RESUMO
BACKGROUND & AIMS: Down-regulation of hepatitis C virus (HCV)-specific CD4(+) T-cell responses is a hallmark of chronic viral persistence in acute hepatitis C. FOXP3(+)CD25(+)CD4(+) regulatory T cells can modulate HCV-specific immune responses in vitro, but the role of virus-specific regulatory T cells in the pathogenesis of chronic viral persistence is unknown. METHODS: Two novel HLA-DR15 tetramers were synthesized to study the kinetics and phenotype of FOXP3(+)-expressing HCV-specific CD4(+) T cells from 10 patients with acute hepatitis C and 15 patients with chronic hepatitis C. RESULTS: In acute hepatitis C, generally only a low percentage of HCV-specific CD4(+) T cells expressed FOXP3(+) (mean of 2.5% in patients with self-limited acute hepatitis C vs 2.4% in patients with evolving chronic hepatitis C). Although distinct but short-lived increases in virus-specific FOXP3(+)CD4(+) T cells occurred in 3 patients (30%, 26%, and 7% of tet(+) CD4(+) T cells, respectively), these did not correlate with the evolution of chronic hepatitis C. HCV-specific FOXP3(+)CD4(+) T cells displayed a distinct phenotype, with only 10% expressing CD25 and 40% being CD127low. Interestingly, this phenotype of FOXP3(+)CD4(+) T cells was already expanded in bulk CD4(+) T cells in patients with chronic hepatitis C. CONCLUSIONS: Although short-lived increases in HCV-specific FOXP3(+)CD4(+) T cells occur during the course of acute hepatitis C, we could not demonstrate an association of HCV-specific regulatory T cells and persistent viremia.
Assuntos
Fatores de Transcrição Forkhead/metabolismo , Hepacivirus/imunologia , Hepatite C Crônica/imunologia , Hepatite C/imunologia , Linfócitos T Reguladores/imunologia , Doença Aguda , Adulto , Idoso , Proliferação de Células , Células Cultivadas , Progressão da Doença , Feminino , Antígenos HLA-DR/imunologia , Cadeias HLA-DRB1 , Hepatite C/diagnóstico , Anticorpos Anti-Hepatite C/sangue , Hepatite C Crônica/diagnóstico , Humanos , Imunofenotipagem , Subunidade alfa de Receptor de Interleucina-2/metabolismo , Subunidade alfa de Receptor de Interleucina-7/metabolismo , Cinética , Masculino , Pessoa de Meia-Idade , Linfócitos T Reguladores/virologia , Viremia/imunologiaRESUMO
In this study, we analyzed if IL-22 displays, similar to other IL-10 like cytokines such as IL-28A, antiviral properties in hepatic cells. Using RT-PCR and immunoblotting, we demonstrated that hepatic cell lines and primary hepatocytes express the functional IL-22 receptor complex consisting of IL-22R1 and IL-10R2. Hepatic IL-22 mRNA expression as measured by quantitative PCR was up-regulated in autoimmune and viral hepatitis compared to cholestatic liver diseases, while IL-22 serum levels did not differ significantly between patients with viral hepatitis and normal controls. IL-22 did not significantly change the expression levels of IFN-alpha/-beta and of the antiviral proteins MxA and 2',5'-OAS. Consequently, it had in comparison to IFN-alpha no relevant antiviral activity in in vitro models of HCV replication and infection. Taken together, hepatic IL-22 expression is up-regulated in viral hepatitis but IL-22 does not directly regulate antiviral proteins and has, in contrast to IFN-alpha, no effect on HCV replication.
Assuntos
Doenças Autoimunes/imunologia , Hepatite C/imunologia , Hepatócitos/imunologia , Interleucinas/metabolismo , Linhagem Celular , Humanos , Immunoblotting , Subunidade beta de Receptor de Interleucina-10/genética , Subunidade beta de Receptor de Interleucina-10/metabolismo , Interleucinas/sangue , Interleucinas/genética , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Receptores de Interleucina/genética , Receptores de Interleucina/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Regulação para Cima , Interleucina 22RESUMO
BACKGROUND: CD4+ T cell help is critical in maintaining antiviral immune responses and such help has been shown to be sustained in acute resolving hepatitis C. In contrast, in evolving chronic hepatitis C CD4+ T cell helper responses appear to be absent or short-lived, using functional assays. METHODOLOGY/PRINCIPAL FINDINGS: Here we used a novel HLA-DR1 tetramer containing a highly targeted CD4+ T cell epitope from the hepatitis C virus non-structural protein 4 to track number and phenotype of hepatitis C virus specific CD4+ T cells in a cohort of seven HLA-DR1 positive patients with acute hepatitis C in comparison to patients with chronic or resolved hepatitis C. We observed peptide-specific T cells in all seven patients with acute hepatitis C regardless of outcome at frequencies up to 0.65% of CD4+ T cells. Among patients who transiently controlled virus replication we observed loss of function, and/or physical deletion of tetramer+ CD4+ T cells before viral recrudescence. In some patients with chronic hepatitis C very low numbers of tetramer+ cells were detectable in peripheral blood, compared to robust responses detected in spontaneous resolvers. Importantly we did not observe escape mutations in this key CD4+ T cell epitope in patients with evolving chronic hepatitis C. CONCLUSIONS/SIGNIFICANCE: During acute hepatitis C a CD4+ T cell response against this epitope is readily induced in most, if not all, HLA-DR1+ patients. This antiviral T cell population becomes functionally impaired or is deleted early in the course of disease in those where viremia persists.
Assuntos
Linfócitos T CD4-Positivos/virologia , Hepacivirus/isolamento & purificação , Hepatite C/imunologia , Linfócitos T Auxiliares-Indutores/virologia , Doença Aguda , Sequência de Aminoácidos , Sequência de Bases , Primers do DNA , Epitopos de Linfócito T/imunologia , Feminino , Genótipo , Antígeno HLA-DR1/química , Antígeno HLA-DR1/imunologia , Hepacivirus/genética , Humanos , Imunidade Celular , Fígado/imunologia , Fígado/virologia , Masculino , Fragmentos de Peptídeos/química , Fragmentos de Peptídeos/imunologia , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Proteínas Virais/análise , Proteínas Virais/química , Proteínas Virais/imunologiaRESUMO
The IL-10-like cytokine IL-22 is produced by activated T cells. In this study, we analyzed the role of this cytokine system in hepatic cells. Expression studies were performed by RT-PCR and quantitative PCR. Signal transduction was analyzed by Western blot experiments and ELISA. Cell proliferation was measured by MTS and [(3)H]thymidine incorporation assays. Hepatocyte regeneration was studied in in vitro restitution assays. Binding of IL-22 to its receptor complex expressed on human hepatic cells and primary human hepatocytes resulted in the activation of MAPKs, Akt, and STAT proteins. IL-22 stimulated cell proliferation and migration, which were both significantly inhibited by the phosphatidylinositol 3-kinase inhibitor wortmannin. IL-22 increased the mRNA expression of suppressor of cytokine signaling (SOCS)-3 and the proinflammatory cytokines IL-6, IL-8, and TNF-alpha. SOCS-1/3 overexpression abrogated IL-22-induced STAT activation and decreased IL-22-mediated liver cell regeneration. Hepatic IL-22 mRNA expression was detectable in different forms of human hepatitis, and hepatic IL-22 mRNA levels were increased in murine T cell-mediated hepatitis in vivo following cytomegalovirus infection, whereas no significant differences were seen in an in vivo model of ischemia-reperfusion injury. In conclusion, IL-22 promotes liver cell regeneration by increasing hepatic cell proliferation and hepatocyte migration through the activation of Akt and STAT signaling, which is abrogated by SOCS-1/3 overexpression.
Assuntos
Hepatócitos/metabolismo , Interleucinas/metabolismo , Regeneração Hepática , Fígado/metabolismo , Transdução de Sinais , Proteínas Supressoras da Sinalização de Citocina/metabolismo , Animais , Técnicas de Cultura de Células , Linhagem Celular Tumoral , Movimento Celular , Proliferação de Células , Modelos Animais de Doenças , Relação Dose-Resposta a Droga , MAP Quinases Reguladas por Sinal Extracelular/metabolismo , Hepatectomia , Hepatite/metabolismo , Hepatócitos/efeitos dos fármacos , Humanos , Mediadores da Inflamação/metabolismo , Interleucinas/farmacologia , Fígado/citologia , Fígado/cirurgia , Ativação Linfocitária , Camundongos , Camundongos Endogâmicos C57BL , Fosfatidilinositol 3-Quinases/metabolismo , Fosforilação , Proteínas Proto-Oncogênicas c-akt/metabolismo , RNA Mensageiro/metabolismo , Receptores de Interleucina/genética , Receptores de Interleucina/metabolismo , Fatores de Transcrição STAT/metabolismo , Transdução de Sinais/efeitos dos fármacos , Proteína 1 Supressora da Sinalização de Citocina , Proteína 3 Supressora da Sinalização de Citocinas , Proteínas Supressoras da Sinalização de Citocina/genética , Linfócitos T/metabolismo , Fatores de Tempo , Transfecção , Regulação para Cima , Interleucina 22RESUMO
BACKGROUND: The peptide hormone relaxin has been demonstrated to exert antifibrotic effects in renal and extrarenal tissues. The aims of this study were to identify potential anti-fibrotic effects of relaxin on human renal fibroblasts in vitro and to analyze their mechanisms. METHODS: All experiments were performed in established renal fibroblast cell lines and in primary cortical fibroblasts. Effects of relaxin were analyzed on cell proliferation, apoptosis, activation of renal fibroblasts, synthesis and secretion of collagen type I and fibronectin, as well as on the secretion of matrix metalloproteinases (MMPs). Effects on transforming growth factor-beta1 (TGF-beta1) receptor binding were analyzed by flow cytometry and on TGF-beta1 signal transduction by immunoblot analyses for Smad4 and 7, translocation from cytosol to nucleus for Smad2 and 3 as well as for phosphorylated and unphosphorylated forms of p38, c-Jun NH2 terminal kinase (JNK) and extracellular-regulated protein kinase (ERK). Finally, specific siRNAs for Smad2 and 3 were applied to assess the signal transduction pathway. RESULTS: After stimulation with relaxin, tyrosine phosphorylation of a 220 kD protein was demonstrated, indicating interaction with the receptor. Relaxin had only modest inhibitory effects on cell proliferation, and no effects on apoptosis. Conversely, relaxin exerted robust effects on TGF-beta1-induced fibroblast to myofibroblast transformation as well as on matrix synthesis and secretion even at the smallest dose tested. The secretion of MMP-2 and MMP-9 was induced noticeably by all investigated relaxin concentrations. TGF-beta1 receptor binding was not influenced by relaxin; however, it prevented Smad2 phosphorylation, translocation to nucleus, and complex formation between Smad2 and 3 indicating a possible interaction with TGF-beta1 signaling. These findings were corroborated by studies using siRNAs to Smad2 and 3 where siRNA to Smad2 but not to Smad3 inhibited the TGF-beta1 induction of fibronectin synthesis. There was no influence of relaxin on intracellular Smad3, Smad4, and Smad7 translocation or phosphorylation of mitogen-activated protein (MAP) kinases. CONCLUSION: Relaxin is a potent inhibitor of TGF-beta1-induced extracellular matrix (ECM) synthesis and secretion as well as fibroblast activation. Furthermore, it induces ECM degradation by induction of MMP-2 and MMP-9. These effects are mediated, at least in part, by inhibition of TGF-beta1 signaling.
Assuntos
Proteínas de Ligação a DNA/antagonistas & inibidores , Fibroblastos/efeitos dos fármacos , Rim/patologia , Sistema de Sinalização das MAP Quinases/efeitos dos fármacos , Relaxina/farmacologia , Transativadores/antagonistas & inibidores , Apoptose/efeitos dos fármacos , Divisão Celular/efeitos dos fármacos , Linhagem Celular , Proteínas de Ligação a DNA/metabolismo , Matriz Extracelular/metabolismo , Matriz Extracelular/patologia , Fibroblastos/metabolismo , Fibroblastos/patologia , Fibrose , Humanos , Metaloproteinase 2 da Matriz/metabolismo , Metaloproteinase 9 da Matriz/metabolismo , Necrose , Fosforilação/efeitos dos fármacos , Receptores de Fatores de Crescimento Transformadores beta/metabolismo , Proteína Smad2 , Proteína Smad3 , Transativadores/metabolismo , Fator de Crescimento Transformador beta/farmacologia , Fator de Crescimento Transformador beta1 , Tirosina/metabolismoRESUMO
Chronic evolution of acute hepatitis C (aHC) occurs in more than 80% of patients but can frequently be prevented by early treatment with interferon (IFN)-alpha. Plasmacytoid dendritic cells (pDCs) are the major endogenous IFN-alpha producers, but their role in aHC is unknown. In this study, frequency, phenotype, and pDC function were analyzed in 13 patients with aHC and 32 patients with chronic hepatitis C (cHC) compared with 20 healthy controls, 33 sustained responders to antiviral treatment, 14 patients with acute hepatitis B (aHB), and 21 patients with nonviral inflammatory disease. In aHC, pDCs in the peripheral blood were significantly reduced compared with healthy controls (median, 0.1% vs. 0.36%, P < .0005) and were inversely correlated to alanine aminotransferase levels (r = -0.823; P < .005). Circulating pDCs in aHC were immature, as determined via reduced expression of HLA-DR and CCR7, and produced little amounts of IFN-alpha (median, 3.5 pg/50,000 peripheral blood mononuclear cells [PBMCs] vs. 498.4 pg/50,000 PBMCs in healthy controls; P < .0005). Less pronounced changes were present in cHC (median, 0.17%, 28.0 pg/50,000 PBMCs IFN-alpha, respectively). However, a significantly reduced frequency and IFN-alpha production was also found in self-limited aHB (median 0.1%, 8.6 pg/50,000 PBMCs) and in patients with nonviral inflammatory disease (median 0.19%, 7.5 pg/50,000 PBMCs). In conclusion, in aHC frequency and IFN-alpha-producing capacity of peripheral blood pDCs are dramatically reduced and inversely correlated with the degree of liver inflammation. In cHC there is incomplete recovery of pDC function, which, however, could be solely due to the chronic inflammatory state.
Assuntos
Células Dendríticas/fisiologia , Hepatite C Crônica/imunologia , Hepatite C/imunologia , Interferon-alfa/biossíntese , Doença Aguda , Adulto , Idoso , Feminino , Antígenos HLA-DR/análise , Humanos , Masculino , Pessoa de Meia-Idade , Receptores CCR7 , Receptores de Quimiocinas/análiseRESUMO
Induction of cytochrome P450 3A (CYP3A) has been suggested as a mechanism of action of ursodeoxycholic acid (UDCA) in cholestasis. CYP3A is of key importance in human drug metabolism, being involved in presystemic extraction of more than 50% of all drugs currently available and of various endogenous compounds. Therefore, we compared the induction potential of UDCA with that of the prototypical inducer rifampicin in a human model study with the CYP3A substrates budesonide and cortisol. Twelve patients with early-stage primary biliary cirrhosis and eight healthy volunteers were treated with UDCA (15 mg/kg daily) for 3 weeks and subsequently with rifampicin (600 mg/d) for 1 week. Extensive pharmacokinetic profiling of oral budesonide (3 mg) was performed by determination of budesonide and phase I metabolites (6beta-hydroxybudesonide, 16alpha-hydroxyprednisolone) in plasma and urine at baseline and at the end of each treatment. In parallel, urinary 6beta-hydroxycortisol, a validated marker of CYP3A induction, was determined. UDCA did not affect biotransformation of budesonide and urinary excretion of 6beta-hydroxycortisol either in patients or in healthy volunteers. Ratios of areas under plasma concentration-time curves (AUC(0-12 h) during UDCA/AUC(0-12 h) before UDCA) of both metabolites were not higher than those of budesonide itself. In contrast, administration of rifampicin markedly induced CYP3A metabolism, resulting in abolished budesonide plasma levels and high urinary excretion of 6beta-hydroxycortisol. Metabolite formation was enhanced by rifampicin, but not by UDCA (e.g., AUC(16alpha-hydroxyprednisolone)/AUC(budesonide) in patients: baseline, 8.6 +/- 3.9; UDCA, 10.7 +/- 7.1; rifampicin, 527.0 +/- 248.7). In conclusion, UDCA is not a relevant inducer of CYP3A enzymes in humans.