WO2019122127A1 - Procédés et moyens de traitement de l'inflammation - Google Patents

Procédés et moyens de traitement de l'inflammation Download PDF

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WO2019122127A1
WO2019122127A1 PCT/EP2018/086195 EP2018086195W WO2019122127A1 WO 2019122127 A1 WO2019122127 A1 WO 2019122127A1 EP 2018086195 W EP2018086195 W EP 2018086195W WO 2019122127 A1 WO2019122127 A1 WO 2019122127A1
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histone
seq
membrane
neutrophils
smooth muscle
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PCT/EP2018/086195
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English (en)
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Gerardus Anna Franciscus Nicolaes
Oliver SÖHNLEIN
Carlos SILVESTRE-ROIG
Kanin WICHAPONG
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Universiteit Maastricht
Academisch Ziekenhuis Maastricht
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    • C—CHEMISTRY; METALLURGY
    • C40—COMBINATORIAL TECHNOLOGY
    • C40B—COMBINATORIAL CHEMISTRY; LIBRARIES, e.g. CHEMICAL LIBRARIES
    • C40B40/00—Libraries per se, e.g. arrays, mixtures
    • C40B40/04—Libraries containing only organic compounds
    • C40B40/10—Libraries containing peptides or polypeptides, or derivatives thereof
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • A61K38/1703—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
    • A61K38/1709—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
    • C07K14/47—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
    • G01N33/6875—Nucleoproteins

Definitions

  • the invention is in the field of medical treatments and provides polypeptides for the treatment of inflammatory diseases.
  • the invention also provides a method for the quantification of histone H4, including in vivo imaging of atherosclerotic lesions using polypeptides.
  • Inflammation is part of the complex biological response of body tissues to harmful stimuli, such as pathogens, damaged cells, or irritants [30] and is a protective response involving immune cells, blood vessels, and molecular mediators.
  • the function of inflammation is to eliminate the initial cause of cell injury, clear out necrotic cells and tissues damaged from the original insult and the inflammatory process, and initiate tissue repair.
  • Inflammation is a generic response, and therefore it is considered as a mechanism of innate immunity, as compared to adaptive immunity, which is specific for each pathogen [42] Too little inflammation could lead to progressive tissue destruction by the harmful stimulus (e.g. bacteria) and compromise the survival of the organism. In contrast, chronic inflammation may lead to a host of diseases, such as hay fever, periodontitis, atherosclerosis, rheumatoid arthritis, and even cancer (e.g., gallbladder carcinoma). Inflammation is therefore normally closely regulated by the body.
  • Inflammation can be classified as either acute or chronic.
  • Acute inflammation is the initial response of the body to harmful stimuli and is achieved by the increased movement of plasma and leukocytes (especially granulocytes) from the blood into the injured tissues.
  • a series of biochemical events propagates and matures the inflammatory response, involving the local vascular system, the immune system, and various cells within the injured tissue.
  • Prolonged inflammation known as chronic inflammation, leads to a progressive shift in the type of cells present at the site of inflammation, such as mononuclear cells, and is characterized by simultaneous destruction and healing of the tissue from the inflammatory process.
  • Inflammation is not a synonym for infection. Infection describes the interaction between the action of microbial invasion and the reaction of the body's inflammatory response—the two components are considered together when discussing an infection, and the word is used to imply a microbial invasive cause for the observed inflammatory reaction. Inflammation on the other hand describes purely the body's immunovascular response, whatever the cause may be.
  • inflammation is not driven by microbial invasion - for example, atherosclerosis, type III hypersensitivity, trauma, ischaemia.
  • pathological situations where microbial invasion does not result in classic inflammatory response— for example, parasitosis, eosinophilia.
  • disorders associated with chronic inflammation include Acne vulgaris, Asthma, Autoimmune diseases, Autoinflammatory diseases, Celiac disease, Chronic prostatitis, Colitis Diverticulitis, Glomerulonephritis, Hidradenitis suppurativa, Hypersensitivities, Inflammatory bowel diseases, Interstitial cystitis, Mast Cell Activation Syndrome, Mastocytosis Otitis, Pelvic inflammatory disease, Reperfusion injury, Rheumatic fever, Rheumatoid arthritis, Rhinitis Sarcoidosis, Transplant rejection and Vasculitis.
  • treatment options for inflammatory diseases like arthritis, including medications, rest, exercise, and surgery to correct joint damage.
  • the type of treatment prescribed will depend on several factors, including the type of disease, the person's age, type of medications he or she is taking, overall health, medical history, and severity of symptoms.
  • neutrophils Being rich in readily-available antimicrobial armory, neutrophils hold an irreplaceable position during host defense. However, it is the same neutrophil-borne mediators that can orchestrate sterile inflammation. In fact, prolonged neutrophil infiltration entertains central processes of inflammation hence boycotting its resolution and ultimately promoting the chronification of the inflammatory response [1] After recruitment to the inflamed tissue, neutrophil activation triggers the release of inflammatory mediators such as cytokines, granule proteins, or DNA structures decorated with antimicrobial and nuclear proteins coined neutrophil extracellular traps (NETs). Excessive accumulation of these neutrophil-borne mediators promotes the destruction of the host tissue and enhances inflammatory leukocyte recruitment and activation, resulting in non-resolving pathogenic inflammation. Such pathogenic role of neutrophils has been exemplified in multiple chronic inflammatory pathologies including neurodegenerative diseases, obesity, arthritis, and chronic obstructive pulmonary disease [2-5].
  • Atherosclerotic plaque formation is a classic example of a chronic inflammation [6]. Rupture of advanced atherosclerotic plaques remains the primary cause of mortality worldwide. Continuous exposure to inflammatory mediators released by infiltrated leukocytes fosters intimal cell death and extracellular matrix degradation thus provoking plaque destabilization [7] Archetypical signs of plaque instability that predict plaque rupture include expanded necrotic cores and thin fibrous caps as a consequence of smooth muscle cell (SMC) decease [8].
  • SMC smooth muscle cell
  • Circulating neutrophils [9] or neutrophil-derived proteins [10-12] predict cardiovascular disease and accumulate in high-risk atherosclerotic plaques [13].
  • the invention provides a polypeptide comprising an amino acid sequence according to SEQ ID NO: 1 and a pharmaceutical composition comprising a polypeptide according to SEQ ID NO:1 and a pharmaceutically acceptable carrier.
  • the invention also provides a composition for use as a medicament wherein the composition comprises a polypeptide according to SEQ ID NO: 1 and a pharmaceutically acceptable carrier.
  • the invention also provides a composition for use in the treatment of inflammation wherein the composition comprises a polypeptide according to SEQ ID NO: 1 and a pharmaceutically acceptable carrier.
  • the invention also provides the use of a polypeptide according to SEQ ID NO: 1 for the detection or quantification of histone H4, such as the use in in vivo imaging of atherosclerotic lesions.
  • Inflammation and especially its chronification, stands out as the most relevant pathophysiological contributor to medical burden. While typically regarded important during acute inflammation, neutrophils, the most abundant circulating white blood cells, have recently been implied in several forms of chronic inflammation. Cell death is a main pathophysiological process fostering chronic inflammation. Here we show that neutrophils orchestrate smooth muscle cell death in atherosclerosis, the major pathology of mortality worldwide. Neutrophils exert direct cytotoxic effects by presenting nuclear histone H4 within extracellular DNA traps to target cells thereby permeabilizing plasma membranes and licensing cell death. Therapeutic neutralization of histone H4 rescues smooth muscle cells and stabilizes atherosclerotic lesions. Thus our data not just shed light on how neutrophils contribute to atherosclerotic plaque destabilization, but also introduce a novel form of cell death into the context of atherosclerosis in particular and into inflammation chronification in general.
  • Phenotypic transition of arterial SMCs towards a pro-inflammatory phenotype is a hallmark of atheroprogression [15].
  • Platelet-derived growth factor (PDGF)- BB signaling in SMCs triggers secretion of chemotactic signals hereby mediating leukocyte infiltration and accelerated atherosclerosis [16].
  • PDGF-BB-activated SMCs Based on the observation that neutrophils predominantly locate in proximity to lesional SMCs, we investigated whether activated SMCs guide neutrophils towards them. Indeed, supernatants obtained from PDGF-BB-activated SMCs exhibited chemotactic activity in neutrophils (figure 2a, figure 10a-c). Moreover, PDGF-BB activation enhanced neutrophil-SMC interaction followed by neutrophil polarization (figure 2b). Since chemokine signaling is a prerequisite for neutrophil activation and NET release [17], we investigated whether secretory products of activated SMCs prime neutrophils to undergo NETosis.
  • Table 4 Genetic inhibition of NET release does not affect blood cell counts, plasma lipid levels, and body weight.
  • Histone H4 neutralization does not affect blood cell counts, plasma lipid levels, and body weight
  • NET-derived histone H4 induces intimal SMC death thereby contributing to plaque instability.
  • NET-derived histones induce endothelial cell cytotoxicity through Toll like receptor (TLR) activation [19,20].
  • TLR Toll like receptor
  • inhibition of TLR signaling did not affect NET-mediated SMC death ( Figure 16).
  • exposure of SMC to histone H4 generated ultra-rapid release of cytoplasmic calcein and uptake of propidium iodide (Figure 3f and 3g). This process was corroborated in whole atherosclerotic lesions incubated with histone H4 ( Figure 3h).
  • the acute time frame of the cytotoxic effect exerted by histone H4 suggested a process independent of programmed cell death pathways.
  • neutrophils and SMCs revealed a direct interaction of histone H4 with the SMC plasma membrane (Figure 3i).
  • subcellular fractionation of SMCs incubated with histone H4 exhibited histone H4 retention specifically in the plasma membrane ( Figure 3j).
  • NETs or histone H4 induced SMC swelling and release of ATP ( Figure 17).
  • the cationic property of histone H4 (zeta potential 26.64 ⁇ 2.82 mV) ensures its interaction with anionic DNA in the nucleus. Given the anionic nature of the plasma membrane we tested whether histone H4-membrane interaction was mediated by electrostatic forces.
  • histone H4 exhibits pore-forming activity and induces cell death.
  • the disturbance of SMC plasma membrane integrity by histone H4 prompted us to investigate its ability to generate membrane pores.
  • Atomic force microscopy on reconstituted membrane bilayers incubated with histone H4 revealed the appearance of pores at low concentrations and complete membrane disruption at higher doses (Figure 4a, Figure 19). Similar pore-like structures were observed within the membranes of SMCs upon exposed to histone H4 ( Figure 4b).
  • Hypercholesterolemic mice were treated as outlined in Figure 19 and blood counts, body weight as well as plasma lipid levels were assessed upon sacrifice (table 6). 77226PC9 10
  • the HIPe peptide was further optimized for inhibition of the histone H4- SMC interaction.
  • Each of the amino acids of SEQ ID NO: 2 was changed to all possible other natural amino acids.
  • the binding free energy of each individual HIPe peptide or analogue thereof, in direct relation to the affinity of the respective peptide for binding to histone H4, is shown in table 7 and preferred
  • the resulting peptide is provided herein as SEQ ID NO: 1.
  • AAGbinding > -3.28 kcal/mol indicates that that position can be mutated and that the resulting mutated peptide has a comparable (AAGbinding between -3.28 kcal/mol and 0 kcal/mol) or higher affinity (AAGbinding > 0 kcal/mol). This may also be interpreted as that the resulting mutated peptide maintains or increases the functional inhibition of histone H4.
  • Table 8 analogues of HIPe peptide (SEQ ID NO: 1 ).
  • Table 9 Preferred analogues of HIPe peptide (SEQ ID NO: 9).
  • the preferred analogues listed in table 9 are provided as SEQ ID NO: 9.
  • the annotation used in table 10 is:“Original amino acid - position number - Mutated amino acid”. For example S13D-N19D denotes that the serine (S) at position 13 was replaced by an aspartic acid residue (D) and in the same peptide an asparagine residue (N) at position 19 was replaced by an aspartic acid (D).
  • the invention relates to a polypeptide as shown in table 8 wherein, in the region corresponding to SEQ ID NO: 2, at most 7 amino acids are different from the peptide according to SEQ ID NO: 2.
  • the term“corresponding to” is used herein to indicate a region in a polypeptide comprising an amino acid sequence according to SEQ ID NO: 1 that when aligned to the amino acid sequence according to SEQ ID NO: 2 would have the highest identity with SEQ ID NO: 2.
  • the invention therewith relates to equivalents of the polypeptides as described herein.
  • Such equivalents may comprise a polypeptide comprising an amino acid sequence that is at least 90% identical to the sequence selected from the group consisting of SEQ ID NO: 1 , SEQ ID NO: 2, SEQ ID NO: 9, and SEQ ID NO: 10 to SEQ ID NO: 70.
  • the term“at least 90%” as used herein includes at least 91 , 92, 93, 94, 95, 96, 97, 98 or even 99% or more. 77226PC9 17
  • Neutrophil contribution to chronic vascular inflammation may so far have been underestimated due to their low abundance at the inflammatory site. Endowed with a short half-life, neutrophils may exhibit a high turnover within the atherosclerotic lesion that involves continuous tissue infiltration followed by a rapid release of NETs and subsequent clearance. Along with these processes, the continued presence of NET-derived proteins in the tissue despite degradation of the DNA scaffold [26] and impaired macrophage phagocytosis upon inflammation [27] has been reported.
  • histones released upon cell damage are noxious and induce tissue damage [29]
  • histones and in particular histone H4 induce a rapid receptor- independent cell death in SMCs based on membrane pore formation, an activity previously ascribed to antimicrobial peptides.
  • histone H4 allows the design of cell-penetrating peptides to therapeutically target microbes or tumor cells.
  • Our findings support that externalized histone H4 originating from intimal NETs induces tissue damage and plaque destabilization.
  • cell necrosis a relevant process occurring at advanced stages of atherosclerosis may also significantly contribute to histone H4 accumulation within the vascular tissue.
  • the invention concerns a polypeptide comprising an amino acid sequence according to SEQ ID NO: 1 or SEQ ID NO: 2.
  • a polypeptide may be in the form of a
  • composition comprising a polypeptide according to claim 1 or 2 and a pharmaceutically acceptable carrier.
  • a polypeptide according to claim 1 or 2 may be used as a medicament, for instance a medicament for the treatment of
  • the disease to be treated is associated with chronic inflammation, such as a disease selected from the group consisting of COPD, asthma, chronic peptide ulcer, tuberculosis, rheumatoid arthritis, arthritis, periodontitis, ulcerative colitis, Crohn’s disease, sinusitis, active hepatitis, lupus erythematosis, scleroderma, atherosclerosis, sarcoidosis, multiple sclerosis, conditions that are associated with increased levels of extracellular cytotoxic histones, sepsis, trauma, ischaemia/reperfusion injury, transfusion related acute lung injury, cancer,
  • chronic inflammation such as a disease selected from the group consisting of COPD, asthma, chronic peptide ulcer, tuberculosis, rheumatoid arthritis, arthritis, periodontitis, ulcerative colitis, Crohn’s disease, sinusitis, active hepatitis, lupus erythematosis, scleroderma, atherosclerosis
  • the peptides and polypeptides as described herein may also be used for detection of histone H4, such as detection in a tissue, in vivo or in vitro, such as the use for in vivo imaging, for instance of atherosclerotic plaques.
  • Figure 1 Neutrophils dictate plague stability.
  • mice were rendered neutropenic (anti-Ly6G, 50 pg i.p., every other day) or neutrophilic (AMD3100, 5mg/kg, s.c.). Neutropenic (white bars) and neutrophilic
  • (I) Percentage of smooth muscle cell area of human atherosclerotic specimens grouped by the number of lesional neutrophils. One way ANOVA with Tukey’s correction. ** p ⁇ 0.01. n 10-25.
  • FC fibrous cap
  • L lumen
  • SMA smooth muscle actin
  • NET-derived histone H4 induces smooth muscle cell lysis and exacerbates
  • Histone H4 blockade improves atherosclerotic plaque stability. Apoe-/- mice with advanced lesions in the carotid artery were treated with isotype control IgG or anti-histone H4 (20 pg/day, i.p.) during the last 4 weeks of high fat diet feeding.
  • Hypercholesterolemic mice with established atherosclerotic lesions were implanted with osmotic minipums delivering vehicle or HIPe (4 mg/kg/day) during the last four weeks.
  • Scale bar 50 pm.
  • Quantification of smooth muscle cell area f
  • Scheme summarizing the cytotoxic activity of neutrophils during atherosclerotic plaque destabilization. Neutrophils guided by
  • Fiqure 7 Modulation of lesional neutrophil counts alters lesion stability.
  • Figure 10 Activated smooth muscle cells stimulate neutrophil migration.
  • Figure 1 Pharmacological inhibition of NET release stabilizes atherosclerotic lesions.
  • MPO myeloperoxidase
  • NE neutrophil elastase
  • SLP secretory leukocyte protease
  • Histone H4 displayed on NETs accumulates in proximity of smooth muscle cells.
  • TLR3 or TLR4 inhibitor (1 gg/ml) for 30 minutes prior NET treatment.
  • FIG. 20 Disruption of Histone H4-cell membrane interaction stabilizes atherosclerotic lesions.
  • guantification lesion size (b), fibrous cap (FC) thickness (c), necrotic core area (d), collagen area (e), and macrophage area (f).
  • n 12 (vehicle) or 1 1 (HIPe). All data is presented as mean ⁇ SEM.
  • mice experiments were performed according to European guidelines for Care and Use of Laboratory Animals. Protocols were approved by the Committee on the Ethics of Animal Experiments of the University of Amsterdam and the Animal
  • mice Animals were housed according to institutional regulations with ad libitum access to food and water. All mice were C57BI6j background. Apolipoprotein E deficient mice (Apoe-/-) were purchased from Jackson Laboratory. Peptidyl Arginine Deiminase 4 (Pad4)-deficient mice were intercrossed with Apoe-/- mice to generate Apoe- /-Pad4-/-.mice. Vulnerable atherosclerotic lesions were induced as described in 1. In brief, eight week-old female mice were fed a high fat diet (HFD; 21 % fat and 0.15 %
  • HFD high fat diet
  • mice received anti- Ly6G or control IgG (50 pg, every other day, BioXcell) intraperitoneally.
  • Neutrophilia was induced by daily subcutaneous injection of AMD3100 (5 mg/kg, Tocris) or PBS as control.
  • Inhibition of NET release was performed by daily subcutaneous administration of Cl- amidine (10 mg/kg, Essen Scientific).
  • mice were treated with intraperitoneal injection of anti-histone H4 (20 pg/day, Biorbyt, orb225483) or control IgG (Dianova).
  • peptide HIPe was administered by using osmotic minipumps (4 mg/kg/day, Alzet, model 2004). All treatments were performed during the last 4 weeks of the experiment to therapeutically treat pre-established atherosclerotic plaques. For the model of spontaneous atheroprogresion, Apoe-/- were fed a HFD for 16 weeks.
  • CCA Carotid endarterectomy
  • mice were euthanized by ketamine/xylacine overdose, the blood was collected by heart puncture after which the mice were flushed with 20 ml of ice cold PBS- EDTA (5 mM EDTA). Subsequently, the left common carotid artery was embedded in Tissue Tek O.C.T. compound (Sakura Finetek) for analysis. For the spontaneous atheroprogression model, aortic arches were isolated, fixed with 4% PFA and embedded in paraffin.
  • Blood was incubated with red blood cell lysis buffer (150 mM NH4CI, 10 mM KHC03, 0.1 mM Na2EDTA) for 5 min at room temperature.
  • Leukocytes were stained with antibodies to CD45 (Biolegend, clone: 30-F1 1 ), CD1 1 b (Biolegend, M1/70), Ly6G (Biolegend, clone: 1 A8), Ly6C (Biolegend, clone: Hk1.4), CD1 15 (eBioscience, clone: AFS98) in staining buffer (20 min, 4 °C).
  • Flow cytometry was performed using the LSR Fortessa (Beckton Dickinson) and data was analysed using FlowJo software (Beckton Dickinson). Haematologic counts were determined with the ScilVet abc Plus analyzer (soil animal care company GmbH).
  • Cryosections (7 pm) or paraffin sections (4 pm) were histologically stained with hematoxylin and eosin (HE) in 70 or 40 pm intervals, respectively. Total collagen content was assessed by pricrosirius red staining in consecutive sections.
  • cryosections were fixed with cold acetone followed by antigen blockade using 5% goat serum/phosphate buffered saline.
  • Paraffin sections underwent antigen retrieval with citrate buffer (10 mM, pH 6.0) before antigen blockade using 5% goat serum/phosphate buffered saline.
  • plaque size and necrotic core area was quantified in 4 pm paraffin sections stained with HE. Consecutive sections were used for histological analysis of neutrophils, neutrophil extracellular traps (NETs) and smooth muscle cells (SMCs). First, antigen retrieval was performed with Lab Vision citrate buffer
  • Plaque vulnerability was assessed as described in reference 31 .
  • intima media and necrotic core area was analyzed in HE-stained sections.
  • Necrotic core was defined as the area devoid of nuclei underneath a formed fibrous cap.
  • Collagen content and fibrous cap thickness was measured on pricosirius red-stained sections.
  • FC thickness was defined as the average of lengths measurements in the positions overlapping with the lines of a square-shaped grid.
  • HE stained sections were scored blinded by two independent, experienced pathologists with little inter- and intra-observer variability. Plaques were classified as early, advanced and complicated lesions regarding Virmani histopathological classification [32] Next, advanced and complicated lesions were divided into regions of FC, shoulders and core using ImageJ software.
  • Plasma cholesterol and triglycerides were determined by CHOD-PAP kit (Roche/Hitachi) and GPO-PAP kit (Roche/Hitachi) respectively, according to
  • MOVAS Mouse vascular aorta/smooth muscle cells
  • DMEM complete medium
  • Gibco complete medium
  • G418 fetal bovine serum
  • 5 mM Sodium pyruvate 5 mM Sodium pyruvate
  • All cells were maintained in an incubator at 37 °C, 5 % C02.
  • To activate MOVAS cells were cultured in complete medium supplemented with 10 ng/ml recombinant murine PDGF-BB (Peprotech) for 6 h. Subsequently cells were washed to remove the stimulus and maintained with fresh complete medium for 24 h. After this time, supernatants were recollected, centrifuged (300 g, 5 min, 4 ' ⁇ ), and frozen until used.
  • Human blood neutrophils were isolated from human blood using Polymorphprep (Axi-Shield) following manufacturer’s instructions.
  • Mouse bone marrow- derived neutrophils were isolated from tibias and femurs from C57BL/6J mice by negative selection using the Neutrophil Isolation Kit (Milteny) according to manufacturer’s instructions.
  • SMCs were incubated with indicated amounts of isolated NETs, histone H2A (Sigma), histone H3, or histone H4 (all Biomol), or equimolecular amounts of histone H4 fragments (Pepscan).
  • Cell viability was measured based on propidium iodide (PI) uptake. PI+ cells were visualized using a climate chamber fluorescence microscope (Leica, DMi8) and quantified by ImageJ software.
  • PI propidium iodide
  • SMC viability was measured using Vybrant MTT cell proliferation assay (ThermoFisher) according to manufacturer’s instructions and measured with by plate reader (Tecan, lnfiniteF200Pro).
  • Live imaging of cell death was performed on SMCs stained with Calcein AM (Thermofisher, 1 :1000). After adding 50 ug/ml of histone H4 or phosphate buffer (control) to the medium, images were acquired every 30 seconds for 30 min using a climate chamber fluorescence microscope to measure PI and calcein signal. PI influx and calcein efflux was measured using ImageJ software.
  • aortas of HFD fed Apoe-/- mice were opened longitudinally and mounted on face on a silicone gel surface.
  • Aortas were incubated with anti-CD31 conjugated to eFIuor 450 (Thermofisher, 1 :50) in combination with Calcein AM (1 :1000) for 1 h.
  • Samples were imaged in z stacks, in intervals of 3 min for a period of 15 min using a LeicaSP5IIMP two-photon laser scanning microscope Leica SP5MP with a pre-chirped and pulsed TLSapphire Laser (Spetra Physics MaiTai Deepsee) tuned at 750 nm and a 20xNA1.00 (Leica) water dipping objective. Image acquisition and processing was performed using LasX software (Leica).
  • NETs Prior to addition to SMCs, NETs (500 ng/ml) were incubated for 1 h with 50 ng/ml of the following antibodies: Ctrl IgG (Santa Cruz), anti-Myeloperoxidase (Merck), anti-LL37 (Santa Cruz), anti-Neutrophil Elastase (Biorbyt), anti-Cathepsin G (Biorbyt), anti-Proteinase 3 (Santa Cruz), anti-Histone H2A (Cell Signalling), anti-Histone H3 (Abeam), or anti-Histone H4 (Cell Signalling).
  • Ctrl IgG Santa Cruz
  • Anti-Myeloperoxidase Merck
  • anti-LL37 Santa Cruz
  • Anti-Neutrophil Elastase Biorbyt
  • Anti-Cathepsin G Biorbyt
  • anti-Proteinase 3 Santa Cruz
  • anti-Histone H2A Cell Signalling
  • SMCs were incubated during 5 min at 4 °C to avoid internalization with 50 pg/ml histone H4 or equimolar amounts of biotinylated histone H4 fragments.
  • histone H4 was pre-treated with 100 pg/ml HIPe during 1 h before incubating with SMCs.
  • cells were extensively washed and fixed with 2 % PFA.
  • Co cultures of SMCs and neutrophils were stimulated by 100 nM PMA for 2 h. After washing, cells were incubated for another 2 h and then fixed by 2 % PFA. Interaction between plasma membranes (Phalloidin) and histone H4 was visualized using confocal STED microscopy.
  • SMCs were incubated with 50 pg/ml of recombinant histone H4 for 1 h or 500 ng/ml of isolated NETs for 48 h. Supernatants were centrifuged (5 min, 300 g) and extracellular ATP was measured using Cell Titer Glo (Promega) and Tecan plate reader according to manufacturer’s instructions.
  • SMCs were treated for 30 min with 200 mM Oleylamine or 200 mM sodium cholesterol sulfate and 1 mM STX64 (both purchased from Sigma).
  • 200 mM Oleylamine or 200 mM sodium cholesterol sulfate and 1 mM STX64 both purchased from Sigma.
  • For cell viability or membrane interaction assays cells were washed and incubated with 50 pg/ml histone H4 for 1 h at 37 ⁇ or 5 min at 4°C, respectively. Cell viability or membrane interaction were measured as describe above.
  • SMCs were washed and resuspended in 10 mM sodium chloride + 38 mM sucrose solution.
  • Recombinant histone H4 and BSA were diluted to 0.1 mg/ml in a 16x diluted PBS solution containing 30 mM sucrose.
  • the zeta potential was measured at 37 °C using Zetasizer Nano (Malvern) in a disposable folded capillary cell (DTS1070, Malvern).
  • Neutrophils (1x10 5 ) were seeded and primed by incubation with conditioned media obtained from control- or PDGF-BB-treated SMCs during 30 min. After washing, neutrophils were incubated with HBSS containing 5 mM SYTOXTM Green Nucleic Acid Stain (Life technologies) and 100 nM PMA. Green fluorescence intensity was measured for 4 h in 30 min intervals using Tecan plate reader.
  • SMCs were incubated with either 50 pg/ml of histone H4 or phosphate buffer (control) in DMEM medium (5 min, 4°C). The cells were washed 3 times with ice cold PBS and collected in ice-cold fractionation buffer by scrapping. Cells were ruptured by passing them through a 25G needle. To pellet the nuclear fraction the ruptured cells were incubated 20 min on ice and centrifuged 5 min at 720 g and 4°C). To separate the mitochondrial fraction from the membrane/cytosolic fraction the supernatant was centrifuged 10 min at 10.000 g and 4°C.
  • the supernatant was ultra-centrifugated (2 times) for 1 h at 100.000 g and 4‘O to separate the cytosolic fraction from the plasma membrane fraction.
  • the nuclear fraction and membrane fraction were lysed with RIPA buffer and protein levels of each fraction were measured using Pierce assay kit
  • Lysates (25 pg protein) were separated by SDS-PAGE and transferred onto Immobilon-P membranes (Millipore Corporation). Membranes were blocked in 4% dry milk and incubated with primary antibodies anti-Histone H4 (Abeam), Glyceraldehyde- 3-Phosphate Dehydrogenase (Merck Milipore), and Anti-Sodium Potassium ATPase (Abeam). Secondary horseradish peroxidase-coupled anti-isotype-specific antibodies (ThermoScientific) were used for detection with enhanced chemiluminescence reagent (Clarity Western ECL substrate, Bio-Rad) and ChemiDoc MP System (BIO-RAD). Scanning electron microscopy.
  • SMCs were incubated with 50 pg/ml of recombinant histone H4 or phosphate buffer for 1 h. Next, cells were washed and the samples were fixed using 4% PFA plus 2.5% glutaraldehyde in PBS (2 h, 4 Q C). Cells were then dehydrated by serial 5 min incubations in increasing concentrations of ethanol
  • Samples were dried in an automated critical point dryer (Leica EM CDP 300) and then coated with chromium in a rotary-pumped coating system (Quorum Technologies Q150RS). Imaging was performed at 15kV with a field emission microscope (JEOL 7600F).
  • neutrophils (2x105) were added to the top compartment of HTS transwell 96 well plates (Corning) with 3 pm pore-size. In the lower compartment, supernatants obtained from non-activated or PDGF-BB-activated SMCs were added. After incubation during 1 h at 37°C, transmigrated neutrophils were analyzed from the bottom compartment by flow cytometry.
  • ROS Reactive oxygen species
  • Bone marrow mouse neutrophils were incubated with 0.1 mM H2-DCF-
  • PDGF-BB-activated SMCs during 30 min. ROS production was measured by flow cytometry.
  • Monolayers of SMCs were treated with vehicle or PDGF-BB (24 h, 10 ng/ml). After washing, bone marrow mouse neutrophils stained with Ly6G-APC (1A8) and CD62L-PE (MEL-14) (Biolegend) were added. After incubating for 15 min, cells were washed and fixed with 4 % PFA. The number of adhered and polarized (CD62L clustering) neutrophils was analyzed by fluorescent microscopy.
  • the histone H4-membrane complex was constructed by assistance of the membrane builder module implemented in the CHARMM-GUI website [34]
  • Histone H4 was placed around 20 Angstrom (A) above membrane layer which composes of 251 DOPC lipids (upper layer) and 50 DOPS/200D DOPC lipids (lower layer).
  • the complex was then solvated by adding water and NaCI to 0.15 M.
  • the water thickness was set to 35 A from the membrane layer which covers the whole H4-membrane complex structure.
  • CHARMM36 force field was assigned for the prepared system (solvated H4-membrane complex). The complex was subjected for MD simulations by AMBER16. Similar energy minimizations and MD simulations protocols as previously described [35] were applied. To relax the system, 2 consecutive steps of energy minimization were employed; first 2500 steps of steepest descent followed by 2500 steps of conjugate gradient algorithm of energy minimization. During this step the histone H4 and membrane were partial fixed by applying soft position constraint (10 and 2.5 kcal/mol for H4 and membrane, respectively).
  • a position-restrained MD phase was carried out for 400 ps by slowly reducing force constraint applied to constraint protein (from 10 to 5, 2.5, 0.5, and 0.1 kcal/mol, respectively) and membrane (from 2.5 to 1 , 0.5, and 0.1 , respectively).
  • the temperature of the system was continuously increased from 0 to 303.15 K and remained at this temperature by application of Langevin dynamics with a collision frequency of 1 ps-1 .
  • free MD simulations were employed for 50 ns and then accelerated molecular dynamics (aMD) were carried out from 50 - 200 ns. In these steps (free MD and aMD), the same protocols were applied; temperature and pressure were set and then kept constant at 303.15 K and 1 bar, respectively.
  • a time step was set to 2 fs with application of a SHAKE algorithm. Electrostatic interactions were calculated by use of the particle-mesh Ewald method, and nonbonded interactions were computed by setting the cut-off at 12 A with the force-based switching at 10 A.
  • H4-peptide complexes were subjected to structure optimization (energy minimization and short MD simulations) and binding free energy (BFE) calculation to predict the binding strength of the designed peptides with histone H4 by using similar protocols and parameters as previous works [35, 37]
  • Molecular mechanics/generalized Born surface area (MM/GBSA) approach using generalized Born model 8 with default parameters was applied to compute binding free energies and only enthalpy values was used to estimate the relative BFE [35].
  • HIPe-H4 complex gave the lowest binding free energy, thus HIPe was chosen for synthesis.
  • the complex between H4-HIPe and membrane was subjected to MD simulation.
  • Peptides used in this study were purchased and synthesized in high purity (>95% HPLC) using solid-phase synthesis (Pepscan). The following peptide sequences were synthesized:
  • H-NIQGITKPAIRRLARRGGVKRISGLIYEETRGVLKVFLENVIRD-OH SEQ ID NO: 5
  • H-NIQGITKPAIRRLARRGGVKRISGLIYEETRGVLKVFLENVIRKDK(Biotin)-NH2 (SEQ ID NO: 6)
  • H-AVTYTEHAKRKTVTAMDVVYALKRQGRTLYGFGG-OH (SEQ ID NO: 7)
  • Biotin-Ahx-AVTYTEHAKRKTVTAMDVVYALKRQGRTLYGFGG-OH SEQ ID NO: 8
  • SAXS Small-angle X-rav scattering
  • peptides were solubilized in nuclease-free water, mixed together with SUVs in fixed peptide-to-lipid charge ratios and equilibrated at room temperature overnight. Precipitated peptide-lipid complexes were loaded into 1.5 mm glass quartz capillaries and hermetically sealed with an oxygen torch. SAXS experiments were conducted at the Stanford Synchrotron Radiation Lightsource (BL 4-2) with monochromatic X-rays of energy 9 keV. Samples were incubated at 37°C and centrifuged before measurement. Scattered radiation was collected using a Rayonix-MX225-HE detector (pixel size 73.2 pm). The 2D powder diffraction patterns were azimuthally integrated into 1 D patterns using the Nika 1.76 package [39] for Igor Pro 7.04
  • EggPC (Avanti Polar Lipids) was purchased as solutions in chloroform (25 mg/ml). Cholesterol (Avanti Polar Lipids) was purchased as a lyophilized powder of 500 mg. A stock solution was prepared by adding 1 ml of chloroform to 25 mg of cholesterol. All stock solutions were stored at -20 q C.
  • the lipid bilayers were prepared by fusion of lipid vesicles.
  • SUVs (diameter ⁇ 100 nm) containing various compositions of lipids were obtained by extrusion.
  • Lipid solutions were prepared in glass tubes using 40 mI of each lipid: EggPC:Cholesterol (80%:20%) (v/v). Chloroform was evaporated under a gas stream and lipids were dried overnight using a vacuum pump.
  • Suspensions of multilamellar vesicles (MLVs) (1 mg/ml) were obtained by resuspending the lipids in 1 ml in 20 mM CaCI2, 20 mM NaCI, 20 mM HEPES. Buffers were filtered through 0.22 pm Millipore membranes.
  • the suspensions were vigorously stirred during 3 min. Four cycles of freeze-thaw were performed to enhance the buffer’s engulfment inside the vesicles. Afterwards, the suspensions were brought to room temperature, stirred, then extruded using LiposoFast-Basic and
  • the 2 filter-supports and the polycarbonate membranes were soaked in the resuspension buffer whereas the 2 syringes were carefully washed using the buffer. Each lipid suspension was extruded 5 times using the 200 nm filter and 19 times using the 100 nm filter.
  • the SUVs were characterized by dynamic light scattering (DynaPro® NanoStar®, Wyatt Technology).
  • the complex mixed lipid membrane containing 10% brain extract PS, 39% brain extract PC, 27% brain extract PE, 19% brain extract sphingomyelin, and 5% liver extract PI on a molar basis, all lipids were mixed at 70 °C temperature prior to extrusion. All the extrusion steps for this complex lipid required that all the material was pre-heated at 70 q C.
  • the SUVs suspensions were diluted in a final volume of 50 mI of deposition buffers (20 mM NaCI, 20 mM HEPES) (1/5, 1/10, 1/20) and deposited onto freshly cleaved mica disks (10 mm in diameter) (O/N, RT).
  • the excess of unbound vesicles was removed by exchanging the solution covering the mica with the deposition buffer (3 times) using a volume of 20 mI.
  • Histone H4 (1 mg/ml) was diluted in the deposition buffer and deposited onto the mica surface for 15 min except stated otherwise. Loosely-bound proteins were gently washed off 3 times by removing 15 mI of the solution and replacing it by the same volume of buffer.
  • AFM measurements were performed at room temperature in buffer using the peak force-tapping mode with a Multimode 8 and a Nanoscope V controller (Bruker).
  • the imaging parameter was: loading force ranged from 250 to 700 pN, scanning rate of 1 Hz, 512 pixels/line and 512 lines.

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Abstract

La présente invention concerne le domaine des traitements médicaux et fournit des polypeptides pour le traitement des maladies inflammatoires. L'invention fournit également un procédé de quantification d'histone H4 tel qu'un procédé d'imagerie in vivo ou in vitro pour les lésions athérosclérotiques utilisant des polypeptides. Plus particulièrement, l'invention fournit un polypeptide comprenant une séquence d'acides aminés selon SEQ ID no : 1.
PCT/EP2018/086195 2017-12-22 2018-12-20 Procédés et moyens de traitement de l'inflammation WO2019122127A1 (fr)

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EP1403639A1 (fr) * 2002-09-30 2004-03-31 G2M Cancer Drugs AG Anticorps en tant que moyen de diagnostic lors d'un traitement par l'administration d'inhibiteurs de l'histone deacetylase
US20070105152A1 (en) * 2005-10-24 2007-05-10 Denu John M Methods for determining protein binding specificity using peptide libraries
US20140234209A1 (en) * 2013-02-15 2014-08-21 Immunomedics, Inc. Chimeric and humanized anti-histone antibodies
WO2017054832A1 (fr) * 2015-10-02 2017-04-06 University Of Copenhagen Petites molécules bloquant les domaines lecteur d'histone

Patent Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP1403639A1 (fr) * 2002-09-30 2004-03-31 G2M Cancer Drugs AG Anticorps en tant que moyen de diagnostic lors d'un traitement par l'administration d'inhibiteurs de l'histone deacetylase
US20070105152A1 (en) * 2005-10-24 2007-05-10 Denu John M Methods for determining protein binding specificity using peptide libraries
US20140234209A1 (en) * 2013-02-15 2014-08-21 Immunomedics, Inc. Chimeric and humanized anti-histone antibodies
WO2017054832A1 (fr) * 2015-10-02 2017-04-06 University Of Copenhagen Petites molécules bloquant les domaines lecteur d'histone

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