RESUMO
CD4+ T cells recognising citrullinated self-epitopes presented by HLA-DRB1 bearing the shared susceptibility epitope (SE) are implicated in rheumatoid arthritis (RA). However, the underlying T cell receptor (TCR) determinants of epitope specificity towards distinct citrullinated peptide antigens, including vimentin-64cit59-71 and α-enolase-15cit10-22 remain unclear. Using HLA-DR4-tetramers, we examine the T cell repertoire in HLA-DR4 transgenic mice and observe biased TRAV6 TCR gene usage across these two citrullinated epitopes which matches with TCR bias previously observed towards the fibrinogen ß-74cit69-81 epitope. Moreover, shared TRAV26-1 gene usage is evident in four α-enolase-15cit10-22 reactive T cells in three human samples. Crystal structures of mouse TRAV6+ and human TRAV26-1+ TCR-HLA-DR4 complexes presenting vimentin-64cit59-71 and α-enolase-15cit10-22, respectively, show three-way interactions between the TCR, SE, citrulline, and the basis for the biased selection of TRAV genes. Position 2 of the citrullinated epitope is a key determinant underpinning TCR specificity. Accordingly, we provide a molecular basis of TCR specificity towards citrullinated epitopes.
Assuntos
Artrite Reumatoide , Linfócitos T CD4-Positivos , Antígeno HLA-DR4 , Camundongos Transgênicos , Vimentina , Humanos , Antígeno HLA-DR4/imunologia , Antígeno HLA-DR4/genética , Artrite Reumatoide/imunologia , Artrite Reumatoide/genética , Camundongos , Animais , Vimentina/imunologia , Vimentina/metabolismo , Vimentina/genética , Linfócitos T CD4-Positivos/imunologia , Citrulinação , Fosfopiruvato Hidratase/imunologia , Fosfopiruvato Hidratase/genética , Fosfopiruvato Hidratase/metabolismo , Epitopos de Linfócito T/imunologia , Citrulina/metabolismo , Citrulina/imunologia , Receptores de Antígenos de Linfócitos T/imunologia , Receptores de Antígenos de Linfócitos T/metabolismo , Epitopos/imunologia , Cristalografia por Raios X , Receptores de Antígenos de Linfócitos T alfa-beta/genética , Receptores de Antígenos de Linfócitos T alfa-beta/imunologia , Receptores de Antígenos de Linfócitos T alfa-beta/metabolismoRESUMO
Since entering the stage 25 years ago as a highly specific serological biomarker for rheumatoid arthritis, anti-citrullinated protein antibodies (ACPAs) have been a topic of extensive research. This hallmark B cell response arises years before disease onset, displays interpatient autoantigen variability, and is associated with poor clinical outcomes. Technological and scientific advances have revealed broad clonal diversity and intriguing features including high levels of somatic hypermutation, variable-domain N-linked glycosylation, hapten-like peptide interactions, and clone-specific multireactivity to citrullinated, carbamylated and acetylated epitopes. ACPAs have been found in different isotypes and subclasses, in both circulation and tissue, and are secreted by both plasmablasts and long-lived plasma cells. Notably, although some disease-promoting features have been reported, results now demonstrate that certain monoclonal ACPAs therapeutically block arthritis and inflammation in mouse models. A wealth of functional studies using patient-derived polyclonal and monoclonal antibodies have provided evidence for pathogenic and protective effects of ACPAs in the context of arthritis. To understand the roles of ACPAs, one needs to consider their immunological properties by incorporating different facets such as rheumatoid arthritis B cell biology, environmental triggers and chronic antigen exposure. The emerging picture points to a complex role of citrulline-reactive autoantibodies, in which the diversity and dynamics of antibody clones could determine clinical progression and manifestations.
Assuntos
Anticorpos Antiproteína Citrulinada , Artrite Reumatoide , Citrulina , Humanos , Artrite Reumatoide/imunologia , Anticorpos Antiproteína Citrulinada/imunologia , Anticorpos Antiproteína Citrulinada/sangue , Animais , Citrulina/imunologia , Autoanticorpos/imunologia , Autoanticorpos/sangue , Linfócitos B/imunologia , Autoantígenos/imunologia , Camundongos , Biomarcadores/sangueRESUMO
Dendritic cells (DCs) represent a heterogeneous family of immune cells that link innate and adaptive immunity and their activation is linked to metabolic changes that are essential to support their activity and function. Hence, targeting the metabolism of DCs represents an opportunity to modify the inflammatory and immune response. Among the natural matrices, Humulus lupulus (Hop) compounds have recently been shown to exhibit immunomodulatory and anti-inflammatory activity. This study aimed to evaluate the ability of specific Hop fractions to modulate DCs metabolism after stimulation with lipopolysaccharide (LPS) by an untargeted metabolomics approach and compare their effect with flavonol quercetin. Following liquid chromatography-based fractionation, three fractions (A, B, and C) were obtained and tested. Cytokine and gene expression were evaluated using ELISA and qPCR, respectively, while the untargeted metabolomics analysis was performed using a combined HILIC-HRMS and DI-FT-ICR approach. The HOP C fraction and quercetin could both reduce the production of several inflammatory cytokines such as IL-6, IL-1α, IL-1ß, and TNF, but differently from quercetin, the HOP C mechanism is independent of extracellular iron-sequestration and showed significant upregulation of the Nrf2/Nqo1 pathway and Ap-1 compared to quercetin. The untargeted analysis revealed the modulation of several key pathways linked to pro-inflammatory and glycolytic phenotypes. In particular, HOP C treatment could modulate the oxidative step of the pentose phosphate pathway (PPP) and reduce the inflammatory mediator succinate, citrulline, and purine-pyrimidine metabolism, differently from quercetin. These results highlight the potential anti-inflammatory mechanism of specific Hop-derived compounds in restoring the dysregulated metabolism in DCs, which can be used in preventive or adjuvant therapies to suppress the undesirable inflammatory response.
Assuntos
Citrulina/metabolismo , Células Dendríticas/metabolismo , Humulus/metabolismo , Inflamação/metabolismo , Pirimidinas/metabolismo , Quercetina/metabolismo , Ácido Succínico/metabolismo , Animais , Anti-Inflamatórios/imunologia , Anti-Inflamatórios/metabolismo , Medula Óssea/imunologia , Medula Óssea/metabolismo , Citrulina/imunologia , Células Dendríticas/imunologia , Modelos Animais de Doenças , Flavonoides , Humulus/imunologia , Inflamação/imunologia , Espectrometria de Massas/métodos , Metabolômica/métodos , Camundongos , Camundongos Endogâmicos C57BL , Extratos Vegetais/imunologia , Extratos Vegetais/metabolismo , Purinas , Pirimidinas/imunologia , Quercetina/imunologia , Ácido Succínico/imunologiaRESUMO
The citrullinated inter-alpha-trypsin inhibitor heavy chain 4 (cit-ITIH4) was identified as its blood level was associated with the arthritis score in peptide glucose-6-phosphate-isomerase-induced arthritis (pGIA) mice and the disease activity in patients with rheumatoid arthritis (RA). This study aimed to clarify its citrullination pathway and function as related to neutrophils. In pGIA-afflicted joints, ITIH4 and cit-ITIH4 levels were examined by immunohistochemistry (IHC), immunoprecipitation (IP) and Western blotting (WB), while peptidylarginine deiminase (PAD) expression was measured by reverse transcription-quantitative polymerase chain reaction (RT-qPCR), IHC and immunofluorescent methods. The pGIA mice received anti-lymphocyte antigen 6 complex locus G6D (Ly6G) antibodies to deplete neutrophils and the expression of cit-ITIH4 was investigated by WB. The amounts of ITIH4 and cit-ITIH4 in synovial fluid (SF) from RA and osteoarthritis (OA) patients were examined by I.P. and W.B. Recombinant ITIH4 and cit-ITIH4 were incubated with sera from healthy volunteers before its chemotactic ability and C5a level were evaluated using Boyden's chamber assay and enzyme-linked immunosorbent assay (ELISA). During peak arthritic phase, ITIH4 and cit-ITIH4 were increased in joints while PAD4 was over-expressed, especially in the infiltrating neutrophils of pGIA mice. Levels of cit-ITIH4 in plasma and joints significantly decreased upon neutrophil depletion. ITIH4 was specifically citrullinated in SF from RA patients compared with OA patients. Native ITIH4 inhibited neutrophilic migration and decreased C5a levels, while cit-ITIH4 increased its migration and C5a levels significantly. Cit-ITIH4 is generated mainly in inflamed joints by neutrophils via PAD4. Citrullination of ITIH4 may change its function to up-regulate neutrophilic migration by activating the complement cascade, exacerbating arthritis.
Assuntos
Artrite Experimental/imunologia , Artrite Reumatoide/imunologia , Movimento Celular/imunologia , Articulações/imunologia , Neutrófilos/imunologia , Proteínas Secretadas Inibidoras de Proteinases/imunologia , Adulto , Idoso , Animais , Artrite Experimental/metabolismo , Artrite Reumatoide/metabolismo , Citrulina/imunologia , Citrulina/metabolismo , Feminino , Humanos , Imuno-Histoquímica , Articulações/metabolismo , Masculino , Camundongos Endogâmicos DBA , Microscopia de Fluorescência , Pessoa de Meia-Idade , Neutrófilos/citologia , Proteínas Secretadas Inibidoras de Proteinases/metabolismo , Adulto JovemRESUMO
Individuals with high anti-citrullinated protein antibody (ACPA) titers have an increased risk of developing rheumatoid arthritis (RA). Although our knowledge of the generation and production of ACPAs has continuously advanced during the past decade, our understanding on the pathogenic mechanisms of how ACPAs interact with immune cells to trigger articular inflammation is relatively limited. Citrullination disorders drive the generation and maintenance of ACPAs, with profound clinical significance in patients with RA. The loss of tolerance to citrullinated proteins, however, is essential for ACPAs to exert their pathogenicity. N-linked glycosylation, cross-reactivity and the structural interactions of ACPAs with their citrullinated antigens further direct their biological functions. Although questions remain in the pathogenicity of ACPAs acting as agonists for a receptor-mediated response, immune complex (IC) formation, complement system activation, crystallizable fragment gamma receptor (FcγR) activation, cross-reactivity to joint cartilage and neutrophil extracellular trap (NET)-related mechanisms have all been suggested recently. This paper presents a critical review of the characteristics and possible biological effects and mechanisms of the immunopathogenesis of ACPAs in patients with RA.
Assuntos
Anticorpos Antiproteína Citrulinada/imunologia , Artrite Reumatoide/etiologia , Artrite Reumatoide/metabolismo , Suscetibilidade a Doenças , Anticorpos Antiproteína Citrulinada/metabolismo , Artrite Reumatoide/patologia , Artrite Reumatoide/terapia , Autoanticorpos/imunologia , Autoantígenos/imunologia , Autoimunidade , Biomarcadores , Citrulina/imunologia , Gerenciamento Clínico , Humanos , Terapia de Alvo MolecularRESUMO
We previously showed that anti-neutrophil extracellular trap (NET) rheumatoid arthritis (RA)-rmAbs derived from CD19+ B cells within RA human synovial tissues frequently react against NETs. In this study, we aimed to characterize the importance of affinity maturation via somatic hypermutation (SHM) within the Ig variable H (VH) and variable L (VL) chains and Fab-N-linked glycosylation in RA synovial B cell clones reactive to NETs and NET-derived Ags such as citrullinated histones. Selected anti-NET RA-rmAbs derived from synovial RA CD19+ B cells were subjected to overlap-PCR to generate germline (GL; VH and VL reverted into GL), hybrid clones (VH/VL region reverted into GL), and N-glycosylation mutants (NâQ) and analyzed for anti-NETs and citrullinated histones (cit-H2B) immunoreactivity. Anti-NET/cit-H2B immunoreactivity of selected RA-rmAbs was abrogated in the VH and VL GL counterpart. In RA B cell hybrid clone RA015/11.88 and RA056/11.23.2, NET and/or cit-H2B immunoreactivity was solely dependent on SHM in the IgVH region whereas RA B cell hybrid clone RA015/11.91 required affinity maturation of both VH and VL for efficient binding to cit-H2B. In 7/80 RA-rmAb, SHM resulted in ex novo N-glycosylation sites in VH and/or VL regions. Removal of Fab-linked glycans in RA056/11.23.2 in the N-mutant counterpart resulted in 90% reduction in immunoreactivity to cit-H2B. Thus, SHM in the IgVH and/or VL regions of RA synovial B cells is necessary for the immunoreactivity to NET-Ags. Fab-N-linked-glycosylation introduction sites are observed in a minority of anti-NET B cell clones but can strongly influence NET-Ag binding.
Assuntos
Antígenos/imunologia , Artrite Reumatoide/imunologia , Linfócitos B/imunologia , Armadilhas Extracelulares/imunologia , Região Variável de Imunoglobulina/imunologia , Neutrófilos/imunologia , Animais , Anticorpos Monoclonais/imunologia , Formação de Anticorpos/imunologia , Citrulina/imunologia , Glicosilação , Histonas/imunologia , Camundongos , Camundongos SCID , Mutação/imunologiaRESUMO
OBJECTIVE: Autoantibodies against antigens carrying distinct post-translational modifications (PTMs), such as citrulline, homocitrulline or acetyllysine, are hallmarks of rheumatoid arthritis (RA). The relation between these anti-modified protein antibody (AMPA)-classes is poorly understood as is the ability of different PTM-antigens to activate B-cell receptors (BCRs) directed against citrullinated proteins (CP). Insights into the nature of PTMs able to activate such B cells are pivotal to understand the 'evolution' of the autoimmune response conceivable underlying the disease. Here, we investigated the cross-reactivity of monoclonal AMPA and the ability of different types of PTM-antigens to activate CP-reactive BCRs. METHODS: BCR sequences from B cells isolated using citrullinated or acetylated antigens were used to produce monoclonal antibodies (mAb) followed by a detailed analysis of their cross-reactivity towards PTM-antigens. Ramos B-cell transfectants expressing CP-reactive IgG BCRs were generated and their activation on stimulation with PTM-antigens investigated. RESULTS: Most mAbs were highly cross-reactive towards multiple PTMs, while no reactivity was observed to the unmodified controls. B cells carrying CP-reactive BCRs showed activation on stimulation with various types of PTM-antigens. CONCLUSIONS: Our study illustrates that AMPA exhibit a high cross-reactivity towards at least two PTMs indicating that their recognition pattern is not confined to one type of modification. Furthermore, our data show that CP-reactive B cells are not only activated by citrullinated, but also by carbamylated and/or acetylated antigens. These data are vital for the understanding of the breach of B-cell tolerance against PTM-antigens and the possible contribution of these antigens to RA-pathogenesis.
Assuntos
Anticorpos Antiproteína Citrulinada/imunologia , Artrite Reumatoide/imunologia , Linfócitos B/imunologia , Processamento de Proteína Pós-Traducional/imunologia , Receptores de Antígenos de Linfócitos B/imunologia , Acetilação , Idoso , Autoanticorpos/imunologia , Citrulinação/imunologia , Citrulina/análogos & derivados , Citrulina/imunologia , Reações Cruzadas/imunologia , Feminino , Humanos , Imunoglobulina G , Masculino , Pessoa de Meia-Idade , Carbamilação de Proteínas/imunologiaRESUMO
Malnutrition is a common problem after allogeneic hematopoietic stem cell transplantation (allo-HSCT) and could impair immune function. Immune dysfunction after allo-HSCT may be linked with infections, GVHD, and relapse and negatively affect the outcome. Aim of this review was to identify malnutrition biomarkers, potentially useful for immune-system monitoring, in the setting of allo-HSCT. After a systematic search, no satisfying biomarker was found, except for citrulline. Citrulline could be useful in monitoring gastrointestinal function after allo-HSCT and its role in the complex relationship with immune-system function ought to be better explored. A multi-omics approach, including biomarkers and PRO (patient reported outcomes) is, in our opinion, the optimal way to study the relationship between malnutrition and transplant outcomes.
Assuntos
Citrulina , Doença Enxerto-Hospedeiro , Transplante de Células-Tronco Hematopoéticas , Desnutrição , Biomarcadores/sangue , Citrulina/sangue , Citrulina/imunologia , Doença Enxerto-Hospedeiro/sangue , Doença Enxerto-Hospedeiro/imunologia , Humanos , Desnutrição/sangue , Desnutrição/imunologia , Transplante HomólogoRESUMO
The mechanisms underlying the transition of rheumatoid arthritis (RA) systemic autoimmunity to the joints remain largely unknown. Here, we demonstrate that macrophages in the secondary lymphoid organs (SLOs) and synovial ectopic lymphoid-like structures (ELSs) express peptidylarginine deiminase 4 (PAD4) in murine collagen induced arthritis (CIA) and synovial biopsies from RA patients. Moreover, peptidyl citrulline colocalized with macrophages in SLOs and ELSs, and depletion of macrophages in CIA decreased lymphoid tissue citrullination and serum anti-citrullinated protein/peptide antibody (ACPA) levels. Furthermore, PAD was released from activated murine and RA synovial tissue and fluid (SF) macrophages which functionally deiminated extracellular proteins/peptides in vitro. Additionally, activated murine and SF macrophages displayed macrophage extracellular trap formation (METosis) and release of intracellular citrullinated histones. Moreover, presentation of citrullinated proteins induced ACPA production in vitro. Thus, lymphoid tissue macrophages contribute to self-antigen citrullination and ACPA production, indicating that their selective targeting would potentially ameliorate citrullination-dependent autoimmune disorders.
Assuntos
Artrite Experimental/imunologia , Autoanticorpos/imunologia , Doenças Autoimunes/imunologia , Citrulinação/imunologia , Armadilhas Extracelulares/imunologia , Macrófagos/imunologia , Proteína-Arginina Desiminase do Tipo 4/imunologia , Animais , Formação de Anticorpos/imunologia , Artrite Reumatoide/imunologia , Autoantígenos/imunologia , Autoimunidade/imunologia , Citrulina/imunologia , Histonas/imunologia , Masculino , Camundongos , Camundongos Endogâmicos DBA , Líquido Sinovial/imunologia , Membrana Sinovial/imunologiaRESUMO
Peptidylarginine deiminases (PADs) are phylogenetically conserved calcium-dependent enzymes which post-translationally convert arginine into citrulline in target proteins in an irreversible manner, causing functional and structural changes in target proteins. Protein deimination causes generation of neo-epitopes, affects gene regulation and also allows for protein moonlighting. Extracellular vesicles are found in most body fluids and participate in cellular communication via transfer of cargo proteins and genetic material. In this study, post-translationally deiminated proteins and extracellular vesicles (EVs) are described for the first time in shark plasma. We report a poly-dispersed population of shark plasma EVs, positive for phylogenetically conserved EV-specific markers and characterised by TEM. In plasma, 6 deiminated proteins, including complement and immunoglobulin, were identified, whereof 3 proteins were found to be exported in plasma-derived EVs. A PAD homologue was identified in shark plasma by Western blotting and detected an expected 70â¯kDa size. Deiminated histone H3, a marker of neutrophil extracellular trap formation, was also detected in nurse shark plasma. This is the first report of deiminated proteins in plasma and EVs, highlighting a hitherto unrecognized post-translational modification in key immune proteins of innate and adaptive immunity in shark.
Assuntos
Arginina/metabolismo , Citrulinação/imunologia , Citrulina/metabolismo , Proteínas de Peixes/imunologia , Desiminases de Arginina em Proteínas/imunologia , Tubarões/imunologia , Animais , Citrulina/imunologia , Vesículas Extracelulares/imunologia , Vesículas Extracelulares/metabolismo , Proteínas de Peixes/sangue , Proteínas de Peixes/metabolismo , Desiminases de Arginina em Proteínas/genética , Tubarões/genéticaRESUMO
Immunonutrition as a therapeutic approach is rapidly gaining interest in the fight against infection. Targeting l-arginine metabolism is intriguing, considering this amino acid is the substrate for antimicrobial NO production by macrophages. The importance of l-arginine during infection is supported by the finding that inhibiting its synthesis from its precursor l-citrulline blunts host defense. During the first few weeks following pulmonary mycobacterial infection, we found a drastic increase in l-citrulline in the lung, even though serum concentrations were unaltered. This correlated with increased gene expression of the l-citrulline-generating (i.e., iNOS) and l-citrulline-using (i.e., Ass1) enzymes in key myeloid populations. Eliminating l-arginine synthesis from l-citrulline in myeloid cells via conditional deletion of either Ass1 or Asl resulted in increased Mycobacterium bovis bacillus Calmette-Guérin and Mycobacterium tuberculosis H37Rv burden in the lungs compared with controls. Our data illustrate the necessity of l-citrulline metabolism for myeloid defense against mycobacterial infection and highlight the potential for host-directed therapy against mycobacterial disease targeting this nutrient and/or its metabolic pathway.
Assuntos
Arginina/metabolismo , Citrulina/metabolismo , Infecções por Mycobacterium/imunologia , Células Mieloides/imunologia , Células Mieloides/metabolismo , Animais , Arginina/imunologia , Citrulina/imunologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Mutantes , Infecções por Mycobacterium/metabolismo , Infecções Respiratórias/imunologia , Infecções Respiratórias/metabolismoRESUMO
For a long time, many types of vaccines have been useful for the prophylaxis of many infectious diseases. Thus far, many adjuvants that enhance the effects of vaccines have been explored. However, very few adjuvants are being used for humans worldwide. In this study, we investigated the adjuvant activity of various substances, and found citrulline to have high potential as an adjuvant. Citrulline is a type of amino acid present in the body of many organisms. A number of biological activities of citrulline have been reported; however, no adjuvant activity has been reported thus far. Aluminum salts, which are commonly used as adjuvants are not water soluble; therefore, some difficulties are encountered while using them as vaccine adjuvants. Citrulline is easy to use because of its water solubility. In this study, we showed for the first time the adjuvant activity of citrulline by using viral antigens and amyloid ß peptide. Water-soluble citrulline, which is present in our body, is a potential adjuvant candidate.
Assuntos
Adjuvantes Imunológicos , Citrulina , Vacinas , Compostos de Alúmen , Peptídeos beta-Amiloides/imunologia , Animais , Arginina/imunologia , Citrulina/imunologia , Feminino , Glicoproteínas de Hemaglutininação de Vírus da Influenza/imunologia , Imunização , Camundongos , Peptídeos/imunologia , Vacinas/imunologia , Vacinas de Produtos InativadosRESUMO
BACKGROUND: Anti-citrullinated peptide antibodies (ACPA) play an important role in rheumatoid arthritis (RA) diagnosis. In our study, we sought to assess the potential diagnostic value of synthetically manufactured peptides that contain epitopes believed to have a pathogenic role in RA. METHODS: Serum samples from RA patients and healthy controls were obtained. Two synthetic peptides were manufactured containing the common epitopes considered to play a pivotal role in the RA pathogenesis including the antigenic epitopes of filaggrin, beta-fibrinogen, collagen, vimentin and enolase. Three different ELISA kits for citrullinated peptides (namely: CCP3, Cit-ME-Vim and Cit-ME-Eno) were tested and compared. To assess the diagnostic value of the three ELISA tests, for each test the optical densities (OD) were recorded. The statistical power of each test was calculated measuring the area under the curve (AUC) corresponding with each peptide. RESULTS: Serum levels of ACPA recognized by the commercial CCP3 in RA and healthy controls were 1.31⯱â¯0.88 optic density units (ODU) and 0.21⯱â¯0.11 ODU, respectively. Cit-ME-Vim levels were 0.55⯱â¯0.46 ODU in RA subjects and 0.17⯱â¯0.182 ODU in healthy controls whereas Cit-ME-Eno was 0.61⯱â¯0.65 ODU in RA subjects and 0.22⯱â¯0.20 ODU in healthy controls. AUC results were as follows: CCP3, 0.89 [95%CI 0.75-0.87]; Cit-ME-Vim, 0.76 [95%CI 0.69-0.82]; Cit-ME-Eno, 0.73 [95%CI 0.65-0.79]. Statistical significance for all results was achieved (pâ¯<â¯.0001). Sensitivity values for each kit are as follow: CCP3 70.42%; Cit-ME-Vim 63.38%; Cit-ME-Eno 40.85%, and specificity 91% for all tests. CONCLUSION: Our study supports the presence of an added value for the Cit-ME-Vim peptides in the diagnosis of RA. Further studies are needed to replicate such findings.
Assuntos
Anticorpos Antiproteína Citrulinada/sangue , Artrite Reumatoide/diagnóstico , Artrite Reumatoide/imunologia , Citrulina/imunologia , Ensaio de Imunoadsorção Enzimática , Epitopos , Proteínas Filagrinas , Humanos , Kit de Reagentes para DiagnósticoAssuntos
Anticorpos Antiproteína Citrulinada/sangue , Doença da Artéria Coronariana/diagnóstico , Idoso , Área Sob a Curva , Citrulina/imunologia , Doença da Artéria Coronariana/tratamento farmacológico , Doença da Artéria Coronariana/patologia , Feminino , Humanos , Inibidores de Hidroximetilglutaril-CoA Redutases/uso terapêutico , Imunoensaio/métodos , Masculino , Pessoa de Meia-Idade , Prognóstico , Curva ROC , Fatores de Risco , Índice de Gravidade de DoençaRESUMO
BACKGROUND The association of periodontitis (PD) with the prevalence of rheumatoid arthritis (RA) remains controversial. Therefore, the aim of this study was to evaluate their correlation and investigate the effects of non-surgical periodontal treatment on RA. MATERIAL AND METHODS A total of 64 patients were enrolled in this study and divided into 4 groups: 18 PD patients (PD+RA-), 18 RA patients (PD-RA+), 18 RA with PD patients (PD+RA+), and 10 healthy controls (PD-RA-). Periodontal and rheumatologic parameters were examined at baseline and 1 month following non-surgical periodontal treatment. RESULTS Our results showed that RA patients had similar periodontal status. However, patients in the PD+RA+ group had significantly higher levels of rheumatologic parameters such as C-reactive protein (CRP), anti-cyclic citrulline peptide antibody (ACPA), erythrocyte sedimentation rate (ESR), and Disease Activity Score 28 (DAS28) than those in the PD-RA+ group. In addition, non-surgical periodontal treatment was efficacious in improving rheumatologic parameters of patients in the PD+RA+ group. CONCLUSIONS The presence of PD might contribute to the progression of RA, while RA might have little effect on accelerating the development of PD. In addition, RA patients with PD receiving non-surgical periodontal treatment resulted in noteworthy improvement in the clinical outcome for RA.
Assuntos
Artrite Reumatoide/complicações , Artrite Reumatoide/patologia , Periodontite/complicações , Periodontite/terapia , Adulto , Artrite Reumatoide/sangue , Sedimentação Sanguínea , Proteína C-Reativa/metabolismo , Citrulina/imunologia , Regulação para Baixo , Feminino , Humanos , Masculino , Periodontite/sangue , Estatísticas não ParamétricasRESUMO
Mast cells play a central role in the early clearance of the intestinal parasite Giardia lamblia. In a previous study, we reported that G. lamblia live trophozoites or trophozoite-derived total soluble extract induced direct activation (IgE-independent) of mast cells and release of IL-6 and TNF-α. To identify the Giardia molecules and the mast cell receptors involved in this activation, trophozoite-derived total soluble proteins separated into three fractions (F1-F3) were evaluated for its ability to activate mast cells in vitro. F2 activated mast cells in a greater extent than F1 and F3. Furthermore, F2 induced the release of IL-6 and TNF-α by mast cells. TLR2 and TLR4 expression increased slightly after mast cell stimulation with either F2 or total soluble extract; however, these receptors were not involved in F2 or total soluble extract-induced proinflammatory cytokine production. Proteins present in F2 as unique and high-intensity bands identified by liquid chromatography coupled with tandem mass spectrometry, include molecules with important biological activities such as enolase and arginine deiminase (ADI). Recombinant ADI and enolase were tested for their ability to activate mast cells, but only ADI induced a significant release of IL-6 and TNF-α. ADI product, citrulline but not ammonium, also induced mast cell release of TNF-α. Interestingly, recombinant ADI still stimulated the secretion of TNF-α by mast cells in a arginine-free medium, although in a lower extend that in the presence of arginine, indicating that either ADI itself can stimulate mast cells or through its metabolic product, citrulline.
Assuntos
Extratos Celulares/imunologia , Citrulina/imunologia , Giardia lamblia/imunologia , Hidrolases/imunologia , Mastócitos/imunologia , Animais , Arginina , Linhagem Celular , Giardíase/imunologia , Giardíase/parasitologia , Interleucina-6/imunologia , Interleucina-6/metabolismo , Fosfopiruvato Hidratase/metabolismo , Ratos , Ratos Sprague-Dawley , Receptor 2 Toll-Like/imunologia , Receptor 4 Toll-Like/imunologia , Trofozoítos/imunologia , Fator de Necrose Tumoral alfa/imunologia , Fator de Necrose Tumoral alfa/metabolismoRESUMO
OBJECTIVE: Anti-citrullinated protein antibodies (ACPAs) have proven highly useful as biomarkers for rheumatoid arthritis (RA). However, composition and functionality of the associated autoreactive B cell repertoire have not been directly assessed. We aimed to selectively investigate citrullinated autoantigen-specific B cell receptors (BCRs) involved in RA and initiate studies on their pathogenicity. METHODS: Blood samples were obtained from patients in a University of Minnesota cohort with ACPA-positive RA (n = 89). Tetramer sets bearing citrullinated filaggrin peptide cfc1 or citrullinated α-enolase peptide were constructed to specifically capture autoreactive B cells from the unaltered, polyclonal repertoire in RA patients. Citrullinated peptide tetramer-bound B cells were subjected to flow cytometric cell sorting and single-cell IGH, IGK, and IGL gene sequencing for B cell lineage determinations. BCR gene sequences were also expressed as recombinant monoclonal antibodies (mAb) for direct evaluation of citrullinated autoantigen binding and effector functionality. RESULTS: Using citrullinated peptide tetramer enrichment to investigate single autoreactive blood B cells, we identified biased V-region gene usage and conserved junction arrangements in BCRs from RA patients. Parsimonious clustering of related immunoglobulin gene nucleotide sequences revealed clonal expansions of rare individual B cell clades, in parallel with divergent sequence mutations. Correspondingly, recombinant mAb generated from such BCR lineages demonstrated citrulline-dependent cross-reactivity extending beyond the citrullinated peptides used for B cell capture. A pair of citrullinated autoantigen-specific mAb with cross-reactive binding profiles also promoted arthritis in mice. CONCLUSION: Our findings suggest that broad ACPA specificities in RA arise from a restricted repertoire of evolving citrulline-multispecific B cell clades with pathogenic potential.
Assuntos
Anticorpos Antiproteína Citrulinada/sangue , Artrite Reumatoide/sangue , Autoanticorpos/sangue , Autoantígenos/sangue , Receptores de Antígenos de Linfócitos B/sangue , Adulto , Anticorpos Antiproteína Citrulinada/imunologia , Anticorpos Monoclonais/sangue , Anticorpos Monoclonais/imunologia , Artrite Reumatoide/imunologia , Autoanticorpos/imunologia , Autoantígenos/imunologia , Biomarcadores/sangue , Estudos de Casos e Controles , Citrulina/imunologia , Reações Cruzadas , Feminino , Proteínas Filagrinas , Humanos , Masculino , Peptídeos Cíclicos/imunologia , Receptores de Antígenos de Linfócitos B/imunologiaRESUMO
Pentraxins are fluid phase pattern recognition molecules that form an important part of the innate immune defence and are conserved between fish and human. In Atlantic cod (Gadus morhua L.), two pentraxin-like proteins have been described, CRP-I and CRP-II. Here we show for the first time that these two CRP forms are post-translationally deiminated (an irreversible conversion of arginine to citrulline) and differ with respect to tissue specific localisation in cod ontogeny from 3 to 84 days post hatching. While both forms are expressed in liver, albeit at temporally differing levels, CRP-I shows a strong association with nervous tissue while CRP-II is strongly associated to mucosal tissues of gut and skin. This indicates differing roles for the two pentraxin types in immune responses and tissue remodelling, also elucidating novel roles for CRP-I in the nervous system. The presence of deimination positive bands for cod CRPs varied somewhat between mucus and serum, possibly facilitating CRP protein moonlighting, allowing the same protein to exhibit a range of biological functions and thus meeting different functional requirements in different tissues. The presented findings may further current understanding of the diverse roles of pentraxins in teleost immune defences and tissue remodelling, as well as in various human pathologies, including autoimmune diseases, amyloidosis and cancer.
Assuntos
Proteína C-Reativa/imunologia , Proteínas de Peixes/imunologia , Gadus morhua/imunologia , Animais , Arginina/genética , Arginina/imunologia , Arginina/metabolismo , Proteína C-Reativa/genética , Proteína C-Reativa/metabolismo , Citrulina/genética , Citrulina/imunologia , Citrulina/metabolismo , Proteínas de Peixes/genética , Proteínas de Peixes/metabolismo , Gadus morhua/genética , Gadus morhua/metabolismo , Humanos , Mucosa/imunologia , Mucosa/metabolismo , Tecido Nervoso/imunologia , Tecido Nervoso/metabolismo , Especificidade de Órgãos/genética , Especificidade de Órgãos/imunologia , Isoformas de Proteínas/genética , Isoformas de Proteínas/imunologia , Isoformas de Proteínas/metabolismo , Processamento de Proteína Pós-Traducional/imunologiaRESUMO
The immune response to citrullinated antigens is found almost exclusively in patients with rheumatoid arthritis (RA). It is a dynamic response that expands before the onset of disease and generates antibodies (anti-citrullinated protein antibodies (ACPAs)) that are extensively glycosylated in the variable domain. This feature of ACPAs is remarkable and warrants detailed investigation, as it can offer insights into the earliest immunologic mechanisms that lead up to the development of RA. The acquisition of variable domain glycans, in fact, could enable ACPA-expressing B cells to breach tolerance. Although the underlying mechanisms are still elusive, data to support this concept are emerging, owing to the reliable identification and isolation of citrullinated antigen-directed B cells from patients with RA. This technological proficiency also allows for the generation of an increasing number of well-defined monoclonal ACPAs, and provides the opportunity to test and define the mechanisms by which the citrullinated antigen-directed B cell response contributes to the onset and persistence of inflammation. Together with a revised perception of the HLA-risk effect and novel insights into how T cells can govern antibody effector functions, these developments shape an increasingly clear picture of the B cell response to citrullinated antigens in the development of RA.
Assuntos
Anticorpos Antiproteína Citrulinada/metabolismo , Artrite Reumatoide/diagnóstico , Linfócitos B/imunologia , Citrulina/imunologia , Anticorpos Antiproteína Citrulinada/química , Artrite Reumatoide/imunologia , Diagnóstico Precoce , Glicosilação , Antígenos HLA/imunologia , HumanosRESUMO
OBJECTIVE: Anticitrullinated protein antibodies (ACPA) have major diagnostic significance in rheumatoid arthritis (RA). ACPA are directed against different citrullinated antigens, including filaggrin, fibrinogen, vimentin, and collagen. The presence of ACPA is associated with joint damage and extraarticular manifestations, suggesting that ACPA may have a significant role in the pathogenesis of RA. METHODS: To verify the effect of ACPA on RA-immune cells, peripheral blood mononuclear cells (PBMC) from cyclic citrullinated peptide (CCP)-positive patients with RA and healthy controls were cocultured in vitro with ACPA. ACPA-positive stained cells were analyzed by flow cytometry and the effect of ACPA on mRNA expression levels was evaluated by real-time PCR. We tested whether the stimulatory effects induced by ACPA could be inhibited by the addition of a new multiepitope citrullinated peptide (Cit-ME). RESULTS: We found that ACPA bind specifically to PBMC from CCP-positive patients with RA through the Fab portion. ACPA induce upregulation of pathogenic cytokine expression (4- to 13-fold increase) in PBMC derived from CCP-positive patients with RA. Moreover, ACPA upregulated IL-1ß and IL-6 mRNA expression levels by 10- and 6-fold, respectively, compared to control IgG. Cit-ME, a genuine ligand of ACPA, inhibited the ACPA-induced upregulation of IL-1ß and IL-6 by 30%. CONCLUSION: ACPA bind to a limited percentage of PBMC and upregulate inflammatory cytokine expression, suggesting that ACPA is involved in RA pathogenesis. Targeting ACPA to decrease their pathogenic effects might provide a novel direction in developing therapeutic strategies for RA.