engleski

OXYSTEROL 7-KETOCHOLESTEROL CAN RE-PROGRAM SYNOVIAL TISSUE MACROPHAGES AND SUPPORT M1 POLARIZATION

Background: Oxidized low-density lipoprotein (oxLDL) particles support lowgrade inflammation and have been found in synovial fluid from osteoarthritis (OA) joints [1]. Their component is 7-ketocholesterol (7-KC), which arises as the result of the oxidation of cholesterol [1]. 7-KC acts proinflammatory and it binds to Toll-like receptor (TLR) 4 expressed on macrophages [1]. Activation of TLR4 stimulates the classical macrophage maturation program, resulting in a specific phenotype of inducible nitric oxide synthase positive (iNOS+) and macrophage (M) 1 function, which produce and secrete proinflammatory chemokines and cytokines [2]. M2 macrophages are associated with wound healing by the production of arginase-1 [2, 3]. Synovial macrophages are of critical importance in the symptomatology and structural progression of OA since M1 polarized macrophages accumulate in human OA synovial tissue during exacerbation [3]. However, it is not known whether 7-KC can re- program synovial tissue macrophages and support M1 polarization. Objectives: We analyzed the influence of 7-KC on the polarization of CD68+ macrophages in the suspension of synovial mononuclear cells (SMCs) in respect to lipopolysaccharide (LPS), as M1 inducer. Methods: Mature synovial tissue samples were obtained during alloarthroplasty of the knee (N = 56). Paraffin embedded tissue sections were labelled by double immunofluorescence using a combination of antibodies directed toward CD68 and iNOS, arginase-1, CCL2 or CCL22. Suspension of SMCs was prepared by enzymatic digestion of tissue samples using collagenase IV and gradient density centrifugation. We analyzed intracellular (iNOS, arginase-1, CCL2, and CCL22) and surface (CD91, mannose receptor, HLA-DR, CD80, CD86 and decoy D6) antigens expression in CD68+ cells in the suspension of freshly isolated or 18 hour-cultured SMCs with 7-KC (25 μM), LPS (10ng/ml), their combination or in the medium only. Results: iNOS and CCL2 were more frequently labelled in lymphocyte clusters, while arginase-1 and CCL22 were labelled in synovial lining CD68+ cells. Phenotype of CD68+ cells did not change significantly after the 18 hour- culture in the medium only, except the decrease of mannose receptor and CD91, when compared with freshly isolated cells. 7-KC increased the percentage of CD86 expressing CD68+ cells, whereas decreased surface expression of CD91 and chemokine decoy D6, like in the culture with LPS, when compared with cells cultured in the medium only. 7-KC decreased the frequency of arginase-1+/CD68+ cells in the suspension and did not change iNOS+ in CD68+ cells, thus increasing the ratio of NOS+/arginase-1+ in CD68+ subset. 7-KC was unable to increase CCL2 like LPS in comparison with cells cultured in the medium only. Neither 7-KC nor LPS affected CCL22 expression in the CD68+ subset. Conclusion: These data provide a new perspective in understanding the polarization of macrophages toward the M1 phenotype mediated with oxysterol 7- KC in vitro.

osteoarthritis ; 7-ketocholesterol ; macrophages ; synovial tissue

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