RESUMO
TNF is important in immune-mediated inflammatory diseases, including spondyloarthritis (SpA). Transgenic (tg) mice overexpressing transmembrane TNF (tmTNF) develop features resembling human SpA. Furthermore, both tmTNF tg mice and SpA patients develop ectopic lymphoid aggregates, but it is unclear whether these contribute to pathology. Therefore, we characterized the lymphoid aggregates in detail and studied potential alterations in the B and T cell lineage in tmTNF tg mice. Lymphoid aggregates developed in bone marrow (BM) of vertebrae and near the ankle joints prior to the first SpA features and displayed characteristics of ectopic lymphoid structures (ELS) including presence of B cells, T cells, germinal centers, and high endothelial venules. Detailed flow cytometric analyses demonstrated more germinal center B cells with increased CD80 and CD86 expression, along with significantly more T follicular helper, T follicular regulatory, and T regulatory cells in tmTNF tg BM compared with non-tg controls. Furthermore, tmTNF tg mice exhibited increased IgA serum levels and significantly more IgA+ plasma cells in the BM, whereas IgA+ plasma cells in the gut were not significantly increased. In tmTNF tg × TNF-RI-/- mice, ELS were absent, consistent with reduced disease symptoms, whereas in tmTNF tg × TNF-RII-/- mice, ELS and clinical symptoms were still present. Collectively, these data show that tmTNF overexpression in mice results in osteitis and ELS formation in BM, which may account for the increased serum IgA levels that are also observed in human SpA. These effects are mainly dependent on TNF-RI signaling and may underlie important aspects of SpA pathology.
Assuntos
Linfócitos B/imunologia , Medula Óssea/metabolismo , Centro Germinativo/imunologia , Proteínas de Membrana/metabolismo , Osteíte/imunologia , Espondilite Anquilosante/imunologia , Linfócitos T/imunologia , Estruturas Linfoides Terciárias/imunologia , Fator de Necrose Tumoral alfa/metabolismo , Animais , Medula Óssea/patologia , Diferenciação Celular , Linhagem da Célula , Células Cultivadas , Modelos Animais de Doenças , Humanos , Imunoglobulina A/metabolismo , Proteínas de Membrana/genética , Camundongos , Transdução de Sinais , Fator de Necrose Tumoral alfa/genéticaRESUMO
The tumor necrosis factor (TNF) and IL-23/IL-17 axes are the main therapeutic targets in spondyloarthritis. Despite the clinical efficacy of blocking either pathway, monotherapy does not induce remission in all patients and its effect on new bone formation remains unclear. We aimed to study the effect of TNF and IL-17A dual inhibition on clinical disease and structural damage using the HLA-B27/human ß2-microglobulin transgenic rat model of SpA. Immunized rats were randomized according to arthritis severity, 1 week after arthritis incidence reached 50%, to be treated twice weekly for a period of 5 weeks with either a dual blockade therapy of an anti-TNF antibody and an anti-IL-17A antibody, a single therapy of either antibody, or PBS as vehicle control. Treatment-blinded observers assessed inflammation and structural damage clinically, histologically and by micro-CT imaging. Both single therapies as well as TNF and IL-17A dual blockade therapy reduced clinical spondylitis and peripheral arthritis effectively and similarly. Clinical improvement was confirmed for all treatments by a reduction of histological inflammation and pannus formation (p < 0.05) at the caudal spine. All treatments showed an improvement of structural changes at the axial and peripheral joints on micro-CT imaging, with a significant decrease for roughness (p < 0.05), which reflects both erosion and new bone formation, at the level of the caudal spine. The effect of dual blockade therapy on new bone formation was more prominent at the axial than the peripheral level. Collectively, our study showed that dual blockade therapy significantly reduces inflammation and structural changes, including new bone formation. However, we could not confirm a more pronounced effect of dual inhibition compared to single inhibition.
Assuntos
Interleucina-17/antagonistas & inibidores , Espondilartrite/etiologia , Espondilartrite/metabolismo , Fator de Necrose Tumoral alfa/antagonistas & inibidores , Animais , Artrite/tratamento farmacológico , Artrite/etiologia , Artrite/metabolismo , Artrite/patologia , Biomarcadores , Modelos Animais de Doenças , Suscetibilidade a Doenças , Imageamento Tridimensional , Imuno-Histoquímica , Masculino , Osteogênese/efeitos dos fármacos , Osteogênese/genética , Ratos , Ratos Transgênicos , Espondilartrite/diagnóstico , Espondilartrite/tratamento farmacológico , Microtomografia por Raio-XRESUMO
Objectives: Excessive bone formation is an important hallmark of AS. Recently it has been demonstrated that axial bony lesions in AS patients can be visualized using 18F-fluoride PET-CT. The aim of this study was to assess whether 18F-fluoride uptake in clinically active AS patients is related to focal bone formation in spine biopsies and is sensitive to change during anti-TNF treatment. Methods: Twelve anti-TNF-naïve AS patients [female 7/12; age 39 years (SD 11); BASDAI 5.5 ± 1.1] were included. 18 F-fluoride PET-CT scans were performed at baseline and in two patients, biopsies were obtained from PET-positive and PET-negative spine lesions. The remaining 10 patients underwent a second 18F-fluoride PET-CT scan after 12 weeks of anti-TNF treatment. PET scans were scored visually by two blinded expert readers. In addition, 18F-fluoride uptake was quantified using the standardized uptake value corrected for individual integrated whole blood activity concentration (SUVAUC). Clinical response to anti-TNF was defined according to a ⩾ 20% improvement in Assessment of SpondyloArthritis international Society criteria at 24 weeks. Results: At baseline, all patients showed at least one axial PET-positive lesion. Histological analysis of PET-positive lesions in the spine confirmed local osteoid formation. PET-positive lesions were found in the costovertebral joints (43%), facet joints (23%), bridging syndesmophytes (20%) and non-bridging vertebral lesions (14%) and in SI joints (75%). After 12 weeks of anti-TNF treatment, 18F-fluoride uptake in clinical responders decreased significantly in the costovertebral (mean SUVAUC -1.0; P < 0.001) and SI joints (mean SUVAUC -1.2; P = 0.03) in contrast to non-responders. Conclusions: 18F-fluoride PET-CT identified bone formation, confirmed by histology, in the spine and SI joints of AS patients and demonstrated alterations in bone formation during anti-TNF treatment.
Assuntos
Antirreumáticos/uso terapêutico , Fluordesoxiglucose F18/farmacologia , Osteogênese/fisiologia , Coluna Vertebral/diagnóstico por imagem , Espondilite Anquilosante/diagnóstico por imagem , Fator de Necrose Tumoral alfa/antagonistas & inibidores , Adolescente , Adulto , Idoso , Biópsia , Feminino , Seguimentos , Humanos , Imageamento Tridimensional , Masculino , Pessoa de Meia-Idade , Tomografia por Emissão de Pósitrons combinada à Tomografia Computadorizada , Compostos Radiofarmacêuticos/farmacologia , Espondilite Anquilosante/tratamento farmacológico , Adulto JovemRESUMO
OBJECTIVE: Melanoma inhibitory activity (MIA) is a small chondrocyte-specific protein with unknown function. MIA knockout mice (MIA(-/-)) have a normal phenotype with minor microarchitectural alterations of cartilage. Our previous study demonstrated that immunodominant epitopes of MIA are actively presented in an HLA-DR4-restricted manner in the inflamed RA joint. The objective of this study was to investigate the potential role of MIA as an autoantigen. METHODS: Collagen-induced arthritis (CIA) and anti-collagen antibody-induced arthritis (CAIA) were induced in MIA(-/-) mice. Anti-type II collagen (anti-CII) antibodies were measured by ELISA. T cell proliferation and cytokine production were assessed by flow cytometry. RESULTS: MIA(-/-) mice had a markedly reduced incidence and severity of CIA and CAIA compared with wild-type (WT) mice. Attenuation of disease was not related to defective binding of anti-CII antibodies to cartilage in the absence of MIA. However, MIA(-/-) mice had significantly reduced anti-CII IgG1 and IgG2a antibody levels accompanied by an increase in FoxP3-expressing CD25(+)CD4(+) regulatory T cells. This was paralleled by a significant reduction in CII-specific IFN-γ production by T cells in MIA(-/-) but not WT animals, suggesting a qualitative impact of MIA on the collagen-induced Th1 response. Furthermore, Ag-specific proliferation of T cells after restimulation with MIA in WT but not MIA(-/-) mice indicated the existence of MIA-specific T cells in the context of CIA. CONCLUSION: These data support a role for MIA as an autoantigen during arthritis development. Whether MIA can influence the balance of pathogenic vs regulatory responses in human RA remains to be investigated.
Assuntos
Artrite Experimental/imunologia , Artrite Experimental/metabolismo , Autoantígenos/metabolismo , Proteínas da Matriz Extracelular/metabolismo , Animais , Anticorpos Anti-Idiotípicos/efeitos adversos , Artrite Experimental/patologia , Autoantígenos/imunologia , Proliferação de Células , Colágeno/efeitos adversos , Colágeno/imunologia , Citocinas/metabolismo , Modelos Animais de Doenças , Proteínas da Matriz Extracelular/deficiência , Proteínas da Matriz Extracelular/genética , Feminino , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Índice de Gravidade de Doença , Linfócitos T/patologiaRESUMO
OBJECTIVE: The presence of disease-specific autoantibodies in RA but not spondyloarthritis (SpA) suggests that B-cell tolerance is preserved in the latter condition despite chronic joint inflammation. Which factors control B-cell tolerance vs autoimmunity in chronic arthritis remains incompletely understood. As single nucleotide polymorphisms in the B-cell scaffold protein with ankyrin repeats (BANK1) gene have recently been associated with various autoantibody-positive autoimmune diseases including RA, we explored whether altered expression of BANK1 was associated with humoral autoimmunity in arthritis. METHODS: Peripheral B-cell subsets and inflamed synovial tissue were obtained from active SpA and RA. Quantitative PCR was used to assess the expression of full-length BANK1 and delta2 BANK1, a splice variant lacking exon 2 that counteracts BANK1 function. B-cell subsets, autoantibody titres and clinical disease were monitored upon CIA induction in Bank1 knockout (KO) mice. RESULTS: Whereas full-length BANK1 was not differentially expressed, the BANK1 delta2 splicing variant was decreased in naïve peripheral B cells as well as in synovial tissue of SpA compared with RA. However, no differences were observed in seropositive vs seronegative RA. Performing functional analysis in mice, we found no differences in B-cell subsets and anti-collagen antibodies upon CIA induction between Bank1 KO mice and littermate controls. Accordingly, the incidence and severity of clinical disease were not altered in Bank1 KO mice. CONCLUSION: This study did not reveal a major role for BANK1 in humoral autoimmunity in chronic arthritis. The decreased levels of BANK1 delta2 in SpA, however, warrant more detailed analysis of the functional consequences of an altered BANK1/BANK1 delta2 balance.
Assuntos
Proteínas Adaptadoras de Transdução de Sinal/genética , Artrite Reumatoide/imunologia , Autoimunidade/genética , Subpopulações de Linfócitos B/imunologia , Regulação da Expressão Gênica/fisiologia , Imunidade Humoral/genética , Proteínas de Membrana/genética , Espondilartrite/imunologia , Animais , Artrite Experimental/imunologia , Autoanticorpos/sangue , Estudos de Casos e Controles , Estudos de Coortes , Primers do DNA/química , Ensaio de Imunoadsorção Enzimática , Citometria de Fluxo , Humanos , Imunofenotipagem , Camundongos , Camundongos Knockout , RNA Mensageiro/metabolismo , Reação em Cadeia da Polimerase em Tempo Real , Membrana Sinovial/imunologia , Membrana Sinovial/metabolismoRESUMO
OBJECTIVE: Inhibition of inflammation and destruction, but not of osteoproliferation, in patients with spondylarthritis (SpA) treated with anti-tumor necrosis factor raises the question of how these three processes are interrelated. This study was undertaken to analyze this relationship in a rat model of SpA. METHODS: Histologic spine and joint samples from HLA-B27/human ß(2) -microglobulin (hß(2) m)-transgenic rats were analyzed for signs of spondylitis and destructive arthritis and semiquantitatively scored as showing mild, moderate, or severe inflammation. RESULTS: In rats exhibiting spondylitis, mildly inflamed sections displayed lymphocyte infiltration in connective tissue adjacent to the junction of the anulus fibrosus and vertebral bone but not at the enthesis. Moderately inflamed tissue samples contained osteoclasts eroding bone outside the cartilage end plate. In sections from rats with severe inflammation, the cartilage end plate and underlying bone marrow were also affected. End-stage disease was characterized by complete destruction of the intervertebral disc and vertebrae, with ongoing infiltration. Osteoproliferation was not observed in samples from rats with no or mild inflammation, but was present at the edge of the vertebrae in sections with moderate inflammation and persisted during severe inflammation and end-stage destruction. Osteoproliferation occurred at the border of inflammation, at a distance from bone destruction. A strong correlation between the extent of inflammation, destruction, and osteoproliferation was observed. Sections from rats with arthritis displayed a similar pattern of synovial inflammation associated with bone destruction, and simultaneous but topographically distinct osteoproliferation starting from the periosteum. CONCLUSION: SpA in B27/hß(2) m-transgenic rats is characterized by destructive inflammatory pannus tissue rather than by enthesitis or osteitis. Destruction and osteoproliferation occur simultaneously but at distinct sites in joints with moderate to severe inflammation.
Assuntos
Osso e Ossos/patologia , Antígeno HLA-B27/genética , Inflamação/patologia , Articulações/patologia , Espondilartrite/patologia , Animais , Osso e Ossos/imunologia , Proliferação de Células , Modelos Animais de Doenças , Antígeno HLA-B27/imunologia , Inflamação/genética , Inflamação/imunologia , Articulações/imunologia , Osteoclastos/imunologia , Osteoclastos/patologia , Ratos , Ratos Transgênicos , Espondilartrite/genética , Espondilartrite/imunologia , Microglobulina beta-2/genética , Microglobulina beta-2/imunologiaRESUMO
OBJECTIVE: Male rats transgenic for HLA-B27 and human ß(2) -microglobulin (hß(2) m) spontaneously develop epididymoorchitis (EO) preceding the development of spondylarthritis (SpA). In the specific B27/hß(2) m-transgenic rat cross-strain (21-3 × 382-2)F(1) , only the males develop SpA, and neither sex develops gut inflammation. This study was undertaken to determine whether EO and SpA in male (21-3 × 382-2)F(1) rats are causally related. In addition, the primary characteristics of EO in this rat arthritis model were assessed. METHODS: Male B27/hß(2) m-transgenic (21-3 × 382-2)F(1) rats underwent bilateral, unilateral, or sham epididymoorchiectomy between ages 36 and 125 days. The castrated rats were given testosterone replacement. Alternatively, the 21-3 and 283-2 transgene loci were crossed with a transgene inducing aspermatogenesis. Rats were observed for the development of EO, arthritis, and spondylitis. RESULTS: In unmanipulated transgenic rats, inflammation was first evident in the ductuli efferentes (DE; ducts linking the rete testis to epididymis) as early as age 30 days. The inflammation was initially neutrophilic, and later became granulomatous. Antisperm and anti-testis cell antibodies appeared in the rat serum after age 70 days. Cells infiltrating the testes were predominantly CD4+ T cells and CD68+ or CD163+ macrophages. Quantitative polymerase chain reaction of the DE, epididymis, and testis showed elevations in the levels of interferon-γ, interleukin-10 (IL-10), and IL-17A. In addition, levels of IL-12A, IL-22, IL-23A, and IL-23 receptor were found to be elevated in the DE. Remarkably, castration of the rats before age 91 days completely prevented the subsequent onset of arthritis and spondylitis, as did transgene-induced azospermia. CONCLUSION: Autoimmune EO develops spontaneously in HLA-B27/hß(2) m-transgenic (21-3 × 283-2)F(1) rats at age 30 days, the age when antigen-positive meiotic germ cells first exit the testis. Persistent testicular inflammation and/or antigenic stimulation are essential prerequisites for the subsequent development of SpA. Thus, dysregulated innate immunity at immune-privileged sites may be an essential mechanism triggering the onset of SpA.
Assuntos
Doenças Autoimunes/complicações , Epididimite/complicações , Antígeno HLA-B27/genética , Orquite/complicações , Caracteres Sexuais , Espondilartrite/etiologia , Espondilartrite/genética , Animais , Doenças Autoimunes/imunologia , Citocinas/metabolismo , Modelos Animais de Doenças , Epididimo/metabolismo , Epididimo/cirurgia , Epididimite/imunologia , Feminino , Imunidade Inata/imunologia , Masculino , Orquite/imunologia , Fenótipo , Proteínas de Ligação a RNA/genética , Ratos , Ratos Sprague-Dawley , Ratos Transgênicos , Espondilartrite/imunologia , Testículo/metabolismo , Testículo/cirurgia , Transgenes/genéticaRESUMO
OBJECTIVE: Spondyloarthritis (SpA) is characterized by pathologic osteogenesis, inflammation, and extensive angiogenesis in axial and peripheral tissues. Current therapies effectively target inflammation, but these therapies lack efficacy in preventing pathologic osteogenesis. Transgenic mice overexpressing transmembrane tumor necrosis factor (tmTNF-Tg mice) exhibit SpA-like features. We hypothesized that type H blood vessels, which are implicated in osteogenesis, are increased and contribute to pathology in this experimental SpA model. METHODS: We analyzed ankles, femora, and vertebrae of tmTNF-Tg mice and nontransgenic littermates and tmTNF-Tg mice on either a TNF receptor type I (TNFRI)-deficient or TNF receptor type II (TNFRII)-deficient background for osteogenesis, angiogenesis, and inflammation using advanced imaging technologies at various stages of disease. RESULTS: Compared to nontransgenic littermates, tmTNF-Tg mice exhibited an increase in vertebral type H vessels and osteoprogenitor cells in subchondral bone. These features of increased angiogenesis and osteogenesis were already present before onset of clinical disease symptoms. Type H vessels and osteoprogenitor cells were in close proximity to inflammatory lesions and ectopic lymphoid structures. The tmTNF-Tg mice also showed perivertebral ectopic type H vessels and osteogenesis, an increased number of vertebral transcortical vessels, and enhanced entheseal angiogenesis. In tmTNF-Tg mice crossed on a TNFRI- or TNFRII-deficient background, no clear reduction in type H vessels was shown, suggesting that type H vessel formation is not exclusively mediated via TNFRI or TNFRII. CONCLUSION: The contribution of type H vessels to pathologic osteogenesis in experimental SpA advances our knowledge of the pathophysiology of this disease and may also provide a novel opportunity for targeted intervention.
Assuntos
Osteogênese , Espondilartrite , Camundongos , Animais , Inflamação , Espondilartrite/tratamento farmacológico , Camundongos Transgênicos , Fator de Necrose Tumoral alfaRESUMO
Objective: IL-17A plays a major role in the pathogenesis of spondyloarthritis (SpA). Here we assessed the impact of inhibition of RAR related orphan receptor-γ (RORC), the key transcription factor controlling IL-17 production, on experimental SpA in HLA-B27 transgenic (tg) rats. Methods: Experimental SpA was induced by immunization of HLA-B27 tg rats with heat-inactivated Mycobacterium tuberculosis. Splenocytes obtained at day 7, 14 and 21 after immunization were restimulated ex vivo to assess the induction of pro-inflammatory cytokines. Rats were then prophylactically treated with a RORC inhibitor versus vehicle control. The biologic effect of RORC inhibition was assessed by pro-inflammatory cytokine expression in draining lymph nodes. Arthritis and spondylitis were monitored clinically, and the degree of peripheral and axial inflammation, destruction and new bone formation was confirmed by histology. Results: Ex vivo mRNA and protein analyses revealed the rapid and selective induction of IL-17A and IL-22 production by a variety of lymphocyte subsets upon disease induction in HLA-B27 tg rats. Prophylactic RORC inhibition in vivo suppressed the expression of IL-17A, IL17F, and IL-22 without affecting the expression of other T helper cell subset related genes. This biological effect did not translate into clinical efficacy as RORC inhibition significantly accelerated the onset of arthritis and spondylitis, and aggravated the clinical severity of arthritis. This worsening of experimental SpA was confirmed by histopathological demonstration of increased inflammation, destruction, and new bone formation. Conclusion: Despite a significant suppression of the IL-17 axis, RORC inhibitor treatment accelerates and aggravates experimental SpA in the HLA-B27 tg rat model.
Assuntos
Membro 3 do Grupo F da Subfamília 1 de Receptores Nucleares/antagonistas & inibidores , Espondilartrite/imunologia , Espondilartrite/patologia , Animais , Modelos Animais de Doenças , Feminino , Antígeno HLA-B27/genética , Masculino , Ratos , Ratos Endogâmicos Lew , Ratos TransgênicosRESUMO
Both Plasmodium and Babesia species are intraerythrocytic protozoans that infect a wide range of hosts, including humans, and they elicit similar inflammatory responses and clinical manifestations that differ markedly in severity. We recently reported that a rhesus macaque that was chronically infected with Babesia microti was able to control infection with Plasmodium cynomolgi (a parasite of macaques with characteristics very similar to those of Plasmodium vivax) better than naïve monkeys. To confirm this and to investigate the underlying immunopathology, six naïve rhesus monkeys were infected with B. microti. After 24 days, four of these monkeys and four naïve rhesus monkeys were challenged with P. cynomolgi blood-stage parasites. B. microti persisted at low levels in all monkeys, and the clinical parameters were comparable to those of noninfected controls. There was a significant decrease in P. cynomolgi parasitemia in animals coinfected with B. microti compared to the parasitemia in animals infected with P. cynomolgi alone. This decrease in P. cynomolgi parasitemia correlated with increases in the levels of proinflammatory monocytes at the time of P. cynomolgi infection and with higher C-reactive protein (CRP) serum levels 1 week after malaria infection. Therefore, we conclude that ongoing infection with B. microti parasites leads to suppression of malaria infection.
Assuntos
Babesia microti/patogenicidade , Babesiose/complicações , Babesiose/patologia , Malária/complicações , Malária/patologia , Doenças dos Macacos/parasitologia , Plasmodium cynomolgi/patogenicidade , Animais , Babesiose/parasitologia , Sangue/imunologia , Sangue/parasitologia , Análise Química do Sangue , Proteína C-Reativa/análise , Modelos Animais de Doenças , Feminino , Humanos , Macaca mulatta , Malária/parasitologia , Monócitos/imunologia , ParasitemiaRESUMO
CD4(+) T cells are important for CD8(+) T-cell priming by providing cognate signals for DC maturation. We analyzed the capacity of CD4(+) T cells to influence CD8(+) T-cell responses induced by activated DC. Surprisingly, mice depleted for CD4(+) cells were able to generate stronger antigen-specific CD8(+) T-cell responses after DC vaccination than non-depleted mice. The same observation was made when mice were vaccinated with MHC class II(-/-) DC, indicating the presence of a MHC class II-dependent CD4(+) T-cell population inhibiting CD8(+) T-cell responses. Recently we described the expansion of DX5(+)CD4(+) T cells, a T-cell population displaying immune regulatory properties, upon vaccination with DC. Intriguingly, we now observe an inverse correlation between CD8(+) T-cell induction and expansion of DX5(+)CD4(+) T cells as the latter cells did not expand after vaccination with MHC class II(-/-) DC. In vitro, DX5(+)CD4(+) T cells were able to limit proliferation, modulate cytokine production and induce Foxp3(+) expression in OVA-specific CD8(+) T cells. Together, our data show an inhibitory role of CD4(+) T cells on the induction of CD8(+) T-cell responses by activated DC and indicate the involvement of DX5(+)CD4(+), but not CD4(+)CD25(+), T cells in this process.
Assuntos
Linfócitos T CD4-Positivos/imunologia , Linfócitos T CD8-Positivos/imunologia , Antígenos de Histocompatibilidade Classe II/imunologia , Integrina alfa2/imunologia , Animais , Anticorpos/administração & dosagem , Anticorpos/imunologia , Linfócitos T CD4-Positivos/citologia , Linfócitos T CD4-Positivos/efeitos dos fármacos , Linfócitos T CD8-Positivos/citologia , Células Dendríticas/imunologia , Feminino , Citometria de Fluxo , Antígenos de Histocompatibilidade Classe II/genética , Subunidade alfa de Receptor de Interleucina-2/imunologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Vacinação/métodosRESUMO
TNF plays a key role in immune-mediated inflammatory diseases including rheumatoid arthritis (RA) and spondyloarthritis (SpA). It remains incompletely understood how TNF can lead to different disease phenotypes such as destructive peripheral polysynovitis in RA versus axial and peripheral osteoproliferative inflammation in SpA. We observed a marked increase of transmembrane (tm) versus soluble (s) TNF in SpA versus RA together with a decrease in the enzymatic activity of ADAM17. In contrast with the destructive polysynovitis observed in classical TNF overexpression models, mice overexpressing tmTNF developed axial and peripheral joint disease with synovitis, enthesitis, and osteitis. Histological and radiological assessment evidenced marked endochondral new bone formation leading to joint ankylosis over time. SpA-like inflammation, but not osteoproliferation, was dependent on TNF-receptor I and mediated by stromal tmTNF overexpression. Collectively, these data indicate that TNF can drive distinct inflammatory pathologies. We propose that tmTNF is responsible for the key pathological features of SpA.
Assuntos
Artrite/metabolismo , Osteogênese , Espondilartrite/metabolismo , Fator de Necrose Tumoral alfa/fisiologia , Proteína ADAM17/metabolismo , Adulto , Animais , Artrite/etiologia , Modelos Animais de Doenças , Feminino , Imunofluorescência , Humanos , Articulações/metabolismo , Masculino , Camundongos , Receptores do Fator de Necrose Tumoral/metabolismo , Espondilartrite/etiologia , Sinovite/etiologia , Sinovite/metabolismo , Fator de Necrose Tumoral alfa/metabolismoRESUMO
Regulatory B cells (Bregs) are immunosuppressive cells that modulate immune responses through multiple mechanisms. The signals required for the differentiation and activation of these cells remain still poorly understood. We have already shown that overexpression of A PRoliferation-Inducing Ligand (APRIL) reduces the incidence and severity of collagen-induced arthritis (CIA) in mice. Furthermore, we have described that APRIL, but not BAFF, promoted IL-10 production and regulatory functions in human B cells. Therefore, we hypothesized that APRIL, but not BAFF, may be involved in the induction and/or activation of IL-10 producing Bregs that suppress inflammatory responses in vitro and in vivo. Here, we describe that APRIL promotes the differentiation of naïve human B cells to IL-10-producing IgA+ B cells. These APRIL-induced IgA+ B cells display a Breg phenotype and inhibit T cell and macrophage responses through IL-10 and PD-L1. Moreover, APRIL-induced IL-10 producing Bregs suppress inflammation in vivo in experimental autoimmune encephalitis (EAE) and contact hypersensitivity (CHS) models. Finally, we showed a strong correlation between APRIL and IL-10 in the inflamed synovial tissue of inflammatory arthritis patients. Collectively, these observations indicate the potential relevance of this novel APRIL-induced IgA+ Breg population for immune homeostasis and immunopathology.
Assuntos
Subpopulações de Linfócitos B/imunologia , Linfócitos B Reguladores/imunologia , Dermatite de Contato/imunologia , Encefalomielite Autoimune Experimental/imunologia , Inflamação/imunologia , Esclerose Múltipla/imunologia , Membro 13 da Superfamília de Ligantes de Fatores de Necrose Tumoral/metabolismo , Animais , Antígeno B7-H1/genética , Antígeno B7-H1/metabolismo , Células Cultivadas , Modelos Animais de Doenças , Regulação da Expressão Gênica , Humanos , Tolerância Imunológica , Imunoglobulina A/metabolismo , Interleucina-10/genética , Interleucina-10/metabolismo , Camundongos , Camundongos Transgênicos , Membro 13 da Superfamília de Ligantes de Fatores de Necrose Tumoral/genéticaRESUMO
OBJECTIVE: It remains unclear if and how inflammation and new bone formation in spondyloarthritis (SpA) are coupled. We undertook this study to assess the hypothesis that interleukin-17A (IL-17A) is a pivotal driver of both processes. METHODS: The effect of tumor necrosis factor (TNF) and IL-17A on osteogenesis was tested in an osteoblastic differentiation assay using SpA fibroblast-like synoviocytes (FLS) differentiated with dexamethasone, ß-glycophosphatase, and ascorbic acid. IL-17A blockade was performed in HLA-B27/human ß2 -microglobulin (hß2 m)-transgenic rats, which served as a model for SpA in both prophylactic and therapeutic settings. Inflammation and new bone formation were evaluated by micro-computed tomography imaging, histologic analysis, and gene expression profiling. RESULTS: TNF and IL-17A significantly increased in vitro osteoblastic differentiation. In vivo, prophylactic blockade of IL-17A significantly delayed spondylitis and arthritis development and decreased arthritis severity. Anti-IL-17A treatment was also associated with prevention of bone loss and periosteal new bone formation. Therapeutic targeting of IL-17A after the initial inflammatory insult also significantly reduced axial and peripheral joint inflammation. This treatment was again associated with a marked reduction in spinal and peripheral structural damage, including new bone formation. RNA sequencing of target tissue confirmed that IL-17A is a key driver of the molecular signature of disease in this model and that therapeutic anti-IL-17A treatment reversed the inflammatory signature and the selected gene expression related to bone damage. CONCLUSION: Both prophylactic and therapeutic inhibition of IL-17A diminished inflammation and new bone formation in HLA-B27/hß2 m-transgenic rats. Taken together with the ability of IL-17A to promote osteoblastic differentiation of human SpA FLS, these data suggest a direct link between IL-17A-driven inflammation and pathologic new bone formation in SpA.
Assuntos
Diferenciação Celular/efeitos dos fármacos , Interleucina-17/fisiologia , Osteogênese/efeitos dos fármacos , Espondilartrite/tratamento farmacológico , Fator de Necrose Tumoral alfa/farmacologia , Animais , Técnicas de Cultura de Células , Modelos Animais de Doenças , Antígeno HLA-B27/metabolismo , Humanos , Inflamação , Osteoblastos/metabolismo , Ratos , Ratos Transgênicos , Espondilartrite/fisiopatologia , Sinoviócitos/efeitos dos fármacos , Microtomografia por Raio-XRESUMO
Introduction: Spondyloarthritis (SpA) is characterized by inflammation, articular bone erosions and pathologic new bone formation. Targeting TNFα or IL-17A with current available therapies reduces inflammation in SpA, however, treatment of the bone pathology in SpA remains an unmet clinical need. Activation of the mammalian target Of rapamycin (mTOR) promotes IL-17A expression and osteogenesis. Therefore, the inhibition of mTOR (with rapamycin) could be a promising therapeutic avenue in SpA. Objectives: To investigate the effect of blocking mTOR on inflammation, bone erosions and new bone formation in SpA. Methods: Peripheral blood mononuclear cells (PBMCs) from patients with SpA were stimulated with anti-CD3/CD28 in the presence or absence of rapamycin and the resulting cytokine expression was assessed. Fibroblast-like synoviocytes (FLS) from SpA patients were assessed for osteogenic differentiation potential in conditions with TNFα, IL-17A, or TNFα plus IL-17A, in the presence or absence of rapamycin. HLA-B27/Huß2m transgenic rats were immunized with low dose heat-inactivated Mycobacterium tuberculosis (M. tub), treated with 1.5 mg/kg rapamycin prophylactically or therapeutically and monitored for arthritis and spondylitis. Histology and mRNA analysis were performed after 5 weeks of treatment to assess inflammation and bone pathology. Results:In vitro TNFα and IL-17A protein production by SpA PBMCs was inhibited in the presence of rapamycin. Rapamycin also inhibited osteogenic differentiation of human SpA FLS. Ex vivo analysis of SpA synovial biopsies indicated activation of the mTOR pathway in the synovial tissue of SpA patients. In vivo, prophylactic treatment of HLA-B27/Huß2m transgenic rats with rapamycin significantly inhibited the development and severity of inflammation in peripheral joints and spine (arthritis and spondylitis), with histological evidence of reduced bone erosions and new bone formation around peripheral joints. In addition, therapeutic treatment with rapamycin significantly decreased severity of arthritis and spondylitis, with peripheral joint histology showing reduced inflammation, bone erosions and new bone formation. IL-17A mRNA expression was decreased in the metacarpophalangeal joints after rapamycin treatment. Conclusion: mTOR blockade inhibits IL-17A and TNFα production by PBMCs, and osteogenic differentiation of FLS from patients with SpA in vitro. In the HLA-B27 transgenic rat model of SpA, rapamycin inhibits arthritis and spondylitis development and severity, reduces articular bone erosions, decreases pathologic new bone formation and suppresses IL-17A expression. These results may support efforts to evaluate the efficacy of targeting the mTOR pathway in SpA patients.
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Osteogênese/efeitos dos fármacos , Sirolimo/administração & dosagem , Espondilartrite/tratamento farmacológico , Serina-Treonina Quinases TOR/imunologia , Animais , Feminino , Humanos , Interleucina-17/genética , Interleucina-17/imunologia , Leucócitos Mononucleares/efeitos dos fármacos , Leucócitos Mononucleares/imunologia , Masculino , Ratos , Ratos Transgênicos , Espondilartrite/genética , Espondilartrite/imunologia , Espondilartrite/fisiopatologia , Sinoviócitos/efeitos dos fármacos , Sinoviócitos/imunologia , Serina-Treonina Quinases TOR/genética , Fator de Necrose Tumoral alfa/genética , Fator de Necrose Tumoral alfa/imunologiaRESUMO
IL-17A is a central driver of spondyloarthritis (SpA), its production was originally proposed to be IL-23 dependent. Emerging preclinical and clinical evidence suggests, however, that IL-17A and IL-23 have a partially overlapping but distinct biology. We aimed to assess the extent to which IL-17A-driven pathology is IL-23 dependent in experimental SpA. Experimental SpA was induced in HLA-B27/Huß2m transgenic rats, followed by prophylactic or therapeutic treatment with an anti-IL23R antibody or vehicle control. Spondylitis and arthritis were scored clinically and hind limb swelling was measured. Draining lymph node cytokine expression levels were analyzed directly ex vivo, and IL-17A protein was measured upon restimulation with PMA/ionomycin. Prophylactic treatment with anti-IL23R completely protected against the development of both spondylitis and arthritis, while vehicle-treated controls did develop spondylitis and arthritis. In a therapeutic study, anti-IL23R treatment failed to reduce the incidence or decrease the severity of experimental SpA. Mechanistically, expression of downstream effector cytokines, including IL-17A and IL-22, was significantly suppressed in anti-IL23R versus vehicle-treated rats in the prophylactic experiments. Accordingly, the production of IL-17A upon restimulation was reduced. In contrast, there was no difference in IL-17A and IL-22 expression after therapeutic anti-IL23R treatment. Targeting the IL-23 axis during the initiation phase of experimental SpA-but not in established disease-inhibits IL-17A expression and suppresses disease, suggesting the existence of IL-23-independent IL-17A production. Whether IL-17A can be produced independent of IL-23 in human SpA remains to be established.
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Spondyloarthritis (SpA) does not display the typical features of auto-immune disease. Despite the strong association with MHC class I, CD8+ T cells are not required for disease induction in the HLA-B27/Huß2m transgenic rats. We used Lewis HLA-B27/Huß2m transgenic rats [21-3 × 283-2]F1, HLA-B7/Huß2m transgenic rats [120-4 × 283-2]F1, and wild-type rats to test our hypothesis that SpA may be primarily driven by the innate immune response. In vitro, splenocytes were stimulated with heat-inactivated Mycobacterium tuberculosis and cytokine expression and production was measured. In vivo, male and female rats were immunized with 30, 60, or 90 µg of heat-inactivated M. tuberculosis and clinically monitored for spondylitis and arthritis development. After validation of the model, we tested whether prophylactic and therapeutic TNF targeting affected spondylitis and arthritis. In vitro stimulation with heat-inactivated M. tuberculosis strongly induced gene expression of pro-inflammatory cytokines such as TNF, IL-6, IL-1α, and IL-1ß, in the HLA-B27 transgenic rats compared with controls. In vivo immunization induced an increased spondylitis and arthritis incidence and an accelerated and synchronized onset of spondylitis and arthritis in HLA-B27 transgenic males and females. Moreover, immunization overcame the protective effect of orchiectomy. Prophylactic TNF targeting resulted in delayed spondylitis and arthritis development and reduced arthritis severity, whereas therapeutic TNF blockade did not affect spondylitis and arthritis severity. Collectively, these data indicate that innate immune activation plays a role in the initiation of HLA-B27-associated disease and allowed to establish a useful in vivo model to study the cellular and molecular mechanisms of disease initiation and progression.
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Adaptive immune responses are orchestrated by specialized professional antigen-presenting cells (APCs), the dendritic cells (DCs), which play crucial roles as initiators and modulators of adaptive immune responses. A main feature of DCs is their phenotypic and functional plasticity. In the absence of any inflammation or pathogenic elements, most DCs in peripheral tissues and lymphoid organs have a resting, immature phenotype characterized by high endocytic capacity and low surface expression of MHC- and costimulatory molecules. However, upon interaction with microbial ligands, pro-inflammatory cytokines or CD40Ligand, DCs rapidly acquire an activated phenotype. These mature DCs have a very efficient T cell-priming ability as a consequence of upregulation of MHC- and costimulatory molecules on their cell surface. For this reason, DC-based vaccines have been used successfully to combat infections and malignancies. Nonetheless, evidence is accumulating that, especially immature, or semi-matured, DCs also have a potent ability to tolerize T cells or prevent undesired immune reactions. Therefore, current and prospective strategies to promote the inherent tolerogenic potential of DCs are a rational approach for the therapy of autoimmune diseases such as rheumatoid arthritis (RA). This review summarizes some aspects of the intriguing ability of DCs to steer the outcome of immunity and their potency to modulate the outcome of various pathological conditions.
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Doenças Autoimunes/prevenção & controle , Células Dendríticas/imunologia , Tolerância Imunológica/fisiologia , Animais , Artrite Reumatoide/imunologia , HumanosRESUMO
INTRODUCTION: Insulin like growth factor (IGF)-I can act on a variety of cells involved in cartilage and bone repair, yet IGF-I has not been studied extensively in the context of inflammatory arthritis. The objective of this study was to investigate whether IGF-I overexpression in the osteoblast lineage could lead to increased reparative or pathological bone formation in rheumatoid arthritis and/or spondyloarthritis respectively. METHODS: Mice overexpressing IGF-I in the osteoblast lineage (Ob-IGF-I+/-) line 324-7 were studied during collagen induced arthritis and in the DBA/1 aging model for ankylosing enthesitis. Mice were scored clinically and peripheral joints were analysed histologically for the presence of hypertrophic chondrocytes and osteocalcin positive osteoblasts. RESULTS: 90-100% of the mice developed CIA with no differences between the Ob-IGF-I+/- and non-transgenic littermates. Histological analysis revealed similar levels of hypertrophic chondrocytes and osteocalcin positive osteoblasts in the ankle joints. In the DBA/1 aging model for ankylosing enthesitis 60% of the mice in both groups had a clinical score 1<. Severity was similar between both groups. Histological analysis revealed the presence of hypertrophic chondrocytes and osteocalcin positive osteoblasts in the toes in equal levels. CONCLUSION: Overexpression of IGF-I in the osteoblast lineage does not contribute to an increase in repair of erosions or syndesmophyte formation in mouse models for destructive and remodeling arthritis.