WO2017077062A1 - A peptide derived from human neutrophile peptide 1 - Google Patents

Abstract

The present invention relates to a peptide derived from human neutrophile peptide 1, to a nucleic acid coding for said peptide, to a pharmaceutical composition comprising said peptide or said nucleic acid, and to uses of said peptide or said nucleic acid.

Description

A peptide derived from human neutrophile peptide 1

The present invention relates to a peptide derived from human neutrophile peptide 1, to a nucleic acid coding for said peptide, to a pharmaceutical composition comprising said peptide or said nucleic acid, and to uses of said peptide or said nucleic acid.

Inflammation is part of a complex biological response of body tissues to harmful stimuli and involves immune cells, blood vessels and molecular mediators. More specifically, it involves inter alia a sequence of phagocyte recruitment to the site of inflammation which comprises an initial extravasation of neutrophils followed by a subsequent emigration of monocytes. That this sequence is not a coincidence but that neutrophils indeed generate signals that recruit monocytes stems from animal models of induced neutropenia (1-4). Several mechanisms that contribute to the transition from neutrophil to monocyte recruitment have been identified, wherein the release of chemotactic granule proteins is of specific importance (5). Lysates of neutrophils exert chemotactic activity on monocytes, suggesting an important role for preformed stores of cellular mediators in launching monocyte extravasation (6). Further support for such causal connection stems from patients with functional neutrophil deficits. Neutrophil lysates of patients suffering from specific granule deficiency lacks their chemotactic effect on monocytes (7). Neutrophils from these patients are devoid of granule proteins such as human neutrophil peptides (HNPs, a-defensins) and human cationic antimicrobial protein 18 (pro- form of LL-37) (8). Subsequently neutrophil-borne, LL-37, azurocidin, cathepsin G, and human neutrophil peptides 1-3 (HNP1-3, a-defensins) were demonstrated to be chemotactic for human and murine monocytes (3,9,19).

Similarly to neutrophils, platelets have been implicated in monocyte recruitment (11). Recent proteomic analyses revealed the abundance of classical chemokines in platelet a and dense granules (12). Deposition of CXCL4 on the endothelium by activated platelets results in subsequent monocyte adhesion (13). Other abundant chemokines in platelet granules are CXCR2 ligands such as CXCL7, MIF, CXCL3, CXCL2, and CXCL5 mediating monocyte activation and adhesion (14,15). Finally, platelets store CCL5, a potent chemokine for monocytes and lymphocytes alike (16). The existence of distinct monocyte subsets has been established in human and mouse blood (17,18). Whereas classical monocytes (CD14⁺CD16^~ in humans, Ly6C^hiCCR2⁺CD62L⁺CX₃CRl^mid in mice) are rapidly recruited to sites of injury and differentiate into inflammatory macrophages following extravasation (19), non-classical monocytes (CD14^l0WCD16⁺ in humans, Ly6C^lowCCR2^~CD62ITCX₃CRl^hi in mice) patrol the vascular lumen (20). Monocyte recruitment and activation has been shown to be detrimental in acute and chronic inflammation, including myocardial infarction, atherosclerosis, sepsis, and acute lung injury (21,22). In these pathologies recruitment of monocytes is accompanied by systemic activation of neutrophils and platelets, thus allowing for cooperation of both cell types in monocyte recruitment.

Treatment of inflammatory diseases may involve different routes. One example is the treatment with corticosteroids, which, however, is hampered by side effects which may be particularly severe if such treatment occurs over a long time.

Atherosclerosis, as an example of a chronic inflammatory disease, is an inflammatory disease of blood vessels, in particular arteries which progresses with age. Atherosclerosis is characterized by the accumulation of immune cells, lipids and calcified components in the arterial wall, the so-called atherosclerotic plaque. The disease remains clinically silent for many decades, but when the atherosclerotic plaque ruptures a thrombus will form on the plaque surface and will encroach the lumen, resulting in (myocardial) infarction or stroke, depending on the vascular bed affected.

HMG-coA-reductase inhibitors (Statins) reduce the burden of cardiovascular disease only by 25%, underscoring an urgent need for pharmaceutical targets suitable for new drug development.

Accordingly, there is a need in the art to provide for new ways of treating inflammatory diseases, such as atherosclerosis, myocardial infarction, acute lung inquiry or sepsis.

These objects are solved by a peptide derived from human neutrophil peptide 1 (HNP1) and consisting of an amino acid sequence selected from formula a) a) (X¹)_nl-RRYGT-X²-X³-YQ-(X⁴)_n2-(X⁵)_n3-(X⁶)_n4 wherein X¹ is C and nl is 0 or 1,

X² is C, A or S,

X³ is I, K or R,

X⁴ is the sequence GRLWAF and n2 is 0 or 1,

X⁵ is C, A or S and n3 is 0 or 1,

X⁶ is C or S and n4 is 0 or 1 , with the proviso that, when n2 is 0, then n3 and n4 are also 0, and when n2 is 1, then n3 and n4 are also 1 , respectively, wherein X and X are independently selected from C and S, wherein optionally, the peptide is covalently linked to a polyalkylene oxide, preferably a polyethylene glycol (PEG) or a polypropylene glycol.

In one embodiment, the peptide consists of an amino acid sequence of formula a), wherein n2

- n4 = 0.

In one embodiment, the peptide consists of an amino acid sequence of formula a), wherein nl is 0.

2

In one embodiment, the peptide consists of an amino acid sequence of formula a), wherein X is S, and X^J is K.

In one embodiment, the peptide consists of an amino acid sequence selected from

CRRYGTCIYQGRLWAFCC SEQ ID NO:l

RRYGTSKYQ SEQ ID NO:2

RRYGTCIYQGRLWAFCC SEQ ID NO:3

CRRYGTAKYQGRLWAFAC SEQ ID NO:4

CRRYGTARYQGRLWAFAC SEQ ID NO:5

RRYGTSIYQ SEQ ID NO:6

CRRYGTS I YQGRLWAF S C SEQ ID NO:7 CRRYGTSKYQGRLWAFSC SEQ ID NO: 8

CRRYGTSRYQGRLWAFSC SEQ ID NO:9

RRYGTSRYQ SEQ ID NO: 10

In one embodiment, the peptide consists of an amino acid sequence selected from

RRYGTSKYQ SEQ ID NO :2

RRYGTSIYQ SEQ ID NO:6

RRYGTSRYQ SEQ ID NO: 10

The objects of the invention are also solved by a nucleic acid coding for a peptide as defined according to the present invention.

In a further aspect, the present invention relates to a peptide according to the present invention or a nucleic acid according to the present invention, for use as a medicament.

The objects of the invention are also solved by a peptide according to the present invention or a nucleic acid according to the present invention for use in the treatment of inflammatory diseases.

In one embodiment, the inflammatory disease is selected from rheumatoid arthritis, ankylosing spondylitis, inflammation post infection, atherosclerosis, myocardial infarction, stroke, acute lung injury, including transfusion related acute lung injury (TRALI), sepsis, pain, dermatitis, psoriasis, atopic skin disease, chronic granulomatous diseases such as tuberculosis, leprosy, sarcoidosis, silicosis, nephritis, amyloidosis, scleroderma, lupus, polymyositis, osteoporosis, inflammatory bowel disease(s), including Crohn's disease, colitis ulcerosa, ulcers, appendicitis, diverticulitis, Sjogren's syndrome, Reiter's syndrome, pelvic inflammatory disease, orbital inflammatory disease, peritonitis, hepatitis , thrombotic disease, inappropriate allergic responses to environmental stimuli such as poison ivy, pollen, insect stings and certain foods, including atopic dermatitis and contact dermatitis, allergic encephalitis, asthma, lung inflammation, COPD, chronic bronchitis. In a further aspect, the present invention relates to a method of treatment of an inflammatory disease, comprising the step of administering the peptide or the nucleic acid according to the present invention to a patient suffering from an inflammatory disease.

In a further aspect, the present invention relates to a pharmaceutical composition comprising the peptide or the nucleic acid according to the present invention, and a pharmaceutically acceptable carrier.

The objects of the present invention are also solved by the use of a peptide according to the present invention or of a nucleic acid according to the present invention for the manufacture of a medicament for the treatment of an inflammatory disease. In one embodiment, the inflammatory disease is as defined above.

The present inventors have surprisingly found that in inflammation, protein heteromers are formed between neutrophil derived human neutrophil peptide 1 (HNP1) and platelet-born cytokine CCL5. They have found that if such protein heteromer formation is prevented, this effectively prevents monocyte recruitment to the site of inflammation and thus allows to alleviate the potentially deleterious effect of such monocyte recruitment to the site of inflammation. To this end, they designed a series of peptides which disturb the interaction between CCL5 and HNP1 and abolish the adverse effects of monocyte recruitment. Hence, effectively, the inventors offer a new set of molecules making use of a novel paradigm of functional het- eromerization of chemotactic molecules released from different cell subsets which offers important therapeutic advantages over direct antagonism or neutralization of chemokines or their receptors. For example CCL5-deficient mice show severely impaired polyclonal and antigen- specific T-cell proliferation. Moreover, mice treated with inhibitors which target at CCR5 or which are deficient in CCL5 show delayed viral clearance by macrophages which is properly due to a lack of anti-apoptotic signals conferred by CCL5-CCR5-interactions to limit cell-to- cell virus dissemination in the host. Therefore, despite the importance of the CCL5-CCR5 axis in atherogenic monocyte recruitment, a direct neutralization of CCL5 may limit the healing after myocardial infarction. In a similar manner, a direct interference with HNP1 may impair host defense as HNP1 does not just stand out as a multifunctional immunomodulatory peptide, but also as a potent host-derived antimicrobial polypeptide. Consequently, patients lacking HNP1 typically present with an increased susceptibility to pyogenic infections. Therefore, the present inventors have found that a selective disruption of the heteromer formation of HNP1 with CCL5 which does not interfere with the interaction of CCL5 with CCR5 offers a therapeutic advantage of preserving normal immune defense mechanisms.

The peptides in accordance with the present invention may be synthesized de novo by any technique known to a person skilled in the art, for example through liquid-phase synthesis or solid-phase synthesis as described in Creighton, Proteins: Structures and molecular Properties, 1993, W.H. Freeman and Company. Alternatively, the peptides may be synthesized through appropriate expression in a host cell. However, peptide synthesis de novo is preferred.

The peptides in accordance with the present invention may be administered as such, or they may also be attached to other entities/molecules in order to prolong the lifetime of the peptides within the metabolism. As an example, they may be attached to polyalkylene oxides, such as polyethylene glycol (PEG) or polypropylene glycol. A person skilled in the art knows how to pegylate proteins and peptides. The use of polyethylene glycol to derivatize peptide therapeutics has been demonstrated to reduce the immunogenicity of the peptides and to prolong the clearance time from circulation. For example, US-patent No. 4,179,337 concerns non-immunogenic peptides, such as enzymes and peptide hormones coupled to polyethylene glycol (PEG) or polypropylene glycol. The principle mode of attachment of PEG and its derivatives to peptides is, in one embodiment, a non-specific bonding to a peptide amino acid residue. Hence, in accordance with the present invention, PEG and its derivatives may be attached to a peptide in accordance with the present invention through a peptide amino acid residue. Such methods of attachments are disclosed in a number of publications, for example US-patent No. 4,496,689, WO 87/00056 and others. Another mode of attaching PEG to peptides is through the specific oxidation of glycosyl residues on a peptide. The oxidized sugar is utilized as a locus for attaching a PEG moiety to the peptide. For example WO 94/05332 discloses the use of a hydrazine- or amino-PEG to add PEG to a glycoprotein. The glycosyl moieties are randomly oxidized to the corresponding aldehydes, which are subsequently coupled to the amino-PEG.

A further way of adding PEG to a peptide is through the use of glycosyltransferases whereby the peptide is exposed to a glycosyltransferase and at least one glycosyl donor under conditions suitable to transfer the at least one glycosyl donor to an amino acid residue of the peptide, wherein the glycosyl donor comprises a polymer, such as polyethylene glycol or polypropylene glycol. This way of pegylation is exemplarily disclosed in WO 03/031464. It pro- vides for a derivatization strategy that results in the formation of a specifically labeled, readily characterizable, essentially homogenous product, wherein the peptide according to the present invention is linked to polyethylene glycol (PEG), polypropylene glycol or other suitable polymeric molecule. In a particularly preferred embodiment, the attached polymeric molecule is polyethylene glycol. In one embodiment, such polyethylene glycol is linear PEG. In another embodiment, such PEG is branched PEG. In a preferred embodiment, such (linear or branched) PEG has a molecular weight in the range of from 10 kDa to 40 kDa.

Pharmaceutical compositions

Administration and Formulation

The production of medicaments containing the peptides or nucleic acids of the invention, and their application can be performed according to well-known pharmaceutical methods.

While the peptides or nucleic acids of the invention, useable according to the invention for use in therapy, may be administered in the form of the raw chemical compound, it is preferred to introduce the active ingredient, optionally in the form of a physiologically acceptable salt in a pharmaceutical composition together with one or more adjuvants, excipients, carriers, buffers, diluents, and/or other customary pharmaceutical auxiliaries. Such salts of the compounds of the invention may be anhydrous or solvated. For storage, the peptides or nucleic acids may also be in lyophilized form.

In a preferred embodiment, the invention provides medicaments comprising a peptide useable according to the invention, or a pharmaceutically acceptable salt or derivative thereof, together with one or more pharmaceutically acceptable carriers therefor, and, optionally, other therapeutic and/or prophylactic ingredients. The carrier(s) must be "acceptable" in the sense of being compatible with the other ingredients of the formulation and not harmful to the recipient thereof

A medicament of the invention may be those suitable for oral, rectal, bronchial, nasal, topical, buccal, sub-lingual, transdermal, vaginal or parenteral (including cutaneous, subcutaneous, intramuscular, intraperitoneal, intravenous, intra-arterial, intracerebral, intraocular injection or infusion) administration, or those in a form suitable for administration by inhalation or insufflation, including powders and liquid aerosol administration, or by sustained release systems. Suitable examples of sustained release systems include semipermeable matrices of solid hydrophobic polymers containing the compound of the invention, which matrices may be in form of shaped articles, e.g. films or microcapsules.

The peptides or nucleic acids useable according to the invention, together with a conventional adjuvant, carrier, or diluent, may thus be placed into the form of medicament and unit dosages thereof. Such forms include solids, and in particular tablets, filled capsules, powder and pellet forms, and liquids, in particular aqueous or non-aqueous solutions, suspensions, emulsions, elixirs, and capsules filled with the same, all for oral use, suppositories for rectal administration, and sterile injectable solutions for parenteral use. Such medicament and unit dosage forms thereof may comprise conventional ingredients in conventional proportions, with or without additional active compounds or principles, and such unit dosage forms may contain any suitable effective amount of the active ingredient commensurate with the intended daily dosage range to be employed.

The peptides or nucleic acids useable according to the invention can be administered in a wide variety of oral and parenteral dosage forms. It will be obvious to those skilled in the art that the following dosage forms may comprise, as the active component, either a pep- tide/nucleic acid(s) useable according to the invention or a pharmaceutically acceptable salt of a peptide/nucleic acid(s) useable according to the invention.

For preparing a medicament from a peptide or nucleic acid useable according to the invention, pharmaceutically acceptable carriers can be either solid or liquid. Solid form preparations include powders, tablets, pills, capsules, cachets, suppositories, and dispersible granules. A solid carrier can be one or more substances which may also act as diluents, flavouring agents, solubilizers, lubricants, suspending agents, binders, preservatives, tablet disintegrating agents, or an encapsulating material.

In powders, the carrier is a finely divided solid which is in a mixture with the finely divided active component. In tablets, the active component is mixed with the carrier having the necessary binding capacity in suitable proportions and compacted in the shape and size desired. Suitable carriers are magnesium carbonate, magnesium stearate, talc, sugar, lactose, pectin, dextrin, starch, gelatin, tragacanth, methylcellulose, sodium carboxymethylcellulose, a low melting wax, cocoa butter, and the like. The term "preparation" is intended to include the formulation of the active compound with encapsulating material as carrier providing a capsule in which the active component, with or without carriers, is surrounded by a carrier, which is thus in association with it. Similarly, cachets and lozenges are included. Tablets, powders, capsules, pills, cachets, and lozenges can be used as solid forms suitable for oral administration.

For preparing suppositories, a low melting wax, such as a mixture of fatty acid glyceride or cocoa butter, is first melted and the active component is dispersed homogeneously therein, as by stirring. The molten homogenous mixture is then poured into convenient sized moulds, allowed to cool, and thereby to solidify. Compositions suitable for vaginal administration may be presented as pessaries, tampons, creams, gels, pastes, foams or sprays containing in addition to the active ingredient such carriers as are known in the art to be appropriate. Liquid preparations include solutions, suspensions, and emulsions, for example, water or water- propylene glycol solutions. For example, parenteral injection liquid preparations can be formulated as solutions in aqueous polyethylene glycol solution.

The peptides or nucleic acid(s) according to the present invention may thus be formulated for parenteral administration (e.g. by injection, for example bolus injection or continuous infusion) and may be presented in unit dose form in ampoules, pre-filled syringes, small volume infusion or in multi-dose containers with an added preservative. The compositions may take such forms as suspensions, solutions, or emulsions in oily or aqueous vehicles, and may contain formulation agents such as suspending, stabilising and/or dispersing agents. Alternatively, the active ingredient may be in powder form, obtained by aseptic isolation of sterile solid or by lyophilization from solution, for constitution with a suitable vehicle, e.g. sterile, pyro- gen-free water, before use.

Aqueous solutions suitable for oral use can be prepared by dissolving the active component in water and adding suitable colorants, flavours, stabilising and thickening agents, as desired. Aqueous suspensions suitable for oral use can be made by dispersing the finely divided active component in water with viscous material, such as natural or synthetic gums, resins, methyl- cellulose, sodium carboxymethylcellulose, or other well-known suspending agents.

Also included are solid form preparations which are intended to be converted, shortly before use, to liquid form preparations for oral administration. Such liquid forms include solutions, suspensions, and emulsions. These preparations may contain, in addition to the active component, colorants, flavours, stabilisers, buffers, artificial and natural sweeteners, dispersants, thickeners, solubilizing agents, and the like.

In one embodiment of the present invention, the medicament is applied topically or systemi- cally or via a combination of the two routes.

For administration, the peptides of the present invention may, in one embodiment, be administered in a formulation containing 0,001% to 70% per weight of the peptide, preferably between 0,0P/o to 70% per weight of the peptide, even more preferred between 0,1%> and 70%> per weight of the peptide. In one embodiment, a suitable amount of peptide administered is in the range of 0.01 mg/kg body weight to 1 g/kg body weight.

Compositions suitable for administration also include lozenges comprising the active agent in a flavoured base, usually sucrose and acacia or tragacanth; pastilles comprising the active ingredient in an inert base such as gelatin and glycerol or sucrose and acacia; and mouthwashes comprising the active ingredient in a suitable liquid carrier.

Solutions or suspensions are applied directly to the nasal cavity by conventional means, for example with a dropper, pipette or spray. The compositions may be provided in single or multi-dose form. In the latter case of a dropper or pipette, this may be achieved by the patient administering an appropriate, predetermined volume of the solution or suspension. In the case of a spray, this may be achieved for example by means of a metering atomising spray pump.

Administration to the respiratory tract may also be achieved by means of an aerosol formulation in which the active ingredient is provided in a pressurised pack with a suitable propellant such as a chlorofluorocarbon (CFC) for example dichlorodifluoromethane, trichlorofluoro- methane, or dichlorotetrafluoroethane, carbon dioxide, or other suitable gas. The aerosol may conveniently also contain a surfactant such as lecithin. The dose of drug may be controlled by provision of a metered valve.

Alternatively the active ingredients may be provided in the form of a dry powder, for example a powder mix of the compound in a suitable powder base such as lactose, starch, starch derivatives such as hydroxypropylmethyl cellulose and polyvinylpyrrolidone (PVP). Conveniently the powder carrier will form a gel in the nasal cavity. The powder composition may be presented in unit dose form for example in capsules or cartridges of, e.g., gelatin, or blister packs from which the powder may be administered by means of an inhaler.

In compositions intended for administration to the respiratory tract, including intranasal compositions, the compound will generally have a small particle size for example of the order of 5 microns or less. Such a particle size may be obtained by means known in the art, for example by micronization.

When desired, compositions adapted to give sustained release of the active ingredient may be employed.

The pharmaceutical preparations are preferably in unit dosage forms. In such form, the preparation is subdivided into unit doses containing appropriate quantities of the active component. The unit dosage form can be a packaged preparation, the package containing discrete quantities of preparation, such as packaged tablets, capsules, and powders in vials or ampoules. Also, the unit dosage form can be a capsule, tablet, cachet, or lozenge itself, or it can be the appropriate number of any of these in packaged form. Tablets or capsules for oral administration and liquids for intravenous administration and continuous infusion are preferred compositions.

If nucleic acids in accordance with the present invention are to be administered, this occurs by way of introducing such nucleic acid into host cells by any means suitable for such purpose, for example using electroporation or a viral vector which is subsequently transfected. Such techniques are known to a person skilled in the art. From such transfected nucleic acid, the respective peptide is expressed in the respective host cell.

Further details on techniques for formulation and administration may be found in the latest edition of Remington's Pharmaceutical Sciences (Maack Publishing Co. Easton, Pa.) and Remington: The science and practice of pharmacy", Lippincott Williams and Wilkins.

Appropriate formulations and ways of manufacturing them are, for example also disclosed in "Arzneiformenlehre, Paul Heinz List, EinLehrbuchfurPharmazeuten, WissenschaftlicheVer- lagsgesellschaft Stuttgart, 4. Auflage, 1985", or "The theory and practice of industrial phar- macy" by Lachman et al, Varghese Publishing House, 1987", or "Modern Pharmaceutics", edited by James Swarbrick, 2. Edition".

The invention is now further described by reference to the following figures which are given to illustrate, not to limit the present invention. Throughout this application, reference to an amino acid sequence is made via the standard one letter code for amino acids. In these figures,

Figure 1 shows that neutrophil and platelet secretory products enhance adhesion of classical monocytes, (a-d) Human classical (a,b,d) or non-classical (c) monocytes were perfused over resting (a-c) or TNF-activated (d) (50 ng, 12 h) HUVEC monolayers and the number of adherent cells was quantified. Neutrophil supernatant (PMN_sup), platelet supernatant (Plt_sup), or a combination of both (PMN/Plt_sup) were deposited 5 minutes prior to perfusion. Green cells in b represent calcein-labelled classical monocytes. n=6. *significant difference compared to ctrl (a) or TNF treatment (d). (e-1) Intravital epifluorescence microscopy of acutely exposed cre- master muscles (e,f) or TNF-activated cremaster muscles (g-1) in Cx₃crl^egf^p/WT mice. To discriminate between monocyte subsets a PE-conjugated antibody to Ly6C (1 μg) was introduced i.v. PMN_SUp, Pltsu (100 μΐ), or a combination of both were injected 15 minutes prior to recording. Quantified are rolling speed (h), rolling flux (e,i,k), and adhesion (f,j,l) of classical monocytes (e,f,h-j) or non-classical monocytes (k,l). g shows representative images, classical monocytes appear in yellow, non-classical monocytes appear in green, scale bar 20 μιη. * significant difference from respective control. n=6. Rolling speed is measured in 5 cells per cremaster muscle. CM, classical monocytes; NCM, non-classical monocytes. Kruskal-Wallis test with post-hoc Dunn test was used in all panels.

Figure 2 shows that neutrophil HNP1 and platelet CCL5 stimulate classical monocyte adhesion in a CCR5 -dependent manner, (a) Classical monocytes were pretreated with antagonists to formyl-peptide receptors (Boc-PLPLP, 10

CCR2 (RS504393, 1.5 μg/ml), CCR5 (maraviroc, 3 μg/ml) or vehicle control and perfused over HUVEC where PMN/Plt_sup was deposited on. n=6. (b) Classical monocytes were perfused over HUVEC where native or CCL5-depleted PMN/Plt_sup were deposited onto. n=6. * significant difference compared to other groups, (c) Representative dot blot with hCCL5, Plt_sup and PMNsu_p spotted on a membrane which was probed with a CCL5 antibody, (d) Classical monocytes were perfused over HUVEC where CCL5 (1 μg/ml) alone (ctrl) or in combination with neutrophil-derived chemotactic proteins HNP1-3, cathepsin G, azurocidin, S100A8, or S100A9 (each 10 μ /ηι1) were deposited on. n=6-8. (e) Classical monocytes were perfused over HUVEC where native PMN/Plt_sup or PMN/Plt_sup depleted of HNPs or HNPs and CCL5 were deposited onto. n=6. Kruskal-Wallis test with post-hoc Dunn test was used in all panels. (f,g) Intravital epifluorescence microscopy of T F-activated cremaster muscles in wild type (f) or Ccr5^' (g) mice injected with a PE-conjugated antibody to Ly6C. Cell adhesion was quantified before and 15 minutes after injection of HNPl (lC^g), CCL5 (1 μg), or a combination of both. Kruskal-Wallis test with post-hoc Dunn test was used in all panels a, b, and e; Mann Whitney test was used in panel d; paired t-test was used in panels f and g.

Figure 3 shows that HNPl and CCL5 employ monocytic CCR5. (a,b) Proximity ligation assay in human classical monocytes treated with HNPl (10 μg/ml)_! CCL5 (1 μg/ml), or a combination of both,in presence (a) or absence (b) of maraviroc (3 μg/ml) . Cells were probed with antibodies to CCR5 and HNPl. n=5-6. (c) CHO cells stably transfected with a CCR5-gfp construct were incubated with rhodamine-conjugated HNPl (10 μg/ml) and CCL5 (lμg/mY) in presence or absence of maraviroc (3 μg/ml). Colocalisation was visualized by confocal microscopy. Scale bar indicates 5 μηι. Kruskal-Wallis test with post-hoc Dunn test was used in all panels. * indicates significant difference from ctrl.

Figure 4 shows that HNP 1 and CCL5 form a targetable heteromer. (a-c) Surface plasmon resonance reveals interaction between HNPl and CCL5. Increasing concentrations of HNPl (a) or of CCL5 (b) were perfused over a biacore sensor chip coated with CCL5 (a) or HNPl (b). CCL5 from various mammalian species were perfused over a HNPl -coated sensor chip and the resulting response was assessed (c). (d) An identified heteromer conformation between human CCL5 (green) -HNPl (magenta) and the interaction between β-strand 1 of CCL5 and β-strand 2-3 of HNPl . (e) A template peptide (green ribbon), used as a starting model to design the SKY peptide, projected on the electrostatic molecular surface of CCL5. Neutral residues such as Gly, Thr and He locate at the negatively-charged surface of CCL5. (f) Binding mode between CCL5 (green ribbon) and the SKY peptide (RRYGTSKYQ) (with key residues SKY) that show interaction with CCL5. H-bond interaction between CCL5 and the SKY peptide are shown in black dashed lines.

Figure 5 shows that disturbance of HNP1-CCL5 interaction abrogates monocyte adhesion evoked by neutrophils and platelets, (a-c) HNPl and CCL5 interaction on endothelial cell surfaces can be inhibited by SKY peptide. Proximity ligation assay on endothelial cells incu- bated with HNPl (10 μ^ηαΐ), CCL5 (^g/ml) or a combination in absence (a) or presence (b,c) of SKY peptide or a scrambled peptide (sSKY). Cells were probed with antibodies to HNPl and CCL5. Representative images of confocal microscopy of untreated, combination of HNPl and CCL5 in absence and presence of SKY peptide (100 μg/ml) endothelial cell surfaces are displayed (c). Scale bar indicates 5 μηι. (d) Presence of HNP1-CCL5 heteromers in cremasteric venules is abrogated by SKY peptide. Mice were treated with vehicle (ctrl), or HNPl (10 μg) and CCL5 (1 μg) in presence or absence of SKY (100 μg) peptide. Heteromers were identified by proximity ligation assay (red) and endothelial cells were stained by CD31 directed antibody (green). Displayed are extended depth of field projections of cremaster venules (left) and transversal 3D reconstructions (right). Scale bar indicates 20 μιη. (e,f) SKY peptide (100 μg/ml) inhibits adhesion of human classical monocytes evoked by immobilized PMN/Pltsup (e) or HNP1/CCL5 (f). n=5-6. (g,h) Intravital epifluorescence microscopy of TNF-activated cremaster muscles in Cxscrl^esfp/WT mice injected with a PE-conjugated antibody to Ly6C. Rolling (g) and adhesion (h) of classical monocytes were quantified before and after HNP1/CCL5 administration in presence or absence of SKY (100 μg). Kruskal-Wallis test with post-hoc Dunn test was used in a, b, e, f; paired t-test was used in panels g and h.

Figure 6 shows that disturbance of HNP1-CCL5 interaction reduces myocardial monocyte and macrophage recruitment, (a) HNPl and CCL5 stimulated monocyte recruitment to the myocardium after ischemia-reperfusion. HNPl and human CCL5 (hCCL5) were overex- pressed in the myocardium by systemic application of a cardiomyotropic AAV2/9 14d before the ischemic event. 72 h after myocardial ischemia-reperfusion injury induced by LAD ligation, classical monocytes adherent to the heart vasculature (left panel) and emigrated into the heart (right panels) as well as macrophages in the heart were quantified by flow cytometry. n=4. (b) SKY peptide (100 μg every 24 hours) abrogates monocyte recruitment into the heart evoked by HNPl and hCCL5. n=8. * indicates significant difference compared to other groups. CM, classical monocytes, (c-e). Displayed are left-ventricle-end-diastolic pressure (LVEDP) (c), contraction velocity (dP/dtmax) (d), and relaxation velocity (dP/dtmin) (e) at baseline and after stimulation with increasing amounts of noradrenaline (10, 25, 50, and 100 ng). n=5-6. * indicates significant effect of treatment. Kruskal-Wallis test with post-hoc Dunn test was used in a and b. Friedman repeated measures test with post-hoc Dunn test was used in c-e. Figure 7 shows that secretory products of neutrophils and platelets do not impact on adhesion of non-classical monocytes, (a) Adhesion of classical monocytes to resting HUVEC after deposition of PMN/Plt_sup. In addition, HUVEC were activated with TNF. n=6. * indicates significant difference compared to control, (b) Intravital epifluorescence microscopy of acutely exposed cremaster muscles in Cx₃crl^egfp/WT mice. PMN_sup, Plt_sup, or a combination of both were injected 15 minutes prior to recording. Displayed is the adhesion of non-classical monocytes (NCM). n=6. Kruskal-Wallis test with post-hoc Dunn test was used in all panels.

Figure 8 shows that HNPl and CCL5 stimulate classical monocyte adhesion via CCR5. (a) Classical monocytes were perfused over HUVEC where CCL5 (^g/ml) together with HNPl, HNP2, HNP3 or a combination of HNPl-3 (10 ng/ml each) were deposited onto and arrest was quantified. n=6. * indicates significant difference compared to Ctrl. (b,c) Classical (b) or non-classical (c) monocytes were perfused over HUVEC where CCL5 (^g/ml), HNPl (10 μg/ml) or a combination of both was immobilized. n=9. * indicates significant difference compared to ctrl. (d) Intravital epifluorescence microscopy of TNF-activated cremaster muscles in wild type mice injected with a PE-conjugated antibody to Ly6C. Cell rolling was quantified before and 15 minutes after injection of HNPl (10 μg), CCL5 (1 μg), or a combination of both. Kruskal-Wallis test with post-hoc Dunn test was used in a-c; paired t-test was used in d.

Figure 9 shows that CCL5 enables HNPl to access CCR5. (a) Human monocytes were incubated with rhodamine-conjugated HNPl (10 μg/ml) and increasing concentrations of CCL5. HNPl binding was measured by flow cytometry, (b) CHO cells stably transfected with a CCR5-gfp construct were incubated with rhodamine-conjugated HNPl (10 μg) and CCL5 (1 μg) in presence or absence of maraviroc ^g/ml). After cross-linking and cell lysis, proteins were separated by SDS PAGE and visualized by specific fluorescence.

Figure 10 shows a design of a HNP1-CCL5 disrupting peptide. Correlation between experimental pK_D (exp. pKo) and calculated binding free energy (BFE) of the identified heteromer conformation of human CCL5-HNP1 complex.

Figure 11 shows that the SKY peptide that does not affect cell viability, (a) Human classical monocytes or HUVEC were incubated with increasing concentrations of SKY peptide and cell viability was measured in a MTT assay (black). Staurosporine (grey) was used as positive control, (b) Human classical monocytes or HUVEC were incubated SKY peptide (100 μg/ml, 24 h) and cell survival was assayed by Annexin V/7-AAD staining. Staurosporine was used as positive control.

Figure 12 shows that the SKY peptide dose-dependently reduces HNP1-CCL5 -mediated monocyte adhesion, (a) Human classical monocytes were perfused over HUVEC in presence of HNP1 (10 μg) and CCL5 (1 μg). SKY peptide was added as indicated, n = 8. (b) Intravital epifluorescence microscopy of TNF-activated cremaster muscles in Cxscrl^eg^^/WT mice injected with a PE-conjugated antibody to Ly6C. Adhesion of classical monocytes was quantified before and after HNP1 (10 μg )/CCL5 (1 μg) administration in presence of SKY peptide in indicated amounts. Kruskal-Wallis test with post-hoc Dunn test was used in a; paired t-test was used in b.

Figure 13 shows that the SKY peptide is non-functional in absence of HNPl and CCL5. (a/b) Intravital epifluorescence microscopy of TNF-activated cremaster muscles in Cxicrl^egfp/m mice injected with a PE-conjugated antibody to Ly6C. Rolling flux (a) and adhesion (b) of classical monocytes were quantified before and after administration of SKY peptide (100 μg). (c) 72 h after myocardial ischemia-reperfusion injury induced by LAD ligation, classical monocytes emigrated into the heart as well as macrophages in the heart were quantified in mice treated with vehicle (ctrl) or SKY peptide (100 μg every 24 hours). n=4.

Figure 14 shows that the SKY peptide is stable and exerts a long lasting effect, (a) SKY peptide was dissolved in buffer and stability of peptide was followed at indicated time points on UPLC-Q-TOF mass spectrometry by integration of SKY peak areas at 220 nm. No changes in mass or fragments of SKY peptide eluting at earlier or later retention times were observed, (b) Intravital epifluorescence microscopy of TNF-activated cremaster muscles in Cxscrl ^g^^/WT mice injected with a PE-conjugated antibody to Ly6C. HNPl (10 μg), CCL5 (1 μg), and SKY peptide (100 μg) were administered 11.5 hours after TNF instillation and adhesion of classical monocytes was quantified every 2 hours afterwards, n = 4 per group. Kruskal-Wallis test with post-hoc Dunn test was used in b.

Figure 15 shows that the SKY peptide inhibits HNP1-CCL5 -evoked monocyte adhesion in large arteries. Apoe ^/'Cxscrl^esfpfWT \CQ were fed a high fat diet for 4 weeks. Adhesion of clas- sical monocytes along the carotid artery was recorded before and after administration of HNPl (10 μg), CCL5 (1 g), and SKY peptide (100 μg). paired t-test was used in all panels.

Figure 16 shows the presence of neutrophils and platelets after ischemia reperfusion. Myocardial ischemia/reperfusion (I/R) injury was induced in wild type mice by LAD ligation. Sham- treated animals were operated, but the LAD was not ligated. After 3 days platelets (CD41⁺) and neutrophils (Ly6G⁺) were identified in the heart homogenate by flow cytometry. Blots are representative of 4 mice.

Figure 17 shows that HNPl, CCL5, and SKY peptide do not impact on monocyte homeostasis in myocardial ischemia reperfusion injury. HNPl and human CCL5 (hCCL5) were overex- pressed in the myocardium by systemic application of a cardiomyotropic AAV2/9 14d before the ischemic event. 72 h after myocardial ischemia-reperfusion injury induced by LAD ligation, apoptosis (a, left) and proliferation (a, right) of classical monocytes (CM) and macrophages in the heart was assessed by flow cytometry. In addition, circulating classical (CM) and non-classical monocytes (NCM) were quantified in the blood. SKY peptide (100 μg) was administered immediately before LAD ligation and every 24 hours after, n = 3-4 for each bar.

Figure 18 shows the purity of neutrophil and platelet preparations. Neutrophils (a) or platelets (b) were isolated from human blood and purity was assessed based on forward (FSC) and side scatter (SSC) properties as well as staining for CD l ib (myeloid cells), CD235a (erythrocytes), and CD41 (platelets).

Figure 19 shows the effect of candidate peptides on monocyte adhesion evoked by a combination of HNPl and CCL5. Human classical monocytes (CM) were perfused over HUVEC where HNPl and CCL5 were deposited onto. Experiments were performed in presence of a vehicle control (ctrl) or in presence of indicated candidate peptides. Bars indicate CM adhesion relative to ctrl which was set to 100%. n=4. n.d., not determined.

Furthermore, reference is made to the following examples which are given to illustrate, not to limit the present invention. Examples

Example 1

Materials and Methods

Isolation of primary cells and cell culture

Leukocytes were isolated from blood of healthy donors with Polymorphprep® density separation (Axis- Shield). Subsequently, classical and non-classical monocytes were purified from the peripheral blood mononuclear cell fraction with Monocyte isolation kit II or CD16⁺ monocyte isolation kit, respectively (Miltenyi Biotec). Neutrophils were obtained as part of the granulocyte cell fraction. Platelets were isolated from venous blood, collected in ACD solution (12 mM citric acid, 15 mM sodium citrate, 25 mM Glucose and 3mM EGTA). Platelet rich plasma (PRP) was obtained by 5 minute centrifugation at 330 g. In order to reduce leukocyte contamination, PRP was diluted 1 : 1 with PBS and centrifuged at 240 g for 10 minutes. The supernatant was centrifuged for 15 minutes at 430 g and pelleted platelets were washed once in PBS. Total platelets were counted with a hematology analyzer (Sysmex XP-300). The purity of neutrophils and platelets was verified by flow cytometry on a FACS Canto II (BD Biosciences) (Fig. 18). Human umbilical vein endothelial cells (HUVEC) (PromoCell) were cultured on collagen-coated plastic surfaces in endothelial growth medium (PromoCell).

Neutrophil and platelet supernatants

Neutrophil and platelet supernatants were generated with a cell density 20 times higher (2 Ox) than the standard average of neutrophil and platelet counts in human blood (150xl0⁶ platelets/ml and 2.5x10⁶ neutrophils/ml). Supernatants were therefore prepared from platelets suspended at 3 x 10⁹/ml and neutrophils suspended at 50xl0⁶/ml in PBS. To induce activation in platelets, the cell suspension was shaken during 30 minutes at room temperature, followed by a centrifugation at 16,000 g to precipitate platelets. Neutrophil degranulation was induced by incubation with fibrinogen-coated Dynabeads ® M-270 Epoxy (Invitrogen), which were coat- ed according to manufacturer's instructions. Briefly, batches of 3.3x10 beads were coated overnight with 100 ug of fibrinogen (Sigma) at room temperature and subsequently washed with PBS. Neutrophils at 50xl0⁶/ml were activated with a bead solution at 48x10⁸ fibrinogen- coated beads/ml during 1 hour at room temperature. Beads were separated from the neutrophil suspension by magnetic force, and cells were subsequently removed by centrifugation at 300g for 5 minutes. In flow chamber assays, supernatants were used at lx final concentration in flow adhesion buffer. In in vivo experiments, lx final concentration was reached by i.v. injection of 100 μΐ of neutrophil and/or platelet supernatant, assuming a total blood volume/mouse of 1.5-2 ml. Immunodepletion of CCL5 and HNP1 in supernatants was performed using magnetic beads (Miltenyi Biotec) conjugated with anti-HNPl (AbD Serotec) or anti-CCL5 polyclonal antibodies (BD bioscience). Depletion was confirmed by dot blot analyses using monoclonal antibodies to the respective protein.

Flow chamber assays

HUVEC were grown to confluence and pre-incubated with neutrophil and/or platelet supernatants or CCL5 (1 μ^ηιΐ) and/or HNP1 (10 μg/ml) for 5 minutes prior to monocyte perfusion. Freshly isolated monocyte subsets were labeled with green calcein, suspended in flow adhesion buffer (10 mM HEPES, 0.5% BSA, 1 mM MgCl₂, 1 mM CaCl₂) and perfused in a parallel wall flow chamber at a concentration of 10⁶ monocytes/ml at a shear rate of 1.5 dyne/cm². In some experiments, monocytes were pretreated with antagonists to formyl -peptide receptors (Boc-PLPLP, 10

or CCR5 (maraviroc, 3 μ^ηιΐ). The adhesion of monocytes under different conditions was quantified as adherent cells per field of view, and calculated as percentage of the untreated condition (control).

Proximity ligation assay

Freshly isolated classical monocytes or cultured HUVEC were incubated with human CCL5 (1 μg/ml) and/or HNP1 (10 g/ml) for 5 minutes. For inhibition experiments, SKY peptide was added at a concentration of 100 μg/ml. Fixation was performed during 10 minutes with 4% PFA at room temperature. Cell preparations were blocked in lx Duolink blocking solution (Sigma- Aldrich) and incubated with anti-CCR5 (R&D) and anti-HNPl (AbD Serotec) primary antibodies for monocytes or anti-CCL5 (Abeam) and anti-HNPl antibodies for HUVEC. Samples were then subjected to incubation with proximity ligation probes and underwent ligation and amplifications steps according to the manufacturer's instructions (Duolink In Situ Detection Reagents Organge). Cell nuclei and HUVEC contour were counterstained with DAPI and with Alexa Fluor 488-conjugated wheat germ agglutinin (Invitrogen), respectively. In the cremaster muscle, human CCL5 and HNPl linked with primary antibodies were injected i.v. together with an AF488-conjugated antibody to CD31 into C57B1/6 mice. After 30 minutes, the mouse was sacrificed and perfused with PBS. The cremaster muscle was exteriorized and fixed with 4% PFA during 30 minutes. After 3 washing steps with PBS, cremaster muscles were incubated with the duolink proximity ligation assay probes (PLA probes) for 3 hours at room temperature. Ligation and amplification steps were run at 37°C during 3 hours and overnight, respectively. Duolink signal in the cremaster venules was imaged with a Leica TCS SP8 3X microscope in confocal mode utilizing a white light laser source for excitation, a 100x1.4 oil objective, and hybrid diode detectors. The system was tuned to minimize channel bleed-through and maximize lateral and spatial resolution. Image acquisition and processing was performed using LasX software (Leica).

Co-localization of CCR5 and HNPl

Chinese hamster ovary cells (CHO) stably transfected with a CCR5-gfp construct (40) were grown in RPMI medium containing FCS (10%), Penicillin/Streptomycin (100 U/ml), Geneti- cin G418 (400 μg ml), and Zeocin (250 μg/ml). Transfected cells were incubated with human CCL5 (1 μg/ml) and rhodamine-conjugated FfNPl (10 μ /ηι1) for 5 minutes in presence or absence of maraviroc (3 μg/ml). After washing with PBS, samples were crosslinked with BS3 (5 mM), and proteins were extracted in cold RIPA buffer (Tris 50 mM, NaCl 150 mM, NP-40 1%, sodium deoxycholate 1%, SDS 0.1%, EDTA 5 mM) supplemented with a protease inhibitor cocktail (Sigma-Aldrich). 20 μg of each sample were loaded and subjected to migration with SDS PAGE. Specific CCR5-gfp and rhodamine-conjugated HNPl fluorescence was visualized using a ChemiDoc™ MP System (BioRad). In separate experiments, CCR5-gfp CHO cells were cultured in Lab-Tek chambered coverglass (Thermo Scientific) and incubated with human CCL5 (1 μg/ml) and rhodamine-conjugated HNPl (10 μ^πύ) for 5 minutes in presence or absence of maraviroc. After washing with PBS, cells were fixed with PFA 4% during 10 minutes. Cell nuclei were counterstained with DAPI and fluorescence was visualized with a Leica TCS SP8 3X microscope in confocal mode utilizing a white light and 405 nm laser source for excitation, a 100x1.4 oil objective, and hybrid diode detectors. The system was tuned to minimize channel bleed-through and maximize lateral and spatial resolution. Image acquisition and processing was performed using LasX software (Leica). Mass spectrometry

Neutrophils or platelets supernatant or both were incubated during 1 hour with an anti-CCL5 antibody (BD bioscience) on protein G magnetic beads (Miltenyi) for each condition. The resulting precipitated proteins were identified by mass spectrometry. Herein, the cysteine residues were reduced by dithiolthreitol and alkylated iodoacetamide. Proteins were digested overnight using porcine trypsin (Promega) and the resulting peptides were reduced under vacuum and desalted by CI 8 zip tip (Millipore). The peptides mixtures were analysed by online nanoflow liquid chromatography tandem mass spectrometry (LC-MS/MS) on an EASY-nLC II™ system (Proxeon) connected to the LTQ Orbitrap Discovery instrument (Thermo Fisher Scientific). 10 μΐ of the peptide mixtures were concentrated onto the 2-cm pre-analytical column (300μηι inner diameter) packed with 5 μηι C₁₈ beads (Dionex). They were separated in a analytical column packed with 3 μιη C₁₈ beads (Dionex). The effluent from the HPLC column was directly electrosprayed into the mass spectrometer. The LTQ Orbitrap instrument was operated in data-dependent mode to automatically switch between full scan MS and MS/MS acquisition. Instrument control was through Tune 2.5.5 and Xcalibur 2.1. A CID-MS/MS top5 method was used with full scan MS spectra (from mlz 400-2000) acquired in the Orbitrap analyzer after accumulation to a target value of 5e6 in the linear ion trap. The 5 most intense peptide ions with charge states >2 were sequentially isolated to a target value of 30,000 and fragmented in the linear ion trap by CID with normalized collision energy of 35% and wideband-activation enabled. Ion selection threshold was 500 counts for MS/MS, and the maximum allowed ion accumulation times were 500 ms for full scans in the orbitrap and 200 ms for CID-MS/MS measurements in the LTQ. Sequences were deduced from the resulting fragment ion spectra using the PEAKS soft (Peaks Studio 5.3, Bioinformatics Solutions Inc.). Then the resulting peptide sequences were submitted to Peaks Search option to perform protein identification using Swiss-Prot database.

Surface plasmon resonance

Interactions between HNPl and CCL5 were studied by surface plasmon resonance on a Biacore XI 00 system (GE Healthcare). Biotinylated CCL5 was immobilized on a streptavidin sensor chip, while HNPl was immobilized on a CM3 chip by amine coupling at a level of 700 response units (RU). HNPl, HNP2, and HNP3 (Bachem), or CCL5 (Peprotech) were used as analytes and diluted in HBS-EP+. In separate experiments, human chemokines CXCL8, CCL1, CCL3, CCL2, CCL11, CCL17, CX₃CL1 (all Peprotech), human neutrophil proteins cathepsin G, azurocidin (both Athens Research), S100A8, or S100A9 (both Prospec), or CCL5 from different species (Mus from Peprotech; Canis, Felis, both Creative Biomart; Equus, Bos, Cavia, Sus, all Genway Biotech Inc.) were used as analytes. Each experiment was performed with a flow of 10 μΐ/min with running buffer. Sensor chip surfaces were regenerated with 50 mM NaOH and equilibrated with running buffer prior to the next injection. Results were analyzed with Biaevalution software.

Protein-Protein Docking and Binding Free Energy Calculations

Sequence alignment between human CCL5 and that of other species (Bos, Canis, Cavia, Equus, Felis, Mus, Sus) revealed a high sequence identity (74-88%) which indicates that these CCL5 orthologs are likely to have the same binding mode to HNP1. Potential configurations of human CCL5-HNP1 heteromeric complex were predicted by application of the protein- protein docking protocol implemented in ICM-pro program. Coordinates of the 3D structures of human CCL5 and FTNP1 were obtained from the Protein Data Bank, PDB code 2L9H and 3HJ2, respectively. Then, homology models for each of the different species of CCL5 were built by using SWISS-MODEL Webserver and evaluated with WHATCHECK. Human CCL5 (2L9H.pdb) was used as a template for construction of CCL5 models. Next, different model structures for the complex between human HNP1 and the individual CCL5 structures were produced based on conformations of human CCL5 and HNP1 derived from protein- protein docking.

Molecular docking of human CCL5 with human HNP1 yielded 21 different conformations, thus we obtained in total 168 different complex structures (21 conformations for each of the 8 species included). The derived structures of the CCL5-HNP1 complexes were refined by application of energy minimization and a short molecular dynamics (MD) simulation (100 ps) using AMBER12 program, while applying a weak force constraint (10 kcal/mol). Prior to performing MD simulations, force fields and charges (Amber 99SB force field) were assigned to the CCL5-HNP1 complexes. Then, the complex was solvated by water (TIP3P model) and counter ions (Na⁺ or CI^") within a 9 A radius. The temperature of the system was set at 300 K. After completion of the MD simulation, snapshots of each complex were extracted for binding free energy calculation using the MM/PBSA approach implemented in AMBER12. Design of small peptide

Employing the obtained structural information of the likely CCL5-HNP1 heteromeric complex as shown in Figure 4d, peptides were designed, based on the sequence of FINP1 between β-strand 2 and 3 (¹⁷RRYGTCIYQGRLWAFCC³³) (SEQ ID NO: 3). This sequence, present at the HNP1 surface that contacts CCL5, was used as a template to design peptides to interfere with the CCL5-FINP1 complex. From the structural analysis, it was concluded that several neutral residues of the wild type FTNP1, such as glycine, threonine and isoleucine, contact the negatively-charged surface of CCL5. These residues were modified to positively-charged residues to improve the binding affinity with CCL5. Moreover, to avoid potential peptide heter- omerization, cysteine at position 22 was modified to serine, which has similar properties yet cannot engage in disulfide bonding. One example of thus designed peptides was a nonapep- tide which was called SKY peptide (RRYGTSKYQ) (SEQ ID NO: 2), and which is characterized by a binding free energy (-21.90±2.04 kcal/mol) which is considerably lower than that of the original HNP1 sequence (-10.95±1.75 kcal/mol). This implies that this peptide (and the others designed by the present inventors) is likely to show high binding affinity with CCL5.

Intravital microscopy

An inflammatory response in Cxjcri^es*^/w/ or Ccr5^'/' mice was induced by acute exposure of the cremaster muscle or by intrascrotal injection of TNF (100 ng, 12 h). The cremaster muscle was exposed and recordings were made using an Olympus BX51 microscope equipped with a Hamamatsu 9100-02 EMCCD camera (Hamamatsu Photonics), a 40x water-dipping objective, and a DualView™ setup for simultaneous detection of two emission light spectra. For image acquisition and analysis Olympus cell^r software (Olympus) was used. A PE-conjugated antibody to Ly6C (eBioscience, 1 μg) was injected i.v. to detect adhesion of Ly6C⁺CX₃CRl⁺ classical monocytes. In each cremaster 5 fields of view were recorded for 30 seconds and the number of adherent cells and the rolling flux (rolling monocytes passing a perpendicular line placed across the observed vessel) from each field were quantified. To reduce the variability between mice, values obtained from each field of view were added and are expressed as absolute values. Rolling speed was quantified by tracking individual cells for 30 seconds. In each cremaster muscle 5 randomly chosen cells were tracked. Leukocyte-endothelial interactions along the carotid artery were analyzed in Apoe ^~ Cx₃crl^es^ ^l< mice having received high fat diet for 4 weeks. Mice were placed in supine position and the right jugular vein was cannulated with a catheter for peptide and antibody injection. The left external carotid artery was surgically exposed as described previously (27). Intravital microscopy was performed after injection of a PE-conjugated antibody to Ly6C (eBi- oscience, 1 μg) using an Olympus BX51 microscope equipped with a Hamamatsu 9100-02 EMC CD camera, a Dual View™ setup, and a lOx saline-immersion objective. Movies of 30 seconds were acquired and analyzed offline. In the carotid artery one field of view was analyzed per mouse.

Virus production

The recombinant rAAV.hCCL5 and rAAV.HNP viruses were produced using triple transfec- tion in HEK293 cells and subsequent purification with cesium sedimentation. One plasmid (5F6) provided the adenoviral helper function, the second plasmid (transplasmid) contained the AAV2 rep and AAV9 cap in trans and a third plasmid (cis-plasmid) supplied the transgene under control of a CMV promoter flanked by cis acting AAV2 internal terminal repeats. After 48 h, cells were harvested and rAAVs were purified using cesium chloride gradients. Quantification of the viral titers was performed via polyA bGH 3' region of the transcript using real-time PCR.

Myocardial ischemia-reperfusion injury

C57B1/6 mice were transduced 2 weeks prior to ischemia-reperfusion injury by injection of 3xl0¹² virus particles (rAAV) coding for either human CCL5 (rAAV.hCCL5) and/or HNP1 (rAAV.HNPl), or an empty virus. SKY peptide (100 μg) was administered immediately prior to surgery and every 24 hours after coronary ligation. Ischemia was induced for 60 minutes by ligation of the left anterior descending coronary artery (LAD). Reperfusion was allowed for three days and followed by invasive monitoring of left ventricular function with the ADV500 Pressure-volume System (Transonic). The catheter was introduced through the left carotid artery and aorta into the left ventricle and base line data were acquired. In order to stimulate the heart function, increasing amounts of noradrenaline (10, 25, 50, and 100 ng) were injected in the jugular vein under continuous monitoring of the pressure curves. Contraction velocity (dP/dt_max), relaxation velocity (dP/dt_mi_n), developed and left-ventricle-end-diastolic pressure (LVEDP) were acquired. In separate experiments, leukocyte infiltration into the myocardium was assessed by flow cytometry. To quantify the adherent leukocytes, anti-CD45-FITC (1 μg) was injected (i.v.) 5 minutes prior to sacrifice. Hearts were harvested, perfused with saline, minced with fine scissors, and digested with collagenase I (450 U/ml), collagenase XI (125 U/ml), hyaluronidase type I-s (60 U/ml) and DNase (60 U/ml) (Sigma-Aldrich) at 37°C for one hour. The resulting single cell suspension was resuspended in PBS/BSA 1% and incubated with antibodies to CD45, CDl lb, Ly6C, F4/80 and CD1 15 (Biolegend). Data were acquired on a FACS Canto II (BD Biosciences) and analysis was performed with FlowJo software. Overexpression of HNP1 and hCCL5 in transduced mice was confirmed by dot blot. 5 μΐ of plasma were spotted onto a nitrocellulose membrane, together with increasing concentrations of recombinant hCCL5 and HNP1 (1 ng/ml to 10 μg/ml) as reference. Anti-CCL5 (Abeam) and anti-HNPl (AbD Serotec) primary antibodies and their corresponding secondary antibodies were used for detection.

Statistics

All data are expressed as mean±SD. Due to variability between blood donors, data from flow adhesion assays are expressed as percent of control. Statistical calculations were performed using GraphPad Prism 5 (GraphPad Software Inc.). After calculating for normality by D'Agostino Pearson omnibus test, Kruskal-Wallis test with post-hoc Dunn test, one way Anova with Holm-Sidak multiple comparison posttest, paired t-test, or Friedman repeated measures test with post-hoc Dunn test was used, p-values < 0.05 were considered significant.

Example 2

Neutrophil and platelet secretory products cooperate in adhesion of classical monocytes

To study the cooperation of neutrophil and platelet secretory products in monocyte adhesion the inventors deposited the supernatant from activated neutrophils (PMN_sup) and platelets (Pltsup) on human umbilical vein endothelial cells (HUVEC) alone or in combination (PMN/Plt_Sup). While monocyte adhesion was not affected in the presence of PMN_sup or Plt_sup, PMN/Plt_SUp enhanced arrest of classical but not non-classical monocytes (Fig. la-c) to a similar degree to that observed after endothelial cytokine activation (Fig. 7a). Deposition of PMN/Plt_SUp evoked similar effects on classical monocyte adhesion when HUVEC were pre- activated with TNF (Fig. Id). To assess the in vivo relevance of these findings the inventors studied monocyte adhesion to postcapillary venules in the cremaster muscle of Cx₃crl^eg^^/wt mice (23). Adhesion of monocyte subsets was tested after trauma-induced activation (Fig. le,f and Fig. 7b) or by intrascrotal TNF activation (Fig. lg-1). In accordance with observations made in vitro, intravenous injection of PMN_sup or Plt_sup alone did not affect adhesion or rolling of either monocyte subset, while administration of PMN/Plt_sup induced a shift from the fraction of rolling classical monocytes towards the adherent fraction (Fig. lh-j). In contrast, rolling and adhesion of non-classical monocytes was not affected (Fig. lk,I).

Platelet CCL5 and neutrophil HNP1 liaise to stimulate monocyte adhesion

Chemoattractants released from granules of neutrophils and platelets can be sensed by monocytes through various chemotactic receptors including formyl-peptide receptors (FPR) and chemokine receptors (5,15). Interestingly, specific inhibition of FPRs or the chemokine receptors CXCR2 and CCR2 had no effect on PMN/Plt_sup-evoked classical monocyte adhesion (Fig. 2a). In contrast, neutralization of CCR5 signaling abrogated stimulated monocyte adhesion. Hence, the inventors suspected that CCL5, a major CCR5 ligand, may be an important factor in this response. In line herewith, PMN/Plt_sup depleted of CCL5 was non-functional (Fig. 2b). However, when comparing PMN_sup and Plt_sup, CCL5 is exclusively released from platelets (Fig. 2c). Thus, the inventors assumed that CCL5 may bind a partner within the PMN_sup to potentiate its effect. To investigate this possibility, the inventors immunoprecipi- tated CCL5 from the PMN/Plt_sup and assessed CCL5-binding partners by mass spectrometry. Among the identified proteins were four which are released from neutrophils and exert a chemotactic response: HNPs, S100A8/A9 (MRP8/14), cathepsin G, and azurocidin. In flow experiments no neutrophil granule protein enhanced classical monocyte adhesion by itself (Fig. 2d). However, when combined with CCL5, HNPs, but not the other granule proteins, specifically evoked enhancement of classical monocyte adhesion (Fig. 2d). The importance of HNPs and CCL5 was confirmed by depletion of HNPs or HNPs and CCL5 which abrogated monocyte adhesion stimulated by PMN/Plt_sup (Fig. 2e). HNPs comprise a group of three proteins which differ in just one amino acid. As HNP1, HNP2, or HNP3 were equally potent in enhancing monocyte adhesion when combined with CCL5 (Fig. 8a) the inventors performed further experiments with HNPl, the most abundant a-defensin family member. Since HNP1 in combination with CCL5 strongly enhanced adhesion of human classical monocytes (Fig. 2d and Figure 8b) but not non-classical monocytes (Figure 8c) the inventors challenged the in vivo relevance of this finding in monocyte reporter Cx₃crl^eg^^/wt mice. With the absence of HNPs in mouse neutrophils (24) the inventors administered human CCL5 and HNPl intravenously and experienced an increase in adhesion with a concomitant decrease in rolling flux of classical monocytes (Fig. 2f and Figure 8d). Interestingly, this effect was lost in Ccr5^"/_ mice (Fig. 2g) suggesting that HNPl and CCL5 act via CCR5.

CCL5 permits HNPl to engage CCR5

In ligand binding assays rhodamine-conjugated HNPl did not bind to classical monocytes. However, CCL5 permitted HNPl binding to human classical monocytes in a dose-dependent fashion (Figure 9a). In proximity ligation assays the inventors could identify that HNPl was in close proximity to CCR5 only in presence of CCL5 (Fig. 3a), suggesting that CCL5 permits HNPl to access CCR5. This notion was supported by experiments where approximation of HNPl to CCR5 in presence of CCL5 was abrogated by a CCR5 antagonist (Fig. 3b). The latter was also confirmed when rhodamine-HNPl was incubated with CHO cells stably trans- fected with a CCR5-gfp construct. Here, CCR5 colocalized with HNPl and - after crosslink- ing - co-migrated in a SDS-PAGE (Fig. 3c and Fig. 9b). Taken together, these data suggest that CCL5 enables HNPl to access CCR5.

HNPl and CCL5 form stable heteromers

The data pointed towards a protein-protein interaction between HNPl and CCL5 which syn- ergize to activate monocytes via CCR5. To further study the interaction between HNPl and CCL5 the inventors performed surface plasmon resonance studies. Biotinylated CCL5 was immobilized on a streptavidin-coated sensor chip and HNPl was superfused resulting in a dose-dependent interaction (Fig. 4a) (Κο=70ηΜ). In a reverse approach, HNPl was immobilized and perfused with increasing concentrations of CCL5 and a similar dose-dependent response was identified (Fig. 4b) ( ο=8.6ηΜ). To better characterize the interaction between HNPl and CCL5 the inventors perfused naturally occurring variants of CCL5 from different species over immobilized HNPl (Fig. 4c). These data were integrated with in silico studies in order to predict a likely heteromeric complex structure of HNPl bound to CCL5, which was subsequently used to design a small peptide to disturb the interaction between HNPl and CCL5. Assuming conservation of the binding mode for the CCL5-HNP1 complex among species, there should be a correlation between calculated binding free energies and experi- mental binding affinities. The CCL5-HNP1 complex (Fig. 4d) represents a likely heteromeric complex as this conformation exhibited the highest correlation between calculated binding free energies and experimental KD (Fig. 10) values identified among 21 different potential conformations derived from protein-protein docking. After analysis of this proposed structure, the inventors found that residues from β-strands 2-3 of HNPl (¹⁷RRYGTCIYQGRLWAFCC³³) (SEQ ID NO: 3) are the key residues that interact with CCL5. Based on the β-strand 2 of HNPl and its binding mode with CCL5 (Fig. 4e) the inventors designed various peptides, e. g. a short peptide (RRYGTSKYQ (SEQ ID NO: 2), also referred to as SKY peptide) exhibiting the highest experimental potential to disturb the interaction between CCL5 and HNPl (Fig. 4f).

Disturbance ofHNPl-CCL5 interaction inhibits monocyte adhesion

In inflammation the endothelium presents proteins secreted from neutrophils and platelets to rolling leukocytes (25,26). Proximity ligation assays clearly showed abundant interactions of HNPl and CCL5 on the endothelial cell surface (Fig. 5a), an effect fully abrogated in presence of the SKY peptide, but not by a scrambled peptide (TYQRRSGKY) (Fig. 5b,c). These observations were confirmed in vivo in the cremaster muscle after intravenous injection of HNPl and CCL5 (Fig. 5d). In flow chamber assays classical monocyte adhesion induced by PMN/Plt_SUp (Fig. 5e) was fully abrogated in the presence of the SKY peptide without affecting the viability of the cells involved (Fig. 11). In addition, adhesion to coimmobilized HNPl and CCL5 was dose-dependently reduced by presence of SKYpeptide (Fig. 5f and Fig. 12a) In line, intravital microscopy of the cremaster muscle showed that intravenous delivery of the SKY peptide dose-dependently prevented adhesion of classical monocytes induced by coadministration of HNPl and CCL5 (Fig. 5g,h and Fig. 12b). To provide an additional control for the specificity of SKY peptide, the inventors tested its effect on monocyte adhesion in mice not receiving HNPl and CCL5. In these experiments, SKY peptide failed to reduce adhesion of classical monocytes (Fig. 13a b)

To assess the duration of the biological effect after a single dose of SKY peptide, the inventors first assessed its chemical stability. Using FPLC the inventors found that the peptide was stable for the entire observation period of 48 hours (Fig. 14a). The biological stability of the effect was tested by intravital microscopy following a single injection of HNPl, CCL5 and SKY peptide. Monocyte adhesion in the cremaster muscle was recorded for 8 hours after ad- ministration of the peptides. HNP1 and CCL5 enhanced monocyte adhesion during the first 6 hours after administration. At 8 hours no biological effect was observed, likely due to peptide clearance. SKY peptide abrogated the effects exerted by HNP1-CCL5 immediately after administration, as well as 2, 4, and 6 hours later (Fig. 15b). Thus, these data indicate a prolonged inhibitory effect in vivo.

Finally, the inventors aimed at studying the effect of HNP1, CCL5, and SKY peptide on adhesion of classical monocytes in a different vascular bed. Given the importance of classical monocytes in atherosclerosis (27), the inventors decided to study monocyte adhesion in the carotid artery. Apoe ^/'Cx₃crl^e8fp/WT mice were fed a high- fat diet for 4 weeks to generate hy- percholesterolemia-induced endothelial dysfunction. Adhesion of classical monocytes was studied following injection of a PE-conjugated antibody to Ly6C using established methodology (27,28). In these experiments, adhesion of classical monocytes was significantly increased by intravenous administration of HNP1 and CCL5, an effect abrogated in the presence of SKY peptide (Fig. 15).

Inhibition ofHNPl-CCL5 interaction reduces myocardial macrophage accumulation

With the importance of neutrophils and platelets in acute myocardial infarction (29), the inventors chose to study adhesion and recruitment of classical monocytes and macrophages three days after ischemia-reperfusion of the left anterior descending (LAD) coronary artery. At this time point neutrophils and platelets can be found in the homogenate of hearts from wild type mice (Fig. 16) and the recruitment of classical monocytes is at its peak (30). In these experiments human CCL5 (hCCL5) and HNP1 were overexpressed in the myocardium by Adeno-associated virus (AAV)-assisted transduction reaching plasma levels of 1.7±0.45 μg/ml and 9.13±3.18 μg/ml, respectively. Overexpression in this way resulted in enhanced endothelial adhesion of classical monocytes and accumulation of macrophages in the heart (Fig. 6a), an effect fully abrogated by repetitive treatment with the SKY peptide (Fig. 6b). This effect was only observed in mice with overexpression of hCCL5 and HNP1 but not wild type mice, thus strengthening the specificity of the peptide (Fig. 13c). Since overexpression of HNP1 and CCL5 and repeated injection of SKY peptide did neither alter counts of monocytes in the blood, nor impact on proliferation and apoptosis of monocytes and macrophages in the heart (Fig. 17), nor reduce plasma CCL5 (2.00±0.74 μg/ml) and HNP1 levels (6.8±3.5 μg/ml ), the inventors conclude that the observed effect on myocardial monocyte and macrophage accumulation primarily stems from alterations of monocyte recruitment. Finally, invasive monitoring of cardiac function revealed that overexpression of hCCL5 and HNPl resulted in impaired cardiac function. Interestingly, this adverse effect was abolished upon repetitive treatment with the SKY peptide (Fig. 6c-e).

Acute and chronic inflammatory conditions frequently associate with neutrophil and platelet activation. These events represent a causal link to subsequent recruitment of monocytes. Through the above-identified experiments, the inventors have shown a novel mechanism of neutrophil-platelet cooperation which rests upon heteromer formation of neutrophil HNPl and platelet CCL5. Both molecules are recognized by monocytes rolling along the endothelium engaging CCR5, thus promoting firm monocyte adhesion. The inventors have furthermore shown that a disturbance of this interaction between HNPl and CCL5 reduces monocyte adhesion and thus allows for an efficient treatment of inflammation triggered by such monocyte adhesion. The selective interference with a formation of the HNPl-CCL5-complex enables therefore customized treatments of inflammatory conditions which are characterized by neutrophil and platelet degranulation, such as acute arterial occlusions, sepsis, atherosclerosis and others.

References and Notes:

1. J. W. Conlan, R. J. North, Neutrophils are essential for early anti-Listeria defense in the liver, but not in the spleen or peritoneal cavity, as revealed by a granulocyte-depleting monoclonal antibody. J Exp Med. 179, 259-68 (1994).

2. A. Chalaris, B. Rabe, K. Paliga, H. Lange, T. Laskay, C. A. Fielding, S. A. Jones, S.

Rose-John, J. Scheller, Apoptosis is a natural stimulus of IL6R shedding and contributes to the proinflammatory trans-signaling function of neutrophils. Blood. 110, 1748-55 (2007).

3. O. Soehnlein, A. Zernecke, E. E. Eriksson, A. G. Rothfuchs, C. T. Pham, H. Herwald, K.

Bidzhekov, M. E. Rottenberg, C. Weber, L. Lindbom, Neutrophil secretion products pave the way for inflammatory monocytes. Blood. 112, 1461-71 (2008).

4. M. Drechsler, R. T. Megens, M. van Zandvoort, C. Weber, O. Soehnlein, Hyperlipidemia- triggered neutrophilia promotes early atherosclerosis. Circulation. 122, 1837-45 (2010).

5. O. Soehnlein, L. Lindbom, C. Weber, Mechanisms underlying neutrophil-mediated monocyte recruitment. Blood. 114, 4613-23 (2009). P. A. Ward, Chemotoxis of mononuclear cells. J Exp Med. 128, 1201-21 (1968). J. I. Gallin, M. P. Fletcher, B. E. Seligmann, S. Hoffstein, K. Cehrs, N. Mounessa, Human neutrophil-specific granule deficiency: a model to assess the role of neutrophil-specific granules in the evolution of the inflammatory response. Blood. 59, 1317-29 (1982). A. F. Gombart, H. P. Koeffler, Neutrophil specific granule deficiency and mutations in the gene encoding transcription factor C/EBP(epsilon). Cwrr Opin Hematol. 9, 36-42 (2002). O. Chertov, H. Ueda, L. L. Xu, K. Tani, W. J. Murphy, J. M. Wang, O. M. Howard, T. J. Sayers, J. J. Oppenheim. Identification of human neutrophil-derived cathepsin G and az- urocidin/CAP37 as chemoattractants for mononuclear cells and neutrophils. J. Exp. Med. 186, 739-47 (1997). M. C. Territo, T. Ganz, M. E. Selsted, R. Lehrer, Monocyte-chemotactic activity of defen- sins from human neutrophils. J. Clin. Invest. 84, 2017-20 (1989). A. Vieira-de-Abreu, R. A. Campbell, A. S. Weyrich, G. A. Zimmerman, Platelets: versatile effector cells in hemostasis, inflammation, and the immune continuum. Semin Immii- nopathol. 34, 5-30 (2012). J. M. Burkhart M. Vaudel, S. Gambaryan, S. Radau, U. Walter, L. Martens, J. Geiger, A. Sickmann, R.P. Zahedi, The first comprehensive and quantitative analysis of human platelet protein composition allows the comparative analysis of structural and functional pathways. Blood. 120, e73-82 (2012). T. Baltus, P. von Hundelshausen, S. F. Mause, W. Buhre, E. Rossaint, and C. Weber, Differential and additive effects of platelet-derived chemokines on monocyte arrest on inflamed endothelium under flow conditions. J Leukoc Biol. 78, 435-41 (2005). J. Bemhagen, R. Krohn, H. Lue, J. L. Gregory, A. Zernecke, R. R. Koenen, M. Dewor, I. Georgiev, A. Schober, L. Leng, T. Kooistra, G. Fingerle-Rowson, P. Ghezzi, R. Klee- mann, S. R. McColl, R. Bucala, M. J. Hickey, C. Weber, MIF is a noncognate ligand of CXC chemokine receptors in inflammatory and atherogenic cell recruitment. Nat Med. 13, 587-96 (2007). E. Karshovska, C. Weber, P. von Hundelshausen, Platelet chemokines in health and disease. Thromb. Haemost. 110, 894-902 (2013). O. Soehnlein, M. Drechsler, Y. Doring, D. Lievens, H. Hartwig, K. Kemmerich, A. Ortega-Gomez, M. Mandl, S. Vijayan, D. Projahn, C. D. Garlichs, R. R. Koenen, M. Hristov, E. Lutgens, A. Zernecke, C. Weber, Distinct functions of chemokine receptor axes in the atherogenic mobilization and recruitment of classical monocytes. EMBO Mol Med. 5, 471-81 (2013). B. Passlick, D. Flieger, H. W. Ziegler-Heitbrock. Identification and characterization of a novel monocyte subpopulation in human peripheral blood. Blood. 74, 2527-34 (1989). F. Geissmann, S. Jung, D. R. Littman. Blood monocytes consist of two principal subsets with distinct migratory properties. Immunity. 19, 71-82 (2003). C. Varol, A. Mildner, S. Jung. Macrophages: development and tissue specialization. Annu Rev Immunol 33, 643-75 (2015). C. Auffray, D. Fogg, M. Garfa, G. Elain, O. Join-Lambert, S. Kayal, S. Sarnacki, A. Cumano, G. Lauvau, F. Geissmann. Monitoring of blood vessels and tissues by a population of monocytes with patrolling behavior. Science. 317, 666-70 (2007). D. Rittirsch, M. A. Flierl, P. A. Ward, Harmful molecular mechanisms in sepsis. Nat Rev Immunol. 8, 776-87 (2008). F. K. Swirski, M. Nahrendorf, Leukocyte behavior in atherosclerosis, myocardial infarction, and heart failure. Science. 339, 161-6 (2013). S. Wantha, J. E. Alard, R. T. Megens, A. M. van der Does, Y. Doring, M. Drechsler, C. T. Pham, M. W. Wang, J. M. Wang, R. L. Gallo, P. von Hundelshausen, L. Lindbom, T. Ha- ckeng, C. Weber, O. Soehnlein, Neutrophil-derived cathelicidin promotes adhesion of classical monocytes. Ore. Res. 112, 792-801 (2013). P. B. Eisenhauer, R. I. Lehrer, Mouse neutrophils lack defensins. Infect. Immun. 60, 3446- 7 (1992). P. von Hundelshausen, K. S. Weber, Y. Huo, A. E. Proudfoot, P. J. Nelson, K. Ley , C. Weber, RANTES deposition by platelets triggers monocyte arrest on inflamed and atherosclerotic endothelium. Circulation. 103, 1772-7 (2001). O. Soehnlein, X. Xie, H. Ulbrich, E. Kenne, P. Rotzius, H. Flodgaard, E. E. Eriksson, L. Lindbom, Neutrophil-derived heparin-binding protein (HBP/CAP37) deposited on endothelium enhances monocyte arrest under flow conditions. J Immunol. 174, 6399-405 (2005). O. Soehnlein, M. Drechsler, Y. Doring, D. Lievens, H. Hartwig, K. Kemmerich, A. Ortega-Gomez, M. Mandl, S. Vijayan, D. Projahn, C. D. Garlichs, R. R. Koenen, M. Hris- tov, E. Lutgens, A. Zernecke, C. Weber. Distinct functions of chemokine receptor axes in the atherogenic mobilization and recruitment of classical monocytes. EMBO Mo! Med. 5, 471-81 (2013).

28. R. Chevre, J. M. Gonzalez-Granado, R. T. Megens, V. Sreeramkumar, C. Silvestre-Roig, P. Molina-Sanchez, C. Weber, 0. Soehnlein, A. Hidalgo, V. Andres. High-resolution imaging of intravascular atherogenic inflammation in live mice. Circ Res. 114, 770-9 (2014).

29. Epelman, P. P. Liu, D. L. Mann, Role of innate and adaptive immune mechanisms in cardiac injury and repair. Nat. Rev. Immunol. 15, 117-29 (2015).

30. M. Nahrendorf, F. K. Swirski, E. Aikawa, L. Stangenberg, T. Wurdinger, J. L. Figueiredo, P. Libby, R. Weissleder, M. J. Pittet. The healing myocardium sequentially mobilizes two monocyte subsets with divergent and complementary functions. J Exp Med. 204, 3037-47 (2007).

The features of the present invention disclosed in the specification, and/or the claims may, both separately and in any combination thereof, be material for realizing the invention in various forms thereof.

Claims

Claims

1. A peptide derived from human neutrophil peptide 1 (HNP1) and consisting of an amino acid sequence selected from formula a) a) (X¹)_nl-RRYGT-X²-X³-YQ-(X⁴)_n2-(X⁵)_n3-(X⁶)_n4 wherein X¹ is C and nl is 0 or 1,

X² is C, A or S,

X³ is I, or R,

X⁴ is the sequence GRLWAF and n2 is 0 or 1,

X⁵ is C, A or S and n3 is 0 or 1,

X⁶ is C or S and n4 is 0 or 1, with the proviso that, when n2 is 0, then n3 and n4 are also 0, and when n2 is 1, then n3 and n4 are also 1, respectively, wherein optionally, the peptide is covalently linked to a polyalkylene oxide, preferably a polyethylene glycol (PEG) or a polypropylene glycol.

2. The peptide according to claim 1, consisting of an amino acid sequence of formula a), wherein n2 - n4 = 0.

3. The peptide according to any of claims 1-2, consisting of an amino acid sequence of formula a), wherein nl is 0.

4. The peptide according to any of claims 1-3, consisting of an amino acid sequence of formula a), wherein X² is S, and X³ is K.

5. The peptide according to any of the foregoing claims, consisting of an amino acid sequence selected from CRRYGTCIYQGRLWAFCC SEQ ID NO:l

RRYGTS YQ SEQ ID NO:2

RYGTCIYQGRLWAFCC SEQ ID NO:3

CRRYGTAKYQGRLWAFAC SEQ ID N0:4

CRRYGTARYQGRLWAFAC SEQ ID NO: 5

RRYGTSIYQ SEQ ID NO:6

CRRYGTS I YQGRLWAF S C SEQ ID NO:7

CRRYGTSKYQGRLWAFSC SEQ ID NO:8

CRRYGTSRYQGRLWAFSC SEQ ID N0:9

RRYGTSRYQ SEQ ID NO: 10

The peptide according to any of the foregoing claims, consisting of an amino acid quence selected from

RRYGTSKYQ SEQ ID NO:2

RRYGTSIYQ SEQ ID NO:6

RRYGTSRYQ SEQ ID NO: 10

7. A nucleic acid coding for a peptide as defined in any of claims 1-6.

8. The peptide according to any of claim 1-6 or the nucleic acid according to claim 7, for use as a medicament.

9. The peptide according to any of claims 1-6 or the nucleic acid according to claim 7, for use in the treatment of inflammatory diseases.

10. The peptide or nucleic acid for use according to claim 9, wherein the inflammatory disease is selected from rheumatoid arthritis, ankylosing spondylitis, inflammation post infection, atherosclerosis, myocardial infarction, stroke, acute lung injury, including transfusion related acute lung injury (TRALI), sepsis, pain, dermatitis, psoriasis, atopic skin disease, chronic granulomatous diseases such as tuberculosis, leprosy, sarcoidosis, silicosis, nephritis, amyloidosis, scleroderma, lupus, polymyositis, osteoporosis, inflammatory bowel disease(s), including Crohn's disease, colitis ulcerosa,, ulcers, appendicitis, diverticulitis, Sjogren's syndrome, Reiter's syndrome, pelvic inflammatory disease, orbital in- flammatory disease, peritonitis, hepatitis , thrombotic disease, inappropriate allergic responses to environmental stimuli such as poison ivy, pollen, insect stings and certain foods, including atopic dermatitis and contact dermatitis, allergic encephalitis, asthma, lung inflammation, COPD, chronic bronchitis.

11. A method of treatment of an inflammatory disease, comprising the step of administering the peptide according to any of claims 1 -6 or the nucleic acid according to claim 7 to a patient suffering from an inflammatory disease.

12. A pharmaceutical composition comprising the peptide according to any of claims 1-6 or the nucleic acid according to claim 7, and a pharmaceutically acceptable carrier.