The role of liver sinusoidal endothelial cells in HCV infection

Papić, Neven (2015) The role of liver sinusoidal endothelial cells in HCV infection. PhD thesis, Sveučilište u Zagrebu.

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Abstract

INTRODUCTION: Liver sinusoidal endothelial cells (LSEC) due to their extraordinary scavenger activity are playing a pivotal role in blood-borne virus clearance. LSEC account for the 20% of hepatic cells and are unique organresident cell population with diverse functions, including degradation of bacterial by-products, antigen presentation and induction of tolerance. While these processes are particularly relevant to HCV infection, the role of LSEC in chronic hepatitis C is not defined. ----- MATERIALS AND METHODS: Aim of this study was to apply systems biology approaches to evaluate the role of LSEC in HCV infection. Poly(A) RNAs from HCV, MOCK or LPS treated primary LSEC cultures were analyzed by RNAsequencing (Illumina) to identify differentially expressed genes (DEG) and biological pathways. Cell transcriptomes were compared to similar analysis with mild (no fibrosis) and severe (cirrhosis) hepatitis C livers, hepatoma and Kupffer cells. ----- RESULTS: Following exposure LSEC internalized HCV, but failed to support HCV replication. LSEC overall displayed 754, 245 and 2543 DEG at 8, 24 and 48h after HCV exposure, respectively. While LPS stimulation triggered exceptionally potent activation, HCV in general induced a downregulation of inflammatory signals. The key innate immune response pathways were significantly downregulated (RIG-I and TLR-signaling pathways), which manifested by the diminished PRR transcripts expression with subsequent tuning down of the expression of the genes encoding for adaptor proteins, JAK-STAT, NF-κB and IRF signaling cascades resulting in the reduced expression of cytokine genes and reflecting in the deficiencies of innate immune response. Molecular mechanisms of these processes involve: upregulation of transcriptional factors essential for the induction of antiinflammatory state (such as MAFB, NUPR1, IL33, KLF15, CEBPD) and downregulation of those shown to promote inflammatory responses (ETV1, MITH, HDAC9, EGR3, IRF6). Meanwhile, a plethora of immunomodulatory genes were significantly upregulated (ACP5, A2M, C1QTNF1, NT5E, SERPING1, BMPER) that might extend to surrounding cells and attenuate liver inflammation. Gene pathway analysis further highlighted enriched KEGG pathways related to cell scavenger functions (e.g. Fc gamma R-mediated phagocytosis, Cell adhesion molecules, Focal adhesion, Leukocyte transendothelial migration, Axon guidance), innate immune responses and cancer development (Pathways in cancer, Apoptosis, p53 signaling, Wnt signaling). In contrast to KC and hepatocytes, TGF- β signaling pathway was significantly downregulated in HCV infected LSEC. The system biology approach helped to identify distinct gene expression profiles in HCV infected LSECs, KCs and hepatocytes. Macrophages demonstrated a broad increase in IL-1β and NFκB-responsive proinflammatory cytokines and chemokines, which correlated with increasing severity of liver disease. In contrast, analysis of genes commonly expressed in LSEC with HCV infected liver showed significant overlap of pathways associated with angiogenesis, adhesion, ECMorganization, and regulation of defense/immune responses. ----- CONCLUSION: This is the first comprehensive gene expression analysis of LSEC that provided insight into the broad portrait of genomic changes associated with HCV infection. These genes are critical components of host immune and inflammatory pathways and provide new evidence that LSEC downregulate inflammation during HCV infection.

Abstract in Croatian

UVOD: Jetrene sinusoidalne endotelne stanice (LSEC) igraju ključnu ulogu u eliminaciji krvlju prenosivih virusa. LSEC čine 20% stanica jetre i predstavljaju jedinstvenu staničnu populaciju važnu za degradaciju bakterijskih produkata, prezentaciju antigena i indukciju «imunološke tolerancije». Iako su ti procesi od posebne važnosti u patogenezi HCV infekcije, uloga LSEC u kroničnom C hepatitisu je nepoznata. ----- MATERIJALI I METODE: Cilj ove studije je primjenom metodologije sistemske biologije istražiti ulogu LSEC u HCV infekciji. Pol II selektirane RNK iz HCV, MOCK ili LPS stimuliranih LSEC bile su analizirane upotrebom RNK-sekvenciranja cijelog staničnog transkriptoma, s ciljem identificikacije diferencijalno eksprimiranih gena (DEG) i staničnih putova. Ekspresija gena je uspoređena s uzorcima jetre blage i uznapredove fibroze, hepatocitima i Kupfferovim stanicama. ----- REZULTATI: LSEC ne podupiru HCV replikaciju, ali virus efikasno ulazi u stanicu. U LSEC identificirano je 754, 245 i 2543 DEG, 8, 24 i 48 sati nakon HCV ekspozicije. Dok je LPS stimulacija potaknula izuzetno snažnu aktivaciju upalnog odgovora, HCV je općenito doveo do utišavanja istog. Ključni putevi prirođene stanične imunosti bili su značajno utišani (RIG-I i TLR-signalni putovi), što se manifestiralo sniženom ekspresijom PRR gena, adaptorskih proteina, JAK-STAT, NF-κB i IRF signalnih kaskada, rezultirajući smanjenom ekspresijom citokina te odražavajući neučinkovitost imunološkog odgovora. Molekularni mehanizmi ovih procesa uključuju: aktivaciju transkripcijskih regulatora ključnih za indukciju protuupalnog odgovora (MAFB, NUPR1, IL33, KLF15, CEBPD) i inhibiciju onih koji promoviraju upalni odgovor (ETV1, MITH, HDAC9, EGR3, IRF6). Istovremeno solubilni imunomodulatori (ACP5, A2M, C1QTNF1, NT5E, SERPING1, BMPER) bili su pojačano eksprimirani, a potencijalno utišivaju upalni proces u jetri. Daljnja analiza je identificirala obogaćenim KEGG stanične putove povezane sa staničnom eliminacijskom funkcijom (Fc gamma R-fagocitozu, stanične adhezijske molekule, tkivna adhezija, transendotelna migracija leukocita, imuni semaforini), prirođenom i stečenom imunošću, karcinogenezom (putovi u razvoju karcinoma, apoptoza, p53- i Wnt- stanični putovi). Za razliku od KC i hepatocita, TGF-β signalni put je bio značajno utišan u LSEC. Različiti profili ekspresije gena su identificirani u LSEC, KC i hepatocitima, sugerirajući njihovu različitu ulogu u patogenezi HCV infekcije. U makrofazima je identificirana pojačana ekspresija proupalnih citokina i kemokina, koja korelira sa stadijem jetrene bolesti. Istovremeno, usporedba LSEC i uzoraka HCV-om inficirane jetre pokazala je značajnu podudarnost u staničnim putovima povezanim s angiogenezom, adhezijom, organizacijom ekstracelularnog matriksa i regulacijom imunološkog odgovora. ----- ZAKLJUČAK: Ovo je prva analiza ekspresije gena u LSEC koja je identificirala promjene u transkriptomu povezane s HCV infekcijom. Identificirani geni čine ključne kompontente staničnog imunološkog i upalnog odgovora i sugeriraju da LSEC utišavaju upalu u tijeku HCV infekcije.

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