Apoptotic biliary epithelial cells and gut dysbiosis in the induction of murine primary biliary cholangitis

Abstract

Primary biliary cholangitis (PBC) is a female-predominant liver autoimmune disease characterized by the specific immune-mediated destruction of the intrahepatic small bile duct. Although apoptosis of biliary epithelial cells (BECs) and alterations in gut microbiota are observed in patients with PBC, it is still unclear whether these events happen in the early stage and cause the breakdown of tolerance in PBC. In this study, we examined the early events in the loss of tolerance in our well-defined 2-OA-OVA-induced murine autoimmune cholangitis (AIC) model. We report herein that apoptosis of BECs was notable in the early stage of murine AIC. An altered gut microbiota, in particular, an increased percentage of gram-positive Firmicutes in AIC mice was also observed. BECs in AIC mice expressed adhesion molecule ICAM-1, cytokines/chemokines TNF-α, CCL2, CXCL9, CXCL10, and toll-like receptor (TLR) 2. Moreover, BECs treated with TLR2 ligand had elevated apoptosis and CXCL10 production. These data collectively suggest a new mechanism of tolerance breakdown in AIC. Altered gut microbiota induces apoptosis of BECs through TLR2 signaling. BECs secrete chemokines to recruit CD8 T cells to damage BECs further.

Keywords: 2-OA, 2-octynoic acid; 2-OA-OVA, 2-octynoic acid conjugated ovalbumin; AIC, autoimmune cholangitis; AMAs, anti-mitochondrial antibodies; Apoptosis; Autoimmune cholangitis; BECs, biliary epithelial cells; Gut microbiota; PBC, primary biliary cholangitis; PDC-E2, E2 component of pyruvate dehydrogenase complex; TLR, toll-like receptor; TUNEL, TdT-mediated deoxyuridine triphosphate nick-end labeling; Toll-like receptor; Xenobiotic.

Fig. 1

Both male and female mice immunized with 2-OA-OVA developed AMAs and liver inflammation. Male and female mice were immunized with 2-OA-OVA or normal saline, and serum levels of AMAs and liver lymphocytes were examined at 3 weeks post-immunization. (A) Serum levels of anti-PDC-E2 IgM and IgG were determined using ELISA. OD, optical density. (B) Representative flow plots show the gating strategies of different subsets of cells. (C) Liver leukocytes and lymphocytes were quantified. (D) The numbers of T, B, NK, and NKT cells in the liver were quantified. (E) The numbers of CD4 T and CD8 T cells in the liver were quantified. (F) The ratio of CD8/CD4 was calculated. M, male mice; F, female mice. The dashed lines in (A) and (C) depict the cut-off for a positive response, calculated as the mean of the results of saline-treated samples plus three standard deviations. Symbols represent one individual mouse, and bars indicate mean ± SEM. n = 5–10 mice per group. *, p < 0.05; **, p < 0.01; ***, p < 0.001; ****, p < 0.0001 (two-way ANOVA followed by Tukey's multiple comparison test).

Fig. 2

Increased expression of liver pro-inflammatory cytokines and TLRs in AIC mice. Male and female mice were immunized with 2-OA-OVA or normal saline and examined at 3 weeks post-immunization. The expression levels of (A) IFN-γ and TNF-α and (B) TLRs in the liver were detected using RT-qPCR. M, male mice; F, female mice. Symbols represent one individual mouse, and bars indicate mean ± SEM. n = 8–12 mice per group. *, p < 0.05; **, p < 0.01; ***, p < 0.001; ****, p < 0.0001 (two-way ANOVA followed by Tukey's multiple comparison test).

Fig. 3

Apoptosis of BECs in AIC mice. Male and female mice were immunized with 2-OA-OVA or normal saline. Three weeks post-immunization, apoptosis of BECs in the liver tissues of mice was measured by TUNEL assay. (A) Representative images of TUNEL assay of liver histopathology (1000× magnification, scale bar, 10 μm). The liver section was triple labeled for apoptotic cells (green), biliary cytokeratin CK-19 (red), and nuclei DAPI (blue). (B) Thirty intrahepatic small bile ducts were counted in each slide to calculate the frequency of bile ducts with TUNEL-positive cells. M, male mice; F, female mice. Symbols represent one individual mouse, and bars indicate mean ± SEM. n = 6–9 mice per group. ***, p < 0.001; ****, p < 0.0001 (two-way ANOVA followed by Tukey's multiple comparison test).

Fig. 4

Adhesion molecules and cytokines/chemokines of BECs in AIC mice. Male and female mice were immunized with 2-OA-OVA or normal saline. Three weeks post-immunization, liver tissues, and intrahepatic small bile ducts were taken. (A) Representative images of MHC class II (IAb) expression in liver histopathology (400× magnification, scale bar, 35 μm). The liver section was triple labeled for IA^b (green), biliary cytokeratin CK-19 (red), and nuclei DAPI (blue). (B) The expression levels of ICAM-1 mRNA in the intrahepatic small bile duct were detected by RT-qPCR. (C) The expression levels of TNF-α, IL-6, CCL2, CXCL9, and CXCL10 mRNA in the intrahepatic small bile duct were detected by RT-qPCR. M, male mice; F, female mice. Symbols represent one individual mouse, and bars indicate mean ± SEM. n = 6–11 mice per group. *, p < 0.05; **, p < 0.01; ***, p < 0.001; ****, p < 0.0001 (two-way ANOVA followed by Tukey's multiple comparison test).

Fig. 5

Marked alternation of gut microbiota after 2-OA-OVA immunization. Male and female mice were immunized with 2-OA-OVA or normal saline. Fecal samples of mice were collected at 3 weeks post-immunization. Gut microbiota compositions were profiled by 16s rRNA next-generation sequencing (NGS). (A) Hierarchical clustering heat map of microbiota abundance at the genus level. Highly relative abundance bacteria were in red. Lowly relative abundance bacteria were in blue. (B) Microbial communities were analyzed by principal coordinate analysis (PCoA) based on the weighted UniFrac distance metric. (C) Relative abundance of fecal microbiota at the phylum level of the taxonomy. (D) Histograms of linear discriminant analysis (LDA) effect size (LEfSe) comparison between fecal microbiota at the family level. Only the taxa having an LDA >3.0 were shown. Log-level changes in LDA scores were displayed on the x-axis. Green bars: taxa found in greater relative abundance in saline controls. Red bars: taxa found in greater relative abundance in 2-OA-OVA immunization. (E) The expression levels of TNF-α mRNA in the intestines were detected by RT-qPCR. Symbols represent one individual mouse, and bars indicate mean ± SEM. n = 6–11 mice per group. *, p < 0.05 (two-way ANOVA followed by Tukey's multiple comparison test).

Fig. 6

Expression of TLRs of the intrahepatic small bile duct in AIC mice. Male and female mice were immunized with 2-OA-OVA or normal saline. The expression levels of TLRs mRNA in the intrahepatic small bile duct of mice at 3 weeks post-immunization were detected by RT-qPCR. M, male mice; F, female mice. Symbols represent one individual mouse, and bars indicate mean ± SEM. n = 6–10 mice per group. *, p < 0.05; **, p < 0.01 (two-way ANOVA followed by Tukey's multiple comparison test).

Fig. 7

Increased apoptosis of TLR2 ligand-stimulated BEC. 603B cells were stimulated with Pam3CSK4 (TLR2 ligand) or not in the presence of TNF-α and SC-514 for 24 h. (A) Representative and graphical summary of flow cytometry analysis of apoptosis of 603B cells. (B) The expression levels of CXCL10 mRNA in treated 603B cells were detected by RT-qPCR. Data were normalized based on 603B cells without stimulation (untreated). Individual symbols represent an independent experiment. **, p < 0.01 (two-tailed paired t-test).

Fig. 8

Overall schema of the initiation of PBC. Exposure to environmental xenobiotics alters gut microbiota composition and induces intestinal inflammation. Gut microbiota membrane components and TNF-α induces apoptosis of BECs, which leads to autoantigen release. Injured BECs express more adhesion molecules ICAM-1 and chemoattractants CCL2, CXCL9, and CXCL10 to recruit immune cells. Among infiltrated immune cells in the portal area, CD8 T cells are responsible for further biliary destruction by secreting more IFN-γ and cytotoxic molecules perforin and granzyme B.