RESUMO
Local and systemic low-grade inflammation, mainly involving the innate immune system, plays an important role in the development of OA. A receptor playing a key role in initiation of this inflammation is the pattern-recognition receptor Toll-like receptor 4 (TLR4). In the joint, various ligands for TLR4, many of which are damage-associated molecular patterns (DAMPs), are present that can activate TLR4 signalling. This leads to the production of pro-inflammatory and catabolic mediators that cause joint damage. In this narrative review, we will first discuss the involvement of TLR4 ligands and signalling in OA. Furthermore, we will provide an overview of methods for inhibit, TLR4 signalling by RNA interference, neutralizing anti-TLR4 antibodies, small molecules and inhibitors targeting the TLR4 co-receptor MD2. Finally, we will focus on possible applications and challenges of these strategies in the dampening of inflammation in OA.
Assuntos
Osteoartrite , Receptor 4 Toll-Like , Humanos , Inflamação , Transdução de Sinais , AlarminasRESUMO
OBJECTIVES: It is well-known that long-term osteoarthritis prognosis is not improved by corticosteroid treatments. Here we investigate what could underlie this phenomenon by measuring the short term corticosteroid response of OA-Mf. METHODS: We determined the genome-wide transcriptomic response to corticosteroids of end-stage osteoarthritic joint synovial macrophages (OA-Mf). This was compared with LPS-tolerized and ß-glucan-trained circulating blood monocyte-derived macrophage models. RESULTS: Upon corticosteroid stimulation, the trained and tolerized macrophages significantly alter the abundance of 201 and 257 RNA transcripts, respectively. By contrast, by the same criteria, OA-Mf have a very restricted corticosteroid response of only 12 RNA transcripts. Furthermore, while metalloproteinases 1, -2, -3 and -10 expression clearly distinguish OA-Mf from both the tolerized and trained macrophage models, OA-Mf Interleukin 1 (IL1), chemokine (CXCL) and cytokine (CCL) family member profiles resemble the tolerized macrophage model, with the exception that OA-Mf show high levels of CCL20. CONCLUSION: Terminal osteoarthritis joints therefore harbor macrophages with an inflammatory state that closely resembles the tolerized macrophage state and this is compounded by a weak corticosteroid response capacity that may explain the lack of positive long-term effects of corticosteroid treatment for osteoarthritis patients.
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OBJECTIVES: OA is characterized by cartilage degeneration and persistent pain. The majority of OA patients present with synovitis, which is associated with increased cartilage damage. Activated synovial macrophages are key contributors to joint destruction. Therefore, a marker that reflects the activation of these cells could be a valuable tool to characterize the destructive potential of synovitis and benefit monitoring of OA. Here, we aimed to investigate the use of CD64 (FcγRI) as a marker to characterize the damaging potential of synovitis in OA. METHODS: Synovial biopsies were obtained from end-stage OA patients that underwent joint replacement surgery. CD64 protein expression and localization was evaluated using immunohistochemistry and immunofluorescence and quantified using flow cytometry. qPCR was performed to measure the expression of FCGR1 and OA-related genes in synovial biopsies, and in primary chondrocytes and primary fibroblasts stimulated with OA conditioned medium (OAS-CM). RESULTS: Our data exposed a wide range of CD64 expression in OA synovium and showed positive correlations between FCGR1 and S100A8, S100A9, IL1B, IL6 and MMP1/2/3/9/13 expression. CD64 protein correlated with MMP1, MMP3, MMP9, MMP13 and S100A9. Furthermore, we observed that synovial CD64 protein levels in source tissue for OAS-CM significantly associated with the OAS-CM-induced expression of MMP1, MMP3 and especially ADAMTS4 in cultured fibroblasts, but not chondrocytes. CONCLUSION: Together, these results indicate that synovial CD64 expression is associated with the expression of proteolytic enzymes and inflammatory markers related to structural damage in OA. CD64 therefore holds promise as marker to characterize the damaging potential of synovitis.
Assuntos
Osteoartrite , Sinovite , Humanos , Metaloproteinase 1 da Matriz/metabolismo , Metaloproteinase 3 da Matriz , Osteoartrite/metabolismo , Sinovite/patologia , Calgranulina B/metabolismo , Membrana Sinovial/metabolismoRESUMO
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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Synovial macrophages are key mediators of OA pathology, and skewing of macrophage phenotype in favour of an M1-like phenotype is thought to underlie the chronicity of synovial inflammation in OA. Components of the metabolic syndrome (MetS), such as dyslipidaemia, can affect macrophage phenotype and function, which could explain the link between MetS and OA development. Recently published studies have provided novel insights into the different origins and heterogeneity of synovial macrophages. Considering these findings, we propose an important role for monocyte-derived macrophages in particular, as opposed to yolk-sac derived residential macrophages, in causing a pro-inflammatory phenotype shift. We will further explain how this can start even prior to synovial infiltration; in the circulation, monocytes can be trained by metabolic factors such as low-density lipoprotein to become extra responsive to chemokines and damage-associated molecular patterns. The concept of innate immune training has been widely studied and implicated in atherosclerosis pathology, but its involvement in OA remains uncharted territory. Finally, we evaluate the implications of these insights for targeted therapy directed to macrophages and metabolic factors.
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Síndrome Metabólica , Osteoartrite , Humanos , Monócitos/metabolismo , Lipoproteínas LDL/metabolismo , Osteoartrite/metabolismo , Inflamação/metabolismo , Síndrome Metabólica/complicações , Fatores de Risco , Membrana Sinovial/metabolismoRESUMO
OA is a complex and highly prevalent degenerative disease affecting the whole joint, in which factors like genetic predisposition, gender, age, obesity and traumas contribute to joint destruction. â¼50-80% of OA patients develop synovitis. OA-associated risk factors contribute to joint instability and the release of cartilage matrix fragments, activating the synovium to release pro-inflammatory factors and catabolic enzymes in turn damaging the cartilage and creating a vicious circle. Currently, no cure is available for OA. Mesenchymal stromal cells (MSCs) have been tested in OA for their chondrogenic and anti-inflammatory properties. Interestingly, MSCs are most effective when administered during synovitis. This review focusses on the interplay between joint inflammation and the immunomodulation by MSCs in OA. We discuss the potential of MSCs to break the vicious circle of inflammation and describe current perspectives and challenges for clinical application of MSCs in treatment and prevention of OA, focussing on preventing post-traumatic OA.
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Imunomodulação , Inflamação/terapia , Transplante de Células-Tronco Mesenquimais/métodos , Osteoartrite/terapia , Sinovite/terapia , Humanos , Inflamação/imunologia , Osteoartrite/imunologia , Sinovite/imunologiaRESUMO
Recently, it was shown that interleukin-1ß (IL-1ß) has diverse stimulatory effects on different murine long bone marrow osteoclast precursors (OCPs) in vitro. In this study, interleukin-1 receptor antagonist deficient (Il1rn-/-) and wild-type (WT) mice were compared to investigate the effects of enhanced IL-1 signaling on the composition of OCPs in long bone, calvaria, vertebra, and jaw. Bone marrow cells were isolated from these sites and the percentage of early blast (CD31hi Ly-6C-), myeloid blast (CD31+ Ly-6C+), and monocyte (CD31- Ly-6Chi) OCPs was assessed by flow cytometry. At the time-point of cell isolation, Il1rn-/- mice showed no inflammation or bone destruction yet as determined by histology and microcomputed tomography. However, Il1rn-/- mice had an approximately two-fold higher percentage of OCPs in long bone and jaw marrow compared to WT. Conversely, vertebrae and calvaria marrow contained a similar composition of OCPs in both strains. Bone marrow cells were cultured with macrophage colony stimulating factor (M-CSF) and receptor of NfκB ligand (RANKL) on bone slices to assess osteoclastogenesis and on calcium phosphate-coated plates to analyze mineral dissolution. Deletion of Il1rn increased osteoclastogenesis from long bone, calvaria, and jaw marrows, and all Il1rn-/- cultures showed increased mineral dissolution compared to WT. However, osteoclast markers increased exclusively in Il1rn-/- osteoclasts from long bone and jaw. Collectively, these findings indicate that a lack of IL-1RA increases the numbers of OCPs in vivo, particularly in long bone and jaw, where rheumatoid arthritis and periodontitis develop. Thus, increased bone loss at these sites may be triggered by a larger pool of OCPs due to the disruption of IL-1 inhibitors.
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Células da Medula Óssea/citologia , Medula Óssea/metabolismo , Proteína Antagonista do Receptor de Interleucina 1/deficiência , Arcada Osseodentária/citologia , Osteoclastos/citologia , Animais , Biomarcadores/metabolismo , Fosfatos de Cálcio/metabolismo , Contagem de Células , Proteína Antagonista do Receptor de Interleucina 1/metabolismo , Arcada Osseodentária/diagnóstico por imagem , Camundongos Endogâmicos BALB C , Minerais/metabolismo , Monócitos/citologia , Crânio/citologia , Microtomografia por Raio-XRESUMO
Bone erosion is one of the central hallmarks of RA and is caused by excessive differentiation and activation of osteoclasts. Presence of autoantibodies in seropositive arthritis is associated with radiographic disease progression. ICs, formed by autoantibodies and their antigens, activate Fcγ-receptor signalling in immune cells, and as such stimulate inflammation-mediated bone erosion. Interestingly, ICs can also directly activate osteoclasts by binding to FcγRs on their surface. Next to autoantibodies, high levels of alarmins, among which is S100A8/A9, are typical for RA and they can further activate the immune system but also directly promote osteoclast function. Therefore, IC-activated FcγRs and S100A8/A9 might act as partners in crime to stimulate inflammation and osteoclasts differentiation and function, thereby stimulating bone erosion. This review discusses the separate roles of ICs, FcγRs and alarmins in bone erosion and sheds new light on the possible interplay between them, which could fuel bone erosion.
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Artrite Reumatoide/metabolismo , Reabsorção Óssea/metabolismo , Calgranulina A/metabolismo , Calgranulina B/metabolismo , Receptores de IgG/metabolismo , Animais , Complexo Antígeno-Anticorpo/metabolismo , Artrite Reumatoide/complicações , Artrite Reumatoide/imunologia , Autoanticorpos/metabolismo , Reabsorção Óssea/etiologia , Reabsorção Óssea/imunologia , Diferenciação Celular , Humanos , Osteoclastos/metabolismoRESUMO
OBJECTIVE: To investigate the role of AXL, a member of the anti-inflammatory TYRO3, AXL MER (TAM) receptor family, in arthritis. METHODS: KRN serum transfer arthritis was induced in Axl-/- and wild-type mice. Knee and ankle joints were scored macro- and microscopically. Synovial gene and protein expression of Axl was determined in naïve and TGF-ß1-overexpressing joints. AXL expression was determined in M1-like or M2-like macrophages and RA synovium. Human macrophages, fibroblasts and synovial micromasses were stimulated with TGF-ß1 or the AXL inhibitor R428. RESULTS: Ankle joints of Axl-/- mice showed exacerbated arthritis pathology, whereas no effect of Axl gene deletion was observed on gonarthritis pathology. To explain this spatial difference, we examined the synovium of naïve mice. In contrast to the knee, the ankle synovial cells prominently expressed AXL. Moreover, the M2-like macrophage phenotype was the dominant cell type in the naïve ankle joint. Human M2-like macrophages expressed higher levels of AXL and blocking AXL increased their inflammatory response. In the murine ankle synovium, gene expression of Tgfb1 was increased and Tgb1 correlated with Axl. Moreover, TGFB1 and AXL expression also correlated in human RA synovium. In human macrophages and synovial micromasses, TGF-ß1 enhanced AXL expression. Moreover, TGF-ß1 overexpression in naïve murine knee joints induced synovial AXL expression. CONCLUSION: Differences in synovial AXL expression are in accordance with the observation that AXL dampens arthritis in ankle, but not in knee joints. We provide evidence that the local differences in AXL expression could be due to TGF-ß1, and suggest similar pathways operate in RA synovium.
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Artrite Experimental/metabolismo , Proteínas Proto-Oncogênicas/metabolismo , Receptores Proteína Tirosina Quinases/metabolismo , Membrana Sinovial/metabolismo , Fator de Crescimento Transformador beta1/metabolismo , Animais , Articulação do Tornozelo/metabolismo , Artrite Experimental/genética , Fibroblastos/metabolismo , Humanos , Articulação do Joelho/metabolismo , Macrófagos/metabolismo , Camundongos , Camundongos Knockout , Proteínas Proto-Oncogênicas/genética , Receptores Proteína Tirosina Quinases/genética , Receptor Tirosina Quinase AxlRESUMO
OBJECTIVES: Previously, we have shown the involvement of Wnt-activated protein Wnt-1-induced signaling protein 1 (WISP1) in the development of OA in mice. Here, we aimed to characterize the relation between WISP1 expression and human OA and its regulatory epigenetic determinants. METHODS: Preserved and lesioned articular cartilage from end-stage OA patients and non-OA-diagnosed individuals was collected. WISP1 expression was determined using immunohistochemistry and damage was classified using Mankin scoring. RNA expression and DNA methylation were assessed in silico from genome-wide datasets (microarray analysis and RNA sequencing, and 450 k-methylationarrays, respectively). Effects of WISP1 were tested in pellet cultures of primary human chondrocytes. RESULTS: WISP1 expression in cartilage of OA patients was increased compared with non-OA-diagnosed controls and, within OA patients, WISP1 was even higher in lesioned compared with preserved regions, with expression strongly correlating with Mankin score. In early symptomatic OA patients with disease progression, higher synovial WISP1 expression was observed as compared with non-progressors. Notably, increased WISP1 expression was inversely correlated with methylation levels of a positional CpG-dinucleotide (cg10191240), with lesioned areas showing strong hypomethylation for this CpG as compared with preserved cartilage. Additionally, we observed that methylation levels were allele-dependent for an intronic single-nucleotide polymorphism nearby cg10191240. Finally, addition of recombinant WISP1 to pellets of primary chondrocytes strongly inhibited deposition of extracellular matrix as reflected by decreased pellet circumference, proteoglycan content and decreased expression of matrix components. CONCLUSION: Increased WISP1 expression is found in lesioned human articular cartilage, and appears epigenetically regulated via DNA methylation. In vitro assays suggest that increased WISP1 is detrimental for cartilage integrity.
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Proteínas de Sinalização Intercelular CCN/metabolismo , Cartilagem Articular/metabolismo , Osteoartrite do Joelho/metabolismo , Proteínas Proto-Oncogênicas/metabolismo , Condrócitos/metabolismo , Metilação de DNA , Epigênese Genética , Humanos , Articulação do Joelho/metabolismoRESUMO
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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Artrite Experimental , Cartilagem Articular , LDL-Colesterol/sangue , Animais , Artrite Experimental/imunologia , Artrite Experimental/metabolismo , Artrite Experimental/patologia , Cartilagem Articular/metabolismo , Cartilagem Articular/patologia , Modelos Animais de Doenças , Inflamação , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Receptores de IgGRESUMO
The general consensus is that milk promotes bone growth and density because is a source of calcium and contains components that enhance intestinal calcium uptake or directly affect bone metabolism. In this study, we investigated the effect of bovine-derived milk 100,000 g pellet (P100), which contains nanoparticles (<220 nm) including extracellular vesicles, on osteoclast differentiation and bone resorption. Bone marrow-derived osteoclast precursor cells were differentiated into osteoclasts by M-CSF and RANKL (control) and in the presence of milk P100. Milk P100 treatment until day 4 increased the number of TRAP-positive mononuclear cells and small (≤5 nuclei) osteoclasts. The number of large (≥6 nuclei) osteoclasts remained the same. These alterations were associated with increased expression of TRAP, NFATc1, and c-Fos. Cells seeded in a calcium-phosphate coated plate or bone slices showed reduced resorption area when exposed to milk P100 during the differentiation phase and even after osteoclast formation. Interestingly, milk P100 treatment enhanced Cathepsin K expression but reduced Carbonic Anhydrase 2 gene expression. Moreover, intracellular acid production was also decreased by milk P100 treatment. Oral delivery of milk P100 to female DBA1/J mice for 7 weeks did not alter bone area; however, increased osteoclast number and area in tibia without changes in serum RANKL and CTX-I levels. We showed for the first time the effect of milk P100 on osteoclast differentiation both in vitro and in vivo and found that milk P100 increased the formation of small osteoclasts but this does not lead to more bone resorption probably due to reduced acid secretion. J. Cell. Physiol. 232: 225-233, 2017. © 2016 Wiley Periodicals, Inc.
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Reabsorção Óssea/tratamento farmacológico , Diferenciação Celular/efeitos dos fármacos , Leite/metabolismo , Nanopartículas/administração & dosagem , Osteoclastos/metabolismo , Animais , Reabsorção Óssea/metabolismo , Fosfatos de Cálcio/farmacologia , Diferenciação Celular/fisiologia , Camundongos Endogâmicos C57BL , Fatores de Transcrição NFATC/metabolismo , Osteoclastos/efeitos dos fármacos , Proteínas Proto-Oncogênicas c-fos/metabolismo , Transdução de Sinais/efeitos dos fármacosRESUMO
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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Condrócitos/metabolismo , Interleucina-1/metabolismo , Interleucina-1beta/farmacologia , Osteoartrite , RNA Mensageiro/metabolismo , Fator de Necrose Tumoral alfa/farmacologia , Adenoviridae , Western Blotting , Condrócitos/efeitos dos fármacos , Desintegrinas/efeitos dos fármacos , Desintegrinas/genética , Desintegrinas/metabolismo , Humanos , Imuno-Histoquímica , Interleucina-1/genética , Interleucina-1beta/efeitos dos fármacos , Interleucina-1beta/genética , Interleucina-1beta/metabolismo , Interleucina-6/genética , Interleucina-6/metabolismo , Interleucina-8/efeitos dos fármacos , Interleucina-8/genética , Interleucina-8/metabolismo , Metaloproteinase 1 da Matriz/efeitos dos fármacos , Metaloproteinase 1 da Matriz/genética , Metaloproteinase 1 da Matriz/metabolismo , Metaloproteinase 13 da Matriz/efeitos dos fármacos , Metaloproteinase 13 da Matriz/genética , Metaloproteinase 13 da Matriz/metabolismo , Metaloproteinase 3 da Matriz/efeitos dos fármacos , Metaloproteinase 3 da Matriz/genética , Metaloproteinase 3 da Matriz/metabolismo , RNA Mensageiro/efeitos dos fármacos , Reação em Cadeia da Polimerase em Tempo RealRESUMO
BACKGROUND: Cells in the intervertebral disc have unique phenotypes and marker genes that separate the nucleus pulposus (NP), annulus fibrosus (AF) and articular cartilage (AC) have been identified. Recently, it was shown that phenotypic marker genes exhibit variable expression in humans. In this study, the bovine tail was used to determine the ability of marker genes to distinguish the outer and inner AF from NP tissue and isolated cells. METHODS: Bovine tail intervertebral discs from 13 donors were dissected and correct isolation of tissue was confirmed. mRNA was isolated directly from tissue or passage 0 monolayer cells and used for gene expression measurements (qPCR). Conventional marker genes (bAcan, bCol1a1, bCol2a1) and novel marker genes (bAdamts17, bBrachyury/T, bCD24, bCol5a1, bCol12a1, bFoxf1, bKrt19, bPax1, bSfrp2) were evaluated. RESULTS: As expected bAcan, bCol2a1 and bCol1a1 distinguished outer AF from NP tissue, while inner AF and NP could not be discriminated. The NP markers bT, bCd24 and bKrt19 were significantly higher expressed in NP than inner and outer AF tissue. bFoxF1 and bPax1 only distinguished IVD tissues from AC. The AF markers bAdamts17, bCol5a1, bCol12a1 and bSfrp2 were higher expressed in the outer AF compared with inner AF and NP tissue. Monolayer culturing strongly decreased bAcan, bCol2a1, bCD24 and bCol5a1 expression, while bCol1a1, bT, bKrt19 and bSfrp2 were not affected. CONCLUSION: The IVD phenotypic marker genes bT, bKrt19, bSfrp2 and bCol12a1 convincingly distinguished NP from outer AF in situ and in vitro.
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Anel Fibroso/metabolismo , Perfilação da Expressão Gênica , Núcleo Pulposo/metabolismo , Transcriptoma , Animais , Bovinos , Marcadores Genéticos , FenótipoRESUMO
There is increasing evidence that low-density lipoprotein (LDL) cholesterol plays a role in the pathology of OA. Specifically, oxidized LDL (oxLDL), which has been shown to play an essential role during development of atherosclerosis, could be involved in processes such as synovial inflammation, cartilage destruction and bone deformations. OxLDL can activate synovial cells such as macrophages, endothelial cells and synovial fibroblasts, resulting in release of growth factors, MMP and pro-inflammatory cytokines. In this review article, we discuss the role of LDL and oxLDL in OA joint pathology and share our viewpoint of possible mechanisms by which these proteins could influence the development and progression of OA. The proposed theory could provide insight into the aetiopathology of OA and give rise to new potential treatments.
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Cartilagem Articular/metabolismo , LDL-Colesterol/metabolismo , Lipoproteínas LDL/metabolismo , Osteoartrite/metabolismo , Membrana Sinovial/metabolismo , Cartilagem Articular/patologia , Citocinas/metabolismo , Humanos , Osteoartrite/patologia , OxirreduçãoRESUMO
OBJECTIVES: Rheumatoid arthritis is a chronic destructive autoimmune disease, but the course is unpredictable in individual patients. An attractive treatment would provide a disease-regulated therapy that offers personalised drug delivery. Therefore, we expressed the anti-inflammatory interleukin-10 (IL-10) gene under the control of inflammation-dependent promoters in a mouse model of arthritis. METHODS: Proximal promoters of S100a8, Cxcl1, Mmp13, Saa3, IL-1b and Tsg6 were selected by whole-genome expression analysis of inflamed synovial tissues from arthritic mice. Mice were injected intraarticularly in knee joints with lentiviral vectors expressing a luciferase reporter or the therapeutic protein IL-10 under control of the Saa3 or Mmp13 promoter. After 4â days, arthritis was induced by intraarticular injection of streptococcal cell walls (SCW). At different time points after arthritis induction, in vivo bioluminescent imaging was performed and knee joints were dissected for histological and RNA analysis. RESULTS: The disease-regulated promoter-luciferase reporter constructs showed different activation profiles during the course of the disease. The Saa3 and Mmp13 promoters were significantly induced at day 1 or day 4 after arthritis induction respectively and selected for further research. Overexpression of IL-10 using these two disease-inducible promoters resulted in less synovitis and markedly diminished cartilage proteoglycan depletion and in upregulation of IL-1Ra and SOCS3 gene expression. CONCLUSIONS: Our study shows that promoters of genes that are expressed locally during arthritis can be candidates for disease-regulated overexpression of biologics into arthritic joints, as shown for IL-10 in SCW arthritis. The disease-inducible approach might be promising for future tailor-made local gene therapy in arthritis.
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Artrite Experimental/terapia , Cartilagem Articular/metabolismo , Terapia Genética , Interleucina-10 , Proteoglicanas/metabolismo , RNA Mensageiro/metabolismo , Membrana Sinovial/imunologia , Sinovite/terapia , Animais , Artrite Experimental/imunologia , Artrite Experimental/patologia , Artrite Reumatoide , Parede Celular/imunologia , Expressão Gênica , Proteína Antagonista do Receptor de Interleucina 1/genética , Masculino , Metaloproteinase 13 da Matriz/genética , Camundongos , Camundongos Endogâmicos C57BL , Regiões Promotoras Genéticas , Proteína Amiloide A Sérica/genética , Streptococcus/imunologia , Proteína 3 Supressora da Sinalização de Citocinas , Proteínas Supressoras da Sinalização de Citocina/genética , Membrana Sinovial/patologia , Sinovite/imunologia , Sinovite/patologiaRESUMO
Rheumatoid arthritis (RA) and osteoarthritis (OA) are serologically and clinically distinctive, but at the local level, both diseases have many molecular pathways in common. In vivo molecular imaging can unravel the local pathologic processes involved in both diseases. In this study, we investigated matrix metalloproteinase (MMP) and cathepsin activity during cartilage destruction, in an RA and an OA mouse model, using biophotonic imaging of substrate-based probes. Mice with collagen-induced arthritis (CIA) or destabilization of the medial meniscus (DMM) were imaged using near-infrared fluorescent probes, activated by several cathepsins or MMPs. Fluorescence signal intensity was compared to synovial gene expression, histology, and cartilage staining of a neoepitope of aggrecan cleaved by MMPs with the amino acids DIPEN. Increased cathepsin and MMP activity was seen during CIA, whereas the DMM model only showed increased MMP activity. DIPEN expression was seen only during CIA. A possible explanation can be differences in gene expressions; MMP3 and -13, known to produce DIPEN neoepitopes, were upregulated in the CIA model, whereas MMP12, known to be involved in elastin degradation and chemokine inhibition, was upregulated in the DMM model. Thus, molecular imaging showed no cathepsin activity at the time of cartilage damage in the DMM model, whereas both cathepsins and MMPs are active in the CIA model during disease progression.
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Artrite Reumatoide/metabolismo , Catepsinas/análise , Metaloproteinases da Matriz/análise , Imagem Molecular/métodos , Osteoartrite/metabolismo , Animais , Artrite Experimental/metabolismo , Catepsinas/metabolismo , Morte Celular , Condrócitos/citologia , Condrócitos/metabolismo , Colágeno Tipo II/efeitos adversos , Colágeno Tipo II/imunologia , Masculino , Metaloproteinases da Matriz/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Joelho de Quadrúpedes/química , Joelho de Quadrúpedes/metabolismo , Joelho de Quadrúpedes/patologiaRESUMO
OBJECTIVE: Mesenchymal stem cells (MSCs) represent a promising tool for cell therapy for several disorders, among them the osteoarticular diseases. For such clinical applications, intraarticular (IA) injection of MSCs may be favored for higher levels of safety and targeting of specific joints. Although the safety of intravenous (IV) administration of MSCs has been reported in a number of clinical trials, the safety and biodistribution of MSCs after IA injection have not been tested. Our objective was to assess the toxicity of clinical-grade human adipose-derived MSCs (AD-MSCs), as well as their biodistribution, after IA injection into SCID mice. METHODS: SCID mice received IA or IV administration of 10(6) human AD-MSCs. Several tissues were recovered at different time points and processed for histologic assessment or real-time polymerase chain reaction (PCR) analysis. A highly sensitive assay was used to monitor the distribution of AD-MSCs, based on amplification of human-specific Alu sequences. RESULTS: Absence of toxicity was observed after AD-MSC infusion. Alu PCR assay revealed a high sensitivity (1 human AD-MSC/10(5) murine cells), with a large linear range (1-5 × 10(4) /10(5) murine cells). Importantly, 15% of the IA-injected AD-MSCs were detectable in the joint for the first month and 1.5% of the AD-MSCs engrafted over the long term, at least 6 months. AD-MSCs were observed in the injected joints and in areas of tissue referred to as stem cell niches, such as the bone marrow, adipose tissue, and muscle. CONCLUSION: These data support the feasibility and safety of using IA delivery of human AD-MSCs in the treatment of rheumatic diseases that affect the joints.
Assuntos
Transplante de Células-Tronco Mesenquimais/métodos , Células-Tronco Mesenquimais/metabolismo , Tecido Adiposo/citologia , Animais , Células Cultivadas , Feminino , Humanos , Infusões Intravenosas , Injeções Intra-Articulares , Masculino , Transplante de Células-Tronco Mesenquimais/efeitos adversos , Camundongos , Camundongos SCIDRESUMO
OBJECTIVE: To generate doxycycline-inducible human tumor necrosis factor α (TNFα)-transgenic mice to overcome a major disadvantage of existing transgenic mice with constitutive expression of TNFα, which is the limitation in crossing them with various knockout or transgenic mice. METHODS: A transgenic mouse line that expresses the human TNFα cytokine exclusively after doxycycline administration was generated and analyzed for the onset of diseases. RESULTS: Doxycycline-inducible human TNFα-transgenic mice developed an inflammatory arthritis- and psoriasis-like phenotype, with fore and hind paws being prominently affected. The formation of "sausage digits" with characteristic involvement of the distal interphalangeal joints and nail malformation was observed. Synovial hyperplasia, enthesitis, cartilage and bone alterations, formation of pannus tissue, and inflammation of the skin epidermis and nail matrix appeared as early as 1 week after the treatment of mice with doxycycline and became aggravated over time. The abrogation of human TNFα expression by the removal of doxycycline 6 weeks after beginning stimulation resulted in fast resolution of the most advanced macroscopic and histologic disorders, and 3-6 weeks later, only minimal signs of disease were visible. CONCLUSION: Upon doxycycline administration, the doxycycline-inducible human TNFα-transgenic mouse displays the major features of inflammatory arthritis. It represents a unique animal model for studying the molecular mechanisms of arthritis, especially the early phases of disease genesis and tissue remodeling steps upon abrogation of TNFα expression. Furthermore, unlimited crossing of doxycycline-inducible human TNFα-transgenic mice with various knockout or transgenic mice opens new possibilities for unraveling the role of various signaling molecules acting in concert with TNFα.
Assuntos
Artrite Experimental/genética , Artrite Psoriásica/genética , Fator de Necrose Tumoral alfa/genética , Animais , Artrite Experimental/metabolismo , Artrite Experimental/patologia , Artrite Psoriásica/metabolismo , Artrite Psoriásica/patologia , Cartilagem/metabolismo , Cartilagem/patologia , Inflamação/patologia , Articulações/metabolismo , Articulações/patologia , Camundongos , Camundongos Transgênicos , Fator de Necrose Tumoral alfa/metabolismoRESUMO
Mesenchymal stem cell (MSC) administration is a promising adjuvant therapy to treat tissue injury. However, MSC survival after administration is often hampered by oxidative stress at the site of injury. Heme oxygenase (HO) generates the cytoprotective effector molecules biliverdin/bilirubin, carbon monoxide (CO) and iron/ferritin by breaking down heme. Since HO-activity mediates anti-apoptotic, anti-inflammatory, and anti-oxidative effects, we hypothesized that modulation of the HO-system affects MSC survival. Adipose-derived MSCs (ASCs) from wild type (WT) and HO-2 knockout (KO) mice were isolated and characterized with respect to ASC marker expression. In order to analyze potential modulatory effects of the HO-system on ASC survival, WT and HO-2 KO ASCs were pre-treated with HO-activity modulators, or downstream effector molecules biliverdin, bilirubin, and CO before co-exposure of ASCs to a toxic dose of H2O2. Surprisingly, sensitivity to H2O2-mediated cell death was similar in WT and HO-2 KO ASCs. However, pre-induction of HO-1 expression using curcumin increased ASC survival after H2O2 exposure in both WT and HO-2 KO ASCs. Simultaneous inhibition of HO-activity resulted in loss of curcumin-mediated protection. Co-treatment with glutathione precursor N-Acetylcysteine promoted ASC survival. However, co-incubation with HO-effector molecules bilirubin and biliverdin did not rescue from H2O2-mediated cell death, whereas co-exposure to CO-releasing molecules-2 (CORM-2) significantly increased cell survival, independently from HO-2 expression. Summarizing, our results show that curcumin protects via an HO-1 dependent mechanism against H2O2-mediated apoptosis, and likely through the generation of CO. HO-1 pre-induction or administration of CORMs may thus form an attractive strategy to improve MSC therapy.