Bidirectional Cross-Talk between Biliary Epithelium and Th17 Cells Promotes Local Th17 Expansion and Bile Duct Proliferation in Biliary Liver Diseases.

There is no effective treatment for autoimmune biliary diseases. Therefore, understanding their immunopathology is crucial. The biliary epithelial cells (BEC), expressing TLR-4, are constantly exposed to gut microbes and bacterial wall LPS, and in settings of inflammation, the immune infiltrate is dense within the peribiliary region of human liver. By dual immunohistochemistry, we affirm human intrahepatic T cell infiltrate includes CCR6+CD4+ and AhR+CD4+ T cells with potential for plasticity to Th17 phenotype. Mechanistically, we demonstrate that Th1 and Th17 inflammatory cytokines and LPS enhance human primary BEC release of the CCR6 ligand CCL20 and BEC secretion of Th17-polarizing cytokines IL-6 and IL-1ß. Cell culture assays with human BEC secretome showed that secretome polarizes CD4 T cells toward a Th17 phenotype and supports the survival of Th17 cells. BEC secretome did not promote Th1 cell generation. Additionally, we give evidence for a mutually beneficial feedback of the type 17 cell infiltrate on BEC, showing that treatment with type 17 cytokines increases BEC proliferation, as monitored by Ki67 and activation of JAK2-STAT3 signaling. This study identifies human BEC as active players in determining the nature of the intrahepatic immune microenvironment. In settings of inflammation and/or infection, biliary epithelium establishes a prominent peribiliary type 17 infiltrate via recruitment and retention and enhances polarization of intrahepatic CD4 cells toward Th17 cells via type 17 cytokines, and, reciprocally, Th17 cells promote BEC proliferation for biliary regeneration. Altogether, we provide new insight into cross-talk between Th17 lymphocytes and human primary biliary epithelium in biliary regenerative pathologies.

Liver homing of clinical grade Tregs after therapeutic infusion in patients with autoimmune hepatitis.

Autoimmune hepatitis (AIH) is an immune-mediated disease with no curative treatment. Regulatory T cell (Treg) therapy is potentially curative in AIH given the critical role of Tregs in preventing autoimmunity. To work effectively, adoptively transferred Tregs must migrate to and survive within the inflamed liver. We conducted a proof-of-concept study aiming to assess the safety and liver-homing properties of good manufacturing practice (GMP)-grade autologous Tregs in patients with AIH. METHODS: Autologous polyclonal GMP-grade Tregs were isolated using leukapheresis and CliniMACS, labelled with indium tropolonate and re-infused intravenously to 4 patients with AIH. GMP-Treg homing to the liver was investigated with longitudinal gamma camera and SPECT-CT scanning. GMP-Treg immunophenotype, function and immunometabolic state were assessed during the study. RESULTS: We observed that the isolated Tregs were suppressive and expressed CXCR3, a chemokine receptor involved in recruitment into the inflamed liver, as well as Treg functional markers CD39, CTLA-4 and the transcription factor Foxp3. Serial gamma camera and SPECT-CT imaging demonstrated that 22-44% of infused Tregs homed to and were retained in the livers of patients with autoimmune hepatitis for up to 72 h. The infused cells did not localise to any off-target organs other than the spleen and bone marrow. GMP-Tregs were metabolically competent and there were no infusion reactions or high-grade adverse effects after Treg infusion. CONCLUSION: Our novel findings suggest that the liver is a good target organ for Treg cellular therapy, supporting the development of clinical trials to test efficacy in autoimmune hepatitis and other autoimmune liver diseases. LAY SUMMARY: Autoimmune liver diseases occur when the body's immune cells target their own liver cells. Regulatory T cells (Tregs) prevent autoimmunity, thus they are a potential therapy for autoimmune liver diseases. In patients with autoimmune hepatitis, Treg infusion is safe, with nearly a quarter of infused Tregs homing to the liver and suppressing tissue-damaging effector T cells. Thus, Tregs are a potentially curative immune cell therapy for early autoimmune liver diseases.

Efficacy of rituximab in difficult-to-manage autoimmune hepatitis: Results from the International Autoimmune Hepatitis Group.

Treatment options remain limited for patients with autoimmune hepatitis (AIH), while there are still concerns over the consequences of long-term corticosteroid use. A few studies have suggested a role for B cell-driven autoimmune liver injury in AIH. This multicentre, international retrospective cohort study from the International Autoimmune Hepatitis Group aims to evaluate the clinical efficacy and safety of rituximab in difficult-to-manage AIH. METHODS: Clinical data from 22 patients who received rituximab between 2007 and 2017 were collected from centres in the United Kingdom, Germany and Canada. Clinical response was assessed using changes in biochemical and immunological parameters up to 24 months post-rituximab infusion. In addition, we compared the doses of prednisolone used 3 months before and 12 months after treatment, and assessed freedom from AIH flares over the post-treatment period. RESULTS: Twenty-two patients with type-1 AIH were included, with a median age of 40 years at diagnosis (range 19-79); 15/22 (68%) were female and 18/22 (82%) were Caucasian. The median period from diagnosis to the end of follow-up in these patients was 11 years (range 3-28). Values of alanine aminotransferase, aspartate aminotransferase and albumin improved significantly following rituximab therapy, and were sustained for up to 2 years (all p ≪0.001). Prednisolone doses were significantly reduced by 12 months post-treatment (p = 0.003), with 13/21 (62%) patients having a dose reduction. Over a median post-treatment follow-up period of 6 years (range 1-10), 5 patients developed AIH flares at a median of 22 months post-treatment, giving an estimated 71% freedom from AIH flare at 2 years. Four of these patients received a second course of treatment, of whom 2 had subsequent further flares. No serious adverse events attributable to rituximab were recorded. CONCLUSION: In patients with difficult-to-manage AIH, rituximab appears to be clinically effective and well tolerated. Rituximab was associated with sustained improvements in serum liver tests, an absence of clinical disease flares, and a reduction in prednisolone dose. Controlled trials are warranted to further evaluate B cell-targeting therapies in patients with AIH. LAY SUMMARY: Autoimmune hepatitis is an autoimmune condition of the liver, usually treated with medications that suppress the immune system, such as steroids. However, some patients do not respond to this treatment. We analysed the safety and efficacy of rituximab in patients who were not responding to first- or second-line therapies. Rituximab was safe and improved liver blood tests in 70% of patients over a 2-year follow-up period, while enabling steroid doses to be reduced in two-thirds of patients, which is a very positive clinical outcome.

Changes in natural killer cells and exhausted memory regulatory T Cells with corticosteroid therapy in acute autoimmune hepatitis.

Autoimmune hepatitis (AIH) is an immune-mediated liver disease currently treated by immunosuppressive medications with significant side effects. Thus, novel mechanistic treatments are greatly needed. We performed prospective deep immunophenotyping of blood immune cells in patients with acute AIH before and after corticosteroid therapy. Blood samples from 26 patients with acute AIH (United Kingdom-AIH Consortium) were phenotyped by flow cytometry at baseline and 4 months after starting corticosteroids. Pretreatment liver tissues were stained for forkhead box P3-positive (FOXP3POS) regulatory T cells (Tregs), clusters of differentiation (CD)56POS natural killer (NK) cells, and chemokine (C-X-C motif) ligand 10. Chemokine secretion by cultured primary hepatocyte and biliary epithelial cells was measured by enzyme-linked immunosorbent assay. Functional coculture assays with stimulated NK cells and Tregs were performed. CD161 ligand, lectin-like transcript-1 expression by intrahepatic immune cells was demonstrated with flow cytometry. Frequencies of NKbright cells declined with therapy (P < 0.001) and correlated with levels of alanine aminotransferase (P = 0.023). The Treg:NKbright ratio was lower pretreatment, and Tregs had an activated memory phenotype with high levels of CD39, cytotoxic T lymphocyte antigen 4, and FOXP3 but also high programmed death ligand 1, indicating exhaustion. Coculture experiments suggested the Tregs could not efficiently suppress interferon-γ secretion by NK cells. Both Tregs and NK cells had high expression of liver infiltration and T helper 17 plasticity-associated marker CD161 (P = 0.04). Pretreatment and CD161pos NK cells expressed high levels of perforin and granzyme B, consistent with an activated effector phenotype (P < 0.05). Lectin-like transcript 1, a ligand for CD161, is expressed on intrahepatic B cells, monocytes, and neutrophils. Conclusion: Activated effector NK cells, which correlate with biochemical measurements of hepatitis, and exhausted memory Tregs are increased in the blood of patients with treatment-naive AIH and decline with corticosteroid therapy. Inadequate regulation of NK cells by exhausted FOXP3pos Tregs may play a role in AIH pathogenesis and contribute to liver injury. (Hepatology Communications 2018;2:421-436).

Human intrahepatic ILC2 are IL-13positive amphiregulinpositive and their frequency correlates with model of end stage liver disease score.

INTRODUCTION: Innate lymphoid cells (ILC) have been implicated in the initiation of inflammation and fibrosis in mice. However, ILC have not been characterized in inflamed human liver tissue. METHODS: Human intrahepatic lymphocytes were isolated by mechanical digestion and phenotyped by flow cytometry. Conditioned medium from cultures of primary human biliary epithelial cells, stellate cells, fibroblasts and inflamed human liver tissue was used to model the effects of the inflammatory liver environment of ILC phenotype and function. RESULTS: All three ILC subsets were present in the human liver, with the ILC1 (CRTH2negCD117neg) subset constituting around 70% of intrahepatic ILCs. Both NCRpos (NKp44+) and NCRneg ILC3 (CRTH2negCD117pos) subsets were also detected. ILC2 (CRTH2pos) frequency correlated with disease severity measured by model of end stage liver disease (MELD) scoring leading us to study this subset in more detail. ILC2 displayed a tissue resident CD69+ CD161++ phenotype and expressed chemokine receptor CCR6 allowing them to respond to CCL20 secreted by cholangiocytes and stellate cells. ILC2 expressed integrins VLA-5 and VLA-6 and the IL-2 and IL-7 cytokine receptors CD25 and CD127 although IL-2 and IL-7 were barely detectable in inflamed liver tissue. Although biliary epithelial cells secrete IL-33, intrahepatic ILC2 had low expression of the ST2 receptor. Intrahepatic ILC2 secreted the immunoregulatory and repair cytokines IL-13 and amphiregulin. CONCLUSIONS: Intrahepatic ILC2 express receptors allowing them to be recruited to bile ducts in inflamed portal tracts. Their frequencies increased with worsening liver function. Their secretion of IL-13 and amphiregulin suggests they may be recruited to promote resolution and repair and thereby they may contribute to ongoing fibrogenesis in liver disease.