The results of our present experiments show that IL-10 inhibited the expression of IL-17 and its transcription factor RORt in F4/80+ macrophages and and (Figures?3E, ?E,3F,3F, ?F,5A5A and ?and5C).5C). genes associated with M1 and M2 markers was analyzed by qRT-PCR. Results Compared to WT mice, IL-10?/? mice had exacerbated CIA development, which was associated with increased production of MIK665 T helper 17 cell (Th17)/Th1 proinflammatory cytokines and CII-specific immunoglobulin G2a antibody after CII immunization. Macrophages in IL-10?/? mice had increased amounts of IL-17 and RORt compared with the amounts in WT mice with CIA. Immunofluorescence microscopy showed that the number of IL-17-producing macrophages in synovial tissues was significantly higher in IL-10?/? mice than in WT mice. IL-10 deficiency might promote macrophage polarization toward the proinflammatory M1 phenotype, which contributes to the rheumatoid arthritis inflammation response. Conclusion IL-10 inhibits IL-17 and RORt expression in macrophages and suppresses macrophages toward the proinflammatory M1 phenotype, which is important for the role of IL-10 in mediating the pathogenesis of CIA. Introduction Rheumatoid arthritis (RA) is an inflammatory autoimmune disease characterized by chronic inflammation within the synovial tissues in multiple joints, and it leads to progressive, erosive destruction of cartilage and joints [1]. Collagen-induced arthritis (CIA) is usually a well-established animal model that has been studied extensively because of its similarities to human RA. Although the etiology and pathogenesis of RA have not been completely elucidated, an imbalance between pro- and anti-inflammatory cytokines has been reported to be a key mechanism for joint inflammation and disease progression in CIA as well as in human RA [2]. Interleukin 10 (IL-10) is an important immunoregulatory cytokine produced by many cell populations, including macrophages, dendritic cells (DCs), T-cell subsets (Th2, Tc2 and Tr1) and B cells [3]. Lipopolysaccharides (LPSs) induce the expression of IL-10 in macrophages and culture [25]. However, whether IL-10 regulates IL-17 expression in macrophages from CIA has not been studied. In our present study, we investigated the functions of IL-10 in RA. IL-10-knockout (IL-10?/?) mice and their WT counterparts were used to establish a RA model. The results show that this development of CIA is usually exacerbated in IL-10?/? mice. Macrophages in IL-10?/? mice significantly upregulate the expression of IL-17 and retinoid-related orphan receptor t (RORt) and MIF (Chondrex). On day 14, these mice were given a second injection of CII dissolved in complete Freunds adjuvant. Clinical arthritis was evaluated using the following scale: grade 0 = no swelling; grade 1 = slight swelling and erythema; grade 2 = pronounced swelling; and grade 3 = joint rigidity. Preparation of peritoneal macrophages The mice were injected intraperitoneally with 2 ml of 5% thioglycollate medium (Sigma-Aldrich, St Louis, MO, USA) for 3 days. They were then killed, and peritoneal macrophages were isolated by lavage with phosphate-buffered saline (PBS). The cells were cultured in Dulbeccos altered Eagles medium made up of 10% fetal bovine serum (FBS) and antibiotics for 2 hours. Next, the cell cultures were washed to remove nonadherent cells before stimulation, and an aliquot was stained with MIK665 F4/80 and sorted by flow cytometry. Preparation of joint macrophages and collection of synovial fluid Joint macrophages and synovial fluid were collected according to a previously described protocol [28,29]. Briefly, after excision of the skin and patellar ligament under a dissecting microscope to expose the synovial membrane, a 30-gauge needle (BD Biosciences, San Jose, CA, USA) was carefully inserted into the membrane, and the synovial cavity was washed by repetitive injections and aspirations with PBS (20 l) to obtain synovial lavage material. This procedure was repeated five times, and a total volume of 100 l of synovial lavage fluid was obtained. After that step, joint and paws samples were removed and kept in RPMI 1640 medium (HyClone Laboratories/Thermo Fisher Scientific, Logan, UT, USA) containing 10% FBS (Sijiqing, Zhejiang, China), 100 IU/ml penicillin, 100 g/ml streptomycin (Beyotime Institute of Biotechnology, Shanghai, China) and 1 mg/ml collagenase (Sigma-Aldrich). The entire mixture was minced and incubated for 1 hour at 37C in a 5% CO2 atmosphere. The procedure was repeated three times, and cell suspensions were filtered with a cell strainer after red blood cell lysis. For macrophage isolation, the total of the above-described cell suspensions in a six-well plate for 2 hours and the adherent cells were harvested as joint macrophages. Synovial fluid MIK665 samples were stored at ?80C prior to performing assays. Quantitative RT-PCR analysis RNA samples were extracted by using TRIzol reagent (Invitrogen, Carlsbad, CA, USA), and cDNA was prepared.

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