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Primary biliary cholangitis (PBC) is a female-predominant liver autoimmune disease characterized by the specific immune-mediated destruction of the intrahepatic small bile duct. Although apoptosis of biliary epithelial cells (BECs) and alterations in gut microbiota are observed in patients with PBC, it is still unclear whether these events happen in the early stage and cause the breakdown of tolerance in PBC. In this study, we examined the early events in the loss of tolerance in our well-defined 2-OA-OVA-induced murine autoimmune cholangitis (AIC) model. We report herein that apoptosis of BECs was notable in the early stage of murine AIC. An altered gut microbiota, in particular, an increased percentage of gram-positive Firmicutes in AIC mice was also observed. BECs in AIC mice expressed adhesion molecule ICAM-1, cytokines/chemokines TNF-α, CCL2, CXCL9, CXCL10, and toll-like receptor (TLR) 2. Moreover, BECs treated with TLR2 ligand had elevated apoptosis and CXCL10 production. These data collectively suggest a new mechanism of tolerance breakdown in AIC. Altered gut microbiota induces apoptosis of BECs through TLR2 signaling. BECs secrete chemokines to recruit CD8 T cells to damage BECs further., Competing Interests: The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (© 2023 The Authors. Published by Elsevier B.V.)

Background & Aims: Increased expression of IFN-stimulated gene 15 (ISG15) and subsequently increased ISGylation are key factors in the host response to viral infection. In this study, we sought to characterize the expression of ISG15, ISGylation, and associated enzymes at each stage of differentiation from induced pluripotent stem cells (iPSCs) to hepatocytes., Methods: To study the regulation of ISGylation, we utilized patient samples and in vitro cell culture models including iPSCs, hepatocytes-like cells, immortalized cell lines, and primary human hepatocytes. Protein/mRNA expression were measured following treatment with poly(I:C), IFNα and HCV infection., Results: When compared to HLCs, we observed several novel aspects of the ISGylation pathway in iPSCs. These include a lower baseline expression of the ISGylation-activating enzyme, UBE1L, a lack of IFN-induced expression of the ISGylation-conjugation enzyme UBE2L6, an attenuated activation of the transcription factor STAT1 and constitutive expression of SOCS1. ISGylation was observed in iPSCs following downregulation of SOCS1, which facilitated STAT1 activation and subsequently increased expression of UBE2L6. Intriguingly, HCV permissive transformed hepatoma cell lines demonstrated higher intrinsic expression of SOCS1 and weaker ISGylation following IFN treatment. SOCS1 downregulation in HCV-infected Huh 7.5.1 cells led to increased ISGylation., Conclusions: Herein, we show that high basal levels of SOCS1 inhibit STAT1 activation and subsequently IFN-induced UBE2L6 and ISGylation in iPSCs. Furthermore, as iPSCs differentiate into hepatocytes, epigenetic mechanisms regulate ISGylation by modifying UBE1L and SOCS1 expression levels. Overall, this study demonstrates that the development of cell-intrinsic innate immunity during the differentiation of iPSCs to hepatocytes provides insight into cell type-specific regulation of host defense responses and related oncogenic processes., Impact and Implications: To elucidate the mechanism underlying regulation of ISGylation, a key process in the innate immune response, we studied changes in ISGylation-associated genes at the different stages of differentiation from iPSCs to hepatocytes. We found that high basal levels of SOCS1 inhibit STAT1 activation and subsequently IFN-induced UBE2L6 and ISGylation in iPSCs. Importantly, epigenetic regulation of SOCS1 and subsequently ISGylation may be important factors in the development of cell type-specific host defense responses in hepatocytes that should be considered when studying chronic infections and oncogenic processes in the liver., Competing Interests: The authors declare no conflict of interest. Please refer to the accompanying ICMJE disclosure forms for further details., (© 2022 The Author(s).)

Background & Aims: XBP1 modulates the macrophage proinflammatory response, but its function in macrophage stimulator of interferon genes (STING) activation and liver fibrosis is unknown. X-box binding protein 1 (XBP1) has been shown to promote macrophage nucleotide-binding oligomerization domain, leucine-rich repeat and pyrin domain-containing 3 (NLRP3) activation in steatohepatitis. Herein, we aimed to explore the underlying mechanism of XBP1 in the regulation of STING signalling and the subsequent NLRP3 activation during liver fibrosis., Methods: XBP1 expression was measured in the human fibrotic liver tissue samples. Liver fibrosis was induced in myeloid-specific Xbp1 -, STING-, and Nlrp3 -deficient mice by carbon tetrachloride injection, bile duct ligation, or a methionine/choline-deficient diet., Results: Although increased XBP1 expression was observed in the fibrotic liver macrophages of mice and clinical patients, myeloid-specific Xbp1 deficiency or pharmacological inhibition of XBP1 protected the liver against fibrosis. Furthermore, it inhibited macrophage NLPR3 activation in a STING/IRF3-dependent manner. Oxidative mitochondrial injury facilitated cytosolic leakage of macrophage self-mtDNA and cGAS/STING/NLRP3 signalling activation to promote liver fibrosis. Mechanistically, RNA sequencing analysis indicated a decreased mtDNA expression and an increased BCL2/adenovirus E1B interacting protein 3 (BNIP3)-mediated mitophagy activation in Xbp1 -deficient macrophages. Chromatin immunoprecipitation (ChIP) assays further suggested that spliced XBP1 bound directly to the Bnip3 promoter and inhibited the transcription of Bnip3 in macrophages. Xbp1 deficiency decreased the mtDNA cytosolic release and STING/NLRP3 activation by promoting BNIP3-mediated mitophagy activation in macrophages, which was abrogated by Bnip3 knockdown. Moreover, macrophage XBP1/STING signalling contributed to the activation of hepatic stellate cells., Conclusions: Our findings demonstrate that XBP1 controls macrophage cGAS/STING/NLRP3 activation by regulating macrophage self-mtDNA cytosolic leakage via BNIP3-mediated mitophagy modulation, thus providing a novel target against liver fibrosis., Lay Summary: Liver fibrosis is a typical progressive process of chronic liver disease, driven by inflammatory and immune responses, and is characterised by an excess of extracellular matrix in the liver. Currently, there is no effective therapeutic strategy for the treatment of liver fibrosis, resulting in high mortality worldwide. In this study, we found that myeloid-specific Xbp1 deficiency protected the liver against fibrosis in mice, while XBP1 inhibition ameliorated liver fibrosis in mice. This study concluded that targeting XBP1 signalling in macrophages may provide a novel strategy for protecting the liver against fibrosis., Competing Interests: The authors declare no conflicts of interest. Please refer to the accompanying ICMJE disclosure forms for further details., (© 2022 The Author(s).)

Non-alcoholic fatty liver disease (NAFLD) is reaching epidemic proportions, with a global prevalence of 25% in the adult population. Non-alcoholic steatohepatitis (NASH), which can lead to cirrhosis, has become the leading indication for liver transplantation in both Europe and the USA. Liver fibrosis is the consequence of sustained, iterative liver injury, and the main determinant of outcomes in NASH. The liver possesses remarkable inherent plasticity, and liver fibrosis can regress when the injurious agent is removed, thus providing opportunities to alter long-term outcomes through therapeutic interventions. Although hepatocyte injury is a key driver of NASH, multiple other cell lineages within the hepatic fibrotic niche play major roles in the perpetuation of inflammation, mesenchymal cell activation, extracellular matrix accumulation as well as fibrosis resolution. The constituents of this cellular interactome, and how the various subpopulations within the fibrotic niche interact to drive fibrogenesis is an area of active research. Important cellular components of the fibrotic niche include endothelial cells, macrophages, passaging immune cell populations and myofibroblasts. In this review, we will describe how rapidly evolving technologies such as single-cell genomics, spatial transcriptomics and single-cell ligand-receptor analyses are transforming our understanding of the cellular interactome in NAFLD/NASH, and how this new, high-resolution information is being leveraged to develop rational new therapies for patients with NASH., Competing Interests: The authors declare no conflicts of interest that pertain to this work. Please refer to the accompanying ICMJE disclosure forms for further details., (© 2022 The Author(s).)

Lipids are a complex and diverse group of molecules with crucial roles in many physiological processes, as well as in the onset, progression, and maintenance of cancers. Fatty acids and cholesterol are the building blocks of lipids, orchestrating these crucial metabolic processes. In the liver, lipid alterations are prevalent as a cause and consequence of chronic hepatitis B and C virus infections, alcoholic hepatitis, and non-alcoholic fatty liver disease and steatohepatitis. Recent developments in lipidomics have also revealed that dynamic changes in triacylglycerols, phospholipids, sphingolipids, ceramides, fatty acids, and cholesterol are involved in the development and progression of primary liver cancer. Accordingly, the transcriptional landscape of lipid metabolism suggests a carcinogenic role of increasing fatty acids and sterol synthesis. However, limited mechanistic insights into the complex nature of the hepatic lipidome have so far hindered the development of effective therapies., Competing Interests: Authors declare no conflicts of interest Please refer to the accompanying ICMJE disclosure forms for further details., (© 2022 The Author(s).)

Alcohol-related liver disease (ARLD) is a primary cause of chronic liver disease in the United States. Despite advances in the diagnosis and management of ARLD, it remains a major public health problem associated with significant morbidity and mortality, emphasising the need to adopt novel approaches to the study of ARLD and its complications. Epigenetic changes are increasingly being recognised as contributing to the pathogenesis of multiple disease states. Harnessing the power of innovative technologies for the study of epigenetics ( e.g. , next-generation sequencing, DNA methylation assays, histone modification profiling and computational techniques like machine learning) has resulted in a seismic shift in our understanding of the pathophysiology of ARLD. Knowledge of these techniques and advances is of paramount importance for the practicing hepatologist and researchers alike. Accordingly, in this review article we will summarise the current knowledge about alcohol-induced epigenetic alterations in the context of ARLD, including but not limited to, DNA hyper/hypo methylation, histone modifications, changes in non-coding RNA, 3D chromatin architecture and enhancer-promoter interactions. Additionally, we will discuss the state-of-the-art techniques used in the study of ARLD ( e.g. single-cell sequencing). We will also highlight the epigenetic regulation of chemokines and their proinflammatory role in the context of ARLD. Lastly, we will examine the clinical applications of epigenetics in the diagnosis and management of ARLD., Competing Interests: The authors declare no conflicts of interest that pertain to this work. Please refer to the accompanying ICMJE disclosure forms for further details., (© 2022 Mayo Foundation https://www.mayo.clinic.org/copyright/.)

Background & Aims: HDV superinfection of chronically HBV-infected patients is the most aggressive form of chronic viral hepatitis, with an accelerated progression towards fibrosis/cirrhosis and increased risk of liver failure, hepatocellular carcinoma, and death. While HDV infection is not susceptible to available direct anti-HBV drugs, suboptimal responses are obtained with interferon-α-based therapies, and the number of investigational drugs remains limited. We therefore analyzed the effect of several innate immune stimulators on HDV replication in infected hepatocytes., Methods: We used in vitro models of HDV and HBV infection based on primary human hepatocytes (PHHs) and the non-transformed HepaRG cell line that are relevant to explore new innate immune therapies., Results: We describe here, for the first time, anti-HDV effects of Pam3CSK4 and BS1, agonists of Toll-like receptor (TLR)-1/2, and the lymphotoxin-β receptor (LTβR), respectively. Both types of agonists induced dose-dependent reductions of total intracellular HDV genome and antigenome RNA and of HDV protein levels, without toxicity in cells monoinfected with HDV or co/superinfected with HBV. Moreover, both molecules negatively affected HDV progeny release and strongly decreased their specific infectivity. The latter effect is particularly important since HDV is thought to persist in humans through constant propagation., Conclusions: Immune-modulators inducing NF-κB pathways in hepatocytes can inhibit HDV replication and should be further evaluated as a possible therapeutic approach in chronically HBV/HDV-infected patients., Lay Summary: Hepatitis delta virus causes the most severe form of viral hepatitis. Despite positive recent developments, effective treatments remain a major clinical need. Herein, we show that immune-modulators that trigger the NF-κB pathways could be effective for the treatment of hepatitis delta infections., Competing Interests: The authors declare no conflicts of interest that pertain to this work. Please refer to the accompanying ICMJE disclosure forms for further details., (© 2021 The Author(s).)

Insights into the immunopathogenesis of chronic HBV infections are fundamental in the quest for novel treatment approaches aimed at a functional cure. While much is known about the ineffective HBV-specific T-cell responses that characterise persistent HBV replication, B cells have been left largely understudied. However, an important role for humoral immunity during the natural history of HBV infections, as well as after functional cure, has been inadvertently revealed by the occurrence of HBV flares following B cell-depleting treatments. Herein, we review our current understanding of the role of the humoral immune response in chronic HBV, both at the level of HBV-specific antibody production and at the phenotypic and broader functional level of B cells. The recent development of fluorescently labelled HBV proteins has given us unprecedented insights into the phenotype and function of HBsAg- and HBcAg-specific B cells. This should fuel novel research into the mechanisms behind dysfunctional HBsAg-specific and fluctuating, possibly pathogenic, HBcAg-specific B-cell responses in chronic HBV. Finally, novel immunomodulatory treatments that partly target B cells are currently in clinical development, but a detailed assessment of their impact on HBV-specific B-cell responses is lacking. We plead for a rehabilitation of B-cell studies related to both the natural history of HBV and treatment development programmes., Competing Interests: TVW has received grants from Gilead Sciences, is a consultant for Janssen Pharmaceuticals, Gilead Sciences, Abbvie, and a sponsored lecturer for Abbvie, Gilead Sciences. HJ has received grants from AbbVie, Arbutus, Gilead Sciences, Janssen, Merck, Roche and served as consultant for: Arbutus, Arena, Enyo, Gilead Sciences, GlaxoSmithKline, Janssen, Merck, Roche, Vir Biotechnology Inc., Viroclinics. TA and SVH have nothing to disclose. Please refer to the accompanying ICMJE disclosure forms for further details., (© 2021 The Author(s).)

Background & Aims: V-set Ig-domain-containing 4 (VSIG4) is an immunomodulatory macrophage complement receptor modulating innate and adaptive immunity and affecting the resolution of bacterial infections. Given its expression on peritoneal macrophages (PMs), we hypothesised a prognostic role of peritoneal VSIG4 concentrations in patients with spontaneous bacterial peritonitis (SBP)., Methods: We isolated PMs from patients with cirrhosis and analysed VSIG4 expression and release by flow cytometry, quantitative real-time PCR, ELISA, and confocal microscopy. We measured soluble VSIG4 concentrations in ascites from 120 patients with SBP and 40 patients without SBP and investigated the association of soluble VSIG4 in ascites with 90-day survival after SBP using Kaplan-Meier statistics, Cox regression, and competing-risks regression analysis., Results: VSIG4 expression was high on resting, large PMs, which co-expressed CD206, CD163, and tyrosine-protein kinase Mer (MERTK). VSIG4 gene expression in PMs decreased in patients with SBP and normalised after resolution. During SBP, VSIG4 hi PMs were depleted (25% vs . 57%; p <0.001) and soluble VSIG4 in ascites were higher in patients with SBP than in patients without (0.73 vs . 0.35 μg/ml; p <0.0001). PM activation by Toll-like receptor (TLR) agonists or infection with live bacteria in vitro resulted in a loss of surface VSIG4 and the release of soluble VSIG4. Mechanistically, shedding of VSIG4 from PMs was protease-dependent and susceptible to microtubule transport inhibition. Soluble VSIG4 in ascites exceeded serum concentrations and correlated with serum creatinine, model for end-stage liver disease score and C-reactive protein during SBP. Concentrations of 1.0206 μg/ml or higher indicated increased 90-day mortality (hazard ratio 1.70; 95% CI 1.01-2.86; p  = 0.046)., Conclusions: VSIG4 is released from activated PMs into ascites during SBP. Higher peritoneal VSIG4 levels indicate patients with organ failure and poor prognosis., Lay Summary: Patients with liver cirrhosis who develop ascites have an increased risk of infection and mortality. Our study shows that in patients with infected ascites, the complement receptor VSIG4 is released by resident macrophages into the abdominal fluid where it can be measured. Patients with elevated levels of this protein in ascites are at high risk of dying within 90 days., Competing Interests: All authors declare no conflict of interest with regard to this manuscript. Please refer to the accompanying ICMJE disclosure forms for further details., (© 2021 The Authors.)

Background & Aims: Acute-on-chronic liver failure (ACLF) is characterised by organ failure(s), high short-term mortality, and, pathophysiologically, deranged inflammatory responses. The extracellular matrix (ECM) is critically involved in regulating the inflammatory response. This study aimed to determine alterations in biomarkers of ECM turnover in ACLF and their association with inflammation, organ failures, and mortality., Methods: We studied 283 patients with cirrhosis admitted for acute decompensation (AD) with or without ACLF, 64 patients with stable cirrhosis, and 30 healthy controls. A validation cohort (25 ACLF, 9 healthy controls) was included. Plasma PRO-C3, PRO-C4, PRO-C5, PRO-C6, and PRO-C8 ( i.e. collagen type III-VI and VIII formation ) and C4M and C6M ( i.e. collagen type IV and VI degradation ) were measured. Immunohistochemistry of PRO-C6 was performed on liver biopsies (AD [n = 7], ACLF [n = 5]). A competing-risk regression analysis was performed to explore the prognostic value of biomarkers of ECM turnover with 28- and 90-day mortality., Results: PRO-C3 and PRO-C6 were increased in ACLF compared to AD ( p  = 0.089 and p <0.001, respectively), whereas collagen degradation markers C4M and C6M were similar. Both PRO-C3 and PRO-C6 were strongly associated with liver function and inflammatory markers. Only PRO-C6 was associated with extrahepatic organ failures and 28- and 90-day mortality (hazard ratio [HR; on log-scale] 6.168, 95% CI 2.366-16.080, p <0.001, and 3.495, 95% CI 1.509-8.093, p  = 0.003, respectively). These findings were consistent in the validation cohort. High PRO-C6 expression was observed in liver biopsies of patients with ACLF., Conclusions: This study shows, for the first time, evidence of severe net interstitial collagen deposition in ACLF and makes the novel observation of the association between PRO-C6 and (extrahepatic) organ failures and mortality. Further studies are needed to define the pathogenic significance of these observations., Lay Summary: This study describes a disrupted turnover of collagen type III and VI in Acute-on-chronic liver failure (ACLF). Plasma biomarkers of these collagens (PRO-C3 and PRO-C6) are associated with the severity of liver dysfunction and inflammation. PRO-C6, also known as the hormone endotrophin, has also been found to be associated with multi-organ failure and prognosis in acute decompensation and ACLF., Competing Interests: AJCK has none to declare. SG has none to declare. EA has none to declare. ID is a full-time employee at Takeda Pharmaceuticals International Co. IL is a full-time employee at Nordic Bioscience. AH has none to declare. SHN is a full-time employee at Nordic Bioscience. MJN is a full-time employee at Nordic Bioscience. HG received an investigator-initiated research grant from Intercept and research grants from AbbVie, ADS AIPHIA Development Services AG (Switzerland), ARLA Food for Health, and the NOVO Nordisk Foundation. HG is on an advisory board at Ipsen. ÀA has none to declare. DY is a full-time employee at Takeda Pharmaceuticals International Co. JM is a co-founder of Yaqrit Ltd. RPM has research collaboration with Yaqrit Ltd., of which he is a co-founder. He also has research collaborations with UCL spin-out Hepyx Ltd. and with Cyberliver Limited. He also has provided a consultancy role to Inventiva Pharmaceuticals. SM has none to declare. FA has none to declare. RM has none to declare. VA is a member of the Yaqrit Scientific Advisory Board. In 2016–2020, PA was on the Biovie Advisory Board and filed a patent application; in 2014–2020, he was invited as a speaker by and received a travel grant from CSL Behring; and in 2018–2019, he was on the Advisory Board at Ferring. DJL is a full-time employee and stockholder at Nordic Bioscience. WT is a full-time employee at Takeda Pharmaceuticals International Co. MAK is a full-time employee and stockholder at Nordic Bioscience. RJ has research collaborations with Yaqrit and Takeda. RJ is the inventor of OPA, which has been patented by the University College London and licensed to Mallinckrodt Pharma. He is also a founder of Yaqrit Ltd., a spinout company from the University College London. He has also co-founded Hepyx Ltd. and Cyberliver Ltd. Please refer to the accompanying ICMJE disclosure forms for further details., (© 2021 The Author(s).)

A growing number of public health bodies, regulators and governments around the world consider electronic vapor products a lower risk alternative to conventional cigarettes. Of critical importance are rapid new approach methodologies to enable the screening of next generation products (NGPs) also known as next generation tobacco and nicotine products. In this study, the activity of conventional cigarette (3R4F) smoke and a range of NGP aerosols (heated tobacco product, hybrid product and electronic vapor product) captured in phosphate buffered saline, were screened by exposing a panel of human cell-based model systems using Biologically Multiplexed Activity Profiling (BioMAP® Diversity PLUS® Panel, Eurofins Discovery). Following exposure, the biological activity for a wide range of biomarkers in the BioMAP panel were compared to determine the presence of toxicity signatures that are associated with specific clinical findings. NGP aerosols were found to be weakly active in the BioMAP Diversity PLUS Panel (≤3/148 biomarkers) whereas significant activity was observed for 3R4F (22/148 biomarkers). Toxicity associated biomarker signatures for 3R4F included immunosuppression, skin irritation and thrombosis, with no toxicity signatures seen for the NGPs. BioMAP profiling could effectively be used to differentiate between complex mixtures of cigarette smoke or NGP aerosol extracts in a panel of human primary cell-based assays. Clinical validation of these results will be critical for confirming the utility of BioMAP for screening NGPs for potential adverse human effects., Competing Interests: Liam Simms, Elizabeth Mason, Fan Yu, Kathryn Rudd, Lukasz Czekala, Edgar Trelles Sticken, Oleg Brinster, Matthew Stevenson and Tanvir Walele were all current employees for Imperial Brands PLC at the time of submission. Imperial Brands PLC, who funded the work and was the sole sponsor of the project. Ellen L. Berg was the Eurofins Discovery contact who conducted the work and provided technical support to the project. None of the Imperial Brands personnel above have any known competing financial interests., (© 2021 The Author(s).)

Background: Several recent studies have stated that glycyrrhizin and licorice extract are present in most traditional Chinese medicine formulas used against SARS-CoV-2 in China. Significant data are showing that glycyrrhizin and licorice extract have multiple beneficial activities in combating most features of SARS-CoV-2., Purpose: The aim of current review was to highlight recent progresses in research that showed the evidence of the potential use of glycyrrhizin and licorice extract against COVID-19., Methodology: We have reviewed the information published from 1979 to October 2020. These studies demonstrated the effects , use and safety of glycyrrhizin and icorice extract against viral infections,bacterial infections, inflammatory disorders of lung ( in vitro and in vivo).  These studies were collated through online electronic databases research (Academic libraries as PubMed, Scopus, Web of Science and Egyptian Knowledge Bank)., Results: Pooled effect size of articles provides information about the rationale for using glycyrrhizin and licorice extract to treat COVID-19. Fifty studies demonstrate antiviral activity of glycyrrhizin and licorice extract. The most frequent mechanism of the antiviral activity is due to disrupting viral uptake into the host cells and disrupting the interaction between receptor- binding domain (RBD) of SARS-COV2 and ACE2 in recent articles. Fifty studies indicate that glycyrrhizin and licorice extract have significant antioxidant, anti-inflammatory and immunomodulatory effects. Twenty five studies provide evidence for the protective effect of glycyrrhizin and licorice extract against inflammation-induced acute lung injury and cardiovascular disorders., Conclusion: The current study showed several evidence regarding the beneficial effects of glycyrrhizin and licorice extract in combating COVID-19. More randomized clinical trials are needed to obtain a precise conclusion., Competing Interests: All authors declare that they have no conflict of interest., (© 2021 The Author(s).)

The recently identified novel cytosolic DNA sensor cyclic GMP-AMP synthase (cGAS) activates the downstream adaptor protein stimulator of interferon genes (STING) by catalysing the synthesis of cyclic GMP-AMP. This in turn initiates an innate immune response through the release of various cytokines, including type I interferon. Foreign DNA (microbial infection) or endogenous DNA (nuclear or mitochondrial leakage) can serve as cGAS ligands and lead to the activation of cGAS-STING signalling. Therefore, the cGAS-STING pathway plays essential roles in infectious diseases, sterile inflammation, tumours, and autoimmune diseases. In addition, cGAS-STING signalling affects the progression of liver inflammation through other mechanisms, such as autophagy and metabolism. In this review, we summarise recent advances in our understanding of the role of cGAS-STING signalling in the innate immune modulation of different liver diseases. Furthermore, we discuss the therapeutic potential of targeting the cGAS-STING pathway in the treatment of liver diseases., Competing Interests: The authors declare no conflicts of interest that pertain to this work. Please refer to the accompanying ICMJE disclosure forms for further details., (© 2021 The Authors.)

Nitric oxide (NO) is a gasotransmitter of great significance to developing the innate immune response to many bacterial and viral infections, while also modulating vascular physiology. The generation of NO from the upregulation of endogenous nitric oxide synthases serves as an efficacious method for inhibiting viral replication in host defense and warrants investigation for the development of antiviral therapeutics. With increased incidence of global pandemics concerning several respiratory-based viral infections, it is necessary to develop broad therapeutic platforms for inhibiting viral replication and enabling more efficient host clearance, as well as to fabricate new materials for deterring viral transmission from medical devices. Recent developments in creating stabilized NO donor compounds and their incorporation into macromolecular scaffolds and polymeric substrates has created a new paradigm for developing NO-based therapeutics for long-term NO release in applications for bactericidal and blood-contacting surfaces. Despite this abundance of research, there has been little consideration of NO-releasing scaffolds and substrates for reducing passive transmission of viral infections or for treating several respiratory viral infections. The aim of this review is to highlight the recent advances in developing gaseous NO, NO prodrugs, and NO donor compounds for antiviral therapies; discuss the limitations of NO as an antiviral agent; and outline future prospects for guiding materials design of a next generation of NO-releasing antiviral platforms., Competing Interests: The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (© 2020 Elsevier Ltd. All rights reserved.)

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection is responsible for the current pandemic coronavirus disease of 2019 (COVID-19). Like other pathogens, SARS-CoV-2 infection can elicit production of the type I and III interferon (IFN) cytokines by the innate immune response. A rapid and robust type I and III IFN response can curb viral replication and improve clinical outcomes of SARS-CoV-2 infection. To effectively replicate in the host, SARS-CoV-2 has evolved mechanisms for evasion of this innate immune response, which could also modulate COVID-19 pathogenesis. In this review, we discuss studies that have reported the identification and characterization of SARS-CoV-2 proteins that inhibit type I IFNs. We focus especially on the mechanisms of nsp1 and ORF6, which are the two most potent and best studied SARS-CoV-2 type I IFN inhibitors. We also discuss naturally occurring mutations in these SARS-CoV-2 IFN antagonists and the impact of these mutations in vitro and on clinical presentation. As SARS-CoV-2 continues to spread and evolve, researchers will have the opportunity to study natural mutations in IFN antagonists and assess their role in disease. Additional studies that look more closely at previously identified antagonists and newly arising mutants may inform future therapeutic interventions for COVID-19., Competing Interests: The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: Marta M. Gaglia reports financial support was provided by American Lung Association., (© 2021 The Authors.)

Leishmania are protozoan parasites that predominantly reside in myeloid cells within their mammalian hosts. Monocytes and macrophages play a central role in the pathogenesis of all forms of leishmaniasis, including cutaneous and visceral leishmaniasis. The present review will highlight the diverse roles of macrophages in leishmaniasis as initial replicative niche, antimicrobial effectors, immunoregulators and as safe hideaway for parasites persisting after clinical cure. These multiplex activities are either ascribed to defined subpopulations of macrophages (e.g., Ly6C high CCR2 + inflammatory monocytes/monocyte-derived dendritic cells) or result from different activation statuses of tissue macrophages (e.g., macrophages carrying markers of of classical [M1] or alternative activation [M2]). The latter are shaped by immune- and stromal cell-derived cytokines (e.g., IFN-γ, IL-4, IL-10, TGF-β), micro milieu factors (e.g., hypoxia, tonicity, amino acid availability), host cell-derived enzymes, secretory products and metabolites (e.g., heme oxygenase-1, arginase 1, indoleamine 2,3-dioxygenase, NOS2/NO, NOX2/ROS, lipids) as well as by parasite products (e.g., leishmanolysin/gp63, lipophosphoglycan). Exciting avenues of current research address the transcriptional, epigenetic and translational reprogramming of macrophages in a Leishmania species- and tissue context-dependent manner., Competing Interests: The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (© 2020 The Author.)

Background & Aims: Thrombospondin 1 (TSP1) is a multifunctional matricellular protein. We previously showed that TSP1 has an important role in obesity-associated metabolic complications, including inflammation, insulin resistance, cardiovascular, and renal disease. However, its contribution to obesity-associated non-alcoholic fatty liver disease/non-alcoholic steatohepatitis (NAFLD or NASH) remains largely unknown; thus, we aimed to determine its role., Methods: High-fat diet or AMLN (amylin liver NASH) diet-induced obese and insulin-resistant NAFLD/NASH mouse models were utilised, in addition to tissue-specific Tsp1 -knockout mice, to determine the contribution of different cellular sources of obesity-induced TSP1 to NAFLD/NASH development., Results: Liver TSP1 levels were increased in experimental obese and insulin-resistant NAFLD/NASH mouse models as well as in obese patients with NASH. Moreover, TSP1 deletion in adipocytes did not protect mice from diet-induced NAFLD/NASH. However, myeloid/macrophage-specific TSP1 deletion protected mice against obesity-associated liver injury, accompanied by reduced liver inflammation and fibrosis. Importantly, this protection was independent of the levels of obesity and hepatic steatosis. Mechanistically, through an autocrine effect, macrophage-derived TSP1 suppressed Smpdl3b expression in liver, which amplified liver proinflammatory signalling (Toll-like receptor 4 signal pathway) and promoted NAFLD progression., Conclusions: Macrophage-derived TSP1 is a significant contributor to obesity-associated NAFLD/NASH development and progression and could serve as a therapeutic target for this disease., Lay Summary: Obesity-associated non-alcoholic fatty liver disease is a most common chronic liver disease in the Western world and can progress to liver cirrhosis and cancer. No treatment is currently available for this disease. The present study reveals an important factor (macrophage-derived TSP1) that drives macrophage activation and non-alcoholic fatty liver disease development and progression and that could serve as a therapeutic target for non-alcoholic fatty liver disease/steatohepatitis., Competing Interests: The authors declare no conflicts of interest that pertain to this study. Please refer to the accompanying ICMJE disclosure forms for further details., (© 2020 The Author(s).)

The term acute-on-chronic liver failure (ACLF) defines an abrupt and life-threatening worsening of clinical conditions in patients with cirrhosis or chronic liver disease. In recent years, different definitions and diagnostic criteria for the syndrome have been proposed by the major international scientific societies. The main controversies relate to the type of acute insult (specifically hepatic or also extrahepatic), the stage of underlying liver disease (cirrhosis or chronic hepatitis) and the concomitant extrahepatic organ failure(s) that should be considered in the definition of ACLF. Therefore, different severity criteria and prognostic scores have been proposed and validated. Current evidence shows that the pathophysiology of ACLF is closely associated with an intense systemic inflammation sustained by circulating pathogen-associated molecular patterns and damage-associated molecular patterns. The development of organ failures may be a result of a combination of tissue hypoperfusion, direct immune-mediated damage and mitochondrial dysfunction. Management of ACLF is currently based on the supportive treatment of organ failures, mainly in an intensive care setting. For selected patients, liver transplantation is an effective treatment that offers a good long-term prognosis. Future studies on potential mechanistic treatments that improve patient survival are eagerly awaited., Competing Interests: E. Weiss reports personal fees form Baxter, MSD France, Biomerieux and Akcea therapeutics, and travel reimbursements from MSD France, outside the present work. The other authors report no conflict of interest related to this work. Please refer to the accompanying ICMJE disclosure forms for further details., (© 2020 The Authors.)

While metabolic syndrome and alcohol consumption are the two main causes of chronic liver disease, one of the two conditions is often predominant, with the other acting as a cofactor of morbimortality. It has been shown that obesity and alcohol act synergistically to increase the risk of fibrosis progression, hepatic carcinogenesis and mortality, while genetic polymorphisms can strongly influence disease progression. Based on common pathogenic pathways, there are several potential targets that could be used to treat both diseases; based on the prevalence and incidence of these diseases, new therapies and clinical trials are needed urgently., Competing Interests: The authors declare no conflicts of interest that pertain to this work. Please refer to the accompanying ICMJE disclosure forms for further details., (© 2020 The Authors.)

Autoantibody production is one of the leading factors of immune infertility, an autoimmune disease of the male reproductive system. The potential involvement of MHC-class II derived self-peptides against bacterial proteins in the antisperm antibody (ASA) production has been reported previously. Apparently, Streptococcus agalactiae has been considered as an important pathogen to impart infection-induced infertility in a bacteriospermia associated leukocytospermia (LCS/BS) state. Hence, the present study attempts to confirm S. agalactiae specific Laminin binding protein (Lmb) derived self-peptide (' KDSYTKKAKAFKKEA') namely human Motile Sperm domain-containing protein 2 (MOSPD2) as an auto-antigen in LCS/BS condition. Semen samples were collected from infertile men with LCS/BS (n ​= ​17) and their fertile counterparts (n ​= ​10). Gram-positive bacteria were predominantly identified in the entire 17 LCS samples using culture method followed by 16S rDNA sequencing technique. TLRs 2 and 4 expression used as markers of immune response in spermatozoa and sperm dysfunction were elevated in the LCS/BS spermatozoa as compared to their fertile counterparts. A significant increase in oxidative stress indices i.e., protein carbonylation, lipid peroxidation and acridine orange test (AOT), was also observed in the LCS/BS spermatozoa. Spermatozoa lysate (both auto and heterologous), bacterial lysate (control) and synthesized MOSPD2 self-peptide were used to test their antigenicity against the autoantibodies by rocket immunoelectrophoresis (RIEP) assay. Seminal plasma from LCS/BS patients with S. agalactiae was used as the source of autoantibodies. Spermatozoa and bacteria lysate; and MOSPD2 self-peptide were able to bind autoantibodies in the seminal plasma. Besides, the self-peptide showed a dose dependent increase in the precipitation of antibody. T-cell epitope mapping of 48 Enterococcus faecalis and 91 Staphylococcus aureus surface proteins confirmed MOSPD2 as a global auto-antigen. Thus, augmentation of TLR expression in LCS/BS spermatozoa inferred MOSPD2 to be a putative immunogen. Altogether, these findings will delineate the significance of MOSPD2 auto-antigen in a bacteria derived immune infertility condition., (© 2019 The Author.)

Programmed death-1 (PD-1), upon engagement by its ligands, programmed death ligand-1 (PD-L1) and programmed death ligand-2 (PD-L2), provides signals that attenuate adaptive immune responses. Here we describe the identification of the Pekin duck PD-L2 (duPD-L2) and its gene structure. The duPD-L2 cDNA encodes a 321 amino acid protein that has an amino acid identity of 76% and 35% with chicken and human PD-L2, respectively. Mapping of the duPD-L2 cDNA with duck genomic sequences revealed an exonic structure similar to that of the human P dcd1lg2 gene. Homology modelling of the duPD-L2 protein was compatible with the murine PD-L2 ectodomain structure. Residues known to be important for PD-1 receptor binding of murine PD-L2 were mostly conserved in duPD-L2 within sheets A and G and partially conserved within sheets C and F. DuPD-L2 mRNA was constitutively expressed in all tissues examined with highest expression levels in lung, spleen, cloaca, bursa, cecal tonsil, duodenum and very low levels of expression in muscle, kidney and brain. Lipopolysaccharide treatment of adherent duck PBMC upregulated duPD-L2 mRNA expression. Our work shows evolutionary conservation of the PD-L2 ectodomain structure and residues important for PD-1 binding in vertebrates including fish. The information provided will be useful for further investigation of the role of duPD-L2 in the regulation of duck adaptive immunity and exploration of PD-1-targeted immunotherapies in the duck hepatitis B infection model.

Formation of neutrophil extracellular traps (NETs) can perpetuate sterile inflammation; thus, it is important to clarify their pathophysiological characteristics. Free heme, derived via hemolysis, is a major contributor to organ damage, and reportedly induces neutrophil activation as well as reactive oxygen species (ROS) production and NET formation. For this study, we examined hemin (Fe 3+ -protoporphyrin IX)-induced NET formation quantitatively in vitro as well as the effects of oxidative stress. NETs formed in vitro from cultured neutrophils were quantitatively detected by using nuclease treatment and Sytox Green, a nucleic acid stain. Hemin-induced NET production was found to be in a dose-dependent manner, NADPH oxidase-dependent and toll-like receptor (TLR)-4 independent. Additionally, the iron molecule in the porphyrin ring was considered essential for the formation of NETs. In the presence of low concentrations of hydrogen peroxide, low concentrations of hemin-induced NETs were enhanced, unlike those of phorbol myristate acetate (PMA)-induced NETs. Quantitative analysis of NET formation may prove to be a useful tool for investigating NET physiology, and hemin could function as a possible therapeutic target for hemolysis-related events.

Dendritic cells (DCs) are major antigen-presenting cells that can efficiently prime and cross-prime antigen-specific T cells. Delivering antigen to DCs via surface receptors is thus an appealing strategy to evoke cellular immunity. Nonetheless, which DC surface receptor to target to yield the optimal CD8(+) and CD4(+) T cell responses remains elusive. Herein, we report the superiority of CD40 over 9 different lectins and scavenger receptors at evoking antigen-specific CD8(+) T cell responses. However, lectins (e.g., LOX-1 and Dectin-1) were more efficient than CD40 at eliciting CD4(+) T cell responses. Common and distinct patterns of subcellular and intracellular localization of receptor-bound αCD40, αLOX-1 and αDectin-1 further support their functional specialization at enhancing antigen presentation to either CD8(+) or CD4(+) T cells. Lastly, we demonstrate that antigen targeting to CD40 can evoke potent antigen-specific CD8(+) T cell responses in human CD40 transgenic mice. This study provides fundamental information for the rational design of vaccines against cancers and viral infections.

Protein-based vaccines offer a safer alternative to live-attenuated or inactivated vaccines but have limited immunogenicity. The identification of adjuvants that augment immunogenicity, specifically in a manner that is durable and antigen-specific, is therefore critical for advanced development. In this study, we use the filovirus virus-like particle (VLP) as a model protein-based vaccine in order to evaluate the impact of four candidate vaccine adjuvants on enhancing long term protection from Ebola virus challenge. Adjuvants tested include poly-ICLC (Hiltonol), MPLA, CpG 2395, and alhydrogel. We compared and contrasted antibody responses, neutralizing antibody responses, effector T cell responses, and T follicular helper (Tfh) cell frequencies with each adjuvant's impact on durable protection. We demonstrate that in this system, the most effective adjuvant elicits a Th1-skewed antibody response and strong CD4 T cell responses, including an increase in Tfh frequency. Using immune-deficient animals and adoptive transfer of serum and cells from vaccinated animals into naïve animals, we further demonstrate that serum and CD4 T cells play a critical role in conferring protection within effective vaccination regimens. These studies inform on the requirements of long term immune protection, which can potentially be used to guide screening of clinical-grade adjuvants for vaccine clinical development.