Isogenic bile duct organoid-macrophage coculture for characterizing the roles of human cytomegalovirus infection in biliary atresia

Abstract

Biliary atresia (BA) is a progressive inflammatory fibrosclerosing disease of the entire biliary system characterized by neonatal bile duct obstruction. Its pathogenesis is still unknown. Recent clinical investigation observed a positive correlation between BA pathogenesis and Human Cytomegalovirus (HCMV) infection; however, it is not clear how HCMV infection contributes to BA. HCMV infection in the liver and macrophage-mediated innate immunity are proposed as one of the key players in BA. In this study, a human isogenic pluripotent stem cell (hiPSC)-derived cholangiocytes (bile duct cells) and macrophages co-culture model was developed to investigate (i) the interactions between HCMV-infected macrophages and cholangiocytes; and (ii) the roles of HCMV-infected macrophages in the pathogenesis of BA. Immunohistochemistry was conducted on human liver biopsies to quantitate HCMV infection in BA and non-BA livers. hiPSC-derived macrophages were infected with HCMV (multiplicity of infection = 1.0). HCMV-infected and uninfected macrophages were co-cultured with the isogenic hiPSC-derived cholangiocytes. Morphological/molecular/single-cell RNA-sequencing (sc-RNA-seq) analyses were conducted on the co-cultures to investigate the impacts of HCMV infection on immune responses and development of macrophages and cholangiocytes. BA patients’ livers (n=40) showed a statistical significantly higher bile duct HCMV infection than that of non-BA livers (n=40) (BA vs non-BA; 45% vs 7.5%). Furthermore, CD86+ pro-inflammatory M1 macrophages were found to accumulate around the HCMV-infected bile ducts in BA patients’ liver. In hiPSC-derived cholangiocytes and macrophages co-culture, in contrast to the development of well-expanded mono-cystic organoids in the uninfected mock culture, multi-cystic, deformed, and poorly expanded organoids were formed in the HCMV-infected co-culture. Upon activation by HCMV infection, macrophages expressed pro-inflammatory mediators (IL6, CCL3, CCL4, CCL8, and CCL20), activated NLRP3 Inflammasome and the complement system. Bile duct cells of the post-infection day 1 (PD1) HCMV infected co-culture were proliferative, expressed elevated levels of pro-inflammatory mediators (IL8, IL-1B, CXCL1, CXCL3, CXCL5) and upregulated pro-inflammatory pathways. Cellchat cell-cell interaction analysis discovered that both the signaling patterns and interaction strength between macrophage and cholangiocytes were reduced in HCMV-infected co-culture. Subsequent ligand-receptor analysis revealed that macrophages derived IL-6 interacts with IL6ST receptors on cholangiocytes, and IL-6/IL6ST interaction induces a robust inflammation in cholangiocytes by upregulation of pro-inflammatory mediators. In PD5 infected co-culture, we observed an expansion of a unique cholangiocyte cluster that exhibited persistent HCMV infection expressing HCMV viral gene UL22A. Together these findings indicated that HCMV infection induces inflammation, abnormal development and proliferation of bile duct cells in the liver. HCMV infection in congenital or prenatal stages could induce bile duct inflammation by robust activation of innate immune systems. Persistent HCMV-mediated innate immune responses could induce destruction of the biliary network and contribute to BA pathogenesis.