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Title:SPREMEMBE HOMEOSTAZE SUBPOPULACIJ CD4+ T LIMFOCITOV IN NJIHOVIH CITOKINSKIH - STAT SIGNALNIH POTI PRI BOLNIKIH S SISTEMSKIM LUPUS ERITEMATOZUSOM
Authors:Goropevšek, Aleš (Author)
Krajnc, Ivan (Mentor) More about this mentor... New window
Pahor, Artur (Co-mentor)
Langerholc, Tomaž (Co-mentor)
Files:.pdf DOK_Goropevsek_Ales_2018.pdf (25,93 MB)
 
Language:Slovenian
Work type:Doctoral dissertation (mb31)
Organization:MF - Faculty of Medicine
Abstract:UVOD Sistemski lupus eritematozus (SLE) je sistemska avtoimunska bolezen, pri kateri kombinacija različnih genetskih dejavnikov in vplivov iz okolja pripelje do porušenja imunoregulatornih mehanizmov, ki omogočajo toleranco do lastnih antigenov. FOXP3+ regulatorni T limfociti (Treg) zavirajo proliferacijo efektorskih T limfocitov in vitro in predstavljajo bistven mehanizem periferne T limfocitne tolerance in vivo. Interferoni (IFN) tipa I, kot je IFN-alfa in homeostatski citokini, kot je IL-7, vplivajo na proliferacijo in funkcijo tako Treg, kot patogenih FOXP3- konvencionalnih T limfocitov (Tcon). Na T limfocite delujejo preko znotrajceličnih signalnih poti, ki vodijo od površinskih receptorjev do STAT proteinov, ki se po aktivaciji-fosforilaciji na specifičnih tirozinskih ostankih, prenesejo v jedro in nadzirajo številne genetske programe, vključene v nadzor proliferacije in diferenciacije CD4+ T limfocitov. Namen naše raziskave je bil preučiti STAT signalizacijo v T limfocitih periferne krvi bolnikov s SLE in ugotoviti, če so STAT signalna neravnotežja povezana s spremembami cirkulirajočih subpopulacij CD4+ T limfocitov, njihove proliferacije in aktivnosti bolezni. BOLNIKI IN METODE Analizirali smo Tcon/Treg subpopulacije, ekspresijo STAT1 proteina (STAT1 MFI) in STAT fosforilacijo (pSTAT) v T limfocitih periferne krvi bolnikov s SLE s pomočjo konvencionalne in slikovne pretočne citometrije. Vključenih je bilo 39 bolnikov s SLE, 33 zdravih kontrol in 29 bolnikov z revmatoidnim artritisom (RA). Aktivnost bolezni SLE smo sledili z določanjem kliničnega indeksa SLEDAI-2000. Z uporabo fosfo-specifične pretočne citometrije je bila naša analiza usmerjena v spremljanje signalinih dogodkov tudi v CD4+FOXP3+ T limfocitih in neposredno primerjavo STAT signalizacije v Tcon in subpopulacijah CD4+FOXP3+ T limfocitov pri bolnikih s SLE. Da smo lahko ocenili odnos med STAT signalnimi spremembami in proliferacijo subpopulacij CD4+ T limfocitov, smo hkrati določali tudi ekspresijo indikatorja proliferacije Ki-67 v Tcon and Treg subpopulacijah. REZULTATI Nivoji STAT1 MFI, ki so bili v primerjavi z bolniki z RA in zdravimi kontrolami statistično značilno višji v CD4+ T limfocitih bolnikov s SLE, so bili v statistično značilni pozitivni korelaciji z indeksom aktivnosti bolezni. STAT1 MFI je bil najvišji v CD45RA-FOXP3hi aktivirani Treg (aTreg) subpopulaciji, ki je kazala tudi najvišji fosforilacijski odgovor STAT1 med CD4+ T limfociti in značilno znižanje ekspresije Ki-67 po stimulaciji z rekombinantnim humanim IFN-alfa in vitro. V primerjavi z zdravimi kontrolami so bile pri bolnikih s SLE statistično značilno nižje tako koncentracije aTreg, kot tudi odstotek Ki-67+ celic med aTreg, ki je bil v statistično značilni negativni korelaciji s STAT1 MFI v CD4+ T limfocitih. CD4+ T limfocite bolnikov s SLE je označevala tudi od IL-7 odvisna STAT5 aktivacija, ki se je kazala z značilno zvišanimi bazalnimi nivoji pSTAT5 v primerjavi s kontrolnima skupinama. Nivoji pSTAT5 so bili statistično značilno višji v Tcon kot v FOXP3+ subpopulaciji CD4+ T limfocitov bolnikov s SLE. V nasprotju z aTreg, so Tcon bolnikov s SLE kazali značilno višjo ekspresijo Ki-67, ki je bila v statistično značilni pozitivni korelaciji z nivoji pSTAT5 v CD4+ T limfocitih. ZAKLJUČEK Naši rezultati kažejo, da je ojačana STAT1 signalizacija vpletena v porušeno homeostazo aTreg pri bolnikih s SLE in bi lahko predstavljala tudi potencialni označevalec aktivnosti bolezni. Od IL-7-odvisna bazalna fosforilacija STAT5, ki je bila značilno višja v Tcon kot v Treg bolnikov s SLE in je bila povezana z ekspresijo Ki-67 v Tcon, pa lahko daje proliferativno prednost Tcon v primerjavi z aTreg. Inhibicija takšne neuravnotežene aktivacije STAT5 bi lahko s selektivnim učinkom na proliferacijo patogenih-konvencionalnih CD4+ T limfocitov ponovno vzpostavila homeostatsko ravnotežje med Tcon in supresivnimi-aktiviranimi Treg in bi lahko predstavljala obetajoč therapevtski pristop pri SLE.
Keywords:avtoimunske bolezni, citokini, interferoni, STAT signalne poti, sistemski lupus eritematozus, CD4+ T limfociti, regulatorni T limfociti
Year of publishing:2018
Source:Maribor
COBISS_ID: 293939456 Link is opened in a new window
NUK URN:URN:SI:UM:DK:HKOSQQIF
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Downloads:102
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Secondary language

Language:English
Title:CHANGES IN HOMEOSTASIS OF CD4+ T CELL SUBSETS AND THEIR CYTOKINE-STAT SIGNALING PATHWAYS IN PATIENTS WITH SYSTEMIC LUPUS ERYTHEMATOSUS
Abstract:INTRODUCTION Systemic lupus erythematosus (SLE) is a systemic autoimmune disease in which several contributing genetic and environmental factors combine to lead to breakdown of immunoregulatory mechanisms assuring tolerance to self-antigens. FOXP3+ regulatory T cells (Tregs) suppress proliferation of effector CD4+ T cells in vitro and constitute an essential mechanism of peripheral T-cell tolerance in vivo. Type I interferons (IFN), such as IFN-alpha and homeostatic cytokines, such as IL-7, influence proliferation and function of both Treg and pathogenic FOXP3- conventional T-cells (Tcon). They act on T cells, through their intracellular signaling pathways that lead from surface receptors to STAT proteins, which upon being activated-phosphorylated at specific tyrosine residues, translocate to the nucleus and control numerous gene programs involved in regulation of proliferation and CD4+ T-cell differentiation. The aim of this study was to investigate STAT signaling in blood T cells from patients with SLE and to determine whether the STAT signaling imbalances are related to changes in circulating CD4+ T cell subsets, their proliferation and disease activity. PATIENTS AND METHODS We assessed Tcon/Treg subsets, STAT1 protein expression (STAT1 MFI) and phosphorylation of STAT (pSTAT) in blood T-cells from SLE patients by using conventional and imaging flow cytometry. 39 SLE patients, 33 healthy controls and 29 rheumatoid arthritis (RA) patients were included. SLE disease activity was assessed by serial SLEDAI-2000 disease scoring system. By using the phospho-specific flow cytometry, our analysis was designed to monitor signaling events also in CD4+FOXP3+ T cells and to directly compare STAT signaling in Tcon and subsets of CD4+FOXP3+ T cells in SLE patients. In order to assess the relationship between STAT signaling changes and proliferation of CD4+ T cell subsets, the expression of Ki-67 was determined as an indicator of proliferation in Tcon and Treg subsets. RESULTS Levels of STAT1 MFI, which were significantly increased in SLE CD4+ T cells when compared to RA patients and healthy controls, were positively correlated with disease activity. The highest STAT1 MFI was found in CD45RA-FOXP3hi activated Treg (aTreg) subset, which demonstrated the highest STAT1 phosphorylation responses among CD4+ T cells and significant decrease in proliferation marker Ki-67 expression after in vitro stimulation with recombinant human IFN-alpha. In addition to decreased absolute aTreg counts, percentage of Ki-67+ aTreg was also significantly decreased in SLE patients compared to healthy controls and was negatively correlated with CD4+ T cell STAT1 MFI. CD4+ T cells from SLE patiens were characterized also by IL-7-dependent STAT5 activation, expressed as significantly increased basal levels of pSTAT5. pSTAT5 levels were significantly higher in Tcon than in FOXP3+ subset of CD4+ T-cells from SLE patients. In contrast to aTreg, Tcon from SLE patients displayed significantly increased expression of Ki-67, which was positively correlated with pSTAT5 levels in CD4+ T cells. CONCLUSION Our findings indicate that augmented STAT1 signaling may be involved in perturbed aTreg homeostasis and could represent a possible marker of SLE disease activity. IL-7-dependent basal STAT5 phosphorylation, which was significantly higher in Tcon than in Treg from SLE patients and was related to Tcon Ki-67 expression, may confer proliferative advantage to Tcon over aTreg. Inhibiton of such imbalanced activation of STAT5 could, by selectively affecting proliferation of pathogenic-conventional CD4+ T cells, restore homeostasis between Tcon and suppressive-activated Treg and may represent a promising therapeutic approach in SLE.
Keywords:autoimmune diseases, cytokines, interferons, STAT signaling pathways, systemic lupus erythematosus, CD4+ T cells, regulatory T cells


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