RESUMO
The high genetic diversity of hepatitis C virus (HCV) complicates effective vaccine development. We screened a cohort of 435 HCV-infected individuals and found that 2%-5% demonstrated outstanding HCV-neutralizing activity. From four of these patients, we isolated 310 HCV antibodies, including neutralizing antibodies with exceptional breadth and potency. High neutralizing activity was enabled by the use of the VH1-69 heavy-chain gene segment, somatic mutations within CDRH1, and CDRH2 hydrophobicity. Structural and mutational analyses revealed an important role for mutations replacing the serines at positions 30 and 31, as well as the presence of neutral and hydrophobic residues at the tip of the CDRH3. Based on these characteristics, we computationally created a de novo antibody with a fully synthetic VH1-69 heavy chain that efficiently neutralized multiple HCV genotypes. Our findings provide a deep understanding of the generation of broadly HCV-neutralizing antibodies that can guide the design of effective vaccine candidates.
Assuntos
Anticorpos Amplamente Neutralizantes/genética , Hepacivirus/imunologia , Anticorpos Anti-Hepatite C/genética , Linfócitos B/imunologia , Anticorpos Amplamente Neutralizantes/química , Anticorpos Amplamente Neutralizantes/imunologia , Regiões Determinantes de Complementaridade/química , Regiões Determinantes de Complementaridade/genética , Regiões Determinantes de Complementaridade/imunologia , Epitopos , Feminino , Genótipo , Hepacivirus/genética , Hepatite C/imunologia , Anticorpos Anti-Hepatite C/química , Anticorpos Anti-Hepatite C/imunologia , Humanos , Cadeias Pesadas de Imunoglobulinas/química , Cadeias Pesadas de Imunoglobulinas/genética , Cadeias Pesadas de Imunoglobulinas/imunologia , Masculino , Pessoa de Meia-Idade , Mutação , Proteínas do Envelope Viral/química , Proteínas do Envelope Viral/imunologiaRESUMO
BACKGROUND: Torque teno virus (TTV) is part of the human virome. TTV load was related to the immune status in patients after organ transplantation. We hypothesize that TTV load could be an additional marker for immune function in people living with HIV (PLWH). METHODS: In this analysis, serum samples of PLWH from the RESINA multicenter cohort were reanalyzed for TTV. Investigated clinical and epidemiological parameters included human pegivirus load, patient age and sex, HIV load, CD4+ T-cell count (Centers for Disease Control and Prevention [CDC] stage 1, 2, or 3), and CDC clinical stage (1993 CDC classification system; stage A, B, or C) before initiation of antiretroviral therapy. Regression analysis was used to detect possible associations among parameters. RESULTS: Our analysis confirmed TTV as a strong predictor of CD4+ T-cell count and CDC class 3. This relationship was used to propose a first classification of TTV load with regard to clinical stage. We found no association with clinical CDC stages A-C. The human pegivirus load was inversely correlated with HIV load but not TTV load. CONCLUSIONS: TTV load was associated with immunodeficiency in PLWH. Neither TTV nor HIV load were predictive for the clinical categories of HIV infection.
Assuntos
Infecções por Vírus de DNA , Infecções por HIV , Torque teno virus , Carga Viral , Humanos , Torque teno virus/isolamento & purificação , Masculino , Feminino , Contagem de Linfócito CD4 , Adulto , Pessoa de Meia-Idade , Infecções por HIV/imunologia , Infecções por HIV/virologia , Infecções por HIV/tratamento farmacológico , Infecções por HIV/complicações , Infecções por Vírus de DNA/virologia , Infecções por Vírus de DNA/imunologia , Infecções por Vírus de DNA/epidemiologia , Estados Unidos/epidemiologia , Centers for Disease Control and Prevention, U.S. , Flaviviridae/imunologia , Estudos de Coortes , Síndrome da Imunodeficiência Adquirida/imunologia , Síndrome da Imunodeficiência Adquirida/virologiaRESUMO
BACKGROUND AND AIMS: Human innate lymphoid cells (ILCs) are critically involved in the modulation of homeostatic and inflammatory processes in various tissues. However, only little is known about the composition of the intrahepatic ILC pool and its potential role in chronic liver disease. Here, we performed a detailed characterization of intrahepatic ILCs in both healthy and fibrotic livers. APPROACH AND RESULTS: A total of 50 livers (nonfibrotic = 22, and fibrotic = 29) were analyzed and compared with colon and tonsil tissue (each N = 14) and peripheral blood (N = 32). Human intrahepatic ILCs were characterized ex vivo and on stimulation using flow cytometry and single-cell RNA sequencing. ILC differentiation and plasticity were analyzed by both bulk and clonal expansion experiments. Finally, the effects of ILC-derived cytokines on primary human HSteCs were studied. Unexpectedly, we found that an "unconventional" ILC3-like cell represented the major IL-13-producing liver ILC subset. IL-13 + ILC3-like cells were specifically enriched in the human liver, and increased frequencies of this cell type were found in fibrotic livers. ILC3-derived IL-13 production induced upregulation of proinflammatory genes in HSteCs, indicating a potential role in the regulation of hepatic fibrogenesis. Finally, we identified KLRG1-expressing ILC precursors as the potential progenitor of hepatic IL-13 + ILC3-like cells. CONCLUSIONS: We identified a formerly undescribed subset of IL-13-producing ILC3-like cells that is enriched in the human liver and may be involved in the modulation of chronic liver disease.
Assuntos
Interleucina-13 , Linfócitos , Humanos , Interleucina-13/metabolismo , Imunidade Inata , Cirrose Hepática/metabolismoRESUMO
BACKGROUND: Primary sclerosing cholangitis (PSC) is a chronic liver disease leading to inflammation with scaring and strictures of bile ducts, which can lead to liver cirrhosis. A subtype of PSC characterized by high serum IgG4 (sIgG4) levels has been reported to be associated with poor outcomes, but the exact role and the longitudinal development of sIgG4 levels in PSC progression remains to be clarified. The aim of this study was to investigate if subsequent analysis of sIgG4 levels allows the identification of the PSC phenotype with high sIgG4. METHODS: sIgG4 values were repeatedly analysed in a well-characterized European PSC cohort of 110 individuals. Biochemical parameters, clinical endpoints, death and liver transplantation were compared between PSC subgroups. RESULTS: 12.7% (n = 14) of PSC patients showed increased sIgG4 levels (PSC-IgG4). The values normalized in 57.1% (n = 8; PSC-IgG4norm) during follow-up measurements, whereas the values remained permanently elevated in 42.9% (n = 6; PSC-IgG4const). Serum values of AP and γGT were significantly higher in PSC-IgG4const compared to PSC-IgG4norm at final blood sampling. Furthermore, mean age at PSC diagnosis was markedly lower in PSC-IgG4const compared to PSC-IgG4norm. CONCLUSIONS: This is the first study analyzing longitudinal development of sIgG4 in PSC. Our data indicate that only sequential determination of sIgG4 levels allow to accurately distinguish between the PSC phenotype with high sIgG4 and PSC with low sIgG4.
Assuntos
Colangite Esclerosante , Imunoglobulina G , Fenótipo , Humanos , Colangite Esclerosante/imunologia , Colangite Esclerosante/sangue , Colangite Esclerosante/diagnóstico , Imunoglobulina G/sangue , Masculino , Feminino , Pessoa de Meia-Idade , Adulto , Biomarcadores/sangue , Idoso , Progressão da Doença , Transplante de FígadoRESUMO
AIM: Primary sclerosing cholangitis (PSC) is a rare cholestatic liver disease characterized by inflammation of the intra- and extrahepatic bile ducts. Pathogenesis of PSC is still enigmatic but is likely to be multifactorial. Recently, we identified an interleukin-6 (IL-6)-dependent signal transducer and activator of transcription 3 (STAT3) activation in CD4+ TH1 and TH17 cells in PSC. The IL-6/STAT3 pathway was shown to be regulated by protease-activated receptor 1 (PAR1) contributing to inflammation. The role of the PAR1 -506 deletion/insertion (Del/Ins) polymorphism in PSC has not yet been investigated. METHODS: Two hundred eighty four PSC patients (200 patients with inflammatory bowel diseases [IBD] and 84 without IBD) and 309 healthy controls were genotyped for PAR1 rs11267092 (-506 Del/Ins -13 bp). Results were correlated with clinical characteristics and transplant-free survival. RESULTS: The frequency of PAR1 -506 Ins allele carriers (Del/Ins and Ins/Ins) was significantly higher in PSC patients (57.0%) compared to healthy controls (39.8%). Furthermore, carriers of PAR1 -506 Ins allele were more likely to have PSC than noncarriers (odds ratio 2.01; 95% confidence interval, 1.45-2.79). Patients with PSC carrying the PAR1 -506 Ins allele showed significantly higher alanine aminotransferase serum levels (p = 0.0357) and a trend toward shorter transplant-free survival time compared to noncarriers (8.9 ± 6.6 years vs. 10.5 ± 7.1 years; p = 0.076). CONCLUSIONS: Our study shows that PAR1 -506 Ins is significantly more frequent in people with PSC. As PAR1 -506 Ins allele carriers tended to have a shorter transplant-free survival, PAR1 might play a role in the development and course of PSC.
RESUMO
BACKGROUND AND AIMS: Liver-associated complications still frequently lead to mortality in people with HIV (PWH), even though combined antiretroviral treatment (cART) has significantly improved overall survival. The quantification of circulating collagen fragments released during collagen formation and degradation correlate with the turnover of extracellular matrix (ECM) in liver disease. Here, we analysed the levels of ECM turnover markers PC3X, PRO-C5, and PRO-C6 in PWH and correlated these with hepatic fibrosis and steatosis. METHODS: This monocentre, retrospective study included 141 PWH. Liver stiffness and liver fat content were determined using transient elastography (Fibroscan) with integrated CAP function. Serum levels of formation of cross-linked type III collagen (PC3X), formation of type V collagen (PRO-C5) and formation type VI collagen (PRO-C6), also known as the hormone endotrophin, were measured with ELISA. RESULTS: Twenty-five (17.7%) of 141 PWH had clinical significant fibrosis with liver stiffness ≥ 7.1 kPa, and 62 PWH (44.0%) had steatosis with a CAP value > 238 dB/m. Study participants with fibrosis were older (p = 0.004) and had higher levels of AST (p = 0.037) and lower number of thrombocytes compared to individuals without fibrosis (p = 0.0001). PC3X and PRO-C6 were markedly elevated in PWH with fibrosis. Multivariable cox regression analysis confirmed PC3X as independently associated with hepatic fibrosis. PRO-C5 was significantly elevated in participants with presence of hepatic steatosis. CONCLUSION: Serological levels of cross-linked type III collagen formation and endotrophin were significantly associated with liver fibrosis in PWH receiving cART and thus may be suitable as a non-invasive evaluation of liver fibrosis in HIV disease.
Assuntos
Colágeno Tipo III , Colágeno Tipo VI , Colágeno Tipo V , Fígado Gorduroso , Infecções por HIV , Cirrose Hepática , Humanos , Biomarcadores/sangue , Biomarcadores/metabolismo , Colágeno Tipo III/sangue , Colágeno Tipo III/metabolismo , Colágeno Tipo VI/sangue , Colágeno Tipo VI/metabolismo , Fígado Gorduroso/sangue , Fígado Gorduroso/complicações , Fígado Gorduroso/diagnóstico por imagem , Fígado Gorduroso/metabolismo , Infecções por HIV/sangue , Infecções por HIV/complicações , Infecções por HIV/tratamento farmacológico , Infecções por HIV/metabolismo , Fígado/diagnóstico por imagem , Fígado/metabolismo , Cirrose Hepática/sangue , Cirrose Hepática/diagnóstico por imagem , Cirrose Hepática/etiologia , Cirrose Hepática/metabolismo , Estudos Retrospectivos , Matriz Extracelular/metabolismo , Terapia Antirretroviral de Alta Atividade , Colágeno Tipo V/sangue , Colágeno Tipo V/metabolismo , Pró-Colágeno/sangue , Pró-Colágeno/metabolismoRESUMO
Hepatocellular carcinoma (HCC) is a severe complication of advanced alcoholic liver disease, which is modulated by genetic predisposition. Identifying new genetic loci might improve screening. Genetic variation of SAMM50 was linked to HCC. We aimed to validate this finding in a large cohort of patients with advanced alcoholic liver disease (ALD). A large, well-characterised cohort of patients with alcoholic cirrhosis without (n = 674) and with (n = 386) HCC, as well as controls with HCC due to viral hepatitis (n = 134), controls with heavy alcohol abuse without liver disease (n = 266) and healthy subjects (n = 237), were genotyped for SAMM50 rs3827385 and rs3761472 and for PNPLA3 rs738409. Genotype frequencies were compared between patients with alcohol-associated cirrhosis with and without HCC by uni- and multivariate analysis. Minor variants in both SAMM50 rs3827385 and rs3761472 were significantly more frequent in patients with alcoholic HCC versus alcoholic cirrhosis and versus the control cohorts. An even stronger association was noted for PNPLA3 rs738409. The univariate analysis resulted in an odds ratio (OR) of 1.8 for carriers of at least one minor variant of SAMM50 rs3827385 and rs3761472 (each p < 0.001), but this association was lost in multivariate analysis with age (OR 1.1/year), male sex (OR 3.2), diabetes (OR 1.9) and carriage of PNPLA3 148M (OR 2.1) remaining in the final model. Although minor variants of both SAMM50 loci are strongly associated with alcoholic HCC, this association is not independent of carriage of the well-known risk variant PNPLA3 148M.
Assuntos
Carcinoma Hepatocelular , Neoplasias Hepáticas , Humanos , Masculino , Carcinoma Hepatocelular/genética , Carcinoma Hepatocelular/patologia , Neoplasias Hepáticas/patologia , Lipase/genética , Polimorfismo de Nucleotídeo Único , Proteínas de Membrana/genética , Cirrose Hepática Alcoólica/genética , Predisposição Genética para Doença , Fatores de Risco , GenótipoRESUMO
BACKGROUND & AIMS: Bacterial translocation drives liver disease progression. We investigated whether functional genetic variants in toll-like receptor 5 (TLR5), the receptor for bacterial flagellin, affect the risk for hepatocellular carcinoma (HCC). METHODS: Healthy controls (n = 212), patients with alcohol abuse without liver disease (n = 382), and patients from a discovery cohort of alcohol-associated cirrhosis (n = 372 including 79 HCC cases), a validation cohort of alcohol-associated cirrhosis (n = 355 including 132 HCC cases), and a cohort of cirrhosis due to nonalcoholic steatohepatitis (NASH) (n = 145 including 62 HCC cases) were genotyped for the TLR5 rs5744174 and rs5744168 polymorphisms. Chemokine levels were measured by ELISA in patients' sera and supernatants of flagellin-stimulated healthy monocytes. RESULTS: Frequency of the TLR5 rs5744174 TT genotype was similar in healthy controls (33%), controls with alcohol abuse (34%), and patients with alcohol-associated cirrhosis in the discovery (28%), validation (33%), and NASH cohort (31%). The TT genotype was enriched in patients with versus without HCC in the discovery, validation, and NASH cohort (41% vs 25%; 39% vs 29%; 40% vs 24%; p < .05 each). This genotype remained a risk factor for HCC (OR = 1.9; p = .01) after multivariate correction for age, gender, diabetes, and carriage of the PNPLA3 148M variant. Interleukin-8 induction in monocytes from healthy controls and serum levels of interleukin-8 and CXCL1 from cirrhotic patients with the TT genotype were significantly increased versus C allele carriers. CONCLUSION: The TLR5 rs5744174 polymorphism, affecting immune response to flagellin, is linked to occurrence of HCC in cirrhosis caused by steatohepatitis.
Assuntos
Carcinoma Hepatocelular , Neoplasias Hepáticas , Hepatopatia Gordurosa não Alcoólica , Carcinoma Hepatocelular/genética , Predisposição Genética para Doença , Humanos , Cirrose Hepática/genética , Neoplasias Hepáticas/genética , Hepatopatia Gordurosa não Alcoólica/genética , Polimorfismo de Nucleotídeo Único , Receptor 5 Toll-Like/genéticaRESUMO
Immune checkpoint inhibition suggests promising progress for the treatment of advanced hepatocellular carcinoma (HCC). However, the underlying cellular mechanisms remain unclear because liver cancer cells apparently do not upregulate inhibitory checkpoint molecules. Here, we analysed whether regulatory T cells (Tregs) can alternatively trigger checkpoint inhibition pathways in HCC. Using flow cytometry we analysed expression of checkpoint molecules (PD-1, PD-L1, CTLA-4, GITR, Tim-3) on peripheral CD4+CD25+Foxp3+ Tregs and their secretion of inhibitory mediators (IL-10, IL-35, TGF-beta, galectin-9) in 116 individuals (50 patients with HCC, 41 non-tumour bearing liver disease controls, 25 healthy controls). Functional activity of Tregs on T effector cells (IFN-gamma production, cytotoxicity) was characterized in vitro using a lectin-dependent cellular cytotoxicity (LDCC) assay against checkpoint inhibitor-negative P815 target cells. Unlike liver patients without malignancy and healthy controls, the frequency of checkpoint inhibitor-positive Tregs inversely correlated to age of patients with HCC (PD-L1, p = 0.0080; CTLA-4, p = 0.0029) and corresponded to enhanced numbers of Tregs producing IL-10 and IL-35 (p < 0.05 each). Tregs inhibited IFN-gamma secretion and cytotoxicity of CD8+ T cells when added to LDCC against P815 cells. Treg-induced inhibition of IFN-gamma secretion could be partially blocked by neutralizing PD-1 and PD-L1 antibodies specifically in HCC patients. In HCC peripheral Tregs upregulate checkpoint inhibitors and contribute to systemic immune dysfunction and antitumoural activity by several inhibitory pathways, presumably facilitating tumour development at young age. Blocking PD-L1/PD-1 interactions in vitro selectively interfered with inhibitory Treg -T effector cell interactions in the patients with HCC and resulted in improved antitumoural activity also against checkpoint inhibitor-negative tumour cells.
Assuntos
Anticorpos Monoclonais/uso terapêutico , Linfócitos T CD8-Positivos/imunologia , Carcinoma Hepatocelular/imunologia , Imunoterapia/métodos , Neoplasias Hepáticas/imunologia , Linfócitos T Reguladores/imunologia , Adulto , Idoso , Idoso de 80 Anos ou mais , Antígeno B7-H1/metabolismo , Citotoxicidade Imunológica , Feminino , Humanos , Tolerância Imunológica , Interferon gama/metabolismo , Ativação Linfocitária , Masculino , Pessoa de Meia-Idade , Receptor de Morte Celular Programada 1/metabolismo , Adulto JovemRESUMO
BACKGROUND & AIMS: CD4+ regulatory T cells (Tregs) expand during chronic hepatitis C virus (HCV) infection, inhibit antiviral immunity and promote fibrosis. Direct-acting antiviral agents (DAA) have revolutionized HCV therapy. However, it is unclear if Tregs are normalized after DAA-induced HCV elimination. METHODS: We analyzed Tregs before (baseline), at end of therapy (EOT), 12 and 24weeks (SVR12, SVR24) and long-term (51±14weeks) after EOT in 26 genotype-1-infected patients who were successfully treated with sofosbuvir (SOF) plus interferon (IFN)/ribavirin (n=12) and IFN-free DAA regimens (SOF plus daclatasvir or simeprevir; n=14). Frequency, phenotype and suppressor function of peripheral Foxp3+ CD25+ CD4+ T cells were studied by multi-color flow cytometry and co-culture inhibition assays. RESULTS: Frequencies and activation status of Foxp3+ CD25+ CD4+ T cells remained elevated above those of normal controls in both treatment groups even long-term after HCV elimination. Co-culture assays indicated a dose-response relationship for functional inhibition of autologous CD4+ effector T cells and confirmed that activation of Tregs remained largely unchanged over the observation period. Unlike IFN-free regimens, SOF plus IFN/ribavirin induced a transiently increased frequency of Foxp3+ CD25+ CD4+ T cells at EOT (5.0% at baseline to 6.1% at EOT; p=0.001). These Foxp3+ CD25+ CD4+ T cells co-expressed the activation markers glycoprotein A repetitions predominant (GARP; p=0.012) and tumor necrosis factor receptor superfamily, member 4 (OX-40; p=0.001) but showed unchanged in vitro inhibitory activity. CONCLUSION: Although IFN-based DAA therapy induced transient expansion of activated Foxp3+ CD25+ CD4+ T cells, neither IFN-based nor IFN-free DAA regimens normalized frequencies and activation status of Tregs one year after viral elimination. Persistence of immunosuppressive Tregs may thus contribute to complications of liver disease even long-term after HCV cure. LAY SUMMARY: In chronic hepatitis C virus (HCV) infection, CD4+ regulatory T cells (Tregs) can reduce antiviral immune responses, promote liver fibrosis and may increase the risk for liver cancer, because they gradually expand during disease. Modern direct-acting antiviral agents (DAA) can "cure" hepatitis C in almost all treated patients. However, our study shows that DAA do not normalize the increased frequency and activation status of Tregs even long-term after HCV elimination. Tregs may persistently modulate functions of the immune system even after "cure" of hepatitis C.
Assuntos
Antivirais/uso terapêutico , Hepatite C Crônica/tratamento farmacológico , Linfócitos T Reguladores/imunologia , Adulto , Idoso , Feminino , Fatores de Transcrição Forkhead/análise , Galectinas/sangue , Hepatite C Crônica/imunologia , Humanos , Masculino , Pessoa de Meia-IdadeRESUMO
BACKGROUND & AIMS: NK cells regulate liver fibrosis by killing activated hepatic stellate cells (HSCs) and are controlled themselves by immune cells and/or soluble factors. Here, we analysed if CD4(+) regulatory T cells (Tregs) modify the interaction between NK cells and HSCs. METHODS: The modification of NK cell activity against HSCs was studied in CD56(high)CD16(-) NK cells, using a flow cytometric CD107a degranulation assay and co-cultures with Tregs from healthy donors and patients with hepatitis C, respectively. We studied the underlying mechanisms in detail, applying Treg supernatants, Treg pretreated HSCs, and recombinant IL-8, TGF-ß1, and IL-10 as well as blocking experiments with neutralizing antibodies and analysed Treg-associated changes in the expression of NK cell receptor ligands on HSCs. RESULTS: Tregs suppressed NK cell activation during HSC co-culture in a cell-contact-dependent manner involving the cytotoxic T-lymphocyte antigen 4 (CTLA-4). NK cell degranulation was further reduced, when HSCs had been pretreated with Tregs (p=0.043), Treg supernatants (p=0.001) or recombinant IL-8 (R=0.630, p=0.001) and TGF-ß1 (R=0.608, p=0.002), respectively. This additional inhibitory effect corresponded to the IL-8/TGF-ß1-mediated downregulation of MIC-A/B and HLA class-I on HSCs. Tregs from hepatitis C likewise inhibited NK cell activity, which was reversed significantly in specific blocking experiments. CONCLUSIONS: Our data indicate that Tregs interfere with NK cell regulation of fibrogenesis via both direct cell-contact-dependent inhibition of NK cells and release of soluble factors, downregulating activating NK cell receptor ligands on HSCs. Our data may be particularly relevant for the intrahepatic accumulation of Tregs in chronic hepatitis C because downregulated NK cell activity against HSCs may blunt their control of fibrogenesis.
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Linfócitos T CD4-Positivos/imunologia , Células Estreladas do Fígado/imunologia , Hepatite C Crônica/imunologia , Imunidade Celular , Células Matadoras Naturais/imunologia , Cirrose Hepática/imunologia , Linfócitos T Reguladores/imunologia , Adulto , Idoso , Comunicação Celular/imunologia , Células Cultivadas , Técnicas de Cocultura , Feminino , Células Estreladas do Fígado/patologia , Hepatite C Crônica/patologia , Humanos , Cirrose Hepática/patologia , Ativação Linfocitária , Masculino , Pessoa de Meia-IdadeRESUMO
INTRODUCTION: Regulatory CD4+ T cells (Tregs) are pivotal for inhibition of autoimmunity. Primary sclerosing cholangitis (PSC) is an autoimmune cholestatic liver disease of unknown etiology where contribution of Tregs is still unclear. Activation of the JAK-STAT pathway critically modifies functions of Tregs. In PSC, we studied activation of STAT proteins and Treg functions in response to cytokines. METHODS: In 51 patients with PSC, 10 disease controls (chronic replicative hepatitis C), and 36 healthy controls we analyzed frequencies of Foxp3+CD25+CD127lowCD4+ Tregs, their expression of ectonucleotidase CD39, and cytokine-induced phosphorylation of STAT1, 3, 5, and 6 using phospho-flow cytometry. In parallel, we measured cytokines IFN-gamma, interleukin (IL)-6, IL-2, and IL-4 in serum via bead-based immunoassays. RESULTS: In patients with PSC, ex vivo frequencies of peripheral Tregs and their expression of CD39 were significantly reduced (p < .05 each). Furthermore, serum levels of IFN-gamma, IL-6, IL-2, and IL-4 were markedly higher in PSC (p < .05 each). Unlike activation of STAT1, STAT5, and STAT6, IL-6 induced increased phosphorylation of STAT3 in Tregs of PSC-patients (p = .0434). Finally, STAT3 activation in Tregs correlated with leukocyte counts. CONCLUSIONS: In PSC, we observed enhanced STAT3 responsiveness of CD4+ Tregs together with reduced CD39 expression probably reflecting inflammatory activity of the disease.
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Colangite Esclerosante , Linfócitos T , Humanos , Interleucina-6 , Interleucina-2 , Interleucina-4 , Janus Quinases , Fatores de Transcrição STAT , Transdução de Sinais , Citocinas , Linfócitos T CD4-PositivosRESUMO
BACKGROUND: The level of type-I interferons (IFNs) in primary sclerosing cholangitis (PSC) was investigated to evaluate its association with disease activity and progression. METHODS: Bioactive type-I IFNs were evaluated in a murine model of PSC and human patients' sera using a cell-based reporter assay and ELISA techniques. In total, 57 healthy participants, 71 PSC, and 38 patients with primary biliary cholangitis were enrolled in this study. RESULTS: Bioactive type-I IFNs were elevated in the liver and serum of multidrug resistance protein 2-deficient animals and showed a correlation with the presence of CD45+ immune cells and serum alanine transaminase levels. Concordantly, bioactive type-I IFNs were elevated in the sera of patients with PSC as compared to healthy controls (sensitivity of 84.51%, specificity of 63.16%, and AUROC value of 0.8267). Bioactive IFNs highly correlated with alkaline phosphatase (r=0.4179, p<0.001), alanine transaminase (r=0.4704, p<0.0001), and gamma-glutamyl transpeptidase activities (r=0.6629, p<0.0001) but not with serum bilirubin. In addition, patients with PSC with advanced fibrosis demonstrated significantly higher type-I IFN values. Among the type-I IFN subtypes IFNα, ß and IFNω could be detected in patients with PSC with IFNω showing the highest concentration among the subtypes and being the most abundant among patients with PSC. CONCLUSIONS: The selectively elevated bioactive type-I IFNs specifically the dominating IFNω could suggest a novel inflammatory pathway that might also have a hitherto unrecognized role in the pathomechanism of PSC.
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Colangite Esclerosante , Interferon Tipo I , Fígado , Animais , Humanos , Camundongos , Alanina Transaminase , Fibrose , Interferon Tipo I/sangue , Fígado/patologiaRESUMO
BACKGROUND & AIMS: Regulatory CD4(+) T cells (Tregs) are considered to affect outcomes of HCV infection, because they increase in number during chronic hepatitis C and can suppress T-cell functions. METHODS: Using microarray analysis, in situ immunofluorescence, ELISA, and flowcytometry, we characterised functional differentiation and localisation of adaptive Tregs in patients with chronic hepatitis C. RESULTS: We found substantial upregulation of IL-8 in Foxp3(+)CD4(+) Tregs from chronic hepatitis C. Activated GARP-positive IL-8(+) Tregs were particularly enriched in livers of patients with chronic hepatitis C in close proximity to areas of fibrosis and their numbers were correlated with the stage of fibrosis. Moreover, Tregs induced upregulation of profibrogenic markers TIMP1, MMP2, TGF-beta1, alpha-SMA, collagen, and CCL2 in primary human hepatic stellate cells (HSC). HSC activation, but not Treg suppressor function, was blocked by adding a neutralizing IL-8 antibody. CONCLUSIONS: Our studies identified Foxp3(+)CD4(+) Tregs as an additional intrahepatic source of IL-8 in chronic hepatitis C acting on HSC. Thus, Foxp3(+)CD4(+) Tregs in chronic hepatitis C have acquired differentiation as regulators of fibrogenesis in addition to suppressing local immune responses.
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Hepatite C Crônica/imunologia , Hepatite C Crônica/patologia , Interleucina-8/biossíntese , Linfócitos T Reguladores/imunologia , Imunidade Adaptativa , Adulto , Idoso , Biomarcadores/metabolismo , Progressão da Doença , Feminino , Fibrose , Fatores de Transcrição Forkhead/metabolismo , Células Estreladas do Fígado/imunologia , Células Estreladas do Fígado/metabolismo , Humanos , Fígado/imunologia , Fígado/patologia , Masculino , Pessoa de Meia-Idade , Linfócitos T Reguladores/classificação , Linfócitos T Reguladores/metabolismo , Regulação para Cima , Adulto JovemRESUMO
INTRODUCTION: Primary sclerosing cholangitis (PSC) is a rare cholestatic liver disease with periductal inflammation and fibrosis. Genetic studies suggest inflammatory cytokines and IL-6-dependent activation of transcription factor STAT3 as pivotal steps in PSC pathogenesis. However, details of inflammatory regulation remain unclear. METHODS: We recruited 50 patients with PSC (36 with inflammatory bowel disease, 14 without inflammatory bowel disease), 12 patients with autoimmune hepatitis, and 36 healthy controls to measure cytokines in the serum, bile, and immune cell supernatant using bead-based immunoassays and flow cytometry and immunohistochemistry to analyze phosphorylation of STATs in immune cells. Finally, we analyzed cytokines and STAT3 phosphorylation of T cells in the presence of JAK1/2 inhibitors. RESULTS: In PSC, IL-6 specifically triggered phosphorylation of STAT3 in CD4 + T cells and lead to enhanced production of interferon (IFN) gamma and interleukin (IL)-17A. Phospho-STAT3-positive CD4 + T cells correlated with systemic inflammation (C-reactive protein serum levels). Combination of immunohistology and flow cytometry indicated that phospho-STAT3-positive cells were enriched in the peribiliary liver stroma and represented CD4 + T cells with prominent production of IFN gamma and IL-17A. JAK1/2 inhibitors blocked STAT3 phosphorylation and production of IFN gamma and IL-6, whereas IL-17A was apparently resistant to this inhibition. DISCUSSION: Our results demonstrate systemic and local activation of the IL-6/STAT3 pathway in PSC. Resistance of IL-17A to STAT3-targeted inhibition points to a more complex immune dysregulation beyond STAT3 activation.
Assuntos
Colangite Esclerosante , Doenças Inflamatórias Intestinais , Humanos , Citocinas/metabolismo , Inflamação , Interleucina-17/genética , Interleucina-17/metabolismo , Interleucina-6/metabolismo , Fator de Transcrição STAT3/genética , Fator de Transcrição STAT3/metabolismoRESUMO
Background & Aims: Progression of alcohol-associated liver disease (ALD) is driven by genetic predisposition. The rs13702 variant in the lipoprotein lipase (LPL) gene is linked to non-alcoholic fatty liver disease. We aimed at clarifying its role in ALD. Methods: Patients with alcohol-associated cirrhosis, with (n = 385) and without hepatocellular carcinoma (HCC) (n = 656), with HCC attributable to viral hepatitis C (n = 280), controls with alcohol abuse without liver damage (n = 366), and healthy controls (n = 277) were genotyped regarding the LPL rs13702 polymorphism. Furthermore, the UK Biobank cohort was analysed. LPL expression was investigated in human liver specimens and in liver cell lines. Results: Frequency of the LPL rs13702 CC genotype was lower in ALD with HCC in comparison to ALD without HCC both in the initial (3.9% vs. 9.3%) and the validation cohort (4.7% vs. 9.5%; p <0.05 each) and compared with patients with viral HCC (11.4%), alcohol misuse without cirrhosis (8.7%), or healthy controls (9.0%). This protective effect (odds ratio [OR] = 0.5) was confirmed in multivariate analysis including age (OR = 1.1/year), male sex (OR = 3.0), diabetes (OR = 1.8), and carriage of the PNPLA3 I148M risk variant (OR = 2.0). In the UK Biobank cohort, the LPL rs13702 C allele was replicated as a risk factor for HCC. Liver expression of LPL mRNA was dependent on LPL rs13702 genotype and significantly higher in patients with ALD cirrhosis compared with controls and alcohol-associated HCC. Although hepatocyte cell lines showed negligible LPL protein expression, hepatic stellate cells and liver sinusoidal endothelial cells expressed LPL. Conclusions: LPL is upregulated in the liver of patients with alcohol-associated cirrhosis. The LPL rs13702 high producer variant confers protection against HCC in ALD, which might help to stratify people for HCC risk. Impact and implications: Hepatocellular carcinoma is a severe complication of liver cirrhosis influenced by genetic predisposition. We found that a genetic variant in the gene encoding lipoprotein lipase reduces the risk for hepatocellular carcinoma in alcohol-associated cirrhosis. This genetic variation may directly affect the liver, because, unlike in healthy adult liver, lipoprotein lipase is produced from liver cells in alcohol-associated cirrhosis.
RESUMO
Group 1 innate lymphoid cells (ILCs) comprise a heterogeneous family of cytotoxic natural killer (NK) cells and ILC1s. We identify a population of "liver-type" ILC1s with transcriptional, phenotypic, and functional features distinct from those of conventional and liver-resident NK cells as well as from other previously described human ILC1 subsets. LT-ILC1s are CD49a+CD94+CD200R1+, express the transcription factor T-BET, and do not express the activating receptor NKp80 or the transcription factor EOMES. Similar to NK cells, liver-type ILC1s produce IFN-γ, TNF-α, and GM-CSF; however, liver-type ILC1s also produce IL-2 and lack perforin and granzyme-B. Liver-type ILC1s are expanded in cirrhotic liver tissues, and they can be produced from blood-derived ILC precursors in vitro in the presence of TGF-ß1 and liver sinusoidal endothelial cells. Cells with similar signature and function can also be found in tonsil and intestinal tissues. Collectively, our study identifies and classifies a population of human cross-tissue ILC1s.
Assuntos
Imunidade Inata , Linfócitos , Humanos , Células Endoteliais , Células Matadoras Naturais , Fígado , Fatores de Transcrição , Análise de Sequência de RNAAssuntos
Antivirais , Coinfecção , HIV , Infecções por HIV , Hepatite C , Humanos , Células Supressoras MieloidesRESUMO
Extrahepatic manifestations of chronic hepatitis C virus (HCV) infection frequently involve the skin. Here, we report the case of a woman, who experienced a psoriasis exacerbation on DAA treatment, which lead to psoriasis resolution upon HCV clearance under continued treatment with elbasvir/grazoprevir.
RESUMO
Hepatic stellate cells (HSCs) represent the main fibrogenic cell type accumulating extracellular matrix in the liver. Recent data suggest that hepatitis C virus (HCV) core protein may directly activate HSCs. Therefore, we examined the influence of recombinant HCV core protein on human HSCs. Primary human HSCs and the human HSC line LX-2 were stimulated with recombinant HCV proteins core and envelope 2 protein. Expression of procollagen type I α-1, α-smooth muscle actin, cysteine- and glycine-rich protein 2, glial fibrillary acidic protein, tissue growth factor ß1, matrix metalloproteinases 2 (MMP2) and 13, tissue inhibitor of metalloproteinases 1 and 2 was investigated by real-time PCR. Intracellular signaling pathways of ERK1/2, p38 and, jun-amino-terminal kinase (JNK) were analyzed by western blot analysis. Recombinant HCV core protein induced upregulation of procollagen type I α-1, α-smooth muscle actin, MMP 2 and 13, tissue inhibitor of metalloproteinases 1 and 2, tissue growth factor ß1, cysteine- and glycine-rich protein 2, and glial fibrillary acidic protein mRNA expression, whereas HCV envelope 2 protein did not exert any significant effect. Blocking of toll-like receptor 2 (TLR2) with a neutralizing antibody prevented mRNA upregulation by HCV core protein confirming that the TLR2 pathway was involved. Furthermore, western blot analysis revealed HCV-induced phosphorylation of the TLR2-dependent signaling molecules ERK1/2, p38 and JNK mitogen-activated kinases. Our in vitro results demonstrate a direct effect of HCV core protein on activation of HSCs toward a profibrogenic state, which is mediated via the TLR2 pathway. Manipulating the TLR2 pathway may thus provide a new approach for antifibrotic therapies in HCV infection.