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OBJECTIVES: Cytotoxic T cells and natural killer (NK) cells are central effector cells in cancer and infections. Their effector response is regulated by activating and inhibitory receptors. The regulation of these cells in systemic autoimmune diseases such as systemic sclerosis (SSc) is less defined. METHODS: We conducted ex vivo analysis of affected skin and blood samples from 4 SSc patient cohorts (a total of 165 SSc vs 80 healthy individuals) using single-cell transcriptomics, flow cytometry and multiplex immunofluorescence staining. We further analysed the effects of costimulatory modulation in functional assays, and in a severely affected SSc patient who was treated on compassionate use with a novel anti-CD3/CD7 immunotoxin treatment. RESULTS: Here, we show that SSc-affected skin contains elevated numbers of proliferating T cells, cytotoxic T cells and NK cells. These cells selectively express the costimulatory molecule CD7 in association with cytotoxic, proinflammatory and profibrotic genes, especially in recent-onset and severe disease. We demonstrate that CD7 regulates the cytolytic activity of T cells and NK cells and that selective depletion of CD7+ cells prevents cytotoxic cell-induced fibroblast contraction and inhibits their profibrotic phenotype. Finally, anti-CD3/CD7 directed depletive treatment eliminated CD7+ skin cells and stabilised disease manifestations in a severely affected SSc patient. CONCLUSION: Together, the findings imply costimulatory molecules as key regulators of cytotoxicity-driven pathology in systemic autoimmune disease, yielding CD7 as a novel target for selective depletion of pathogenic cells.
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Esclerodermia Sistémica , Linfocitos T , Humanos , Antígenos CD7/metabolismo , Células Asesinas NaturalesRESUMEN
OBJECTIVES: Macrophages are key orchestrators of the osteoarthritis (OA)-associated inflammatory response. Macrophage phenotype is dependent on environmental cues like the inflammatory factor S100A8/A9. Here, we investigated how S100A9 exposure during monocyte-to-macrophage differentiation affects macrophage phenotype and function. METHODS: OA synovium cellular composition was determined using flow cytometry and multiplex immunohistochemistry. Healthy donor monocytes were differentiated towards M1- and M2-like macrophages in presence of S100A9. Macrophage markers were measured using flow cytometry and phagocytic activity was determined using pHrodo Red Zymosan A BioParticles. Gene expression was determined using qPCR. Protein secretion was measured using Luminex and ELISA. RESULTS: Macrophages were the dominant leucocyte subpopulation in OA synovium. They mainly presented with a M2-like phenotype, although the majority also expressed M1-like macrophage markers. Long-term exposure to S100A9 during monocyte-to-macrophage differentiation increased M2-like macrophage markers CD163 and CD206 in M1-like and M2-like differentiated cells. In addition, M1-like macrophage markers were increased in M1-like, but decreased in M2-like differentiated macrophages. In agreement with this mixed phenotype, S100A9 stimulation modestly increased expression and secretion of pro-inflammatory markers and catabolic enzymes, but also increased expression and secretion of anti-inflammatory/anabolic markers. In accordance with the upregulation of M2-like macrophage markers, S100A9 increased phagocytic activity. Finally, we indeed observed a strong association between S100A8 and S100A9 expression and the M2-like/M1-like macrophage ratio in end-stage OA synovium. CONCLUSION: Chronic S100A8/A9 exposure during monocyte-to-macrophage differentiation favours differentiation towards a M2-like macrophage phenotype. The properties of these cells could help explain the catabolic/anabolic dualism in established OA joints with low-grade inflammation.
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OBJECTIVE: Activated synovial fibroblasts are key effector cells in RA. Selectively depleting these based upon their expression of fibroblast activation protein (FAP) is an attractive therapeutic approach. Here we introduce FAP imaging of inflamed joints using 68Ga-FAPI-04 in a RA patient, and aim to assess feasibility of anti-FAP targeted photodynamic therapy (FAP-tPDT) ex vivo using 28H1-IRDye700DX on RA synovial explants. METHODS: Remnant synovial tissue from RA patients was processed into 6 mm biopsies and, from several patients, into primary fibroblast cell cultures. Both were treated using FAP-tPDT. Cell viability was measured in fibroblast cultures and biopsies were evaluated for histological markers of cell damage. Selectivity of the effect of FAP-tPDT was assessed using flow cytometry on primary fibroblasts and co-cultured macrophages. Additionally, one RA patient intravenously received 68Ga-FAPI-04 and was scanned using PET/CT imaging. RESULTS: In the RA patient, FAPI-04 PET imaging showed high accumulation of the tracer in arthritic joints with very low background signal. In vitro, FAP-tPDT induced cell death in primary RA synovial fibroblasts in a light dose-dependent manner. An upregulation of cell damage markers was observed in the synovial biopsies after FAP-tPDT. No significant effects of FAP-tPDT were noted on macrophages after FAP-tPDT of neighbouring fibroblasts. CONCLUSION: In this study the feasibility of selective FAP-tPDT in synovium of rheumatoid arthritis patients ex vivo is demonstrated. Furthermore, this study provides the first indication that FAP-targeted PET/CT can be used to image arthritic joints, an important step towards application of FAP-tPDT as a targeted locoregional therapy for RA.
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Artritis Reumatoide , Fotoquimioterapia , Artritis Reumatoide/diagnóstico por imagen , Artritis Reumatoide/tratamiento farmacológico , Artritis Reumatoide/metabolismo , Fibroblastos/metabolismo , Humanos , Tomografía Computarizada por Tomografía de Emisión de Positrones , Membrana Sinovial/metabolismoRESUMEN
OBJECTIVE: High levels of IL-22 are present in serum and synovial fluid of patients with RA. As both pro- and anti-inflammatory roles for IL-22 have been described in studies using animal models of RA, its exact function in arthritis remains poorly defined. With this study we aimed to further unravel the mechanism by which IL-22 exerts its effects and to decipher its therapeutic potential by overexpression of IL-22 either locally or systemically during experimental arthritis. METHODS: CIA was induced in DBA-1 mice by immunization and booster injection with type II collagen (col II). Before arthritis onset, IL-22 was overexpressed either locally by intra-articular injection or systemically by i.v. injection using an adenoviral vector and clinical arthritis was scored for a period of 10 days. Subsequently, joints were isolated for histological analysis of arthritis severity and mRNA and protein expression of various inflammatory mediators was determined in the synovium, spleen and serum. RESULTS: Local IL-22 overexpression did not alter arthritis pathology, whereas systemic overexpression of IL-22 potently reduced disease incidence, severity and pathology during CIA. Mice systemically overexpressing IL-22 showed strongly reduced serum cytokine levels of TNF-α and macrophage inflammatory protein 1α that correlated significantly with the enhanced expression of the negative immune regulator SOCS3 in the spleen. CONCLUSION: With this study, we revealed clear anti-inflammatory effects of systemic IL-22 overexpression during CIA. Additionally, we are the first to show that the protective effect of systemic IL-22 during experimental arthritis is likely orchestrated via upregulation of the negative regulator SOCS3.
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Artritis Experimental/terapia , Interleucinas/metabolismo , Proteína 3 Supresora de la Señalización de Citocinas/metabolismo , Animales , Artritis Experimental/inmunología , Artritis Experimental/metabolismo , Artritis Experimental/patología , Modelos Animales de Enfermedad , Femenino , Articulaciones/patología , Ratones , Ratones Endogámicos C57BL , Ratones Endogámicos DBA , Reacción en Cadena en Tiempo Real de la Polimerasa , Proteína 3 Supresora de la Señalización de Citocinas/inmunología , Interleucina-22RESUMEN
Systemic sclerosis (SSc) is a rare, severe, auto-immune disease characterized by inflammation, vasculopathy and fibrosis. Activated (myo)fibroblasts are crucial drivers of this fibrosis. By exploiting their expression of fibroblast activation protein (FAP) to perform targeted photodynamic therapy (tPDT), we can locoregionally deplete these pathogenic cells. In this study, we explored the use of FAP-tPDT in primary skin fibroblasts from SSc patients, both in 2D and 3D cultures. Method: The FAP targeting antibody 28H1 was conjugated with the photosensitizer IRDye700DX. Primary skin fibroblasts were obtained from lesional skin biopsies of SSc patients via spontaneous outgrowth and subsequently cultured on plastic or collagen type I. For 2D FAP-tPDT, cells were incubated in buffer with or without the antibody-photosensitizer construct, washed after 4 h and exposed to λ = 689 nm light. Cell viability was measured using CellTiter Glo®®. For 3D FAP-tPDT, cells were seeded in collagen plugs and underwent the same treatment procedure. Contraction of the plugs was followed over time to determine myofibroblast activity. Results: FAP-tPDT resulted in antibody-dose dependent cytotoxicity in primary skin fibroblasts upon light exposure. Cells not exposed to light or incubated with an irrelevant antibody-photosensitizer construct did not show this response. FAP-tPDT fully prevented contraction of collagen plugs seeded with primary SSc fibroblasts. Even incubation with a very low dose of antibody (0.4 nM) inhibited contraction in 2 out of 3 donors. Conclusions: Here we have shown, for the first time, the potential of FAP-tPDT for the treatment of fibrosis in SSc skin.
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Endopeptidasas/administración & dosificación , Matriz Extracelular/metabolismo , Fibroblastos/efectos de los fármacos , Fibrosis/prevención & control , Proteínas de la Membrana/administración & dosificación , Miofibroblastos/efectos de los fármacos , Fotoquimioterapia/métodos , Esclerodermia Sistémica/tratamiento farmacológico , Colágeno Tipo I/metabolismo , Fibroblastos/patología , Fibrosis/metabolismo , Fibrosis/patología , Humanos , Miofibroblastos/patología , Esclerodermia Sistémica/metabolismo , Esclerodermia Sistémica/patologíaRESUMEN
OBJECTIVE: In RA, synovial fibroblasts become activated. These cells express fibroblast activation protein (FAP) and contribute to the pathogenesis by producing cytokines, chemokines and proteases. Selective depletion in inflamed joints could therefore constitute a viable treatment option. To this end, we developed and tested a new therapeutic strategy based on the selective destruction of FAP-positive cells by targeted photodynamic therapy (tPDT) using the anti-FAP antibody 28H1 coupled to the photosensitizer IRDye700DX. METHODS: After conjugation of IRDye700DX to 28H1, the immunoreactive binding and specificity of the conjugate were determined. Subsequently, tPDT efficiency was established in vitro using a 3T3 cell line stably transfected with FAP. The biodistribution of [111In]In-DTPA-28H1 with and without IRDye700DX was assessed in healthy C57BL/6N mice and in C57BL/6N mice with antigen-induced arthritis. The potential of FAP-tPDT to induce targeted damage was determined ex vivo by treating knee joints from C57BL/6N mice with antigen-induced arthritis 24 h after injection of the conjugate. Finally, the effect of FAP-tPDT on arthritis development was determined in mice with collagen-induced arthritis. RESULTS: 28H1-700DX was able to efficiently induce FAP-specific cell death in vitro. Accumulation of the anti-FAP antibody in arthritic knee joints was not affected by conjugation with the photosensitizer. Arthritis development was moderately delayed in mice with collagen-induced arthritis after FAP-tPDT. CONCLUSION: Here we demonstrate the feasibility of tPDT to selectively target and kill FAP-positive fibroblasts in vitro and modulate arthritis in vivo using a mouse model of RA. This approach may have therapeutic potential in (refractory) arthritis.
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Artritis Experimental/tratamiento farmacológico , Fibroblastos/efectos de los fármacos , Fotoquimioterapia/métodos , Células 3T3/efectos de los fármacos , Animales , Femenino , Indoles/uso terapéutico , Ratones , Ratones Endogámicos C57BL , Ratones Endogámicos DBA , Compuestos de Organosilicio/uso terapéuticoRESUMEN
OBJECTIVES: In this study, we used hypercholesterolaemic apolipoprotein E-deficient (Apoe-/-) mice to investigate LDL/oxLDL effect on synovial inflammation and cartilage destruction during antigen-induced arthritis (AIA). Further, as macrophage FcγRs are crucial to immune complex-mediated AIA, we investigated in vitro the effects of high cholesterol levels on the expression of FcγRs on macrophages. METHODS: AIA was induced by intra-articular injection of mBSA into knee joints of immunised Apoe-/- and wild type (WT) control mice. Joint swelling was measured by uptake of 99mTc pertechnetate (99mTc). Joint inflammation and cartilage destruction were assessed by histology. Anti-mBSA IgGs were measured by ELISA and specific T-cell response by lymphocyte stimulation test. Upon oxLDL stimulation of WT macrophages, protein levels of FcγRs were measured by flow cytometry. RESULTS: Local induction of AIA resulted in less joint swelling, synovial infiltrate and exudate in the joint cavity in Apoe-/- mice compared to WT controls, even though both their humoral and adaptive immune response were comparable. Whereas Apoe deficiency alone did not affect macrophage expression of FcγRs, oxLDL sharply reduced the protein levels of activating FcγRs, crucial in mediating cartilage damage. In agreement with the reduced inflammation in Apoe-/- mice, we observed decreased MMP activity and destruction in the articular cartilage. CONCLUSIONS: Taken together, our findings suggest that high levels of LDL/oxLDL during inflammation, dampen the initiation and chronicity of joint inflammation and cartilage destruction in AIA by regulating macrophage FcγR expression.
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Artritis Experimental , Cartílago Articular , LDL-Colesterol/sangre , Animales , Artritis Experimental/inmunología , Artritis Experimental/metabolismo , Artritis Experimental/patología , Cartílago Articular/metabolismo , Cartílago Articular/patología , Modelos Animales de Enfermedad , Inflamación , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Receptores de IgGRESUMEN
Specialized proresolving mediators (SPRM), which arise from n-3 long-chain polyunsaturated fatty acids (n-3FA), promote resolution of inflammation and may help to prevent progression of an acute inflammatory response into chronic inflammation in patients with arthritis. Thus, this study is aimed at determining whether systemic RvE1 treatment reduces arthritis onset and severity in murine collagen-induced arthritis (CIA) and spontaneous cytokine production by human rheumatoid arthritis (RA) synovial explants. 10-week-old DBA1/J male mice were subjected to CIA and treated systemically with 0.1 µg RvE1, 1 µg RvE1, 5 mg/kg anti-TNF (positive control group), PBS (negative control group), or with a combination of 1 µg of RvE1 plus 5 mg/kg anti-TNF using prophylactic or therapeutic strategies. After CIA immunization, mice were treated twice a week by RvE1 or anti-TNF for 10 days. Arthritis development was assessed by visual scoring of paw swelling and histology of ankle joints. Moreover, human RA synovial explants were incubated with 1 nM, 10 nM, or 100 nM of RvE1, and cytokine levels (IL-1ß, IL-6, IL-8, IL-10, INF-γ, and TNF-α) were measured using Luminex bead array. CIA triggered significant inflammation in the synovial cavity, proteoglycan loss, and cartilage and bone destruction in the ankle joints of mice. Prophylactic and therapeutic RvE1 regimens did not ameliorate CIA incidence and severity. Anti-TNF treatment significantly abrogated signs of joint inflammation, bone erosion, and proteoglycan depletion, but additional RvE1 treatment did not further reduce the anti-TNF-mediated suppression of the disease. Treatment with different concentrations of RvE1 did not decrease the expression of proinflammatory cytokines in human RA synovial explants in the studied conditions. Collectively, our findings demonstrated that RvE1 treatment was not an effective approach to treat CIA in DBA1/J mice in both prophylactic and therapeutic strategies. Furthermore, no effects were noticed when human synovial explants were incubated with different concentrations of RvE1.
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Artritis Experimental/sangre , Artritis Experimental/tratamiento farmacológico , Artritis Reumatoide/sangre , Artritis Reumatoide/tratamiento farmacológico , Citocinas/sangre , Ácido Eicosapentaenoico/análogos & derivados , Animales , Ácido Eicosapentaenoico/uso terapéutico , Interleucina-10/sangre , Interleucina-1beta/sangre , Interleucina-6/sangre , Interleucina-8/sangre , Masculino , Espectrometría de Masas , Ratones , Ratones Endogámicos DBA , Estudios Prospectivos , Resultado del Tratamiento , Factor de Necrosis Tumoral alfa/sangreRESUMEN
Objectives: RA is a chronic autoimmune disease leading to progressive destruction of cartilage and bone. RA patients show elevated IL-22 levels and the amount of IL-22-producing Th cells positively correlates with the extent of erosive disease, suggesting a role for this cytokine in RA pathogenesis. The purpose of this study was to determine the feasibility of SPECT/CT imaging with 111In-labelled anti-fibroblast activation protein antibody (28H1) to monitor the therapeutic effect of neutralizing IL-22 in experimental arthritis. Methods: Mice (six mice/group) with CIA received anti-IL-22 or isotype control antibodies. To monitor therapeutic effects after treatment, SPECT/CT images were acquired 24 h after injection of 111In-28H1. Imaging results were compared with macroscopic, histologic and radiographic arthritis scores. Results: Neutralizing IL-22 before CIA onset effectively prevented arthritis development, reaching a disease incidence of only 50%, vs 100% in the control group. SPECT imaging showed significantly lower joint tracer uptake in mice treated early with anti-IL-22 antibodies compared with the control-treated group. Reduction of disease activity in those mice was confirmed by macroscopic, histological and radiographic pathology scores. However, when treatment was initiated in a later phase of CIA, progression of joint pathology could not be prevented. Conclusion: These findings suggest that IL-22 plays an important role in CIA development, and neutralizing this cytokine seems an attractive new strategy in RA treatment. Most importantly, SPECT/CT imaging with 111In-28H1 can be used to specifically monitor therapy responses, and is potentially more sensitive in disease monitoring than the gold standard method of macroscopic arthritis scoring.
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Artritis/diagnóstico por imagen , Cartílago Articular/diagnóstico por imagen , Gelatinasas/genética , Regulación de la Expresión Génica , Interleucinas/genética , Proteínas de la Membrana/genética , ARN Mensajero/genética , Serina Endopeptidasas/genética , Tomografía Computarizada por Tomografía Computarizada de Emisión de Fotón Único/métodos , Animales , Artritis/tratamiento farmacológico , Artritis/genética , Cartílago Articular/efectos de los fármacos , Cartílago Articular/metabolismo , Colágeno/toxicidad , Modelos Animales de Enfermedad , Endopeptidasas , Gelatinasas/biosíntesis , Inmunohistoquímica , Interleucinas/biosíntesis , Masculino , Proteínas de la Membrana/biosíntesis , Ratones , Ratones Endogámicos DBA , Reacción en Cadena en Tiempo Real de la Polimerasa , Serina Endopeptidasas/biosíntesis , Membrana Sinovial/metabolismo , Membrana Sinovial/patología , Interleucina-22RESUMEN
Increasing epidemiologic evidence supports a link between periodontitis and rheumatoid arthritis. The actual involvement of periodontitis in the pathogenesis of rheumatoid arthritis and the underlying mechanisms remain, however, poorly understood. We investigated the influence of concomitant periodontitis on clinical and histopathologic characteristics of T cell-mediated experimental arthritis and evaluated modulation of type II collagen (CII)-reactive Th cell phenotype as a potential mechanism. Repeated oral inoculations of periodontal pathogens Porphyromonas gingivalis and Prevotella nigrescens induced periodontitis in mice, as evidenced by alveolar bone resorption. Interestingly, concurrent periodontitis induced by both bacteria significantly aggravated the severity of collagen-induced arthritis. Exacerbation of arthritis was characterized by increased arthritic bone erosion, whereas cartilage damage remained unaffected. Both P. gingivalis and P. nigrescens skewed the CII-specific T cell response in lymph nodes draining arthritic joints toward the Th17 phenotype without affecting Th1. Importantly, the levels of IL-17 induced by periodontal pathogens in CII-specific T cells directly correlated with the intensity of arthritic bone erosion, suggesting relevance in pathology. Furthermore, IL-17 production was significantly correlated with periodontal disease-induced IL-6 in lymph node cell cultures. The effects of the two bacteria diverged in that P. nigrescens, in contrast to P. gingivalis, suppressed the joint-protective type 2 cytokines, including IL-4. Further in vitro studies showed that the Th17 induction strongly depended on TLR2 expression on APCs and was highly promoted by IL-1. Our data provide evidence of the involvement of periodontitis in the pathogenesis of T cell-driven arthritis through induction of Ag-specific Th17 response.
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Artritis Experimental/complicaciones , Artritis Experimental/inmunología , Artritis Reumatoide/inmunología , Enfermedades Periodontales/complicaciones , Enfermedades Periodontales/inmunología , Animales , Artritis Experimental/patología , Artritis Reumatoide/complicaciones , Artritis Reumatoide/patología , Interleucina-1/inmunología , Ratones , Ratones Endogámicos BALB C , Enfermedades Periodontales/microbiología , Células Th17/inmunología , Receptor Toll-Like 2/inmunologíaRESUMEN
OBJECTIVE: Previous studies have demonstrated a protective role of Toll-like receptor 2 (TLR-2) and a proinflammatory function of TLR-4 during chronic T cell-driven arthritis. The involvement of TLRs in T cell-independent arthritic processes, however, remains unclear. This study was undertaken to determine the functional significance of TLR-2 and TLR-4 in T cell-independent immune complex-driven arthritis. METHODS: Serum-transfer arthritis was induced in wild-type and TLR-deficient mice by intraperitoneal injections of arthritogenic K/BxN mouse serum. Arthritis was assessed macroscopically and by histologic analysis. The influence of TLR-2 on macrophage cytokine profile, Fcγ receptor (FcγR) expression, and response to immune complexes was determined. RESULTS: While TLR-4, unexpectedly, did not play any significant role, TLR-2 deficiency accelerated the onset and markedly increased the severity of acute immune complex-driven arthritis in mice. TLR-2 deficiency resulted in a substantial increase in joint inflammation, bone erosion, and cartilage pathology, indicating a protective function of TLR-2 in passive FcγR-driven disease. Ex vivo study of the macrophage inflammatory phenotype revealed increased production of tumor necrosis factor α (TNFα) and interleukin-6 (IL-6) despite similar levels of IL-10, along with a significant increase in FcγR-specific response, in TLR-2-/- mouse macrophages early in the disease. Although distinct FcγR messenger RNA expression was not affected, cell surface protein expression of the inhibitory FcγRIIB in TLR-2-/- naive primary macrophages was specifically diminished, resulting in a higher proinflammatory response. Accordingly, TLR-2 stimulation specifically up-regulated FcγRIIB, but not the activating FcγR, on macrophages. CONCLUSION: TLR-2 regulates acute immune complex-driven arthritis by controlling macrophage FcγR response. Our findings indicate that the protective role of TLR-2 is extended beyond its previously described role in promoting Treg cells during T cell-mediated arthritis.
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Complejo Antígeno-Anticuerpo/fisiología , Artritis Experimental/fisiopatología , Receptores de IgG/fisiología , Transducción de Señal/fisiología , Receptor Toll-Like 2/fisiología , Animales , Artritis Experimental/inmunología , Artritis Experimental/patología , Células Cultivadas , Modelos Animales de Enfermedad , Femenino , Técnicas In Vitro , Interleucina-6/metabolismo , Macrófagos/metabolismo , Macrófagos/patología , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Suero/inmunología , Índice de Severidad de la Enfermedad , Receptor Toll-Like 2/deficiencia , Receptor Toll-Like 2/genética , Receptor Toll-Like 4/deficiencia , Receptor Toll-Like 4/genética , Receptor Toll-Like 4/fisiología , Factor de Necrosis Tumoral alfa/metabolismoRESUMEN
OBJECTIVE: Increasing evidence indicates the involvement of Toll-like receptors (TLRs) in the progression of arthritis; however, the contribution of the two signaling pathways used by TLRs, which are mediated by myeloid differentiation factor 88 (MyD88) and TRIF, remains unclear. The objective of this study was to investigate the specific roles of MyD88 and TRIF in chronic experimental arthritis and the accompanying adaptive immune responses. METHODS: Chronic arthritis was induced in wild-type, MyD88(-/-) , and Trif(lps2) (TRIF(-/-) ) mice by repetitive intraarticular injections of streptococcal cell wall (SCW) fragments. SCW-specific T cell and B cell responses, joint swelling, and histopathologic changes were analyzed during chronic arthritis. RESULTS: Both MyD88 and TRIF pathways contributed to antigen-specific T cell proliferation and antibody production, with the MyD88 pathway playing the dominant role. The severity of joint swelling and synovial inflammation, as well as the histopathologic damage to cartilage and bone, was strongly dependent on MyD88 signaling, whereas TRIF was redundant. MyD88 signaling was critical for the development of pathogenic T cell response (i.e., interleukin-17 [IL-17] production) in response to SCW antigen. Interestingly, when the T cell-dependent phase was prolonged, TRIF signaling appeared to down-regulate bone erosion, an effect accompanied by an inhibitory effect on IL-17 production. CONCLUSION: This study reveals a central role of MyD88 and a counterregulatory function of TRIF in T cell-driven arthritis. The findings provide a rationale for a pathway-specific interference in order to block the pathogenic features and to preserve or stimulate the beneficial aspects of TLR signaling.
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Inmunidad Adaptativa , Proteínas Adaptadoras del Transporte Vesicular/metabolismo , Artritis Experimental/metabolismo , Factor 88 de Diferenciación Mieloide/metabolismo , Transducción de Señal/inmunología , Receptores Toll-Like/metabolismo , Animales , Artritis Experimental/inmunología , Artritis Experimental/patología , Cartílago Articular/inmunología , Cartílago Articular/metabolismo , Cartílago Articular/patología , Proliferación Celular , Interleucina-17/inmunología , Interleucina-17/metabolismo , Articulaciones/inmunología , Articulaciones/metabolismo , Articulaciones/patología , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Índice de Severidad de la Enfermedad , Membrana Sinovial/inmunología , Membrana Sinovial/metabolismo , Membrana Sinovial/patología , Linfocitos T/metabolismo , Linfocitos T/patologíaRESUMEN
Joint pain severity in arthritic diseases differs between sexes and is often more pronounced in women. This disparity is thought to stem from biological mechanisms, particularly innate immunity, yet the understanding of sex-specific differences in arthritic pain remains incomplete. This study aims to investigate these disparities using an innate immunity-driven inflammation model induced by intra-articular injections of Streptococcus Cell Wall fragments to mimic both acute and pre-sensitized joint conditions. Nociceptive behavior was evaluated via gait analysis and static weight-bearing, and inflammation was evaluated via joint histology and the synovial gene expression involved in immune response. Although acute inflammation and pain severity were comparable between sexes, distinct associations between synovial inflammatory gene expression and static nociceptive behavior emerged. These associations delineated sex-specific relationships with pain, highlighting differential gene interactions (Il6 versus Cybb on day 1 and Cyba/Gas6 versus Nos2 on day 8) between sexes. In conclusion, our study found that, despite similar pain severity between sexes, the association of inflammatory synovial genes revealed sex-specific differences in the molecular inflammatory mechanisms underlying pain. These findings suggest a path towards more personalized treatment strategies for pain management in arthritis and other inflammatory joint diseases.
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Sinovitis , Masculino , Humanos , Ratones , Femenino , Animales , Sinovitis/metabolismo , Dolor , Inflamación/complicaciones , Artralgia , Inmunidad InnataRESUMEN
Background: Osteoarthritis (OA) is a progressive joint disease and a major cause of chronic pain in adults. The prevalence of OA is higher in female patients, who tend to have worse OA outcomes, partially due to pain. The association between joint pain and OA pathology is often inconclusive. Preclinical research studies have largely overlooked sex as a potential determinant in joint pain during OA. This study aimed to investigate the role of sex in joint pain in the collagenase-induced OA (CiOA) model and its link with joint pathology. Methods: Multiple aspects of pain were evaluated during identically executed experiments of CiOA in male and female C57BL/6J mice. Cartilage damage, osteophyte formation, synovial thickness, and cellularity were assessed by histology on day 56. The association between pain and pathology was investigated, disaggregated by sex. Results: Differences in pain behavior between sexes were found in the majority of the evaluated pain methods. Females displayed lower weight bearing ability in the affected leg compared to males during the early phase of the disease, however, the pathology at the end stage was comparable between sexes. In the second cohort, males displayed increased mechanical sensitivity in the affected joint compared to females but also showed more cartilage damage at the end stage of the model. Within this cohort, gait analysis showed varied results. Males used the affected paw less often and displayed dynamic weight-bearing compensation in the early phase of the model. These differences were not observed in females. Other evaluated parameters displayed comparable gait behavior between males and females. A detailed analysis of individual mice revealed that seven out of 10 pain measurements highly correlated with OA histopathology in females (Pearson r range: 0.642-0.934), whereas in males this measurement was only two (Pearson r range: 0.645-0.748). Conclusion: Our data show that sex is a determinant in the link between pain-related behavior with OA features. Therefore, to accurately interpret pain data it is crucial to segregate data analysis by sex to draw the correct mechanistic conclusion.
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Osteoartritis , Ratones , Masculino , Femenino , Animales , Ratones Endogámicos C57BL , Osteoartritis/etiología , Dolor/etiología , Artralgia/complicaciones , MarchaRESUMEN
TGF-ß1 is an important growth factor to promote the differentiation of T helper 17 (Th17) and regulatory T cells (Treg). The potential of TGF-ß1 as therapeutic target in T cell-mediated diseases like rheumatoid arthritis (RA) is unclear. We investigated the effect of TGF-ß1 inhibition on murine Th17 differentiation in vitro, on human RA synovial explants ex vivo, and on the development of experimental arthritis in vivo. Murine splenocytes were differentiated into Th17 cells, and the effect of the TGF-ßRI inhibitor SB-505124 was studied. Synovial biopsies were cultured in the presence or absence of SB-505124. Experimental arthritis was induced in C57Bl6 mice and treated daily with SB-505124. Flow cytometry analysis was performed to measure different T cell subsets. Histological sections were analysed to determine joint inflammation and destruction. SB-505124 potently reduced murine Th17 differentiation by decreasing Il17a and Rorc gene expression and IL-17 protein production. SB-505124 significantly suppressed IL-6 production by synovial explants. In vivo, SB-505124 reduced Th17 numbers, while increased numbers of Tregs were observed. Despite this skewed Th17/Treg balance, SB-505124 treatment did not result in suppression of joint inflammation and destruction. Blocking TGF-ß1 signalling suppresses Th17 differentiation and improves the Th17/Treg balance. However, local SB-505124 treatment does not suppress experimental arthritis.
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Artritis Experimental/metabolismo , Citocinas/metabolismo , Linfocitos T Reguladores/metabolismo , Células Th17/metabolismo , Factor de Crecimiento Transformador beta1/metabolismo , Animales , Benzodioxoles/farmacología , Diferenciación Celular/efectos de los fármacos , Femenino , Humanos , Imidazoles/farmacología , Interleucina-17/metabolismo , Ratones , Ratones Endogámicos C57BL , Miembro 3 del Grupo F de la Subfamilia 1 de Receptores Nucleares/metabolismo , Piridinas/farmacología , Transducción de Señal , Linfocitos T Reguladores/efectos de los fármacos , Células Th17/efectos de los fármacos , Técnicas de Cultivo de Tejidos/métodosRESUMEN
Macrophages play a crucial role in the initiation and progression of rheumatoid arthritis (RA). Liposomes can be used to deliver therapeutics to macrophages by exploiting their phagocytic ability. However, since macrophages serve as the immune system's first responders, it is inadvisable to systemically deplete these cells. By loading the liposomes with the photosensitizer IRDye700DX, we have developed and tested a novel way to perform photodynamic therapy (PDT) on macrophages in inflamed joints. PEGylated liposomes were created using the film method and post-inserted with micelles containing IRDye700DX. For radiolabeling, a chelator was also incorporated. RAW 264.7 cells were incubated with liposomes with or without IRDye700DX and exposed to 689 nm light. Viability was determined using CellTiterGlo. Subsequently, biodistribution and PDT studies were performed on mice with collagen-induced arthritis (CIA). PDT using IRDye700DX-loaded liposomes efficiently induced cell death in vitro, whilst no cell death was observed using the control liposomes. Biodistribution of the two compounds in CIA mice was comparable with excellent correlation of the uptake with macroscopic and microscopic arthritis scores. Treatment with 700DX-loaded liposomes significantly delayed arthritis development. Here we have shown the proof-of-principle of performing PDT in arthritic joints using IRDye700DX-loaded liposomes, allowing locoregional treatment of arthritis.
RESUMEN
Osteoarthritis (OA) is a destructive disease of the joint with age and obesity being its most important risk factors. Around 50% of OA patients suffer from inflammation of the synovial joint capsule, which is characterized by increased abundance and activation of synovial macrophages that produce reactive oxygen species (ROS) via NADPH-oxidase 2 (NOX2). Both ROS and high blood levels of low-density lipoprotein (LDL) are implicated in OA pathophysiology, which may interact to form oxidized LDL (oxLDL) and thereby promote disease. Therefore, targeting NOX2 could be a viable treatment strategy for OA. Collagenase-induced OA (CiOA) was used to compare pathology between wild-type (WT) and Nox2 knockout (Nox2-/-) C57Bl/6 mice. Mice were either fed a standard diet or Western diet (WD) to study a possible interaction between NOX2-derived ROS and LDL. Synovial inflammation, cartilage damage and ectopic bone size were assessed on histology. Extracellular ROS production by macrophages was measured in vitro using the Amplex Red assay. Nox2-/- macrophages produced basal levels of ROS but were unable to increase ROS production in response to the alarmin S100A8 or the phorbol ester PMA. Interestingly, Nox2 deficiency reduced cartilage damage, synovial lining thickness and ectopic bone size, whereas these disease parameters were not affected by WD-feeding. These results suggest that NOX2-derived ROS are involved in CiOA development.
RESUMEN
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.
Asunto(s)
Artritis Experimental/patología , Microbioma Gastrointestinal/efectos de los fármacos , Proteína Antagonista del Receptor de Interleucina 1/genética , Oligosacáridos/farmacología , Animales , Densidad Ósea/efectos de los fármacos , Suplementos Dietéticos , Heces/microbiología , Femenino , Proteína Antagonista del Receptor de Interleucina 1/deficiencia , Lactobacillus/genética , Lactobacillus/aislamiento & purificación , Masculino , Ratones , Ratones Endogámicos BALB C , Ratones Noqueados , Prebióticos , Receptores de Interleucina-1 , Subgrupos de Linfocitos T/citología , Subgrupos de Linfocitos T/metabolismoRESUMEN
Background: Injection of adipose-derived mesenchymal stromal cells (ASCs) into murine knee joints after induction of inflammatory collagenase-induced osteoarthritis (CiOA) reduces development of joint pathology. This protection is only achieved when ASCs are applied in early CiOA, which is characterized by synovitis and high S100A8/A9 and IL-1ß levels, suggesting that inflammation is a prerequisite for the protective effect of ASCs. Our objective was to gain more insight into the interplay between synovitis and ASC-mediated amelioration of CiOA pathology. Methods: CiOA was induced by intra-articular collagenase injection. Knee joint sections were stained with hematoxylin/eosin and immunolocalization of polymorphonuclear cells (PMNs) and ASCs was performed using antibodies for NIMP-R14 and CD271, respectively. Chemokine expression induced by IL-1ß or S100A8/A9 was assessed with qPCR and Luminex. ASC-PMN co-cultures were analyzed microscopically and with Luminex for inflammatory mediators. Migration of PMNs through transwell membranes toward conditioned medium of non-stimulated ASCs (ASCNS-CM) or IL-1ß-stimulated ASCs (ASCIL-1ß-CM) was examined using flow cytometry. Phagocytic capacity of PMNs was measured with labeled zymosan particles. Results: Intra-articular saline injection on day 7 of CiOA increased synovitis after 6 h, characterized by PMNs scattered throughout the joint cavity and the synovium. ASC injection resulted in comparable numbers of PMNs which clustered around ASCs in close interaction with the synovial lining. IL-1ß-stimulation of ASCs in vitro strongly increased expression of PMN-attracting chemokines CXCL5, CXCL7, and KC, whereas S100A8/A9-stimulation did not. In agreement, the number of clustered PMNs per ASC was significantly increased after 6 h of co-culturing with IL-1ß-stimulated ASCs. Also migration of PMNs toward ASCIL-1ß-CM was significantly enhanced (287%) when compared to ASCNS-CM. Interestingly, association of PMNs with ASCs significantly diminished KC protein release by ASCs (69% lower after 24 h), accompanied by reduced release of S100A8/A9 protein by the PMNs. Moreover, phagocytic capacity of PMNs was strongly enhanced after priming with ASCIL-1ß-CM. Conclusions: Local application of ASCs in inflamed CiOA knee joints results in clustering of attracted PMNs with ASCs in the synovium, which is likely mediated by IL-1ß-induced up-regulation of chemokine release by ASCs. This results in enhanced phagocytic capacity of PMNs, enabling the clearance of debris to attenuate synovitis.
Asunto(s)
Interleucina-1beta/fisiología , Trasplante de Células Madre Mesenquimatosas , Células Madre Mesenquimatosas/fisiología , Neutrófilos/fisiología , Osteoartritis de la Rodilla/terapia , Fagocitosis , Animales , Artritis Experimental/terapia , Células Cultivadas , Quimiocinas/fisiología , Femenino , Humanos , Ratones , Ratones Endogámicos C57BL , Neutrófilos/inmunologíaRESUMEN
[This corrects the article DOI: 10.1371/journal.pone.0219366.].