was continued for a further day (day 8) following which animals were subjected to low dose endotoxemia by systemic injection of LPS via the jugular vein catheter. Peripheral blood was collected to quantify LPS-induced plasma cytokine and SPM concentration in peripheral plasma and hematological and clinical biochemistry parameters.

[00391] The dose of LPS used in the current study was validated in a preliminary cohort of animals implanted only with the vascular access catheter (n=4; non-surgical (NS) sham), and was initially selected on experience with anesthetized LPS experiments [57] and evidence in literature. The dose selected (0.025 μg/kg, i.v.) was shown to evoke a robust increase in plasma TNF-a levels (Fig. 9A; grey trace) in the presence of only mild to moderate clinical behaviors (see Methods).

[00392] Following systemic injection of LPS, typical clinical behaviors (lethargy and shivering) were seen in all animals; these started around 45 min post-injection and persisted for around 45 min in the case of shivering and 4 h for the lethargy. These behaviors resolved without the need for medical intervention. No difference was noted in clinical behaviors between neuromodulated or sham animals. In all pigs, LPS injection caused an upregulation of systemic TNF-a levels (Fig. 9 A) peaking at 1 h post injection. However, this dynamic upregulation of TNF-a was smaller in SpNS animals in comparison to sham as well as NS sham animals. Quantification of the total amount of TNF-a, measured as area under the curve (AUC) between 0.5 and 2.0 h post-LPS injection (the time period including TNF-a increase, peak and subsequent decline occurred), revealed a significant reduction of TNF-a in SpNS vs. sham animals (Fig. 9B); there was a reduction of approximately 40% in SpNS vs. sham animals (P=0.029; 345.0±42.62 vs. 566.0±213.0 pg/ml.h, respectively). The amount of TNF-a was also significantly reduced compared to NS sham (P=0.046), while no significant difference was found between sham and NS sham animals.

[00393] Analysis of peripheral circulating leukocytes showed LPS-induced dynamic changes in total WBC, neutrophils, monocytes (Fig. 9C-E) and lymphocytes (see Supplementary data). In all animals there was a significant effect of time in all the four parameters (P<0.0001 for all). Total circulating WBCs showed an initial leukopenia, followed by leukocytosis. There was approximately a 40% reduction from baseline values of WBCs which reached a minimum around 1 h, followed by an elevation at 4 h (130-160 % of baseline) which resolved to baseline values by 24 h (Fig. 9C). These changes were mainly driven by variation in neutrophil number (Fig. 9D). Additionally, monocytes showed an initial reduction, with a minimum around 1.5 h, which then resolved by 24 h (Fig. 9E). When comparing SpNS and sham animals, there was a significant effect of neuromodulation on the number of monocytes at 3 and 4 h post-LPS (overall ANCOVA P=0.021; post-hoc analysis P=0.004 and P=0.009 respectively), with a higher proportion of circulating monocytes in SpNS animals versus sham. There was no significant effect of SpNS on WBCs or neutrophils. Furthermore, there were no changes in blood biochemistry parameters following LPS administration in either SpNS or sham animals.

[00394] Flow cytometry analysis also revealed an effect of LPS administration on the proportion of peripheral leukocyte subsets at 3 h post LPS, before returning towards baseline levels at 24 h (in line with hematological analysis). There was however a significantly smaller LPS-induced reduction in circulating monocytes in SpNS animals, quantified as CD16⁺ monocytes (P=0.039), with post-hoc analysis revealing a significant effect at the 3 h timepoint (P=0.031 ; Fig. 9F and G). Analysis of CD172a^T monocytes revealed no overall significant effect of SpNS (P=0.065), however post-hoc analysis revealed a significantly smaller LPS-induced reduction at the 3 h timepoint in stimulated animals (P=0.029; Fig. 9H and I). Smaller changes from baseline in stimulated animals were also seen for CD14⁺ monocytes and CD163⁺ monocytes (Fig. 16D and E). Additionally, when assessing the expression of CD 14 on CD 16⁺ monocytes, stimulated animals presented with a reduced CD14 expression (MFI) level (Fig. 9J and K; overall ANCOVA P=0.047; post-hoc analysis P=0.040 at the 3 h timepoint); no difference was observed for CD16⁺CD14^low. No differences between groups were observed for CD4⁺ and CD8^T lymphocytes (Fig. 16A-C).

[00395] Effects of chronic neuromodulation on specialized pro-resolving mediators

[00396] In order to understand if SpN neuromodulation could affect inflammation resolution processes, other than modulating cytokine production, the concentration of SPMs in peripheral plasma was quantified using liquid chromatography with tandem mass spectrometry (LC-MS/MS)- based lipid mediator profiling. Lipid mediators from all four major essential fatty acid metabolomes were identified, including the docosahexaenoic acid and n-3 docosapentaenoic acid metabolome. These mediators were detected in accordance with published criteria that include matching retention time and at least six diagnostic ions in the MS/MS spectrum [62], Assessment of plasma mediator concentrations using partial least square discriminant analysis (PLS -DA) demonstrated that chronic neuromodulation led to a shift in plasma lipid mediator profile. This was highlighted by a shift in the cluster of lipid mediators representing plasma collected from pigs subjected to chronic modulation when compared with sham stimulated animals (Fig. 10). Of note, this shift in plasma lipid mediator profiles was present at 8 days after initiation of chronic neuromodulation (Fig. 10A), prior to LPS administration, and was retained throughout the 24 h time course of LPS challenge (Fig. 10B-D). In order to evaluate the lipid mediators that contributed to this separation between the two experimental groups, the variable in importance (VIP) score was assessed; this determines the contribution of each mediator in the observed group separation. Here, several pro-inflammatory eicosanoids including PGD2 and PGE2, as well as SPMs from the protectin and resolvin bioactive metabolomes, were amongst those that contributed to the separation between SpNS and sham animals (Fig. 10A-D; right panels). Of note, concentrations of the inflammatory eicosanoids were decreased in the stimulated group, whereas SPM concentrations were increased (Fig. 10).

[00397] Lipid mediator pathway analysis was conducted by assessing the flux down each of the four bioactive metabolomes, comparing between SpNS and sham-stimulated pigs at 24 h post LPS. An upregulation of several ALOX15-derived mediators, including the DHA-derived RvDl, RvD2, PD1, the n-3 DPA derived RvD5_n-3 DP A, the EPA-derived RvEl, RvE2, RvE3 and the AA-derived LXB4, was observed in SpNS pigs compared to Sham (Fig. 11). Interestingly, upregulation of the ALOX- 15 pathway was also observed prior to LPS administration (8 days after initiation of chronic neuromodulation) characterised by increased levels of family D and E resolvins (RvDl, RvD5 and RvEl, RvE3) and LXB4 (Fig. 17).

[00398] Finally, to determine whether SpNS stimulation directly regulates plasma lipid mediator profiles the impact of acute stimulation on plasma SPM concentrations was investigated using samples collected from previously performed experiments (see Example 1, low dose endotoxemia) under terminal anesthesia [57], In line with chronic stimulation experiments, the lipid mediator profiles from acutely SpN stimulated pigs clustered separately from those obtained from sham animals when using PLS-DA across 2 h time window after LPS administration (Fig. 6).

Assessment of VIP scores demonstrated that acute SpNS downregulated plasma prostaglandin concentrations, and upregulated resolvin and protectin concentration (Fig. 6A-C; right panels). Similarly to chronically stimulated pigs, acute stimulation promoted SPM profile separation already prior to LPS injection (2 hours after initiation of stimulation) mainly via upregulation of a number of ALOX15-derived SPM (including RvD3 and RvE3) at both 0 (Fig. 7) and 2 h (Fig. 8) after LPS time point.

[00399] Table 4. Antibody panels used for Flow Cytometry.

Antibody Bio-Rad Catalogue Conjugated Isotype Dilution no. probe

CD14 MCA1218F FITC IgG2b 1 :10

CD 16 MCA1971PE PE IgGl 1 :10

CD 172a MCA2312F FITC IgGl 1 :10

CD 163 MCA2311PE PE IgGl 1 :10

[00400] Discussion

[00401] The results presented here, using a conscious chronic human-relevant anatomical and functional model, provide a novel preclinical surgical procedure to implant a SpN circumferential cuff electrode and IPG and a robust demonstration of the immunomodulatory effects of SpN neuromodulation (induction of pro-resolutive SPMs, suppression of LPS-induced cytokine levels and monocyte mobilization and activation). Together these findings support the use of chronic neuromodulation of the SpN in human clinical trials to investigate the effects of SpN neuromodulation for the treatment of acute or chronic inflammatory disorders.

[00402] In some embodiments, translation to human use is based on the observation that stimulation of the SpN resulted in no observable sensation in conscious animals. Using the burst stimulation parameters there was no evidence of behavioral changes during SpN neuromodulation up to 40 pC. This is in line with other work showing that only a small proportion (<5%) of SpN axons in pigs and humans are afferent in phenotype, based on calcitonin gene-related peptide staining [57, 26], Furthermore, literature evidence of splenic afferent fibers has described roles only associated with mechanical and pressure sensing [48],

[00403] Stimulation of the SpN in conscious pigs showed a clear suppression of peripheral TNF- a in the systemic circulation following systemic LPS-challenge, in line with the results obtained in similar models used in anesthetized mice, rats and pigs [4, 54, 7], In the foregoing results of the present example, however, the stimulation resulted in a stronger (ca. 50%) reduction as compared to that previously observed by our group in pigs under terminal anesthesia (ca. 20-30%) [57], Without being limited to any one theory of operation, this could reflect either an unmasked effect of stimulation in a setting devoid of anesthetic agents, known to affect the nervous and immune system, or a cumulative immunomodulatory effect due to the delivery of multiple stimulations over several days as compared to acute stimulation.

[00404] The data presented here also provide an extended understanding of the immunomodulatory effect induced by SpN neuromodulation, beyond regulation of cytokine production. Modulation of TNF-a production by SpN stimulation was associated with a different response in peripheral monocytes when compared to sham animals. Endotoxin administration has been reported to cause recruitment of peripheral leukocytes into marginal pools, with consequent reduction of circulating levels [69], as observed herein. However, SpN stimulated animals showed a less pronounced change in peripheral monocytes following LPS injection, as measured by hematology and flow cytometry. In particular, CD163⁺ and CD172a⁺ monocytes were more strongly reduced in sham animals at 3 h post-LPS. These populations in the pig are the closest representation of the human (CD14^dimCD16⁺) and mouse (CX3CR1^highGr1-CCR2) “non-classical monocytes” [70-72], These monocytes have been shown in mice to patrol blood vessels and rapidly infiltrate tissues during inflammation where they produce cytokines and chemokines [73], and have been associated with resolution functions (available online at cbi.nlm.nih.gov/pubmed/25838429). In parallel, a reduced accumulation of CD16⁺CD14^high monocytes was observed in stimulated animals, which in pigs represent the population of “classical monocytes”, characterized in humans by CD14^highCD16‘ and in mice by CX3CR1^lowGrl^TCCR2⁺ [70, 73-75], These monocytes are known instead to be slowly activated in response to inflammatory stimuli and then infiltrate inflamed tissues and produce pro-inflammatory mediators [73], Taken together these data indicate that splenic nerve stimulation reduces the monocyte response to LPS, resulting in a reduction of classical pro-inflammatory monocyte peripheral accumulation (CD16^TCD14^high) and an increased (compare to sham) proportion of non-classical, pro-resolving monocytes (00163⁺, CD172a⁺). This effect could be directly mediated by SpN stimulation, possibly affecting monocyte-endothelium interactions and cell recruitment, or as a cascade consequence of the modulation of cytokine (and other mediators) production.

[00405] In addition to reducing classical pro-inflammatory responses, SpN neuromodulation led to the peripheral accumulation of specialized pro-resolutive lipid mediators able to influence leukocyte responses. The data described in the present example provide evidence that the SpN may play a role in the circuitry regulating resolution mechanisms, including vagal connections to abdominal organs, and that acute and chronic stimulation can modulate SPM production. These findings are lacking in the prior art [60, 36], The observed upregulation of ALOX15-derived SPMs in stimulated animals further provides evidence that the splenic circuit may be activating specific mechanisms in resolution processes.

[00406] The endotoxemia model used here replicates some of the key features of acute and chronic inflammatory diseases. Thus, SpNS may have beneficial effects in inflammatory conditions, in particular where monocytes and splenic leukocytes play a crucial role in the pathogenesis and maintenance of disease activity. In addition, pro-resolving SPMs are known to play a crucial role in pre-clinical models and patients with acute and chronic inflammatory conditions, such as RA, atherosclerosis and myocardial infarction. For example, some of the ALOX-15 derived SPMs specifically regulated by SpNS, like RvD3 and RvDl, have been shown to have therapeutic effect in arthritis models and to be produced by macrophages abundant in drug- free remission patients [76, 77]; RvD2 and MaRl prevent atheroprogression in mice [78]; and RvDl produced by spleen-derived leukocytes drives resolution in the heart after infarction [79], In line with the above, early evidence exists that modulating SpN activity in mice with arthritis can reduce disease activity [24],

[00407] While the SpN stimulation directly targets the spleen, it is of note that changes were observed in the systemic circulation. Some of these changes were observed in LPS-naive animals. Specifically, acute and chronic stimulation were able to modulate the expression of SPMs prior to inflammatory challenge. Most of these changes were linked to ALOX15 pathway, also seen during LPS challenge, thus confirming this as specifically activated by SpN neuromodulation. For example, outflow of NA and other factors from the spleen may potentially cause downstream effects on peripheral cells and other organs. However, it is known that NA secreted from the spleen into peripheral circulation is rapidly removed by circulating metabolizing enzymes and should have very little effects on other organs/cells. Indeed, in a previous study following SpN neuromodulation, an increase in plasma NA was observed only in blood sampled locally from the splenic vein and not from the systemic circulation. In addition, during 7-days of stimulation no changes in non-classical CD163⁺ or CD172a⁺ monocytes were observed. Non-classical monocytes have been shown to be mobilized from peripheral pools by systemic catecholamine release [46], Therefore, although some NA exits the spleen parenchyma, the concentration may not be enough to cause systemic effects. Clinical trials will examine the magnitude of effect of SpN neuromodulation on these pathway-specific mediators and their utility as a measure of target engagement. While SpA BF can be used intraoperatively, defining a chronic conscious peripheral biomarker of SpN-mediated activation of immunological pathways within the spleen would allow the clinician to optimally adjust stimulation parameters to maximize therapeutic effect while minimizing off-target effects.

[00408] Importantly, prior to LPS challenge systemic immune suppression was not observed, as assessed by ex vivo TNF-a assays, peripheral blood hematology and flow cytometry. This strengthens original observations in acutely-stimulated animals where no changes in peripheral white blood cells were observed following SpNS (or VN stimulation), as opposed to the leukopenia induced by pharmacological treatment with dexamethasone [57], Thus, in some embodiments, SpN stimulation, rather than globally suppressing the immune system, may be able to prime it towards a more balanced and pro-resolutive phenotype that translates into reduced inflammatory responses upon endotoxin challenge.

[00409] In summary, the foregoing examples demonstrate that, in some embodiments, the method comprises performing chronic conscious splenic neuromodulation by implanting electrodes around the splenic NVB in a minimally-invasive manner that can safely deliver highly tolerable selective activation of splenic immunomodulatory circuits. In some embodiments, SpN neuromodulation primes the immune system towards a pro-resolutive phenotype such that following systemic immune challenge, there is a pronounced, effective and balanced anti- inflammatory effect. In some embodiments, the systems and methods disclosed herein provide significant advancements towards a better understanding of neuro-immune modulatory mechanisms in large animal species. Furthermore, in some embodiments, the systems and methods disclosed herein are used to develop a novel bioelectronic medicine for patients with acute or chronic inflammatory conditions, by targeting the near-organ autonomic nervous system of the spleen.

[00410] EXAMPLE 3 - Splenic Nerve Stimulation in Human Spleen Model

[00411] An example assay was performed to assess the effect of splenic nerve stimulation on ALOX15-derived Resolvin DI concentration, as illustrated in Fig. 18. Human spleens were retrieved from deceased organ donors. Total leukocytes were isolated from segments of the spleens and characterized by flow cytometry. Splenocytes were composed of T cells, B cells and monocytes/macrophages, thus replicating the populations of cells typically observed in the spleen. Total splenocytes were challenged with LPS with or without Noradrenaline (NA) to mimic splenic nerve stimulation (which releases NA and ultimately drives immune modulatory effects). In order to mimic different level of stimulations, various concentrations of NA were used. Three hours after incubation with LPS ± NA the conditioned medium was collected and the concentration of AL0X15-derived Resolvin DI (RvDl) was measured using a commercially available kit (Cayman chemicals). Data showed that while LPS reduced the production of RvDl as compared to naive (not challenged with LPS or NA) control cells, NA caused a dose-dependent increase in the concentration of RvDl. All NA concentrations tested (0.008-800 pM) caused a 2- times increase vs LPS and 1.5-2 times increase vs naive controls. This data demonstrate that NA is potentially a direct mediator of RvDl production by splenocytes and that the effect of splenic nerve stimulation on ALOX 15 pathway activation translate to humans.

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[00413] CONCLUSION

[00414] Plural instances may be provided for components, operations or structures described herein as a single instance. Finally, boundaries between various components, operations, and data stores are somewhat arbitrary, and particular operations are illustrated in the context of specific illustrative configurations. Other allocations of functionality are envisioned and may fall within the scope of the implementation(s). In general, structures and functionality presented as separate components in the example configurations may be implemented as a combined structure or component. Similarly, structures and functionality presented as a single component may be implemented as separate components. These and other variations, modifications, additions, and improvements fall within the scope of the implementation(s).

[00415] It will also be understood that, although the terms first, second, etc. may be used herein to describe various elements, these elements should not be limited by these terms. These terms are used to distinguish one element from another. For example, a first subject could be termed a second subject, and, similarly, a second subject could be termed a first subject, without departing from the scope of the present disclosure. The first subject and the second subject are both subjects, but they are not the same subject.

[00416] The terminology used in the present disclosure is for the purpose of describing particular embodiments and is not intended to be limiting of the present disclosure. As used in the description of the present disclosure and the appended claims, the singular forms “a”, “an” and “the” are intended to include the plural forms as well, unless the context clearly indicates otherwise. It will also be understood that the term “and/or” as used herein refers to and encompasses any and all possible combinations of one or more of the associated listed items. It will be further understood that the terms “comprises” and/or “comprising,” when used in this specification, specify the presence of stated features, integers, steps, operations, elements, and/or components, but do not preclude the presence or addition of one or more other features, integers, steps, operations, elements, components, and/or groups thereof. [00417] As used herein, the term “if’ may be construed to mean “when” or “upon” or “in response to determining” or “in response to detecting,” depending on the context. Similarly, the phrase “if it is determined” or “if [a stated condition or event] is detected” may be construed to mean “upon determining” or “in response to determining” or “upon detecting (the stated condition or event)” or “in response to detecting (the stated condition or event),” depending on the context.

[00418] The foregoing description included example systems, methods, techniques, instruction sequences, and computing machine program products that embody illustrative implementations. For purposes of explanation, numerous specific details were set forth in order to provide an understanding of various implementations of the inventive subject matter. It will be evident, however, to those skilled in the art that implementations of the inventive subject matter may be practiced without these specific details. In general, well-known instruction instances, protocols, structures and techniques have not been shown in detail.

[00419] The foregoing description, for purposes of explanation, has been described with reference to specific implementations. However, the illustrative discussions above are not intended to be exhaustive or to limit the implementations to the precise forms disclosed. Many modifications and variations are possible in view of the above teachings. The implementations were chosen and described in order to best explain the principles and their practical applications, to thereby enable others skilled in the art to best utilize the implementations and various implementations with various modifications as are suited to the particular use contemplated.

Claims

What is claimed is:

1. A method of treating an inflammatory condition in a subject, the method comprising: placing one or more signal-conducting interfaces in signaling contact with one or more splenic nerves of the subject; forming a connection between a signal-generating source and the one or more signal- conducting interfaces; and generating a stimulation at the one or more signal-conducting interfaces with the signal- generating source, wherein the stimulation activates the ALOX15 pathway to achieve a change in a parameter.

2. The method of claim 1 , wherein the inflammatory condition is a chronic inflammatory condition.

3. The method of claim 1 or 2, wherein the inflammatory condition is an acute inflammatory condition.

4. The method of any one of claims 1-3, wherein the inflammatory condition is arthritis, inflammatory bowel disease, rheumatoid arthritis, or Crohn’s disease.

5. The method of any one of claims 1-4, wherein the subject has been diagnosed with the inflammatory condition.

6. The method of any one of claims 1-5, the method further comprising isolating the one or more splenic nerves from connective tissue and the splenic vein of the subject.

7. The method of claim 6, the method further comprising placing the one or more signal- conducting interfaces in physical contact with the one or more splenic nerves.

8. The method of any one of claims 1-7, wherein the connection between the signal-generating source and the one or more signal- conducting interfaces is a wireless connection. The method of any one of claims 1-7, wherein the connection between the signal-generating source and the one or more signal-conducting interfaces is through a lead. The method of any one of claims 1-9, wherein the generating the stimulation that activates the ALOX15 pathway produces an improvement in a physiological parameter in the subject, wherein the improvement in the physiological parameter is selected from the group consisting of: a reduction in one or more pro-inflammatory cytokines, an increase in one or more anti-inflammatory cytokines and/or one or more pro-resolving mediators, an increase in one or more catecholamines, a change in one or more immune cell populations or one or more immune cell surface co-stimulatory molecules, a reduction in one or more factors involved in the inflammation cascade, and a reduction in one or more immune response mediators. The method of any one of claims 1-10, wherein the activation of the ALOX 15 pathway comprises a change in a physiological level of one or more substrates selected from the group consisting of: linoleic acid, alpha-linolenic acid, gamma-linolenic acid, arachidonic acid (AA), eicosapentaenoic acid (EP A), and docosahexaenoic acid (DHA). The method of any one of claims 1-11, wherein the activation of the ALOX 15 pathway comprises an increase in enzymatic activity of arachidonate 15-lipoxygenase (ALOX15). The method of any one of claims 1-12, wherein the activation of the ALOX 15 pathway comprises a change in a physiological level of one or more metabolites in the arachidonic acid (AA) metabolism pathway, wherein the metabolite is selected from the group consisting of: 15S-HpETE, 15S-HETE, LTA4, 15S-Epoxytetraene, LXA4, LXB4, AT- LXA4, AT-LXB4, hepoxilin isomers, and eoxins. The method of any one of claims 1-13, wherein the activation of the ALOX 15 pathway comprises a change in a physiological level of one or more metabolites in the eicosapentaenoic acid (EP A) metabolism pathway, wherein the metabolite is selected from the group consisting of: 18R-H(p)EPE, 5S-H(p)-18R-HEPE, 15S-HpEPA, and 15-S-HEPA.

15. The method of any one of claims 1-14, wherein the activation of the ALOX 15 pathway comprises a change in a physiological level of one or more metabolite in the docosahexaenoic acid (DHA) metabolism pathway, wherein the metabolite is 17S- H(p)DHA or 17S-HDHA.

16. The method of any one of claims 1-15, wherein the activation of the ALOX15 pathway comprises a change in a physiological level of one or more specialized pro-resolving mediator (SPM).

17. The method of claim 16, wherein the SPM is a D-series resolvin, an E-series resolvin, or a T-series resolvin.

18. The method of claim 16, wherein the SPM is RvDl or RvD2.

19. The method of any one of claims 1-18, wherein the activation of the ALOX 15 pathway comprises an increase in enzymatic activity of arachidonate 15-lipoxygenase type II (ALOX15B).

20. The method of any one of claims 1-19, wherein: the one or more signal-conducting interfaces comprises one or more electrodes; the forming a connection forms an electrical connection; the signal -generating source is an electrical source; and the generating a stimulation at the one or more electrodes generates an electrical stimulation.

21. The method of claim 20, wherein the electrical source is a pulse generator; the electrical stimulation is a pulse sequence that comprises an active period followed by an inactive period, the active period comprises a plurality of electrical pulses applied by the pulse generator to the one or more electrodes, and the inactive period comprises a predetermined amount of time in which no pulse is applied by the pulse generator to the one or more electrodes.

22. The method of claim 21, the method further comprising implanting the pulse generator within the subject.

23. The method of claim 21 or 22, wherein the plurality of electrical pulses consists of between two and 1500 electrical pulses.

24. The method of any one of claims 21-23, wherein a pulse in the plurality of pulses has pulse width of between 500 microseconds (ps) and 2000 ps.

25. The method of any one of claims 21-24, wherein each pulse in the plurality of pulses has a pulse width of between 500 microseconds (ps) and 2000 ps.

26. The method of any one of claims 21-25, wherein a pulse in the plurality of pulses has a bipolar waveform, a biphasic waveform or a symmetrical waveform.

27. The method of any one of claims 21-26, wherein the plurality of electrical pulses have a frequency that is between 0.1 Hz and 300 Hz.

28. The method of claim 27, wherein the frequency is between 6 Hz and 14 Hz.

29. The method of any one of claims 21-28, wherein the active period has a duration of between 0.1 seconds and 30 seconds, and the predetermined amount of time of the inactive period is between 0.3 seconds and 30 seconds.

30. The method of any one of claims 21-29, wherein the generating the pulse sequence is repeated a plurality of times.

31. The method of claim 20, wherein the electrical source is a pulse generator, the electrical stimulation at the one or more electrodes is a continuous stimulation pulse sequence applied by the pulse generator, the continuous stimulation pulse sequence has a frequency of 15 Hz or less, and the continuous stimulation pulse sequence has a duration of greater than one minute.

32. The method of claim 31, the method further comprising implanting the pulse generator within the subject.

33. The method of claim 31 or 32, wherein a pulse in the continuous stimulation pulse sequence has a pulse width of between 200 ps and 1500 ps.

34. The method of any one of claims 31-33, wherein the continuous stimulation pulse sequence has a duration of between one minute and 15 minutes.

35. The method of any one of claims 31-34, wherein each pulse in the continuous stimulation pulse sequence has a pulse width of between 300 ps and 1200 ps.

36. The method of any one of claims 31-35, wherein a pulse in the continuous stimulation pulse sequence has a bipolar waveform, a biphasic waveform or a symmetrical waveform.

37. The method of any one of claims 31-36, wherein the frequency is between 3 Hz and 14 Hz.

38. The method of any one of claims 31-37, wherein the frequency is between 6 Hz and 14 Hz.

39. The method of claim 20, wherein the inflammatory condition is an acute inflammatory condition; the electrical stimulation is a continuous stimulation pulse sequence, wherein the continuous stimulation pulse sequence has a frequency of 15 Hz or less; and the continuous stimulation pulse sequence has a duration of greater than one minute.

40. The method of claim 39, wherein a pulse in the continuous stimulation pulse sequence has a pulse width of between 200 ps and 1500 ps.

41. The method of claim 39 or 40, wherein the continuous stimulation pulse sequence has a duration of between one minute and 15 minutes.

42. The method of any one of claims 39-41, wherein each pulse in the continuous stimulation pulse sequence has a pulse width of between 300 ps and 1200 ps.

43. The method of any one of claims 39-42, wherein a pulse in the continuous stimulation pulse sequence has a total charge of between 25pC and 45 pC.

44. The method of any one of claims 39-43, wherein a pulse in the continuous stimulation pulse sequence has a bipolar waveform, a biphasic waveform or a symmetrical waveform.

45. The method of any one of claims 39-43, wherein the frequency is between 3 Hz and 14 Hz.

46. The method of any one of claims 39-45, wherein the frequency is between 6 Hz and 14 Hz.

47. The method of claim 20, wherein the inflammatory condition is a chronic inflammatory condition; the electrical stimulation is applied periodically, wherein the electrical stimulation has a frequency of 50 Hz or less, preferably wherein the electrical stimulation has a frequency of < 10 Hz, optionally wherein the electrical stimulation has a frequency of < 2 Hz, optionally wherein the electrical stimulation has a frequency of < 1 Hz.

48. The method of claim 47, wherein the electrical stimulation has a frequency of between 3 Hz and 14 Hz.

49. The method of any one of claims 47-48, wherein the electrical stimulation has a frequency of between 6 Hz and 14 Hz.

50. The method of any one of claims 47-49, wherein the electrical stimulation is a pulse sequence, wherein the pulse sequence has a plurality of electrical pulses applied by the pulse generator to the one or more electrodes, wherein a pulse in the polarity of pulses has a pulse width of between 200 ps and 2000 ps. The method of any one of claims 47-50, wherein a pulse has a bipolar waveform, a biphasic waveform or a symmetrical waveform. The method of any one of claims 47-51, wherein a pulse has a biphasic charge-balanced rectangular waveform. The method of any one of claims 20-52 herein an electrode in the one or more electrodes is a cuff electrode, a circumferential cuff electrode, a catheter intravascular electrode, a stent, or a patch. The method of any one of claims 1-19, wherein the generating a stimulation at the one or more signal-conducting interfaces comprises generating a signal, at the signal-generating source, that is selected from the group consisting of: an electrical signal, an infrared signal, an ultrasound signal, a mechanical signal, and a magnetic signal.