RESUMO
Glycosylation of antibodies, particularly in the Fc domain, critically modulate the ability of antibodies to bind to FcRs, maintaining immune quiescence to achieve a finely orchestrated immune response. The removal of sialic acid and galactose residues dramatically alters the physiological function of IgGs, and alterations of Ig glycosylation have been associated with several autoimmune disorders. However, Ig glycosylation has not been extensively studied in autoimmune cholangitis. We applied triple quadruple mass spectroscopy with subsequent multiple reaction monitoring to elucidate the profile, composition and linkage of sugar residues of antibody glycans in patients with primary biliary cholangitis (PBC), primary sclerosing cholangitis (PSC) and healthy controls (HC). Agalactosylated, HexNAc terminated IgG1 glycoforms were enriched in both PBC and PSC. Levels of IgM glycans at site N439 and fucosylated glycans in J chain, were significantly decreased in PBC compared to PSC and HC. PSC patients had decreased bisecting glycoforms and increased biantennary glycoforms on IgA compared to PBC. Importantly, our data demonstrate the association of distinct branching and composition patterns of Ig glycoforms with disease severity and liver cirrhosis, which highlight the importance of glycan biology as a potential mechanism and/or a disease specific signal of inflammation.
Assuntos
Autoanticorpos/metabolismo , Doenças Autoimunes/diagnóstico , Colangite Esclerosante/diagnóstico , Imunoglobulina G/metabolismo , Cirrose Hepática Biliar/diagnóstico , Adulto , Idoso , Autoanticorpos/sangue , Autoanticorpos/imunologia , Doenças Autoimunes/sangue , Doenças Autoimunes/imunologia , Biomarcadores/sangue , Biomarcadores/metabolismo , Estudos de Casos e Controles , Colangite Esclerosante/sangue , Colangite Esclerosante/imunologia , Feminino , Glicômica/métodos , Glicosilação , Voluntários Saudáveis , Humanos , Imunoglobulina G/sangue , Imunoglobulina G/imunologia , Cirrose Hepática Biliar/sangue , Cirrose Hepática Biliar/imunologia , Masculino , Pessoa de Meia-Idade , Polissacarídeos/imunologia , Polissacarídeos/metabolismo , Índice de Gravidade de DoençaRESUMO
Primary biliary cholangitis (PBC) is a classic autoimmune disease in which humoral, cytotoxic, and innate immune responses have been implicated with the specific targeting of a mitochondrial antigen. The mainstay of treatment remains the bile acid ursodeoxycholic acid (UDCA). Corticosteroids may have some benefits, but to date, clinical trials of biologics targeting B cells and IL-12/23 have not shown any efficacy. Because activated T cells target the intrahepatic bile ducts in PBC and pre-clinical models suggested that blocking CD80/CD86 with CTLA-4 Ig might have therapeutic benefit in PBC, we performed an open-label trial to determine if CTLA-4 Ig (abatacept) is safe and potentially efficacious in PBC patients with an incomplete response to UDCA. PBC patients with an alkaline phosphatase (ALP) > 1.67 × the upper limit of normal after 6 months on UDCA treatment or who were intolerant of UDCA received abatacept 125 mg s.q. weekly for 24 weeks. The co-primary endpoint was ALP normalization or a >40% reduction from baseline. Among 16 subjects enrolled and who received at least 1 dose of abatacept, 1 (6.3%) met the co-primary endpoint. Absolute and percent changes in ALP [median (95% CI)] were +2.8 U/L (-90.9-96.6) and -0.28% (-21.1-15.5), respectively. No significant changes were observed in ALP, ALT, total bilirubin, albumin, immunoglobulins, or liver stiffness. Abatacept treatment decreased several non-terminally differentiated CD4+ but not CD8+ T cell populations, including decreases in CD4+ CCR5+ (p = 0.02) and CD4+ PD1+ (p = 0.03) lymphocytes. In contrast there were increases in CD4+ CCR7+ lymphocytes (p = 0.034). Treatment emergent adverse events occurred in 4 subjects. Abatacept was well tolerated in this population of PBC patients but like other biologics in PBC was ineffective in achieving biochemical responses associated with improved clinical outcomes.
Assuntos
Produtos Biológicos/uso terapêutico , Cirrose Hepática Biliar/tratamento farmacológico , Abatacepte/administração & dosagem , Abatacepte/efeitos adversos , Abatacepte/uso terapêutico , Adulto , Produtos Biológicos/administração & dosagem , Produtos Biológicos/efeitos adversos , Biomarcadores , Ensaios Clínicos como Assunto , Suscetibilidade a Doenças , Feminino , Humanos , Imunossupressores/administração & dosagem , Imunossupressores/efeitos adversos , Imunossupressores/uso terapêutico , Mediadores da Inflamação/metabolismo , Cirrose Hepática Biliar/diagnóstico , Cirrose Hepática Biliar/etiologia , Cirrose Hepática Biliar/metabolismo , Masculino , Pessoa de Meia-Idade , Terapia de Alvo Molecular/efeitos adversos , Terapia de Alvo Molecular/métodosRESUMO
We have reported on a murine model of autoimmune cholangitis, generated by altering the AU-rich element (ARE) by deletion of the interferon gamma (IFN-γ) 3' untranslated region (coined ARE-Del-/- ), that has striking similarities to human primary biliary cholangitis (PBC) with female predominance. Previously, we suggested that the sex bias of autoimmune cholangitis was secondary to intense and sustained type I and II IFN signaling. Based on this thesis, and to define the mechanisms that lead to portal inflammation, we specifically addressed the hypothesis that type I IFNs are the driver of this disease. To accomplish these goals, we crossed ARE-Del-/- mice with IFN type I receptor alpha chain (Ifnar1) knockout mice. We report herein that loss of type I IFN receptor signaling in the double construct of ARE-Del-/- Ifnar1-/- mice dramatically reduces liver pathology and abrogated sex bias. More importantly, female ARE-Del-/- mice have an increased number of germinal center (GC) B cells as well as abnormal follicular formation, sites which have been implicated in loss of tolerance. Deletion of type I IFN signaling in ARE-Del-/- Ifnar1-/- mice corrects these GC abnormalities, including abnormal follicular structure. CONCLUSION: Our data implicate type I IFN signaling as a necessary component of the sex bias of this murine model of autoimmune cholangitis. Importantly these data suggest that drugs that target the type I IFN signaling pathway would have potential benefit in the earlier stages of PBC. (Hepatology 2018;67:1408-1419).
Assuntos
Doenças Autoimunes/imunologia , Colangite/imunologia , Interferon Tipo I/genética , Fígado/patologia , Animais , Linfócitos B/imunologia , Modelos Animais de Doenças , Feminino , Citometria de Fluxo , Fígado/imunologia , Fígado/metabolismo , Masculino , Camundongos , Camundongos Knockout , Fatores Sexuais , Transdução de Sinais/imunologiaRESUMO
A major problem in autoimmunity has been identification of the earliest events that lead to breach of tolerance. Although there have been major advances in dissecting effector pathways and the multilineage immune responses to mitochondrial self-antigens in primary biliary cholangitis, the critical links between environmental factors and tolerance remain elusive. We hypothesized that environmental xenobiotic modification of the E2 subunit of the pyruvate dehydrogenase (PDC-E2) inner lipoyl domain can lead to loss of tolerance to genetically susceptible hosts. Previously we demonstrated that serum anti-PDC-E2 autoantibodies cross-react with the chemical xenobiotics 2-octynoic acid and 6,8-bis (acetylthio) octanoic acid and further that there is a high frequency of PDC-E2-specific peripheral plasmablasts. Herein we generated 104 recombinant monoclonal antibodies (mAbs) based on paired heavy-chain and light-chain variable regions of individual plasmablasts derived from primary biliary cholangitis patients. We identified 32 mAbs reactive with native PDC-E2, including 20 specific for PDC-E2 and 12 cross-reactive with both PDC-E2 and 2-octynoic acid and 6,8-bis (acetylthio) octanoic acid. A lower frequency of replacement somatic hypermutations, indicating a lower level of affinity maturation, was observed in the complementarity-determining regions of the cross-reactive mAbs in comparison to mAbs exclusively recognizing PDC-E2 or those for irrelevant antigens. In particular, when the highly mutated heavy-chain gene of a cross-reactive mAb was reverted to the germline sequence, the PDC-E2 reactivity was reduced dramatically, whereas the xenobiotic reactivity was retained. Importantly, cross-reactive mAbs also recognized lipoic acid, a mitochondrial fatty acid that is covalently bound to PDC-E2. CONCLUSION: Our data reflect that chemically modified lipoic acid or lipoic acid itself, through molecular mimicry, is the initial target that leads to the development of primary biliary cholangitis. (Hepatology 2017;66:885-895).
Assuntos
Anticorpos Monoclonais/imunologia , Autoantígenos/imunologia , Autoimunidade/genética , Colangite/imunologia , Colangite/patologia , Xenobióticos/imunologia , Anticorpos Monoclonais/metabolismo , Autoantígenos/genética , Autoimunidade/imunologia , Feminino , Amplificação de Genes , Humanos , Immunoblotting , Masculino , Mimetismo Molecular/genética , Reação em Cadeia da Polimerase em Tempo Real , Sensibilidade e Especificidade , Ácido Tióctico/imunologia , Ácido Tióctico/metabolismoRESUMO
UNLABELLED: In most autoimmune diseases the serologic hallmarks of disease precede clinical pathology by years. Therefore, the use of animal models in defining early disease events becomes critical. We took advantage of a "designer" mouse with dysregulation of interferon gamma (IFNγ) characterized by prolonged and chronic expression of IFNγ through deletion of the IFNγ 3'-untranslated region adenylate uridylate-rich element (ARE). The ARE-Del(-/-) mice develop primary biliary cholangitis (PBC) with a female predominance that mimics human PBC that is characterized by up-regulation of total bile acids, spontaneous production of anti-mitochondrial antibodies, and portal duct inflammation. Transfer of CD4 T cells from ARE-Del(-/-) to B6/Rag1(-/-) mice induced moderate portal inflammation and parenchymal inflammation, and RNA sequencing of liver gene expression revealed that up-regulated genes potentially define early stages of cholangitis. Interestingly, up-regulated genes specifically overlap with the gene expression signature of biliary epithelial cells in PBC, implying that IFNγ may play a pathogenic role in biliary epithelial cells in the initiation stage of PBC. Moreover, differentially expressed genes in female mice have stronger type 1 and type 2 IFN signaling and lymphocyte-mediated immune responses and thus may drive the female bias of the disease. CONCLUSION: Changes in IFNγ expression are critical for the pathogenesis of PBC. (Hepatology 2016;64:1189-1201).
Assuntos
Doenças Autoimunes/etiologia , Colangite/imunologia , Interferon gama/biossíntese , Animais , Doenças Autoimunes/metabolismo , Colangite/metabolismo , Feminino , Masculino , Camundongos , Fatores SexuaisRESUMO
BACKGROUND: Adenoviruses are important pathogens with the potential for interspecies transmission between humans and non-human primates. Although many adenoviruses have been identified in monkeys, the knowledge of these viruses from the Colobinae members is quite limited. FINDINGS: We conducted a surveillance of viral infection in endangered golden snub-nosed monkeys (Rhinopithecus roxellana) in the subfamily Colobinae in China, and found that 5.1% of sampled individuals were positive for adenovirus. One of the adenoviruses (SAdV-WIV19) was successfully isolated and its full-length genome was sequenced. The full-length genome of WIV19 is 33,562 bp in size, has a G + C content of 56.2%, and encodes 35 putative genes. Sequence analysis revealed that this virus represents a novel species in the genus Mastadenovirus. Diverse cell lines, including those of human origin, were susceptible to WIV19. CONCLUSION: We report the first time the isolation and full-length genomic characterization of an adenovirus from the subfamily Colobinae.
Assuntos
Infecções por Adenoviridae/veterinária , Adenoviridae/classificação , Adenoviridae/isolamento & purificação , Colobinae/virologia , Doenças dos Primatas/epidemiologia , Doenças dos Primatas/virologia , Adenoviridae/genética , Infecções por Adenoviridae/epidemiologia , Infecções por Adenoviridae/virologia , Animais , Composição de Bases , China/epidemiologia , Análise por Conglomerados , Ordem dos Genes , Genes Virais , Genoma Viral , Filogenia , Prevalência , Análise de Sequência de DNARESUMO
UNLABELLED: The interleukin (IL)-12/IL-23-mediated Th1/Th17 signaling pathway has been associated with the etiopathogenesis of primary biliary cirrhosis (PBC). To address the cytokine microenvironment specifically in the liver, we examined the localized expression of cytokine subunits and their corresponding receptors using previously optimized immunohistochemistry with an extensive panel of antibodies directed at IL-12p70, IL-12p35, interferon-gamma (IFN-γ), IL-12RB2, IL-23p40, IL-23p19, IL-17, and IL-23R using liver from PBC (n = 51) and non-PBC (n = 80) control liver disease patients. Multiple portal tracts in each patient were blindly evaluated and individually scored. We report herein that although IL-12/Th1 and IL-23/Th17 staining was detected in all of the liver sections, they were primarily localized around the damaged interlobular bile ducts in PBC. Most important, Th17 skewing was prominent in advanced PBC patients with intensive secretion of IL-23p19 by inflamed hepatocytes around IL-23R, IL-12RB2, and IFN-γ expressing degenerated cholangiocytes. Our novel finding on the direct association of Th17 skewing and disease severity illustrates the significance of the IL-23/Th17 pathway in the perpetuation of IL-12/Th1-mediated immunopathology in PBC. Furthermore, localized IL-23p19 production by hepatocytes may enhance profibrotic Th17 signaling and proinflammatory IFN-γ production that contribute to PBC pathology. CONCLUSION: Our data emphasize the pathogenic relevance of IL-12/Th1 and IL-23/Th17 in the evolution of PBC. Of significance, however, the shift from a Th1 to a Th17 imbalance at advanced stages of the disease suggests the necessity to consider modulation of the IL-23/Th17 pathway as a potential target for therapeutic intervention.
Assuntos
Interleucina-12/fisiologia , Interleucina-23/fisiologia , Cirrose Hepática Biliar/etiologia , Células Th1/fisiologia , Células Th17/fisiologia , Adulto , Idoso , Feminino , Humanos , Imuno-Histoquímica , Interferon gama/metabolismo , Cirrose Hepática Biliar/patologia , Cirrose Hepática Biliar/terapia , Masculino , Pessoa de Meia-Idade , Transdução de Sinais/fisiologiaRESUMO
UNLABELLED: The serologic hallmark of primary biliary cirrhosis (PBC), the antimitochondrial response to the E2 component of the pyruvate dehydrogenase complex (PDC-E2), has unique features, including continuous high titers of immunoglobulin M (IgM) and IgG reactivity throughout all stages of disease, capable not only of target enzyme inhibition, but also crossreactive with chemical xenobiotics that share molecular homology with the inner lipoyl domain of PDC-E2; such chemicals have been proposed as potential etiological agents. We used flow cytometry and enzyme-linked immunospot assay (ELISPOT) to examine B-cell subsets in 59 subjects, including 28 with PBC, 13 with primary sclerosing cholangitis (PSC), and 18 healthy controls. Strikingly, in PBC, although there were no significant differences in B-cell phenotype subpopulations, 10% of the total IgG and IgA plasmablast population and 23% of the IgM plasmablast population were uniquely reactive with PDC-E2, detected in the CXCR7+ CCR10low plasmablast population. In contrast, plasmablast reactivity to a control antigen, tetanus toxoid, was minimal and similar in all groups. Additionally, we isolated plasmablast-derived polyclonal antibodies and compared reactivity with plasma-derived antibodies and noted a distinct noncirculating tissue source of xenobiotic crossreacting antibodies. The high levels of autoantigen specific peripheral plasmablasts indicate recent activation of naive or memory B cells and a continuous and robust activation. The presence of CXCR7+ CCR10low PDC-E2-specific ASCs suggests a mechanistic basis for the migration of circulating antigen specific plasmablasts to the mucosal epithelial ligands CXCL12 and CCL28. CONCLUSION: Our findings suggest a sustained rigorous B-cell response in PBC, likely activated and perpetuated by cognate autoantigen.
Assuntos
Autoantígenos/imunologia , Linfócitos B/imunologia , Di-Hidrolipoil-Lisina-Resíduo Acetiltransferase/imunologia , Cirrose Hepática Biliar/imunologia , Adulto , Idoso , Idoso de 80 Anos ou mais , Especificidade de Anticorpos , Células Produtoras de Anticorpos/metabolismo , Estudos de Casos e Controles , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Receptores CCR10/metabolismo , Receptores CXCR/metabolismo , Adulto JovemRESUMO
Within the last decade, several mouse models that manifest characteristic features of primary biliary cirrhosis (PBC) with antimitochondrial antibodies (AMAs) and immune-mediated biliary duct pathology have been reported. Here, the authors discuss the current findings on two spontaneous (nonobese diabetic autoimmune biliary disease [NOD.ABD] and dominant negative transforming growth factor-ß receptor II [dnTGFßRII]) and two induced (chemical xenobiotics and microbial immunization) models of PBC. These models exhibit the serological, immunological, and histopathological features of human PBC. From these animal models, it is evident that the etiology of PBC is multifactorial and requires both specific genetic predispositions and environmental insults (either xenobiotic chemicals or microbial), which lead to the breaking of tolerance and eventually liver pathology. Human PBC is likely orchestrated by multiple factors and hence no single model can fully mimic the immunopathophysiology of human PBC. Nevertheless, knowledge gained from these models has greatly advanced our understanding of the major immunological pathways as well as the etiology of PBC.
Assuntos
Autoimunidade , Ductos Biliares Intra-Hepáticos/imunologia , Modelos Animais de Doenças , Cirrose Hepática Biliar/imunologia , Animais , Ductos Biliares Intra-Hepáticos/metabolismo , Ductos Biliares Intra-Hepáticos/microbiologia , Ductos Biliares Intra-Hepáticos/patologia , Biomarcadores/sangue , Escherichia coli/patogenicidade , Humanos , Cirrose Hepática Biliar/sangue , Cirrose Hepática Biliar/induzido quimicamente , Cirrose Hepática Biliar/genética , Cirrose Hepática Biliar/metabolismo , Cirrose Hepática Biliar/microbiologia , Cirrose Hepática Biliar/patologia , Camundongos Endogâmicos NOD , Camundongos Transgênicos , Proteínas Serina-Treonina Quinases/genética , Proteínas Serina-Treonina Quinases/metabolismo , Receptor do Fator de Crescimento Transformador beta Tipo II , Receptores de Fatores de Crescimento Transformadores beta/genética , Receptores de Fatores de Crescimento Transformadores beta/metabolismo , Transdução de Sinais , Sphingomonadaceae/patogenicidade , XenobióticosRESUMO
Primary biliary cirrhosis (PBC) is an enigmatic disease mediated by autoimmune destruction of cholangiocytes in hepatic bile ducts. The early immunological events leading to PBC are poorly understood; clinical signs of disease occur very late in the pathological process. We have used our unique murine model of PBC in dominant-negative TGF-ß receptor type II transgenic mice to delineate critical early immunopathological pathways, and previously showed that dnTGFßRII CD8 T cells transfer biliary disease. Herein we report significantly increased numbers of hepatic dnTGFßRII terminally differentiated (KLRG1(+)) CD8 T cells, a CD8 subset previously shown to be enriched in antigen specific cells during hepatic immune response to viral infections. We performed bone marrow chimera studies to assess whether dnTGFßRII CD8 mediated disease was cell intrinsic or extrinsic. Unexpectedly, mixed (dnTGFßRII and B6) bone marrow chimeric (BMC) mice were protected from biliary disease compared to dnTGFßRII single bone marrow chimerics. To define the protective B6 cell subset, we performed adoptive transfer studies, which showed that co-transfer of B6 Tregs prevented dnTGFßRII CD8 T cell mediated cholangitis. Treg mediated disease protection was associated with significantly decreased numbers of hepatic KLRG1(+) CD8 T cells. In contrast, co-transfer of dnTGFßRII Tregs offered no protection, and dnTGFßRII Treg cells were functionally defective in suppressing effector CD8 T cells in vitro compared to wild type B6 Tregs. In vitro cholangiocyte cytotoxicity assays demonstrated significantly increased numbers of cytotoxic hepatic dnTGFßRII KLRG1(+) CD8 cells compared to B6. Protection from disease by B6 Tregs was associated with elimination of hepatic dnTGFßRII CD8 mediated cholangiocyte cytotoxicity. These results emphasize that autoimmune cholangitis requires defects in both the T effector and regulatory compartments, and that an intrinsic T cell effector defect is not sufficient to mediate autoimmune biliary disease in the setting of intact immune regulation. These results have important implications for understanding the early pathogenesis of human PBC.
Assuntos
Linfócitos T CD8-Positivos/imunologia , Colangite/imunologia , Receptores Imunológicos/imunologia , Linfócitos T Reguladores/imunologia , Transferência Adotiva , Animais , Doenças Autoimunes , Medula Óssea/imunologia , Medula Óssea/patologia , Linfócitos T CD8-Positivos/patologia , Linfócitos T CD8-Positivos/transplante , Linhagem da Célula/imunologia , Quimera/genética , Quimera/imunologia , Colangite/genética , Colangite/patologia , Citotoxicidade Imunológica , Modelos Animais de Doenças , Feminino , Expressão Gênica , Humanos , Lectinas Tipo C , Cirrose Hepática Biliar/genética , Cirrose Hepática Biliar/imunologia , Cirrose Hepática Biliar/patologia , Camundongos , Camundongos Transgênicos , Receptores Imunológicos/genética , Receptores de Fatores de Crescimento Transformadores beta/genética , Receptores de Fatores de Crescimento Transformadores beta/imunologia , Linfócitos T Reguladores/patologia , Linfócitos T Reguladores/transplanteRESUMO
UNLABELLED: Collectively, the data in both humans and murine models of human primary biliary cirrhosis (PBC) suggest that activated T cells, particularly CD8 T cells, play a critical role in biliary cell destruction. Under physiological conditions, T-cell activation involves two critical signals that involve the major histocompatibility complex and a set of costimulatory molecules, which include a receptor on T cells termed cytotoxic T lymphocyte antigen 4 (CTLA-4). Germane to the studies reported herein, signaling by CTLA-4 has the potential to modulate costimulation and induce inhibitory signals. In this study, we have taken advantage of our well-defined murine model of PBC, in which mice are immunized with 2-octynoic acid coupled to bovine serum albumin (2OA-BSA), leading to the production of high-titer antimitochondrial autoantibodies (AMAs) and portal cellular infiltrates. To investigate the potential of CTLA-4-Ig (immunoglobulin) as an immunotherapeutic agent, we treated mice both before and after induction of autoimmune cholangitis. First, we demonstrate that CTLA-4-Ig treatment, begun 1 day before 2OA-BSA immunization, completely inhibits the manifestations of cholangitis, including AMA production, intrahepatic T-cell infiltrates, and bile duct damage. However, and more critically, treatment with CTLA-4-Ig, initiated after the development of autoimmune cholangitis in previously immunized mice, also resulted in significant therapeutic benefit, including reduced intrahepatic T-cell infiltrates and biliary cell damage, although AMA levels were not altered. CONCLUSION: These data suggest that an optimized regimen with CTLA-4-Ig has the potential to serve as an investigative therapeutic tool in patients with PBC.
Assuntos
Linfócitos T CD8-Positivos/imunologia , Imunoconjugados/uso terapêutico , Cirrose Hepática Biliar/tratamento farmacológico , Abatacepte , Animais , Colangite/tratamento farmacológico , Colangite/imunologia , Modelos Animais de Doenças , Ácidos Graxos Monoinsaturados , Feminino , Humanos , Cirrose Hepática Biliar/imunologia , Camundongos , Mitocôndrias/imunologiaRESUMO
UNLABELLED: There are several murine models described with features similar to human primary biliary cirrhosis (PBC). Among these models, the one which has the closest serologic features to PBC is a mouse with a T-cell-restricted expression of the dominant negative transforming growth factor ß receptor type II (dnTGFßRII). Our work has demonstrated that CD8(+) T cells from dnTGFßRII mice transfer autoimmune cholangitis to Rag1(-/-) recipients. However, it remained unclear whether the autoimmune cholangitis was secondary to an intrinsic function within CD8(+) T cells or due to the abnormal TGFßR environment within which CD8(+) T cells were generated. To address this mechanistic issue, we used our dnTGFßRII, OT-I/Rag1(-/-) , OT-II/Rag1(-/-) mice and in addition generated OT-I/dnTGFßRII/Rag1(-/-) , and OT-II/dnTGFßRII/Rag1(-/-) mice in which the entire T-cell repertoire was replaced with ovalbumin (OVA)-specific CD8(+) or CD4(+) T cells, respectively. Importantly, neither the parental OT-I/dnTGFßRII/Rag1(-/-) mice and/or OT-II/dnTGFßRII/Rag1(-/-) mice developed cholangitis. However, adoptive transfer demonstrated that only transfer of CD8(+) T cells from dnTGFßRII mice but not CD8(+) T cells from OT-I/Rag1(-/-) mice or from OT-I/dnTGFßRII/Rag1(-/-) mice transferred disease. These data were not secondary to an absence of CD4(+) T cell help since a combination of CD8(+) T cells from OT-I/dnTGFßRII/Rag1(-/-) and CD4(+) T cells from OT II/dnTGFßRII/Rag1(-/-) or CD8(+) T cells from OT-I/dnTGFßRII/Rag1(-/-) with CD4(+) T cells from OT-II/Rag1(-/-) mice failed to transfer disease. CONCLUSION: Defective TGFßRII signaling, in addition to clonal CD8(+) T cells that target biliary cells, are required for induction of autoimmune cholangitis.
Assuntos
Doenças Autoimunes/fisiopatologia , Linfócitos T CD8-Positivos/patologia , Colangite/fisiopatologia , Modelos Animais de Doenças , Proteínas Serina-Treonina Quinases/genética , Receptores de Fatores de Crescimento Transformadores beta/genética , Transferência Adotiva , Animais , Doenças Autoimunes/imunologia , Doenças Autoimunes/metabolismo , Linfócitos T CD8-Positivos/imunologia , Linfócitos T CD8-Positivos/metabolismo , Colangite/imunologia , Colangite/metabolismo , Feminino , Proteínas de Homeodomínio/genética , Proteínas de Homeodomínio/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Ovalbumina/metabolismo , Proteínas Serina-Treonina Quinases/deficiência , Proteínas Serina-Treonina Quinases/metabolismo , Receptor do Fator de Crescimento Transformador beta Tipo II , Receptores de Fatores de Crescimento Transformadores beta/deficiência , Receptores de Fatores de Crescimento Transformadores beta/metabolismo , Transdução de Sinais/fisiologia , Especificidade do Receptor de Antígeno de Linfócitos T/fisiologiaRESUMO
UNLABELLED: Antimitochondrial antibodies (AMAs) directed against the lipoyl domain of the E2 subunit of pyruvate dehydrogenase (PDC-E2) are detected in 95% of patients with primary biliary cirrhosis (PBC) and are present before the onset of clinical disease. The recent demonstration that AMAs recognize xenobiotic modified PDC-E2 with higher titers than native PDC-E2 raises the possibility that the earliest events involved in loss of tolerance are related to xenobiotic modification. We hypothesized that reactivity to such xenobiotics would be predominantly immunoglobulin M (IgM) and using sera from a large cohort of PBC patients and controls (n = 516), we examined in detail sera reactivity against either 6,8-bis(acetylthio) octanoic acid (SAc)-conjugated bovine serum albumin (BSA), recombinant PDC-E2 (rPDC-E2) or BSA alone. Further, we also defined the relative specificity to the SAc moiety using inhibition enzyme-linked immunosorbent assay (ELISA); SAc conjugate and rPDC-E2-specific affinity-purified antibodies were also examined for antigen specificity, isotype, and crossreactivity. Reactivity to SAc conjugates is predominantly IgM; such reactivity reflects a footprint of previous xenobiotic exposure. Indeed, this observation is supported by both direct binding, crossreactivity, and inhibition studies. In both early and late-stage PBC, the predominant Ig isotype to SAc is IgM, with titers higher with advanced stage disease. We also note that there was a higher level of IgM reactivity to SAc than to rPDC-E2 in early-stage versus late-stage PBC. Interestingly, this finding is particularly significant in light of the structural similarity between SAc and the reduced form of lipoic acid, a step which is similar to the normal physiological oxidation of lipoic acid. CONCLUSION: Specific modifications of the disulfide bond within the lipoic-acid-conjugated PDC-E2 moiety, i.e., by an electrophilic agent renders PDC-E2 immunogenic in a genetically susceptible host.
Assuntos
Anticorpos Anti-Idiotípicos/imunologia , Cirrose Hepática Biliar/etiologia , Cirrose Hepática Biliar/imunologia , Mitocôndrias/imunologia , Xenobióticos/efeitos adversos , Especificidade de Anticorpos , Autoantígenos/imunologia , Estudos de Casos e Controles , Colangite Esclerosante/sangue , Colangite Esclerosante/imunologia , Di-Hidrolipoil-Lisina-Resíduo Acetiltransferase/imunologia , Hepatite Autoimune/sangue , Hepatite Autoimune/imunologia , Humanos , Imunoglobulina M/sangue , Cirrose Hepática Biliar/sangue , Proteínas Mitocondriais/imunologia , Proteínas Recombinantes/imunologia , Soroalbumina Bovina/imunologiaRESUMO
Mice with a dominant-negative transforming growth factor ß receptor restricted to T cells (dnTGFßRII mice) develop an inflammatory biliary ductular disease that strongly resembles human primary biliary cirrhosis (PBC). Furthermore, deletion of the gene encoding interleukin (IL)-12p40 resulted in a strain (IL-12p40(-/-) dnTGFßRII) with dramatically reduced autoimmune cholangitis. To further investigate the role of the IL-12 cytokine family in dnTGFßRII autoimmune biliary disease, we deleted the gene encoding the IL-12p35 subunit from dnTGFßRII mice, resulting in an IL-12p35(-/-) dnTGFßRII strain which is deficient in two members of the IL-12 family, IL-12 and IL-35. In contrast to IL-12p40(-/-) mice, the IL-12p35(-/-) mice developed liver inflammation and bile duct damage with similar severity but delayed onset as the parental dnTGFßRII mice. The p35(-/-) mice also demonstrated a distinct cytokine profile characterized by a shift from a T-helper 1 (Th1) to a Th17 response. Strikingly, liver fibrosis was frequently observed in IL-12p35(-/-) mice. In conclusion, IL-12p35(-/-) dnTGFßRII mice, histologically and immunologically, reflect key features of PBC, providing a useful generic model to understand the immunopathology of human PBC.
Assuntos
Subunidade p35 da Interleucina-12/genética , Cirrose Hepática/patologia , Proteínas Serina-Treonina Quinases/genética , Receptores de Fatores de Crescimento Transformadores beta/genética , Animais , Modelos Animais de Doenças , Hepatite Animal/etiologia , Hepatite Animal/patologia , Interleucina-12/deficiência , Interleucina-12/fisiologia , Subunidade p35 da Interleucina-12/deficiência , Subunidade p40 da Interleucina-12/genética , Cirrose Hepática Biliar/genética , Cirrose Hepática Biliar/patologia , Camundongos , Receptor do Fator de Crescimento Transformador beta Tipo II , Células Th1/fisiologia , Células Th17/fisiologiaRESUMO
Dominant-negative TGF-ß receptor II (dnTGF-ßRII) mice spontaneously develop an autoimmune cholangitis resembling human primary biliary cirrhosis (PBC). Interestingly, the dominant-negative TGF-ß receptor is expressed by both CD4(+) and CD8(+) T cells and leads to greatly reduced (but not absent) TGF-ß signaling resulting in T cell intrinsic cell mediated autoimmunity. However, the mechanisms of the T cell dysregulation remain unclear. Recently it has been shown that TGF-ß signaling is intimately involved with miRNA biogenesis and control. Herein we show that lack of T cell TGF-ß signaling leads to down regulation of T cell miRNAs but up-regulation of the key inflammatory miRNA 21. Furthermore, the expression of miR-21 from hepatic effector CD8(+) T cells is significantly higher than in the same subsets isolated from spleen and mesenteric lymph nodes of the dnTGF-ßRII mice. Previous studies indicate that miR-21 increases the synthesis of IFN-γ and IL-17A by T cells and suppresses apoptosis via programmed cell death protein 4 (PDCD4). Data presented herein demonstrate that transfecting w.t. B6 T cell subsets with miR-21 resulted in up-regulation of the inflammatory cytokines TNF-α and IFN-γ, thus partly replicating the dnTGF-ßRII T cell phenotype. In conclusion, these data suggest miR-21 plays a critical role in the production of pro-inflammatory cytokines in dnTGF-ßRII mice, which could be a contributing factor for the development of the organ-specific autoimmune cholangitis and colitis in this murine model of human PBC.
Assuntos
Citocinas/imunologia , Mediadores da Inflamação/imunologia , MicroRNAs/imunologia , Proteínas Serina-Treonina Quinases/imunologia , Receptores de Fatores de Crescimento Transformadores beta/imunologia , Animais , Proteínas Reguladoras de Apoptose/imunologia , Proteínas Reguladoras de Apoptose/metabolismo , Doenças Autoimunes/genética , Doenças Autoimunes/imunologia , Doenças Autoimunes/metabolismo , Linfócitos T CD4-Positivos/imunologia , Linfócitos T CD4-Positivos/metabolismo , Linfócitos T CD8-Positivos/imunologia , Linfócitos T CD8-Positivos/metabolismo , Colangite/genética , Colangite/imunologia , Colangite/metabolismo , Citocinas/metabolismo , Citometria de Fluxo , Expressão Gênica/imunologia , Humanos , Mediadores da Inflamação/metabolismo , Interferon gama/imunologia , Interferon gama/metabolismo , Interleucina-17/imunologia , Interleucina-17/metabolismo , Fígado/imunologia , Fígado/metabolismo , Fígado/patologia , Cirrose Hepática Biliar/genética , Cirrose Hepática Biliar/imunologia , Cirrose Hepática Biliar/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , MicroRNAs/genética , Proteínas Serina-Treonina Quinases/genética , Proteínas Serina-Treonina Quinases/metabolismo , Proteínas de Ligação a RNA/imunologia , Proteínas de Ligação a RNA/metabolismo , Receptor do Fator de Crescimento Transformador beta Tipo II , Receptores de Fatores de Crescimento Transformadores beta/genética , Receptores de Fatores de Crescimento Transformadores beta/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase ReversaRESUMO
UNLABELLED: The aim of this study was to determine the safety and potential efficacy of B-cell depletion with the anti-CD20 monoclonal antibody rituximab in patients with primary biliary cirrhosis (PBC) and an incomplete response to ursodeoxycholic acid (UDCA). This open-label study enrolled six patients with PBC and incomplete responses to UDCA to be treated with 2 doses of 1000 mg rituximab separated by 2 weeks and followed for 52 weeks. The primary endpoints were safety and changes in B-cell function. Two patients received only 1 dose of rituximab, one due to activation of latent varicella and the other due to a viral upper respiratory infection. Serum levels of total IgG, IgM, and IgA as well as anti-mitochondrial autoantibodies (AMAs) IgA and IgM decreased significantly from baseline by 16 weeks and returned to baseline levels by 36 weeks. Stimulation of B cells with CpG produced significantly less IgM at 52 weeks after treatment compared with B cells at baseline. In addition, transient decreases in memory B-cell and T-cell frequencies and an increase in CD25(high) CD4(+) T cells were observed after treatment. These changes were associated with significant increases in mRNA levels of FoxP3 and transforming growth factor-ß (TGF-ß) and a decrease in tumor necrosis factor-α (TNF-α) in CD4(+) T cells. Notably, serum alkaline phosphatase levels were significantly reduced up to 36 weeks following rituximab treatment. CONCLUSION: These data suggest that depletion of B cells influences the induction, maintenance, and activation of both B and T cells and provides a potential mechanism for treatment of patients with PBC with an incomplete response to UDCA.
Assuntos
Anticorpos Monoclonais Murinos/uso terapêutico , Linfócitos B/efeitos dos fármacos , Fatores Imunológicos/uso terapêutico , Cirrose Hepática Biliar/tratamento farmacológico , Adulto , Fosfatase Alcalina/sangue , Anticorpos Monoclonais Murinos/farmacologia , Autoanticorpos/sangue , Contagem de Linfócito CD4 , Colagogos e Coleréticos/uso terapêutico , Feminino , Humanos , Fatores Imunológicos/farmacologia , Imunofenotipagem , Fígado/enzimologia , Cirrose Hepática Biliar/imunologia , Cirrose Hepática Biliar/metabolismo , Pessoa de Meia-Idade , RNA Mensageiro/metabolismo , Rituximab , Linfócitos T/metabolismo , Falha de Tratamento , Ácido Ursodesoxicólico/uso terapêuticoRESUMO
Dominant negative form of transforming growth factor beta receptor type II (dnTGFßRII) mice, expressing a dominant negative form of TGFß receptor II under control of the CD4 promoter, develop autoimmune colitis and cholangitis. Deficiency in interleukin (IL)-12p40 lead to a marked diminution of inflammation in both the colon and the liver. To distinguish whether IL-12p40 mediates protection by the IL-12 or IL-23 pathways, we generated an IL-23p19(-/-) dnTGFßRII strain deficient in IL-23, but not in IL-12; mice were longitudinally followed for changes in the natural history of disease and immune responses. Interestingly, IL-23p19(-/-) mice demonstrate dramatic improvement in their colitis, but no changes in biliary pathology; mice also manifest reduced T-helper (Th)17 cell populations and unchanged IFN-γ levels. We submit that the IL-12/Th1 pathway is essential for biliary disease pathogenesis, whereas the IL-23/Th17 pathway mediates colitis. To further assess the mechanism of the IL-23-mediated protection from colitis, we generated an IL-17A(-/-) dnTGFßRII strain deficient in IL-17, a major effector cytokine produced by IL-23-dependent Th17 cells. Deletion of the IL-17A gene did not affect the severity of either cholangitis or colitis, suggesting that the IL-23/Th17 pathway contributes to colon disease in an IL-17-independent manner. These results affirm that the IL-12/Th1 pathway is critical to biliary pathology in dnTGFßRII mice, whereas colitis is caused by a direct effect of IL-23.
Assuntos
Colangite/imunologia , Colite/imunologia , Interleucina-17/genética , Subunidade p19 da Interleucina-23/metabolismo , Receptores de Fatores de Crescimento Transformadores beta/metabolismo , Análise de Variância , Animais , Biomarcadores/sangue , Biópsia por Agulha , Colangite/genética , Colangite/fisiopatologia , Colite/genética , Colite/fisiopatologia , Citocinas/análise , Citocinas/sangue , Modelos Animais de Doenças , Progressão da Doença , Citometria de Fluxo , Deleção de Genes , Imuno-Histoquímica , Interleucina-17/imunologia , Interleucina-17/metabolismo , Subunidade p19 da Interleucina-23/genética , Subunidade p19 da Interleucina-23/imunologia , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Mutantes , Reação em Cadeia da Polimerase/métodos , Distribuição Aleatória , Receptores de Fatores de Crescimento Transformadores beta/genética , Receptores de Fatores de Crescimento Transformadores beta/imunologia , Sensibilidade e Especificidade , Estatísticas não ParamétricasRESUMO
We previously described the NOD.c3c4 mouse, which is protected from type 1 diabetes (T1D) because of protective alleles at multiple insulin-dependent diabetes (Idd) genes, but develops autoimmune biliary disease (ABD) resembling primary biliary cirrhosis (PBC). In this paper, we characterize the NOD.ABD strain, which is genetically related to the NOD.c3c4 strain but develops both ABD and T1D. Histologically, NOD.ABD biliary disease is indistinguishable from that in NOD.c3c4 mice. The frequency of effector memory (CD44(+)CD62L(-)) and central memory (CD44(+)CD62L(+)) CD8 T cells is significantly increased in the intrahepatic lymphocyte fraction of NOD.ABD mice, and NOD.ABD CD8 T cells produce more IFN-γ and TNF-α, compared with controls. NOD.ABD splenocytes can transfer ABD and T1D to NOD.c3c4 scid mice, but only T1D to NOD scid mice, suggesting that the genetic origin of the target organ and/or its innate immune cells is critical to disease pathogenesis. The disease transfer model, importantly, shows that biliary duct damage (characteristic of PBC) and inflammation precede biliary epithelial cell proliferation. Unlike T1D where both CD4 and CD8 T cells are required for disease transfer, purified NOD.ABD CD8 T cells can transfer liver inflammation into NOD.c3c4 scid recipients, and disease transfer is ameliorated by cotransferring T regulatory cells. Unlike NOD.c3c4 mice, NOD.ABD mice do not develop anti-nuclear or anti-Smith autoantibodies; however, NOD.ABD mice do develop the antipyruvate dehydrogenase Abs typical of human PBC. The NOD.ABD strain is a model of immune dysregulation affecting two organ systems, most likely by mechanisms that do not completely coincide.
Assuntos
Ductos Biliares/imunologia , Ductos Biliares/patologia , Linfócitos T CD8-Positivos/imunologia , Linfócitos T CD8-Positivos/patologia , Diabetes Mellitus Tipo 1/imunologia , Diabetes Mellitus Tipo 1/patologia , Cirrose Hepática Biliar/imunologia , Cirrose Hepática Biliar/patologia , Transferência Adotiva , Animais , Cruzamentos Genéticos , Diabetes Mellitus Tipo 1/genética , Modelos Animais de Doenças , Feminino , Humanos , Células K562 , Cirrose Hepática Biliar/genética , Masculino , Camundongos , Camundongos Congênicos , Camundongos Endogâmicos C57BL , Camundongos Endogâmicos NOD , Camundongos SCID , Especificidade de Órgãos/genética , Especificidade de Órgãos/imunologiaRESUMO
C-type lectin receptors are pattern recognition receptors that are critical for autoimmunity and the immune response. Mincle is a C-type lectin receptor expressed by a variety of antigen presenting cells including macrophages, neutrophils, dendritic cells and B cells; a variety of stimuli including stress are known to induce the expression of Mincle. Mincle is an FcRγ-associated activation receptor that senses damaged cells and upon ligation induces activated macrophages to produce inflammatory cytokines. Recently, while several studies have reported that Mincle plays an important role in macrophage responses to fungal infection its function on B cells remains to be defined. In efforts to elucidate the function of Mincle expressed by B cells, we studied the expression of Mincle on subsets of B cells and analyzed cytokines and synthesized immunoglobulin upon ligation of Mincle. The expression of Mincle on CD27-CD19(+) naïve B cells is significantly higher than CD27 + CD19(+) memory B cells. The stimulation of TLR9 ligand induced Mincle expression on B cells. Furthermore, co-stimulation of TLR9 and Mincle ligand reduced IgG and IgA production from B cells without a significant change in the inflammatory cytokines TNF-α, IL-6, IL-8 and IL-10. Our data identifies Mincle as a potentially critical player in human B cell responses.
Assuntos
Subpopulações de Linfócitos B/imunologia , Regulação da Expressão Gênica/imunologia , Lectinas Tipo C/imunologia , Leucócitos Mononucleares/imunologia , Receptores Imunológicos/imunologia , Adulto , Idoso , Antígenos CD19/genética , Antígenos CD19/imunologia , Subpopulações de Linfócitos B/citologia , Subpopulações de Linfócitos B/efeitos dos fármacos , Células Cultivadas , Feminino , Regulação da Expressão Gênica/efeitos dos fármacos , Glicolipídeos/farmacologia , Humanos , Imunoglobulina A , Imunoglobulina G/biossíntese , Imunoglobulina G/imunologia , Memória Imunológica/efeitos dos fármacos , Memória Imunológica/imunologia , Interleucina-10/biossíntese , Interleucina-6/biossíntese , Interleucina-8/biossíntese , Lectinas Tipo C/genética , Leucócitos Mononucleares/citologia , Leucócitos Mononucleares/efeitos dos fármacos , Lipopolissacarídeos/farmacologia , Masculino , Pessoa de Meia-Idade , Oligodesoxirribonucleotídeos/farmacologia , Ligação Proteica , Receptores Imunológicos/genética , Receptor Toll-Like 9/genética , Receptor Toll-Like 9/imunologia , Membro 7 da Superfamília de Receptores de Fatores de Necrose Tumoral/genética , Membro 7 da Superfamília de Receptores de Fatores de Necrose Tumoral/imunologia , Fator de Necrose Tumoral alfa/biossínteseRESUMO
UNLABELLED: Primary biliary cirrhosis (PBC) is considered a model autoimmune disease due to the clinical homogeneity of patients and the classic hallmark of antimitochondrial antibodies (AMAs). Indeed, the presence of AMAs represents the most highly directed and specific autoantibody in autoimmune diseases. However, the contribution of B cells to the pathogenesis of PBC is unclear. Therefore, although AMAs appear to interact with the biliary cell apotope and contribute to biliary pathology, there is no correlation of disease severity and titer of AMAs. The recent development of well-characterized monoclonal antibodies specific for the B cell populations, anti-CD20 and anti-CD79, and the development of a well-defined xenobiotic-induced model of autoimmune cholangitis prompted us to use these reagents and the model to address the contribution of B cells in the pathogenesis of murine PBC. Prior to the induction of autoimmune cholangitis, mice were treated with either anti-CD20, anti-CD79, or isotype-matched control monoclonal antibody and followed for B cell development, the appearance of AMAs, liver pathology, and cytokine production. Results of the studies reported herein show that the in vivo depletion of B cells using either anti-CD20 or anti-CD79 led to the development of a more severe form of cholangitis than observed in control mice, which is in contrast with results from several other autoimmune models that have documented an important therapeutic role of B cell-specific depletion. Anti-CD20/CD79-treated mice had increased liver T cell infiltrates and higher levels of proinflammatory cytokines. CONCLUSION: Our results reflect a novel disease-protective role of B cells in PBC and suggest that B cell depletion therapy in humans with PBC should be approached with caution.