TNFα, PDGF, and TGFß synergistically induce synovial lining hyperplasia via inducible PI3Kδ.

OBJECTIVES: To determine the mechanism underlying hypertrophic synovium in rheumatoid arthritis (RA). METHODS: We examined micromass cultures of fibroblast-like synoviocytes (FLSs) stimulated with tumor necrosis factor α (TNFα), platelet-derived growth factor (PDGF), and/or transforming growth factor ß (TGFß). The hypertrophic architecture of the micromasses, expression of phosphoinositide 3 kinase (PI3K) isoforms, and persistent activation of PI3K-Akt pathways were investigated. FLSs transfected with siRNA were also examined in the micromass cultures. RESULTS: The combination of TNFα, PDGF, and TGFß (TPT condition) induced obvious hypertrophic architecture of the intimal lining layer in FLSs in micromass cultures, and was accompanied by upregulated expression of matrix metalloproteinase-3 (MMP3), Cadherin-11, and PI3Kδ. In monolayer FLSs, the TPT condition enhanced the expression of PI3Kδ and persistent activation of the PI3K-Akt pathway. Knockdown of PI3Kδ significantly inhibited the formation of the hypertrophic synovial lining in the TPT condition. CONCLUSIONS: These results collectively indicate that inducible PI3Kδ plays a crucial role in persistent activation of PI3K-Akt in FLSs, and in the formation of a hypertrophic synovial lining. PI3Kδ may be an alternative treatment target for the regulation of proliferative synovium in RA.

A distinct human CD4+ T cell subset that secretes CXCL13 in rheumatoid synovium.

OBJECTIVE: A subset of CD4+ T cells in the synovium of patients with rheumatoid arthritis (RA) produce CXCL13, a chemokine that is crucial for the formation of germinal centers. This study was undertaken to determine the relevance of this population to known subsets of T helper cells and to proinflammatory cytokines, and how these cells are generated. METHODS: The expression of Th markers and CXCL13 by CD4+ T cells in RA synovium and the involvement of proinflammatory cytokines in CXCL13 production were assessed. We also investigated whether CXCL13+CD4+ T cells could be newly induced. RESULTS: CXCL13+CD4+ T cells in RA synovium were negative for interferon-γ (IFNγ), interleukin-4 (IL-4), IL-17, FoxP3, and CXCR5 and expressed low levels of inducible T cell costimulator, indicating that this population is a distinct human CD4 subset. T cell receptor (TCR) stimulation of CD4+ T cells, obtained from RA synovium with low expression of CXCL13, promptly induced CXCL13 production and addition of proinflammatory cytokines supported the long-term production of CXCL13. These findings indicate that CXCL13-producing CD4+ T cells can be in a memory state ready to be reactivated upon TCR stimulation and that proinflammatory cytokines are involved in persistent CXCL13 production. TCR stimulation of CD4+ T cells from the blood of healthy volunteers, together with proinflammatory cytokine supplementation, induced a population that produced CXCL13, but not IFNγ. Synovial T cells recruited CXCR5+ cells in a CXCL13-dependent manner. CONCLUSION: CXCL13-producing CD4+ T cells induced in RA synovium may play a role in the recruitment of CXCR5+ cells, such as B cells and circulating follicular helper T cells, and in ectopic lymphoid neogenesis at sites of inflammation.

Lectin-like oxidized low-density lipoprotein receptor 1 signal is a potent biomarker and therapeutic target for human rheumatoid arthritis.

OBJECTIVE: To determine whether lectin-like oxidized low-density lipoprotein (ox-LDL) receptor 1 (LOX-1) and the soluble form of LOX-1 (sLOX-1) are novel target molecules for the diagnosis and treatment of rheumatoid arthritis (RA). METHODS: Expression of ox-LDL and LOX-1 proteins in human RA synovium was evaluated by immunohistochemistry. Human RA fibroblast-like synoviocytes (FLS) were assessed for ox-LDL-induced expression of LOX-1 and ox-LDL-induced production of matrix metalloproteinase 1 (MMP-1) and MMP-3. Levels of sLOX-1 in the plasma and synovial fluid of patients with RA, compared with patients with osteoarthritis (OA), were determined by a specific chemiluminescence enzyme-linked immunoassay. In animal experiments, ox-LDL was injected into the knee joints of mice, with or without an anti-LOX-1 neutralizing antibody or sLOX-1, and the severity of arthritis was analyzed by histology and immunohistochemistry. RESULTS: Oxidized LDL and LOX-1 proteins were detected in the RA synovial tissue. Levels of MMP-1 and MMP-3 were enhanced by stimulation of RA FLS with ox-LDL, and the production of both MMPs was inhibited by blockade of the ox-LDL-LOX-1 interaction with the anti-LOX-1 neutralizing antibody or sLOX-1. Levels of sLOX-1 in the plasma and synovial fluid of RA patients were significantly higher than those in OA patients and healthy controls and were positively correlated with inflammation markers and the extent of RA disease activity. In the knees of mice, blockade of the ox-LDL-LOX-1 interaction suppressed arthritic changes and reduced the expression of MMP-3 induced by ox-LDL. CONCLUSION: These findings strongly indicate that sLOX-1 is a novel biomarker that may be useful for the diagnosis of RA and for the evaluation of disease activity in RA. Furthermore, the results suggest that LOX-1 may be a potent therapeutic target for RA.

IL-27-producing CD14(+) cells infiltrate inflamed joints of rheumatoid arthritis and regulate inflammation and chemotactic migration.

Interleukin (IL)-27, a heterodimeric cytokine, has been reported to be involved in the pathogenesis of autoimmune diseases through mediating differentiation of Th1 or Th17 cells and immune cell activity or survival. However, the origin and effects of IL-27 in joints of rheumatoid arthritis (RA) remain unclear. In this study, we investigated the distribution and anti-inflammatory roles of IL-27 in RA synovium. The IL-27 levels in plasma of RA patients, osteoarthritis (OA) patients, or healthy volunteers (n=15 per group) were equivalent and were at most 1 ng/ml, but the IL-27 level in synovial fluid of RA patients (n=15, mean 0.13 ng/ml; range 0.017-0.37 ng/ml) was significantly higher than that in synovial fluid of OA patients (n=15, mean 0.003 ng/ml; range 0-0.033 ng/ml) and potentially lower than in plasma. We analyzed the protein level of IL-27 produced by RA fibroblast-like synoviocytes (FLSs) or mononuclear cells (MNCs) from RA or OA synovial fluid or peripheral blood and showed that IL-27 in RA joints was derived from MNCs but not from FLSs. We also found by flow cytometry that IL-27-producing MNCs were CD14(+), and that these CD14(+)IL-27(+) cells were clearly detected in RA synovium but rarely in OA synovium by immunohistochemistry. Furthermore, we demonstrated that a relatively physiological concentration of IL-27 below 10 ng/ml suppressed the production of IL-6 and CCL20 from RA FLSs induced by proinflammatory cytokines through the IL-27/IL-27R axis. In the synovial fluid of RA, the IL-27 level interestingly had positive correlation with the IFN-γ level (r=0.56, p=0.03), but weak negative correlation with the IL-17A level (r=-0.30, p=0.27), implying that IL-27 in inflammatory joints of RA induces Th1 differentiation and suppresses the development or the migration of Th17 cells. These findings indicate that circulating IL-27-producing CD14(+) cells significantly infiltrate into inflamed regions such as RA synovium and have anti-inflammatory effects in several ways: both directly through the reduction of IL-6 production, and possibly through the induction of Th1 development and the suppression of Th17 development; and indirectly by regulation of recruitment of CCR6(+) cells, such as Th17 cells, through the suppression of CCL20 production. Our results suggest that such a serial negative feedback system could be applied to RA therapy.

MicroRNA-451 down-regulates neutrophil chemotaxis via p38 MAPK.

OBJECTIVE: MicroRNAs (miRNAs) are endogenous small noncoding RNAs that regulate the activity of target messenger RNAs (mRNAs) and cellular processes. MicroRNA-451 (miR-451) is one of the miRNAs that is conserved perfectly among vertebrates, and it regulates cell proliferation, invasion, and apoptosis in tumors. However, the role of miR-451 in autoimmune arthritis is unknown. This study was undertaken to identify the role of miR-451 in autoimmune arthritis. METHODS: We compared the expression of miR-451 in neutrophils from patients with rheumatoid arthritis (RA) and healthy controls. We also evaluated the role of miR-451 in neutrophil chemotaxis in vivo and in vitro using murine neutrophils. The regulation of p38 MAPK by miR-451 was assessed. Double-stranded miR-451 was administered to SKG mice, the arthritis score was determined, and histologic examination was performed. RESULTS: MicroRNA-451 expression in neutrophils isolated from patients with RA was lower than that in healthy controls. Systemic administration of miR-451 significantly disrupted the infiltration of neutrophils in an air-pouch model of local inflammation without affecting apoptosis of neutrophils. Overexpression of miR-451 significantly suppressed the migration of neutrophils to fMLP. We identified CPNE3 and Rab5a as direct targets of miR-451. Overexpression of miR-451 suppressed the phosphorylation of p38 MAPK via 14-3-3ζ, a known target of miR-451, and Rab5a. In SKG mice, miR-451 treatment reduced the severity of arthritis and the number of infiltrating cells. CONCLUSION: These results suggest that miR-451 suppresses neutrophil chemotaxis via p38 MAPK and is a potential target in the treatment of RA.

Inhibition of miR-92a enhances fracture healing via promoting angiogenesis in a model of stabilized fracture in young mice.

MicroRNAs (miRNAs) are endogenous small noncoding RNAs regulating the activities of target mRNAs and cellular processes. Although no miRNA has been reported to play an important role in the regulation of fracture healing, several miRNAs control key elements in tissue repair processes such as inflammation, hypoxia response, angiogenesis, stem cell differentiation, osteogenesis, and chondrogenesis. We compared the plasma concentrations of 134 miRNAs in 4 patients with trochanteric fractures and 4 healthy controls (HCs), and the levels of six miRNAs were dysregulated. Among these miRNAs, miR-92a levels were significantly decreased 24 hours after fracture, compared to HCs. In patients with a trochanteric fracture or a lumbar compression fracture, the plasma concentrations of miR-92a were lower on days 7 and 14, but had recovered on day 21 after the surgery or injury. To determine whether systemic downregulation of miR-92a can modulate fracture healing, we administered antimir-92a, designed using locked nucleic acid technology to inhibit miR-92a, to mice with a femoral fracture. Systemic administration of antimir-92a twice a week increased the callus volume and enhanced fracture healing. Enhancement of fracture healing was also observed after local administration of antimir-92a. Neovascularization was increased in mice treated with antimir-92a. These results suggest that plasma miR-92a plays a crucial role in bone fracture healing in human and that inhibition of miR-92a enhances fracture healing through angiogenesis and has therapeutic potential for bone repair.

MCP/CCR2 signaling is essential for recruitment of mesenchymal progenitor cells during the early phase of fracture healing.

OBJECTIVE: The purpose of this study was to investigate chemokine profiles and their functional roles in the early phase of fracture healing in mouse models. METHODS: The expression profiles of chemokines were examined during fracture healing in wild-type (WT) mice using a polymerase chain reaction array and histological staining. The functional effect of monocyte chemotactic protein-1 (MCP-1) on primary mouse bone marrow stromal cells (mBMSCs) was evaluated using an in vitro migration assay. MCP-1-/- and C-C chemokine receptor 2 (CCR2)-/- mice were fractured and evaluated by histological staining and micro-computed tomography (micro-CT). RS102895, an antagonist of CCR2, was continuously administered in WT mice before or after rib fracture and evaluated by histological staining and micro-CT. Bone graft exchange models were created in WT and MCP-1-/- mice and were evaluated by histological staining and micro-CT. RESULTS: MCP-1 and MCP-3 expression in the early phase of fracture healing were up-regulated, and high levels of MCP-1 and MCP-3 protein expression observed in the periosteum and endosteum in the same period. MCP-1, but not MCP-3, increased migration of mBMSCs in a dose-dependent manner. Fracture healing in MCP-1-/- and CCR2-/- mice was delayed compared with WT mice on day 21. Administration of RS102895 in the early, but not in the late phase, caused delayed fracture healing. Transplantation of WT-derived graft into host MCP-1-/- mice significantly increased new bone formation in the bone graft exchange models. Furthermore, marked induction of MCP-1 expression in the periosteum and endosteum was observed around the WT-derived graft in the host MCP-1-/- mouse. Conversely, transplantation of MCP-1-/- mouse-derived grafts into host WT mice markedly decreased new bone formation. CONCLUSIONS: MCP-1/CCR2 signaling in the periosteum and endosteum is essential for the recruitment of mesenchymal progenitor cells in the early phase of fracture healing.

Comprehensive microRNA analysis identifies miR-24 and miR-125a-5p as plasma biomarkers for rheumatoid arthritis.

MicroRNAs (miRNAs) are present in human plasma and known as a non-invasive biomarker for cancer detection. Our study was designed to identify plasma miRNAs specific for rheumatoid arthritis (RA) by a comprehensive array approach. We performed a systematic, array-based miRNA analysis on plasma samples from three RA patients and three healthy controls (HCs). Plasma miRNAs with more than four times change or with significant (P<0.05) change in expression, or detectable only in RA plasma, were confirmed with plasma from eight RA patients and eight HCs using real-time quantitative PCR. Consistently detectable miRNAs that were significantly different between RA patients and HCs were chosen for further validation with 102 RA patients and 104 HCs. The area under curves (AUC) were calculated after plotting the receiver operating characteristic (ROC) curves. To determine if these miRNAs are specific for RA, the concentrations of these miRNAs were analyzed in 24 patients with osteoarthritis (OA), and 11 patients with systemic lupus erythematosus (SLE). The array analysis and the subsequent confirmation in larger patient cohort identified significant alterations in plasma levels of seven miRNAs. The highest AUC was found for miR-125a-5p, followed in order by miR-24 and miR-26a. Multivariable logistic regression analysis showed that miR-24, miR-30a-5p, and miR-125a-5p were crucial factors for making detection model of RA and provided a formula for Estimated Probability of RA by plasma MiRNA (ePRAM), employing miR-24, miR-30a-5p and miR-125a-5p, which showed increased diagnostic accuracy (AUC: 0.89). The level of miR-24, miR-125a-5p, and ePRAM in OA and SLE patients were lower than that in RA. There was no significant difference in detection for anti-citrullinated protein antibody (ACPA)-positive and ACPA-negative RA patients. These results suggest that the plasma concentrations of miR-24 and miR-125a-5p, and ePRAM are potential diagnostic markers of RA even if patients were ACPA-negative.

Stromal cell-derived factor 1 regulates the actin organization of chondrocytes and chondrocyte hypertrophy.

Stromal cell-derived factor 1 (SDF-1/CXCL12/PBSF) plays important roles in the biological and physiological functions of haematopoietic and mesenchymal stem cells. This chemokine regulates the formation of multiple organ systems during embryogenesis. However, its roles in skeletal development remain unclear. Here we investigated the roles of SDF-1 in chondrocyte differentiation. We demonstrated that SDF-1 protein was expressed at pre-hypertrophic and hypertrophic chondrocytes in the newly formed endochondral callus of rib fracture as well as in the growth plate of normal mouse tibia by immunohistochemical analysis. Using SDF-1(-/-) mouse embryo, we histologically showed that the total length of the whole humeri of SDF-1(-/-) mice was significantly shorter than that of wild-type mice, which was contributed mainly by shorter hypertrophic and calcified zones in SDF-1(-/-) mice. Actin cytoskeleton of hypertrophic chondrocytes in SDF-1(-/-) mouse humeri showed less F-actin and rounder shape than that of wild-type mice. Primary chondrocytes from SDF-1(-/-) mice showed the enhanced formation of philopodia and loss of F-actin. The administration of SDF-1 to primary chondrocytes of wild-type mice and SDF-1(-/-) mice promoted the formation of actin stress fibers. Organ culture of embryonic metatarsals from SDF-1(-/-) mice showed the growth delay, which was recovered by an exogenous administration of SDF-1. mRNA expression of type X collagen in metatarsals and in primary chondrocytes of SDF-1(-/-) mouse embryo was down-regulated while the administration of SDF-1 to metatarsals recovered. These data suggests that SDF-1 regulates the actin organization and stimulates bone growth by mediating chondrocyte hypertrophy.