RESUMO
A longstanding goal has been to find an antigen-specific preventive therapy, i.e., a vaccine, for autoimmune diseases. It has been difficult to find safe ways to steer the targeting of natural regulatory antigen. Here, we show that the administration of exogenous mouse major histocompatibility complex class II protein bounding a unique galactosylated collagen type II (COL2) peptide (Aq-galCOL2) directly interacts with the antigen-specific TCR through a positively charged tag. This leads to expanding a VISTA-positive nonconventional regulatory T cells, resulting in a potent dominant suppressive effect and protection against arthritis in mice. The therapeutic effect is dominant and tissue specific as the suppression can be transferred with regulatory T cells, which downregulate various autoimmune arthritis models including antibody-induced arthritis. Thus, the tolerogenic approach described here may be a promising dominant antigen-specific therapy for rheumatoid arthritis, and in principle, for autoimmune diseases in general.
Assuntos
Artrite Reumatoide , Doenças Autoimunes , Animais , Camundongos , Vacinas de Subunidades Antigênicas , Linfócitos T Reguladores , AnticorposRESUMO
Animal models for inflammatory arthritides such as rheumatoid arthritis (RA) and psoriatic arthritis are widely accepted and frequently used to identify pathological mechanisms and validate novel therapeutic strategies. Unfortunately, many publications reporting on these animal studies lack detailed description and appropriate assessment of the distinct histopathological features of arthritis: joint inflammation, cartilage damage and bone erosion. Therefore, the European consortium BeTheCure, consisting of 38 academic and industrial partners from 15 countries, set as goal to standardise the histological evaluation of joint sections from animal models of inflammatory arthritis. The consensual approach of a task force including 16 academic and industrial scientists as well as laboratory technicians has resulted in the development of the Standardised Microscopic Arthritis Scoring of Histological sections ('SMASH') recommendations for a standardised processing and microscopic scoring of the characteristic histopathological features of arthritis, exemplified by four different rodent models for arthritis: murine collagen-induced arthritis, collagen-antibody-induced arthritis, human tumour necrosis factor transgenic Tg197 mice and rat pristane-induced arthritis, applicable to any other inflammatory arthritis model. Through standardisation, the SMASH recommendations are designed to improve and maximise the information derived from in vivo arthritis experiments and to promote reproducibility and transparent reporting on such studies. In this manuscript, we will discuss and provide recommendations for analysis of histological joint sections: identification of the regions of interest, sample preparation, staining procedures and quantitative scoring methods. In conclusion, awareness of the different features of the arthritis pathology in animal models of inflammatory arthritis is of utmost importance for reliable research outcome, and the standardised histological processing and scoring methods in these SMASH recommendations will help increase uniformity and reproducibility in preclinical research on inflammatory arthritis.
Assuntos
Artrite Experimental , Artrite Reumatoide , Animais , Artrite Experimental/patologia , Artrite Reumatoide/patologia , Modelos Animais de Doenças , Humanos , Camundongos , Camundongos Transgênicos , Ratos , Reprodutibilidade dos TestesRESUMO
Rheumatoid Arthritis (RA) represents a chronic T cell-mediated inflammatory autoimmune disease. Studies have shown that epigenetic mechanisms contribute to the pathogenesis of RA. Histone deacetylases (HDACs) represent one important group of epigenetic regulators. However, the role of individual HDAC members for the pathogenesis of arthritis is still unknown. In this study we demonstrate that mice with a T cell-specific deletion of HDAC1 (HDAC1-cKO) are resistant to the development of Collagen-induced arthritis (CIA), whereas the antibody response to collagen type II was undisturbed, indicating an unaltered T cell-mediated B cell activation. The inflammatory cytokines IL-17 and IL-6 were significantly decreased in sera of HDAC1-cKO mice. IL-6 treated HDAC1-deficient CD4+ T cells showed an impaired upregulation of CCR6. Selective inhibition of class I HDACs with the HDAC inhibitor MS-275 under Th17-skewing conditions inhibited the upregulation of chemokine receptor 6 (CCR6) in mouse and human CD4+ T cells. Accordingly, analysis of human RNA-sequencing (RNA-seq) data and histological analysis of synovial tissue samples from human RA patients revealed the existence of CD4+CCR6+ cells with enhanced HDAC1 expression. Our data indicate a key role for HDAC1 for the pathogenesis of CIA and suggest that HDAC1 and other class I HDACs might be promising targets of selective HDAC inhibitors (HDACi) for the treatment of RA.
Assuntos
Artrite Reumatoide/etiologia , Artrite Reumatoide/metabolismo , Suscetibilidade a Doenças , Histona Desacetilase 1/metabolismo , Linfócitos T/imunologia , Linfócitos T/metabolismo , Animais , Artrite Reumatoide/patologia , Biomarcadores , Colágeno/efeitos adversos , Citocinas/metabolismo , Modelos Animais de Doenças , Regulação da Expressão Gênica , Histona Desacetilase 1/genética , Humanos , Mediadores da Inflamação/metabolismo , Camundongos , Camundongos Knockout , Subpopulações de Linfócitos T/imunologia , Subpopulações de Linfócitos T/metabolismoRESUMO
Rheumatoid arthritis (RA) is associated with amino acid variants in multiple MHC molecules. The association to MHC class II (MHC-II) has been studied in several animal models of RA. In most cases these models depend on T cells restricted to a single immunodominant peptide of the immunizing Ag, which does not resemble the autoreactive T cells in RA. An exception is pristane-induced arthritis (PIA) in the rat where polyclonal T cells induce chronic arthritis after being primed against endogenous Ags. In this study, we used a mixed genetic and functional approach to show that RT1-Ba and RT1-Bb (RT1-B locus), the rat orthologs of HLA-DQA and HLA-DQB, determine the onset and severity of PIA. We isolated a 0.2-Mb interval within the MHC-II locus of three MHC-congenic strains, of which two were protected from severe PIA. Comparison of sequence and expression variation, as well as in vivo blocking of RT1-B and RT1-D (HLA-DR), showed that arthritis in these strains is regulated by coding polymorphisms in the RT1-B genes. Motif prediction based on MHC-II eluted peptides and structural homology modeling suggested that variants in the RT1-B P1 pocket, which likely affect the editing capacity by RT1-DM, are important for the development of PIA.
Assuntos
Artrite Experimental/genética , Artrite Reumatoide/genética , Predisposição Genética para Doença , Antígenos de Histocompatibilidade/genética , Sequência de Aminoácidos , Aminoácidos/genética , Animais , Anticorpos Bloqueadores/imunologia , Anticorpos Bloqueadores/farmacologia , Artrite Experimental/imunologia , Artrite Reumatoide/imunologia , Sítios de Ligação/genética , Peso Corporal/efeitos dos fármacos , Peso Corporal/imunologia , Modelos Animais de Doenças , Genótipo , Haplótipos/imunologia , Antígenos de Histocompatibilidade/química , Antígenos de Histocompatibilidade/imunologia , Humanos , Modelos Moleculares , Dados de Sequência Molecular , Polimorfismo Genético/imunologia , Estrutura Terciária de Proteína , Ratos , Índice de Gravidade de Doença , Terpenos/imunologiaRESUMO
Pemphigus vulgaris (PV) is considered as a model for an autoantibody-mediated organ-specific autoimmune disorder. IgG autoantibodies directed against the desmosomal cadherin desmoglein 3 (Dsg3), the major autoantigen in PV, cause loss of epidermal keratinocyte adhesion, resulting in blisters and erosions of the skin and mucous membranes. The association of human autoimmune diseases with distinct HLA alleles is a well-known phenomenon, such as the association with HLA-DRB1*04:02 in PV. However, direct evidence that HLA-DRB1*04:02-restricted autoreactive CD4(+) T cells recognizing immunodominant epitopes of Dsg3 initiate the production of Dsg3-reactive IgG autoantibodies is still missing. In this study, we show in a humanized HLA-DRB1*04:02-transgenic mouse model that HLA-DRB1*04:02-restricted T cell recognition of human Dsg3 epitopes leads to the induction of pathogenic IgG Abs that induce loss of epidermal adhesion, a hallmark in the immune pathogenesis of PV. Activation of Dsg3-reactive CD4(+) T cells by distinct human Dsg3 peptides that bind to HLA-DRß1*04:02 is tightly regulated by the HLA-DRB1*04:02 allele and leads, via CD40-CD40L-dependent T cell-B cell interaction, to the production of IgG Abs that recognize both N- and COOH-terminal epitopes of the human Dsg3 ectodomain. These findings demonstrate key cellular and humoral immune events in the autoimmune cascade of PV in a humanized HLA-transgenic mouse model. We show that CD4(+) T cells recognizing immunodominant Dsg3 epitopes in the context of the PV-associated HLA-DRB1*04:02 induce the secretion of Dsg3-specific IgG in vivo. Finally, these results identify Dsg3-reactive CD4(+) T cells as potential therapeutic targets in the future.
Assuntos
Autoanticorpos/imunologia , Autoantígenos/imunologia , Desmogleína 3/imunologia , Cadeias HLA-DRB1/imunologia , Imunoglobulina G/imunologia , Pênfigo/imunologia , Linfócitos T/imunologia , Linfócitos T/metabolismo , Sequência de Aminoácidos , Animais , Especificidade de Anticorpos/imunologia , Linfócitos B/imunologia , Linfócitos B/metabolismo , Comunicação Celular/imunologia , Células Dendríticas/imunologia , Células Dendríticas/metabolismo , Modelos Animais de Doenças , Epitopos/química , Epitopos/imunologia , Cadeias HLA-DRB1/genética , Humanos , Imunização , Camundongos , Camundongos Transgênicos , Pênfigo/genética , Peptídeos/química , Peptídeos/imunologia , Ligação Proteica , Pele/imunologia , Pele/metabolismo , Pele/patologia , Subpopulações de Linfócitos T/imunologia , Subpopulações de Linfócitos T/metabolismoRESUMO
INTRODUCTION: Collagen-induced arthritis (CIA) has traditionally been performed in MHC class II A(q)-expressing mice, whereas most genetically modified mice are on the C57BL/6 background (expressing the b haplotype of the major histocompatibility complex (MHC) class II region). However, C57BL/6 mice develop arthritis after immunisation with chicken-derived collagen type II (CII), but arthritis susceptibility has been variable, and the immune specificity has not been clarified. OBJECTIVE: To establish a CIA model on the C57BL/6 background with a more predictable and defined immune response to CII. RESULTS: Both chicken and rat CII were arthritogenic in C57BL/6 mice provided they were introduced with high doses of Mycobacterium tuberculosis adjuvant. However, contaminating pepsin was strongly immunogenic and was essential for arthritis development. H-2(b)-restricted T cell epitopes on chicken or rat CII could not be identified, but expression of A(q) on the C57BL/6 background induced T cell response to the CII260-270 epitope, and also prolonged the arthritis to be more chronic. CONCLUSIONS: The putative (auto)antigen and its arthritogenic determinants in C57BL/6 mice remains undisclosed, questioning the value of the model for addressing T cell-driven pathological pathways in arthritis. To circumvent this impediment, we recommend MHC class II congenic C57BL/6N.Q mice, expressing A(q), with which T cell determinants have been thoroughly characterised.
Assuntos
Artrite Experimental/genética , Artrite Reumatoide/genética , Colágeno Tipo II/imunologia , Modelos Animais de Doenças , Genes MHC da Classe II/genética , Linfócitos T/imunologia , Animais , Artrite Experimental/imunologia , Artrite Reumatoide/imunologia , Galinhas , Epitopos de Linfócito T/imunologia , Haplótipos , Imunização , Camundongos , Camundongos Congênicos , Camundongos Endogâmicos C57BL , Camundongos Endogâmicos , Mycobacterium/imunologia , RatosRESUMO
OBJECTIVE: Type II collagen (CII) is a cartilage-specific protein to which a loss of immune tolerance may trigger autoimmune reactions and cause arthritis. The major T cell epitope on CII, amino acids 259-273, can be presented by several HLA-DRB1 04 alleles in its native or posttranslational glycosylated form. The present study was undertaken to functionally explore and compare CII-autoreactive T cells from blood and synovial fluid of patients with rheumatoid arthritis (RA). METHODS: Peripheral blood was obtained from HLA-DRB1 04-positive RA patients (n = 10) and control subjects (n = 10) and stimulated in vitro with several variants of the CII(259-273) epitope, i.e., unmodified, glycosylated on Lys-264, glycosylated on Lys-270, or glycosylated on both Lys-264 and Lys-270. Up-regulation of CD154 was used to identify responding T cells. These cells were further characterized by intracellular staining for interleukin-17 (IL-17), interferon-γ (IFNγ), and IL-2 by flow cytometry. Synovial T cells from RA patients were investigated in parallel. RESULTS: Multifunctional T cell responses toward all examined variants of the CII(259-273) peptide could be detected in RA patients and, to a lesser extent, also in healthy HLA-matched controls (P < 0.001). In RA patients, a comparison between blood- and joint-derived T cell function revealed a significant increase in levels of the proinflammatory cytokine IFNγ in synovial T cells (P = 0.027). Studies of longitudinally obtained samples showed that T cell responses were sustained over the course of disease, and even included epitope spreading. CONCLUSION: The identification of inflammatory T cell responses to both glycosylated and nonglycosylated variants of the major CII epitope in RA patients suggests that CII autoreactivity in RA may be more common than previously recognized.
Assuntos
Artrite Reumatoide/imunologia , Artrite Reumatoide/patologia , Colágeno Tipo II/imunologia , Epitopos de Linfócito T/imunologia , Linfócitos T/imunologia , Linfócitos T/patologia , Artrite Reumatoide/metabolismo , Células Sanguíneas/patologia , Estudos de Casos e Controles , Progressão da Doença , Glicosilação , Cadeias HLA-DRB1/metabolismo , Humanos , Interferon gama/metabolismo , Interleucina-17/metabolismo , Interleucina-2/metabolismo , Estudos Longitudinais , Líquido Sinovial/citologia , Linfócitos T/metabolismoRESUMO
OBJECTIVE: To establish a new animal model in DRB1*0401 (DR4)-transgenic mice in which T cell tolerance to self type II collagen (CII) can be broken and allow for the development of autoimmune arthritis, to investigate the role of posttranslational modifications of the CII(259-273) epitope in the induction and breaking of tolerance of DR4-restricted T cells, and to characterize DR4-restricted T cell recognition of the immunodominant CII(259-273) epitope. METHODS: DR4-transgenic mice expressing either the entire human CII protein (HuCII) or only the immunodominant T cell epitope of heterologous CII (MMC) in joint cartilage were established on different genetic backgrounds, and susceptibility to collagen-induced arthritis (CIA) was tested. RESULTS: HuCII mice displayed stronger T cell tolerance to heterologous CII than did MMC mice. On the B10 background, arthritis developed only in MMC mice with a defective oxidative burst. However, MMC mice on the C3H background were susceptible to arthritis also with a functional oxidative burst. Significant recall responses in tolerized mice were detected only against the nonglycosylated CII(259-273) epitope. Recognition of the CII(259-273) epitope was heterogeneous, but the majority of T cells in DR4 mice specifically recognized the nonglycosylated side chain of lysine at position 264. CONCLUSION: It is possible to break tolerance to self CII and induce arthritis in DR4 mice. However, arthritis susceptibility is tightly controlled by the genetic background and by the source of the transgenic element for expressing the heterologous CII peptide as a self CII protein in the joint. In contrast to CIA in A(q)-expressing mice, the nonglycosylated CII(259-273) epitope is clearly immunodominant in both tolerized and nontolerized DR4 mice.
Assuntos
Artrite Experimental/imunologia , Colágeno Tipo II/imunologia , Antígeno HLA-DR4/imunologia , Tolerância a Antígenos Próprios/imunologia , Linfócitos T/imunologia , Animais , Artrite Experimental/genética , Feminino , Predisposição Genética para Doença , Antígeno HLA-DR4/genética , Humanos , Masculino , Camundongos , Camundongos Transgênicos , Ratos , Tolerância a Antígenos Próprios/genéticaRESUMO
Reactive oxygen species (ROS) are important in the immune defense against invading pathogens, but they are also key molecules in the regulation of inflammatory reactions. Low levels of ROS production due to a polymorphism in the neutrophil cytosolic factor 1 (Ncf1) gene are associated with autoimmunity and arthritis severity in mouse models induced with adjuvant. We established an adjuvant-free arthritis model in which disease is induced by injection of the autoantigen collagen type II (CII) and depends on IL-5-producing T cells and eosinophils. In addition, the transgenic expression of mutated mouse CII allowed us to investigate an autoreactive immune response to an autologous Ag and by that natural tolerance mechanism. We show that a deficient ROS production, due to a spontaneous mutation in Ncf1, leads to increased autoantibody production and expansion of IL-33R-expressing T cells, impaired T cell tolerance toward tissue-specific CII, and severe arthritis in this unique model without disturbing adjuvant effects. These results demonstrate that the insufficient production of ROS promotes the breakdown of immune tolerance and development of autoimmune and adjuvant-free arthritis through an IL-5- and IL33R-dependent T cell activation pathway.
Assuntos
Artrite Experimental/etiologia , Interleucina-5/metabolismo , NADPH Oxidases/genética , Receptores de Interleucina-1/metabolismo , Receptores de Interleucina/metabolismo , Explosão Respiratória/fisiologia , Linfócitos T/patologia , Animais , Artrite Experimental/induzido quimicamente , Artrite Experimental/imunologia , Colágeno Tipo II/administração & dosagem , Colágeno Tipo II/efeitos adversos , Proteína 1 Semelhante a Receptor de Interleucina-1 , Interleucina-33 , Interleucinas , Camundongos , Camundongos Endogâmicos DBA , Camundongos Knockout , NADPH Oxidases/fisiologia , Espécies Reativas de Oxigênio/metabolismo , Linfócitos T/imunologiaRESUMO
The introduction of the CTLA-4 recombinant fusion protein has demonstrated therapeutic effects by selectively modulating T-cell activation in rheumatoid arthritis. Here we show, using a forward genetic approach, that a mutation in the SH3gl1 gene encoding the endocytic protein Endophilin A2 is associated with the development of arthritis in rodents. Defective expression of SH3gl1 affects T cell effector functions and alters the activation threshold of autoreactive T cells, thereby leading to complete protection from chronic autoimmune inflammatory disease in both mice and rats. We further show that SH3GL1 regulates human T cell signaling and T cell receptor internalization, and its expression is upregulated in rheumatoid arthritis patients. Collectively our data identify SH3GL1 as a key regulator of T cell activation, and as a potential target for treatment of autoimmune diseases.
Assuntos
Aciltransferases/deficiência , Artrite Reumatoide/enzimologia , Artrite Reumatoide/imunologia , Doenças Autoimunes/enzimologia , Doenças Autoimunes/imunologia , Ativação Linfocitária/imunologia , Linfócitos T/imunologia , Aciltransferases/genética , Aciltransferases/metabolismo , Animais , Artrite Reumatoide/prevenção & controle , Autoimunidade , Endocitose , Feminino , Humanos , Células Jurkat , Linfonodos/metabolismo , Linfonodos/patologia , Masculino , Camundongos , Mutação/genética , Ratos , Receptores de Antígenos de Linfócitos T/metabolismo , Transdução de Sinais , Regulação para Cima/genéticaRESUMO
Establishing effective central tolerance requires the promiscuous expression of tissue-restricted antigens by medullary thymic epithelial cells. However, whether central tolerance also extends to post-translationally modified proteins is not clear. Here we show a mouse model of autoimmunity in which disease development is dependent on post-translational modification (PTM) of the tissue-restricted self-antigen collagen type II. T cells specific for the non-modified antigen undergo efficient central tolerance. By contrast, PTM-reactive T cells escape thymic selection, though the PTM variant constitutes the dominant form in the periphery. This finding implies that the PTM protein is absent in the thymus, or present at concentrations insufficient to induce negative selection of developing thymocytes and explains the lower level of tolerance induction against the PTM antigen. As the majority of self-antigens are post-translationally modified, these data raise the possibility that T cells specific for other self-antigens naturally subjected to PTM may escape central tolerance induction by a similar mechanism.
Assuntos
Artrite Experimental/imunologia , Tolerância Central/imunologia , Colágeno Tipo II/imunologia , Processamento de Proteína Pós-Traducional/imunologia , Linfócitos T/imunologia , Animais , Autoantígenos/imunologia , Autoimunidade/imunologia , Modelos Animais de Doenças , Camundongos , Camundongos Transgênicos , Timócitos/imunologia , Timo/imunologiaRESUMO
OBJECTIVE: To develop a new chronic rheumatoid arthritis model that is driven by the innate immune system. METHODS: Injection of a cocktail of 4 monoclonal antibodies against type II collagen, followed on days 5 and 60 by intraperitoneal injections of mannan (from Saccharomyces cerevisiae), was used to induce development of chronic arthritis in B10.Q mice. The role of the innate immune system as compared to the adaptive immune system in this arthritis model was investigated using genetically modified mouse strains. RESULTS: A new model of chronic relapsing arthritis was characterized in B10.Q mice, in which a persistently active, chronic disease was found. This relapsing disease was driven by macrophages lacking the ability to mount a reactive oxygen species response against pathogens, and was associated with the classical/alternative pathway, but not the lectin pathway, of complement activation. The disease was independent of Fcγ receptor type III, and also independent of the activity of adaptive immune cells (B and T cells), indicating that the innate immune system, involving complement activation, could be the sole driver of chronicity. CONCLUSION: Chronic active arthritis can be driven innately by macrophages without the involvement of T and B cells in the adaptive immune system.
Assuntos
Artrite Experimental/imunologia , Artrite Reumatoide/imunologia , Imunidade Inata/imunologia , Macrófagos/imunologia , Linfócitos T/imunologia , Animais , Anticorpos Monoclonais/imunologia , Linfócitos B/imunologia , Colágeno Tipo II/imunologia , Modelos Animais de Doenças , CamundongosRESUMO
BACKGROUND: To facilitate the development of therapies for rheumatoid arthritis (RA), the Innovative Medicines Initiative BTCure has combined the experience from several laboratories worldwide to establish a series of protocols for different animal models of arthritis that reflect the pathogenesis of RA. Here, we describe chronic pristane-induced arthritis (PIA) model in DA rats, and provide detailed instructions to set up and evaluate the model and for reporting data. METHODS: We optimized dose of pristane and immunization procedures and determined the effect of age, gender, and housing conditions. We further assessed cage-effects, reproducibility, and frequency of chronic arthritis, disease markers, and efficacy of standard and novel therapies. RESULTS: Out of 271 rats, 99.6% developed arthritis after pristane-administration. Mean values for day of onset, day of maximum arthritis severity and maximum clinical scores were 11.8±2.0 days, 20.3±5.1 days and 34.2±11 points on a 60-point scale, respectively. The mean frequency of chronic arthritis was 86% but approached 100% in long-term experiments over 110 days. Pristane was arthritogenic even at 5 microliters dose but needed to be administrated intradermally to induce robust disease with minimal variation. The development of arthritis was age-dependent but independent of gender and whether the rats were housed in conventional or barrier facilities. PIA correlated well with weight loss and acute phase reactants, and was ameliorated by etanercept, dexamethasone, cyclosporine A and fingolimod treatment. CONCLUSIONS: PIA has high incidence and excellent reproducibility. The chronic relapsing-remitting disease and limited systemic manifestations make it more suitable than adjuvant arthritis for long-term studies of joint-inflammation and screening and validation of new therapeutics.
Assuntos
Artrite Experimental/induzido quimicamente , Artrite Experimental/patologia , Artrite Reumatoide/induzido quimicamente , Artrite Reumatoide/patologia , Modelos Animais de Doenças , Imunossupressores/toxicidade , Terpenos/toxicidade , Animais , Feminino , Masculino , RatosRESUMO
The most widely used model for rheumatoid arthritis is the collagen-induced arthritis (CIA) in mice. This model has gained acceptance since it is reproducible, well defined and has proven useful for development of new therapies for rheumatoid arthritis, as exemplified by the most recent advancement using TNFalpha neutralization treatment. The collagen-induced arthritis model, however, represents only certain pathways leading to arthritis and there is no consensus on how they operate. Nevertheless, we are beginning to understand the immune recognition structures, such as MHC molecules, lymphocyte receptors and type II collagen epitopes, which are of crucial importance for the development of this disease. These provide useful tools for further investigations of the pathogenesis of CIA as well as for understanding the pathogenesis of rheumatoid arthritis.
Assuntos
Artrite Reumatoide/induzido quimicamente , Artrite Reumatoide/genética , Artrite/induzido quimicamente , Artrite/genética , Colágeno , Animais , Artrite/fisiopatologia , Artrite Reumatoide/fisiopatologia , Modelos Animais de Doenças , Genes MHC da Classe II/genética , Genes MHC da Classe II/fisiologia , Humanos , Camundongos , Linfócitos T/fisiologiaRESUMO
INTRODUCTION: Immune responses against collagen type II (CII) are crucial for the development of collagen-induced arthritis (CIA). The aim of the present study was to evaluate and compare the CII-directed T cell and antibody specificity at different time points in the course of CIA using two mouse strains on the B10 genetic background - B10.Q, expressing Aq MHC class II molecules, and B10.DR4.Ncf1*/*, expressing human rheumatoid arthritis-associated MHC II DR4 molecules (DRA*0101/DRB*0401). METHODS: B10.Q and B10.DR4.Ncf1*/* mice were immunized with CII emulsified in adjuvant and development of CIA was assessed. T cells from draining lymph nodes were restimulated in vitro with CII peptides and interferon-gamma (IFN-γ) levels in culture supernatants were evaluated by ELISA. CII-specific antibody levels in serum samples were measured by ELISA. RESULTS: At four different CIA time points we analyzed T cell specificity to the immunodominant CII epitope 259-273 (CII259-273) and several posttranslationally modified forms of CII259-273 as well as antibody responses to three B cell immunodominant epitopes on CII (C1, U1, J1). Our data show that CII-specific T and B cell responses increase dramatically after disease onset in both strains and are sustained during the disease course. Concerning anti-CII antibody fine specificity, during all investigated stages of CIA the B10.Q mice responded predominantly to the C1 epitope, whereas the B10.DR4.Ncf1*/* mice also recognized the U1 epitope. In the established disease phase, T cell reactivity toward the galactosylated CII259-273 peptide was similar between the DR4- and the Aq-expressing strains whereas the response to the non-modified CII peptide was dramatically enhanced in the DR4 mice compared with the B10.Q. In addition, we show that the difference in the transgenic DR4-restricted T cell specificity to CII259-273 is not dependent on the degree of glycosylation of the collagen used for immunization. CONCLUSIONS: The present study provides important evaluation of CII-specific immune responses at different phases during CIA development as well as a comparative analysis between two CIA mouse models. We indicate significant differences in CII T cell and antibody specificities between the two strains and highlight a need for improved humanized B10.DR4 mouse model for rheumatoid arthritis.
Assuntos
Especificidade de Anticorpos/imunologia , Artrite Experimental/imunologia , Colágeno Tipo II/imunologia , Linfócitos T/imunologia , Animais , Formação de Anticorpos/imunologia , Ensaio de Imunoadsorção Enzimática , Epitopos de Linfócito T/imunologia , Feminino , Humanos , Interferon gama/imunologia , Interferon gama/metabolismo , Masculino , Camundongos Transgênicos , Especificidade da Espécie , Linfócitos T/metabolismo , Fatores de TempoRESUMO
INTRODUCTION: The Vß12-transgenic mouse was previously generated to investigate the role of antigen-specific T cells in collagen-induced arthritis (CIA), an animal model for rheumatoid arthritis. This mouse expresses a transgenic collagen type II (CII)-specific T-cell receptor (TCR) ß-chain and consequently displays an increased immunity to CII and increased susceptibility to CIA. However, while the transgenic Vß12 chain recombines with endogenous α-chains, the frequency and distribution of CII-specific T cells in the Vß12-transgenic mouse has not been determined. The aim of the present report was to establish a system enabling identification of CII-specific T cells in the Vß12-transgenic mouse in order to determine to what extent the transgenic expression of the CII-specific ß-chain would skew the response towards the immunodominant galactosylated T-cell epitope and to use this system to monitor these cells throughout development of CIA. METHODS: We have generated and thoroughly characterized a clonotypic antibody, which recognizes a TCR specific for the galactosylated CII(260-270) peptide in the Vß12-transgenic mouse. Hereby, CII-specific T cells could be quantified and followed throughout development of CIA, and their phenotype was determined by combinatorial analysis with the early activation marker CD154 (CD40L) and production of cytokines. RESULTS: The Vß12-transgenic mouse expresses several related but distinct T-cell clones specific for the galactosylated CII peptide. The clonotypic antibody could specifically recognize the majority (80%) of these. Clonotypic T cells occurred at low levels in the naïve mouse, but rapidly expanded to around 4% of the CD4+ T cells, whereupon the frequency declined with developing disease. Analysis of the cytokine profile revealed an early Th1-biased response in the draining lymph nodes that would shift to also include Th17 around the onset of arthritis. Data showed that Th1 and Th17 constitute a minority among the CII-specific population, however, indicating that additional subpopulations of antigen-specific T cells regulate the development of CIA. CONCLUSIONS: The established system enables the detection and detailed phenotyping of T cells specific for the galactosylated CII peptide and constitutes a powerful tool for analysis of the importance of these cells and their effector functions throughout the different phases of arthritis.
Assuntos
Anticorpos Monoclonais/imunologia , Artrite Experimental/imunologia , Colágeno Tipo II/genética , Imunofenotipagem/métodos , Receptores de Antígenos de Linfócitos T alfa-beta/genética , Animais , Especificidade de Anticorpos/imunologia , Artrite Experimental/metabolismo , Colágeno Tipo II/imunologia , Citocinas/imunologia , Citocinas/metabolismo , Epitopos de Linfócito T/imunologia , Citometria de Fluxo , Galactose/imunologia , Galactose/metabolismo , Hibridomas , Camundongos , Camundongos Endogâmicos DBA , Camundongos Transgênicos , Ratos , Receptores de Antígenos de Linfócitos T alfa-beta/imunologia , Proteínas Recombinantes/genética , Proteínas Recombinantes/imunologia , Linfócitos T/citologia , Linfócitos T/imunologia , Linfócitos T/metabolismoRESUMO
OBJECTIVE: To develop a new mouse model for arthritis using cartilage oligomeric matrix protein (COMP) and to study the role of major histocompatibility complex (MHC) and Ncf1 genes in COMP-induced arthritis (COMPIA). METHODS: Native (pentameric) and denatured (monomeric) COMP purified from a rat chondrosarcoma was injected into mice with Freund's adjuvant to induce arthritis. C3H.NB, C3H.Q, B10.P, B10.Q, (B10.Q x DBA/1)F1, (BALB/c x B10.Q)F1, Ncf1 mutated, H-2Aq, H-2Ap, and human DR4+-transgenic mice were used. Anti-COMP antibodies and COMP levels in the immune sera were analyzed, and passive transfer of arthritis with purified immune sera was tested. RESULTS: Immunization with rat COMP induced a severe, chronic, relapsing arthritis, with a female preponderance, in the mice. The disease developed in C3H.NB mice, but not in B10.P mice, although they share the same MHC haplotype. Both H-2q and H-2p MHC haplotypes allowed the initiation of COMPIA. Using H-2Aq-transgenic and H-2Ap-transgenic mice, we demonstrated a role of both the Aq and Ep class II molecules in this model. Interestingly, the introduction of a mutation in the Ncf1 gene, which is responsible for the reduced oxidative burst phenotype, into the COMPIA-resistant B10.Q mouse strain rendered them highly susceptible to arthritis. In addition, the transfer of anti-COMP serum was found to induce arthritis in naive mice. Mice transgenic for the rheumatoid arthritis (RA)-associated DR4 molecule were found to be highly susceptible to COMPIA. CONCLUSION: Using rat COMP, we have developed a new and unique mouse model of chronic arthritis that resembles RA. This model will be useful as an appropriate and alternative model for studying the pathogenesis of RA.
Assuntos
Artrite/imunologia , Proteínas da Matriz Extracelular/imunologia , Glicoproteínas/imunologia , Complexo Principal de Histocompatibilidade/genética , NADPH Oxidases/metabolismo , Animais , Anticorpos/sangue , Artrite/sangue , Artrite/induzido quimicamente , Proteína de Matriz Oligomérica de Cartilagem , Modelos Animais de Doenças , Proteínas da Matriz Extracelular/efeitos adversos , Proteínas da Matriz Extracelular/sangue , Feminino , Glicoproteínas/efeitos adversos , Glicoproteínas/sangue , Humanos , Proteínas Matrilinas , Camundongos , RatosRESUMO
The view on reactive oxygen species (ROS) in inflammation is currently shifting from being considered damaging toward having a more complex role in regulating inflammatory reactions. We recently demonstrated a role of ROS in regulation of animal models for the autoimmune disease rheumatoid arthritis. Low levels of ROS production, due to a mutation in the Ncf1 gene coding for the Ncf1 (alias p47(phox)) subunit of the NADPH oxidase complex, was shown to be associated with increased autoimmunity and arthritis severity in both rats and mice. To further investigate the role of ROS in autoimmunity, we studied transgenic mice expressing collagen type II (CII) with a mutation (D266E) in the immunodominant epitope that mimics the rat and human CII (i.e., mutated mouse collagen or MMC). This mutation results in a stronger binding of the epitope to the MHC class II molecule and leads to more pronounced tolerance and resistance to arthritis induced with rat CII. When the Ncf1 mutation was bred into these mice, tolerance was broken, resulting in enhanced T cell autoreactivity, high titers of anti-CII Abs, and development of severe arthritis. These findings highlight the importance of a sufficient ROS production in maintenance of tolerance to self-Ags, a central mechanism in autoimmune diseases such as rheumatoid arthritis. This is important as we, for the first time, can follow the effect of ROS on molecular mechanisms where T cells are responsible for either protection or promotion of arthritis depending on the level of oxygen species produced.
Assuntos
Artrite Experimental/imunologia , Artrite Experimental/metabolismo , Colágeno Tipo II/imunologia , Regulação para Baixo , Tolerância Imunológica , Espécies Reativas de Oxigênio , Linfócitos T/imunologia , Linfócitos T/metabolismo , Animais , Artrite Experimental/genética , Colágeno Tipo II/administração & dosagem , Colágeno Tipo II/genética , Regulação para Baixo/genética , Predisposição Genética para Doença , Tolerância Imunológica/genética , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Mutantes , Camundongos Transgênicos , Ratos , Espécies Reativas de Oxigênio/metabolismoRESUMO
Collagen induced arthritis (CIA) is the most studied animal model for rheumatoid arthritis and is associated with the MHC class II molecule Aq. T-cell recognition of a peptide from type II collagen, CII256-270, bound to Aq is a requirement for development of CIA. Lysine 264 is the major T-cell recognition site of CII256-270 and CIA is in particular associated with recognition of lysine 264 after posttranslational hydroxylation and subsequent attachment of a beta-D-galactopyranosyl moiety. In this paper we have studied the structural requirements of collagenous glycopeptides required for T-cell stimulation, as an extension of earlier studies of the recognition of the galactose moiety. Synthesis and evaluation of alanine substituted glycopeptides revealed that there are T-cells that only recognise the galactosylated hydroxylysine 264, and no other amino acid side chains in the peptide. Other T-cells also require glutamic acid 266 as a T-cell contact point. Introduction of a methylene ether isostere instead of the amide bond between residues 260 and 261 allowed weaker recognition by some, but not all, of the T-cells. Altogether, these results allowed us to propose a model for glycopeptide recognition by the T-cells, where recognition from one or the other side of the galactose moiety could explain the different binding patterns of the T-cells.