A. Boonstra

Re-evaluation of hepatitis B virus clinical phases by systems biology identifies unappreciated roles for the innate immune response and B cells

To identify immunological mechanisms that govern distinct clinical phases of a chronic hepatitis B virus (HBV) infection—immune tolerant (IT), immune active (IA), inactive carrier (IC), and hepatitis B e antigen (HBeAg)‐negative (ENEG) hepatitis phases—we performed a systems biology study. Serum samples from untreated chronic HBV patients (nu2009=u200971) were used for multiplex cytokine measurements, quantitative hepatitis B surface antigen (HBsAg), HBeAg levels, HBV genotype, and mutant analysis. Leukocytes were phenotyped using multicolor flow cytometry, and whole‐blood transcriptome profiles were generated. The latter were compared with liver biopsy transcriptomes from IA (nu2009=u200916) and IT (nu2009=u20093) patients. HBV viral load as well as HBeAg and HBsAg levels (Pu2009<u20090.001), but not leukocyte composition, differed significantly between distinct phases. Serum macrophage chemotactic protein 1, interleukin‐12p40, interferon (IFN)‐gamma‐inducible protein 10, and macrophage inflammatory protein 1 beta levels were different between two or more clinical phases (Pu2009<u20090.05). Comparison of blood transcriptomes identified 64 differentially expressed genes. The gene signature distinguishing IA from IT and IC patients was predominantly composed of highly up‐regulated immunoglobulin‐encoding genes. Modular repertoire analysis using gene sets clustered according to similar expression patterns corroborated the abundant expression of B‐cell function‐related genes in IA patients and pointed toward increased (ISG) transcript levels in IT patients, compared to subsequent phases. Natural killer cell activities were clustered in clinical phases with biochemical liver damage (IA and ENEG phases), whereas T‐cell activities were higher in all phases, compared to IT patients. B‐cell‐related transcripts proved to be higher in biopsies from IA versus IT patients. Conclusion: HBV clinical phases are characterized by distinct blood gene signatures. Innate IFN and B‐cell responses are highly active during the IT and IA phases, respectively. This suggests that the presumed immune tolerance in chronic HBV infections needs to be redefined. (Hepatology 2015;62:87‐100)

Liver monocytes and kupffer cells remain transcriptionally distinct during chronic viral infection

Due to the scarcity of immunocompetent animal models for chronic viral hepatitis, little is known about the role of the innate intrahepatic immune system during viral replication in the liver. These insights are however fundamental for the understanding of the inappropriate adaptive immune responses during the chronic phase of the infection. We apply the Lymphocytic Choriomenigitis Virus (LCMV) clone 13 mouse model to examine chronic virus-host interactions of Kupffer cells (KC) and infiltrating monocytes (IM) in an infected liver. LCMV infection induced overt clinical hepatitis, with rise in ALT and serum cytokines, and increased intrahepatic F4/80 expression. Despite ongoing viral replication, whole liver transcriptome showed baseline expression levels of inflammatory cytokines, interferons, and interferon induced genes during the chronic infection phase. Transcriptome analyses of sorted KC and IMs using NanoString technology revealed two unique phenotypes with only minimal overlap. At the chronic viral infection phase, KC showed no increased transcription of activation markers Cd80 and Cd86, but an increased expression of genes related to antigen presentation, whereas monocytes were more activated and expressed higher levels of Tnf transcripts. Although both KCs and intrahepatic IM share the surface markers F4/80 and CD11b, their transcriptomes point towards distinctive roles during virus-induced chronic hepatitis.

Biochemical measures and frailty in people with intellectual disabilities

INTRODUCTIONnPeople with intellectual disabilities (ID) are earlier frail than people in the general population. Although this may be explained by lifelong unfavourable social, psychological and clinical causes, underlying physiological pathways might be considered too. Biological measures can help identify pathophysiological pathways. Therefore, we examined the association between frailty and a range of serum markers on inflammation, anaemia, the metabolic system, micronutrients and renal functioning.nnnMETHODSnParticipants (n = 757) with borderline to severe ID (50+) were recruited from three Dutch ID care and support services.nnnRESULTSnFrailty was measured with a frailty index, a measure based on the accumulation of deficits. Linear regression analyses were performed to identify associations between frailty and biochemical measures independent of age, gender, level of ID and the presence of Down syndrome. Frailty appears associated with inflammation (IL-6 and CRP), anaemia, metabolic markers (glucose, cholesterol and albumin) and renal functioning (cystatin-C and creatinine).nnnDISCUSSIONnThese results are in line with results observed in the general population. Future research needs to investigate the causal relation between biochemical measures and frailty, with a special focus on inflammation and nutrition. Furthermore, the possibility to screen for frailty using biochemical measures needs to be used.

Interferon-alpha treatment rapidly clears Hepatitis E virus infection in humanized mice

Antiviral treatment options for chronic Hepatitis E Virus (HEV) infections are limited and immunological determinants of viral persistence remain largely unexplored. We studied the antiviral potency of pegylated interferon-α (pegIFNα) against HEV infections in humanized mice and modelled intrahepatic interferon stimulated gene (ISG) responses. Human gene expression levels in humanized mouse livers were analyzed by qPCR and Nanostring. Human CXCL10 was measured in mouse serum. HEV genotype 3 (gt3) infections were cleared from liver and feces within 8 pegIFNα doses in all mice and relapsed after a single pegIFNα injection in only half of treated animals. Rapid viral clearance by pegIFNα was confirmed in HEV gt1, but not in Hepatitis B Virus infected animals. No ISG induction was observed in untreated HEV gt3 and gt1 infected humanized livers compared to control chimeric mice, irrespective of the human hepatocyte donor, viral isolate or HEV infection duration. Human specific ISG transcript levels in mouse liver increased significantly after pegIFNα treatment and induced high circulating human CXCL10 in mouse serum. In conclusion, HEV gt1 and gt3 infections do not elicit innate intrahepatic immune responses and remain highly sensitive to pegIFNα in immunocompromised humanized mice.

Role of microbial translocation in soluble CD14 up-regulation in HIV-, but not in HCV-, infected chimpanzees

During human immunodeficiency virus (HIV) infection, soluble CD14 (sCD14) is up-regulated as a consequence of pathological disruption of the gut epithelial barrier, and subsequent increased microbial translocation. Also in hepatitis C virus (HCV)-infected patients with advanced liver fibrosis, increased levels of sCD14 have been reported. Since the liver plays an important role in clearance of translocated bacterial products, hepatic fibrosis may negatively affect clearance and thus contribute to higher sCD14 levels. Chimpanzees (Pan troglodytes) infected with HCV typically show no signs of liver fibrosis. Here, we have tested the hypothesis that increased levels of sCD14 occur in the absence of hepatic fibrosis or microbial translocation in chimpanzees chronically infected with HCV. sCD14 was up-regulated in both HIV/simian immunodeficiency virus (SIV)- and HCV-infected chimpanzees. In HIV/SIV-infected chimpanzees, intestinal fatty acid-binding protein, a marker for gut perturbation, lipopolysaccharide (LPS)-binding-protein and LPS core antibodies, confirm that sCD14 up-regulation was caused by increased microbial translocation. In HCV-infected chimpanzees, no evidence was found for increased microbial translocation despite up-regulation of sCD14. Additionally, the impact of liver fibrosis on microbial translocation was addressed by direct comparison of chimpanzees with a high HCV load and human patients with advanced fibrosis. These data suggest that only in a small minority of HCV patients, hepatic fibrosis corroborates microbial translocation.

Spontaneous and NCR mediated cytotoxicity are effector functions of distinct NK subsets in HCV infected chimpanzees

In humans, CD16 and CD56 are used to identify functionally distinct natural killer (NK) subsets. Due to ubiquitous CD56 expression, this marker cannot be used to distinguish between NK cell subsets in chimpanzees. Therefore, functional analysis of distinct NK subsets during hepatitis C virus (HCV) infection has never been performed in these animals. In the present study an alternative strategy was used to identify four distinct NK subsets on the basis of the expression of CD16 and CD94. The expression of activating and inhibiting surface receptors showed that these subsets resemble human NK subsets. CD107 expression was used to determine degranulation of the different subsets in naive and HCV‐infected chimpanzees. In HCV‐infected chimpanzees increased spontaneous cytotoxicity was observed in CD94high/dimCD16pos and CD94lowCD16pos subsets. By contrast, increased natural cytotoxicity receptor (NCR)‐ mediated degranulation after NKp30 and NKp44 triggering was demonstrated in the CD94dimCD16neg subset. Our findings suggest that spontaneous and NCR‐mediated cytotoxicity are effector functions of distinct NK subsets in HCV‐infected chimpanzees.

Spontaneous and natural cytotoxicity receptor-mediated cytotoxicity are effector functions of distinct natural killer subsets in hepatitis C virus-infected chimpanzees

In humans, CD16 and CD56 are used to identify functionally distinct natural killer (NK) subsets. Due to ubiquitous CD56 expression, this marker cannot be used to distinguish between NK cell subsets in chimpanzees. Therefore, functional analysis of distinct NK subsets during hepatitis C virus (HCV) infection has never been performed in these animals. In the present study an alternative strategy was used to identify four distinct NK subsets on the basis of the expression of CD16 and CD94. The expression of activating and inhibiting surface receptors showed that these subsets resemble human NK subsets. CD107 expression was used to determine degranulation of the different subsets in naive and HCV‐infected chimpanzees. In HCV‐infected chimpanzees increased spontaneous cytotoxicity was observed in CD94high/dimCD16pos and CD94lowCD16pos subsets. By contrast, increased natural cytotoxicity receptor (NCR)‐ mediated degranulation after NKp30 and NKp44 triggering was demonstrated in the CD94dimCD16neg subset. Our findings suggest that spontaneous and NCR‐mediated cytotoxicity are effector functions of distinct NK subsets in HCV‐infected chimpanzees.