Intergenic risk variant rs56258221 skews the fate of naive CD4+ T cells via miR4464-BACH2 interplay in primary sclerosing cholangitis.

Primary sclerosing cholangitis (PSC) is an immune-mediated liver disease of unknown pathogenesis, with a high risk to develop cirrhosis and malignancies. Functional dysregulation of T cells and association with genetic polymorphisms in T cell-related genes were previously reported for PSC. Here, we genotyped a representative PSC cohort for several disease-associated risk loci and identified rs56258221 (BACH2/MIR4464) to correlate with not only the peripheral blood T cell immunophenotype but also the functional capacities of naive CD4+ T (CD4+ TN) cells in people with PSC. Mechanistically, rs56258221 leads to an increased expression of miR4464, in turn causing attenuated translation of BACH2, a major gatekeeper of T cell quiescence. Thereby, the fate of CD4+ TN is skewed toward polarization into pro-inflammatory subsets. Clinically, people with PSC carrying rs56258221 show signs of accelerated disease progression. The data presented here highlight the importance of assigning functional outcomes to disease-associated genetic polymorphisms as potential drivers of diseases.

IFNγ and CTLA-4 Drive Hepatic CD4 T-Cell Tolerance and Protection From Autoimmunity in Mice.

BACKGROUND & AIMS: The liver has a distinct capacity to induce immune tolerance to hepatic antigens. Although liver tolerance can be advantageous for preventing autoimmune and inflammatory diseases, it also can be detrimental by preventing immune surveillance of infected or malignant cells. Here, we investigated the immune mechanisms that establish hepatic tolerance. METHODS: Tolerance was investigated in C-reactive protein (CRP)-myelin basic protein (MBP) mice expressing the neuroantigen MBP in hepatocytes, providing profound resistance to MBP-induced neuroinflammation. Tolerance induction was studied after transfer of MBP-specific CD4 T cells into CRP-MBP mice, and tolerance mechanisms were tested using depleting or blocking antibodies. RESULTS: Although tolerant CRP-MBP mice display increased numbers of forkhead box P3+ regulatory T cells, we here found them not essential for the maintenance of hepatic tolerance. Instead, upon MBP recognition in the liver, MBP-specific T cells became activated to produce interferon (IFN)γ, which, in turn, induced local up-regulation of recruitment molecules, including Chemokine (C-X-C motif) ligand9 and its receptor C-X-C motif chemokine receptor3, facilitating endothelial translocation and redirection of MBP-specific T cells into the hepatic parenchyma. There, the translocated MBP-specific CD4 T cells partly converted into interleukin 10-producing type 1 regulatory T cells, and significantly up-regulated the expression of immune checkpoint molecules, notably cytotoxic T-lymphocyte-associated protein 4 (CTLA-4). Intriguingly, although liver tolerance was not affected by impairment of interleukin 10 signaling, concomitant blockade of IFNγ and CTLA-4 abrogated hepatic tolerance induction to MBP, resulting in neuroinflammatory autoimmune disease in these mice. CONCLUSIONS: IFNγ-mediated redirection of autoreactive CD4 T cells into the liver and up-regulation of checkpoint molecules, including CTLA-4, were essential for tolerance induction in the liver, hence representing a potential treatment target for boosting or preventing liver tolerance.

Serum CXCL5 Detects Early Hepatocellular Carcinoma and Indicates Tumor Progression.

Chemokines or chemotactic cytokines play a pivotal role in the immune pathogenesis of liver cirrhosis and hepatocellular carcinoma (HCC). Nevertheless, comprehensive cytokine profiling data across different etiologies of liver diseases are lacking. Chemokines might serve as diagnostic and prognostic biomarkers. In our study, we analyzed serum concentrations of 12 inflammation-related chemokines in a cohort of patients (n = 222) with cirrhosis of different etiologies and/or HCC. We compared 97 patients with cirrhosis and treatment-naïve HCC to the chemokine profile of 125 patients with cirrhosis but confirmed absence of HCC. Nine out of twelve chemokines were significantly elevated in sera of cirrhotic patients with HCC compared to HCC-free cirrhosis controls (CCL2, CCL11, CCL17, CCL20, CXCL1, CXCL5, CXCL9, CXCL10, CXCL11). Among those, CXCL5, CXCL9, CXCL10, and CXCL11 were significantly elevated in patients with early HCC according to the Barcelona Clinic Liver Cancer (BCLC) stages 0/A compared to cirrhotic controls without HCC. In patients with HCC, CXCL5 serum levels were associated with tumor progression, and levels of CCL20 and CXCL8 with macrovascular invasion. Importantly, our study identified CXCL5, CXCL9, and CXCL10 as universal HCC markers, independent from underlying etiology of cirrhosis. In conclusion, regardless of the underlying liver disease, patients with cirrhosis share an HCC-specific chemokine profile. CXCL5 may serve as a diagnostic biomarker in cirrhotic patients for early HCC detection as well as for tumor progression.

Pathogen detection in RNA-seq data with Pathonoia.

BACKGROUND: Bacterial and viral infections may cause or exacerbate various human diseases and to detect microbes in tissue, one method of choice is RNA sequencing. The detection of specific microbes using RNA sequencing offers good sensitivity and specificity, but untargeted approaches suffer from high false positive rates and a lack of sensitivity for lowly abundant organisms. RESULTS: We introduce Pathonoia, an algorithm that detects viruses and bacteria in RNA sequencing data with high precision and recall. Pathonoia first applies an established k-mer based method for species identification and then aggregates this evidence over all reads in a sample. In addition, we provide an easy-to-use analysis framework that highlights potential microbe-host interactions by correlating the microbial to the host gene expression. Pathonoia outperforms state-of-the-art methods in microbial detection specificity, both on in silico and real datasets. CONCLUSION: Two case studies in human liver and brain show how Pathonoia can support novel hypotheses on microbial infection exacerbating disease. The Python package for Pathonoia sample analysis and a guided analysis Jupyter notebook for bulk RNAseq datasets are available on GitHub.

Preoperative Immunotherapy in Hepatocellular Carcinoma: Current State of the Art.

Hepatocellular carcinoma (HCC) is a malignancy that requires multidisciplinary evaluation to develop individualized and tailored treatment concepts. While liver resection and transplantation represent the mainstay of curative treatment in patients with early-stage HCC, disease recurrence remains an important burden. Immune checkpoint inhibitors (ICI) have become standard of care in the palliative setting, achieving promising response rates with overall good tolerability. Accordingly, ICIs are being evaluated in (neo)adjuvant concepts in order to improve survival. Nevertheless, neoadjuvant therapies are not recommended by current guidelines as they have not been proven to improve the outcome in large Phase III trials yet. Especially in the context of liver transplantation (LT), perioperative ICI usage is in need of a particularly critical risk-benefit assessment, as the immunotherapy may significantly increase the risk of rejection. In this review, we summarize available data on ICI-based perioperative treatment strategies in HCC. We discuss current drawbacks and challenges of this treatment concept and specifically highlight the risk of allograft rejection when ICI are given in patients (subsequently) considered for liver transplantation.

Histological activity despite normal ALT and IgG serum levels in patients with autoimmune hepatitis and cirrhosis.

BACKGROUND & AIMS: In autoimmune hepatitis (AIH), normal levels of transaminases and IgG define biochemical remission and are considered the best surrogate markers for histological remission. This study assessed whether this also applies to patients with AIH cirrhosis. METHODS: In this European multicentric study, we included 125 biopsies from 113 patients with AIH and histologically proven cirrhosis; 105 biopsies from 104 patients with AIH without cirrhosis served as controls. Biochemical parameters were available within 4 weeks of biopsy. AIH activity was graded according to the modified Hepatitis Activity Index (mHAI), with mHAI ≥4/18 considered to indicate risk of disease progression. RESULTS: In total, 47 out of 125 liver biopsies were obtained from patients with AIH cirrhosis and normal ALT levels at time of biopsy. Only 26% (12/47) of those livers showed histological remission (mHAI <4/18), whereas 36% (17/47) showed moderate to high histological activity (mHAI ≥6/18). In patients with noncirrhotic AIH, 88% (46/52 biopsies) of cases with normal ALT levels had histological remission and only 4% (2/52) had an mHAI ≥6/18 (p <0.001). The addition of IgG to define complete biochemical remission only slightly improved the association with histological remission in the limited number of patients with AIH cirrhosis available for analysis [29% (5/17) of biopsies with mHAI <4/18]. ALT correlated closely with mHAI in AIH without cirrhosis but poorly in AIH with cirrhosis. CONCLUSIONS: In contrast to patients with noncirrhotic AIH, in patients with AIH cirrhosis, who are at risk of disease progression, normal ALT levels and potentially also complete biochemical remission are poor surrogate markers of histological remission. Thus, new biomarkers are needed to monitor disease activity and progression in patients with AIH cirrhosis. LAY SUMMARY: Autoimmune hepatitis (AIH) is an inflammatory disease of the liver that usually responds to immunosuppressive therapy. Serum transaminases and IgG levels within the normal ranges define complete biochemical remission and are considered as surrogate markers for histological disease activity. Here, we show that those biochemical markers are not sufficient to indicate low disease activity in patients with AIH and already established cirrhosis. Consequently, until better biomarkers for disease activity are found, only liver biopsy can reliably indicate disease activity in the presence of cirrhosis. Additional investigations, such as measurements of liver stiffness, should be undertaken to monitor non-invasively for disease progression in patients with AIH and established cirrhosis.

Genetic aspects of adult and pediatric autoimmune hepatitis: A concise review.

Autoimmune Hepatitis (AIH) is a heterogenous, mostly chronic liver disease that affects people of all age groups, women more often than men. The aim of therapy is to prevent cirrhosis, as it mainly accounts for liver-related mortality in patients with AIH. Rates of remission are high in patients with AIH, but life-long immunosuppressive therapy is required. AIH is hypothesized to originate from immunologic reactivity targeted against mostly unknown self-antigens, potentially triggered by viral infections among other factors. While AIH does not follow a Mendelian inheritance pattern, part of the risk of developing AIH or worse disease course, is attributed to specific genetic risk factors. Major associations for the risk of development of AIH were found for HLA-DRB1*03:01 and HLA-DRB1*04:01 in adult AIH in the only genome-wide association study on AIH. However, other potential risk loci in SH2B3, CARD10 and KIR genes were described. This review covers the current knowledge on genetic risk factors in adult and pediatric AIH.

Treatment and re-treatment results of HCV patients in the DAA era.

BACKGROUND: Re-treatment in patients with a chronic hepatitis C virus (HCV) infection and a previous failure to direct-acting antiviral (DAA) treatment remains a challenge. Therefore, we investigated the success rate of treatment and re-treatment regimens used at our center from October 2011 to March 2018. METHODS: A retrospective analysis of DAA-based HCV therapies of 1096 patients was conducted. Factors associated with a virological relapse were identified by univariable and multivariable logistic regression, treatment success of the re-treatment regimens was evaluated by an analysis of sustained virological response (SVR) rates in patients with a documented follow-up 12 weeks after the end of treatment. RESULTS: Of 1096 patients treated with DAA-based regimens, 91 patients (8%) were lost to follow-up, 892 of the remaining 1005 patients (89%) achieved an SVR12. Most patients (65/113, 58%) who experienced a virological relapse received an interferon-based DAA regimen. SVR rates were comparable in special cohorts like liver transplant recipients (53/61, 87%) and people with a human immunodeficiency virus (HIV) coinfection (41/45, 91%). On multivariable analysis, interferon-based DAA therapy was associated with treatment failure (odds ratio 0.111, 95%-confidence interval 0.054-0.218) among others. One hundred seventeen patients with multiple DAA treatment courses were identified, of which 97 patients (83%) experienced a single relapse, but further relapses after two (18/117, 15%) or even three (2/117, 2%) treatment courses were also observed. Eighty-two of 96 (85%) re-treatment attempts with all-oral DAA regimens were successful after an initial treatment failure. CONCLUSION: Overall, DAA re-treatments were highly effective in this real-world cohort and only a minority of patients failed more than two treatment courses. Switching to-or addition of-a new drug class seem to be valid options for the re-treatment of patients especially after failure of an interferon-based regimen.

Diagnosis and treatment of primary biliary cholangitis.

Primary biliary cholangitis is a cholestatic, chronic autoimmune liver disease with a wide individual variation in disease progression. The diagnosis is predominantly based on chronic elevation of alkaline phosphatase and the presence of anti-mitochondrial antibodies or other specific antinuclear antibodies (i.e. anti-gp210 and anti-sp100). Even in early-stage disease, health-related quality of life can be severely impaired by symptoms such as pruritus, fatigue, and sicca syndrome and metabolic bone disease should be assessed and treated. The prognosis of the disease is, however, largely determined by the development of cirrhosis and its complications. Ursodeoxycholic acid is associated with an improved prognosis and should be initiated and continued in all patients. Clinical outcome is related to the biochemical response to ursodeoxycholic acid, but the prognosis of those with an incomplete response is still better than those who remain untreated. Obeticholic acid was recently approved as second-line treatment and bezafibrate may serve as an adequate off-label alternative, particularly in patients with pruritus. Preliminary data suggest an additive effect of triple therapy with ursodeoxycholic acid, obeticholic acid, and bezafibrate, whereas other promising drugs are being evaluated in clinical trials.

TGF-ß-dependent induction of CD4⁺CD25⁺Foxp3⁺ Tregs by liver sinusoidal endothelial cells.

BACKGROUND & AIMS: CD4(+) CD25(+) Foxp3(+) regulatory T cells (Tregs) have a profound ability to control immune responses. We have previously shown that the liver is a major source of peripherally induced Tregs. Here, we investigate the liver cell types and molecular mechanisms responsible for hepatic Treg induction. METHODS: To assess the Treg-inducing potential of liver resident antigen-presenting cell types, we studied the conversion of Foxp3(-) non-Tregs into Foxp3(+) Tregs induced by liver dendritic cells (DCs), liver sinusoidal endothelial cells (LSECs), or Kupffer cells (KCs). The dependency of Treg induction on TGF-ß was tested in Treg conversion assays using T cells with reduced TGF-ß sensitivity. The suppressive potential of liver cell-induced Tregs was assessed by an in vitro suppression assay and in vivo, in the model of experimental autoimmune encephalomyelitis (EAE). RESULTS: All tested liver cell types were capable of inducing Foxp3(+) Tregs; however, LSECs were most efficient in inducing Tregs. Treg-induction was antigen-specific and depended on TGF-ß. LSECs featured membrane-bound LAP/TGF-ß and the anchor molecule GARP, which is required for tethering LAP/TGF-ß to the cell membrane. LSEC-induced Tregs suppressed proliferation and cytokine secretion of effector T cells in vitro. LSEC-induced Tregs were also functional suppressors in vivo, as neuroantigen-specific Tregs induced by LSECs were able to suppress EAE. CONCLUSIONS: We demonstrate that LSECs are the major liver cell type responsible for TGF-ß dependent hepatic Treg induction. The extraordinary capacity of LSECs to induce Tregs was associated with their unique ability to tether TGF-ß to their membrane.