The role of interleukin 7 and immune auto-reactivity to self-antigen LL-37/CRAMP in atherosclerosis

Povzetek: Ateroskleroza je bolezen kopičenja maščobnih delcev v žilno steno arterij. Po svoji naravi je progresivna bolezen, ki jo zaznamuje večanje aterosklerotičnega plaka do točke ko le-ta poči in vsebina izpostavi krvnemu obtoku. To na mestu razpoke povzroči nastanek strdka, ki lahko posledično zapre ali zoži arterijo do te mere, da lahko privede do ishemije tkiva katerega taka arterija prehranjuje. Pri srcu to pomeni ishemijo srčne mišičnine – miokardni infarkt. Ob miokardnem infarktu se v sistemsko cirkulacijo zaradi nekroze srčne mišice izločajo vnetni mediatorji, ti pa vplivajo na večje vnetje v aterosklerotičnih lehah na kar kažejo slikovne študije ožilja po ishemičnem dogodku. Klinično to vidimo tudi v povečanem tveganju za ponovni ishemični dogodek, najvišje v roku enega leta po miokardnem infarktu. Na to kažejo podatki iz številnih epidemioloških študij s celega sveta. Modulacija vnetja po miokardnem infarktu lahko zmanjša tveganje za ponovni dogodek, kar so pokazali podatki tako iz predkliničnih kot tudi iz kliničnih študij. V prvem delu doktorske teze predstavim novo pot imunomodulacije po miokardnem infarktu z inhibicijo receptorja za interlevkin 7. V živalski študiji na miših z aterosklerotično boleznijo in pridruženim miokardnim infarktom pokažem, da interlevkin 7 deluje kot monocitni kemotaktični dejavnik, da je količina interlevkina 7 po miokardnem infarktu v žilni steni povišana ter, da se po miokardnem infarktu poveča sinteza mRNA interlevkina 7 v samem ishemično prizadetem miokardu. Imunomodulacija vnetja z inhibitornim monoklonskim protitelesom proti receptorju interlevkina 7 zmanjša kopičenje maščob v žilni steni ter infiltracijo vnetnih celic, predvsem monocitov in makrofagov, kar pomeni zmanjšanje vnetja in stabilizacijo plaka. V prvem delu doktorskega dela tako pokažem doprinos in pomen vnetja in prirojenega imunskega sistema v proces ateroskleroze. Imunski sistem v grobem sestavljata prirojeni imunski sistem, katerega sestavni del so celice kot so monociti in makrofagi, udeleženi v proces ateroskleroze tako v akutni kot kronični fazi bolezni ter pridobljeni imunski sistem. Po začetni, nespecifični fazi vnetja pride do aktivacije specifičnega, pridobljenega dela imunskega sistema, katerega predstavniki so med drugim limfociti T, limfociti B ter antigen predstavitvene celice. Značilnost tega dela imunskega sistema je tarčno, antigensko specifično vnetje, za katerega so značilne interakcije med receptorji na celicah T in MHC molekulami na antigen predstavitvenih celicah. Znano je, da pri procesu ateroskleroze pride do aktivacije avto-reaktivnih limfocitov T in B in specifičnega odziva le-teh proti maščobnim delcem v aterosklerotičnih lehah. En takih delcev je peptid p210, ki je del molekule ApoB100, sestavni del delcev LDL, glavnih krivcev za nastanek vnetja v žilni steni. Raziskave so pokazale obstoj LDL in apoB-100 reaktivnih celic T, vendar do sedaj specifični epitopi pomembni pri aterosklerozi še niso bili potrjeni. V drugem delu doktorskega dela pokažem prisotnost avtoreaktivnih, antigen specifičnih CD8+ limfocitov T, pri katerih stimulacija s peptidom p210 izzove avto-imunski odziv. Avto-imunski odziv temelji na prepoznavi kompleksa antigen-MHC-I molekula s strani celičnega receptorja T. S pomočjo IEDB baze (Immune Epitope Data Base) smo preverili predvideno vezavo peptidnega fragmenta p210 na mišje molekule MHC-I. Na podlagi izbranega peptidnega fragmenta z visoko predvideno vezavno afiniteto na MHC-I smo izdelali fluorescenčno označen pentamer sestavljen iz petih MHC-I molekul, specifičnih za p210 fragment in preverili prisotnost CD8+ celic T, ki se na pentamer vežejo ob specifični prepoznavi epitopa p210. Miši, ki so prejemale aterogeno dieto z visoko vsebnostjo maščob, so izražale večji delež pentamer pozitivnih CD8+ celic T, ki so imele izražen efektorski fenotip. Te celice se ob stimulaciji s peptidom p210 klonalno razmnožijo, kar je značilnost specifične razpoznave antigena s strani T-celičnega receptorja (TCR). Imunomodulacija z imunizacijo s p210 je povečala delež p210-specifičnih pentamer pozitivnih CD8+ limfocitov T in zmanjšala breme ateroskleroze. Interakcija CD8+ limfocitov T z antigen predstavitvenimi celicami temelji na vezavi antigenskega peptida z vezavnim mestom MHC-I molekule na površini antigen predstavitvenih celic in prepoznavi kompleksa antigen/MHC-I s strani T-celičnega receptorja CD8+ limfocitov T. Znano je, da se MHC-I molekule pri bolezenskih stanjih izločajo v krvni obtok. Te topne MHC-I molekule na svoji površini oz. vezavnem mestu nosijo peptidne fragmente, ki predstavljajo potencialne avto-antigene udeležene v boleznska stanja. Z analizo in identifikacijo peptidov, vezanih na topne MHC-I molekule, lahko prepoznamo antigene pomembne pri procesu ateroskleroze in miokardnega infarkta. V tretjem delu teze predstavim novo metodo odkrivanja potencialnih avto-antigenov udeleženih v proces ateroskleroze in po miokardnem infarktu in prikažem translacijski pristop testiranja relevantnosti odkritih peptidov v biološkem modelu ateroskleroze in miokardnega infarkta. Z metodo imunoprecipitacije smo iz krvi pacientov z akutnim koronarnim sindromom pridobljene v AZACS študiji, ki imajo dokazano aterosklerozo, izolirali peptide, ki so bili ob primerjavi z zdravimi kontrolami specifični za paciente. Peptide smo identificirali s pomočjo masne spektrometrije in primerjave aminokislinskega zaporedja z bazo človeških proteinov Uniprot. S to metodo smo odkrili več potencialnih avto-antigenov udeleženih v proces ateroskleroze in miokardnega infarkta. Za nadaljno testiranje biološkega pomena smo izbrali peptide specifične za paciente, ki so imeli na masni spektrometriji najvišjo intenzivnost signala, ali pa so bili skupni največjem številu pacientov (Keratin 8, ARID1A, Bleomicin hidrolaza ter LL-37). Mišje homologe teh peptidov smo testirali na živalskem modelu ateroskleroze ter ateroskleroze s pridruženim miokardnim infarktom. Od izbranih peptidov sta keratin II (mišji analog človeškemu keratinu 8) ter CRAMP (mišji analog človeškemu LL-37) izzvala največji imunski odziv. Keratin II je v mišjem ApoE(-/-) modelu ateroskleroze izzval predvsem efektorski odziv CD8+ limfocitov T, katerega ni bilo videti v mišjem ApoE(-/-) modelu ateroskleroze s pridruženim miokardnim infarktom. To je bilo razvidno tudi ob stimulaciji perifernih mononuklearnih celic izoliranih iz krvi pacientov s stabilno koronarno boleznijo in pacientov z akutnim koronarnim sindromom. Antimikrobni peptid LL-37, fragment proteina hCAP18, ki se nahaja v granulah nevrofilnih granulocitov in igra pomembno vlogo pri nevtrofilnem vnetju, je znan avto-antigen pri avtoimunih boleznih kot je luskavica. Pacienti z luskavico imajo večje breme ateroskleroze kot splošno, zdravo prebivalstvo in pridruženo višje tveganje za srčno-žilne dogodke. Bolniki z dokazano aterosklerozo imajo po drugi strani dvakrat višjo prevalenco luskavice kot zdravo prebivalstvo. Poleg tega je LL-37 moč najti v aterosklerotičnih lehah. Pomanjkanje mišjega ekvivalenta LL-37, imenovanega CRAMP (Cathelicidin Related Antimicrobial Peptide) v mišjem modelu zmanjša aterosklerotično breme. V namen proučevanja vpletenosti CRAMP v proces ateroskleroze pri miših smo sintetizirali skrajšano različico molekule (tCRAMP – truncated CRAMP), ki je vsebovala predvidena vezavna mesta za mišje MHC-I molekule. Stimulacija mišjih imunskih celic je izzvala močan efektorski odziv celic T, ki je bil še posebej poudarjen pri miših z razširjenim aterosklerotičnim bremenom po hranjenju z dieto bogato z maščobami. V odziv sta bili udeleženi tako CD4 kot tudi CD8 T-celična vrsta. Z imunizacijo kot način imunomodulacije smo preverili pomen tCRAMP antigena pri aterosklerozi. Izbrali smo dve imunizacijski dozi, višjo (100ug) in nižjo (20ug), ki sta imeli zelo različen biološki odziv. Nižja imunizacijska doza je preko povečanja deleža CD8+ celic T centralnega spomina ter znižanja deleža vnetnih dendritičnih celic CD11b+ zmanjšala breme aterosklerotične bolezni. Višja imunizacijska doza pa je, ravno obratno, povečala breme aterosklerotične bolezni in povzročila avto-imunsko preobrazbo imunskega sistema. Videti je bilo povišanje avto-reaktivnosti, predvsem z večjim deležem tako CD4+ kot CD8+ efektorskih celic T kot tudi povečanje deleža dendritičnih celic CD11b+, klasično celic vnetnega fenotipa. V doktorski tezi pokažem pomen tako prirojenega kot pridobljenega imunskega sistema v procesu ateroskleroze in vnetja po miokardnem infarktu ter potencialne nove načine imunomodulacije, ki se lahko uporabljajo za zmanjšanje bremena ateroskleroze. Teza predstavi pomen interlevkin 7 vnetne poti in vpletenost le-te v vnetje po miokardnem infarktu. Inhibicija receptorja interlevkina 7 zmanjša posti-ishemično vnetje v aterosklerotičnih lehah. Monoklonska protitelesa proti interlevkinu 7 so bila že prestala varnostno klinično študijo in predstavljajo zanimivo priložnost za klinično uporabo imunomodulacije po miokardnem infarktu. Doktorsko delo je pomembno tudi zaradi predstavitve novega načina raziskovanja avto-antigenov udeleženih v proces ateroskleroze. Določanje imunopeptidoma iz krvi pacientov s koronarno boleznijo je izvedljivo in privede do odkrivanja antigenov biološkega pomena. Predstavim tudi pomembno translacijsko pot raziskovanja in testiranja teh antigenov v pristopu, ki vodi od pacienta do laboratorijskega testiranja hipoteze in nazaj do pacienta in omogoča pridobivanje potencialno pomembnih kliničnih odkritij. V svojem delu tako s translacijskim pristopom pokažem poglobljen pomen avto-antigena LL-37 odkritega v vzorcih krvi pacientov po akutnem koronarnem sindromu v procesu ateroskleroze.Summary: Atherosclerosis is a chronic progressive disease process characterized by lipid accumulation inside the the intimal layer of the arterial wall. The progressive course of the disease can lead to eventual plaque rupture or erosion, causing exposure of the pro-inflammatory plaque contents to the bloodstream. This is followed by thrombus formation on the rupture site and ischemia of the tissue supplied by the affected artery. If the tissue in question is the myocardium this arterial occlusion causes myocardial infarction. Injury to the myocardium causes a systemic inflammatory reaction with pro-inflammatory cytokine release which impacts atherosclerotic plaque inflammation shown by imaging studies in both preclinical and clinical studies. Epidemiological data show that patient with myocardial infarction are at an increased risk of a recurrent ischemic event in the first year following an index event. Immunomodulation following myocardial infarction has shown to reduce the risk of a recurrent event as evidenced by both preclinical as well as clinical studies. The first part of this thesis is dedicated to a novel pathway of immunomodulation after myocardial infarction by interleukin-7 receptor inhibition. The study was conducted on a mouse ApoE(-/-) model of myocardial infarction and atherosclerosis. We show that interleukin-7 acts as a chemoattractant for monocytes in vivo, its synthesis is increased inside the ishemic myocardium following myocardial infarction and increased levels are found inside the arterial wall following a myocardial infarction as well. Immunomodulation by interleukin-7 receptor blocking by monoclonal antibody reduces the lipid content of atherosclerotic plaque as well as monocyte and macrophage infiltration into the plaque thus reducing inflammation and causing plaque stabilization. The first part of the thesis thus shows the importance of a novel innate inflammatory pathway in atherosclerosis. The human immune system is composed roughly of the innate immune system, represented by cells such as neutrophils, monocytes and macrophages, involved in atherosclerosis in both the acute and chronic stages of disease and the specific, adaptive immune system which is activated after the nonspecific phase of inflammation. The main drivers of this specific part of the immune system are lymphocytes, both T cells and B cells, as well as antigen presenting cells. The MHC/T-cell receptor coupling is a hallmark of the adaptive immune system and is caused by targeted, antigen specific binding of short peptides to both antigen recognition molecules on the cell surfaces of T cells and antigen presenting cells. Data show that activation of auto-reactive T and B cells and antigen specific responses of these cells to lipid particles occur in atherosclerosis. One of the aforementioned particles is p210, part of the apoliprotein ApoB100, a component of LDL, the culprit cholesterol particle involved in plaque inflammation. Research has shown the existence of LDL and ApoB-100 reactive T cells, but specific epitopes involved in atherosclerosis have not yet been discerned. In the second part of this thesis I show the presence of autoreactive, antigen specific CD8+ T lymphocytes, activated by p210 stimulation in an auto-immune manner. This involves recognition and binding of a T-cell receptor to an antigen loaded MHC-I molecule. Potential binding of the p210 peptide to mouse MHC-I was assessed using the Immune Epitope Data Base (IEDB) prediction site and based on binding prediction score a fluorescent pentamer composed of five bound MHC-I molecules was constructed with specificity for p210 epitope recognition. This was used to test for p210 specific CD8+ T cells in a mouse model of atherosclerosis. Mice, fed a high fat, pro-atherogenic diet showed an increase of pentamer positive CD8+ T cells, exhibiting an effector T cell phenotype. Following stimulation, these cells show clonal proliferation, a hallmark of antigen specific recognition by T cell receptor (TCR). Immunomodulation by way of immunization showed a further increase in p210 specific pentamer positive CD8+ T lymphocytes as well as a reduction in atherosclerotic burden in the p210 immunized mice. The CD8+ T cell interaction with antigen presenting cells is based on antigen binding on MHC-I molecules on the surface of antigen presenting cells and recognition of the antigen-MHC-I complex by T cell receptor on the surface of CD8+ T lymphocytes. It\u27s been shown that certain disease states cause MHC-I molecule shedding off the surface of cells. These can be detected in the bloodstream of patients and the peptides bound to these circulating, soluble MHC-I molecules represent possible auto-antigen involved in disease. Analysis of these peptides in patients with coronary artery disease and myocardial infarction can reveal previously unknown antigens important in atherosclerosis and its acute ischemic complication. The third part of this thesis shows a novel way of immunopeptidome profiling in order to reveal new potential antigens involved in atherosclerosis. Presented here is also a translational approach to testing the relevance of these novel peptide antigen in a biological model of atherosclerosis and combined atherosclerosis and myocardial infarction. Immunoprecipitation of soluble MHC-I complexes obtained from blood samples of patients with acute coronary syndrome collected from the AZACS trial revealed peptides specific to patients relative to matched control blood samples. The peptides were identified using mass spectrommetry and the amino acid sequence matched to known peptides from the Uniprot human protein database. The method enabled the identification of potential auto-antigen involved in atherosclerosis and myocardial infarction. In order to further test the biological relevance of the discovery, the peptides, specific to patients with the highest intensity on mass spectrometry were selected as well as peptides common and specific to most of the patient samples. The peptides tested were Keratin 8, ARID1A, Bleomycin Hyrolase and LL-37. Mouse homologues to these peptides were chosen to test auto-reactive responses in an Apo(-/-) mouse model of atherosclerosis as well as a combined ApoE (-/-) mouse model of atherosclerosis and myocardial infarction. Keratin II (murine homolog to human Keratin 8) and CRAMP (murine homolog to LL-37) showed highest immune responses upon stimulation and were chosen for further testing. Keratin II elicited a predominately effector CD8+ T lymphocyte response in the ApoE (-/-) model of atherosclerosis but not in a combined ApoE (-/-) murine model of atherosclerosis and myocardial infarction. This was evident also after testing human peripheral blood mononuclear cells isolated from blood samples taken from patient with either stable coronary artery disease or acute coronary syndrome. The antimicrobial peptide LL-37, a fragment of the larger hCAP-18 and a constituent of neutrophil granules is an important component of neutrophil mediated inflammation. It is a known auto-antigen in psoriasis, a classic auto-immune disease. Patients with psoriasis have increased burden of atherosclerosis compared to the healthy population and a higher risk of cardiovascular complication due to increased risk of plaque rupture. LL-37 has also been shown to be present in atherosclerotic plaque. Deficiency of mouse homolog to LL-37, named CRAMP (Cathelicidin Related Antimicrobial Peptide) in a CRAMP (-/-) murine model with atherosclerosis has shown to reduce atherosclerotic burden. To study the effect of CRAMP in a murine model of atherosclerosis, and to avoid its inherent immune properties, we synthesized a truncated version of CRAMP containing the highest predicted binding sites to mouse MHC-I molecules. Stimulation of murine immune cell populations elicited a strong effector T cell response in both CD4+ and CD8+ T cell populations which was amplified in mice fed a pro-atherogenic high fat diet. Immunization as a way of immunomodulation was used to test the biologic relevance as auto-antigen. Two different immunization doses were used, a higher 100ug dose and a lower 20 ug dose. These two doses exhibited a differential biological effect on atherosclerosis and immune activation. The lower immunization dose elicited a CD8+ Central Memory T cell response and reduced the pro-inflammatory CD11b+ Dendritic Cell populations along with a reduction in the burden of atherosclerotic disease. The higher immunization dose on the other hand increased the level of CD11b+ dendritic cells as well as caused an increase and activation of CD4+ and CD8+ Effector Memory T cells along with a significant increase in atherosclerosis. The higher immunization dose thus caused an increase in auto-reactivity to the auto-antigen and exhibited a seemingly auto-immune transformation of the murine immune system. The thesis outlines the importance of both the innate and adaptive immune systems in atherosclerosis and inflammation following myocardial infarction and potential new ways of immunomodulation effectively reducing the burden of atherosclerotic disease. It shows the importance of the interleukin-7 inflammatory pathway following myocardial infarction. Immunomodulation by inhibition of the IL-7 receptor mitigates the effect of the post-ishemic inflammation on atherosclerotic plaque. Monoclonal antibodies targeting IL-7 have already been tested in a clinical trial and thus represent a promising opportunity for immunomodulation after myocardial infarction in a clinical situation. The thesis outlines an important novel way of self-antigen discovery. Immunopeptidome analysis from patient blood samples is feasible and yields biologically relevant self-peptides involved in atherosclerosis. Furthermore we delineate an important translational pathway to discovery and testing of these antigen in a bedside-bench-bedside approach which produces data relevant to clinical practice and patient health. Our translational pathway revealed the involvement of LL-37, a known auto-antigen in psoriasis as an auto-antigen in atherosclerosis