TLR4-mediated IL-12 production enhances IFN-gamma and IL-1 beta production, which inhibits TGF-beta production and promotes antibody-induced joint inflammation

Introduction: Toll-like receptor (TLR)4 promotes joint inflammation in mice. Despite that several studies report a functional link between TLR4 and interleukin-(IL-)1 beta in arthritis, TLR4-mediated regulation of the complicated cytokine network in arthritis is poorly understood. To address this, we investigated the mechanisms by which TLR4 regulates the cytokine network in antibody-induced arthritis. Methods: To induce arthritis, we injected mice with K/BxN serum. TLR4-mediated pathogenesis in antibody-induced arthritis was explored by measuring joint inflammation, cytokine levels and histological alteration. Results: Compared to wild type (WT) mice, TLR4(-/-) mice showed attenuated arthritis and low interferon (IFN)-gamma, IL-12p35 and IL-1 beta transcript levels in the joints, but high transforming growth factor (TGF)-beta expression. Injection of lipopolysaccharide (LPS) enhanced arthritis and exaggerated joint cytokine alterations in WT, but not TLR4(-/-) or IL-12p35(-/-) mice. Moreover, STAT4 phosphorylation in joint cells and intracellular IL-12p35 expression in macrophages, mast cells and Gr-1(+) cells were detected in WT mice with arthritis and enhanced by LPS injection. Therefore, IL-12p35 appears to act downstream of TLR4 in antibody-induced arthritis. TLR4-mediated IL-12 production enhanced IFN-gamma and IL-1 beta production via T-bet and pro-IL-1 beta production. Recombinant IL-12, IFN-gamma and IL-1 beta administration restored arthritis, but reduced joint TGF-beta levels in TLR4(-/-) mice. Moreover, a TGF-beta blockade restored arthritis in TLR4(-/-) mice. Adoptive transfer of TLR4-deficient macrophages and mast cells minimally altered joint inflammation and cytokine levels in macrophage- and mast cell-depleted WT mice, respectively, whereas transfer of WT macrophages or mast cells restored joint inflammation and cytokine expression. Gr-1(+) cell-depleted splenocytes partially restored arthritis in TLR4(-/-) mice. Conclusion: TLR4-mediated IL-12 production by joint macrophages, mast cells and Gr-1(+) cells enhances IFN-gamma and IL-1 beta production, which suppresses TGF-beta production, thereby promoting antibody-induced arthritis.