Activating transcription factor 3 (ATF3) is a stress-inducible transcription factor that regulates inflammatory responses; however, its cell-type-specific role in liver fibrosis remains incompletely defined. We investigated the function of myeloid cell-specific ATF3 in carbon tetrachloride (CCl4)-induced liver fibrosis. ATF3 induction was confirmed in bone marrow-derived macrophages following lipopolysaccharide stimulation in vitro. Myeloid-specific Atf3 knockout mice (Atf3fl/fl LysM-Cre+) and littermate controls were subjected to CCl4 treatment to determine the impact of ATF3 loss in vivo. Myeloid ATF3 deficiency significantly exacerbated CCl4-induced liver injury, reflected by elevated serum ALT and AST levels, increased collagen deposition, and higher fibrosis scores. ATF3 loss enhanced hepatic stellate cell activation, as evidenced by increased α-SMA protein and elevated Acta2 and Col1a1 expression. Although total macrophage abundance was unchanged, ATF3 deficiency shifted macrophage composition toward increased Ly6C+ infiltrating monocyte-derived macrophages and reduced Kupffer cell markers. Mechanistically, Atf3-deficient livers displayed increased inflammatory chemokine expression (Ccl3, Ccl4, Ccl5, Ccl8), reduced matrix metalloproteinases (Mmp2, Mmp8, and Mmp9), increased tissue inhibitor of metalloproteinases 1 (Timp1), and enhanced activation of transforming growth factor-beta1 (TGF-β1)/SMAD signaling. These findings identify myeloid ATF3 as a protective regulator that restrains inflammatory amplification and extracellular matrix accumulation during liver fibrosis. Modulation of ATF3-dependent pathways may represent a potential therapeutic strategy for fibrotic liver disease.

RNA-sensing TLRs are strategically positioned in the endolysosome to detect incoming nonself RNA. RNase T2 plays a critical role in processing long, structured RNA into short oligoribonucleotides that engage TLR7 or TLR8. In addition to its positive regulatory role, RNase T2 also restricts RNA recognition through unknown mechanisms, as patients deficient in RNase T2 suffer from neuroinflammation. Consistent with this, mice lacking RNase T2 exhibit interferon-dependent neuroinflammation, impaired hematopoiesis, and splenomegaly. However, the mechanism by which RNase T2 deficiency unleashes inflammation in vivo remains unknown. Here, we report that the inflammatory phenotype found in Rnaset2-/- mice is completely reversed in the absence of TLR13, suggesting aberrant accumulation of an RNA ligand for this receptor. Interestingly, this TLR13-driven inflammatory phenotype is also fully present in germ-free mice, suggesting a role for RNase T2 in limiting erroneous TLR13 activation by an as yet unidentified endogenous ligand. These results establish TLR13 as a potential self-sensor that is kept in check by RNase T2.

The liver is innervated by primary sensory nerve fibres releasing the neuropeptide calcitonin gene-related peptide (CGRP). Elevated plasma levels of CGRP have been found in patients with liver fibrosis or cirrhosis. We hypothesised that signalling of CGRP and its receptors might regulate liver fibrosis and propose a novel potential target for the treatment. In this study, hepatic expression of CGRP and its receptor component, the receptor activity-modifying protein 1 (RAMP1), was dramatically increased in diseased livers of patients. In a murine liver fibrosis model, deficiency of RAMP1 resulted in attenuated fibrogenesis characterized by less collagen deposition and decreased activity of hepatic stellate cells (HSC). Mechanistically, activity of the TGFβ1 signalling core component Smad2 was severely impaired in the absence of RAMP1, and Yes-associated protein (YAP) activity was found to be diminished in RAMP1-deficient liver parenchyma. In vitro, stimulation of the HSC line LX-2 cells with CGRP induces TGFβ1 production and downstream signalling as well as HSC activation documented by increased α-SMA expression and collagen synthesis. We further demonstrate in LX-2 cells that CGRP promotes YAP activation and its nuclear translocation subsequent to TGFβ1/Smad2 signals. These data support a promotive effect of CGRP signalling in liver fibrosis via stimulation of TGFβ1/Smad2 and YAP activity.

Chronic hepatitis B virus (HBV) infection affects 300 million patients worldwide1,2, in whom virus-specific CD8 T cells by still ill-defined mechanisms lose their function and cannot eliminate HBV-infected hepatocytes3-7. Here we demonstrate that a liver immune rheostat renders virus-specific CD8 T cells refractory to activation and leads to their loss of effector functions. In preclinical models of persistent infection with hepatotropic viruses such as HBV, dysfunctional virus-specific CXCR6+ CD8 T cells accumulated in the liver and, as a characteristic hallmark, showed enhanced transcriptional activity of cAMP-responsive element modulator (CREM) distinct from T cell exhaustion. In patients with chronic hepatitis B, circulating and intrahepatic HBV-specific CXCR6+ CD8 T cells with enhanced CREM expression and transcriptional activity were detected at a frequency of 12-22% of HBV-specific CD8 T cells. Knocking out the inhibitory CREM/ICER isoform in T cells, however, failed to rescue T cell immunity. This indicates that CREM activity was a consequence, rather than the cause, of loss in T cell function, further supported by the observation of enhanced phosphorylation of protein kinase A (PKA) which is upstream of CREM. Indeed, we found that enhanced cAMP-PKA-signalling from increased T cell adenylyl cyclase activity augmented CREM activity and curbed T cell activation and effector function in persistent hepatic infection. Mechanistically, CD8 T cells recognizing their antigen on hepatocytes established close and extensive contact with liver sinusoidal endothelial cells, thereby enhancing adenylyl cyclase-cAMP-PKA signalling in T cells. In these hepatic CD8 T cells, which recognize their antigen on hepatocytes, phosphorylation of key signalling kinases of the T cell receptor signalling pathway was impaired, which rendered them refractory to activation. Thus, close contact with liver sinusoidal endothelial cells curbs the activation and effector function of HBV-specific CD8 T cells that target hepatocytes expressing viral antigens by means of the adenylyl cyclase-cAMP-PKA axis in an immune rheostat-like fashion.

The liver is innervated by primary sensory nerve fibres releasing the neuropeptide calcitonin gene-related peptide (CGRP). Elevated plasma levels of CGRP have been found in patients with liver fibrosis or cirrhosis. We hypothesised that signalling of CGRP and its receptors might regulate liver fibrosis and propose a novel potential target for the treatment. In this study, hepatic expression of CGRP and its receptor component, the receptor activity-modifying protein 1 (RAMP1), was dramatically increased in diseased livers of patients. In a murine liver fibrosis model, deficiency of RAMP1 resulted in attenuated fibrogenesis characterized by less collagen deposition and decreased activity of hepatic stellate cells (HSC). Mechanistically, activity of the TGF(31 signalling core component Smad2 was severely impaired in the absence of RAMP1, and Yes-associated protein (YAP) activity was found to be diminished in RAMP1-deficient liver parenchyma. In vitro, stimulation of the HSC line LX-2 cells with CGRP induces TGF(31 production and downstream signalling as well as HSC activation documented by increased a-SMA expression and collagen synthesis. We further demonstrate in LX-2 cells that CGRP promotes YAP activation and its nuclear translocation subsequent to TGF(31/Smad2 signals. These data support a promotive effect of CGRP signalling in liver fibrosis via stimulation of TGF(31/Smad2 and YAP activity.

Einleitung Die Darm-Leber-Achse wird mit Lebererkrankungen und deren Pathophysiologie in Verbindung gebracht, obwohl die zugrundeliegenden Mechanismen noch unklar sind. Wir haben den funktionellen Beitrag des Darmmikrobioms und seiner Metaboliten zur Leberregeneration in einem präklinischen Mausmodell und in Patientenproben untersucht.

Background & Aims Hepatocyte growth and proliferation is dependent on the synthesis of membrane phospholipids. Lipid synthesis, in turn, requires short chain fatty acids (SCFA) generated by bacterial fermentation, delivered through the gut- liver axis. We therefore hypothesized that dysbiotic insults like antibiotics treatment not only negatively affect gut microbiota, but also impair hepatic lipid synthesis and liver regeneration. Methods Stable isotope labelling and 70% partial hepatectomy (PHx) was carried out in C57Bl/6J wildtype mice, in mice treated with broad-spectrum antibiotics, in germfree mice and gnotobiotic mice colonized with minimal microbiota. Microbiome was analysed by 16S rRNA gene sequencing and microbial culture. Gut content, liver and blood were tested by lipidomics mass spectrometry, qRT-PCR, immunoblot and immunohistochemistry for expression of proliferative and lipogenic markers. Matched biopsies from hyperplastic and hypoplastic liver tissue of human patients subjected to portal vein embolization were analysed by qRT-PCR for lipogenic enzymes and results were correlated with liver volumetry. Results Three days of antibiotics treatment induced persistent dysbiosis with significantly decreased beta-diversity and richness, but massive increase of Proteobacteria , accompanied by decreased colonic SCFA. After PHx, antibiotics- treated mice showed delayed liver regeneration, increased mortality, impaired hepatocyte proliferation and decreased hepatic phospholipid synthesis. Expression of the key lipogenic enzyme SCD1 was upregulated after PHx, but delayed by antibiotics-treatment. Germfree mice essentially recapitulated the phenotype of antibiotics-treatment. Importantly, phospholipid synthesis, hepatocyte proliferation, liver regeneration and survival were rescued in gnotobiotic mice colonized with a minimal SCFA-producing microbial community. SCD1 was required for human hepatoma cell proliferation, and its hepatic expression was associated with liver regeneration and hyperproliferation in human patients. Conclusion Gut microbiota are pivotal for hepatic membrane phospholipid synthesis and liver regeneration. Lay Summary Gut microbiota affects the liver lipid metabolism through the gut-liver axis, and microbial metabolites promote liver regeneration. Perturbations of the microbiome, e.g., by antibiotics treatment, impair the production of bacterial metabolites, which serve as building blocks for new membrane lipids in liver cells. As a consequence, hepatocyte growth and proliferation, and ultimately, liver regeneration and survival after liver surgery is impaired. Highlights Partial hepatectomy in mice pretreated with antibiotics is associated with impaired hepatocyte proliferation and phospholipid synthesis, delayed liver regeneration and increased mortality The delay in liver regeneration and impaired lipogenesis upon antibiotics treatment is preceded by dysbiosis of gut microbiota, increase of Proteobacteria and decreased short-chain fatty acids in the gut Partial hepatectomy in germfree mice essentially phenocopies the detrimental effects of antibiotic treatment Liver regeneration and mortality, as well as phospholipid synthesis and hepatocyte proliferation in germfree mice are fully rescued upon colonisation with a minimal gut bacterial consortium capable of short-chain fatty acid production In human patients, the intrahepatic expression of lipid synthesis enzymes positively correlates with proliferation and liver regeneration in the clinic Thus, liver regeneration is affected by composition of gut microbiota Clinically, pre-operative analysis of the gut microbiome may serve as biomarker to determine the extent of liver resections

Supplementary Tables 1-2 from Multiple Gene Expression Classifiers from Different Array Platforms Predict Poor Prognosis of Colorectal Cancer

Introduction The gut-liver axis has been implicated in liver disease and physiology although the underlying mechanisms remain unclear. We determined the functional contribution of the gut microbiome and its metabolites to liver regeneration in a preclinical mouse model and patient samples.

Background & aims: Chronic liver damage can ultimately lead to liver cirrhosis. Sensory nerves innervating the liver secrete the neuropeptide calcitonin gene-related peptide (CGRP), which binds to receptor activity-modifying protein (RAMP)1. We investigated whether the loss of RAMP1 affects liver fibrosis in chronic liver inflammation. Methods: Wild-type and RAMP1-deficient mice were injected with carbon tetrachloride (CCl4) to induce liver injury. CGRP/RAMP1 levels in CCl4-treated livers were determined by RT-PCR. Fibrosis was assessed by Sirius red staining and Western blot (α-SMA and collagen). The influence of CGRP/RAMP1 on the Hippo signaling pathway was assessed by Western blot. In addition, we stimulated the human hepatic stellate cell line (LX-2) in vitro with CGRP. The expression of α-SMA, collagen and YAP proteins was quantified by Western blot. Results: Chronic liver injury resulted in upregulation of hepatic CGRP/RAMP1 mRNA expression. In the absence of RAMP1, murine livers exhibited less fibrosis. In addition, expression of markers for hepatocyte proliferation was decreased. Also, the expression of YAP was diminished in RAMP1-deficient mice. At the same time, phosphorylation of YAP on Ser127, which promotes inactivation of YAP, was increased in RAMP1-deficient livers. In vitro, stimulation of LX-2 cells with CGRP promoted hepatic stellate cell activation. Moreover, treatment with CGRP resulted in inactivation of YAP, confirming our in vivo results. Conclusion: RAMP1/CGRP signaling promotes liver fibrosis and controls YAP activity, as well as the activation of LX-2 cells in vitro. The CGRP receptor RAMP1 may serve as a potential target of antifibrotic therapy in liver disease.

Leishmania parasites, endemic in (sub)tropical regions, cause a neglected tropical disease affecting ∼12 million cases worldwide. The spectrum of pathologies ranges from cutaneous leishmaniasis to progressive fatal visceral disease. Both in humans and mice, healing is associated with successful development of IFN-γ‒producing T helper type (Th)1/Tc1 cells, whereas Th2-, Th17-, and regulatory T-cell‒predominant responses are associated with progression and/or nonhealing lesions ( Kautz-Neu et al., 2011 Kautz-Neu K. Noordegraaf M. Dinges S. Bennett C.L. John D. Clausen B.E. et al. Langerhans cells are negative regulators of the anti-Leishmania response. J Exp Med. 2011; 208: 885-891 Google Scholar ; Sacks and Noben-Trauth, 2002 Sacks D. Noben-Trauth N. The immunology of susceptibility and resistance to Leishmania major in mice. Nat Rev Immunol. 2002; 2: 845-858 Google Scholar ). After transmission, Leishmania major primarily resides in skin-resident macrophages. Lesion infiltrating IFN-γ+ T cells activate macrophages to eliminate parasites through nitric oxide ( Sacks and Noben-Trauth, 2002 Sacks D. Noben-Trauth N. The immunology of susceptibility and resistance to Leishmania major in mice. Nat Rev Immunol. 2002; 2: 845-858 Google Scholar ). In contrast to the silent invasion of macrophages, parasite internalization by dendritic cells (DCs) triggers DC activation ( Woelbing et al., 2006 Woelbing F. Susanna L.K. Moelle K. Belkaid Y. Sunderkoetter C. Verbeek S. et al. Uptake of Leishmania major by dendritic cells is mediated by Fcgamma receptors and facilitates acquisition of protective immunity. J Exp Med. 2006; 203: 177-188 Google Scholar ), and infected DCs are considered to induce protection by IL-12 ( von Stebut et al., 1998 von Stebut E. Belkaid Y. Jakob T. Sacks D.L. Udey M.C. Uptake of Leishmania major amastigotes results in activation and interleukin 12 release from murine skin-derived dendritic cells: implications for the initiation of anti-Leishmania immunity. J Exp Med. 1998; 188: 1547-1552 Google Scholar ) and by priming T cells against L. major antigens. These early events after parasite inoculation are critical for disease outcome.

The cytoplasmic phosphatase DUSP6 and its nuclear counterpart DUSP5 are negative regulators of RAS/ERK signalling. Here we use deletion of either Dusp5 or Dusp6 to explore the roles of these phosphatases in a murine model of KRAS G12D -driven pancreatic cancer. By 56-days, loss of either DUSP5 or DUSP6 causes a significant increase in KRAS G12D -driven pancreatic hyperplasia. This is accompanied by increased pancreatic acinar to ductal metaplasia (ADM) and the development of pre-neoplastic pancreatic intraepithelial neoplasia (PanINs). In contrast, by 100-days, pancreatic hyperplasia is reversed with significant atrophy of pancreatic tissue and weight loss observed in animals lacking either DUSP5 or DUSP6. On further ageing, Dusp 6 −/− mice display accelerated development of metastatic pancreatic ductal adenocarcinoma (PDAC), while in Dusp5 −/− animals, although PDAC development is increased this process is attenuated by atrophy of pancreatic acinar tissue and severe weight loss in some animals before cancer could progress. Our data suggest that despite a common target in the ERK MAP kinase, DUSP5 and DUSP6 play partially non-redundant roles in suppressing oncogenic KRAS G12D signalling, thus retarding both tumour initiation and progression. Our data suggest that loss of either DUSP5 or DUSP6, as observed in certain human tumours, including the pancreas, could promote carcinogenesis.

Stringent regulation of the inflammatory response is crucial for normal tissue regeneration. Here, we analyzed the role of Toll‐like receptor 3 (TLR3) in pancreatic regeneration after acute pancreatitis (AP). AP was induced by caerulein treatment in mice with global TLR3 deficiency (TLR3OFF) or in mice re‐expressing TLR3 exclusively in the myeloid cell lineage (TLR3Mye). Compared to WT mice, TLR3OFF mice had a markedly increased formation of acinar‐to‐ductal metaplasia (ADM) that persisted until day 7 after initiation of AP. Pancreatic tissue of WT mice was completely regenerated after 5 days with no detectable ADM structures. The enhancing effect of TLR3‐deficiency on ADM formation was closely linked with an increased and prolonged accumulation of macrophages in pancreata of TLR3OFF mice. Importantly, the phenotype of TLR3OFF mice was rescued in TLR3Mye mice, demonstrating the causative role of myeloid cell selective TLR3 signaling. Moreover, in vitro stimulation of macrophages through TLR3 initiated cell death by a caspase‐8‐associated mechanism. Therefore, these findings provide evidence that TLR3 signaling in myeloid cells is sufficient to limit inflammation and ADM formation and to promote regeneration after AP. Notably, resolution of inflammation after AP was associated with macrophage sensitivity to TLR3‐mediated cell death.

BACKGROUND & AIMS Hepatic innate immune control of viral infections has largely been attributed to Kupffer cells, the liver macrophages. However, also hepatocytes, the parenchymal cells of the liver, possess potent immunological functions in addition to their known metabolic functions. Owing to their abundance in the liver and known immunological functions, we aimed to investigate the direct anti-viral mechanisms employed by hepatocytes. METHODS Using lymphocytic choriomeningitis virus (LCMV) as a model of liver infection, we first assessed the role of myeloid cells by depletion prior to infection. We investigated the role of hepatocyte-intrinsic innate immune signaling by infecting mice lacking canonical NF-κB signaling (IKKβΔHep) specifically in hepatocytes. In addition, mice lacking hepatocyte-specific interferon-α/β signaling-(IFNARΔHep), or interferon-α/β signaling in myeloid cells-(IFNARΔMyel) were infected. RESULTS Here, we demonstrate that LCMV activates NF-κB signaling in hepatocytes. LCMV-triggered NF-κB activation in hepatocytes did not depend on Kupffer cells or TNFR1- but rather on TLR-signaling. LCMV-infected IKKβΔHep livers displayed strongly elevated viral titers due to LCMV accumulation within hepatocytes, reduced interferon-stimulated gene (ISG) expression, delayed intrahepatic immune cell influx and delayed intrahepatic LCMV-specific CD8+ T-cell responses. Notably, viral clearance and ISG expression were also reduced in LCMV-infected primary hepatocytes lacking IKKβ, demonstrating a hepatocyte-intrinsic effect. Similar to livers of IKKβΔHep mice, enhanced hepatocytic LCMV accumulation was observed in livers of IFNARΔHep, whereas IFNARΔMyel mice were able to control LCMV-infection. Hepatocytic NF-κB signaling was also required for efficient ISG induction in HDV-infected dHepaRG cells and interferon-α/β-mediated inhibition of HBV replication in vitro. CONCLUSIONS Together, these data show that hepatocyte-intrinsic NF-κB is a vital amplifier of interferon-α/β signaling pivotal for early, strong ISG responses, influx of immune cells and hepatic viral clearance.

Conclusion:Our study demonstrates that the neuropeptide CGRP via signaling to its receptor RAMP1 promotes liver regeneration through controling YAP activity.This previously unknown signaling pathway might be highly relevant for patients with liver diseases.

TLR3 is implicated in anti-viral immune responses, but may also act as a sensor of tissue damage in the absence of infection. Here, we provide evidence for an essential role of TLR3 in liver regeneration after an acute loss of tissue due to partial hepatectomy. Mice lacking TLR3 had a severe and sustained defect in the restoration of liver tissue with reduced liver-to-body weight ratios even after an extended recovery period of 2 weeks. Hepatocyte cell cycle progression into S phase was impaired in TLR3-deficient mice. Mechanistic analyses revealed that TLR3-deficient mice had markedly reduced systemic levels of active HGF, but had increased amounts of inactive tissue-bound HGF. Importantly, expression of uPA, which orchestrates the processing and release of HGF from the hepatic extracellular matrix, was reduced in regenerating livers of TLR3-deficient mice. In addition, expression of the HGF maturation factor HGFAC was transiently diminished in TLR3-deficient mice. In vitro, engagement of TLR3 directly stimulated expression of uPA by hepatic stellate cells. Thus, TLR3 supports liver regeneration through upregulation of uPA, which promotes the release of preformed HGF from extracellular matrix stores.

Conventional Ly6Chi monocytes have developmental plasticity for a spectrum of differentiated phagocytes. Here we show, using conditional deletion strategies in a mouse model of Toll-like receptor (TLR) 7-induced inflammation, that the spectrum of developmental cell fates of Ly6Chi monocytes, and the resultant inflammation, is coordinately regulated by TLR and Notch signaling. Cell-intrinsic Notch2 and TLR7-Myd88 pathways independently and synergistically promote Ly6Clo patrolling monocyte development from Ly6Chi monocytes under inflammatory conditions, while impairment in either signaling axis impairs Ly6Clo monocyte development. At the same time, TLR7 stimulation in the absence of functional Notch2 signaling promotes resident tissue macrophage gene expression signatures in monocytes in the blood and ectopic differentiation of Ly6Chi monocytes into macrophages and dendritic cells, which infiltrate the spleen and major blood vessels and are accompanied by aberrant systemic inflammation. Thus, Notch2 is a master regulator of Ly6Chi monocyte cell fate and inflammation in response to TLR signaling.