| 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 (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. |
