Regulation of immune responses in primary biliary cholangitis: a transcriptomic analysis of peripheral immune cells

BACKGROUND AIMS: In patients with primary biliary cholangitis (PBC), the serum liver biochemistry measured during treatment with ursodeoxycholic acid-the UDCA response-accurately predicts long-term outcome. Molecular characterization of patients stratified by UDCA response can improve biological understanding of the high-risk disease, thereby helping to identify alternative approaches to disease-modifying therapy. In this study, we sought to characterize the immunobiology of the UDCA response using transcriptional profiling of peripheral blood mononuclear cell subsets. METHODS: We performed bulk RNA-sequencing of monocytes and TH1, TH17, TREG, and B cells isolated from the peripheral blood of 15 PBC patients with adequate UDCA response ("responders"), 16 PBC patients with inadequate UDCA response ("nonresponders"), and 15 matched controls. We used the Weighted Gene Co-expression Network Analysis to identify networks of co-expressed genes ("modules") associated with response status and the most highly connected genes ("hub genes") within them. Finally, we performed a Multi-Omics Factor Analysis of the Weighted Gene Co-expression Network Analysis modules to identify the principal axes of biological variation ("latent factors") across all peripheral blood mononuclear cell subsets. RESULTS: Using the Weighted Gene Co-expression Network Analysis, we identified modules associated with response and/or disease status (q<0.05) in each peripheral blood mononuclear cell subset. Hub genes and functional annotations suggested that monocytes are proinflammatory in nonresponders, but antiinflammatory in responders; TH1 and TH17 cells are activated in all PBC cases but better regulated in responders; and TREG cells are activated-but also kept in check-in responders. Using the Multi-Omics Factor Analysis, we found that antiinflammatory activity in monocytes, regulation of TH1 cells, and activation of TREG cells are interrelated and more prominent in responders. CONCLUSIONS: We provide evidence that adaptive immune responses are better regulated in patients with PBC with adequate UDCA response

A protocol paper for the MOTION Study—A longitudinal study in a cohort aged 60 years and older to obtain mechanistic knowledge of the role of the gut microbiome during normal healthy ageing in order to develop strategies that will improve lifelong health and wellbeing

Background: Advances in medicine and public health mean that people are living longer; however, a significant proportion of that increased lifespan is spent in a prolonged state of declining health and wellbeing which places increasing pressure on medical, health and social services. There is a social and economic need to develop strategies to prevent or delay age-related disease and maintain lifelong health. Several studies have suggested links between the gut microbiome and age-related disease, which if confirmed would present a modifiable target for intervention development. The MOTION study aims to determine whether and how changes in the gut microbiome are associated with physical and mental capacity. A comprehensive longitudinal multiparameter study such as this has not been previously undertaken. Methods: MOTION is a longitudinal prospective cohort study with a focus on gut health and cognitive function. 360 healthy individuals aged 60 years and older, living in East Anglia, UK will be recruited to the study, stratified into one of three risk groups (cohorts) for developing dementia based on their cognitive function. Participants will attend study appointments every six months over four years, providing stool and blood samples and a health questionnaire. Participants will also undergo physical measurements and cognitive tests at alternating appointments, and undergo Optical Coherence Tomography scans at 3 timepoints. Two subgroups of participants in the study will provide colonic tissue biopsies (n = ≥30 from each cohort), and brain imaging (n = 30) at two timepoints. Discussion: This study will provide new insights into the gut-(microbiota)-brain axis and the relationship between age-associated changes in gut microbe populations and cognitive health. Such insights could help develop new microbe-based strategies to improve lifelong health and wellbeing

A randomized, placebo-controlled, phase II study of obeticholic acid for primary sclerosing cholangitis

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Fine-tuning of SIRT1 expression is essential to protect the liver from cholestatic liver disease

Cholestasis comprises aetiologically heterogeneous conditions characterized by accumulation of bile acids in the liver that actively contribute to liver damage. Sirtuin 1 (SIRT1) regulates liver regeneration and bile acid metabolism by modulating farnesoid X receptor (FXR); we here investigate its role in cholestatic liver disease. We determined SIRT1 expression in livers from patients with cholestatic disease, in two experimental models of cholestasis, as well as in human and murine liver cells in response to bile acid loading. SIRT1-overexpressing (SIRT oe ) and hepatocyte-specific SIRT1-KO (knockout) mice (SIRT hep–/– ) were subjected to bile duct ligation (BDL) and were fed with a 0.1% DDC (3,5-diethoxycarbonyl-1,4-dihydrocollidine) diet to determine the biological relevance of SIRT1 during cholestasis. The effect of NorUDCA (24-norursodeoxycholic acid) was tested in BDL/SIRT oe mice. We found that SIRT1 was highly expressed in livers from cholestatic patients, mice after BDL, and Mdr2 knockout mice (Mdr2 –/– ) animals. The detrimental effects of SIRT1 during cholestasis were validated in vivo and in vitro. SIRT oe mice showed exacerbated parenchymal injury whereas SIRT hep–/– mice evidenced a moderate improvement after BDL and 0.1% DDC feeding. Likewise, hepatocytes isolated from SIRT oe mice showed increased apoptosis in response to bile acids, whereas a significant reduction was observed in SIRT hep–/– hepatocytes. Importantly, the decrease, but not complete inhibition, of SIRT1 exerted by norUDCA treatment correlated with pronounced improvement in liver parenchyma in BDL/SIRT oe mice. Interestingly, both SIRT1 overexpression and hepatocyte-specific SIRT1 depletion correlated with inhibition of FXR, whereas modulation of SIRT1 by NorUDCA associated with restored FXR signaling. Conclusion: SIRT1 expression is increased during human and murine cholestasis. Fine-tuning expression of SIRT1 is essential to protect the liver from cholestatic liver damage

Top research priorities in liver and gallbladder disorders in the UK

OBJECTIVES: There is a mismatch between research questions considered important by patients, carers and healthcare professionals and the research performed in many fields of medicine. The non-alcohol-related liver and gallbladder disorders priority setting partnership was established to identify the top research priorities in the prevention, diagnostic and treatment of gallbladder disorders and liver disorders not covered by the James-Lind Alliance (JLA) alcohol-related liver disease priority setting partnership. DESIGN: The methods broadly followed the principles of the JLA guidebook. The one major deviation from the JLA methodology was the final step of identifying priorities: instead of prioritisation by group discussions at a consensus workshop involving stakeholders, the prioritisation was achieved by a modified Delphi consensus process. RESULTS: A total of 428 unique valid diagnostic or treatment research questions were identified. A literature review established that none of these questions were considered 'answered' that is, high-quality systematic reviews suggest that further research is not required on the topic. The Delphi panel achieved consensus (at least 80% Delphi panel members agreed) that a research question was a top research priority for six questions. Four additional research questions with highest proportion of Delphi panel members ranking the question as highly important were added to constitute the top 10 research priorities. CONCLUSIONS: A priority setting process involving patients, carers and healthcare professionals has been used to identify the top 10priority areas for research related to liver and gallbladder disorders. Basic, translational, clinical and public health research are required to address these uncertainties

British Society of Gastroenterology guidelines for the diagnosis and management of cholangiocarcinoma

These guidelines for the diagnosis and management of cholangiocarcinoma (CCA) were commissioned by the British Society of Gastroenterology liver section. The guideline writing committee included a multidisciplinary team of experts from various specialties involved in the management of CCA, as well as patient/public representatives from AMMF (the Cholangiocarcinoma Charity) and PSC Support. Quality of evidence is presented using the Appraisal of Guidelines for Research and Evaluation (AGREE II) format. The recommendations arising are to be used as guidance rather than as a strict protocol-based reference, as the management of patients with CCA is often complex and always requires individual patient-centred considerations

Intestinal microbiome-macrophage crosstalk contributes to cholestatic liver disease by promoting intestinal permeability in mice

Background and Aims: Mounting evidence supports an association between cholestatic liver disease and changes in the composition of the microbiome. Still, the role of the microbiome in the pathogenesis of this condition remains largely undefined.  Approach and Results: To address this, we have used two experimental models, administering alpha-naphtylisocyanate or feeding a 0.1% 3,5-diethoxycarbonyl-1,4-dihydrocollidine diet, to induce cholestatic liver disease in germ-free mice and germ-free mice conventionalized with the microbiome from wild-type, specific pathogen-free animals. Next, we have inhibited macrophage activation by depleting these cells using clodronate liposomes and inhibiting the inflammasome with a specific inhibitor of NOD-, LRR-, and pyrin domain-containing protein 3. Our results demonstrate that cholestasis, the accumulation of bile acids in the liver, fails to promote liver injury in the absence of the microbiome in vivo. Additional in vitro studies supported that endotoxin sensitizes hepatocytes to bile-acid–induced cell death. We also demonstrate that during cholestasis, macrophages contribute to promoting intestinal permeability and to altered microbiome composition through activation of the inflammasome, overall leading to increased endotoxin flux into the cholestatic liver.  Conclusions: We demonstrate that the intestinal microbiome contributes to cholestasis-mediated cell death and inflammation through mechanisms involving activation of the inflammasome in macrophages

Factors Associated With Outcomes of Patients With Primary Sclerosing Cholangitis and Development and Validation of a Risk Scoring System.

We sought to identify factors that are predictive of liver transplantation or death in patients with primary sclerosing cholangitis (PSC), and to develop and validate a contemporaneous risk score for use in a real-world clinical setting. Analyzing data from 1,001 patients recruited to the UK-PSC research cohort, we evaluated clinical variables for their association with 2-year and 10-year outcome through Cox-proportional hazards and C-statistic analyses. We generated risk scores for short-term and long-term outcome prediction, validating their use in two independent cohorts totaling 451 patients. Thirty-six percent of the derivation cohort were transplanted or died over a cumulative follow-up of 7,904 years. Serum alkaline phosphatase of at least 2.4 × upper limit of normal at 1 year after diagnosis was predictive of 10-year outcome (hazard ratio [HR] = 3.05; C = 0.63; median transplant-free survival 63 versus 108 months; P < 0.0001), as was the presence of extrahepatic biliary disease (HR = 1.45; P = 0.01). We developed two risk scoring systems based on age, values of bilirubin, alkaline phosphatase, albumin, platelets, presence of extrahepatic biliary disease, and variceal hemorrhage, which predicted 2-year and 10-year outcomes with good discrimination (C statistic = 0.81 and 0.80, respectively). Both UK-PSC risk scores were well-validated in our external cohort and outperformed the Mayo Clinic and aspartate aminotransferase-to-platelet ratio index (APRI) scores (C statistic = 0.75 and 0.63, respectively). Although heterozygosity for the previously validated human leukocyte antigen (HLA)-DR*03:01 risk allele predicted increased risk of adverse outcome (HR = 1.33; P = 0.001), its addition did not improve the predictive accuracy of the UK-PSC risk scores. Conclusion: Our analyses, based on a detailed clinical evaluation of a large representative cohort of participants with PSC, furthers our understanding of clinical risk markers and reports the development and validation of a real-world scoring system to identify those patients most likely to die or require liver transplantation.Financial support has been received by National Institute of Health Research (RD-TRC and Birmingham Biomedical Research Centre), Isaac Newton Trust, Addenbrooke’s charitable trust, Norwegian PSC Research Center and PSC Support. GMH is supported by the Lily and Terry Horner Chair in Autoimmune Liver Disease Research, Toronto Centre for Liver Disease, Toronto

Analysis of FOXP3+ Regulatory T Cells That Display Apparent Viral Antigen Specificity during Chronic Hepatitis C Virus Infection

We reported previously that a proportion of natural CD25+ cells isolated from the PBMC of HCV patients can further upregulate CD25 expression in response to HCV peptide stimulation in vitro, and proposed that virus-specific regulatory T cells (Treg) were primed and expanded during the disease. Here we describe epigenetic analysis of the FOXP3 locus in HCV-responsive natural CD25+ cells and show that these cells are not activated conventional T cells expressing FOXP3, but hard-wired Treg with a stable FOXP3 phenotype and function. Of ∼46,000 genes analyzed in genome wide transcription profiling, about 1% were differentially expressed between HCV-responsive Treg, HCV-non-responsive natural CD25+ cells and conventional T cells. Expression profiles, including cell death, activation, proliferation and transcriptional regulation, suggest a survival advantage of HCV-responsive Treg over the other cell populations. Since no Treg-specific activation marker is known, we tested 97 NS3-derived peptides for their ability to elicit CD25 response (assuming it is a surrogate marker), accompanied by high resolution HLA typing of the patients. Some reactive peptides overlapped with previously described effector T cell epitopes. Our data offers new insights into HCV immune evasion and tolerance, and highlights the non-self specific nature of Treg during infection