Translation of IRF-1 Restricts Hepatic Interleukin-7 Production to Types I and II Interferons: Implications for Hepatic Immunity.

Interleukin-7 (IL-7) is an important cytokine with pivotal pro-survival functions in the adaptive immune system. However, the role of IL-7 in innate immunity is not fully understood. In the present study, the impact of hepatic IL-7 on innate immune cells was assessed by functional experiments as well as in patients with different stages of liver cirrhosis or acute-on-chronic liver failure (ACLF). Human hepatocytes and liver sinusoidal endothelial cells secreted IL-7 in response to stimulation with interferons (IFNs) of type I and II, yet not type III. De novo translation of interferon-response factor-1 (IRF-1) restricted IL-7 production to stimulation with type I and II IFNs. LPS-primed human macrophages were identified as innate immune target cells responding to IL-7 signaling by inactivation of Glycogen synthase kinase-3 (GSK3). IL-7-mediated GSK3 inactivation augmented LPS-induced secretion of pro-inflammatory cytokines and blunted LPS tolerance of macrophages. The IFN-IRF-1-IL-7 axis was present in liver cirrhosis patients. However, liver cirrhosis patients with or without ACLF had significantly lower concentrations of IL-7 in serum compared to healthy controls, which might contribute to LPS-tolerance in these patients. In conclusion, we propose the presence of an inflammatory cascade where IFNs of type I/II induce hepatocellular IL-7 in an IRF-1-restriced way. Beyond its role in adaptive immune responses, IL-7 appears to augment the response of macrophages to LPS and to ameliorate LPS tolerance, which may improve innate immune responses against invading pathogens.

Dysregulated Adaptive Immunity Is an Early Event in Liver Cirrhosis Preceding Acute-on-Chronic Liver Failure.

Introduction: Acute-on-chronic liver failure (ACLF) is characterized by high levels of systemic inflammation and parallel suppression of innate immunity, whereas little is known about adaptive immune immunity in ACLF. We therefore aimed to characterize the development of the adaptive immune system during the progression of liver cirrhosis to ACLF. Patients with compensated/stable decompensated liver cirrhosis, acute decompensation of liver cirrhosis, or ACLF were recruited from a prospective cohort study. Comprehensive immunophenotyping was performed using high dimensional flow cytometry. Replication of Torque teno (TT) virus was quantified as a marker of immunosuppression. High frequencies of detectable TT virus were observed already in patients with compensated/stable decompensated liver cirrhosis compared to healthy controls (>50% vs. 19%), suggesting relatively early occurrence of immunosuppression in cirrhosis. In line, profoundly reduced numbers of distinct innate and adaptive immune cell populations were observed before ACLF development. These changes were accompanied by parallel upregulation of co-stimulatory (e.g. CD40L, OX40, CD69, GITR, TIM-1) and inhibitory immune checkpoints (e.g. PDPN, PROCR, 2B4, TIGIT) on CD4+ and CD8+ T cells, which again preceded the development of ACLF. On a functional basis, the capacity of CD4+ and CD8+ T cells to produce pro-inflammatory cytokines upon stimulation was strongly diminished in patients with acute decompensation of liver cirrhosis and ACLF. Conclusion: Impaired innate and-in particular-adaptive cellular immunity occurs relatively early in the pathogenesis of liver cirrhosis and precedes ACLF. This may contribute to the development of ACLF by increasing the risk of infections in patients with liver cirrhosis.

Ten quick tips for getting the most scientific value out of numerical data.

Most studies in the life sciences and other disciplines involve generating and analyzing numerical data of some type as the foundation for scientific findings. Working with numerical data involves multiple challenges. These include reproducible data acquisition, appropriate data storage, computationally correct data analysis, appropriate reporting and presentation of the results, and suitable data interpretation. Finding and correcting mistakes when analyzing and interpreting data can be frustrating and time-consuming. Presenting or publishing incorrect results is embarrassing but not uncommon. Particular sources of errors are inappropriate use of statistical methods and incorrect interpretation of data by software. To detect mistakes as early as possible, one should frequently check intermediate and final results for plausibility. Clearly documenting how quantities and results were obtained facilitates correcting mistakes. Properly understanding data is indispensable for reaching well-founded conclusions from experimental results. Units are needed to make sense of numbers, and uncertainty should be estimated to know how meaningful results are. Descriptive statistics and significance testing are useful tools for interpreting numerical results if applied correctly. However, blindly trusting in computed numbers can also be misleading, so it is worth thinking about how data should be summarized quantitatively to properly answer the question at hand. Finally, a suitable form of presentation is needed so that the data can properly support the interpretation and findings. By additionally sharing the relevant data, others can access, understand, and ultimately make use of the results. These quick tips are intended to provide guidelines for correctly interpreting, efficiently analyzing, and presenting numerical data in a useful way.

The portal vein as a distinct immunological compartment - A comprehensive immune phenotyping study.

Advanced liver diseases are associated with impaired intestinal barrier function, which results in bacterial influx via the portal vein to the liver, causing hepatic and systemic inflammation. Little is known about possible concomitant trafficking of immune cells from the intestines to the liver. We therefore performed a comprehensive immunophenotyping study of the portal venous versus peripheral blood compartment in patients with liver cirrhosis who received a transjugular intrahepatic portosystemic stent shunt (TIPS). Our analysis suggests that the portal vein constitutes a distinct immunological compartment resembling that of the intestines, at least in patients with advanced liver cirrhosis. In detail, significantly lower frequencies of naïve CD4+ T cells, monocytes, dendritic cells and Vδ2 T cells were observed in the portal vein, whereas frequencies of activated CD4+ and CD8+ T cells, as well as of mucosa-associated Vδ1 T cells were significantly higher in portal venous compared to peripheral blood. In conclusion, our data raises interesting questions, e.g. whether liver cirrhosis-associated chronic inflammation of the intestines and portal hypertension promote an influx of activated intestinal immune cells like γδ T cells into the liver.

Differential modulation of hepatitis C virus replication and innate immune pathways by synthetic calcitriol-analogs.

BACKGROUND AND AIMS: Vitamin D signaling is involved in infectious and non-infectious liver diseases, yet the natural vitamin D metabolites are suboptimal therapeutic agents. In the present study, we therefore aimed to explore the potential and mechanism of selected calcitriol analogs to regulate the hepatocellular transcriptome and to inhibit hepatitis C virus (HCV) in comparison with calcitriol. METHODS: Human hepatoma cell lines and primary human macrophages were stimulated with calcitriol and selected calcitriol analogs. The effect of calcitriol and its derivatives on hepatocellular gene expression and vitamin D receptor (VDR) signaling as well as on replication of HCV were assessed by quantitative PCR, microarray analyses and in silico analyses of ligand-VDR complexes. RESULTS: The structurally related vitamin D analogs calcipotriol and tacalcitiol, but not calcitriol itself, suppressed HCV replication in a VDR-dependent manner. Using a residue-interaction network approach we outline structural and functional differences between VDR-ligand complexes. In particular we find characteristics in the VDR structure bound to calcipotriol with distinct local residue interaction patterns that affect key functional residues that pertain to the VDR charge clamp, H397 and F422, a VDR regulatory element for interaction with co-activators and -repressors. As a consequence, we show calcipotriol in comparison to calcitriol to induce stronger regulatory actions on the transcriptome of hepatocytes and macrophages including key antimicrobial peptides. CONCLUSION: Calcipotriol induces local structure rearrangements in VDR that could possibly translate into a superior clinical potential to execute important non-classical vitamin D effects such as inhibition of HCV replication.

Macrophage-Derived Extracellular Vesicles Induce Long-Lasting Immunity Against Hepatitis C Virus Which Is Blunted by Polyunsaturated Fatty Acids.

Extracellular vesicles (EVs) are increasingly recognized as important mediators of intercellular communication. In this study, we aimed to further characterize the role of macrophage-derived EVs in immune responses against hepatitis C virus (HCV) and the potential of polyunsaturated fatty acids (PUFAs) to modulate this modality of innate immunity. To this end, EVs were isolated from interferon-stimulated macrophage cultures or from serum of patients with acute or chronic hepatitis C. EVs were characterized by electron microscopy, flow cytometry, RNA-sequencing, and Western blot analysis. The effect of EVs on replication of HCV was assessed in coculture models. Functional analyses were performed to assess the impact of PUFAs on EV-mediated antiviral immunity. We found that macrophages secreted various cytokines shortly after stimulation with type I and II IFN, which orchestrated a fast but short-lasting antiviral state. This rapid innate immune answer was followed by the production of macrophage-derived EVs, which induced a late, but long-lasting inhibitory effect on HCV replication. Of note, exposure of macrophages to PUFAs, which are important regulators of immune responses, dampened EV-mediated antiviral immune responses. Finally, EVs from patients with hepatitis C exhibited long-lasting antiviral activities during IFN therapy as well. The antiviral effect of EVs from Caucasian and Japanese patients differed, which may be explained by different nutritional uptake of PUFAs. In conclusion, our data indicate that macrophage-derived EVs mediate long-lasting inhibitory effects on HCV replication, which may bridge the time until efficient adaptive immune responses are established, and which can be blunted by PUFAs.

Phospho-dependent Regulation of SAMHD1 Oligomerisation Couples Catalysis and Restriction.

SAMHD1 restricts HIV-1 infection of myeloid-lineage and resting CD4+ T-cells. Most likely this occurs through deoxynucleoside triphosphate triphosphohydrolase activity that reduces cellular dNTP to a level where reverse transcriptase cannot function, although alternative mechanisms have been proposed recently. Here, we present combined structural and virological data demonstrating that in addition to allosteric activation and triphosphohydrolase activity, restriction correlates with the capacity of SAMHD1 to form "long-lived" enzymatically competent tetramers. Tetramer disruption invariably abolishes restriction but has varied effects on in vitro triphosphohydrolase activity. SAMHD1 phosphorylation also ablates restriction and tetramer formation but without affecting triphosphohydrolase steady-state kinetics. However phospho-SAMHD1 is unable to catalyse dNTP turnover under conditions of nucleotide depletion. Based on our findings we propose a model for phosphorylation-dependent regulation of SAMHD1 activity where dephosphorylation switches housekeeping SAMHD1 found in cycling cells to a high-activity stable tetrameric form that depletes and maintains low levels of dNTPs in differentiated cells.