Anti tnf

Murine BMDMs were obtained as previously described (Xu et al., 2012 (link)) and maintained in DMEM (HyClone) supplemented with 10% FBS (Gibco) and 10% supernatant of L929 cell as conditioned medium providing macrophage colony-stimulating factor (M-CSF, identified as complete medium). Briefly, bone marrow cells were extracted and cultured with complete medium for 5 days to derive BMDMs. Cell culture grade LPS and Pam3CSK4 were purchased from Invivogen and were used at a concentration of 10 ng/ml unless otherwise specified. SF1670 were from Selleck. anti-IL10R, anti-IgG, and anti-TNF were purchased from BioXCell. anti-IL10R or anti-IgG was added 30 min prior to LPS stimulation and were present throughout LPS exposure.

CFA-induced inflammation was induced by i.p. injection of 200–400 μl of a 1:1 emulsion of DPBS and CFA (Sigma-Aldrich). Mice were analyzed at the stated time points after CFA injection. PolyI:C treatments were performed intravenously at 1.4 mg/Kg/d. LTβR signaling was blocked with 200 μg of LTβR-Ig consisting of the LTβR ectodomain fused with the Fc domain of a mouse IgG1 antibody specific for hen egg lysozyme (Hel-Ig). Control experiments were performed by treatment with 200 μg HEL-Ig. Inflammatory cytokines TNF-α, IFN-γ, and IL-1β were blocked with anti-TNF (#BE0058; Bio-X-Cell), anti-INF-γ (#BE0055; Bio-X-Cell), or anti-IL-1β (#BE0246; Bio-X-Cell) antibodies at 100 μg/mouse injected intravenously via the tail vein or retro-orbital sinus immediately before CFA injection. For LTβR-Ig treatment at steady state, mice were injected with 100 μg LTβR-Ig i.v. once a week for 3 wk and analyzed 3 wk after the first injection. In vivo BrdU incorporation was performed by BrdU injection i.v. (1 mg/mouse). Flow cytometry detection of incorporated BrdU was performed with FITC or APC BrdU Flow Kit (BD Biosciences) by following the manufacturer’s protocol.