RESUMO
OBJECTIVE: To investigate mechanistically the reported beneficial effects of immune-activated mesenchymal stromal cell (MSC) therapy to treat equine septic arthritis, leveraging Nanostring technology. ANIMALS: 8 Quarter Horses with induced tibiotarsal Staphylococcus aureus septic arthritis treated IA with either Toll-like receptor-3 agonist polyinosinic:polycytidylic acid-activated MSCs + vancomycin antimicrobials (TLR-MSC-VAN; n = 4) or antimicrobials (VAN; 4). METHODS: Synovial tissues were collected and fixed in neutral-buffered 10% formalin, and formalin-fixed paraffin-embedded synovial and osteochondral tissues were sequenced using a custom-designed 200-gene equine Nanostring nCounter immune panel to directly quantify expression of key immune and cartilage-related genes. Immunohistochemistry to detect CD3+ T cells was performed on synovial tissues to further quantify T-cell infiltration in TLR-MSC-VAN- versus VAN-treated joints. RESULTS: Comparison of synovial transcriptomes between groups revealed moderate changes in differential gene expression, with upregulated expression of 9 genes and downregulated expression of 17 genes with fold change ≥ 2 or ≤ -2 and a significant false discovery rate-adjusted P value of ≤ .05. The most upregulated genes in TLR-MSC-VAN-treated horses included those related to T-lymphocyte recruitment and function, while pathways related to innate immune activation and inflammation were significantly downregulated. Immunohistochemistry and quantitation of CD3+ T-cell infiltrates revealed a numerically greater infiltrate in synovial tissues of TLR-MSC-VAN-treated horses, which did not reach statistical significance in this small sample set (P = .20). CLINICAL RELEVANCE: Targeted transcriptomic analyses using an equine Nanostring immune and cartilage health panel provided new mechanistic insights into how innate and adaptive immune cells within synovial tissues respond to TLR-activated MSC treatment when used to treat septic arthritis.
Assuntos
Artrite Infecciosa , Doenças dos Cavalos , Membrana Sinovial , Linfócitos T , Animais , Cavalos , Artrite Infecciosa/veterinária , Doenças dos Cavalos/terapia , Doenças dos Cavalos/imunologia , Membrana Sinovial/citologia , Células-Tronco Mesenquimais , Transcriptoma , Infecções Estafilocócicas/veterinária , Perfilação da Expressão Gênica/veterinária , Feminino , Masculino , Transplante de Células-Tronco Mesenquimais/veterináriaRESUMO
BACKGROUND: Mesenchymal stem cells (MSCs) are multipotent cells with broad immunosuppressive capacities. Recently, it has been reported that MSCs can transfer mitochondria to various cell types, including fibroblast, cancer, and endothelial cells. It has been suggested that mitochondrial transfer is associated with a physiological response to cues released by damaged cells to restore and regenerate damaged tissue. However, the role of mitochondrial transfer to immune competent cells has been poorly investigated. METHODS AND RESULTS: Here, we analyzed the capacity of MSCs from the bone marrow (BM) of healthy donors (BM-MSCs) to transfer mitochondria to primary CD4+CCR6+CD45RO+ T helper 17 (Th17) cells by confocal microscopy and fluorescent-activated cell sorting (FACS). We then evaluated the Th17 cell inflammatory phenotype and bioenergetics at 4 h and 24 h of co-culture with BM-MSCs. We found that Th17 cells can take up mitochondria from BM-MSCs already after 4 h of co-culture. Moreover, IL-17 production by Th17 cells co-cultured with BM-MSCs was significantly impaired in a contact-dependent manner. This inhibition was associated with oxygen consumption increase by Th17 cells and interconversion into T regulatory cells. Finally, by co-culturing human synovial MSCs (sMSCs) from patients with rheumatoid arthritis (RA) with Th17 cells, we found that compared with healthy BM-MSCs, mitochondrial transfer to Th17 cells was impaired in RA-sMSCs. Moreover, artificial mitochondrial transfer also significantly reduced IL-17 production by Th17 cells. CONCLUSIONS: The present study brings some insights into a novel mechanism of T cell function regulation through mitochondrial transfer from stromal stem cells. The reduced mitochondrial transfer by RA-sMSCs might contribute to the persistence of chronic inflammation in RA synovitis.
Assuntos
Células-Tronco Mesenquimais/citologia , Mitocôndrias/transplante , Células Th17/metabolismo , Artrite Reumatoide/metabolismo , Artrite Reumatoide/patologia , Células da Medula Óssea/citologia , Células Cultivadas , Técnicas de Cocultura , Humanos , Interleucina-17/metabolismo , Células-Tronco Mesenquimais/efeitos dos fármacos , Células-Tronco Mesenquimais/metabolismo , Mitocôndrias/metabolismo , Consumo de Oxigênio , Membrana Sinovial/citologia , Linfócitos T Reguladores/citologia , Linfócitos T Reguladores/imunologia , Linfócitos T Reguladores/metabolismo , Células Th17/citologia , Células Th17/imunologia , Fator de Necrose Tumoral alfa/farmacologiaRESUMO
Osteoarthritis is an incapacitating disease characterized by pain and a progressive decrease in joint mobility. The implantation of mesenchymal stem cells (MSCs) has shown promising results for its treatment. The challenge remains to keep the cells longer at the site of action, increasing their therapeutic potential. The aim of this study was to evaluate the effectiveness of the Qtracker® 655 nanocrystal marking on allogeneic synovial membrane (SM) MSCs, encapsulated in alginate hydrogel, evaluating the migration of these cells. The 10 radiocarpal joints were submitted to arthroscopic surgery (D0), divided into two groups. The chondral defect was treated according to the group: GA free-labelled MSCSM and GB labelled MSCSM microcapsules. Seven days after lesion induction and implantation of labelled cells, biopsies of the lesion site were performed in two animals, and fragments of SM and joint capsule also collected, which were frozen and later processed for fluorescence microscopy. The synovial fluid of the three animals was analyzed by flow cytometry three times - 3, 7 and 21â¯days after application. The cellular marking with the nanocrystals allowed the visualization of the cells in cartilage, synovial membrane, synovial fluid and articular capsule, but with a predilection for the synovial membrane and the lesion site was scarce. The labelled MSCSM in microcapsules were scarce in the synovial fluid and could be related to the small quantity of MSCs leaving the pores of the microcapsules, also favorable results, as the cells release paracrine effects acting for a long period until the cellular differentiation.
Assuntos
Alginatos/administração & dosagem , Movimento Celular , Doenças dos Cavalos/terapia , Hidrogéis/administração & dosagem , Células-Tronco Mesenquimais/fisiologia , Osteoartrite/veterinária , Animais , Artroscopia/veterinária , Feminino , Cavalos , Masculino , Osteoartrite/terapia , Membrana Sinovial/citologiaRESUMO
Background: Cartilage restoration is a desperately needed bridge for patients with symptomatic cartilage lesions. Chondral lesion is a pathology with high prevalence, reaching as much as 63% of general population and 36% among athletes. Despite autologous chondrocyte implantation versatility, it still fails to fully reproduce hyaline articular cartilage characteristics. Mesenchymal stem cells (MSCs) may be isolated from various known tissues, including discarded fragments at arthroscopy such as synovial membrane. Choice of harvesting site is motivated by MSCs' abilities to modulate immunologic and inflammatory response through paracrine communication. Synovial MSCs have a greater proliferation and strong chondrogenic potential than bone and adipose MSCs and a less hypertrophic differentiation than bone MSCs. Good manufacturing practice (GMP) laboratory techniques for human clinical trials are still novel. To our knowledge, there are only two clinical trials in humans published since today. Purpose: Therefore, this work aimed to isolate and characterize synovial MSCs and evaluated their differentiation properties according to GMP standards. Materials and Methods: One-gram tissue sample from three patients of synovia was harvested at the beginning of arthroscopy surgery. MSCs were isolated, expanded, and characterized by flow cytometry. Results: It was possible to isolate and expand MSCs cultures from synovia, characterize MSCs by flow cytometry using proper monoclonal antibodies, and differentiate MSCs by coloring technique after chondrogenic, adipogenic, and osteogenic differentiations. Cartilage treatment may benefit from these tissue engineering protocols since arthroscopic procedures are routinely performed for different purposes in a previous stage and a favorable chondronegic differentiation cell lineage may be collected and stored in a less invasive way. Conclusion: Laboratory protocols established according to presented GMP were able to isolate and characterize MSCs obtained from synovia. Impact Statement Articular cartilage restoration is a desperately needed bridge for patients with symptomatic cartilage lesions and it rises as a socioeconomic issue with a considerable economic burden. Synovial mesenchymal stem cells (MSCs) have a greater proliferation rate and strong chondrogenic potential than bone and adipose MSCs and a less hypertrophic differentiation than bone MSCs. To our knowledge, there are only two human clinical trials with good manufacturing practice laboratory techniques for synovial MSCs harvesting and differentiation. Cartilage treatment may benefit from these tissue engineering protocols since arthroscopic procedures are routinely performed for different purposes in a previous stage.
Assuntos
Cartilagem Articular/fisiologia , Células-Tronco Mesenquimais/citologia , Regeneração/fisiologia , Membrana Sinovial/citologia , Engenharia Tecidual/métodos , Adipogenia , Adolescente , Adulto , Células Cultivadas , Condrogênese , Feminino , Humanos , Masculino , Osteogênese , Adulto JovemRESUMO
This study aims to investigate the role of P-glycoprotein (P-gp) expression level in drug resistance to disease-modifying anti-rheumatic drugs in refractory rheumatoid arthritis (RRA). We evaluated and compared the expression levels of P-gp in fibroblast-like synoviocyte (FLS) cells in patients with rheumatoid arthritis (RA) and osteoarthritis (OA), and investigated the potential mechanism of P-gp-induced multidrug resistance in RRA. Ten patients were enrolled and divided into two groups: six in the RA group and four in the OA group. The expression level of P-gp in FLS cells was detected by western blotting following cell culture. A linear correlation algorithm was used to assess the association between the level of P-gp and disease activity (using DAS28 scoring), as well as the duration of methotrexate (MTX) treatment in the RRA patients. The level of P-gp in the RRA patients was markedly higher than that in the OA patients (P < 0.05, t = -4.179). There was a positive linear correlation between the P-gp level in FLS cells and the duration of MTX treatment in the RRA group (Ð = 0.733, P < 0.05), whereas there was no significant correlation between the P-gp level and DAS28 scoring (Ð = 0.206, P > 0.05). P-gp might be upregulated during the progression of RRA, which possibly correlates with the development of resistance to MTX.
Assuntos
Membro 1 da Subfamília B de Cassetes de Ligação de ATP/fisiologia , Artrite Reumatoide/metabolismo , Resistência a Múltiplos Medicamentos , Osteoartrite/metabolismo , Membrana Sinovial/metabolismo , Membro 1 da Subfamília B de Cassetes de Ligação de ATP/biossíntese , Idoso , Antirreumáticos/uso terapêutico , Artrite Reumatoide/tratamento farmacológico , Artrite Reumatoide/microbiologia , Células Cultivadas , Feminino , Fibroblastos , Humanos , Masculino , Metotrexato/uso terapêutico , Pessoa de Meia-Idade , Osteoartrite/tratamento farmacológico , Membrana Sinovial/citologiaRESUMO
BACKGROUND: Osteoarthritis (OA) is a common arthritic disease and multifactorial whole-joint disease. Interactions of chemokines and OA is inadequately documented. RESULTS: In vivo and in vitro studies were conducted to investigate monocyte chemoattractant protein 1 (MCP-1) and receptor chemokine (C-C motif) receptor 2 (CCR2) in chondrocyte degradation and cartilage degeneration. Chondrocytes from 16 OA patients and 6 normal controls were involved in this study. After stimulation of MCP-1, the expression of MCP-1 and CCR2 increased significantly (P < 0.001) and the expression of MMP-13 also increased (P < 0.05). MCP-1 stimulation also induced (or enhanced) the apoptosis of OA chondrocytes (P < 0.05). Additionally, the degradation of cartilage matrix markers (metalloproteinase 3 and 13, MMP3 and MMP13) in the culture medium of normal chondrocytes was also assessed. Furthermore, intra-articular injection of MCP-1 in mouse knees induced cartilage degradation and the CCR2 antagonist did not impede cartilage destroy in rats knees of monosodium iodoacetate (MIA) model. CONCLUSIONS: The results of this study demonstrate that the MCP-1-CCR2 ligand-receptor axis plays a special role in the initiation and progression of OA pathology. Patients with ambiguous etiology can gain some insight from the MCP-1-CCR2 ligand-receptor axis.
Assuntos
Quimiocina CCL2/metabolismo , Condrócitos/metabolismo , Osteoartrite do Joelho/fisiopatologia , Receptores CCR2/metabolismo , Adolescente , Idoso , Animais , Apoptose/fisiologia , Quimiocina CCL2/genética , Condrócitos/enzimologia , Progressão da Doença , Ensaio de Imunoadsorção Enzimática , Feminino , Fibroblastos/metabolismo , Humanos , Técnicas In Vitro , Ácido Iodoacético , Masculino , Proteínas Matrilinas/metabolismo , Metaloproteinase 13 da Matriz/metabolismo , Metaloproteinase 3 da Matriz/metabolismo , Camundongos Endogâmicos C57BL , Pessoa de Meia-Idade , Ratos Sprague-Dawley , Receptores CCR2/antagonistas & inibidores , Receptores CCR2/genética , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Membrana Sinovial/citologia , Adulto JovemRESUMO
BACKGROUND: Isolation of mesenchymal stem cells (MSCs) in equines, has been reported for different tissues including bone marrow, adipose, umbilical cord, peripheral blood, and yolk sac. In regard to the MSCs derived from synovial fluid (SF) or membrane (SM), there is data available for humans, dogs, pigs, goats and horses. Especially in equines, these cells have being considered promising candidates for articular regeneration. Herein, we established and characterized MSCs obtained from equine SF and SM. Samples were obtained during arthroscopy and cultured using MEM (Minimum Essential Medium). MSCs were characterized by morphology and expression of specific markers for stem cells, pluripotency, inflammation, and cell cycle. RESULTS: The medium MEM was more effective (97% ± 2) to maintain both cultures. The cultures were composed by adherent cells with fibroblast-like shape, which had a growth pattern represented by a sigmoidal curve. After the expansion, the cells were analyzed by flow cytometry for stem cells, inflammatory, and cell cycle markers, and both lineages showed significant expression of CD45, Oct3/4, Nanog, CD105, CD90, CD34, CD117, CD133, TRA-1-81, VEGF, and LY6a. In contrast, there were differences in the cell cycle phases between the lineages, which was not observed in relation to the mitochondrial electrical potential. CONCLUSION: Given the large impact that joint pathology has on the athletic performance horses, our results suggested that the SF and SM are promising sources of stem cells with satisfactory characteristics of growth and gene expression that can be used in equine regenerative medicine.
Assuntos
Cavalos , Células-Tronco Mesenquimais/fisiologia , Líquido Sinovial/citologia , Membrana Sinovial/citologia , Animais , Ciclo Celular , Proliferação de Células , Células Cultivadas , Potencial da Membrana Mitocondrial , Células-Tronco Mesenquimais/citologiaRESUMO
Brazilin is an active compound of Caesalpinia sappan L. (Leguminosae), which possesses pro-apoptotic and anti-inflammation potentials depending on the specific cell type. However, it is largely unknown whether autophagy is implicated in the mechanism underlying its chemotherapeutic and anti-inflammatory effects in rheumatoid arthritis (RA). Here, we show that treatment of RA fibroblast-like synoviocytes (FLS) with brazilin results in enhanced level of autophagic flux, evidenced by accumulation of autophagosome and increased level of lipidated LC3 (LC3-II), which is mainly mediated by enhanced production of reactive oxygen species (ROS). Interestingly, long-term exposure of brazilin was able to restore cell survival against the cytotoxity, exclusively in RA FLS, but not in normal fibroblast. Importantly, such a restoration from brazilin-induced cytotoxity in RA FLS was completely abrogated after co-treatment with autophagy inhibitors including NH4Cl or chloroquine. Furthermore, we found that the pretreatment of RA FLS with brazilin reduced LPS- or TNF-induced NF-κB activation and the secretion of inflammatory cytokines in parallel with the enhanced autophagic flux. Such anti-NF-κB potentials of brazilin were drastically masked in RA FLS when autophagy was suppressed. These results suggest that brazilin is capable of activating autophagy exclusively in RA FLS, and such inducible autophagy promotes cell survival and limits inflammatory response.
Assuntos
Anti-Inflamatórios/farmacologia , Artrite Reumatoide/tratamento farmacológico , Autofagia/efeitos dos fármacos , Benzopiranos/farmacologia , Fibroblastos/efeitos dos fármacos , Fibroblastos/patologia , Membrana Sinovial/citologia , Animais , Anti-Inflamatórios/química , Artrite Reumatoide/imunologia , Artrite Reumatoide/patologia , Benzopiranos/química , Caesalpinia/química , Células Cultivadas , Fibroblastos/citologia , Fibroblastos/imunologia , Humanos , Camundongos , NF-kappa B/imunologia , Células NIH 3T3 , Espécies Reativas de Oxigênio/imunologia , Membrana Sinovial/efeitos dos fármacos , Membrana Sinovial/imunologia , Membrana Sinovial/patologiaRESUMO
BACKGROUND: Osteoarthritis (OA) is a common arthritic disease and multifactorial whole-joint disease. Interactions of chemokines and OA is inadequately documented. RESULTS: In vivo and in vitro studies were conducted to investigate monocyte chemoattractant protein 1 (MCP-1) and receptor chemokine (C-C motif) receptor 2 (CCR2) in chondrocyte degradation and cartilage degeneration. Chondrocytes from 16 OA patients and 6 normal controls were involved in this study. After stimulation of MCP-1, the expression of MCP-1 and CCR2 increased significantly (P < 0.001) and the expression of MMP-13 also increased (P < 0.05). MCP-1 stimulation also induced (or enhanced) the apoptosis of OA chondrocytes (P < 0.05). Additionally, the degradation of cartilage matrix markers (metalloproteinase 3 and 13, MMP3 and MMP13) in the culture medium of normal chondrocytes was also assessed. Furthermore, intra-articular injection of MCP-1 in mouse knees induced cartilage degradation and the CCR2 antagonist did not impede cartilage destroy in rats knees of monosodium iodoacetate (MIA) model. CONCLUSIONS: The results of this study demonstrate that the MCP-1-CCR2 ligand-receptor axis plays a special role in the initiation and progression of OA pathology. Patients with ambiguous etiology can gain some insight from the MCP-1-CCR2 ligand-receptor axis.
Assuntos
Humanos , Animais , Masculino , Feminino , Adolescente , Pessoa de Meia-Idade , Idoso , Camundongos , Ratos , Adulto Jovem , Quimiocina CCL2/metabolismo , Condrócitos/metabolismo , Osteoartrite do Joelho/fisiopatologia , Receptores CCR2/metabolismo , Membrana Sinovial/citologia , Técnicas In Vitro , Ensaio de Imunoadsorção Enzimática , Ratos Sprague-Dawley , Apoptose/fisiologia , Progressão da Doença , Quimiocina CCL2/genética , Metaloproteinase 3 da Matriz/metabolismo , Condrócitos/enzimologia , Ácido Iodoacético , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Metaloproteinase 13 da Matriz/metabolismo , Receptores CCR2/antagonistas & inibidores , Receptores CCR2/genética , Fibroblastos/metabolismo , Proteínas Matrilinas/metabolismo , Camundongos Endogâmicos C57BLRESUMO
BACKGROUND: Dact gene family encodes multifunctional proteins that are important modulators of Wnt and TGF-ß signaling pathways. Given that these pathways coordinate multiple steps of limb development, we investigated the expression pattern of the two chicken Dact genes (Dact1 and Dact2) from early limb bud up to stages when several tissues are differentiating. RESULTS: During early limb development (HH24-HH30) Dact1 and Dact2 were mainly expressed in the cartilaginous rudiments of the appendicular skeleton and perichondrium, presenting expression profiles related, but distinct. At later stages of development (HH31-HH35), the main sites of Dact1 and Dact2 expression were the developing synovial joints. In this context, Dact1 expression was shown to co-localize with regions enriched in the nuclear ß-catenin protein, such as developing joint capsule and interzone. In contrast, Dact2 expression was restricted to the interzone surrounding the domains of bmpR-1b expression, a TGF-ß receptor with crucial roles during digit morphogenesis. Additional sites of Dact expression were the developing tendons and digit blastemas. CONCLUSIONS: Our data indicate that Dact genes are good candidates to modulate and, possibly, integrate Wnt and TGF-ß signaling during limb development, bringing new and interesting perspectives about the roles of Dact molecules in limb birth defects and human diseases.