BACKGROUND:Miscarriage, defined as the spontaneous loss of a pregnancy before 24 weeks' gestation, is common with approximately 25% of women experiencing a miscarriage in their lifetime. An estimated 15% of pregnancies end in miscarriage. Miscarriage can lead to serious morbidity, including haemorrhage, infection, and even death, particularly in settings without adequate healthcare provision. Early miscarriages occur during the first 14 weeks of pregnancy, and can be managed expectantly, medically or surgically. However, there is uncertainty about the relative effectiveness and risks of each option. OBJECTIVES:To estimate the relative effectiveness and safety profiles for the different management methods for early miscarriage, and to provide rankings of the available methods according to their effectiveness, safety, and side-effect profile using a network meta-analysis. SEARCH METHODS:We searched the Cochrane Pregnancy and Childbirth's Trials Register (9 February 2021), ClinicalTrials.gov and the WHO International Clinical Trials Registry Platform (ICTRP) (12 February 2021), and reference lists of retrieved studies. SELECTION CRITERIA:We included all randomised controlled trials assessing the effectiveness or safety of methods for miscarriage management. Early miscarriage was defined as less than or equal to 14 weeks of gestation, and included missed and incomplete miscarriage. Management of late miscarriages after 14 weeks of gestation (often referred to as intrauterine fetal deaths) was not eligible for inclusion in the review. Cluster- and quasi-randomised trials were eligible for inclusion. Randomised trials published only as abstracts were eligible if sufficient information could be retrieved. We excluded non-randomised trials. DATA COLLECTION AND ANALYSIS:At least three review authors independently assessed the trials for inclusion and risk of bias, extracted data and checked them for accuracy. We estimated the relative effects and rankings for the primary outcomes of complete miscarriage and composite outcome of death or serious complications. The certainty of evidence was assessed using GRADE. Relative effects for the primary outcomes are reported subgrouped by the type of miscarriage (incomplete and missed miscarriage). We also performed pairwise meta-analyses and network meta-analysis to determine the relative effects and rankings of all available methods. MAIN RESULTS:Our network meta-analysis included 78 randomised trials involving 17,795 women from 37 countries. Most trials (71/78) were conducted in hospital settings and included women with missed or incomplete miscarriage. Across 158 trial arms, the following methods were used: 51 trial arms (33%) used misoprostol; 50 (32%) used suction aspiration; 26 (16%) used expectant management or placebo; 17 (11%) used dilatation and curettage; 11 (6%) used mifepristone plus misoprostol; and three (2%) used suction aspiration plus cervical preparation. Of these 78 studies, 71 (90%) contributed data in a usable form for meta-analysis. Complete miscarriage Based on the relative effects from the network meta-analysis of 59 trials (12,591 women), we found that five methods may be more effective than expectant management or placebo for achieving a complete miscarriage: · suction aspiration after cervical preparation (risk ratio (RR) 2.12, 95% confidence interval (CI) 1.41 to 3.20, low-certainty evidence), · dilatation and curettage (RR 1.49, 95% CI 1.26 to 1.75, low-certainty evidence), · suction aspiration (RR 1.44, 95% CI 1.29 to 1.62, low-certainty evidence), · mifepristone plus misoprostol (RR 1.42, 95% CI 1.22 to 1.66, moderate-certainty evidence), · misoprostol (RR 1.30, 95% CI 1.16 to 1.46, low-certainty evidence). The highest ranked surgical method was suction aspiration after cervical preparation. The highest ranked non-surgical treatment was mifepristone plus misoprostol. All surgical methods were ranked higher than medical methods, which in turn ranked above expectant management or placebo. Composite outcome of death and serious complications Based on the relative effects from the network meta-analysis of 35 trials (8161 women), we found that four methods with available data were compatible with a wide range of treatment effects compared with expectant management or placebo: · dilatation and curettage (RR 0.43, 95% CI 0.17 to 1.06, low-certainty evidence), · suction aspiration (RR 0.55, 95% CI 0.23 to 1.32, low-certainty evidence), · misoprostol (RR 0.50, 95% CI 0.22 to 1.15, low-certainty evidence), · mifepristone plus misoprostol (RR 0.76, 95% CI 0.31 to 1.84, low-certainty evidence). Importantly, no deaths were reported in these studies, thus this composite outcome was entirely composed of serious complications, including blood transfusions, uterine perforations, hysterectomies, and intensive care unit admissions. Expectant management and placebo ranked the lowest when compared with alternative treatment interventions. Subgroup analyses by type of miscarriage (missed or incomplete) agreed with the overall analysis in that surgical methods were the most effective treatment, followed by medical methods and then expectant management or placebo, but there are possible subgroup differences in the effectiveness of the available methods.  AUTHORS' CONCLUSIONS: Based on relative effects from the network meta-analysis, all surgical and medical methods for managing a miscarriage may be more effective than expectant management or placebo. Surgical methods were ranked highest for managing a miscarriage, followed by medical methods, which in turn ranked above expectant management or placebo. Expectant management or placebo had the highest chance of serious complications, including the need for unplanned or emergency surgery. A subgroup analysis showed that surgical and medical methods may be more beneficial in women with missed miscarriage compared to women with incomplete miscarriage. Since type of miscarriage (missed and incomplete) appears to be a source of inconsistency and heterogeneity within these data, we acknowledge that the main network meta-analysis may be unreliable. However, we plan to explore this further in future updates and consider the primary analysis as separate networks for missed and incomplete miscarriage.

Background & Aims: 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. (C) 2019 Published by Elsevier B.V.

Background and Aims: Ascites and spontaneous bacterial peritonitis (SBP) are frequent complications of liver cirrhosis. In spite of the clinical impact, knowledge about ascites as an immune cell compartment in liver disease is limited. Therefore, we analyzed NK cells in blood, ascites, and liver. Methods: Mononuclear cells from blood, ascites, and liver explants of patients with advanced liver disease were extracted by density gradient centrifugation. Phenotyping and analysis of functional responses were carried out using flow cytometry. Migratory potential was investigated with transwell chamber assays. NK cell metabolism was assessed by Seahorse technology. Results: NK cell frequency was increased in uninfected ascites compared to blood, but not to liver. Ascites NK cells were predominantly CD16positive. CD56bright ascites NK cells did not share the typical phenotype of their liver counterparts. In contrast to the inhibitory receptor NKG2A, expression of the activating receptor NKG2D was decreased on ascites and liver CD16positive NK cells. Ascites NK cells expressed higher levels of CXCR3 than blood or liver NK cells, corresponding to increased ascites levels of CXCL10. Blood NK cells migrated toward ascites. Stimulation of mononuclear cells with Escherichia coli led to downregulation of NKG2D expression and IL-12 and IL-18 mediated secretion of interferon-γ by ascites and liver, but not blood NK cells. In-vivo, ascites NK cells expressed higher levels of the activation marker CD69 and lower levels of NKG2D during SBP compared to uninfected ascites. Conclusion: Ascites NK cells display a particular phenotype and are implicated in local immune defense against translocating bacteria.

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-1β. 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.

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).

Background & Aims:cd T cells comprise a substantial proportion of tissue-associated lymphocytes. However, our current understanding of human cd T cells is primarily based on peripheral blood subsets, while the immunobiology of tissueassociated subsets remains largely unclear. Therefore, we aimed to elucidate the T cell receptor (TCR) diversity, immunophenotype and function of cd T cells in the human liver. Methods:We characterised the TCR repertoire, immunophenotype and function of human liver infiltrating cd T cells, by TCR sequencing analysis, flow cytometry, in situ hybridisation and immunohistochemistry. We focussed on the predominant tissue-associated Vd2 cd subset, which is implicated in liver immunopathology. Results: Intrahepatic Vd2 cd T cells were highly clonally focussed, with single expanded clonotypes featuring complex, private TCR rearrangements frequently dominating the compartment. Such T cells were predominantly CD27 effector lymphocytes, whereas naïve CD27, TCR-diverse populations present in matched blood were generally absent in the liver. Furthermore, while a CD45RA Vd2 cd effector subset present in both liver and peripheral blood contained overlapping TCR clonotypes, the liver Vd2 cd T cell pool also included a phenotypically distinct CD45RA effector compartment that was enriched for expression of the tissue tropism marker CD69, the hepatic homing chemokine receptors CXCR3 and CXCR6, and liver-restricted TCR clonotypes, suggestive of intrahepatic tissue residency. Liver infiltrating Vd2 cd cells were capable of polyfunctional cytokine secretion, and unlike peripheral blood subsets, were responsive to both TCR and innate stimuli.

Background & Aims: gamma delta T cells comprise a substantial proportion of tissue-associated lymphocytes. However, our current understanding of human gamma delta T cells is primarily based on peripheral blood subsets, while the immunobiology of tissue-associated subsets remains largely unclear. Therefore, we aimed to elucidate the T cell receptor (TCR) diversity, immunophenotype and function of gamma delta T cells in the human liver. Methods: We characterised the TCR repertoire, immunophenotype and function of human liver infiltrating gamma delta T cells, by TCR sequencing analysis, flow cytometry, in situ hybridisation and immunohistochemistry. We focussed on the predominant tissue-associated V delta 2(-) gamma delta subset, which is implicated in liver immunopathology. Results: Intrahepatic V delta 2(-) gamma delta T cells were highly clonally focussed, with single expanded clonotypes featuring complex, private TCR rearrangements frequently dominating the compartment. Such T cells were predominantly CD27(lo/-) effector lymphocytes, whereas naive CD27(hi), TCR-diverse populations present in matched blood were generally absent in the liver. Furthermore, while a CD45RA(hi) V delta 2(-) gamma delta effector subset present in both liver and peripheral blood contained overlapping TCR clonotypes, the liver V delta 2(-) gamma delta T cell pool also included a phenotypically distinct CD45RA(lo) effector compartment that was enriched for expression of the tissue tropism marker CD69, the hepatic homing chemokine receptors CXCR3 and CXCR6, and liver-restricted TCR clonotypes, suggestive of intrahepatic tissue residency. Liver infiltrating V delta 2(-) gamma delta cells were capable of polyfunctional cytokine secretion, and unlike peripheral blood subsets, were responsive to both TCR and innate stimuli. Conclusion: These findings suggest that the ability of V delta 2(-) gamma delta T cells to undergo clonotypic expansion and differentiation is crucial in permitting access to solid tissues, such as the liver, which results in functionally distinct peripheral and liver-resident memory gamma delta T cell subsets. They also highlight the inherent functional plasticity within the V delta 2(-) gamma delta T cell compartment and provide information that could be used for the design of cellular therapies that suppress liver inflammation or combat liver cancer. Lay summary: gamma delta T cells are frequently enriched in many solid tissues, however the immunobiology of such tissue-associated subsets in humans has remained unclear. We show that intrahepatic gamma delta T cells are enriched for clonally expanded effector T cells, whereas naive gamma delta T cells are largely excluded. Moreover, whereas a distinct proportion of circulating T cell clonotypes was present in both the liver tissue and peripheral blood, a functionally and clonotypically distinct population of liver-resident gamma delta T cells was also evident. Our findings suggest that factors triggering gamma delta T cell clonal selection and differentiation, such as infection, can drive enrichment of gamma delta T cells into liver tissue, allowing the development of functionally distinct tissue-restricted memory populations specialised in local hepatic immunosurveillance. (C) 2018 European Association for the Study of the Liver. Published by Elsevier B.V.

Die spontane bakterielle Peritonitis (SBP) ist eine häufige Komplikation von Patienten mit Leberzirrhose. T-Zellen unterstützen Makrophagen bei der Eliminierung von Bakterien. Über Unterschiede zwischen den T-Zell-Subpopulationen in relevanten Geweben von Patienten mit Leberzirrhose ist bisher wenig bekannt.

The microenvironment of lymphoid organs can aid healthy immune function through provision of both structural and molecular support. In mice, fibroblastic reticular cells (FRCs) create an essential T-cell support structure within lymph nodes, while human FRCs are largely unstudied. Here, we show that FRCs create a regulatory checkpoint in human peripheral T-cell activation through 4 mechanisms simultaneously utilised. Human tonsil and lymph node-derived FRCs constrained the proliferation of both naïve and pre-activated T cells, skewing their differentiation away from a central memory T-cell phenotype. FRCs acted unilaterally without requiring T-cell feedback, imposing suppression via indoleamine-2,3-dioxygenase, adenosine 2A Receptor, prostaglandin E2, and transforming growth factor beta receptor (TGFβR). Each mechanistic pathway was druggable, and a cocktail of inhibitors, targeting all 4 mechanisms, entirely reversed the suppressive effect of FRCs. T cells were not permanently anergised by FRCs, and studies using chimeric antigen receptor (CAR) T cells showed that immunotherapeutic T cells retained effector functions in the presence of FRCs. Since mice were not suitable as a proof-of-concept model, we instead developed a novel human tissue-based in situ assay. Human T cells stimulated using standard methods within fresh tonsil slices did not proliferate except in the presence of inhibitors described above. Collectively, we define a 4-part molecular mechanism by which FRCs regulate the T-cell response to strongly activating events in secondary lymphoid organs while permitting activated and CAR T cells to utilise effector functions. Our results define 4 feasible strategies, used alone or in combinations, to boost primary T-cell responses to infection or cancer by pharmacologically targeting FRCs.

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 (CRTH2(neg)CD117(neg)) subset constituting around 70% of intrahepatic ILCs. Both NCRpos (NKp44(+)) and NCRneg ILC3 (CRTH2(neg) CD117(pos)) subsets were also detected. ILC2 (CRTH2(pos)) 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.

with primary biliary cirrhosis based on serum albumin levels, platelet count and serum alkaline phosphatase level. A easy accessible online tool is available where values can be entered and score greater than 50% is considered to predict the presence of varices, thereby warranting oesophago-gastro-duodenoscopy (OGD). The aim of this study was to validate this score in an external validation cohort from Liverpool. Methods study Of OGD any clinical were identified and findings of the OGD noted. Results of blood tests to allow calcu-lation of the NVP score were recorded. An NVP probability of 0.5 was used as the cut-off to analyse the performance of the score. Results involved in the study had mean albumin levels of 36, platelets of 260 with an ALP ranging between 58 and 811. 97% female and median age of patients was 67 years. 30 PBC patients who an OGD were identified. 10 of 30 patients had varices on endoscopy. The NVP Score performed well in identifying those in whom varices were absent in this cohort (sensitivity of 100%, specificity 69%, Negative Predictive Value 100% and Positive Predictive Value 10%; overall accuracy 84.5%) and had a good discriminating power with 0.89.

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AIM:Imaging of blood flow in narrow channels and close to vessel walls is important in cardiovascular research for understanding pathogenesis. Our aim was to provide novel nanoprobes with visible emission and long lifetimes as trackers of flow.MATERIALS & METHODS:Gold nanoparticles coated with an iridium complex were prepared. Luminescence imaging was used to monitor their flows in different hematocrit blood and in murine tissues.RESULTS:The velocities are independent of hematocrit level and the nanoparticles entering blood circulation can be clearly detected in vessels in lungs, mesentery and the skeletal muscle.CONCLUSION:The work introduces for the first time iridium-based yellow-green luminescence with nanoparticle size of 100 nm for visualizing and monitoring flows with much higher resolution than conventional alternatives.

The liver is a unique immunological organ, which has both tolerogenic and immunogenic properties. It resides strategically between the portal venous and systemic circulation and thus receives a dual blood supply. The portal venous circulation constantly carries antigen- and microbiota-rich blood to the hepatic sinusoids. Pathogens from the gastrointestinal barrier are filtered through the liver and scanned by antigen-presenting cells and presented to lymphocytes before arriving in the systemic circulation. Since the liver is the first organ these pathogens reach, it acts like a firewall to protect against the pathogens invading the systemic circulation. The human liver is populated by different immune cells, which are both resident and recruited from the circulation. Most immune cells reside in the portal tract, with a few scattered throughout the parenchyma lobules. The lymphoid repertoire includes significant numbers of T, B and natural killer cells. T lymphocytes can be divided into conventional (CD4, CD8) and unconventional lymphocytes (invariant NK T, γδ T, and mucosal-associated invariant T cells). Many of the liver-resident lymphocytes differ phenotypically and functionally from circulating lymphocytes. The frequencies of these immune cells and/or their phenotypic makers could be applied as predictors in liver diseases. Many immunological manipulations to treat patients with liver diseases are now underway such as cytokine blockade therapies for IL-17 and the tumor necrosis factor family B-cell-activating factor (BAFF), along with a future potential application of cell-based therapy in autoimmune liver diseases and immune-based vaccination in liver cancer.

CD4+ CD25high CD127low forkhead box protein 3 (FoxP3+ ) regulatory T cells (Treg ) are essential for the maintenance of peripheral tolerance. Impaired Treg function and an imbalance between effector and Tregs contribute to the pathogenesis of autoimmune diseases. We reported recently that the hepatic microenvironment is deficient in interleukin (IL)-2, a cytokine essential for Treg survival and function. Consequently, few liver-infiltrating Treg demonstrate signal transducer and activator of transcription-5 (STAT-5) phosphorylation. To establish the potential of IL-2 to enhance Treg therapy, we investigated the effects of very low dose Proleukin (VLDP) on the phosphorylation of STAT-5 and the subsequent survival and function of Treg and T effector cells from the blood and livers of patients with autoimmune liver diseases. VLDP, at less than 5 IU/ml, resulted in selective phosphorylation of STAT-5 in Treg but not effector T cells or natural killer cells and associated with increased expression of cytotoxic T lymphocyte antigen-4 (CTLA-4), FoxP3 and CD25 and the anti-apoptotic protein Bcl-2 in Treg with the greatest enhancement of regulatory phenotype in the effector memory Treg population. VLDP also maintained expression of the liver-homing chemokine receptor CXCR3. VLDP enhanced Treg function in a CTLA-4-dependent manner. These findings open new avenues for future VLDP cytokine therapy alone or in combination with clinical grade Treg in autoimmune liver diseases, as VLDP could not only enhance regulatory phenotype and functional property but also the survival of intrahepatic Treg .

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.

Introduction Innate lymphoid cells (ILCs) involve in the initiation, regulation and resolution of inflammation. Although the role of ILCs in murine liver fibrosis and biliary proliferation has been reported, the phenotypic characteristics and functional role of these cells in human liver disease remains undefined. We explored the detailed phenotype of human intrahepatic ILCs to gain insight on their function. Methods Liver infiltrating lymphocytes from normal liver tissue, autoimmune liver diseases (AILD), alcoholic liver disease (ALD) and non-alcoholic steatohepatitis (NASH) explants were phenotyped with flow cytometry. Results Total intrahepatic ILC (CD3− lineage− CD45− CD127+ ) comprised 1% (IQR 0.4–1.7%) of the CD3− CD45+ population in normal liver and 0.4% for ALD/NASH and autoimmune livers. The ILC1 subset constituted the majority of intrahepatic ILC and its frequency is higher in normal liver compared to diseased livers (85% vs. 67%) (p < 0.05). In contrast, CD161+CRTH2+ ILC2 subset and ILC3 subset frequency are higher in diseased livers compared to normal livers. Interestingly, frequency of ILC3 is significantly higher in ALD compared to normal liver (p < 0.01). Intrahepatic ILC subsets are in activated and tissue resident state (CD69 75–90%). All subsets expressed the liver tissue homing chemokine receptor, CXCR3. Expression frequency is highest on diseased ILC1 (AILD 50%, ALD/NASH 56%,) compared to ILC2 (AILD 24%; ALD/NASH 15%) and ILC3 (AILD 38%, ALD/NASH 28%). Biliary tropic receptor CCR6 was expressed more highly by ILC subsets in diseased compared to normal livers. The fibronectin receptor, VLA5 and laminin receptor VLA6 were both highly expressed on all intrahepatic ILC subsets (60–90% and 55–80% respectively). In terms of the cytokine receptors expressed, on all ILC subsets, IL-6R was near undetectable (2%) and low expressions of ST2 (3–6%), IL-25R (2–5%) and IL-23R (2–7%) were detected. In contrast there was almost ubiquitous expression of IL18Ralpha. CD25 was also very highly expressed. Of note, CD25 was significantly higher in ILC1 and ILC3 subsets from normal compared to AILD (p < 0.01) livers (40% vs. 80%; 66% vs. 93%) while intrahepatic ILC2 and ILC3 subsets in diseased states highly expressed CD25 (>90%). Of likely functional importance, intrahepatic ILC1 expressed IFN-γ (40%) while ILC2 expressed IL-13 (20–50%) in diseased states. Conclusion We report for the first time the presence of all three ILC subsets within the human liver immune cell infiltrates. CXCR3+ IFN-γ expressing ILC1 subset is enriched in both normal and inflamed diseased livers. Higher frequencies of CCR6+ VLA5+ VLA6+ IL-13 expressing ILC2 are observed in diseased livers suggesting that this subset may play a role in biliary pathology and peri-biliary fibrosis. Disclosure of Interest None Declared