Mucosa-associated invariant T cells link intestinal immunity with antibacterial immune defects in alcoholic liver disease

Abstract

Background/aims: Intestinal permeability with systemic distribution of bacterial products are central in the immunopathogenesis of alcoholic liver disease (ALD), yet links with intestinal immunity remain elusive. Mucosa-associated invariant T cells (MAIT) are found in liver, blood and intestinal mucosa and are a key component of antibacterial host defences. Their role in ALD is unknown.

Methods/design: We analysed frequency, phenotype, transcriptional regulation and function of blood MAIT cells in severe alcoholic hepatitis (SAH), alcohol-related cirrhosis (ARC) and healthy controls (HC). We also examined direct impact of ethanol, bacterial products from faecal extracts and antigenic hyperstimulation on MAIT cell functionality. Presence of MAIT cells in colon and liver was assessed by quantitative PCR and immunohistochemistry/gene expression respectively.

Results: In ARC and SAH, blood MAIT cells were dramatically depleted, hyperactivated and displayed defective antibacterial cytokine/cytotoxic responses. These correlated with suppression of lineage-specific transcription factors and hyperexpression of homing receptors in the liver with intrahepatic preservation of MAIT cells in ALD. These alterations were stronger in SAH, where surrogate markers of bacterial infection and microbial translocation were higher than ARC. Ethanol exposure in vitro, in vivo alcohol withdrawal and treatment with Escherichia coli had no effect on MAIT cell frequencies, whereas exposure to faecal bacteria/antigens induced functional impairments comparable with blood MAIT cells from ALD and significant MAIT cell depletion, which was not observed in other T cell compartments.

Conclusions: In ALD, the antibacterial potency of MAIT cells is compromised as a consequence of contact with microbial products and microbiota, suggesting that the 'leaky' gut observed in ALD drives MAIT cell dysfunction and susceptibility to infection in these patients.

Keywords: alcoholic liver disease; bacterial translocation; inflammation; mucosal immunity; t lymphocytes.

Figure 1

Blood MAIT cell depletion in patients with ALD. (A) Gating strategy to identify MAIT cells by FACS. (B) Blood MAIT cell frequencies in controls (n=13), ARC (n=9) and SAH (n=13) patients. (C) MAIT cell depletion is selective, as TCR_Vα7.2(+) GEM cells and TCR_Vα7.2(+)/CD161(−) are unchanged (counts as above). (D) No MAIT cell depletion in healthy PBMC treated with control/ALD plasma (ARC: n=5; SAH: n=5) or blood MAIT cells from controls (n=9), ARC (n=9) and SAH (n=9) stimulated with high bacterial load. (E) Lack of MAIT cell depletion upon in vitro ethanol treatment of healthy PBMC. ALD, alcohol-related liver disease; ARC, alcohol-related cirrhosis; FACS, flow cytometry; GEM, germline-encoded mycolyl lipid-reactive T cells; MAIT, mucosa-associated invariant T cells; PBMC, peripheral blood mononuclear cells; SAH, severe alcoholic hepatitis.

Figure 2

Preservation of MAIT cells in the liver in ALD. Intrahepatic hyperexpression of homing markers (A) and MR1 (B) within total liver tissue, public microarray dataset analysis (Ctrl: n=7; SAH: n=15). (C) Liver parenchymal and portal TCR_Vα7.2 (clone 3C10) immunohistochemistry to identify intrahepatic TCR_Vα7.2(+) cells; fresh-frozen biopsies from ALD livers (n=5) and healthy liver donors (n=5). ALD, alcohol-related liver disease; MAIT, mucosa-associated invariant T cells; MR1, MHC-related molecule 1; SAH, severe alcoholic hepatitis.

Figure 3

Blood MAIT cells in ALD display a hyperactivated phenotype. (A) CD69/HLA-DR hyperexpression by ALD blood MAIT cells compared with controls (n=9 for all groups). (B) ALD plasma (ARC plasma: n=5; SAH plasma: n=5) can increase CD69/HLA-DR expression in healthy PBMC compared with control plasma. (C) MAIT cell activation status is not induced by in vitro ethanol treatment of healthy PBMC. (D) Gal1 expression is increased in SAH liver tissue (control: n=7; SAH: n=15), public microarray dataset analysis. (E) Immunoinhibitory checkpoints are not upregulated on hyperactivated blood MAIT cells in ALD (n=9 for all groups). (F) Comparable levels of cell proliferation/senescence markers (Ki67/CD57) between groups (n=5 for all groups). ALD, alcohol-related liver disease; ARC, alcohol-related cirrhosis; Gal1, Galectin 1; MAIT, mucosa-associated invariant T cells; PBMC, peripheral blood mononuclear cells; SAH, severe alcoholic hepatitis.

Figure 4

Blood MAIT cells show defective antibacterial cytokine/cytotoxic responses in ALD. (A)–(C) MAIT-specific IFNγ, TNFα and IL-17 responses upon in vitro Escherichia coli stimulation of ALD/control PBMC, absolute frequencies, and Δ-changes versus unstimulated samples. (D)–(F) MAIT cell GranzymeB, CD107a and Perforin responses upon in vitro E. coli stimulation of ALD/control PBMC; absolute frequencies and fold-changes versus unstimulated samples (n=9 for all groups). ALD, alcohol-related liver disease; ARC, alcohol-related cirrhosis; IFN, interferon; IL, interleukin; MAIT, mucosa-associated invariant T cells; PBMC, peripheral blood mononuclear cells; SAH, severe alcoholic hepatitis; TNF, tumour necrosis factor.

Figure 5

Defective expression of innate-like/type-17 transcription factors by blood MAIT cells and intrahepatically in ALD. (A) Blood MAIT cells from ALD display reduced expression of RORC and PLZF compared with controls (n=5 for all groups). (B) SAH liver tissue (n=15) underexpresses RORC, PLZF and Eomesodermin compared with healthy liver tissue (n=7). ALD, alcohol-related liver disease; ARC, alcohol-related cirrhosis; MAIT, mucosa-associated invariant T cells; SAH, severe alcoholic hepatitis.

Figure 6

Contact between healthy PBMC and faecal extracts of bacterial antigens, toxins and metabolites (FEB) from ALD patients and controls causes quantitative and functional impairments of MAIT. (A) FEB-induced MAIT cell depletion. Apoptosis rates are not different between the groups, illustrating that FEB-induced MAIT cell depletion is apoptosis independent. Control stools: n=12; ARC stools: n=20; SAH stools: n=7. The bar plot represents mean±SD. The black bars represent % of apoptotic VybrantFAM(+) MAIT cells; the white/shaded bars represent MAIT cell frequencies; both quantities are measured on the total CD8 T cell population, as described in ‘Materials and Methods’. (B) FEB-induced hyperactivated state on MAIT, CD69/HLA-DR. (C) FEB-induced immunoinhibitory checkpoint upregulation, PD1/TIM3/LAG3. FEB-induced suppression of MAIT cell antibacterial cytokine (D) and cytotoxic (E) responses; zebra bars represent Escherichia coli-stimulated results. In panels B–E, control stools: n=8; ARC stools: n=18; SAH stools: n=5. ALD, alcohol-related liver disease; ARC, alcohol-related cirrhosis; MAIT, mucosa-associated invariant T cells; PMBC, peripheral blood mononuclear cells; SAH, severe alcoholic hepatitis.