Title THE ROLE OF NKG2D-MEDIATED IMMUNOSENSING IN THE
PATHOGENESIS OF NON-ALCOHOLIC FATTY LIVER
DISEASE Title (english) ULOGA IMUNOSNOG PREPOZNAVANJA POSREDOVANOG
RECEPTOROM NKG2D U PATOGENEZI NEALKOHOLNE
MASNE BOLESTI JETRE Author Karlo Mladenić Mentor Bojan Polić (mentor)Committee member Felix Martinus Wensveen (predsjednik povjerenstva)Committee member Anna Mrzljak (član povjerenstva)Committee member Hrvoje Jakovac (član povjerenstva)Granter University of Rijeka Defense date and country 2023-12-05, Croatia Scientific / art field, BIOMEDICINE AND HEALTHCARE Universal decimal classificationUDC ) 61 - Medical sciences ThesaurusMESH - Medical Subject Headings )
Non-alcoholic Fatty Liver Disease
etiology
Non-alcoholic Fatty Liver Disease
immunology
Inflammation
NK Cell Lectin-Like Receptor Subfamily K
Interleukin-17
Abstract Objectives: Non-alcoholic fatty liver disease (NAFLD) comprises a spectrum of clinical entities ranging from benign steatosis to cirrhosis. Transition from non-alcoholic fatty liver (NAFL) to non alcoholic steatohepatitis (NASH) represents a key event in the pathophysiology of NAFLD that may lead to fibrosis and hepatocellular carcinoma development. The general consensus is that activation of pro-inflammatory immune effectors plays a crucial role in the transition from NAFL to NASH [1]. However, what triggers inflammation in NAFLD is unknown. Preliminary findings suggested that NKG2D signaling contributes to the development of NASH. Our objective therefore was to elucidate the role of NKG2D mediated immune-sensing in early NASH pathogenesis. Materials and methods: To investigate NAFLD pathogenesis, age- and sex- matched mice were fed ad libitum with normal chow diet (NCD) or steatosis-steatohepatitis diet (SSD) for indicated periods. To determine the impact of SSD feeding on NASH pathogenesis, histological changes in the levels of fibrosis and steatosis were determined by the Picro-Sirius Red staining of the liver tissue, along with activation of hepatic stellate cells by specific immunohistochemical staining to αSMA. Expression of the ligands for the activating receptor NKG2D was measured directly on ex vivo isolated hepatocytes from SSD-fed mice by flow cytometry, or by qPCR after in vitro cultivation of primary hepatocytes in different metabolic conditions. The roles of different immune cells and cytokines in NKG2D-mediated NASH development were determined by antibody mediated neutralization or in different genetically modified mice. Changes on total transcriptome levels between C57BL/6J and Klrk1-/- mice which were fed with NCD and SSD, were determined by liver bulk RNA-sequencing. Mass spectrometry was used to determine changes in the lipidomic profile of the murine liver upon SSD. Human liver biopsies of NAFLD patients were analyzed for NKG2D ligand expression and IL-17 production by immunohistochemical staining, while liver biopsies and blood was analyzed by flow cytometry to determine immune changes. Results: Cholesterol accumulation in metabolically-stressed hepatocytes induce expression of ligands for the activating immune receptor NKG2D. Tissue-resident innate-like T cells, especially γδ T cells, are activated through NKG2D and secrete IL-17A. IL-17A induces production of chemokines by hepatocytes that recruit pro-inflammatory cells into the liver, causing NASH and fibrosis. NKG2D-deficient, Tcrd-/- and AlbCre Il17rafl/fl mice had significantly reduced fibrosis in dietary models for NASH. Notably, the frequency of IL-17A+ γδ T cells in the blood of NAFLD patients positively correlated with liver pathology.
Conclusion: Our findings identify a key molecular mechanism through which stressed hepatocytes trigger inflammation in context of NAFLD, with great potential as therapeutic targets and/or disease biomarkers in the future.
Abstract (english) Ciljevi: Nealkoholna masna bolest jetre (NAFLD, od engl. Non-alcoholic fatty liver disease)
obuhvaća niz kliničkih entiteta u rasponu od benigne steatoze do ciroze. Prijelaz iz nealkoholne
masne jetre (NAFL, od engl. Non-alcoholic fatty liver) u nealkoholni steatohepatitis (NASH)
predstavlja ključni događaj u patofiziologiji NAFLD-a koji može dovesti do razvoja fibroze i
hepatocelularnog karcinoma. Opći je konsenzus da aktivacija proupalnih imunosnih efektora igra
ključnu ulogu u tranziciji NAFL-a u NASH [1]. Međutim, nije poznato što izaziva upalu kod NAFLDa. Preliminarni rezultati sugeriraju da NKG2D signalizacija doprinosi razvoju NASH-a. Stoga je
naš cilj bio razjasniti ulogu NKG2D posredovanog prepoznavanja u ranoj patogenezi NASH-a.
Materijali i metode: Kako bi se istražila patogeneza NAFLD-a, miševi iste dobi i spola hranjeni
su ad libitum normalnom prehranom (NCD) ili steatoza-steatohepatitis dijetom (SSD) tijekom
naznačenih razdoblja. Kako bi se odredio utjecaj hranjenja SSD-om na patogenezu NASH-a,
histološke promjene u razinama fibroze i steatoze određene su bojenjem jetrenog tkiva PicroSirius Red-om, zajedno s aktivacijom zvjezdastih stanica jetre specifičnim imunohistokemijskim
bojenjem na αSMA. Ekspresija liganada za aktivirajući receptor NKG2D mjerena je izravno na ex
vivo izoliranim hepatocitima iz miševa hranjenih SSD-om metodom protočne citometrije ili qPCRom nakon uzgoja primarnih hepatocita u različitim metaboličkim uvjetima in vitro. Uloge različitih
imunoloških stanica i citokina u razvoju NKG2D-posredovanog NASH-a određene su
neutralizacijom specifičnim protutijelima ili korištenjem različitih genetski modificiranih miševa.
Promjene na ukupnim razinama transkriptoma između C57BL/6J i Klrk1-/- miševa koji su hranjeni
s NCD-om i SSD-om, određene su cijelogenomskim sekvenciranjem i analizom RNA jetre.
Masena spektrometrija korištena je za određivanje promjena u profilu lipidoma mišje jetre nakon
SSD-a. Na biopsijama ljudske jetre pacijenata s NAFLD-om ekspresija NKG2D liganda i
proizvodnja IL-17 određena je imunohistokemijskim bojenjem, dok su biopsije jetre i krv
analizirane protočnom citometrijom kako bi se odredile imunološke promjene.
Rezultati: Akumulacija kolesterola u hepatocitima pod metaboličkim stresom inducira izražaj
liganada za aktivacijski imunološki receptor NKG2D. T-stanice nalik urođenima, posebno γδ Tstanice, aktiviraju se putem NKG2D receptora i izlučuju IL-17A. IL-17A potiče hepatocite da
proizvode kemokine koji privlače proupalne stanice u jetru, uzrokujući NASH i fibrozu. NKG2Ddeficijentni, Tcrd-/-
i AlbCre Il17rafl/fl miševi nisu razvili fibrozu u modelu prehrane koja inducira
NASH. Značajno, učestalost IL-17A+ γδ T stanica u krvi pacijenata s NAFLD-om pozitivno je
korelirala s patologijom jetre.
Zaključak: Naša otkrića identificiraju ključni molekularni mehanizam putem kojeg hepatociti uslijed metaboličkog stresa pokreću upalu u kontekstu NAFLD-a, s velikim potencijalom kao terapeutske mete i/ili prognostički biomarkeri u budućnosti.
Keywords
Inflammation
Interleukin-17A
Nonalcoholic Fatty Liver Disease
Nonalcoholic Steatohepatitis
NKG2D Receptor
Keywords (english)
Interleukin-17A
Nealkoholna masna bolest jetre
Nealkoholni Steatohepatitis
NKG2D receptor
Upala.
Language english URN:NBN urn:nbn:hr:184:739346 Promotion 2024 Study programme Title: Biomedicine Postgraduate (doctoral) study programme Type of resource Text File origin Born digital Access conditions Access restricted to students and staff of home institution Terms of use Created on 2024-02-15 09:54:46
