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OBJECTIVES: Systemic sclerosis (SSc) is characterized by multiple clinical manifestations. Vasculopathy is a main disease hallmark and ranges in severity from an exacerbated Raynaud phenomenon to pulmonary arterial hypertension (PAH). The potential involvement of the immune system in SSc-associated vascular abnormalities is not clear. Here, we set out to study SSc-related immune parameters and determine whether and which peripheral T cell subsets associate with vascular severity in SSc patients. METHODS: Peripheral blood and clinical data were collected from 30 SSc patients, 5 patients with idiopathic PAH and 15 age and sex-matched healthy donors (HD). In this cross-sectional cohort, SSc patients with PAH (n = 15) were matched for their age, sex and medication with SSc patients with no signs of PAH (n = 15). Lymphocyte subsets were quantified by multi-colour flow cytometry. RESULTS: SSc patients exhibited elevated percentages of T peripheral helper cells (Tph), CD4+GZMB+ T cells and decreased levels of Th1 cells compared with HD. Increased presence of both CD4+ and CD8+ exhausted-like (CD28-) T cells, characterized by raised cytokine and cytotoxic signature, was also observed in SSc compared with HD blood. Furthermore, IL-4 expressing CD4+CD8+ T cells were significantly increased in SSc peripheral blood. Interestingly, the presence of PAH in SSc was accompanied by a distinct T helper profile, characterized by raised percentages of Th17 and Tph cells. CONCLUSION: SSc patients with severe vasculopathy (presence of PAH) exhibited a distinct T cell profile, suggesting a potential role of auto-immune inflammation in SSc vascular complications.
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Esclerodermia Sistémica , Humanos , Esclerodermia Sistémica/inmunología , Esclerodermia Sistémica/complicaciones , Femenino , Masculino , Persona de Mediana Edad , Estudios Transversales , Adulto , Linfocitos T Colaboradores-Inductores/inmunología , Hipertensión Arterial Pulmonar/inmunología , Hipertensión Arterial Pulmonar/etiología , Estudios de Casos y Controles , Anciano , Hipertensión Pulmonar/etiología , Hipertensión Pulmonar/inmunología , Citometría de FlujoRESUMEN
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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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
OBJECTIVE: Transforming growth factor-ß (TGF-ß) signaling via SMAD2/3 is crucial to control cartilage homeostasis. However, TGF-ß can also have detrimental effects by signaling via SMAD1/5/9 and thereby contribute to diseases like osteoarthritis (OA). In this study, we aimed to block TGF-ß-induced SMAD1/5/9 signaling in primary human OA chondrocytes, while maintaining functional SMAD2/3 signaling. DESIGN: Human OA chondrocytes were pre-incubated with different concentrations of ALK4/5/7 kinase inhibitor SB-505124 before stimulation with TGF-ß. Changes in SMAD C-terminal phosphorylation were analyzed using Western blot and response genes were measured with quantitative Polymerase Chain Reaction. To further explore the consequences of our ability to separate pathways, we investigated TGF-ß-induced chondrocyte hypertrophy. RESULTS: Pre-incubation with 0.5 µM SB-505124, maintained ±50% of C-terminal SMAD2/3 phosphorylation and induction of JUNB and SERPINE1, but blocked SMAD1/5/9-C phosphorylation and expression of ID1 and ID3. Furthermore, TGF-ß, in levels comparable to those in the synovial fluid of OA patients, resulted in regulation of hypertrophic and dedifferentiation markers in OA chondrocytes; i.e. an increase in COL10, RUNX2, COL1A1, and VEGF and a decrease in ACAN expression. Interestingly, in a subgroup of OA chondrocyte donors, blocking only SMAD1/5/9 caused stronger inhibition on TGF-ß-induced RUNX2 than blocking both SMAD pathways. CONCLUSION: Our findings indicate that using low dose of SB-505124 we maintained functional SMAD2/3 signaling that blocks RUNX2 expression in a subgroup of OA patients. We are the first to show that SMAD2/3 and SMAD1/5/9 pathways can be separately modulated using low and high doses of SB-505124 and thereby split TGF-ß's detrimental from protective function in chondrocytes.
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Cartílago Articular , Osteoartritis , Humanos , Condrocitos/metabolismo , Fosforilación , Factor de Crecimiento Transformador beta/farmacología , Factor de Crecimiento Transformador beta/metabolismo , Subunidad alfa 1 del Factor de Unión al Sitio Principal , Cartílago Articular/metabolismo , Osteoartritis/metabolismo , Proteína Smad2/metabolismoRESUMEN
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
During osteoarthritis (OA), hypertrophy-like chondrocytes contribute to the disease process. TGF-ß's signaling pathways can contribute to a hypertrophy(-like) phenotype in chondrocytes, especially at high doses of TGF-ß. In this study, we examine which transcription factors (TFs) are activated and involved in TGF-ß-dependent induction of a hypertrophy-like phenotype in human OA chondrocytes. We found that TGF-ß, at levels found in synovial fluid in OA patients, induces hypertrophic differentiation, as characterized by increased expression of RUNX2, COL10A1, COL1A1, VEGFA and IHH. Using luciferase-based TF activity assays, we observed that the expression of these hypertrophy genes positively correlated to SMAD3:4, STAT3 and AP1 activity. Blocking these TFs using specific inhibitors for ALK-5-induced SMAD signaling (5 µM SB-505124), JAK-STAT signaling (1 µM Tofacitinib) and JNK signaling (10 µM SP-600125) led to the striking observation that only SB-505124 repressed the expression of hypertrophy factors in TGF-ß-stimulated chondrocytes. Therefore, we conclude that ALK5 kinase activity is essential for TGF-ß-induced expression of crucial hypertrophy factors in chondrocytes.
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Condrocitos , Osteoartritis , Condrocitos/metabolismo , Humanos , Hipertrofia/metabolismo , Osteoartritis/genética , Osteoartritis/metabolismo , Fenotipo , Factor de Crecimiento Transformador beta/metabolismo , Factores de Crecimiento Transformadores/genética , Factores de Crecimiento Transformadores/metabolismoRESUMEN
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
Osteoarthritis (OA) is characterized by progressive articular cartilage loss. Human mesenchymal stromal cells (MSCs) can be used for cartilage repair therapies based on their potential to differentiate into chondrocytes. However, the joint microenvironment is a major determinant of the success of MSC-based cartilage formation. Currently, there is no tool that is able to predict the effect of a patient's OA joint microenvironment on MSC-based cartilage formation. Our goal was to develop a molecular tool that can predict this effect before the start of cartilage repair therapies. Six different promoter reporters (hIL6, hIL8, hADAMTS5, hWISP1, hMMP13, and hADAM28) were generated and evaluated in an immortalized human articular chondrocyte for their responsiveness to an osteoarthritic microenvironment by stimulation with OA synovium-conditioned medium (OAs-cm) obtained from 32 different knee OA patients. To study the effect of this OA microenvironment on MSC-based cartilage formation, MSCs were cultured in a three-dimensional pellet culture model, while stimulated with OAs-cm. Cartilage formation was assessed histologically and by quantifying sulfated glycosaminoglycan (sGAG) production. We confirmed that OAs-cm of different patients had significantly different effects on sGAG production. In addition, significant correlations were obtained between the effect of the OAs-cm on cartilage formation and promoter reporter outcome. Furthermore, we validated the predictive value of measuring two promoter reporters with an independent cohort of OAs-cm and the effect of 87.5% of the OAs-cm on MSC-based cartilage formation could be predicted. Together, we developed a novel tool to predict the effect of the OA joint microenvironment on MSC-based cartilage formation. This is an important first step toward personalized cartilage repair strategies for OA patients. Impact statement We describe the development of a novel molecular tool to predict if an osteoarthritis joint microenvironment is permissive for cartilage repair or not. Such a tool is of great importance in determining the success of mesenchymal stromal cell-based cartilage repair strategies.
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Cartílago Articular , Células Madre Mesenquimatosas , Osteoartritis de la Rodilla , Cartílago Articular/patología , Condrocitos/patología , Condrogénesis/fisiología , Humanos , Osteoartritis de la Rodilla/patologíaRESUMEN
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: 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
BACKGROUND: Chondrogenic differentiation of mesenchymal stem cells (MSC) requires transforming growth factor beta (TGFß) signaling. TGFß binds to the type I receptor activin-like kinase (ALK)5 and results in C-terminal SMAD2/3 phosphorylation (pSMAD2/3C). In turn pSMAD2/3C translocates to the nucleus and regulates target gene expression. Inflammatory mediators are known to exert an inhibitory effect on MSC differentiation. In this study we investigated the effect of interleukin 1 ß (IL1ß) on SMAD2/3 signaling dynamics and post-translational modifications. RESULTS: Co-stimulation of MSC with TGFß and IL1ß did not affect peak pSMAD2C levels at 1h post-stimulation. Surprisingly, SMAD3 transcriptional activity, as determined by the CAGA12-luciferase reporter construct, was enhanced by co-stimulation of TGFß and IL1ß compared to TGFß alone. Furthermore, IL1ß stimulation induced CAGA12-luciferase activity in a SMAD dependent way. As SMAD function can be modulated independent of canonical TGFß signaling through the SMAD linker domain, we studied SMAD2 linker phosphorylation at specific threonine and serine residues. SMAD2 linker threonine and serine modifications were observed within 1h following TGFß, IL1ß or TGFß and IL1ß stimulation. Upon co-stimulation linker modified SMAD2 accumulated in the cytoplasm and SMAD2/3 target gene transcription (ID1, JUNB) at 2-4h was inhibited. A detailed time course analysis of IL1ß-induced SMAD2 linker modifications revealed a distinct temperospatial pattern compared to TGFß. Co-stimulation with both factors resulted in a similar kinetic profile as TGFß alone. Nevertheless, IL1ß did subtly alter TGFß-induced pSMAD2C levels between 8 and 24h post-stimulation, which was reflected by TGFß target gene expression (PAI1, JUNB). Direct evidence for the importance of SMAD3 linker modifications for the effect of IL1ß on TGFß signaling was obtained by over-expression of SMAD3 or a SMAD3 linker phospho-mutant. Finally, an inhibitor screening was performed to identify kinases involved in SMAD2/3 linker modifications. We identified TAK1 kinase activity as crucial for IL1ß-induced SMAD2 linker modifications and CAGA12-luciferase activity. CONCLUSIONS: TGFß and IL1ß signaling interact at the SMAD2/3 level in human primary MSC. Down-stream TGFß target genes were repressed by IL1ß independent of C-terminal SMAD2 phosphorylation. We demonstrate that SMAD2/3 linker modifications are required for this interplay and identified TAK1 as a crucial mediator of IL1ß-induced TGFß signal modulation.
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Interleucina-1beta/genética , Quinasas Quinasa Quinasa PAM/genética , Proteína Smad2/genética , Proteína smad3/genética , Factor de Crecimiento Transformador beta/genética , Diferenciación Celular/genética , Condrogénesis/genética , Regulación del Desarrollo de la Expresión Génica/efectos de los fármacos , Humanos , Interleucina-1beta/administración & dosificación , Interleucina-1beta/metabolismo , Células Madre Mesenquimatosas/metabolismo , Fosforilación , Procesamiento Proteico-Postraduccional , Transducción de Señal/genética , Factor de Crecimiento Transformador beta/administración & dosificación , Factor de Crecimiento Transformador beta/metabolismoRESUMEN
To improve cartilage formation by bone marrow-derived mesenchymal stem cells (BMSCs), the signaling mechanism governing chondrogenic differentiation requires better understanding. We previously showed that the transforming growth factor-ß (TGFß) receptor ALK5 is crucial for chondrogenesis induced by TGFß. ALK5 phosphorylates SMAD2 and SMAD3 proteins, which then form complexes with SMAD4 to regulate gene transcription. By modulating the expression of SMAD2, SMAD3 and SMAD4 in human BMSCs, we investigated their role in TGFß-induced chondrogenesis. Activation of TGFß signaling, represented by SMAD2 phosphorylation, was decreased by SMAD2 knockdown and highly increased by SMAD2 overexpression. Moreover, TGFß signaling via the alternative SMAD1/5/9 pathway was strongly decreased by SMAD4 knockdown. TGFß-induced chondrogenesis of human BMSCs was strongly inhibited by SMAD4 knockdown and only mildly inhibited by SMAD2 knockdown. Remarkably, both knockdown and overexpression of SMAD3 blocked chondrogenic differentiation. Chondrogenesis appears to rely on a delicate balance in the amount of SMAD3 and SMAD4 as it was not enhanced by SMAD4 overexpression and was inhibited by SMAD3 overexpression. Furthermore, this study reveals that TGFß-activated phosphorylation of SMAD2 and SMAD1/5/9 depends on the abundance of SMAD4. Overall, our findings suggest a more dominant role for SMAD3 and SMAD4 than SMAD2 in TGFß-induced chondrogenesis of human BMSCs.
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Diferenciación Celular , Condrogénesis , Células Madre Mesenquimatosas/fisiología , Proteína Smad2/metabolismo , Proteína smad3/metabolismo , Proteína Smad4/metabolismo , Factor de Crecimiento Transformador beta/metabolismo , Células Cultivadas , Expresión Génica , Técnicas de Silenciamiento del Gen , Humanos , Fosforilación , Procesamiento Proteico-Postraduccional , Transducción de SeñalRESUMEN
Objective: A crucial feature of OA is cartilage degradation. This process is mediated by pro-inflammatory cytokines, among other factors, via induction of matrix-degrading enzymes. Interleukin 37 (IL37) is an anti-inflammatory cytokine and is efficient in blocking the production of pro-inflammatory cytokines during innate immune responses. We hypothesize that IL37 is therapeutic in treating the inflammatory cytokine cascade in human OA chondrocytes and can act as a counter-regulatory cytokine to reduce cartilage degradation in OA. Methods: Human OA cartilage was obtained from patients undergoing total knee or hip arthroplasty. Immunohistochemistry was applied to study IL37 protein expression in cartilage biopsies from OA patients. Induction of IL37 expression by IL1ß, OA synovium-conditioned medium and TNFα was investigated in human OA chondrocytes. Adenoviral overexpression of IL37 followed by IL1ß stimulation was performed to investigate the anti-inflammatory potential of IL37. Results: IL37 expression was detected in cartilage biopsies of OA patients and induced by IL1ß. After IL1ß stimulation, increased IL1ß, IL6 and IL8 expression was observed in OA chondrocytes. Elevated IL37 levels diminished the IL1ß-induced IL1ß , IL6 and IL8 gene levels and IL1ß and IL8 protein levels. In addition to the reduction in pro-inflammatory cytokine expression, IL37 reduced MMP1 , MMP3 , MMP13 and disintegrin and metalloproteinase with thrombospondin motifs 5 gene levels and MMP3 and MMP13 protein levels. Conclusion: IL37 is induced by IL1ß, and IL37 itself reduced IL1ß, IL6 and IL8 production, indicating that IL37 is able to induce a counter-regulatory anti-inflammatory feedback loop in chondrocytes. In addition, IL37 dampens catabolic enzyme expression. This supports IL37 as a potential therapeutic target in OA.
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Condrocitos/metabolismo , Interleucina-1/metabolismo , Interleucina-1beta/farmacología , Osteoartritis , ARN Mensajero/metabolismo , Factor de Necrosis Tumoral alfa/farmacología , Adenoviridae , Western Blotting , Condrocitos/efectos de los fármacos , Desintegrinas/efectos de los fármacos , Desintegrinas/genética , Desintegrinas/metabolismo , Humanos , Inmunohistoquímica , Interleucina-1/genética , Interleucina-1beta/efectos de los fármacos , Interleucina-1beta/genética , Interleucina-1beta/metabolismo , Interleucina-6/genética , Interleucina-6/metabolismo , Interleucina-8/efectos de los fármacos , Interleucina-8/genética , Interleucina-8/metabolismo , Metaloproteinasa 1 de la Matriz/efectos de los fármacos , Metaloproteinasa 1 de la Matriz/genética , Metaloproteinasa 1 de la Matriz/metabolismo , Metaloproteinasa 13 de la Matriz/efectos de los fármacos , Metaloproteinasa 13 de la Matriz/genética , Metaloproteinasa 13 de la Matriz/metabolismo , Metaloproteinasa 3 de la Matriz/efectos de los fármacos , Metaloproteinasa 3 de la Matriz/genética , Metaloproteinasa 3 de la Matriz/metabolismo , ARN Mensajero/efectos de los fármacos , Reacción en Cadena en Tiempo Real de la PolimerasaRESUMEN
OBJECTIVE: RNA-binding protein with multiple splicing (RBPMS) has been shown to physically interact with Smads and enhance transforming growth factor-ß (TGF-ß)-mediated Smad2/3 transcriptional activity in mammalian cells. Objective of this study was to examine whether expression of RBPMS is regulated by interleukin-1ß (IL)-1ß and TGF-ß superfamily growth factors and whether expression of RBPMS is altered during aging and experimental osteoarthritis. METHODS: Expression of RBPMS protein was investigated in chondrocyte cell lines of murine (H4) and human (G6) origin using Western blot analysis. Regulation of RBPMS expression in H4 chondrocytes at mRNA level was done by reverse transcriptase-quantitative polymerase chain reaction. Furthermore, characterization of Smad signaling pathways regulating RBPMS expression was performed by blocking studies using small molecule inhibitors or by transfection studies with adenoviral vector constructs (constitutive-active ALK1 and constitutive-active ALK5). Expression of RBPMS in cartilage of different age groups of C57BL/6N mice (6 months and 20 months) and in a surgically induced osteoarthritis (OA) mouse model was analyzed using immunohistochemistry. RESULTS: RBPMS was shown to be expressed in chondrocytes and cartilage of murine, human, and bovine origin. TGF-ß inhibited RBPMS expression while BMP2 and IL-1ß increased its expression. TGF-ß-induced inhibition was blocked by ALK5 inhibitor. Overexpression of ca-ALK1 stimulated RBPMS expression. Moreover, RBPMS expression was found to be reduced with ageing and in OA pathogenesis. CONCLUSIONS: Expression of RBPMS in chondrocytes is regulated by TGF-ß superfamily members and IL-1ß, indicating a counter-regulatory mechanism. Expression of RBPMS, in cartilage and its reduction during ageing and OA might suggest its potential role in the maintenance of normal articular cartilage.
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OBJECTIVE: To rescue chondrogenic differentiation of human mesenchymal stem cells (hMSCs) in osteoarthritic conditions by inhibition of protein kinases. METHODS: hMSCs were cultured in pellets. During early chondrogenic differentiation, these were exposed to osteoarthritic synovium-conditioned medium (OAS-CM), combined with the Janus kinase (JAK)-inhibitor tofacitinib and/or the transforming growth factor ß-activated kinase 1 (TAK1)-inhibitor oxozeaenol. To evaluate effects on chondrogenesis, the glycosaminoglycan (GAG) content of the pellets was measured at the time that chondrogenesis was manifest in control cultures. Moreover, mRNA levels of matrix molecules and enzymes were measured during this process, using real-time polymerase chain reaction (RT-PCR). Initial experiments were performed with hMSCs from a fetal donor, and results of these studies were confirmed with hMSCs from adult donors. RESULTS: Exposure to OAS-CM resulted in pellets with a much lower GAG content, reflecting inhibited chondrogenic differentiation. This was accompanied by decreased mRNA levels of aggrecan, type II collagen, and Sox9, and increased levels of matrix metalloproteinase (MMP)1, MMP3, MMP13, ADAMTS4, and ADAMTS5. Both tofacitinib (JAK-inhibitor) and oxozeaenol (TAK1 inhibitor) significantly increased the GAG content of the pellets in osteoarthritis (OA)-like conditions. The combination of both protein kinase inhibitors showed an additive effect on GAG content. In agreement with this, in the presence of OAS-CM, both tofacitinib and oxozeaenol increased mRNA expression of sox9. The expression of aggrecan and type II collagen was also up-regulated, but this only reached significance for aggrecan after TAK1 inhibition. Both inhibitors decreased the mRNA levels of MMP1, 3, and 13 in the presence of OAS-CM. Moreover, oxozeaenol also significantly down-regulated the mRNA levels of aggrecanases ADAMTS4 and ADAMTS5. When combined, the inhibitors caused additive reduction of OA-induced MMP1 mRNA expression. Counteraction of OAS-CM-induced inhibition of chondrogenesis by these protein kinase inhibitors was confirmed with hMSCs of two different adult donors. Both tofacitinib and oxozeaenol significantly improved GAG content in cell pellets from these adult donors. CONCLUSIONS: Tofacitinib and oxozeaenol partially prevent the inhibition of chondrogenesis by factors secreted by OA synovium. Their effects are additive. This indicates that these protein kinase inhibitors can potentially be used to improve cartilage formation under the conditions occurring in osteoathritic, or otherwise inflamed, joints.
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Diferenciación Celular/efectos de los fármacos , Condrogénesis/efectos de los fármacos , Quinasas Janus/antagonistas & inhibidores , Quinasas Quinasa Quinasa PAM/antagonistas & inhibidores , Células Madre Mesenquimatosas/patología , Osteoartritis/patología , Inhibidores de Proteínas Quinasas/farmacología , Adulto , Cartílago Articular/efectos de los fármacos , Cartílago Articular/crecimiento & desarrollo , Cartílago Articular/patología , Feto/citología , Humanos , Quinasas Janus/metabolismo , Quinasas Quinasa Quinasa PAM/metabolismo , Células Madre Mesenquimatosas/enzimología , Piperidinas/farmacología , Pirimidinas/farmacología , Pirroles/farmacología , Factores de Tiempo , Zearalenona/farmacologíaRESUMEN
Haploinsufficiency of Euchromatin histone methyltransferase 1 (EHMT1), a chromatin modifying enzyme, is the cause of Kleefstra syndrome (KS). KS is an intellectual disability (ID) syndrome, with general developmental delay, hypotonia, and craniofacial dysmorphisms as additional core features. Recent studies have been focused on the role of EHMT1 in learning and memory, linked to the ID phenotype of KS patients. In this study we used the Ehmt1(+/-) mouse model, and investigated whether the core features of KS were mimicked in these mice. When comparing Ehmt1(+/-) mice to wildtype littermates we observed delayed postnatal growth, eye opening, ear opening, and upper incisor eruption, indicating a delayed postnatal development. Furthermore, tests for muscular strength and motor coordination showed features of hypotonia in young Ehmt1(+/-) mice. Lastly, we found that Ehmt1(+/-) mice showed brachycephalic crania, a shorter or bent nose, and hypertelorism, reminiscent of the craniofacial dysmorphisms seen in KS. In addition, gene expression analysis revealed a significant upregulation of the mRNA levels of Runx2 and several other bone tissue related genes in P28 Ehmt1(+/-) mice. Runx2 immunostaining also appeared to be increased. The mRNA upregulation was associated with decreased histone H3 lysine 9 dimethylation (H3K9me2) levels, the epigenetic mark deposited by Ehmt1, in the promoter region of these genes. Together, Ehmt1(+/-) mice indeed recapitulate KS core features and can be used as an animal model for Kleefstra syndrome. The increased expression of bone developmental genes in the Ehmt1(+/-) mice likely contributes to their cranial dysmorphisms and might be explained by diminished Ehmt1-induced H3K9 dimethylation.
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Huesos/metabolismo , Anomalías Craneofaciales/enzimología , Anomalías Craneofaciales/patología , Regulación del Desarrollo de la Expresión Génica/fisiología , Cardiopatías Congénitas/enzimología , Cardiopatías Congénitas/patología , N-Metiltransferasa de Histona-Lisina/deficiencia , Discapacidad Intelectual/enzimología , Discapacidad Intelectual/patología , Cráneo/anomalías , Análisis de Varianza , Animales , Inmunoprecipitación de Cromatina , Deleción Cromosómica , Cromosomas Humanos Par 9/enzimología , Discapacidades del Desarrollo/genética , Discapacidades del Desarrollo/patología , Masculino , Ratones , Ratones Noqueados , Hipotonía Muscular/genética , Hipotonía Muscular/patología , Osteopontina , Reacción en Cadena en Tiempo Real de la PolimerasaRESUMEN
Lysyl hydroxylase 2b (LH2b) is known to increase pyridinoline cross-links, making collagen less susceptible to enzymatic degradation. Previously, we observed a relationship between LH2b and osteoarthritis-related fibrosis in murine knee joint. For this study, we investigate if transforming growth factor-beta (TGF-ß) and connective tissue growth factor (CTGF) regulate procollagen-lysine, 2-oxoglutarate 5-dioxygenase 2 (PLOD2) (gene encoding LH2b) and LH2b expression differently in osteoarthritic human synovial fibroblasts (hSF). Furthermore, we investigate via which TGF-ß route (Smad2/3P or Smad1/5/8P) LH2b is regulated, to explore options to inhibit LH2b during fibrosis. To answer these questions, fibroblasts were isolated from knee joints of osteoarthritis patients. The hSF were stimulated with TGF-ß with or without a kinase inhibitor of ALK4/5/7 (SB-505124) or ALK1/2/3/6 (dorsomorphin). TGF-ß, CTGF, constitutively active (ca)ALK1 and caALK5 were adenovirally overexpressed in hSF. The gene expression levels of PLOD1/2/3, CTGF and COL1A1 were analyzed with Q-PCR. LH2 protein levels were determined with western blot. As expected, TGF-ß induced PLOD2/LH2 expression in hSF, whereas CTGF did not. PLOD1 and PLOD3 were not affected by either TGF-ß or CTGF. SB-505124 prevented the induction of TGF-ß-induced PLOD2, CTGF and COL1A1. Surprisingly, dorsomorphin completely blocked the induction of CTGF and COL1A1, whereas TGF-ß-induced PLOD2 was only slightly reduced. Overexpression of caALK5 in osteoarthritic hSF significantly induced PLOD2/LH2 expression, whereas caALK1 had no effect. We showed, in osteoarthritic hSF, that TGF-ß induced PLOD2/LH2 via ALK5 Smad2/3P. This elevation of LH2b in osteoarthritic hSF makes LH2b an interesting target to interfere with osteoarthritis-related persistent fibrosis.
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Factor de Crecimiento del Tejido Conjuntivo/metabolismo , Fibroblastos/metabolismo , Osteoartritis/metabolismo , Procolágeno-Lisina 2-Oxoglutarato 5-Dioxigenasa/genética , Proteínas Serina-Treonina Quinasas/metabolismo , Receptores de Factores de Crecimiento Transformadores beta/metabolismo , Factor de Crecimiento Transformador beta/metabolismo , Células Cultivadas , Fibroblastos/patología , Fibrosis/genética , Fibrosis/metabolismo , Fibrosis/patología , Regulación de la Expresión Génica , Humanos , Osteoartritis/genética , Osteoartritis/patología , Procolágeno-Lisina 2-Oxoglutarato 5-Dioxigenasa/metabolismo , Proteínas Serina-Treonina Quinasas/antagonistas & inhibidores , Proteínas Serina-Treonina Quinasas/genética , Receptor Tipo I de Factor de Crecimiento Transformador beta , Receptores de Factores de Crecimiento Transformadores beta/antagonistas & inhibidores , Receptores de Factores de Crecimiento Transformadores beta/genética , Transducción de Señal , Proteína Smad1/metabolismo , Proteína Smad2/metabolismoRESUMEN
Articular cartilage has a very limited intrinsic repair capacity leading to progressive joint damage. Therapies involving tissue engineering depend on chondrogenic differentiation of progenitor cells. This chondrogenic differentiation will have to survive in a diseased joint. We postulate that catabolic factors in this environment inhibit chondrogenesis of progenitor cells. We investigated the effect of a catabolic environment on chondrogenesis in pellet cultures of human mesenchymal stem cells (hMSCs). We exposed chondrogenically differentiated hMSC pellets, to interleukin (IL)-1α, tumor necrosis factor (TNF)-α or conditioned medium derived from osteoarthritic synovium (CM-OAS). IL-1α and TNF-α in CM-OAS were blocked with IL-1Ra or Enbrel, respectively. Chondrogenesis was determined by chondrogenic markers collagen type II, aggrecan, and the hypertrophy marker collagen type X on mRNA. Proteoglycan deposition was analyzed by safranin o staining on histology. IL-1α and TNF-α dose-dependently inhibited chondrogenesis when added at onset or during progression of differentiation, IL-1α being more potent than TNF-α. CM-OAS inhibited chondrogenesis on mRNA and protein level but varied in extent between patients. Inhibition of IL-1α partially overcame the inhibitory effect of the CM-OAS on chondrogenesis whereas the TNF-α contribution was negligible. We show that hMSC chondrogenesis is blocked by either IL-1α or TNF-α alone, but that there are additional factors present in CM-OAS that contribute to inhibition of chondrogenesis, demonstrating that catabolic factors present in OA joints inhibit chondrogenesis, thereby impairing successful tissue engineering.
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Condrogénesis/efectos de los fármacos , Medios de Cultivo Condicionados/farmacología , Células Madre Mesenquimatosas/citología , Células Madre Mesenquimatosas/efectos de los fármacos , Osteoartritis/metabolismo , Anciano , Proteína Morfogenética Ósea 2/farmacología , Diferenciación Celular/efectos de los fármacos , Femenino , Humanos , Interleucina-1alfa/farmacología , Masculino , Células Madre Mesenquimatosas/metabolismo , Persona de Mediana Edad , Factor de Crecimiento Transformador beta/farmacología , Factor de Necrosis Tumoral alfa/farmacologíaRESUMEN
The aim of this study was to investigate the roles of Smad2/3 and Smad1/5/8 phosphorylation in transforming growth factor-beta-induced chondrogenic differentiation of bone-marrow-derived mesenchymal stem cells (BMSCs) to assess whether specific targeting of different Smad signaling pathways offers possibilities to prevent terminal differentiation and mineralization of chondrogenically differentiated BMSCs. Terminally differentiated chondrocytes produced in vitro by chondrogenic differentiation of BMSCs or studied ex vivo during murine embryonic limb formation stained positive for both Smad2/3P and Smad1/5/8P. Hyaline-like cartilage produced in vitro by articular chondrocytes or studied in ex vivo articular cartilage samples that lacked expression for matrix metalloproteinase 13 and collagen X only expressed Smad2/3P. When either Smad2/3 or Smad1/5/8 phosphorylation was blocked in BMSC culture by addition of SB-505124 or dorsomorphin throughout culture, no collagen II expression was observed, indicating that both pathways are involved in early chondrogenesis. Distinct functions for these pathways were demonstrated when Smad signaling was blocked after the onset of chondrogenesis. Blocking Smad2/3P after the onset of chondrogenesis resulted in a halt in collagen II production. On the other hand, blocking Smad1/5/8P during this time period resulted in decreased expression of matrix metalloproteinase 13, collagen X, and alkaline phosphatase while allowing collagen II production. Moreover, blocking Smad1/5/8P prevented mineralization. This indicates that while Smad2/3P is important for continuation of collagen II deposition, Smad1/5/8 phosphorylation is associated with terminal differentiation and mineralization.