RESUMO
Fibroblast-like synoviocytes (FLS) are joint-lining cells that promote rheumatoid arthritis (RA) pathology. Current disease-modifying antirheumatic agents (DMARDs) operate through systemic immunosuppression. FLS-targeted approaches could potentially be combined with DMARDs to improve control of RA without increasing immunosuppression. Here, we assessed the potential of immunoglobulin-like domains 1 and 2 (Ig1&2), a decoy protein that activates the receptor tyrosine phosphatase sigma (PTPRS) on FLS, for RA therapy. We report that PTPRS expression is enriched in synovial lining RA FLS and that Ig1&2 reduces migration of RA but not osteoarthritis FLS. Administration of an Fc-fusion Ig1&2 attenuated arthritis in mice without affecting innate or adaptive immunity. Furthermore, PTPRS was down-regulated in FLS by tumor necrosis factor (TNF) via a phosphatidylinositol 3-kinase-mediated pathway, and TNF inhibition enhanced PTPRS expression in arthritic joints. Combination of ineffective doses of TNF inhibitor and Fc-Ig1&2 reversed arthritis in mice, providing an example of synergy between FLS-targeted and immunosuppressive DMARD therapies.
Assuntos
Antirreumáticos , Artrite Reumatoide , Sinoviócitos , Animais , Antirreumáticos/uso terapêutico , Células Cultivadas , Fibroblastos/metabolismo , Camundongos , Sinoviócitos/metabolismo , Sinoviócitos/patologia , Fator de Necrose Tumoral alfa/metabolismoRESUMO
The hematopoietic-specific protein tyrosine phosphatase nonreceptor type 22 (PTPN22) is encoded by a major autoimmunity risk gene. PTPN22 inhibits T cell activation by dephosphorylating substrates involved in proximal T cell receptor (TCR) signaling. Here, we found by mass spectrometry that PTPN22 was phosphorylated at Ser751 by PKCα in Jurkat and primary human T cells activated with phorbol ester/ionomycin or antibodies against CD3/CD28. The phosphorylation of PTPN22 at Ser751 prolonged its half-life by inhibiting K48-linked ubiquitination and impairing recruitment of the phosphatase to the plasma membrane, which is necessary to inhibit proximal TCR signaling. Additionally, the phosphorylation of PTPN22 at Ser751 enhanced the interaction of PTPN22 with the carboxyl-terminal Src kinase (CSK), an interaction that is impaired by the PTPN22 R620W variant associated with autoimmune disease. The phosphorylation of Ser751 did not affect the recruitment of PTPN22 R620W to the plasma membrane but protected this mutant from degradation. Together, out data indicate that phosphorylation at Ser751 mediates a reciprocal regulation of PTPN22 stability versus translocation to TCR signaling complexes by CSK-dependent and CSK-independent mechanisms.
Assuntos
Proteína Tirosina Fosfatase não Receptora Tipo 22/metabolismo , Receptores de Antígenos de Linfócitos T/metabolismo , Serina/metabolismo , Transdução de Sinais , Doenças Autoimunes/genética , Doenças Autoimunes/metabolismo , Proteína Tirosina Quinase CSK/metabolismo , Células Cultivadas , Células HEK293 , Humanos , Células Jurkat , Espectrometria de Massas/métodos , Mutação de Sentido Incorreto , Fosforilação , Proteína Tirosina Fosfatase não Receptora Tipo 22/genética , Serina/genética , Linfócitos T/metabolismoRESUMO
Receptor-type protein tyrosine phosphatase α (RPTPα) is an important positive regulator of SRC kinase activation and a known promoter of cancer growth, fibrosis, and arthritis. The domain structure of RPTPs comprises an extracellular region, a transmembrane helix, and two tandem intracellular catalytic domains referred to as D1 and D2. The D2 domain of RPTPs is believed to mostly play a regulatory function; however, no regulatory model has been established for RPTPα-D2 or other RPTP-D2 domains. Here, we solved the 1.8 Å resolution crystal structure of the cytoplasmic region of RPTPα, encompassing D1 and D2, trapped in a conformation that revealed a possible mechanism through which D2 can allosterically inhibit D1 activity. Using a D2-truncation RPTPα variant and mutational analysis of the D1/D2 interfaces, we show that D2 inhibits RPTPα phosphatase activity and identified a 405PFTP408 motif in D1 that mediates the inhibitory effect of D2. Expression of the gain-of-function F406A/T407A RPTPα variant in HEK293T cells enhanced SRC activation, supporting the relevance of our proposed D2-mediated regulation mechanism in cell signaling. There is emerging interest in the development of allosteric inhibitors of RPTPs but a scarcity of validated allosteric sites for RPTPs. The results of our study not only shed light on the regulatory role of RPTP-D2 domains, but also provide a potentially useful tool for the discovery of chemical probes targeting RPTPα and other RPTPs.
Assuntos
Membrana Celular/metabolismo , Proteínas Tirosina Fosfatases/metabolismo , Proteínas Tirosina Fosfatases Classe 4 Semelhantes a Receptores/química , Proteínas Tirosina Fosfatases Classe 4 Semelhantes a Receptores/metabolismo , Regulação Alostérica , Sequência de Aminoácidos , Domínio Catalítico , Cristalografia por Raios X , Humanos , Modelos Moleculares , Ligação Proteica , Conformação Proteica , Proteínas Tirosina Fosfatases/química , Homologia de SequênciaRESUMO
BACKGROUND: Signalling through insulin-like growth factor 1 receptor (IGF-1R) is essential for cell survival, but may turn pathogenic in uncontrolled tissue growth in tumours. In rheumatoid arthritis (RA), the IGF-1R signalling is activated and supports expansion of the inflamed synovia. AIM: In the present study, we assess if disruption of IGF-1R signalling resolves arthritis. MATERIAL AND METHODS: Clinical associations of IGF-1R expression in leukocytes of the peripheral blood were studied in 84 RA patients. Consequences of the IGF-1R signalling inhibition for arthritis were studied in mBSA immunised Balb/c mice treated with NT157 compound promoting degradation of insulin receptor substrates. RESULTS: In RA patients, high expression of IGF-1R in leukocytes was associated with systemic inflammation as verified by higher expression of NF-kB, serum levels of IL6 and erythrocyte sedimentation rate, and higher pain perception. Additionally, phosphorylated IGF-1R and STAT3 enriched T cells infiltrate in RA synovia. Treatment with NT157 inhibited the phosphorylation of IGF-1R and STAT3 in synovia, and alleviated arthritis and joint damage in mice. It also reduced expression of IGF-1R and despaired ERK and Akt signalling in spleen T cells. This limited IL-6 production, changed RoRgt/FoxP3 balance and IL17 levels. CONCLUSION: IGF-1R signalling contributes to T cell dependent inflammation in arthritis. Inhibition of IGF-1R on the level of insulin receptor substrates alleviates arthritis by restricting IL6-dependent formation of Th17 cells and may open for new treatment strategies in RA.
Assuntos
Artrite Reumatoide/imunologia , Interleucina-6/imunologia , Receptor IGF Tipo 1/imunologia , Transdução de Sinais/imunologia , Membrana Sinovial/imunologia , Células Th17/imunologia , Adulto , Animais , Artrite Reumatoide/metabolismo , Artrite Reumatoide/patologia , Humanos , Inflamação/imunologia , Inflamação/metabolismo , Inflamação/patologia , Interleucina-6/metabolismo , Camundongos , Camundongos Endogâmicos BALB C , Pessoa de Meia-Idade , Receptor IGF Tipo 1/metabolismo , Fator de Transcrição STAT3/imunologia , Fator de Transcrição STAT3/metabolismo , Membrana Sinovial/metabolismo , Membrana Sinovial/patologia , Células Th17/metabolismo , Células Th17/patologiaRESUMO
OBJECTIVE: During rheumatoid arthritis (RA), fibroblast-like synoviocytes (FLS) critically promote disease pathogenesis by aggressively invading the extracellular matrix of the joint. The focal adhesion kinase (FAK) signaling pathway is emerging as a contributor to the anomalous behavior of RA FLS. The receptor protein tyrosine phosphatase α (RPTPα), which is encoded by the PTPRA gene, is a key promoter of FAK signaling. The aim of this study was to investigate whether RPTPα mediates FLS aggressiveness and RA pathogenesis. METHODS: Through RPTPα knockdown, we assessed FLS gene expression by quantitative polymerase chain reaction analysis and enzyme-linked immunosorbent assay, invasion and migration by Transwell assays, survival by annexin V and propidium iodide staining, adhesion and spreading by immunofluorescence microscopy, and activation of signaling pathways by Western blotting of FLS lysates. Arthritis development was examined in RPTPα-knockout (KO) mice using the K/BxN serum-transfer model. The contribution of radiosensitive and radioresistant cells to disease was evaluated by reciprocal bone marrow transplantation. RESULTS: RPTPα was enriched in the RA synovial lining. RPTPα knockdown impaired RA FLS survival, spreading, migration, invasiveness, and responsiveness to platelet-derived growth factor, tumor necrosis factor, and interleukin-1 stimulation. These phenotypes correlated with increased phosphorylation of Src on inhibitory Y(527) and decreased phosphorylation of FAK on stimulatory Y(397) . Treatment of RA FLS with an inhibitor of FAK phenocopied the knockdown of RPTPα. RPTPα-KO mice were protected from arthritis development, which was due to radioresistant cells. CONCLUSION: By regulating the phosphorylation of Src and FAK, RPTPα mediates proinflammatory and proinvasive signaling in RA FLS, correlating with the promotion of disease in an FLS-dependent model of RA.
Assuntos
Artrite Experimental/genética , Artrite Reumatoide/genética , Fibroblastos/metabolismo , Proteína-Tirosina Quinases de Adesão Focal/metabolismo , Proteínas Tirosina Fosfatases Classe 4 Semelhantes a Receptores/genética , Quinases da Família src/metabolismo , Animais , Articulação do Tornozelo , Apoptose/efeitos dos fármacos , Apoptose/genética , Artrite Experimental/metabolismo , Artrite Reumatoide/metabolismo , Western Blotting , Adesão Celular/efeitos dos fármacos , Adesão Celular/genética , Movimento Celular/efeitos dos fármacos , Movimento Celular/genética , Sobrevivência Celular/efeitos dos fármacos , Sobrevivência Celular/genética , Progressão da Doença , Ensaio de Imunoadsorção Enzimática , Fibroblastos/efeitos dos fármacos , Perfilação da Expressão Gênica , Técnicas de Silenciamento de Genes , Interleucina-1/farmacologia , Camundongos , Camundongos Knockout , Fosforilação/efeitos dos fármacos , Fosforilação/genética , Fator de Crescimento Derivado de Plaquetas/farmacologia , Reação em Cadeia da Polimerase , Transdução de Sinais/efeitos dos fármacos , Transdução de Sinais/genética , Membrana Sinovial/citologia , Fator de Necrose Tumoral alfa/farmacologia , Quinases da Família src/efeitos dos fármacosRESUMO
Switched antibody classes are important for efficient immune responses. Aberrant antibody production to otherwise harmless antigens may result in autoimmunity. The protein kinase fms-like tyrosine kinase 3 receptor (Flt3) has an important role during early B-cell development, but the role of Flt3 in peripheral B cells has not been assessed before. Herein we describe a previously unappreciated role for Flt3 in IgG1 class-switch recombination (CSR) and production. We show that Flt3 is reexpressed on B-cell lymphoma 6(+) germinal center B cells in vivo and following LPS activation of peripheral B cells in vitro. Absence of Flt3 signaling in Flt3 ligand-deficient mice results in impaired IgG1 CSR and accumulation of IgM-secreting plasma cells. On activated B cells, Flt3 is coexpressed and functions in synergy with the common-gamma chain receptor family. B cells from Flt3 ligand-deficient mice have impaired IL-4R signaling, with reduced phosphorylation of signal transducer and activator of transcription (Stat) 6, and demonstrate a failure to initiate CSR to IgG1 with low expression of γ1 germ-line transcripts, resulting in impaired IgG1 production. Thus, functional synergy between Flt3 and IL-4R signaling is critical for Stat-mediated regulation of sterile γ1 germ-line transcripts and CSR to IgG1.
Assuntos
Linfócitos B/imunologia , Centro Germinativo/imunologia , Switching de Imunoglobulina , Imunoglobulina G/imunologia , Tirosina Quinase 3 Semelhante a fms/fisiologia , Animais , Apoptose , Regulação da Expressão Gênica , Imunoglobulina M/imunologia , Ligantes , Ativação Linfocitária , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Camundongos Knockout , Plasmócitos/imunologia , Receptores de Interleucina-4/metabolismo , Transdução de Sinais , Tirosina Quinase 3 Semelhante a fms/genéticaRESUMO
Follicular T helper (Tfh) cells are recognized by the expression of CXCR5 and the transcriptional regulator Bcl-6. Tfh cells control B cell maturation and antibody production, and if deregulated, may lead to autoimmunity. Here, we study the role of the proto-oncogene survivin in the formation of Tfh cells. We show that blood Tfh cells of patients with the autoimmune condition rheumatoid arthritis, have intracellular expression of survivin. Survivin was co-localized with Bcl-6 in the nuclei of CXCR5+CD4 lymphocytes and was immunoprecipitated with the Bcl-6 responsive element of the target genes. Inhibition of survivin in arthritic mice led to the reduction of CXCR5+ Tfh cells and to low production of autoantibodies. Exposure to survivin activated STAT3 and induced enrichment of PD-1+Bcl-6+ subset within Tfh cells. Collectively, our study demonstrates that survivin belongs to the Tfh cell phenotype and ensures their optimal function by regulating transcriptional activity of Bcl-6.
Assuntos
Linfócitos T CD4-Positivos/imunologia , Proteínas de Ligação a DNA/imunologia , Proteínas Inibidoras de Apoptose/imunologia , Animais , Autoimunidade/imunologia , Proteínas de Ligação a DNA/química , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Genes p53 , Humanos , Proteínas Inibidoras de Apoptose/química , Proteínas Inibidoras de Apoptose/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos DBA , Modelos Moleculares , Fator 1 de Ligação ao Domínio I Regulador Positivo , Estrutura Secundária de Proteína , Proto-Oncogene Mas , Proteínas Proto-Oncogênicas c-bcl-6 , Proteínas Repressoras/genética , Proteínas Repressoras/imunologia , SurvivinaRESUMO
BACKGROUND: The development of biologics has greatly increased the quality of life and the life expectancy of many patients with rheumatoid arthritis. However, a large number of these patients have an increased risk of developing serious infections. The aim of this study was to examine differential effects of anti-tumor necrosis factor (TNF) treatment and CTLA4 immunoglobulin (Ig) treatment on both immunological response and host defense in a murine model of septic arthritis. METHODS: Abatacept (CTLA4-Ig), etanercept (anti-TNF), or phosphate-buffered saline were given to NMRI mice intravenously inoculated with Staphylococcus aureus. The clinical course of septic arthritis and histopathological and radiological changes of joints were compared among the groups. RESULTS: Mice receiving CTLA4-Ig treatment had more-severe septic arthritis, compared with controls and mice receiving anti-TNF treatment. Anti-TNF treatment led to more-severe weight loss and kidney abscesses, as well as a higher bacterial burden in the kidneys. Mice receiving CTLA4-Ig therapy had lower serum levels of interleukin 4, whereas mice receiving anti-TNF therapy had higher levels of TNF-α. Both iNOS and arginase-1 expression were reduced in peritoneal macrophages from mice receiving CTLA4-Ig, compared with expression in the anti-TNF group. CONCLUSIONS: CTLA4-Ig therapy significantly increased the susceptibility to S. aureus septic arthritis in mice, whereas anti-TNF therapy deteriorated host bacterial clearance, resulting in more-severe weight loss and kidney abscesses.
Assuntos
Antirreumáticos/uso terapêutico , Artrite Infecciosa/imunologia , Artrite Reumatoide/imunologia , Infecções Estafilocócicas/imunologia , Staphylococcus aureus/imunologia , Abatacepte/uso terapêutico , Animais , Artrite Infecciosa/microbiologia , Artrite Reumatoide/microbiologia , Etanercepte/uso terapêutico , Feminino , Articulações/imunologia , Articulações/patologia , Camundongos , Infecções Estafilocócicas/microbiologia , Fator de Necrose Tumoral alfa/imunologiaRESUMO
Fms-like tyrosine kinase 3 ligand (Flt3L) is known as the primary differentiation and survival factor for dendritic cells (DCs). Furthermore, Flt3L is involved in the homeostatic feedback loop between DCs and regulatory T cell (Treg). We have previously shown that Flt3L accumulates in the synovial fluid in rheumatoid arthritis (RA) and that local exposure to Flt3L aggravates arthritis in mice, suggesting a possible involvement in RA pathogenesis. In the present study we investigated the role of Flt3L on DC populations, Tregs as well as inflammatory responses in experimental antigen-induced arthritis. Arthritis was induced in mBSA-immunized mice by local knee injection of mBSA and Flt3L was provided by daily intraperitoneal injections. Flow cytometry analysis of spleen and lymph nodes revealed an increased formation of DCs and subsequently Tregs in mice treated with Flt3L. Flt3L-treatment was also associated with a reduced production of mBSA specific antibodies and reduced levels of the pro-inflammatory cytokines IL-6 and TNF-α. Morphological evaluation of mBSA injected joints revealed reduced joint destruction in Flt3L treated mice. The role of DCs in mBSA arthritis was further challenged in an adoptive transfer experiment. Transfer of DCs in combination with T-cells from mBSA immunized mice, predisposed naïve recipients for arthritis and production of mBSA specific antibodies. We provide experimental evidence that Flt3L has potent immunoregulatory properties. Flt3L facilitates formation of Treg cells and by this mechanism reduces severity of antigen-induced arthritis in mice. We suggest that high systemic levels of Flt3L have potential to modulate autoreactivity and autoimmunity.
Assuntos
Artrite/metabolismo , Doenças Autoimunes/metabolismo , Proteínas de Membrana/metabolismo , Linfócitos T Reguladores/metabolismo , Animais , Artrite/induzido quimicamente , Doenças Autoimunes/induzido quimicamente , Células Dendríticas/imunologia , Células Dendríticas/metabolismo , Células Dendríticas/patologia , Humanos , Interleucina-6/imunologia , Interleucina-6/metabolismo , Proteínas de Membrana/genética , Camundongos , Soroalbumina Bovina/toxicidade , Linfócitos T Reguladores/imunologia , Linfócitos T Reguladores/patologia , Fator de Necrose Tumoral alfa/imunologia , Fator de Necrose Tumoral alfa/metabolismo , Tirosina Quinase 3 Semelhante a fms/metabolismoRESUMO
Survivin is known as an inhibitor of apoptosis and a positive regulator of cell division. We have recently identified survivin as a predictor of joint destruction in patients with rheumatoid arthritis (RA). Flt3 ligand (Flt3L) is expressed in the inflamed joints and has adjuvant properties in arthritis. Studies on 90 RA patients (median age 60.5 years [range, 24-87], disease duration 10.5 years [range, 0-35]) show a strong positive association between the levels of survivin and Flt3L in blood. Here, we present experimental evidence connecting survivin and Flt3L signaling. Treatment of BALB/c mice with Flt3L led to an increase of survivin in the bone marrow and in splenic dendritic cells. Flt3L changed the profile of survivin splice variants, increasing transcription of the short survivin40 in the bone marrow. Treatment with an Flt3 inhibitor reduced total survivin expression in bone marrow and in the dendritic cell population in spleen. Inhibition of survivin transcription in mice, by shRNA lentiviral constructs, reduced the gene expression of Flt3L. We conclude that expression of survivin is a downstream event of Flt3 signaling, which serves as an essential mechanism supporting survival of leukocytes during their differentiation, and maturation of dendritic cells, in RA.