Gut Microbiota Contributes To Dimorphism In Primary Biliary Cholangitis

Primary biliary cholangitis(PBC)is an autoimmune disease,characterized by small and medium-sized intrahepatic bile ducts injury,granulomatous lymphocytic infiltration,and high titer specific anti-automitochondrial antibody(AMA)in serum.However,the etiology is unknown.With the loss of immune tolerance,the liver is attacked by an autoimmune system,which causes biliary obstruction and chronic cholestasis,resulting in biliary epithelial cells injury.The disease can progress to liver fibrosis,cirrhosis,and liver failure.The disease occurs at a female-dominant phenotype.Currently,the generally accepted ratio of female to male patients is 9:1.Epidemiological studies show that at least one in 1000 women over the age of40 years have PBC,but its mechanism is still unclear.Sex differences exist in innate and adaptive immune responses,resulting in sex-specific outcomes from autoimmune and infectious diseases,malignancies,and vaccines.Therefore,it is important in clinical treatment to develop sex-specific treatments for autoimmune diseases.Our previous study showed that the infiltration of liver immune cells in PBC murine model,dnTGFβRⅡ mice was significantly increased compared with wild type mice.Pathological staining demonstrates that the liver of dnTGFβRⅡ mice showed liver injury,such as inflammation in the portal area,bile duct injury and granuloma formation,and high titer of specific anti-automitochondrial antibody(AMA)in the serum,similar with clinical PBC patients.Our study explored the mechanism of sexual dimorphism in PBC through dnTGFβRⅡ mice.Our results are listed below:(1)Female dnTGFβRⅡ mice developed a more severe autoimmune disease that was similar to clinical PBC patientsWe found dnTGFβRⅡ mice showed female dominance similarly to PBC patients,including more severe lymphocytic infiltration and a higher relative expression of inflammatory cytokines in the female liver.Furthermore,female dnTGFβRⅡ mice developed higher levels of inflammatory cytokines in serum,including IFN-γ and TNF-α,compared to the male mice,indicating that female dnTGFβRⅡ mice show more severe autoimmune disease than male littermates.These results suggest that dnTGFβRⅡ mice can mimic the clinical phenotype and sexual dimorphism of PBC disease.(2)The sexual dimorphism of dnTGFβRⅡ mice was not dependent on the protection of androgen.Many studies have shown that androgens play a protective role in autoimmune diseases.To investigate whether androgens protect male dnTGFβRⅡ mice from development of PBC disease,we removed the testicles of male dnTGFβRⅡ mice at the age of 4 weeks to eliminate the influence of androgen before the age of sexual maturation.Intriguingly,elimination of androgen through gonadectomy in male dnTGFβRⅡ mice did not influence disease severity,including no change in the infiltration levels of immune cells and histopathological features in liver,supporting that androgen is an unessential factor in sustaining the female dominance.(3)The commensal gut microbiome played a critical role in determining the observed sexual differences in dnTGFβRⅡ mice.Previous studies indicate that the composition and abundance of the gut microbiome changes affect the development of liver disease.To explore whether the gut microbiome is involved in regulating sexual dimorphism of dnTGFβRⅡ mice,we add a mixture of four kinds of antibiotics into drinking water to clear the commensal gut microbiota.We found that most of the differences between male and female dnTGFβRⅡ mice disappeared by feeding dnTGFβRⅡ mice with antibiotic,including lymphocyte infiltration,most part of inflammatory cytokine levels in the liver,and the level of IFN-γ and TNF-α in serum,suggesting that intestinal microbes play a key role in determining the observed sexual differences in dnTGFβRⅡ mice.Lastly,to explore the potential mechanism of the gut microbiota in sexual differences of dnTGFβRⅡ mice,we performed metagenomic sequencing of gut microbiota based on the gut content samples from dnTGFβRⅡ mice.We found there were female-specific or male-specific microflora in dnTGFβRⅡ mice.Simultaneously,the gut microbiota composition was similar in female and male dnTGFβRⅡ mice,including Bacteroides,Prevotella,and Lactobacillus.However,the abundance among them was different.The community abundance of Bacteroides and Prevotella decreased in the female sample compared with that in the male sample,and that of Lactobacillus increased.We also performed functional pathways analysis and observed significant enrichment of glycerolipid metabolism in female dnTGFβRⅡ mice while observing increased functions related to butanoate metabolism,thiamine metabolism,and fatty acid metabolism in male dnTGFβRⅡ mice.In conjunction,our results revealed that PBC is androgen protection-independent and related to commensal gut microbiota-mediated female predominance.It provides a theoretical basis for PBC disease’s occurrence and development and provides a theoretical basis for customized treatment programs.