BACKGROUND & AIMS:The susceptibility of patients with cirrhosis to infection, a major determinant of prognosis, relates to the development of immuneparesis, a complex interplay of different immunosuppressive cells and soluble factors. The mechanisms underlying the dynamics of immuneparesis of innate immunity remain unclear. We aimed to dissect the heterogeneity of circulating monocyte states in different stages of cirrhosis, and to determine the function of selected differentially expressed genes. METHODS:We systematically investigated circulating monocytes in health and compensated/non-acutely decompensated cirrhosis using single-cell RNA sequencing. Selective genes were confirmed by flow cytometry and diverse functional assays on monocytes ex vivo. RESULTS:We partitioned monocytes into seven clusters. Their abundances varied between cirrhosis stages, confirming previously reported changes, i.e. the reduction in CD14lowCD16++ and emergence of monocytic myeloid-derived suppressor cells in advanced stages. Differentially expressed genes between health and disease and among stages were detected, including CD52 for the first time. CD52 expression on monocytes significantly increased through compensated and non-acutely decompensated cirrhosis. In patients with cirrhosis, CD52highCD14+CD16highHLA-DRhigh monocytes had a functional phenotype of active phagocytes, with enhanced migratory potential and increased cytokine production capacity but limited ability to activate T cells. Following acute decompensation, CD52 was cleaved by elevated PLC, and soluble CD52 was detected in the circulation. Inhibition and cleavage of CD52 significantly suppressed monocyte functions ex vivo and in vitro, while the predominance of immunosuppressive CD52low circulating monocytes in patients with acute decompensation was associated with infection and low transplant-free survival. CONCLUSION:CD52 may represent a biologically relevant target for future immunotherapy. Stabilising CD52 may enhance monocyte functions and infection control in the context of cirrhosis, guided by soluble CD52/PLC as biomarkers of immuneparesis. IMPACT AND IMPLICATIONS:Monocyte dysfunction substantially contributes to infection susceptibility, which is a major determinant of the prognosis of patients with cirrhosis and represents a major unmet therapeutic need. Its underlying mechanisms remain poorly understood, although, among hepatologists, it is thought that the therapeutic reconstitution of monocyte function could enhance defence against infection and thus reduce morbidity and mortality of patients with cirrhosis. By systematically delineating the heterogeneity and function of circulating monocytes ex vivo, we identified that the absence of CD52 expression on monocytes represented a distinct biomarker of monocyte dysfunction in patients with cirrhosis, discriminating patients at substantial risk of infectious complications. Otherwise, given the beneficial antimicrobial functions of CD52-expressing monocytes, CD52 stablisation may also represent a therapeutic approach worth exploring.

Introduction Primary sclerosing cholangitis (PSC) is the classical hepatobiliary manifestation of inflammatory bowel disease (IBD). The strong association between gut and liver inflammation has driven several pathogenic hypotheses to which the intestinal microbiome is proposed to contribute. Pilot studies of faecal microbiota transplantation (FMT) in PSC and IBD are demonstrated to be safe and associated with increased gut bacterial diversity. However, the longevity of such changes and the impact on markers of disease activity and disease progression have not been studied. The aim of this clinical trial is to determine the effects of repeated FMT as a treatment for PSC-IBD.Methods and analysis FAecal micRobiota transplantation in primary sclerosinG chOlangitis (FARGO) is a phase IIa randomised placebo-controlled trial to assess the efficacy and safety of repeated colonic administration of FMT in patients with non-cirrhotic PSC-IBD. Fifty-eight patients will be recruited from six sites across England and randomised in a 1:1 ratio between active FMT or FMT placebo arms. FMT will be manufactured by the University of Birmingham Microbiome Treatment Centre, using stool collected from rigorously screened healthy donors. A total of 8 weekly treatments will be delivered; the first through colonoscopic administration (week 1) and the remaining seven via once-weekly enema (up to week 8). Participants will then be followed on a 12-weekly basis until week 48 from the first treatment visit. The primary efficacy outcome will be to determine the effect of FMT on serum alkaline phosphatase values over time (end of study at 48 weeks). Key secondary outcomes will be to evaluate the impact of FMT on other liver biochemical parameters, PSC risk scores, circulating and imaging markers of liver fibrosis, health-related quality of life measures, IBD activity and the incidence of PSC-related clinical events. Key translational objectives will be to identify mucosal metagenomic, metatranscriptomic, metabolomic and immunological pathways associated with the administration of FMT.Ethics and dissemination The protocol was approved by the South Central—Hampshire B Research Ethics Committee (REC 23/SC/0147). Participants will be required to provide written informed consent. The results of this trial will be disseminated through national and international presentations and peer-reviewed publications.Trial registration number The trial was registered at ClinicalTrials.gov on 23 February 2024 (NCT06286709). Weblink: Study Details | FAecal Microbiota Transplantation in primaRy sclerosinG chOlangitis | ClinicalTrials.gov.

The presence of CD8 + T cells in the cytoplasm of biliary epithelial cells (BEC) has been correlated with biliary damage associated with primary biliary cholangitis (PBC). Here, we characterise the mechanism of CD8 + T cell invasion into BEC. CD8 + T cells observed within BEC were large, eccentric, and expressed E-cadherin, CD103 and CD69. They were also not contained within secondary vesicles. Internalisation required cytoskeletal rearrangements which facilitated contact with BEC. Internalised CD8 + T cells were observed in both non-cirrhotic and cirrhotic diseased liver tissues but enriched in PBC patients, both during active disease and at the time of transplantation. E-cadherin expression by CD8 + T cells correlated with frequency of internalisation of these cells into BEC. E-cadherin + CD8 + T cells formed β-catenin-associated interactions with BEC, were larger than E-cadherin - CD8 + T cells and invaded into BEC more frequently. Overall, we unveil a distinct cell-in-cell structure process in the liver detailing the invasion of E-cadherin + CD103 + CD69 + CD8 + T cells into BEC.

Abstract Background LiMAx is a novel test for assessing the metabolic capacity and dynamic function of the liver. The current review aims to investigate the predictive role of LiMAx for postoperative outcomes in patients undergoing liver resection. Methods A systematic review was conducted using EMBASE, Web of Science, Medline and Cochrane Library databases. Preoperative LiMAx values and various postoperative outcomes were extracted (e.g. mortality, post-hepatectomy liver failure). Pooled averages were calculated where appropriate. Postoperative complications in relation to the extent of resection and LiMAx values were the outcomes assessed. Results 13 studies, comprising 1668 participants and 20 separate cohorts in total, were included. The meta-regression of preoperative LiMAx and postoperative outcomes yielded non-significant results (p>0.05). A second meta-regression highlighted significant correlations between extra major surgery rate and various outcomes (p<0.05), suggesting its confounding role in the first regression. A further analysis found a non-significant tendency for mortality to correlate with LiMAx as expected in patients undergoing minor resection. Conclusion In this review, there was no statistical correlation between LiMAx values and post-hepatectomy outcomes. The LiMAx test was relatively more helpful in predicting mortality in patients undergoing minor resection; this was likely confounded by selection bias in the low quality studies. Larger multi-centre studies with standardised reporting of outcomes are required.

Background: Primary sclerosing cholangitis is a progressive inflammatory liver disease characterized by biliary and liver fibrosis. Vascular adhesion protein-1 (VAP-1) is important in the inflammatory process driving liver fibrosis. We evaluated the safety and efficacy of VAP-1 blockade with a monoclonal antibody (timolumab, BTT1023) in patients with primary sclerosing cholangitis. Methods: BUTEO was a prospective, single-arm, open-label, multicenter, phase II trial, conducted in 6 centers in the United Kingdom. Patients with primary sclerosing cholangitis aged 18-75 years had an alkaline phosphatase value of >1.5 times the upper limit of normal. The dose-confirmatory stage aimed to confirm the safety of timolumab through the incidence of dose-limiting toxicity and sufficient trough levels of circulating antibody to block VAP-1 function. The primary outcome of the dose-expansion portion of the trial was patient's response to timolumab at day 99, as measured by a reduction in serum alkaline phosphatase by 25% or more from baseline to day 99. Results: Twenty-three patients were recruited: 7 into the initial dose-confirmatory stage and a further 16 into an expansion stage. Timolumab (8 mg/kg) was confirmed to be safe for the duration of administration with sufficient circulating levels. Only 2 of the 18 evaluable patients (11.1%) achieved a reduction in alkaline phosphatase levels of 25% or more, and both the proportion of circulating inflammatory cell populations and biomarkers of fibrosis remained unchanged from baseline. Conclusions: The BUTEO trial confirmed 8 mg/kg timolumab had no short-term safety signals and resulted in sufficient circulating levels of VAP-1 blocking timolumab. However, the trial was stopped after an interim assessment due to a lack of efficacy as determined by no significant change in serum liver tests.

Background & Aims Infectious complications determine the prognosis of cirrhosis patients. Their infection susceptibility relates to the development of immuneparesis, a complex interplay of different immunosuppressive cells and soluble factors. Mechanisms underlying the dynamics of immuneparesis of innate immunity remain inconclusive. We aimed to dissect the heterogeneity of circulating monocyte states in different cirrhosis stages, and pursued the function of selected differentially expressed (DE) genes. Methods We systematically investigated circulating monocytes in health, compensated and not-acutely decompensated (NAD) cirrhosis using single cell RNA sequencing. Selective genes were confirmed by flow cytometry and diverse functional assays on monocytes ex vivo . Results We identified seven monocyte clusters. Their abundances varied between cirrhosis stages, confirming previously reported changes i.e. reduction in CD14lowCD16++ and emergence of M-MDSC in advanced stages. DE genes between health and disease and among stages were detected, including for the first time CD52. CD52-expression on monocytes significantly enhanced throughout compensated and NAD cirrhosis. Heretofore the biological significance of CD52-expression on monocytes remained unknown. CD52highCD14+CD16highHLA-DRhigh monocytes in patients with cirrhosis revealed a functional phenotype of active phagocytes with enhanced migratory potential, increased cytokine production, but poor T cell activation. Following acute decompensation (AD), CD52 was cleaved by elevated phospholipase C (PLC), and soluble CD52 (sCD52) was detected in the circulation. Inhibition and cleavage of CD52 significantly suppressed monocyte functions ex vivo and in vitro , and the predominance of immunosuppressive CD52low circulating monocytes in patients with AD was associated with infection and low transplant-free survival. Conclusion CD52 may represent a biologically relevant target for future immunotherapy. Stabilising CD52 may enhance monocyte functions and infection control in the context of cirrhosis, guided by sCD52/PLC as biomarkers indicating immuneparesis. Lay summary Recurrent infections are a major cause of death in patients with liver cirrhosis. A fundamental understanding of the mechanisms that suppress immune responses in patients with cirrhosis is lacking, but required for the development of strategies to restore innate immunity in cirrhosis patients and prevent infection. The current study identified a novel marker for deficient immune responses and a potential target for such a future immune-based therapy. ![Graphical Abstract][1]</img> Graphical Abstract scRNA-seq identified seven circulating monocyte states, changing in cirrhosis patients at different stages of disease. Circulating monocytes overexpress CD52 in cirrhosis, but are absent in AD/ACLF due to PLC. CD52-expressing monocytes show high capability for phagocytosis, cytokine production, adhesion and migration potential and T cell suppression. Created with [BioRender.com][2] ### Competing Interest Statement The authors have declared no competing interest. [1]: pending:yes [2]: http://BioRender.com

Modeling immune cell recruitment by liver endothelial cells in vitro is important to better understand the pathology of chronic inflammatory liver diseases and cancers. Here, we present a protocol for the study of monocyte transmigration across activated primary human liver endothelial cells, under physiological flow conditions. We describe primary endothelial cell isolation from human liver tissues and monocyte isolation from human blood. We then detail the shear flow-based assay and subsequent analysis of the different stages of monocyte transmigration. For complete details on the use and execution of this protocol, please refer to Wilkinson et al.1.

Gremlin-1 has been implicated in liver fibrosis in metabolic dysfunction-associated steatohepatitis (MASH) via inhibition of bone-morphogenetic protein (BMP) signalling and has thereby been identified as a potential therapeutic target. Using rat in vivo and human in vitro and ex vivo model systems of MASH fibrosis, we show that neutralisation of Gremlin-1 activity with monoclonal therapeutic antibodies does not reduce liver inflammation or liver fibrosis. Still, Gremlin-1 was upregulated in human and rat MASH fibrosis, but expression was restricted to a small subpopulation of COL3A1/THY1 + myofibroblasts. Lentiviral overexpression of Gremlin-1 in LX-2 cells and primary hepatic stellate cells led to changes in BMP-related gene expression, which did not translate to increased fibrogenesis. Furthermore, we show that Gremlin-1 binds to heparin with high affinity, which prevents Gremlin-1 from entering systemic circulation, prohibiting Gremlin-1-mediated organ crosstalk. Overall, our findings suggest a redundant role for Gremlin-1 in the pathogenesis of liver fibrosis, which is unamenable to therapeutic targeting.

The liver has a unique ability to regenerate1,2; however, in the setting of acute liver failure (ALF), this regenerative capacity is often overwhelmed, leaving emergency liver transplantation as the only curative option3-5. Here, to advance understanding of human liver regeneration, we use paired single-nucleus RNA sequencing combined with spatial profiling of healthy and ALF explant human livers to generate a single-cell, pan-lineage atlas of human liver regeneration. We uncover a novel ANXA2+ migratory hepatocyte subpopulation, which emerges during human liver regeneration, and a corollary subpopulation in a mouse model of acetaminophen (APAP)-induced liver regeneration. Interrogation of necrotic wound closure and hepatocyte proliferation across multiple timepoints following APAP-induced liver injury in mice demonstrates that wound closure precedes hepatocyte proliferation. Four-dimensional intravital imaging of APAP-induced mouse liver injury identifies motile hepatocytes at the edge of the necrotic area, enabling collective migration of the hepatocyte sheet to effect wound closure. Depletion of hepatocyte ANXA2 reduces hepatocyte growth factor-induced human and mouse hepatocyte migration in vitro, and abrogates necrotic wound closure following APAP-induced mouse liver injury. Together, our work dissects unanticipated aspects of liver regeneration, demonstrating an uncoupling of wound closure and hepatocyte proliferation and uncovering a novel migratory hepatocyte subpopulation that mediates wound closure following liver injury. Therapies designed to promote rapid reconstitution of normal hepatic microarchitecture and reparation of the gut-liver barrier may advance new areas of therapeutic discovery in regenerative medicine.

Activation of cardiac fibroblasts and differentiation to myofibroblasts underlies development of pathological cardiac fibrosis, leading to arrhythmias and heart failure. Myofibroblasts are characterised by increased α-smooth muscle actin (α-SMA) fibre expression, secretion of collagens and changes in proliferation. Transforming growth factor-beta (TGF-β) and increased mechanical stress can initiate myofibroblast activation. Reversibility of the myofibroblast phenotype has been observed in murine cells but has not been explored in human cardiac fibroblasts. In this study, chronically activated adult primary human ventricular cardiac fibroblasts and human induced pluripotent stem cell derived cFbs (hiPSC-cFbs) were used to investigate the potential for reversal of the myofibroblast phenotype using either subculture on soft substrates or TGF-β receptor inhibition. Culture on softer plates (25 or 2 kPa Young’s modulus) did not alter proliferation or reduce expression of α-SMA and collagen 1. Similarly, culture of myofibroblasts in the presence of TGF-β inhibitor did not reverse myofibroblasts back to a quiescent phenotype. Chronically activated hiPSC-cFbs also showed attenuated response to TGF-β receptor inhibition and inability to reverse to quiescent fibroblast phenotype. Our data demonstrate substantial loss of TGF-β signalling plasticity as well as a loss of feedback from the surrounding mechanical environment in chronically activated human myofibroblasts.

BACKGROUND & AIMS: AXL and MERTK expression on circulating monocytes modulated immune responses in pa-tients with cirrhosis (CD14(+) HLA-DR+ AXL(+) ) and acute-on -chronic liver failure (CD14(+) MERTK+ ). AXL expression involved enhanced efferocytosis, sustained phagocytosis, but reduced tumor necrosis factor-alpha/interleukin-6 production and T-cell activation, suggesting a homeostatic function. Axl was expressed on murine airway in tissues contacting the external environment, but not interstitial lung-and tissue-resident synovial lining macrophages. Here, we assessed AXL expression on tissue macrophages in patients with cirrhosis. METHODS: Using multiplexed immunofluorescence we compared AXL expression in liver biopsies in cirrhosis (n = 22), chronic liver disease (n = 8), non-cirrhotic portal hypertension (n = 4), and healthy controls (n = 4). Phenotype and function of isolated primary human liver macrophages were character-ized by flow cytometry (cirrhosis, n = 11; control, n = 14) ex vivo. Also, AXL expression was assessed on peritoneal (n = 29) and gut macrophages (n = 16) from cirrhotic patients. Regulation of AXL expression was analyzed in vitro and ex vivo using primary hepatic stellate cells (HSCs), LX-2 cells, and GAS6 in co-culture experiments. RESULTS: AXL was expressed on resident (CD68(+)) but not tissue-infiltrating (MAC387(+)) liver macrophages, hepatocytes, HSCs, or sinusoidal endothelial cells. Prevalence of hepatic CD68(+)AXL(+) cells significantly decreased with cirrhosis pro-gression: (healthy, 90.2%; Child-Pugh A, 76.1%; Child-Pugh B, 64.5%; and Child-Pugh C, 18.7%; all P < .05) and negatively correlated with Model for End-Stage Liver Disease and C-reactive protein (all P < .05). AXL-expressing hepatic mac-rophages were CD68(high)HLA-DR(high)CD16(high)CD206(high). AXL expression also decreased on gut and peritoneal macrophages from cirrhotic patients but increased in regional lymph nodes. GAS6, enriched in the cirrhotic liver, appeared to be secreted by HSCs and down-regulate AXL in vitro. CONCLUSIONS: Decreased AXL expression on resident liver macrophages in advanced cirrhosis, potentially in response to activated HSC-secreted GAS6, suggests a role for AXL in the regulation of hepatic immune homeostasis.

ˆCCL24 is a pro-fibrotic, pro-inflammatory chemokine expressed in several chronic fibrotic diseases. In the liver, CCL24 plays a role in fibrosis and inflammation, and blocking CCL24 led to reduced liver injury in experimental models. We studied the role of CCL24 in primary sclerosing cholangitis (PSC) and evaluated the potential therapeutic effect of blocking CCL24 in this disease. Multidrug resistance gene 2-knockout (Mdr2-/-) mice demonstrated CCL24 expression in liver macrophages and were used as a relevant experimental PSC model. CCL24-neutralizing monoclonal antibody, CM-101, significantly improved inflammation, fibrosis, and cholestasis-related markers in the biliary area. Moreover, using spatial transcriptomics, we observed reduced proliferation and senescence of cholangiocytes following CCL24 neutralization. Next, we demonstrated that CCL24 expression was elevated under pro-fibrotic conditions in primary human cholangiocytes and macrophages, and it induced proliferation of primary human hepatic stellate cells and cholangiocytes, which was attenuated following CCL24 inhibition. Correspondingly, CCL24 was found to be highly expressed in liver biopsies of patients with PSC. CCL24 serum levels correlated with Enhanced Liver Fibrosis score, most notably in patients with high alkaline phosphatase levels. These results suggest that blocking CCL24 may have a therapeutic effect in patients with PSC by reducing liver inflammation, fibrosis, and cholestasis.

Following myocardial infarction (MI), elderly patients have a poorer prognosis than younger patients, which may be linked to increased coronary microvessel susceptibility to injury. Interleukin-36 (IL-36), a newly discovered proinflammatory member of the IL-1 superfamily, may mediate this injury, but its role in the injured heart is currently not known. We first demonstrated the presence of IL-36(α/β) and its receptor (IL-36R) in ischemia/reperfusion-injured (IR-injured) mouse hearts and, interestingly, noted that expression of both increased with aging. An intravital model for imaging the adult and aged IR-injured beating heart in real time in vivo was used to demonstrate heightened basal and injury-induced neutrophil recruitment, and poorer blood flow, in the aged coronary microcirculation when compared with adult hearts. An IL-36R antagonist (IL-36Ra) decreased neutrophil recruitment, improved blood flow, and reduced infarct size in both adult and aged mice. This may be mechanistically explained by attenuated endothelial oxidative damage and VCAM-1 expression in IL-36Ra–treated mice. Our findings of an enhanced age-related coronary microcirculatory dysfunction in reperfused hearts may explain the poorer outcomes in elderly patients following MI. Since targeting the IL-36/IL-36R pathway was vasculoprotective in aged hearts, it may potentially be a therapy for treating MI in the elderly population.