WO2021209465A1 - Peptide for the treatment of net-associated diseases - Google Patents

Abstract

The invention is in the field of medical treatments. It provides means and methods for the treatments of diseases related to or caused by neutrophil extracellular traps (NETs). More in particular, it relates to a peptide comprising an amino acid sequence H-CEPLSEVEDYLDSSLKYNAKDTINYC-OH (SEQ ID NO: 1) or an equivalent thereof that differs from SEQ ID NO: 1 by not more than 3 amino acids.

Description

Title: PEPTIDE FOR THE TREATMENT OF NET-ASSOCIATED DISEASES

Field of the invention

The invention is in the field of medical treatments. It provides means and methods for he treatments of diseases related to or caused by neutrophil extracellular traps (NETs).

Background of the invention

Acute infection is a well-established risk factor of cardiovascular inflammation increasing the risk for a cardiovascular complication within the first three weeks after infection. However, the nature of the processes underlying such aggravation remains unclear. Lipopolysaccharide (LPS) derived from Gram-negative bacteria is a potent activator of circulating immune cells including neutrophils, which foster inflammation through discharge of neutrophil extracellular traps (NETs).

Atherosclerosis is a lipid-driven chronic inflammation of the arterial vessel wall. In its late stages, atherosclerosis is the underlying pathophysiology of myocardial infarction and stroke and hence the leading cause of mortality worldwide. Monocytes and their descendants hold crucial roles throughout all stages of atherosclerosis as they contribute to lipid modification and respond with a pronounced inflammatory response upon uptake of modified lipids [1] With only limited numbers of macrophages residing in large arteries, the vast majority of monocytic cells needs to be de novo recruited in a process known as the leukocyte recruitment cascade, which is orchestrated by fine-tuned interactions of selectins, chemokines, and integrins and their respective receptors [2] In recent years, studies have provided evidence for the contribution of neutrophils to the arterial monocyte recruitment. Herein, neutrophils deposit preformed chemoattractants on endothelial cells or activate endothelial cells directly to stimulate monocyte adhesion [3-6]

Multiple bacterial and viral pathogens have been associated with atherosclerosis by seroepidemiologial studies and identification of the infectious agent in human atherosclerotic tissue. Moreover, there is strong clinical evidence for the acceleration of arterial inflammation by acute infection [7] As an example, urinary tract infection and bacteremia associate with an increase in the short-term risk of myocardial infarction [8, 9] Most striking data, however are available from patients with pneumonia, with the risk of myocardial infarction peaking at the onset of infection and is proportional to the severity of illness [10-12] Division of Gram-negative bacteria or their elimination leads to release of lipopolysaccharide (LPS) into the blood stream, i. e. endotoxinemia. Neutrophils are rapidly activated by LPS leading to the release of neutrophil extracellular traps (NETs), a complex structure composed of nuclear chromatin and proteins of nuclear cytoplasmatic and granule origin [13] Proteins reported to attract monocytes are localized within NETs. Moreover, NETs have been detected at the luminal side of large arteries [14]

The role of NETs in inflammation has still to be elucidated in more detail so that therapies for NET-related diseases can be developed and improved.

Summary of the invention

The invention relates to a peptide comprising an amino acid sequence H-CEPLSEVEDYLDSSLKYNAKDTINYC-OH (SEQ ID NO: 1) or equivalents thereof and their use in medical therapy.

Detailed description of the invention

Here we utilize a model of endotoxinemia to link acute infection and subsequent neutrophil activation with acceleration of vascular inflammation.

To investigate the effect of endotoxinemia on myeloid cell recruitment during atheroprogression, Apoe-/- mice were fed a high fed diet for 4 weeks and injected with LPS. Treatment in this way resulted in the striking expansion of atherosclerotic lesion sizes (Figure 1). While plasma cholesterol and triglyceride levels were not affected by treatment in this way, counts of circulating neutrophils and monocytes were depleted, likely due to margination and extravasation of activated myeloid cells. Indeed, the number of neutrophils, monocytes and macrophages recruited to atherosclerotic lesions as well as to inflamed aorta was drastically increased in LPS-treated mice (Figures 2 and 3). This notion was confirmed by use of intravital microscopy of carotid arteries performed in Apoe-/-Cx3cr1eGFP mice. There, LPS enhanced adhesion of both GFP+ monocytes as well as of antibody-labeled neutrophils.

LPS is known to trigger the release of NETs [20] and hence we investigated whether in our experimental setting NETs may contribute to heightened neutrophil and monocyte recruitment. Intravital microscopy permitted to visualize DAPI+ structures with a NET-like shape in the lumen of mice receiving LPS (Figure 4). Additionally, cell-free DNA as well as DNA-MPO complexes, a plasma marker of NETs, are strikingly increased in the plasma of mice with endotoxinemia (Figures 5 - 8). Importantly, plasma DNA-MPO complexes correlated with plasma endotoxin levels indicating that LPS directly induces NET release (Figures 5 - 8).

To test if NETs along the arterial lumen would contribute to the atherosclerosis phenotype observed in endotoxinemic mice, we treated mice with BB Cl-amidine, a potent inhibitor of protein arginine deiminases (PADs) with favorable pharmacokinetics in terms of plasma half-life and EC50. Intravital imaging confirmed that BB Cl-amidine treatment abrogated luminal NET release (Figure 4). In addition, heightened lesion formation as well as lesional accumulation of myeloid cells in response to LPS was completely abolished (Figure 1 - 3). In agreement herewith, BB Cl-amidine treatment led to a vast reduction of arterial myeloid cell adhesion (Figure 9, 10) suggesting that luminal NETs promote arterial myeloid cell adhesion during endotoxinemia.

We next investigated how NETs promote monocyte adhesion independent of receptor signaling. Therefore, we allowed classical monocytes to sediment on NETs in vitro. Indeed, we witnessed a significant adhesion of monocytes to NETting neutrophils (Figure 11). Fluorescence imaging revealed that these monocytes predominantly adhered directly to the NET scaffold and inhibition of monocyte adhesion after NET degradation with DNase I confirmed the importance of NETs in static monocyte adhesion (Figure 11). To transfer these findings to a physiologically relevant setting, we initiated NET release in adherent neutrophils and perfused classical monocytes in a flow chamber assay. As for the static adhesion assay, we found that monocytes primarily bound to DNA fibers and that degradation of NETs abolished adhesion evoked by activated neutrophils (Figure 12).

These observations are reminiscent of earlier work showing that proteins typically found in NETs or NETs themselves can increase cell adhesion by engaging chemokine receptor signaling and leukocyte integrin activation [2, 21-24] Thus, we neutralized chemokine receptors (Figure 13), receptors of alarmins (Figure 14), or integrins (Figure 15) prior to incubation with NETs.

Much to our surprise, none of these treatments impacted on adhesion evoked by NETs. Thus we concluded that the adhesion evoked by NETs may be signaling independent. Indeed, depletion of calcium by use of a chelator did not affect NET-mediated adhesion. In addition, abrogation of signaling of G-protein coupled receptors by pertussis toxin or even fixation of monocytes with 4% paraformaldehyde (PFA) did not impair adhesion of monocytes to NETs (Figure 16, 17). Taken together, these data show that monocyte adhesion to NETs is independent of receptor signaling.

We went on to show that NET-resident histone H2a attracts monocytes electrostatically. Given the signaling-independent adhesion of monocytes to NETs, we assumed that biophysical interactions such as electrostatic charges could be important in this process. In fact, monocytes present an overall negative surface charge (z potential -10.51±0.69 mV), while NETs are decorated with highly cationic proteins. To test the importance of charge interaction in monocyte adhesion to NETs, monocytes were incubated with either cholesterol sulfate or oleylamine. Cholesterol sulfate is a negatively-charged steroid lipid, whereas oleylamine is a positively-charged unsaturated fatty acid. Both lipids integrate with their lipophilic part into the phospholipid bilayer of the cell membrane and hence allow manipulating cell surface charge. In our hands, incubation with cholesterol sulfate or oleyelamine rendered monocyte surface charges more negative or more positive, respectively (Figure 18, 19).

To assess the relevance of the monocyte surface charge during adhesion to NETs we allowed monocytes of different surface charges to adhere to NETs. In these experiments we were able to generate a stringent correlation with more negative surface charges resulting in higher monocyte adhesion and vice versa (Figure 20). To assess the physical properties of charge-dependent adhesion in more depth, we performed atomic force spectroscopy with a monocyte immobilized at the tip of the cantilever. Atomic force microscopy (AFM) is a scanning probe microscope with piezoelectric elements to move the spring like cantilever. The deflecting cantilever is used to directly measure forces acting on the monocyte. A force-distance curve reports on the contact force (I), the force until maximum adhesion (II), the forces needed to fully detach the monocyte from the NET (III) and the distance of this detachment (IV) (Figure 21). Monocytes with different surface charges were immobilized at the tip of the AFM cantilever and in this position probed on NETs. Manipulation of the monocyte surface charges impacted on the maximum adhesion force, the detachment force and the detachment distance to separate monocytes from NETs, the energy required to separate the monocyte from the NET, and the adhesion frequency. Overall, NET-adhesion strength and the ability to adhere to NETs was heightened when monocytes were rendered more negatively charged and opposite effects were found in less negatively charged monocytes (Figure 22, 23), thus confirming the concept of charge-driven monocyte adhesion.

Neutrophil extracellular traps are decorated with a large array of granule- derived, cytoplasmatic, and nuclear proteins. Proteomics of NETs allowed the identification of 73 enriched polypeptides of which two clusters exhibited cationic surface charge as deferred from the peptides isoelectric point. One cluster was centered on nuclear histones of which histone H2a is the most abundant one. A second cluster revealed antimicrobial polypeptides with myeloperoxidase (MPO) and cathepsin G being the two candidates characterized by both cationic charge and high abundance (Figure 24). To assess the contribution of these NET-resident proteins towards monocyte adhesion, we treated NETs with antibodies towards these polypeptides and recorded monocyte adhesion. In these studies, neutralization of antimicrobial polypeptides abundantly found in NETs (MPO, cathepsin G) and of such previously found to contribute to monocyte adhesion (LL37, elastase, proteinase 3, HNP1-3) yielded no effect (Figure 25) [4, 5, 25] However, neutralization of histone H2a, the most abundant nuclear protein found in NETs, fully abrogated NET-driven monocyte adhesion (Figure 25). High resolution confocal microscopy revealed that monocytes indeed bound to NET-resident histone H2a. Finally, we found that histone H2a binds to monocytes in a charge dependent fashion, thus confirming our understanding of charge-mediated monocyte adhesion.

Then we set out to investigate whether therapeutic neutralization of histone H2a diminishes arterial monocyte adhesion and atheroprogression during endotoxinemia. Thus far our data suggest that histone H2a presented in NETs released during endotoxinemia causes myeloid cell adhesion, a process dramatically accelerating atheroprogression. Hence, we devised a protocol aimed at halting histone H2a-induced monocyte adhesion in vivo. Herein, a histone H2a neutralizing antibody or an isotype control IgG were administered in hypercholesterolemic, LPS-treated mice and luminal adhesive events were studied by intravital microscopy. Whereas treatment with histone H2a targeting antibodies did not impact on the presence of luminal NET-like structures and the number of circulating leukocytes, adhesion of myeloid cells was significantly reduced (Figures 26 - 28).

We also set out to design peptide inhibitors to neutralize histone H2a. Peptides contain several advantages such as ease of synthesis and optimization to improve pharmacokinetic and binding properties, large-scale and cost-effective production and applicability to conjugate with specific probes for drug delivery or for using as imaging tools [26] Moreover, we have previously demonstrated and proven that monocyte adhesion can be interrupted by peptide inhibitors [5, 17] and the highly positively charged N-terminus of histone H4 can be neutralized by a peptide [18] We in silico designed and developed peptides targeting histone H2a as described in the Examples section. The most potent peptide called Cyclical Histone 2A Interference Peptide (CHIP), with amino acid sequence H-CEPLSEVEDYLDSSLKYNAKDTINYC- OH, (SEQ ID NO: 1) bound to the N-terminal domain of histone 2A, and the electrostatic interactions between positively charged residues (Arg and Lys) of histone H2a with negatively charged residues (Asp and Glu) of CHIP and H-bond interactions promoted a stable complex formation (Figure 29).

To test the functionality of this peptide we first performed in vitro experiments. Herein, pretreatment of NETs with CHIP reduced adhesion significantly (Figure 30). Biophysically, CHIP reduced the interaction strength of monocytes and NETs as shown by atomic force microscopy (Figures 31 - 33).

We then set out to testing CHIP in vivo. Delivery of CHIP to hypercholesterolemic, LPS-treated mice, did not impact on the presence of NET-like structures in the arterial lumen, but significantly diminished arterial adhesion of neutrophils and monocytes (Figures 34 - 37). Beyond impacting on luminal events, CHIP allowed to drastically reduce atherosclerotic lesion sizes (Figure 38). These changes in lesion size were not associated with differences in blood counts or plasma lipid levels. The lesions of mice treated with CHIP were characterized by the accumulation of less neutrophils and macrophages (Figures 39, 40). In line herewith, aortic neutrophil and monocyte numbers were strikingly reduced by CHIP (Figures 41, 42). Taken together, neutralization of histone H2a by a peptide according to SEQ ID NO: 1 results in the reduction of arterial myeloid cell recruitment and accelerated atherosclerosis evoked by endotoxinemia.

It will be clear from the above results that other NETs-associated diseases can be treated equally well with the peptide according to SEQ ID NO: 1. Such NETs-associated diseases include for example autoimmune disorders [40], stroke [41], (transfusion-related) acute lung injury [36,37,38,42], ischaemia/reperfusion injury (IRI) [38,39,43], kidney injury [39,44], liver injury [45,46], trauma [47] and sepsis [48,49] Histones are not only released as a consequence of cellular injury, they also actively contribute towards a self-sustaining cascade of ongoing cell death [50] The invention may also be applied in the treatment of Acute respiratory distress syndrome such as ARDS caused by infection with coronavirus [51 - 53]

The peptide according to SEQ ID NO: 1 can therefore also be used to treat inflammation.

The teaching as provided herein should not be interpreted so narrowly that it relates only to a peptide comprising the sequence according to SEQ ID NO: 1. An alanine scan of the peptide according to SEQ ID NO: 1 can be easily performed by the skilled person and will reveal that at least 1 , such as 2 and 3 amino acids in SEQ ID NO: 1 can be replaced by another amino acid without affecting the function of the peptide.

Moreover, when we tested the peptide according to SEQ ID NO: 1 as a circular peptide wherein the N-terminal cysteine residue and the C-terminal cysteine residue were used to circularise the peptide, results were even improved. Hence, the invention also relates to a peptide comprising an amino acid sequence H- CEPLSEVEDYLDSSLKYNAKDTINYC-OH (SEQ ID NO: 1) or an equivalent thereof that differs from SEQ ID NO: 1 by not more than 3 amino acids that is circular.

In summary, the following may be noted from this study. Atherosclerosis is the leading cause of mortality in Western society. Excess mortality from cardiovascular disease during influenza epidemics was first recognized early in the 20th century, but the specific association of influenza and other infections with myocardial infarction was not characterized until decades later. In fact, over the last 20 years epidemiological studies have consistently demonstrated the association between acute infection with multiple bacterial and viral pathogens and the short term increase in cardiovascular complications [7] As an example, a self-controlled case series involving U.S. veterans showed a remarkable increase in the risk of myocardial infarction during the first 15 days after hospitalization for acute bacterial pneumonia, to a risk that was 48 times higher than that in any 15-day period during the year before or after the onset of infection [27] An increase in the short-term risk of myocardial infarction has also been described in association with urinary tract infection and bacteremia [8, 9] The strength and temporal pattern of the association between acute infections and an increased risk of myocardial infarction suggest a causal relationship.

Because the association has been shown with a variety of pathogens, sites of infection, and the association is stronger and lasts longer when the infection is more severe, it is likely that the infection and the host response to infection are major determinants in this relationship. Here we used a model of endotoxinemia to mimic acute, severe infection. In these experiments, administration of endotoxin induces a rapid expansion of atherosclerotic lesions characterized by extension of the lesional myeloid cell compartment. This increase was fully abrogated when release of NETs was pharmacologically inhibited. Our studies identify a mechanism that centers on monocyte adhesion to luminal NETs in general and to NET-resident histone H2a in particular. Neutralization of histone H2a by antibodies or by in silico designed peptides permits inhibition of endotoxin-accelerated monocyte adhesion and lesional myeloid cell accumulation, thus providing a potential strategy for improved care of patients at risk of cardiovascular events and experiencing an acute infection.

Since our study was performed with endotoxin stimulation only, this may be perceived as limitation in the applicability of our study to clinical scenarios. However, an increased risk for cardiovascular complications after an acute infections has been observed for numerous pathogens, including viruses such as influenza virus, corona virus, or respiratory syncytial virus [7], Gram-positive bacteria including Streptococcus pneumonia and Staphylococcus aureus, as well as Gram-negative bacteria like Escherichia coli and Haemophilus influenza [9, 10, 12, 28] Interestingly, NET release is triggered by a variety of stimuli including all of the pathogens listed above [29] and hence the NET-centered mechanism identified herein may in part be applicable to a large variety of pathogen-associated cardiovascular complications although confirmation in additional studies is required.

Neutrophils have previously been reported to pave the way for inflammatory monocytes into developing atherosclerotic lesions. Rendering mice neutropenic during initial stages of atherosclerosis diminishes lesion sizes as well as lesion composition, with lowered macrophage accumulation [30] Neutrophils stimulate various mechanisms that promote monocyte recruitment. Among these, secretion of chemotactic proteins stands out as an important mechanism for arterial monocyte recruitment. Cathepsin G, cathelicidin, complexes formed of neutrophil-derived a- defensin and platelet-borne CCL5, and CCL2 released from neutrophils in a circadian fashion were shown to induce firm monocyte adhesion in mouse models of vascular inflammation when immobilized on arterial endothelial cells [3-5, 31] However, all these ligands bind to specific receptors and increase adhesion through increased integrin affinity and avidity. Herein we identify electrostatic interactions between cationic histone H2a and negatively charged monocyte surfaces as a novel mechanism driving infection-associated monocyte recruitment into atherosclerotic lesions. Histone H2a combines abundance within NETs and surface charge and may hence stand out as an important epitope for monocyte adhesion. However, other histone isotypes may engage similar activities as they are comparable in abundance and charge within NETs. Of note, a recent study has shown that activated smooth muscle cells residing in the fibrous cap of advanced atherosclerotic lesions stimulate neutrophils to release NETs. These are rich in cytotoxic histone H4 that can puncture human and murine SMCs leading to their death and, when re-occurring, thinning of the fibrous cap [18] Thus, therapeutic neutralization of histone epitopes may exert a dual beneficial effect allowing to limit macrophage burden and fibrous cap thinning.

The mechanism of NET-driven monocyte recruitment is receptor independent and hence lacks specificity. In fact, surgical removal of tumors (especially colorectal cancer) is frequently associated with bacteremia and NETs triggered in such settings have been suggested to promote immobilization of tumor cells in vascular beds thus promoting formation of metastases [32-34] Mechanistically, some studies reported betal integrins on tumor cells to mediate adhesion to NETs while other studies found unspecific binding to NETs underlying the process of NET-driven metastasis [2, 21 , 22] Consequently, degradation of NETs using DNase I has been found to limit metastases formation in animal models combining infection and tumor development [2, 21] Observations made in the here presented study suggest that histone H2a can also promote adhesion of tumor cells and promote metastases and perioperative treatment with histone-neutralizing therapies may provide an efficient way to reduce cancer spreading.

Infections in cardiovascular risk patients increase the chance of cardiovascular complications within the first three weeks after an infection several fold. We here identify a mechanism centered on extracellular histone H2a which induces adhesion and recruitment of monocytes. While data from the CANTOS trial reveal the overall positive effects of anti-inflammation therapy in the context of atherosclerosis, neutralization of ILIbeta was also associated to heightened risk of bacterial infections35. Therapeutic neutralization of histone H2a will likely not be linked to such adverse side effects as the antimicrobial actions of histone H2a can be compensated by the abundance of other antimicrobial polypeptides residing within NETs. In addition, the strict focus on infection-associated cardiovascular complications will be relevant to a very short time window of not more than three weeks after infection and can be combined with standard antibiotic treatment. Thus, targeting histone H2a may stand out as an innovative way to lower arterial monocyte recruitment accelerated by infection while coming with limited intrinsic side effects.

The invention therefore relates to a peptide comprising an amino acid sequence H-CEPLSEVEDYLDSSLKYNAKDTINYC-OH (SEQ ID NO: 1). More in particular it relates to a peptide as described herein consisting of the amino acid sequence H-CEPLSEVEDYLDSSLKYNAKDTINYC-OH (SEQ ID NO: 1). Such a peptide may advantageously be used in the treatment of a disease, more in particular in the treatment of a NETs-related disease. In one embodiment of the invention, the treatment is related to the treatment of a NETs-related disease selected from the group consisting of atherosclerosis, autoimmune disorders, stroke, transfusion -related acute lung injury, acute lung injury, lung injury, ischaemia/reperfusion injury (IRI), kidney injury, liver injury, trauma, sepsis and inflammation.

The teaching as provided herein should not be so narrowly interpreted as that only peptides comprising the exact sequences of SEQ ID NO: 1 would work in a therapy as described in the claims. Equivalents of such a peptide will work as well and can be found by the skilled person with only little experimental effort. So is it well within range of the skills of that person to produce and test peptides that differ only one, two or even three amino acids from the sequence as provided in SEQ ID NO: 1 and still work. Such peptides are termed herein “equivalents” or “equivalent peptides”.

Legend to the figures

Figures 1 - 4: Endotoxinemia accelerates atherosclerosis. Experimental setup. Apoe or Apoe^Cxacrl^ mice were fed a HFD for 4 weeks and treated with either PBS (Ctrl) or with LPS (1 mg/kg BW). Another LPS-treated group received a BB Cl-amidine (BB Cl-A, 1mg/kg BW) 12 h before and along with LPS injection. Figure 1 : Aortic root lesion size analyzed in HE-stained sections. Representative images, scale bar 300 pm. Figure 2: Lesional neutrophils (Ly6G⁺ cells) quantified in root sections. Figure 3: Macrophages (Mac2⁺ cells) quantified in root sections. Figure 4: Count of luminal NET-like structures in left common carotid artery. Data are analyzed by one- way ANOVA with Dunnett's multiple comparisons test; *p£0.05, **p£0.01 , ***p£0.001 , ****p£0.0001. All data are presented as meaniSEM.

Figures 5 - 8: Endotoxinemia-triggered NET release stimulates arterial myeloid cell recruitment. Apoe mice were fed a HFD for 4 weeks and treated with either PBS (Ctrl) or with LPS (1 mg/kg BW). Another LPS-treated group received a BB Cl-amidine (BB Cl-A, 1 mg/kg BW) 12 h before and along with LPS injection. Quantification of cell free DNA in plasma (Figure 5), plasma endotoxin units (Figure 6), and circulating MPO-DNA complexes (Figure 7) as measure of NET release. Pearson correlation of NETs (MPO-DNA complexes) with plasma endotoxin units (Figure 8). Data are analyzed by one-way ANOVA with Dunnett's multiple comparisons test, Kurskal-Wallis test with Dunn’s post test or unpaired t-test; *p£0.05, **p£0.01 , ***p£0.001, ****p£0.0001. All data are presented as mean ±SEM.

Figures 9 - 10: Endotoxinemia accelerates atherosclerosis.

Experimental setup. Apoe or Apoe^Cxacrl^ mice were fed a HFD for 4 weeks and treated with either PBS (Ctrl) or with LPS (1 mg/kg BW). Another LPS-treated group received a BB Cl-amidine (BB Cl-A, 1 mg/kg BW) 12 h before and along with LPS injection. Quantification of luminally adherent neutrophils (Figure 9) and monocytes (Figure 10).

Figures 11 - 17: Monocyte adhesion to NETs is receptor independent. In vitro monocyte adhesion to expelled NETs under static or flow conditions. Figure 11 : Monocytes were added to neutrophils (Ctrl) or NETting neutrophils (induced by A23187) and adhesion was quantified by a fluorescence plate reader. NETs were also degraded by DNase I. Figure 12: Monocytes were perfused at 0.5 dyne/cm² over neutrophils, NETs, or degraded NETs and their adhesion was quantified manually. Monocytes where pre-treated with antagonists or antibodies to chemokine receptors (Figure 13), Toll-like receptors (Figure 14), or integrins (Figure 15) prior to incubation with NETs. Monocytes fixed with PFA or treated with pertussis toxin (PTx) were used in static (Figure 16) or flow (Figure 17) adhesion assays. Data are analyzed by one way ANOVA with Dunnett's multiple comparisons test or Kruskal-Wallis test with Dunn's multiple comparisons test; *p£0.05, **p£0.01 , ***p£0.001 , **** p£0.0001. All data are presented as meaniSEM.

Figures 18 - 25: Monocyte adhesion to NETs in regulated by cationic histone H2a. z-potential analysis of isolated monocytes treated with Ch-sulfate (Figure 18) or oleylamine (Figure 19). Figure 20: Pearson correlation of monocyte adhesion to NETs versus monocyte z-potential. Figure 21 : Scheme of single cell atomic force microscopy (AFM) force spectroscopy. Monocytes were probed on expelled NETs at 200 pN contact force. Figures 22 and 23: Quantification of the area under the curve reflecting the energy required to rupture the monocyte-NET interaction. Figure 24: Proteome analysis of NET resident proteins. Circle size reflects protein abundance, while color codes for charge (red cationic, green anionic). Figure 25: Monocyte adhesion to NETs pre-incubated with antibodies to indicated NET-associated proteins. Data are analyzed by unpaired t-test, Kruskal-Wallis test with Dunn's multiple comparisons test or one-way ANOVA with Dunnett's multiple comparisons test; *p£0.05, **p£0.01 , ***p£0.001. All data are presented as mean ±SEM. HNP, human neutrophil peptides; MPO, myeloperoxidase; PR3, proteinase 3; NE, neutrophil elastase; CatG, cathepsin G.

Figure 26 - 40: Neutralization of histone H2a inhibits endotoxin-induced arterial myeloid cell recruitment and atheroprogression. Experimental outline: Apoe Cx₃cr1^GFP were fed a HFD for 4 weeks, treated with LPS (1 mg/kg, 4h) and injected with isotype respective control (IgG), or a histone H2a-targeting antibody (anti-H2a). (Figures 26 - 28) Intravital microscopy was used to quantify luminal NET-like structures (Figure 26) in the left carotid artery as well as adherent neutrophils (Figure 27) and monocytes (Figure 28). Figure 29: The structure of histone H2a (magenta), CHIP (orange) and the histone H2a-CHIP complex which was derived from protein-protein docking and molecular dynamic simulation. CHIP bound and interacted with the N- terminal part of histone H2a. The electrostatic interactions between Arg or Lys of Histone H2a (green sticks) with Glu or Asp of CHIP (cyan sticks) as well as hydrogen bonds (displayed as dash lines) help to stabilize the complex formation between histone H2a and CHIP. Figures 30 - 33: Pharmacological interruption of histone H2a monocyte-binding was validated in static adhesion assays (Figure 30) as well as by single cell force spectroscopy (Figures 31 - 33). Figures 34 - 40: In vivo validation of therapeutic effect of CHIP in endotoxin-accelerated atherosclerosis. Experimental outline. Apoe^~/~Cx₃cr1^GFP were fed a HFD for 4 weeks, treated with LPS (1 mg/kg, 4h) and injected with vehicle or CHIP (5 mg/kg). Figures 35 - 37: Intravital microscopy was used to quantify luminal NET-like structures (Figure 35) in the left carotid artery as well as adherent neutrophils (Figure 37) and monocytes (Figure 36). Figure 38: Aortic root lesion size analyzed after HE staining. Quantification of Lesional neutrophils (Ly6G⁺ cells) (Figure 39), and macrophages (Mac2⁺ cells) (Figure 39). Figure 40: Representative immunofluorescence images showing lesional Mac2⁺ cells (grey) and nuclei (DAPI, blue), scale bar 50 pm. Data are analyzed by Mann-Whitney test; *p£0.05, **p£0.01 , ***p£0.001. All data are presented as mean ±SEM.

Figures 41 and 42: Blocking histone H2a does not affect plasma lipids or cell counts in blood but rather diminishes aortic myeloid cell accumulation. Apoe Cx₃cr1^GFP were fed a HFD for 4 weeks, treated with LPS (1 mg/kg, 4h) and injected with isotype respective control (IgG), or a histone H2a-targeting antibody (anti-H2a). Figure 41- 42: Apoe Cx₃cr1^GFP were fed a HFD for 4 weeks, treated with LPS (1 mg/kg, 4h) and injected with vehicle or CHIP (5 mg/kg). Flow cytometry was used to assess neutrophils (G) and classical monocytes (H) in aortic homogenates. Data were analyzed with Mann-Whitney test (A-C) or Unpaired t test (D-H); **p£0.01. All data are presented as mean ±SEM.

Examples

Example 1 : Animal experiments

We surveyed atheroprogression in Apoe or Apoe^Cxscrl^ reporter mice on C57BI/6J background after 4 weeks of high-fat diet (HFD) (21% fat, Ssniff). Endotoxinemia was induced by intraperitoneal (i.p.) injection of lipopolysaccharide (LPS, Escherichia coii, 0111 :B4, Sigma Aldrich, 1 mg/kg BW, i.p.). Control mice received vehicle (PBS, 100 pi, i.p.). Thereafter, atherosclerotic lesions were analyzed in aortic root sections or cell adhesion was studied by intravital microscopy of the left carotid artery [4] To visualize neutrophils and NET-like structures, mice were injected i.v. with a PE-conjugated antibody to Ly6G (1A8, 1 pg, Biolegend) and 4',6-Diamidino- 2-phenylindol (DAPI, Thermo Fischer). To assess the effect of NETs we blocked NET- formation with BB Cl-amidine (1 mg/kg BW, Cayman Chemical Company). In additional experiments, mice received antibodies to histone H2a (20 pg/mouse, Biorbyt), its respective IgG isotype control (Jackson ImmunoResearch), or a cyclical histone 2A interference peptide (CHIP, 5 mg/kg BW). All animal experiments were approved by the local ethics committee and performed in accordance with institutional guidelines.

Example 2: Flow cytometry

Leukocyte counts were quantified by flow cytometry. Staining of single cell suspensions of blood and bone marrow were conducted using combinations of antibodies specific for CD45 (A20, eBioscience), CD11b (M1/70, eBioscience), CD115 (AFS98, eBioscience), Gr1 (RB6-8C5, BioLegend). Before cell staining, red blood cell lysis was performed. Furthermore, aortas were digested by liberase (1.25mg/ml, Roche) and single cells were labeled with antibodies to CD11b (M1/70, bioLegend), Ly6G (1A8, BioLegend), MHC II (M5/114.15.2, BD Bioscience), Gr1 (RB6-8C5, BioLegend), F4/80 (BM8, BioLegend), CD45 (A20, eBioscience). Cells were washed with Hanks Balanced Salt Solution (HBSS) and directly analyzed by flow cytometry using a FACSCanto II (BD). Absolute cell numbers were assessed by use of CountBright™ absolute counting beads (Invitrogen). Data were analyzed with FlowJo Software (10.1 Flowjo LLC).

Example 3: Cell free DNA measurement

Cell free DNA was measured in the plasma with Quant-iT PicoGreen dsDNA assay (Life Technologies) the mean fluorescence intensity was quantified and according to the standard curve, results are expressed as ng/ml . Samples were measured with a plate reader (Tecan infinite F200 pro).

Example 4: NET-ELISA

Neutrophil extracellular traps were measured in plasma by detecting myeloperoxidase and cell free DNA as described elsewhere [15] Briefly, MPO-DNA complexes were quantified by modified MPO ELISA (Hycult Biotech) combined with cell death detection kit (Roche).

Example 5: Endotoxin measurement

LPS plasma levels were assessed with Pierce Chromogenic Endotoxin Quant kit (Thermo Fisher Scientific). Plasma samples were diluted 1 :50 in endotoxin free water and heat inactivated for 15 min at 70°C. Amebocyte lysate reagent was added to each well, incubated for 30 min followed by chromogenic substrate solution incubation. After 6 min of incubation, the stop solution was added, and the absorption was measured with Tecan infinite F200 pro (OD 405 nm).

Example 6: Histology and Immunohistochemistry

The size of atherosclerotic plaques was measured in aortic roots after staining with Hematoxylin (Merck) and Eosin (Roth) followed by computerized image analysis and quantification (ImageJ, 1.48v). Aortic roots were stained with antibodies to Ly6G (1A8, BD Biosciences) and Mac2 (M3/38, biozol). Nuclei were counter-stained with DAPI (Thermo Fischer). A Leica DM4000 microscope with a 20x objective (Leica Microsystems) and a Leica DFC 365FX camera were used to capture images.

Example 7: In vitro adhesion assay

Blood was drawn from healthy volunteers. Neutrophils were isolated by polymorphprep according to the manufacturer’s instructions. Monocytes were isolated via negative selection using monocyte isolation kit II (MACS Miltenyi biotec). Adhesion of monocytes to NETs was studied under static and flow conditions. Isolated monocytes were labeled with CellTrace™ calcein violet AM (Thermo fisher scientific). Monocytes were added (0.5x10⁵ cell/well) to flat bottom 96-well plates (Falcon corning) or flow chambers (m-slide VI 0.1 ibiTreat (Ibidi)) containing either neutrophils (2x10⁵ cells/well, labeled with cell tracker red CMTPX (Thermo fisher scientific)) or NETing neutrophils (25 mM calcium ionophore A23187 (Sigma Aldrich)) for 15 min or 3 min under applied flow (0.5 dyne/ cm²; PHD ULTRA syringe pump, Harvard Apparatus). Non-adherent monocytes were washed off. Statically adherent monocytes were quantified in a microplate reader (Tecan infinite™ 200 pro). Monocytes adherent under flow conditions were counted manually. For each flow channel three fields were quantified and averaged. NETs were visualized by co-staining DNA with DAPI and citrullinated H3 or Sytox green nucleic acid stain (Thermo Fisher Scientific). To study monocyte adhesion to NETs, traps were treated with DNAse (10U, Sigma Aldrich), antibodies to Histone H2a (10 pg/ml; Cell Signaling), Myeloperoxidase (10 pg/ml; AB1224, Merck), Proteinase 3 (10 pg/ml; MAB6134, R&D systems), Neutrophil elastase (10 pg/ml; Biorbyt), Cathepsin G (10 pg/ml; Biorbyt), LL-37 (10 pg/ml; Santa Cruz Biotechnology), a-defensin (10 pg/ml; Hycult Biotech), CHIP (200 pg/ml, Pepscan). In another experimental setting monocytes were pre-incubated with antagonists to CCR1 (1 pM; BX471 , Tocris), CCR2 (1 mM; RS504393, Tocris), CCR5 (1 pM; SB32437, Tocris), CCR5 (0.1 mM; DAPTA, Tocris), CXCR2 (1 mM; SB225002, Tocris), CXCR4 (1 mM; AMD3100, Sigma Aldrich), FPR1 (10 mM: Cyclosporin H, Tocris), FPR2 (10 mM; WRW4, Tocris), TLR1-2 (100 mM; CU CPT 22, Tocris), TLR4 (100 mM; C34, Tocris), TLR9 (100 mM; Hydroxychloroquine sulfate, Tocris), VLA-4 (10 mM; BI01211 , Tocris), P2x7 (0.1 mM; A 74 0003, Tocris), or P2y2 (10 mM; AR- C118925xx, Tocris). To block chemokine receptor signaling, cells were pretreated with Pertussis toxin (0.8 pg/ml, Sigma-Aldrich); Ca²⁺ mobilization was abrogated using the calcium chelator BAPTA AM (2 mM; Thermo Fisher Scientific). Finally, antibodies to Mac1 (M1/70, 10 mg, Biolegend) or LFA1 (10 mg, R7.1 , Biolegend) were used to test the relevance of integrin activation.

Example 8: Molecular Dynamics (MD) Simulations and Peptide Design

Three-dimensional (3D) structures of protein-protein complexes were used as a starting point for rationally design and develop bioactive compounds to modulate protein-protein interactions [16-18] Thus, the complex between human BRPF1 bromodomain and histone H2a peptide (PDB code: 4QYL) was selected and utilized to design potential histone H2a peptide inhibitors. The loop regions (residue 31-43 and 78-91) of bromodomain binding with the N-terminal tail of histone H2a were extracted and used as a starting peptide which were then docked onto the N-termini histone H2a (chain C of the nucleosome structure, PDB code: 1 KX5). Based on the derived complex between the template peptide and histone H2a several peptides were designed and then docked onto the N-terminal tail of histone H2a as well by application of protein-protein docking program (HADDOCK2.2 Webserver [19]). The derived histone H2a -peptide complexes were subsequently subjected to structural optimization and binding free energy calculations to predict binding strength of peptides with histone H2a. Similar protocols and standard parameters as in our previous work were applied for these purposes [17, 18] Briefly, energy minimization was carried out for 10,000 steps (5,000 steps of steepest descent followed by 5,000 steps of conjugate gradient algorithm) and subsequently a short MD simulation (500 ps) was performed by using TIP3P water model and AMBER14SB force field for peptides and protein (histone H2a) and setting essential parameters at the standard values (e.g. temperature at 300 K, pressure at 1 bar, time step at 2 fs with SHAKE constraint). Binding free energy was calculated by using molecular mechanics/generalized Born surface area (MM/GBSA) approach (generalized Born model 8 with default parameters). MD simulations and binding calculations were carried out by using AMBER16 program. Binding affinity of the designed peptides were prioritized according to their predicted binding free energy and the peptide with the lowest binding free energy, which is CHIP (H-CEPLSEVEDYLDSSLKYNAKDTINYC- OH, SEQ ID NO: 1), containing an S-S bond between the cysteine residues at the N- and C-terminus, was selected for synthesis and further experimental testing. Example 9: Zeta (Ci potential

Cell surface charge of either untreated monocytes, monocytes incubated with sodium cholesterol sulfate (200 mM, Sigma-Aldrich) and sulfatase inhibitor STX64 (1 mM, Sigma-Aldrich), or monocytes incubated with oleylamine (200 mM, Sigma- Aldrich) was measured using a Malvern Zetasizer Nano. For z potential measurements, 10 mI cell suspension (1x10⁶ cells/ml) was with mixed with 90 mI 300 mM sucrose (Sigma-Aldrich) and the sample was loaded at the bottom of a DTS1070 cuvette (Malvern Instruments) that was prefilled with 10 mM sodium chloride (Sigma- Aldrich). Measurements 30 runs per measurement, monomodal mode) were recorded at 37 °C as technical triplicates.

Example 10: Atomic force microscopy

Neutrophils (0.5 x 10⁶) were seeded on a flouro dish (WPI Inc.) with a glass bottom and left for adherence and NET-formation as described above. A single monocyte was captured on a cantilever (MLCT-010, Bruker) coated with 0.1 mg/ml concavalin A (Merck). Before capturing monocytes, the dish and cantilever were washed 3x (HBSS, Gibco) and the cantilever was routinely calibrated on a clean area in the probing dish. Monocyte viability was controlled by propidiumiodid (5 mI/sample, eBioscience) staining. AFM force spectroscopy was only performed with living cells (at least 8 cells per experimental setup). The single monocyte on the cantilever tip was brought above a single NET-structure and probed with an approach and retraction speed of 10 pm/s, the pulling range was set to 25 pm, and the maximal contact force applied to the monocyte was 200 pN. The monocytes were probed on an area of 10 pm² resulting in ten acquired force curves. Data were analyzed by using JPK Data Pocessing software (Version spm-5.0.96).

Example 11 : Sample preparation for MS

Isolated human neutrophils were stimulated to produce NETs. Alu I was added to each well to cut the NETs DNA into smaller soluble pieces. Samples were centrifuged to eliminate cell debris and keep the supernatant containing the DNA and associated NET proteins in solution. A commercially-available mass spectrometry sample preparation kit was utilized to ensure minimal sample loss and reproducibility. The supernatant containing the DNA and associated NET proteins in solution was prepared with the iST Kit for proteomic sample preparation (PreOmics GmbH). In brief, this involved denaturation, alkylation, digestion and peptide purification. Reduction of disulfide bridges, cysteine alkylation and protein denaturation was performed at 95°C for 10 min. After a 5 min cooling step at room temperature, proteins were propionylated, trypsin and LysC were added to the mixture at a ratio of 1 :100 micrograms of enzyme to micrograms of protein. Digestion was performed at 37°C for 1 h. The cartridges were washed twice and eluted with 200 ul of 80%ACN + 0.25% TFA solution. The elution was speed vacuumed until dryness and stored until MS analysis.

Example 12: LC-MS/MS analysis

Peptides were re-suspended in 17 pi of 0.1 % TFA. A total of 5.0 mI were injected into a nano-HPLC device (Thermo Fisher Scientific) using a gradient from 4% B to 90% B (solvent A 0.1% FA in water, solvent B 80% ACN, 0.1% FA in water) over 90 min at a flow rate of 300 nl/min. Data was acquired in DDA positive mode using a Q Exactive HF spectrometer (Thermo Fisher Scientific). MS1 spectra were acquired at 60K resolution and AGC target value of 3e6 within a m/z range of 250 to 1600. The top 10 precursor ions were isolated with a window of 2.0 m/z and fragmented with NCE of 27 eV. Dynamic exclusion windows of of 12.0 ppm and 20 s were used. MS2 spectra were obtained at 15K resolution and AGC target value of 1e5 within a m/z range of 200 to 2000.

Example 13: Statistics

All statistics analyses were performed by using GraphPad Prism 8 software. Outliers have been determined by Grubbs' test with a=0.05. To test normal distribution, the D’Agostino-Pearson omnibus test was used. If normality was passed, data were tested by two-tailed unpaired t-test or one-way ANOVA with Dunnet’s correction. The Mann-Whitney test or Kurskal-Wallis test with Dunn’s correction was performed when data were not normally distributed. In all used tests a 95% confidence interval was utilized with p<0.05 was assumed as significant difference. All data are represented as mean ±SEM. References

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Claims

1. Peptide comprising an amino acid sequence

H-CEPLSEVEDYLDSSLKYNAKDTINYC-OH (SEQ ID NO: 1) or an equivalent thereof that differs from SEQ ID NO: 1 by not more than 3 amino acids.

2. Peptide according to claim 1 consisting of the amino acid sequence H-CEPLSEVEDYLDSSLKYNAKDTINYC-OH (SEQ ID NO: 1) or an equivalent thereof that differs from SEQ ID NO: 1 by not more than 3 amino acids.

3. Peptide according to claim 1 that is circular.

4. Peptide according to any one of claims 1 - 3 for use in the treatment of a disease.

5. Peptide for use according to claim 4 wherein the disease is a NETs- related disease.

6. Peptide for use according to claim 5 wherein the NETs-related disease is selected from the group consisting of atherosclerosis, autoimmune disorders, stroke, transfusion-related)acute lung injury, lung injury, ischaemia/reperfusion injury (IRI), kidney injury, liver injury, trauma, sepsis and inflammation.