Oxidative stress and chronic inflammation in osteoarthritisMarchev, Andrey S.; Dimitrova, Petya A.; Burns, Andrew J. ; Kostov, Rumen; DinkovaKostova, Albena; Georgiev, Milen I.

Leflunomide, an inhibitor of de novo pyrimidine biosynthesis, has recently been introduced as a treatment for rheumatoid arthritis in an attempt to ameliorate inflammation by inhibiting lymphocyte activation. Although the immunosuppressive ability of leflunomide has been well described in several experimental animal models, the precise effects of a limited pyrimidine supply on T cell differentiation and effector functions have not been elucidated. We investigated the impact of restricted pyrimidine biosynthesis on the activation and differentiation of CD4 T cells in vivo and in vitro. Decreased activation of memory CD4 T cells in the presence of leflunomide resulted in impaired generation and outgrowth of Th1 effectors without an alteration of Th2 cell activation. Moreover, priming of naive T cells in the presence of leflunomide promoted Th2 differentiation from uncommitted precursors in vitro and enhanced Th2 effector functions in vivo, as indicated by an increase in Ag-specific Th2 cells and in the Th2-dependent Ag-specific Ig responses (IgG1) in immunized mice. The effects of leflunomide on T cell proliferation and differentiation could be antagonized by exogenous UTP, suggesting that they were related to a profound inhibition of de novo pyrimidine biosynthesis. These results indicate that leflunomide might exert its anti-inflammatory activities in the treatment of autoimmune diseases by preventing the generation of proinflammatory Th1 effectors and promoting Th2 cell differentiation. Moreover, the results further suggest that differentiation of CD4 T cells can be regulated at the level of nucleotide biosynthesis.

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Several immune cell populations are involved in cartilage damage, bone erosion, and resorption processes during osteoarthritis. The purpose of this study was to investigate the role of NK cells in the pathogenesis of experimental osteoarthritis and whether and how neutrophils can regulate their synovial localization in the disease. Experimental osteoarthritis was elicited by intra-articular injection of collagenase in wild type and 8-wk old mice. To follow osteoarthritis progression, cartilage damage, synovial thickening, and osteophyte formation were measured histologically. To characterize the inflammatory cells involved in osteoarthritis, synovial fluid was collected early after disease induction, and the cellular and cytokine content were quantified by flow cytometry and ELISA, respectively. We found that NK cells and neutrophils are among the first cells that accumulate in the synovium during osteoarthritis, both exerting a pathogenic role. Moreover, we uncovered a crucial role of the CXCL10/CXCR3 axis, with CXCL10 increasing in synovial fluids after injury and mice being protected from disease development. Finally, in vivo depletion experiments showed that neutrophils are involved in an NK cell increase in the synovium, possibly by expressing CXCL10 in inflamed joints. Thus, neutrophils and NK cells act as important disease-promoting immune cells in experimental osteoarthritis and their functional interaction is promoted by the CXCL10/CXCR3 axis.

Pregnancy is a state where high and stage-dependent plasticity of the maternal immune system is necessary in order to equilibrate between immunosuppression of harmful responses towards the fetus and ability to fight infections. TCR γδ cells have been implicated in the responses in infectious diseases, in the regulation of immune responses, and in tissue homeostasis and repair. The variety of functions makes γδ T cells a particularly interesting population during pregnancy. In this study, we investigated the proportion, phenotype and TCR γ and δ repertoires of γδ T cells at the maternal–fetal interface and in the blood of pregnant women using FACS, immunohistochemistry and spectratyping. We found an enrichment of activated and terminally differentiated pro-inflammatory γδ T-cell effectors with specific location in the human decidua during early pregnancy, while no significant changes in their counterparts in the blood of pregnant women were observed. Our spectratyping data revealed polyclonal CDR3 repertoires of the δ1, δ2 and δ3 chains and γ2, γ3, γ4 and γ5 chains and oligoclonal and highly restricted CDR3γ9 repertoire of γδ T cells in the decidua and blood of pregnant women. Early pregnancy induces recruitment of differentiated pro-inflammatory γδ T-cell effectors with diverse TCR repertoires at the maternal–fetal interface.

IntroductionGlucosamine is an amino-monosaccharide and precursor of glycosaminoglycans, major components of joint cartilage. Glucosamine has been clinically introduced for the treatment of osteoarthritis but the data about its protective role in disease are insufficient. The goal of this study was to investigate the effect of long term administration of glucosamine on bone resorption and remodeling.MethodsThe effect of glucosamine on bone resorption and remodeling was studied in a model of collagenase-induced osteoarthritis (CIOA). The levels of macrophage-inflammatory protein (MIP)-1α, protein regulated upon activation, normal T-cell expressed, and secreted (RANTES), soluble receptor activator of nuclear factor kappa-B ligand (RANKL), tumor necrosis factor (TNF)-α, and interleukin (IL)-6, 4 and 10 in synovial fluid were measured by enzyme-linked immunosorbent assay (ELISA). Cell populations in synovial extracts and the expression of RANKL, of receptors for TNF-α (TNF-αR) and interferon γ (IFN-γR) on clusters of differentiation (CD) three positive T cells were analyzed by flow cytometry. Transforming growth factor (TGF)-β3, bone morphogenetic protein (BMP)-2, phosphorylated protein mothers against decapentaplegic homolog 2 (pSMAD-2), RANKL and Dickkopf-1 protein (DKK-1) positive staining in CIOA joints were determined by immunohistochemistry.ResultsThe administration of glucosamine hydrochloride in CIOA mice inhibited loss of glycosaminoglycans (GAGs) and proteoglycans (PGs) in cartilage, bone erosion and osteophyte formation. It decreased the levels of soluble RANKL and IL-6 and induced IL-10 increase in the CIOA joint fluids. Glucosamine limited the number of CD11b positive Ly6G neutrophils and RANKL positive CD3 T cells in the joint extracts. It suppressed bone resorption via down-regulation of RANKL expression and affected bone remodeling in CIOA by decreasing BMP-2, TGF-β3 and pSMAD-2 expression and up-regulating DKK-1 joint levels.ConclusionsOur data suggest that glucosamine hydrochloride inhibits bone resorption through down-regulation of RANKL expression in the joints, via reduction of the number of RANKL positive CD3 T cells and the level of sRANKL in the joints extracts. These effects of glucosamine appear to be critical for the progression of CIOA and result in limited bone remodeling of the joints.

We investigated the ability of neutrophils to express receptor activator of nuclear factor kappa-B ligand (RANKL), to secrete osteoprotegerin (OPG), and to produce IL-17. Arthritis was induced by intra-articular injection of zymosan, a ligand for Toll-like receptor 2 (TLR2). Frequencies of neutrophils in bone marrow (BM), blood and synovial fluid (SF), receptor expression, and cytokine production were evaluated by flow cytometry. 1A8 antibody (1A8 Ab) was used to deplete neutrophils in zymosan-injected SCID mice. IL-17, RANKL, and OPG amounts in SF, serum, or cell cultures were determined by ELISA. The development of arthritis was associated with increased secretion of IL-17, RANKL, and OPG in serum and SF, elevated frequencies of Ly6G+CD11b+ cells in BM, blood, and SF and upregulated RANKL expression. Both IL-17 and OPG were absent in serum and SF after neutrophil depletion; therefore we assume that they were released by neutrophils. In vitro blood Ly6G+CD11b+ cells from arthritic mice produced spontaneously IL-17, IFN-γ, and OPG and expressed RANKL. This phenotype was sustained by IL-17. TLR2 engagement increased IL-17 and IFN-γ production, potentiated IL-17-mediated RANKL expression, and inhibited OPG secretion. We conclude that TLR2 regulates the destructive potential of neutrophils and its targeting might limit joint alterations in arthritis.

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