Spatial and functional links between cellular virological state and progression of liver fibrosis in chronic hepatitis B

Abstract

Abbreviations: 38 HBV, Hepatitis B Virus; CHB, chronic hepatitis B; ECM, extracellular matrix; HepPar-1, 39 hepatocyte Paraffin 1; HNF4a, Hepatocyte nuclear factor 4 alpha; hepatitis B e 40 antigen (HBeAg); covalently closed circular DNA (cccDNA); IT, immune tolerant; IA, 41 immune active; ENH, hepatitis B e antigen negative hepatitis; ISH, in situ 42 hybridization; HSC, hepatic stellate cells; HBsAg, Hepatitis B Surface Antigen; AgNOR, 43 argyrophilic nucleolar organizer region, IP-10, Interferon gamma-induced protein 10. 44 45 46 3 Abstract 47 Chronic Hepatitis B Virus (HBV) infection is strongly associated with the progression of 48 liver fibrosis, cirrhosis and hepatocellular carcinoma. Despite intensive study, the 49 detailed mechanisms leading to HBV induced liver disease have not been fully 50 elucidated. Previously, we reported a mosaic distribution of viral antigens and nucleic 51 acids at single-cell level in liver tissues of chronic hepatitis B (CHB) patients and 52 proposed a 'three-stage model' of HBV infection in vivo. Here, we explored whether 53 the different stages at cellular level is functionally linked with fibrogenesis. We 54 observed a tight spatial relationship between the invasion of collagen fibers and 55 transitions from S-rich to DNA-rich stage. While S-rich cells mainly localized within 56 minimally fibrotic tissue, DNA-rich cells were often closely surrounded by a milieu of 57 stiffened extracellular matrix (ECM). cDNA microarray and subsequent validation 58 analyses revealed that S-rich cells manifested elevated ribosomal proteins and 59 oxidative phosphorylation genes in a disease phase-dependent manner. On the other 60 hand, DNA-rich cells exhibited gradually deteriorated expression of hepatocyte-61 specific antigen and transcriptional regulator in parallel with the progression of hepatic 62 fibrosis. Finally, during fibrogenesis, inflammatory genes such as IP-10 were found to 63 be expressed in both portal infiltrated cells and surrounding parenchymal cells which 64 resulted in suppressed antigen expression. Taken together, we propose that liver 65 inflammation and accompanying fibrogenesis is spatially and functionally linked with 66 the transition of virological stages at cellular level. These transitions occur possibly due 67 to an altered hepatocyte transcription profile in response to a transformed ECM 68 4 environment. The collective viral and host activities shape the histological alterations 69 and progression of liver disease during CHB infection. (262 words) 70 71 72 5 Introduction 73 Globally, hepatitis B virus (HBV) chronically infects over 240 million people. Patients 74 with chronic hepatitis B (CHB) often develop liver fibrosis, cirrhosis and hepatocellular 75 carcinoma, which results in over 780,000 deaths annually(1). Currently available 76 antivirals such as nulceot(s)ide analogs and pegylated interferon can effectively control 77 viremia and lower the risk of CHB related liver disease, but sustained off-treatment 78 responses are still rare(2). Novel therapies a...