Study On The DUOX2~+ACE2~+ Cholangiocyte Subpopulation In Primary Biliary Cholangitis

Backgroud:Primary biliary cholangitis(PBC)is an autoimmune liver disease characterized by selective destruction of small-and medium-sized intrahepatic bile ducts with intrahepatic granuloma formation.It is believed that genetic factors,environmental inducements,intestinal flora and individual immune status are involved in the pathogenesis of PBC.The single cell RNA sequencing(sc RNA-seq)is an advanced technique for us to analyze the expression of transcriptome at single cell perspective.In less than 10 years,it has been widely applied to the fields about tumor,immunology,embryology,neurobiology,and stem cell research.It is necessary to study the pathophysiological mechanism of PBC based on the single cell RNA perspective to provide new targets and theoretical basis for the treatment of PBC.Methods:Liver samples from control(CTR)and PBC patients were used for sc RNA-seq and bioinformatics analysis was performed.Fluorescence activated cell sorting(FACS),quantitative real time polymerase chain reaction(q-PCR)and multiplex immunofluorescence(IF)analyses were futher used to verify the specific DUOX2~+ACE2~+(dual-oxidase-2,DUOX2;angiotensin-converting-enzyme-2,ACE2)cholangiocyte subpopulation expression and explore the correlation analysis between the cholangioctes and diseases.The mouse PBC model was constructed by coupling 2-Octynoic acid(2-OA)with Bovine Serum Albumin(BSA).Moreover,Partial deletion of DUOX2~+ACE2~+cholangiocytes by adeno-associated virus 8-double-floxed inverse open reading frame(AAV8-Cre-DIO)system were used in mouse PBC model to research the function of DUOX2~+ACE2~+cholangiocytes in the PBC progression.Results:We constructed the liver cell atlas of CTR and PBC,which suggesting that the liver cell could be divided into even 30 subgroups and11 cell lines.It also revealed the proportion of each cell line in normal and disease where the proportion of bile duct cells and liver cells decrease but immune cells increase.Bioinformatics showed that T and B cells were activated in PBC,and the immune response mediated by bile duct cell subsets was enhanced.Furthermore,sc RNA-seq showed that bile duct cells could be divided into 8 subgroups in which DUOX2~+ACE2~+cholangiocytes have the function of high bile secretion and bile acid transport and sepcifically reduced in PBC disease.The unique DUOX2~+ACE2~+cholangiocytes were consistently verified in normal human and mouse livers.A selective decrease of DUOX2~+ACE2~+cholangiocytes was observed in the liver of PBC patients,but not in patients with secondary sclerosing cholangitis(SSC),obstructive cholestasis(OC),and nonalcoholic steatohepatitis(NASH).The PBC mouse model was further constructed and DUOX2~+cholangiocyte subgroup specific knockout was performed.Together,it was found that DUOX2~+ACE2~+could promote the development of PBC to a certain extent.Conclusions:The liver cell map of CTR and PBC were constructed and cell subsets were functionally analyzed by bioinformatics.DUOX2~+ACE2~+cholangoicytes were identified and their expression and function were verified in human and mouse livers.The selective reduction of DUOX2~+ACE2~+cholangoicytes in the liver of PBC patients was also found.Partial selective knockdown of DUOX2~+ACE2~+cholangoicyte subsets was associated with severity and cholestasis in PBC model mice.These results suggest that DUOX2~+ACE2~+bile duct subsets may play a role as the target cells of PBC disease.