RESUMO
BACKGROUND: Excessive osteoclast activity, which is strongly stimulated by pro-inflammatory mediators, results in bone and cartilage degeneration as central features of many arthritides. Levels of the alarmin S100A8/A9 and interleukin (IL)-1ß are both increased in arthritis patients and correlate with disease activity and progression of tissue erosion. We previously presented S100A8/A9 as a good biomarker for joint inflammation and arthritis pathology under circumstances of high IL-1 signaling in mice that lack the gene encoding IL-1 receptor antagonist (Il1rn-/- mice). Here, we investigated whether S100A8/A9 is also actively involved in the development of joint inflammation and both cartilage and bone pathology under these conditions by comparing Il1rn-/- mice with mice that have an additional deficiency for S100a9 (Il1rn-/-XS100a9-/-). METHODS: Il1rn-/-XS100a9-/- on a BALB/c background were obtained by crossing S100a9-/- mice and Il1rn-/- mice. Arthritis incidence and severity were macroscopically scored. Myeloid cell populations in the bone marrow and spleen were determined using flow cytometry. In vitro osteoclastogenesis of bone marrow cells was evaluated with TRAP staining. Microscopic joint inflammation, cartilage degeneration, and bone destruction were evaluated using histology of ankle joints of 12- and 20-week-old mice. RESULTS: Macroscopically scored arthritis severity was comparable between Il1rn-/- and Il1rn-/-XS100a9-/- mice. Inflammation, cartilage erosion, and bone erosion were clearly present in 12-week-old mice of both strains lacking Il1rn-/-, but not significantly different between Il1rn-/-XS100a9-/- and Il1rn-/-. Moreover, we observed that the numbers of neutrophils and monocytes were increased by the absence of Il1rn, which was affected by the absence of S100a9 only in the spleen but not in the bone marrow. In line with our other findings, the absence of S100a9 did not affect the osteoclastogenic potential of osteoclast precursors in the absence of Il1rn. Finally, in agreement with the findings in early arthritis development in 12-week-old mice, cartilage and bone erosion in 20-week-old mice was significantly higher in both Il1rn-/- strains, but the additional absence of S100a9 did not further affect tissue pathology. CONCLUSION: S100A8/A9 deficiency does not significantly affect inflammation and joint destruction in mice with high IL1ß signaling suggesting that S100A8/A9 is not essential for the development of arthritis under these conditions.
Assuntos
Artrite Experimental , Calgranulina A , Calgranulina B , Proteína Antagonista do Receptor de Interleucina 1 , Animais , Artrite Experimental/genética , Calgranulina A/genética , Calgranulina A/metabolismo , Calgranulina B/genética , Calgranulina B/metabolismo , Humanos , Inflamação/genética , Proteína Antagonista do Receptor de Interleucina 1/genética , Camundongos , Camundongos Endogâmicos BALB C , Camundongos KnockoutRESUMO
BACKGROUND: Synovitis-associated pain is mediated by inflammatory factors that may include S100A8/9, which is able to stimulate nociceptive neurons via Toll-like receptor 4. In this study, we investigated the role of S100A9 in pain response during acute synovitis. METHODS: Acute synovitis was induced by streptococcal cell wall (SCW) injection in the knee joint of C57Bl/6 (WT) and S100A9-/- mice. The expression of S100A8/A9 was determined in serum and synovium by ELISA and immunohistochemistry. Inflammation was investigated by 99mTc accumulation, synovial cytokine release, and histology at days 1, 2, and 7. To assess pain, weight distribution, gait analysis, and mechanical allodynia were monitored. Activation markers in afferent neurons were determined by qPCR and immunohistochemistry in the dorsal root ganglia (DRG). Differences between groups were tested using a one-way or two-way analysis of variance (ANOVA). Differences in histology were tested with a non-parametric Mann-Whitney U test. p values lower than 0.05 were considered significant. RESULTS: Intra-articular SCW injection resulted in increased synovial expression and serum levels of S100A8/A9 at day 1. These increased levels, however, did not contribute to the development of inflammation, since this was equal in S100A9-/- mice. WT mice showed a significantly decreased percentage of weight bearing on the SCW hind paw on day 1, while S100A9-/- mice showed no reduction. Gait analysis showed increased "limping" behavior in WT, but not S100A9-/- mice. Mechanical allodynia was observed but not different between WT and S100A9-/- when measuring paw withdrawal threshold. The gene expression of neuron activation markers NAV1.7, ATF3, and GAP43 in DRG was significantly increased in arthritic WT mice at day 1 but not in S100A9-/- mice. CONCLUSIONS: S100A8/9, released from the synovium upon inflammation, is an important mediator of pain response in the knee during the acute phase of inflammation.
Assuntos
Dor Aguda , Artrite Experimental , Sinovite , Alarminas , Animais , Artrite Experimental/genética , Calgranulina A/genética , Calgranulina B/genética , Camundongos , Sinovite/genéticaRESUMO
The intestinal microbiome is perturbed in patients with new-onset and chronic autoimmune inflammatory arthritis. Recent studies in mouse models suggest that development and progression of autoimmune arthritis is highly affected by the intestinal microbiome. This makes modulation of the intestinal microbiota an interesting novel approach to suppress inflammatory arthritis. Prebiotics, defined as non-digestible carbohydrates that selectively stimulate the growth and activity of beneficial microorganisms, provide a relatively non-invasive approach to modulate the intestinal microbiota. The aim of this study was to assess the therapeutic potential of dietary supplementation with a prebiotic mixture of 90% short-chain galacto-oligosaccharides and 10% long-chain fructo-oligosaccharides (scGOS/lcFOS) in experimental arthritis in mice. We here show that dietary supplementation with scGOS/lcFOS has a pronounced effect on the composition of the fecal microbiota. Interestingly, the genera Enterococcus and Clostridium were markedly decreased by scGOS/lcFOS dietary supplementation. In contrast, the family Lachnospiraceae and the genus Lactobacillus, both associated with healthy microbiota, increased in mice receiving scGOS/lcFOS diet. However, the scGOS/lcFOS induced alterations of the intestinal microbiota did not induce significant effects on the intestinal and systemic T helper cell subsets and were not sufficient to reproducibly suppress arthritis in mice. As expected, we did observe a significant increase in the bone mineral density in mice upon dietary supplementation with scGOS/lcFOS for 8 weeks. Altogether, this study suggests that dietary scGOS/lcFOS supplementation is able to promote presumably healthy gut microbiota and improve bone mineral density, but not inflammation, in arthritis-prone mice.
Assuntos
Artrite Experimental/patologia , Microbioma Gastrointestinal/efeitos dos fármacos , Proteína Antagonista do Receptor de Interleucina 1/genética , Oligossacarídeos/farmacologia , Animais , Densidade Óssea/efeitos dos fármacos , Suplementos Nutricionais , Fezes/microbiologia , Feminino , Proteína Antagonista do Receptor de Interleucina 1/deficiência , Lactobacillus/genética , Lactobacillus/isolamento & purificação , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Knockout , Prebióticos , Receptores de Interleucina-1 , Subpopulações de Linfócitos T/citologia , Subpopulações de Linfócitos T/metabolismoRESUMO
Objective: Rheumatoid arthritis (RA) is a chronic and progressive joint disease. It appears that anti-inflammatory feedback mechanisms that could restrain joint inflammation and restore homeostasis are insufficient to perform this control. In this study, we investigated the contribution of the MER tyrosine kinase-mediated anti-inflammatory response on arthritis and whether targeting MER could be a valid approach to treat RA. Methods: KRN serum transfer arthritis (KRN STA) was induced in either Mertk-deficient mice or in mice that adenovirally overexpressed Pros1. Human synovial micromasses were treated with MER-specific antibodies or PROS1. Collagen-induced arthritis (CIA) mice were treated with MER-specific agonistic antibodies or by viral overexpression of Pros1. Results: Mertk-/- mice showed exacerbated arthritis pathology, whereas Pros1 overexpression diminished joint pathology in KRN STA. Human synovial micromasses challenged with MER-specific antibodies enhanced the secretion of inflammatory cytokines, whereas stimulating MER with PROS1 reduced the secretion of these cytokines, confirming the protective role of MER. Next, we treated CIA mice with MER-specific agonistic antibodies, and this unexpectedly resulted in exacerbated arthritis pathology. This was associated with increased numbers of apoptotic cells in their knee joints and higher serum levels of interleukin (IL)-16C, a cytokine released by secondary necrotic neutrophils. Apoptotic cell numbers and IL-16C levels were enhanced during arthritis in Mertk-/- mice and reduced in Pros1-overexpressing mice. Conclusion: MER plays a protective role during joint inflammation and activating MER by its ligand PROS1 ameliorates disease. Treatment of mice with MER receptor agonistic antibodies is deleterious due to its counterproductive effect of blocking efferocytosis in the arthritic joint.
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Artrite Experimental/imunologia , Artrite Reumatoide/imunologia , Proteínas de Transporte/fisiologia , c-Mer Tirosina Quinase/fisiologia , Animais , Artrite Experimental/patologia , Artrite Reumatoide/patologia , Proteínas de Ligação ao Cálcio , Linhagem Celular , Citocinas/imunologia , Modelos Animais de Doenças , Feminino , Humanos , Articulação do Joelho/imunologia , Articulação do Joelho/patologia , Masculino , Camundongos Endogâmicos C57BL , Camundongos Endogâmicos DBA , Camundongos Knockout , Membrana Sinovial/imunologiaRESUMO
BACKGROUND: Osteoclast-mediated bone erosion is a central feature of rheumatoid arthritis (RA). Immune complexes, present in a large percentage of patients, bind to Fcγ receptors (FcγRs), thereby modulating the activity of immune cells. In this study, we investigated the contribution of FcγRs, and FcγRIV in particular, during antigen-induced arthritis (AIA). METHODS: AIA was induced in knee joints of wild-type (WT), FcγRI,II,III-/-, and FcγRI,II,III,IV-/- mice. Bone destruction, numbers of tartrate-resistant acid phosphatase-positive (TRAP+) osteoclasts, and inflammation were evaluated using histology; expression of the macrophage marker F4/80, neutrophil marker NIMPR14, and alarmin S100A8 was evaluated using immunohistochemistry. The percentage of osteoclast precursors in the bone marrow was determined using flow cytometry. In vitro osteoclastogenesis was evaluated with TRAP staining, and gene expression was assessed using real-time PCR. RESULTS: FcγRI,II,III,IV-/- mice showed decreased bone erosion compared with WT mice during AIA, whereas both the humoral and cellular immune responses against methylated bovine serum albumin were not impaired in FcγRI,II,III,IV-/- mice. The percentage of osteoclast precursors in the bone marrow of arthritic mice and their ability to differentiate into osteoclasts in vitro were comparable between FcγRI,II,III,IV-/- and WT mice. In line with these observations, numbers of TRAP+ osteoclasts on the bone surface during AIA were comparable between the two groups. Inflammation, a process that strongly activates osteoclast activity, was reduced in FcγRI,II,III,IV-/- mice, and of note, mainly decreased numbers of neutrophils were present in the joint. In contrast to FcγRI,II,III,IV-/- mice, AIA induction in knee joints of FcγRI,II,III-/- mice resulted in increased bone erosion, inflammation, and numbers of neutrophils, suggesting a crucial role for FcγRIV in the joint pathology by the recruitment of neutrophils. Finally, significant correlations were found between bone erosion and the number of neutrophils present in the joint as well as between bone erosion and the number of S100A8-positive cells, with S100A8 being an alarmin strongly produced by neutrophils that stimulates osteoclast resorbing activity. CONCLUSIONS: FcγRs play a crucial role in the development of bone erosion during AIA by inducing inflammation. In particular, FcγRIV mediates bone erosion in AIA by inducing the influx of S100A8/A9-producing neutrophils into the arthritic joint.
Assuntos
Artrite Experimental/imunologia , Osso e Ossos/imunologia , Calgranulina A/imunologia , Calgranulina B/imunologia , Neutrófilos/imunologia , Receptores de IgG/imunologia , Animais , Artrite Experimental/genética , Artrite Experimental/metabolismo , Osso e Ossos/metabolismo , Osso e Ossos/patologia , Calgranulina A/metabolismo , Calgranulina B/metabolismo , Articulação do Joelho/imunologia , Articulação do Joelho/metabolismo , Articulação do Joelho/patologia , Macrófagos/imunologia , Macrófagos/metabolismo , Camundongos Endogâmicos C57BL , Camundongos Knockout , Neutrófilos/metabolismo , Osteoclastos/imunologia , Osteoclastos/metabolismo , Receptores de IgG/genética , Receptores de IgG/metabolismo , Fosfatase Ácida Resistente a Tartarato/imunologia , Fosfatase Ácida Resistente a Tartarato/metabolismoRESUMO
BACKGROUND: Monocytes are dominant cells present within the inflamed synovium during osteoarthritis (OA). In mice, two functionally distinct monocyte subsets are described: pro-inflammatory Ly6Chigh and patrolling Ly6Clow monocytes. Alarmins S100A8/A9 locally released by the synovium during inflammatory OA for prolonged periods may be dominant proteins involved in stimulating recruitment of Ly6Chigh monocytes from the circulation to the joint. Our objective was to investigate the role of S100A8/A9 in the mobilization of Ly6Chigh and Ly6Clow monocytic populations to the inflamed joint in collagenase-induced OA (CiOA). METHOD: S100A8 was injected intra-articularly to investigate monocyte influx. CiOA was induced by injection of collagenase into knee joints of wild-type C57BL/6 (WT), and S100a9-/- mice. Mice were sacrificed together with age-matched saline-injected control mice (n = 6/group), and expression of monocyte markers, pro-inflammatory cytokines, and chemokines was determined in the synovium using ELISA and RT-qPCR. Cells were isolated from the bone marrow (BM), spleen, blood, and synovium and monocytes were identified using FACS. RESULTS: S100A8/A9 was highly expressed during CiOA. Intra-articular injection of S100A8 leads to elevated expression of monocyte markers and the monocyte-attracting chemokines CCL2 and CX3CL1 in the synovium. At day 7 (d7) after CiOA induction in WT mice, numbers of Ly6Chigh, but not Ly6Clow monocytes, were strongly increased (7.6-fold) in the synovium compared to saline-injected controls. This coincided with strong upregulation of CCL2, which preferentially attracts Ly6Chigh monocytes. In contrast, S100a9-/- mice showed a significant increase in Ly6Clow monocytes (twofold) within the synovium at CiOA d7, whereas the number of Ly6Chigh monocytes remained unaffected. In agreement with this finding, the Ly6Clow mobilization marker CX3CL1 was significantly higher within the synovium of S100a9-/- mice. Next, we studied the effect of S100A8/A9 on release of Ly6Chigh monocytes from the BM into the circulation. A 14% decrease in myeloid cells was found in WT BM at CiOA d7. No decrease in myeloid cells in S100a9-/- BM was found, suggesting that S100A8/A9 promotes the release of myeloid populations from the BM. CONCLUSION: Induction of OA locally leads to strongly elevated S100A8/A9 expression and an elevated influx of Ly6Chigh monocytes from the BM to the synovium.
Assuntos
Artrite Experimental/imunologia , Calgranulina A/imunologia , Calgranulina B/imunologia , Quimiotaxia de Leucócito/imunologia , Monócitos/imunologia , Osteoartrite/imunologia , Animais , Artrite Experimental/metabolismo , Artrite Experimental/patologia , Calgranulina A/metabolismo , Calgranulina B/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Monócitos/metabolismo , Osteoartrite/metabolismo , Osteoartrite/patologia , Membrana Sinovial/imunologia , Membrana Sinovial/metabolismo , Membrana Sinovial/patologiaRESUMO
BACKGROUND: Perturbation of commensal intestinal microbiota has been associated with several autoimmune diseases. Mice deficient in interleukin-1 receptor antagonist (Il1rn -/- mice) spontaneously develop autoimmune arthritis and are susceptible to other autoimmune diseases such as psoriasis, diabetes, and encephalomyelitis; however, the mechanisms of increased susceptibility to these autoimmune phenotypes are poorly understood. We investigated the role of interleukin-1 receptor antagonist (IL-1Ra) in regulation of commensal intestinal microbiota, and assessed the involvement of microbiota subsets and innate and adaptive mucosal immune responses that underlie the development of spontaneous arthritis in Il1rn -/- mice. RESULTS: Using high-throughput 16S rRNA gene sequencing, we show that IL-1Ra critically maintains the diversity and regulates the composition of intestinal microbiota in mice. IL-1Ra deficiency reduced the intestinal microbial diversity and richness, and caused specific taxonomic alterations characterized by overrepresented Helicobacter and underrepresented Ruminococcus and Prevotella. Notably, the aberrant intestinal microbiota in IL1rn -/- mice specifically potentiated IL-17 production by intestinal lamina propria (LP) lymphocytes and skewed the LP T cell balance in favor of T helper 17 (Th17) cells, an effect transferable to WT mice by fecal microbiota. Importantly, LP Th17 cell expansion and the development of spontaneous autoimmune arthritis in IL1rn -/- mice were attenuated under germ-free condition. Selective antibiotic treatment revealed that tobramycin-induced alterations of commensal intestinal microbiota, i.e., reduced Helicobacter, Flexispira, Clostridium, and Dehalobacterium, suppressed arthritis in IL1rn -/- mice. The arthritis phenotype in IL1rn -/- mice was previously shown to depend on Toll-like receptor 4 (TLR4). Using the ablation of both IL-1Ra and TLR4, we here show that the aberrations in the IL1rn -/- microbiota are partly TLR4-dependent. We further identify a role for TLR4 activation in the intestinal lamina propria production of IL-17 and cytokines involved in Th17 differentiation preceding the onset of arthritis. CONCLUSIONS: These findings identify a critical role for IL1Ra in maintaining the natural diversity and composition of intestinal microbiota, and suggest a role for TLR4 in mucosal Th17 cell induction associated with the development of autoimmune disease in mice.
Assuntos
Artrite/imunologia , Microbioma Gastrointestinal , Doenças Hereditárias Autoinflamatórias/imunologia , Proteína Antagonista do Receptor de Interleucina 1/fisiologia , Interleucina-17/imunologia , Receptor 4 Toll-Like/imunologia , Animais , Antibacterianos/administração & dosagem , Artrite/microbiologia , Doenças Autoimunes/imunologia , Doenças Autoimunes/microbiologia , Variação Genética , Helicobacter/genética , Doenças Hereditárias Autoinflamatórias/microbiologia , Sequenciamento de Nucleotídeos em Larga Escala , Proteína Antagonista do Receptor de Interleucina 1/deficiência , Proteína Antagonista do Receptor de Interleucina 1/genética , Proteína Antagonista do Receptor de Interleucina 1/imunologia , Mucosa Intestinal/imunologia , Mucosa Intestinal/microbiologia , Camundongos , Camundongos Knockout , Mucosa/imunologia , Mucosa/microbiologia , Prevotella/genética , RNA Ribossômico 16S , Ruminococcus/genética , Células Th17/imunologia , Receptor 4 Toll-Like/genéticaRESUMO
Synovial fibroblasts are key cells orchestrating the inflammatory response in arthritis. Here we demonstrate that loss of miR-146a, a key epigenetic regulator of the innate immune response, leads to increased joint destruction in a TNF-driven model of arthritis by specifically regulating the behavior of synovial fibroblasts. Absence of miR-146a in synovial fibroblasts display a highly deregulated gene expression pattern and enhanced proliferation in vitro and in vivo. Deficiency of miR-146a induces deregulation of tumor necrosis factor (TNF) receptor associated factor 6 (TRAF6) in synovial fibroblasts, leading to increased proliferation. In addition, loss of miR-146a shifts the metabolic state of fibroblasts towards glycolysis and augments the ability of synovial fibroblasts to support the generation of osteoclasts by controlling the balance of osteoclastogenic regulatory factors receptor activator of NF-κB ligand (RANKL) and osteoprotegerin (OPG). Bone marrow transplantation experiments confirmed the importance of miR-146a in the radioresistant mesenchymal compartment for the control of arthritis severity, in particular for inflammatory joint destruction. This study therefore identifies microRNA-146a as an important local epigenetic regulator of the inflammatory response in arthritis. It is a central element of an anti-inflammatory feedback loop in resident synovial fibroblasts, who are orchestrating the inflammatory response in chronic arthritis. MiR-146a restricts their activation, thereby preventing excessive tissue damage during arthritis.
Assuntos
Artrite/genética , Artrite/metabolismo , Fibroblastos/metabolismo , Articulações/metabolismo , Articulações/patologia , MicroRNAs/genética , Animais , Artrite/patologia , Artrite Experimental , Reabsorção Óssea/genética , Proliferação de Células , Modelos Animais de Doenças , Fibroblastos/patologia , Expressão Gênica , Regulação da Expressão Gênica , Humanos , Camundongos , Camundongos Transgênicos , Interferência de RNA , Membrana Sinovial/citologia , Membrana Sinovial/metabolismo , Fator 6 Associado a Receptor de TNF/genética , Fator 6 Associado a Receptor de TNF/metabolismo , Fator de Necrose Tumoral alfa/genética , Fator de Necrose Tumoral alfa/metabolismoRESUMO
BACKGROUND: Seronegative joint diseases are characterized by a lack of well-defined biomarkers since autoantibodies are not elevated. Calprotectin (S100A8/A9) is a damage-associated molecular pattern (DAMP) which is released by activated phagocytes, and high levels are found in seronegative arthritides. In this study, we investigated the biomarker potential of systemic and local levels of these S100 proteins to assess joint inflammation and joint destruction in an experimental model for seronegative arthritis. METHODS: Serum levels of S100A8/A9 and various cytokines were monitored during disease development in interleukin-1 receptor antagonist (IL-1Ra)-/- mice using ELISA and multiplex bead-based immunoassay, and were correlated to macroscopic and microscopic parameters for joint inflammation, bone erosion, and cartilage damage. Local expression of S100A8 and S100A9 and matrix metalloproteinase (MMP)-mediated cartilage damage in the ankle joints were investigated by immunohistochemistry. In addition, local S100A8 and activated MMPs were monitored in vivo by optical imaging using anti-S100A8-Cy7 and AF489-Cy5.5, a specific tracer for activated MMPs. RESULTS: Serum levels of S100A8/A9 were significantly increased in IL-1Ra-/- mice and correlated with macroscopic joint swelling and histological inflammation, while serum levels of pro-inflammatory cytokines did not correlate with joint swelling. In addition, early serum S100A8/A9 levels were prognostic for disease outcome at a later stage. The increased serum S100A8/A9 levels were reflected by an increased expression of S100A8 and S100A9 within the ankle joint, as visualized by molecular imaging. Next to inflammatory processes, serum S100A8/A9 also correlated with histological parameters for bone erosion and cartilage damage. In addition, arthritic IL-1Ra-/- mice with increased synovial S100A8 and S100A9 expression showed increased cartilage damage that coincided with MMP-mediated neoepitope expression and in vivo imaging of activated MMPs. CONCLUSIONS: Expression of S100A8 and S100A9 in IL-1Ra-/- mice strongly correlates with synovial inflammation, bone erosion, and cartilage damage, underlining the potential of S100A8/A9 as a systemic and local biomarker in seronegative arthritis not only for assessing inflammation but also for assessing severity of inflammatory joint destruction.
Assuntos
Artrite Experimental/patologia , Biomarcadores/análise , Calgranulina A/biossíntese , Calgranulina B/biossíntese , Animais , Calgranulina A/análise , Calgranulina B/análise , Ensaio de Imunoadsorção Enzimática , Feminino , Imuno-Histoquímica , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Camundongos KnockoutRESUMO
OBJECTIVES: In the present study, we generated a new protein, recombinant human alpha-1-anti-trypsin (AAT)-IgG1 Fc fusion protein (AAT-Fc), and evaluated its properties to suppress inflammation and interleukin (IL)-1ß in a mouse model of gouty arthritis. METHODS: A combination of monosodium urate (MSU) crystals and the fatty acid C16.0 (MSU/C16.0) was injected intra-articularly into the knee to induce gouty arthritis. Joint swelling, synovial cytokine production and histopathology were determined after 4â h. AAT-Fc was evaluated for inhibition of MSU/C16.0-induced IL-1ß release from human blood monocytes and for inhibition of extracellular IL-1ß precursor processing. RESULTS: AAT-Fc markedly suppressed MSU/C16.0-induced joint inflammation by 85-91% (p<0.001). Ex vivo production of IL-1ß and IL-6 from cultured synovia were similarly reduced (63% and 65%, respectively). The efficacy of 2.0â mg/kg AAT-Fc in reducing inflammation was comparable to 80â mg/kg of plasma-derived AAT. Injection of AAT-Fc into mice increased circulating levels of endogenous IL-1 receptor antagonist by fourfold. We also observed that joint swelling was reduced by 80%, cellular infiltration by 95% and synovial production of IL-1ß by 60% in transgenic mice expressing low levels of human AAT. In vitro, AAT-Fc reduced MSU/C16.0-induced release of IL-1ß from human blood monocytes and inhibited proteinase-3-mediated extracellular processing of the IL-1ß precursor into active IL-1ß. CONCLUSIONS: A single low dose of AAT-Fc is highly effective in reducing joint inflammation in this model of acute gouty arthritis. Considering the long-term safety of plasma-derived AAT use in humans, subcutaneous AAT-Fc emerges as a promising therapy for gout attacks.
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Artrite Experimental/tratamento farmacológico , Artrite Gotosa/tratamento farmacológico , Supressores da Gota/uso terapêutico , Fragmentos Fc das Imunoglobulinas/uso terapêutico , Proteína Antagonista do Receptor de Interleucina 1/biossíntese , Interleucina-1beta/antagonistas & inibidores , Proteínas Recombinantes de Fusão/uso terapêutico , alfa 1-Antitripsina/uso terapêutico , Animais , Artrite Experimental/imunologia , Artrite Experimental/patologia , Artrite Gotosa/imunologia , Artrite Gotosa/patologia , Células Cultivadas , Relação Dose-Resposta a Droga , Avaliação Pré-Clínica de Medicamentos/métodos , Supressores da Gota/administração & dosagem , Supressores da Gota/farmacologia , Humanos , Fragmentos Fc das Imunoglobulinas/administração & dosagem , Fragmentos Fc das Imunoglobulinas/farmacologia , Injeções Intra-Articulares , Injeções Intraperitoneais , Interleucina-1beta/metabolismo , Receptores de Lipopolissacarídeos/análise , Masculino , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Monócitos/efeitos dos fármacos , Monócitos/imunologia , Proteínas Recombinantes de Fusão/administração & dosagem , Proteínas Recombinantes de Fusão/farmacologia , alfa 1-Antitripsina/administração & dosagem , alfa 1-Antitripsina/farmacologiaRESUMO
INTRODUCTION: Type 17 T helper cells and interleukin (IL)-17 play important roles in the pathogenesis of human and murine arthritis. Although there is a clear link between IL-17 and granulocyte macrophage colony-stimulating factor (GM-CSF) in the inflammatory cascade, details about their interaction in arthritic synovial joints are unclear. In view of the introduction of GM-CSF and IL-17 inhibitors to the clinic, we studied how IL-17 and GM-CSF orchestrate the local production of inflammatory mediators during experimental arthritis. METHODS: To allow detection of additive, complementary or synergistic effects of IL-17 and GM-CSF, we used two opposing experimental approaches: treatment of arthritic mice with neutralising antibodies to IL-17 and GM-CSF and local overexpression of these cytokines in naive synovial joints. Mice were treated for 2 weeks with antibodies against IL-17 and/or GM-CSF after onset of collagen-induced arthritis. Naive mice were injected intraarticularly with adenoviral vectors for IL-17 and/or GM-CSF, resulting in local overexpression. Joint inflammation was monitored by macroscopic scoring, X-rays and histology. Joint washouts, synovial cell and lymph node cultures were analysed for cytokines, chemokines and inflammatory mediators by Luminex analysis, flow cytometry and quantitative polymerase chain reaction. RESULTS: Combined therapeutic anti-IL-17 and anti-GM-CSF ameliorated arthritis progression, and joint damage was dramatically reduced compared with treatment with anti-IL-17 or anti-GM-CSF alone. Anti-IL-17 specifically reduced synovial IL-23 transcription, whereas anti-GM-CSF reduced transcription of matrix metalloproteinases (MMPs) and receptor activator of nuclear factor κB ligand (RANKL). Overexpression of IL-17 or GM-CSF in naive knee joints elicited extensive inflammatory infiltrate, cartilage damage and bone destruction. Combined overexpression revealed additive and synergistic effects on the production of MMPs, RANKL and IL-23 in the synovium and led to complete destruction of the joint structure within 7 days. CONCLUSIONS: IL-17 and GM-CSF differentially mediate the inflammatory process in arthritic joints and show complementary and local additive effects. Combined blockade in arthritic mice reduced joint damage not only by direct inhibition of IL-17 and GM-CSF but also by indirect inhibition of IL-23 and RANKL. Our results provide a rationale for combination therapy in autoinflammatory conditions, especially for patients who do not fully respond to inhibition of the separate cytokines.
Assuntos
Artrite Experimental/imunologia , Artrite Reumatoide/imunologia , Fator Estimulador de Colônias de Granulócitos e Macrófagos/imunologia , Interleucina-17/imunologia , Transdução de Sinais/imunologia , Animais , Artrite Experimental/patologia , Artrite Reumatoide/patologia , Osso e Ossos/patologia , Cartilagem Articular/patologia , Citometria de Fluxo , Interleucina-23/imunologia , Masculino , Metaloproteinases da Matriz/imunologia , Camundongos , Reação em Cadeia da Polimerase em Tempo Real , Receptor Ativador de Fator Nuclear kappa-B/imunologia , Células Th17/imunologiaRESUMO
BACKGROUND: Superparamagnetic Iron Oxide Nanoparticles (SPION) are used in diagnostic imaging of a variety of different diseases. For such in-vivo application, an additional coating with a polymer, for example polyvinyl alcohol (PVA), is needed to stabilize the SPION and prevent aggregation. As the particles are foreign to the body, reaction against the SPION could occur. In this study we investigated the effects that SPION may have on experimental arthritis after intra-articular (i.a.) or intravenous (i.v.) injection. METHODS: PVA-coated SPION were injected either i.a. (6 or 24 µg iron) or i.v. (100 µg or 1 mg iron) into naïve Toll-like receptor-4 deficient (TLR4-/-) or wild-type C57Bl/6 mice, or C57Bl/6 mice with antigen-induced arthritis. As control, some mice were injected with PVA or PBS. MR imaging was performed at 1 and 7 days after injection. Mice were sacrificed 2 hours and 1, 2, 7, 10 and 14 days after injection of the SPION, and RNA from synovium and liver was isolated for pro-inflammatory gene expression analysis. Serum cytokine measurements and whole knee joint histology were also performed. RESULTS: Injection of a high dose of SPION or PVA into naïve knee joints resulted in an immediate upregulation of pro-inflammatory gene expression in the synovium. A similar gene expression profile was observed after SPION or PVA injection into knee joints of TLR4-/- mice, indicating that this effect is not due to LPS contamination. Histological analysis of the knee joints also revealed synovial inflammation after SPION injection. Two hours after i.v. injection of SPION or PVA into naïve mice, an upregulation of pro-inflammatory gene expression was detected in the liver. Administration of SPION or PVA into arthritic mice via i.a. injection did not result in an upregulation in gene expression and also no additional effects were observed on histology. MR imaging and histology showed long-term retention of SPION in the inflamed joint. However, 14 days after the injections no long-term effects were evident for gene expression, histology or serum cytokine concentrations. CONCLUSIONS: Injection of SPION, either locally or systemically, gives an acute inflammatory response. In the long term, up to 14 days after the injection, while the SPION reside in the joint, no further activating effects of SPION were observed. Hence, we conclude that SPION do not aggravate arthritis and can therefore be used safely to detect joint inflammation by MR imaging.
Assuntos
Artrite Experimental/imunologia , Citocinas/metabolismo , Compostos Férricos/metabolismo , Nanopartículas de Magnetita/administração & dosagem , Animais , Artrite Experimental/patologia , Citocinas/genética , Injeções Intra-Articulares , Injeções Intravenosas , Imageamento por Ressonância Magnética , Nanopartículas de Magnetita/química , Camundongos , Álcool de Polivinil/químicaRESUMO
In autoimmune diseases, a disturbance of the balance between T helper 17 (Th17) and regulatory T cells (Tregs) is often observed. This disturbed balance is also the case in rheumatoid arthritis (RA). Genetic predisposition to RA confers the presence of several polymorphisms mainly regulating activation of T lymphocytes. However, the presence of susceptibility factors is neither necessary nor sufficient to explain the disease development, emphasizing the importance of environmental factors. Multiple studies have shown that commensal gut microbiota is of great influence on immune homeostasis and can trigger the development of autoimmune diseases by favoring induction of Th17 cells over Tregs. However the mechanism by which intestinal microbiota influences the Th cell balance is not completely understood. Here we review the current evidence supporting the involvement of commensal intestinal microbiota in rheumatoid arthritis, along with a potential role of Toll-like receptors (TLRs) in modulating the relevant Th cell responses to trigger autoimmunity. A better understanding of TLR triggering by intestinal microbiota and subsequent T cell activation might offer new perspectives for manipulating the T cell response in RA patients and may lead to the discovery of new therapeutic targets or even preventive measures.
Assuntos
Artrite/imunologia , Artrite/metabolismo , Doenças Autoimunes/imunologia , Doenças Autoimunes/metabolismo , Microbioma Gastrointestinal/imunologia , Subpopulações de Linfócitos T/imunologia , Subpopulações de Linfócitos T/metabolismo , Receptores Toll-Like/metabolismo , Animais , Antígenos de Bactérias/imunologia , Antígenos de Bactérias/metabolismo , Artrite Reumatoide/imunologia , Artrite Reumatoide/metabolismo , Humanos , Imunomodulação , Mucosa Intestinal/imunologia , Mucosa Intestinal/metabolismo , Mucosa Intestinal/microbiologia , Ligação Proteica , Células Th17/imunologia , Células Th17/metabolismoRESUMO
INTRODUCTION: Rheumatoid arthritis (RA) is a chronic disease causing recurring inflammatory joint attacks. These attacks are characterized by macrophage infiltration contributing to joint destruction. Studies have shown that RA treatment efficacy is correlated to synovial macrophage number. The aim of this study was to experimentally validate the use of in vivo superparamagnetic iron oxide nanoparticle (SPION) labeled macrophages to evaluate RA treatment by MRI. METHODS: The evolution of macrophages was monitored with and without dexamethasone (Dexa) treatment in rats. Two doses of 3 and 1 mg/kg Dexa were administered two and five days following induction of antigen induced arthritis. SPIONs (7 mg Fe/rat) were injected intravenously and the knees were imaged in vivo on days 6, 10 and 13. The MR images were scored for three parameters: SPION signal intensity, SPION distribution pattern and synovial oedema. Using 3D semi-automated software, the MR SPION signal was quantified. The efficacy of SPIONs and gadolinium chelate (Gd), an MR contrast agent, in illustrating treatment effects were compared. Those results were confirmed through histological measurements of number and area of macrophages and nanoparticle clusters using CD68 immunostaining and Prussian blue staining respectively. RESULTS: Results show that the pattern and the intensity of SPION-labeled macrophages on MRI were altered by Dexa treatment. While the Dexa group had a uniform elliptical line surrounding an oedema pocket, the untreated group showed a diffused SPION distribution on day 6 post-induction. Dexa reduced the intensity of SPION signal 50-60% on days 10 and 13 compared to controls (P = 0.00008 and 0.002 respectively). Similar results were found when the signal was measured by the 3D tool. On day 13, the persisting low grade arthritis progression could not be demonstrated by Gd. Analysis of knee samples by Prussian blue and CD68 immunostaining confirmed in vivo SPION uptake by macrophages. Furthermore, CD68 immunostaining revealed that Dexa treatment significantly decreased the area and number of synovial macrophages. Prussian blue quantification corresponded to the macrophage measurements and both were in agreement with the MRI findings. CONCLUSIONS: We have demonstrated the feasibility of MRI tracking of in vivo SPION-labeled macrophages to assess RA treatment effects.
Assuntos
Dexametasona/farmacologia , Macrófagos/efeitos dos fármacos , Macrófagos/diagnóstico por imagem , Imageamento por Ressonância Magnética/métodos , Nanopartículas de Magnetita/química , Animais , Anti-Inflamatórios/farmacologia , Antígenos CD/metabolismo , Antígenos de Diferenciação Mielomonocítica/metabolismo , Artrite Experimental/diagnóstico por imagem , Artrite Experimental/tratamento farmacológico , Artrite Experimental/metabolismo , Meios de Contraste , Relação Dose-Resposta a Droga , Monitoramento de Medicamentos/métodos , Edema/diagnóstico por imagem , Edema/tratamento farmacológico , Edema/metabolismo , Feminino , Ferrocianetos/química , Gadolínio DTPA , Imuno-Histoquímica , Articulação do Joelho/química , Articulação do Joelho/diagnóstico por imagem , Articulação do Joelho/efeitos dos fármacos , Macrófagos/química , Radiografia , Ratos Endogâmicos Lew , Reprodutibilidade dos Testes , Coloração e Rotulagem/métodos , Membrana Sinovial/diagnóstico por imagem , Membrana Sinovial/efeitos dos fármacos , Membrana Sinovial/patologiaRESUMO
INTRODUCTION: The protein platform called the NOD-like-receptor -family member (NLRP)-3 inflammasome needs to be activated to process intracellular caspase-1. Active caspase-1 is able to cleave pro-Interleukin (IL)-1ß, resulting in bioactive IL-1ß. IL-1ß is a potent proinflammatory cytokine, and thought to play a key role in the pathogenesis of Lyme arthritis, a common manifestation of Borrelia burgdorferi infection. The precise pathways through which B. burgdorferi recognition leads to inflammasome activation and processing of IL-1ß in Lyme arthritis has not been elucidated. In the present study, we investigated the contribution of several pattern recognition receptors and inflammasome components in a novel murine model of Lyme arthritis. METHODS: Lyme arthritis was elicited by live B. burgdorferi, injected intra-articularly in knee joints of mice. To identify the relevant pathway components, the model was applied to wild-type, NLRP3-/-, ASC-/-, caspase-1-/-, NOD1-/-, NOD2-/-, and RICK-/- mice. As a control, TLR2-/-, Myd88-/- and IL-1R-/- mice were used. Peritoneal macrophages and bone marrow-derived macrophages were used for in vitro cytokine production and inflammasome activation studies. Joint inflammation was analyzed in synovial specimens and whole knee joints. Mann-Whitney U tests were used to detect statistical differences. RESULTS: We demonstrate that ASC/caspase-1-driven IL-1ß is crucial for induction of B. burgdorferi-induced murine Lyme arthritis. In addition, we show that B. burgdorferi-induced murine Lyme arthritis is less dependent on NOD1/NOD2/RICK pathways while the TLR2-MyD88 pathway is crucial. CONCLUSIONS: Murine Lyme arthritis is strongly dependent on IL-1 production, and B. burgdorferi induces inflammasome-mediated caspase-1 activation. Next to that, murine Lyme arthritis is ASC- and caspase-1-dependent, but NLRP3, NOD1, NOD2, and RICK independent. Also, caspase-1 activation by B. burgdorferi is dependent on TLR2 and MyD88. Based on present results indicating that IL-1 is one of the major mediators in Lyme arthritis, there is a rationale to propose that neutralizing IL-1 activity may also have beneficial effects in chronic Lyme arthritis.
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Proteínas Reguladoras de Apoptose/metabolismo , Artrite/metabolismo , Proteínas de Transporte/metabolismo , Caspase 1/metabolismo , Doença de Lyme/metabolismo , Animais , Proteínas Reguladoras de Apoptose/genética , Artrite/genética , Artrite/microbiologia , Western Blotting , Borrelia burgdorferi/fisiologia , Proteínas Adaptadoras de Sinalização CARD , Proteínas de Transporte/genética , Caspase 1/genética , Células Cultivadas , Feminino , Interações Hospedeiro-Patógeno , Inflamassomos/genética , Inflamassomos/metabolismo , Interleucina-1beta/metabolismo , Articulação do Joelho/metabolismo , Articulação do Joelho/microbiologia , Células L , Doença de Lyme/genética , Doença de Lyme/microbiologia , Macrófagos/metabolismo , Macrófagos/microbiologia , Camundongos , Camundongos da Linhagem 129 , Camundongos Endogâmicos C57BL , Camundongos Knockout , Fator 88 de Diferenciação Mieloide/genética , Fator 88 de Diferenciação Mieloide/metabolismo , Proteína 3 que Contém Domínio de Pirina da Família NLR , Receptor 2 Toll-Like/genética , Receptor 2 Toll-Like/metabolismoRESUMO
OBJECTIVE: To examine whether synovial interleukin-17 (IL-17) expression promotes tumor necrosis factor (TNF)-induced joint pathologic processes in vivo, and to analyze the surplus ameliorative value of neutralizing IL-17 in addition to TNF during collagen-induced arthritis (CIA). METHODS: Adenoviral vectors were used to induce overexpression of IL-17 and/or TNF in murine knee joints. In addition, mice with CIA were treated, at different stages of arthritis, with soluble IL-17 receptor (sIL-17R), TNF binding protein (TNFBP), or the combination. RESULTS: Overexpression of IL-17 and TNF resulted in joint inflammation and bone erosion in murine knees. Interestingly, IL-17 strikingly enhanced both the joint-inflammatory and joint-destructive capacity of TNF. Further analysis revealed a strongly enhanced up-regulation of S100A8, IL-1ß, and matrix metalloproteinase (MMP) messenger RNA, only when both TNF and IL-17 were present. Moreover, the increase in irreversible cartilage destruction was not merely the result of enhanced inflammation, but also was associated with a direct synergistic effect of these cytokines in the joint. S100A9 deficiency in mice protected against IL-17/TNF-induced expression of cartilage NITEGE neoepitopes. During established arthritis, the combination of sIL-17R and TNFBP was more effective than the anticytokine treatments alone, and significantly inhibited further joint inflammation and cartilage destruction. CONCLUSION: Local synovial IL-17 expression enhances the role of TNF in joint destruction. Synergy between TNF and IL-17 in vivo results in striking exaggeration of cartilage erosion, in parallel with a synergistic up-regulation of S100A8, IL-1ß, and erosive MMPs. Moreover, neutralizing IL-17 in addition to TNF further improves protection against joint damage and is still effective during late-stage CIA. Therefore, compared with anti-TNF alone, combination blocking of TNF and IL-17 may have additional therapeutic value for the treatment of destructive arthritis.
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Artrite Experimental/metabolismo , Calgranulina A/metabolismo , Cartilagem Articular/metabolismo , Interleucina-17/metabolismo , Interleucina-1beta/metabolismo , Metaloproteinases da Matriz/metabolismo , Fator de Necrose Tumoral alfa/metabolismo , Animais , Artrite Experimental/patologia , Cartilagem Articular/patologia , Articulação do Joelho/metabolismo , Articulação do Joelho/patologia , Masculino , Camundongos , Camundongos TransgênicosRESUMO
Eicosanoids are essential mediators of the inflammatory response and contribute both to the initiation and the resolution of inflammation. Leukocyte-type 12/15-lipoxygenase (12/15-LO) represents a major enzyme involved in the generation of a subclass of eicosanoids, including the anti-inflammatory lipoxin A(4) (LXA(4)). Nevertheless, the impact of 12/15-LO on chronic inflammatory diseases such as arthritis has remained elusive. By using two experimental models of arthritis, the K/BxN serum-transfer and a TNF transgenic mouse model, we show that deletion of 12/15-LO leads to uncontrolled inflammation and tissue damage. Consistent with these findings, 12/15-LO-deficient mice showed enhanced inflammatory gene expression and decreased levels of LXA(4) within their inflamed synovia. In isolated macrophages, the addition of 12/15-LO-derived eicosanoids blocked both phosphorylation of p38MAPK and expression of a subset of proinflammatory genes. Conversely, 12/15-LO-deficient macrophages displayed significantly reduced levels of LXA(4), which correlated with increased activation of p38MAPK and an enhanced inflammatory gene expression after stimulation with TNF-alpha. Taken together, these results support an anti-inflammatory and tissue-protective role of 12/15-LO and its products during chronic inflammatory disorders such as arthritis.