Methods for managing miscarriage: a network meta-analysis.

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.

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.

NK Cells in Ascites From Liver Disease Patients Display a Particular Phenotype and Take Part in Antibacterial Immune Response.

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.

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.

The human lymph node microenvironment unilaterally regulates T-cell activation and differentiation.

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 (TGFßR). 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.

Human liver infiltrating γδ T cells are composed of clonally expanded circulating and tissue-resident populations.

BACKGROUND & AIMS: γδ T cells comprise a substantial proportion of tissue-associated lymphocytes. However, our current understanding of human γδ 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 γδ T cells in the human liver. METHODS: We characterised the TCR repertoire, immunophenotype and function of human liver infiltrating γδ T cells, by TCR sequencing analysis, flow cytometry, in situ hybridisation and immunohistochemistry. We focussed on the predominant tissue-associated Vδ2- γδ subset, which is implicated in liver immunopathology. RESULTS: Intrahepatic Vδ2- γδ T cells were highly clonally focussed, with single expanded clonotypes featuring complex, private TCR rearrangements frequently dominating the compartment. Such T cells were predominantly CD27lo/- effector lymphocytes, whereas naïve CD27hi, TCR-diverse populations present in matched blood were generally absent in the liver. Furthermore, while a CD45RAhi Vδ2- γδ effector subset present in both liver and peripheral blood contained overlapping TCR clonotypes, the liver Vδ2- γδ T cell pool also included a phenotypically distinct CD45RAlo 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 Vδ2- γδ 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 Vδ2- γδ 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 γδ T cell subsets. They also highlight the inherent functional plasticity within the Vδ2- γδ 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: γδ 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 γδ T cells are enriched for clonally expanded effector T cells, whereas naïve γδ 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 γδ T cells was also evident. Our findings suggest that factors triggering γδ T cell clonal selection and differentiation, such as infection, can drive enrichment of γδ T cells into liver tissue, allowing the development of functionally distinct tissue-restricted memory populations specialised in local hepatic immunosurveillance.

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).

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

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.

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.

Tailoring iridium luminescence and gold nanoparticle size for imaging of microvascular blood flow.

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.

Corrigendum to "Autoimmune Hepatitis: Progress from Global Immunosuppression to Personalised Regulatory T Cell Therapy".

[This corrects the article DOI: 10.1155/2016/7181685.].

Clinical Potential of Regulatory T Cell Therapy in Liver Diseases: An Overview and Current Perspectives.

The increasing demand for liver transplantation and the decline in donor organs has highlighted the need for alternative novel therapies to prevent chronic active hepatitis, which eventually leads to liver cirrhosis and liver cancer. Liver histology of chronic hepatitis is composed of both effector and regulatory lymphocytes. The human liver contains different subsets of effector lymphocytes that are kept in check by a subpopulation of T cells known as Regulatory T cells (Treg). The balance of effector and regulatory lymphocytes generally determines the outcome of hepatic inflammation: resolution, fulminant hepatitis, or chronic active hepatitis. Thus, maintaining and adjusting this balance is crucial in immunological manipulation of liver diseases. One of the options to restore this balance is to enrich Treg in the liver disease patients. Advances in the knowledge of Treg biology and development of clinical grade isolation reagents, cell sorting equipment, and good manufacturing practice facilities have paved the way to apply Treg cells as a potential therapy to restore peripheral self-tolerance in autoimmune liver diseases (AILD), chronic rejection, and posttransplantation. Past and on-going studies have applied Treg in type-1 diabetes mellitus, systemic lupus erythematosus, graft versus host diseases, and solid organ transplantations. There have not been any new therapies for the AILD for more than three decades; thus, the clinical potential for the application of autologous Treg cell therapy to treat autoimmune liver disease is an attractive and novel option. However, it is fundamental to understand the deep immunology, genetic profiles, biology, homing behavior, and microenvironment of Treg before applying the cells to the patients.

Autoimmune Hepatitis: Progress from Global Immunosuppression to Personalised Regulatory T Cell Therapy.

Autoimmune hepatitis (AIH) is an immune mediated liver injury. The precise aetiology of AIH is still unknown but current evidence suggests both genetic and environmental factors are involved. Breakdown in peripheral self-tolerance, and impaired functions of FOXP3(+) regulatory T cell along with effector cell resistance to suppression at the tissue level seem to play an important role in AIH immunopathogenesis. AIH is predominantly a T lymphocytes driven disease but B lymphocytes are also involved in the immunopathology. Innate immune cells are crucial in the initial onset of disease and their response is followed by adaptive T (Th1, Th17, and cytotoxic T cells) and B cell responses evidenced by liver histology and peripheral blood serology. Standard treatment regimens involving steroid and immunosuppressive medications lead to global immune suppression requiring life-long therapy with many side effects. Biologic therapies have been attempted but duration of remission is short-lived. Future direction of diagnosis and treatment for AIH should be guided by "omics" and the immunology profile of the individual patient and clinicians should aim to deliver personalised medicine for their patients. Cell therapy such as infusion of autologous, antigen-specific, and liver-homing regulatory T cells to restore hepatic immune tolerance may soon be a potential future treatment for AIH patients.

Human intrahepatic regulatory T cells are functional, require IL-2 from effector cells for survival, and are susceptible to Fas ligand-mediated apoptosis.

UNLABELLED: Regulatory T cells (Treg ) suppress T effector cell proliferation and maintain immune homeostasis. Autoimmune liver diseases persist despite high frequencies of Treg in the liver, suggesting that the local hepatic microenvironment might affect Treg stability, survival, and function. We hypothesized that interactions between Treg and endothelial cells during recruitment and then with epithelial cells within the liver affect Treg stability, survival, and function. To model this, we explored the function of Treg after migration through human hepatic sinusoidal-endothelium (postendothelial migrated Treg [PEM Treg ]) and the effect of subsequent interactions with cholangiocytes and local proinflammatory cytokines on survival and stability of Treg . Our findings suggest that the intrahepatic microenvironment is highly enriched with proinflammatory cytokines but deficient in the Treg survival cytokine interleukin (IL)-2. Migration through endothelium into a model mimicking the inflamed liver microenvironment did not affect Treg stability; however, functional capacity was reduced. Furthermore, the addition of exogenous IL-2 enhanced PEM Treg phosphorylated STAT5 signaling compared with PEMCD8. CD4 and CD8 T cells are the main source of IL-2 in the inflamed liver. Liver-infiltrating Treg reside close to bile ducts and coculture with cholangiocytes or their supernatants induced preferential apoptosis of Treg compared with CD8 effector cells. Treg from diseased livers expressed high levels of CD95, and their apoptosis was inhibited by IL-2 or blockade of CD95. CONCLUSION: Recruitment through endothelium does not impair Treg stability, but a proinflammatory microenvironment deficient in IL-2 leads to impaired function and increased susceptibility of Treg to epithelial cell-induced Fas-mediated apoptosis. These results provide a mechanism to explain Treg dysfunction in inflamed tissues and suggest that IL-2 supplementation, particularly if used in conjunction with Treg therapy, could restore immune homeostasis in inflammatory and autoimmune liver disease. (Hepatology 2016;64:138-150).

Biliary epithelium and liver B cells exposed to bacteria activate intrahepatic MAIT cells through MR1.

BACKGROUND & AIMS: Mucosal-Associated Invariant T (MAIT) cells are innate-like T cells characterised by the invariant TCR-chain, Vα7.2-Jα33, and are restricted by MR1, which presents bacterial vitamin B metabolites. They are important for antibacterial immunity at mucosal sites; however, detailed characteristics of liver-infiltrating MAIT (LI-MAIT) and their role in biliary immune surveillance remain unexplored. METHODS: The phenotype and intrahepatic localisation of human LI-MAIT cells was examined in diseased and normal livers. MAIT cell activation in response to E. coli-exposed macrophages, biliary epithelial cells (BEC) and liver B cells was assessed with/without anti-MR1. RESULTS: Intrahepatic MAIT cells predominantly localised to bile ducts in the portal tracts. Consistent with this distribution, they expressed biliary tropic chemokine receptors CCR6, CXCR6, and integrin αEß7. LI-MAIT cells were also present in the hepatic sinusoids and possessed tissue-homing chemokine receptor CXCR3 and integrins LFA-1 and VLA-4, suggesting their recruitment via hepatic sinusoids. LI-MAIT cells were enriched in the parenchyma of acute liver failure livers compared to chronic diseased livers. LI-MAIT cells had an activated, effector memory phenotype, expressed α4ß7 and receptors for IL-12, IL-18, and IL-23. Importantly, in response to E. coli-exposed macrophages, liver B cells and BEC, MAIT cells upregulated IFN-γ and CD40 Ligand and degranulated in an MR1-dependent, cytokine-independent manner. In addition, diseased liver MAIT cells expressed T-bet and RORγt and the cytokines IFN-γ, TNF-α, and IL-17. CONCLUSIONS: Our findings provide the first evidence of an immune surveillance effector response for MAIT cells towards BEC in human liver; thus they could be manipulated for treatment of biliary disease in the future.

Direct infection of primary endothelial cells with human cytomegalovirus prevents angiogenesis and migration.

Human cytomegalovirus (hCMV) is a beta herpesvirus that establishes lifelong infection. Although the virus does not usually cause overt clinical symptoms in immunocompetent individuals it can have deleterious effects in immunocompromised patients, such as those on post-transplant medication or with HIV infection. hCMV is the most common congenital infection and can lead to serious fetal sequelae. Endothelial cells (ECs) are natural hosts for hCMV in vivo, therefore, investigations of how this cell type is modulated by infection are key to understanding hCMV pathogenesis. Previous studies have examined the effect of secretomes from hCMV-infected cells on EC angiogenesis, whereas the effect of direct infection on this process has not been so well investigated. Here, we show that placental ECs are viral targets during congenital infection and that vessels in infected tissue appear morphologically abnormal. We demonstrate that the clinical hCMV strain VR1814 impaired EC tube assembly in in vitro angiogenesis assays and inhibited wound healing ability in scratch assays. Secretomes from infected cultures did not impair angiogenesis of uninfected ECs, suggesting that cell-intrinsic changes, as opposed to secreted factors, were responsible. We observed viral gene transcription dependent downregulation of the expression of angiogenesis-associated genes, including angiopoietin-2, TEK receptor and vascular endothelial growth factor receptors. An alternative clinical hCMV stain, TB40E showed similar effects on EC angiogenesis. Together, our data indicate that direct infection with hCMV can induce an anti-migratory and anti-angiogenic EC phenotype, which could have a detrimental effect on the vasculature development in infected tissues.

Analysis of the effects of stromal cells on the migration of lymphocytes into and through inflamed tissue using 3-D culture models.

Stromal cells may regulate the recruitment and behaviour of leukocytes during an inflammatory response, potentially through interaction with the endothelial cells (EC) and the leukocytes themselves. Here we describe new in vitro methodologies to characterise the effects of stromal cells on the migration of lymphocytes through endothelium and its underlying matrix. Three-dimensional tissue-like constructs were created in which EC were cultured above a stromal layer incorporating fibroblasts either as a monolayer on a porous filter or dispersed within a matrix of collagen type 1. A major advantage of these constructs is that they enable each step in leukocyte migration to be analysed in sequence (migration through EC and then stroma), as would occur in vivo. Migrated cells can also be retrieved from the constructs to identify which subsets traffic more effectively and how their functional responses evolve during migration. We found that culture of EC with dermal fibroblasts promoted lymphocyte transendothelial migration but not onward transit through matrix. A critical factor influencing the effect of fibroblasts on recruitment proved to be their proximity to the EC, with direct contact tending to disrupt migration. Comparison of the different approaches indicates that choice of an appropriate 3-D model enables the steps in lymphocyte entry into tissue to be studied in sequence, the regulatory mechanism to be dissected, and the effects of changes in stroma to be investigated.

Infection and transmission dynamics of rKSHV.219 in primary endothelial cells.

Kaposi's sarcoma-associated herpesvirus (KSHV) is the aetiologic agent of Kaposi's sarcoma (KS), a tumour of endothelial cell origin. The study of KS development was aided by the generation of a recombinant GFP (latent)/RFP (lytic)-expressing KSHV (rKSHV.219) by Vieira and O'Hearn (2004). In this study the first data characterising primary endothelial cell infection and transmission with this virus is presented. Infection was predominantly latent and the percentage of GFP-positive cells increased over time. Neither horizontal transmission of infection, nor cellular proliferation, explained this increase. Analysis of latency-associated nuclear antigen (LANA-1) expression revealed that a threshold level of infection was required for GFP expression early post infection. At later time points GFP correlated more closely with LANA-1 expression, likely due to the accumulation of GFP over time. This study provides methodological guidance for the use of rKSHV.21. In addition, it highlights potential problems associated with the use of fluorescent proteins as markers of viral infection.

Delay of migrating leukocytes by the basement membrane deposited by endothelial cells in long-term culture.

We investigated the migration of human leukocytes through endothelial cells (EC), and particularly their underlying basement membrane (BM). EC were cultured for 20days on 3µm-pore filters or collagen gels to form a distinct BM, and then treated with tumour necrosis factor-α, interleukin-1ß or interferon-γ. Neutrophil migration through the cytokine-treated EC and BM was delayed for 20-day compared to 4-day cultures. The BM alone obstructed chemotaxis of neutrophils, and if fresh EC were briefly cultured on stripped BM, there was again a hold-up in migration. In studies with lymphocytes and monocytes, we could detect little hold-up of migration for 20-day versus 4-day cultures, in either the filter- or gel-based models. Direct microscopic observations showed that BM also held-up neutrophil migration under conditions of flow. Treatment of upper and/or lower compartments of filters with antibodies against integrins, showed that neutrophil migration through the endothelial monolayer was dependent on ß(2)-integrins, but not ß1- or ß(3)-integrins. Migration from the subendothelial compartment was supported by ß1- and ß(2)-integrins for all cultures, but blockade of ß(3)-integrin only inhibited migration effectively for 20-day cultures. Flow cytometry indicated that there was no net increase in expression of ß1- or ß3-integrins during neutrophil migration, and that their specific subendothelial function was likely dependent on turnover of integrins during migration. These studies show that BM is a distinct barrier to migration of human neutrophils, and that ß(3)-integrins are particularly important in crossing this barrier. The lesser effect of BM on lymphocytes and monocytes supports the concept that crossing the BM is a separate, leukocyte-specific, regulated step in migration.