Hepatitis B and Hepatitis C Virus Infection Promote Liver Fibrogenesis through a TGF-β1–Induced OCT4/Nanog Pathway

Wenting Li; Xiaoqiong Duan; Chuanlong Zhu; Xiao Liu; Andre J. Jeyarajan; Min Xu; Zeng Tu; Qiuju Sheng; Dong Chen; Chuanwu Zhu; Tuo Shao; Zhimeng Cheng; Shadi Salloum; Esperance A. Schaefer; Annie J. Kruger; Jacinta A. Holmes; Raymond T. Chung; Wenyu Lin

doi:10.4049/jimmunol.2001453

Key Points

HBV/HCV coinfection enhanced fibrogenesis compared with HBV or HCV monoinfection.
HBV, HCV, and HBV/HCV induced liver fibrogenesis through the TGF-β1–OCT4/Nanog pathway.

Visual Abstract

Abstract

Hepatitis B virus (HBV)/hepatitis C virus (HCV) coinfection accelerates liver fibrosis progression compared with HBV or HCV monoinfection. Octamer binding transcription factor 4 (OCT4) and Nanog are direct targets of the profibrogenic TGF-β1 signaling cascade. We leveraged a coculture model to monitor the effects of HBV and HCV coinfection on fibrogenesis in both sodium taurocholate cotransporting polypeptide–transfected Huh7.5.1 hepatoma cells and LX2 hepatic stellate cells (HSCs). We used CRISPR-Cas9 to knock out OCT4 and Nanog to evaluate their effects on HBV-, HCV-, or TGF-β1–induced liver fibrogenesis. HBV/HCV coinfection and HBx, HBV preS2, HCV Core, and HCV NS2/3 overexpression increased TGF-β1 mRNA levels in sodium taurocholate cotransporting polypeptide–Huh7.5.1 cells compared with controls. HBV/HCV coinfection further enhanced profibrogenic gene expression relative to HBV or HCV monoinfection. Coculture of HBV and HCV monoinfected or HBV/HCV coinfected hepatocytes with LX2 cells significantly increased profibrotic gene expression and LX2 cell invasion and migration. OCT4 and Nanog guide RNA independently suppressed HBV-, HCV-, HBV/HCV-, and TGF-β1–induced α-SMA, TIMP-1, and Col1A1 expression and reduced Huh7.5.1, LX2, primary hepatocyte, and primary human HSC migratory capacity. OCT4/Nanog protein expression also correlated positively with fibrosis stage in liver biopsies from patients with chronic HBV or HCV infection. In conclusion, HBV and HCV independently and cooperatively promote liver fibrogenesis through a TGF-β1–induced OCT4/Nanog-dependent pathway.

Footnotes

This work was supported by grants from Natural Science Foundation of China (NSFC 82102383 to X.D., NSFC 81871661 to W. Lin, and NSFC 81770591 to Chuanlong Zhu), the Fundamental Research Funds for the Central Universities of China (WK9110000048 to W. Li), Natural Science Foundation of Anhui Province (Grant 1908085MH242), Sichuan Provincial Science and Technology Department (Grant 2019YJ0281), Key Project Foundation of Anhui Province, China (Grant S202104j07020097 to W. Li), Project Foundation of Academic Leader Anhui Province, China (Grant 2018H178 to W. Li), a project supported by Hainan Province Clinical Medical Center (to W. Li), U.S. Department of Health and Human Services, National Institutes of Health, National Institute of Allergy and Infectious Diseases (Grant R01AI155140 to R.T.C. and W. Lin), and Foundation for the National Institutes of Health (Grants AI069939, AI082630, DK098079, and DK108370 to R.T.C.).
W. Li., X.D., Chuanlong Zhu, R.T.C., and W. Lin designed and directed the project. W. Li, X.D., Chuanlong Zhu, X.L., and Z.T. performed experiments and analyzed data. X.L., A.J.J., M.X., Z.T., Q.S., D.C., Chuanwu Zhu, T.S., Z.C., A.J.K., J.A.H., E.A.S., and S.S. provided reagents and advice. W. Li., X.D., A.J.J., A.J.K., J.A.H., R.T.C., and W. Lin wrote and edited the manuscript with comments from all authors.
The online version of this article contains supplemental material.
Abbreviations used in this article
Dac
daclatasvir
ECM
extracellular matrix
ETV
entecavir
gRNA
guide RNA
HBV
hepatitis B virus
HBVvp
purified HBV particle
HCC
hepatocellular carcinoma
HCV
hepatitis C virus
HCVvp
purified HCV particle
HSC
hepatic stellate cell
NTCP
sodium taurocholate cotransporting polypeptide
OCT4
octamer binding transcription factor 4
PHH
primary human hepatocyte
pHSC
primary human hepatic stellate cell
qPCR
quantitative PCR