Several studies have demonstrated that TLR4-mediated signals induce macrophages, dendritic cells and synovial cells from RA patients to produce IL-12 in vitro [21–23], indicating that TLR4-mediated signals induce IL-12 production by various immune and non-immune cells. Moreover, another study demonstrated that an IL-12p35/IFN-γ axis promotes antibody-induced joint inflammation by suppressing TGF-β production in joint tissues . These findings led us to hypothesize that a TLR4-mediated IL-12p35/IFN-γ axis regulates antibody-induced arthritis by suppressing TGF-β production. Consistent with this hypothesis, our current experiments revealed that IFN-γ, IL-12p35 and IL-1β transcript levels in joint tissues increased in WT mice compared with TLR4-/- mice following K/BxN serum transfer, whereas TGF-β transcript levels decreased. These findings suggest that IL-1β in addition to the IL-12p35/IFN-γ axis promotes TLR4-mediated joint inflammation. Several lines of evidence in our experiments suggest that IL-12 acts downstream of TLR4, triggering the production of both IFN-γ and IL-1β in joint tissues during antibody-induced arthritis, but suppressing TGF-β production. First, TLR4-/- mice produce minimal amounts of IL-12p35 in their joints during antibody-induced arthritis compared with WT mice. Moreover, injection of recombinant IL-12 into TLR4-/- mice restored joint inflammation. In vitro experiments revealed that LPS induced IL-12 production by joint immune cells, a response dependent on MyD88 and TRIF. Injection of LPS into WT mice increased the phosphorylation of the IL-12-inducing transcription factor STAT4 in joint immune cells during antibody-induced arthritis. Collectively, these findings suggest that TLR4-mediated signals induce the production of IL-12 by joint immune cells during antibody-induced arthritis. Second, injection of LPS enhanced antibody-induced arthritis and the production of IFN-γ and IL-1β in the joints of WT mice, but not IL-12p35-/- mice. Furthermore, injection of recombinant IL-12 into TLR4-/- mice enhanced the production of IFN-γ and IL-1β in the joints during antibody-induced arthritis, whereas recombinant IFN-γ and IL-1β did not enhance IL-12p35 production. Moreover, LPS-induced production of IL-12 by joint immune cells increased IFN-γ and IL-1β production by enhancing T-bet expression and pro-IL-1β production. These findings suggest that TLR4-mediated IL-12 production enhances the production of both IL-1β and IFN-γ in the joints during antibody-induced arthritis. However, that IL-12 induces IL-1β production by enhancing pro-IL-1β production during joint inflammation has not previously been reported. Third, injection of recombinant IFN-γ, IL-1β or IL-12 into TLR4-/- mice reduced TGF-β production in joint tissues during antibody-induced arthritis, while a TGF-β blockade restored antibody-induced arthritis in TLR4-/- mice. These findings indicate that TLR4-mediated IL-12/IL-1β and IL12-/IFN-γ axes in the joints suppress TGF-β production, thereby promoting antibody-induced arthritis. As no previous reports have addressed functional links between TLR4 and IL-12 regulatory axes in the pathogenesis of antibody-induced arthritis, this study provides the first demonstration that TLR4-mediated IL-12 promotes arthritis by regulating the production of both IL-1β and IFN-γ, thereby suppressing TGF-β production.
It has been suggested that TLR4-mediated signals promote joint inflammation by increasing levels of either IL-17 or IL-1β in murine arthritis models [10, 11, 24, 25]. However, WT and IL-17-/- mice showed similar joint inflammation and cytokine production in the K/BxN serum transfer model , suggesting that IL-17 may have minimal involvement in the TLR4-mediated regulation of antibody-induced arthritis. With regard to IL-1β, Choe et al. suggested that TLR4 regulation of joint inflammation bypasses the need for IL-1, although TLR4 and IL-1R play crucial roles in promoting antibody-induced arthritis . In their experiments, IL-1R-/- mice showed attenuated arthritis compared with WT mice upon K/BxN serum transfer, while LPS injection did not alter joint inflammation in IL-1R-/- or WT mice. Based on these findings, they suggested that LPS-mediated TLR4 signals do not regulate joint inflammation in WT or IL-1R-/- mice. In contrast to their results, our experiments demonstrated that injection of WT mice with LPS aggravated arthritis, when sub-maximal joint swelling was induced by injection of an appropriate amount of K/BxN serum, whereas LPS did not alter full-blown arthritis in WT mice (Figure 1E), a result consistent with the results of Choe et al. These findings suggest that LPS-mediated TLR4 signals regulate antibody-induced arthritis, depending on the severity of joint inflammation, which might also account for contradictory results that TLR4-/- mice showed K/BxN serum-induced arthritis comparable to WT mice , although these divergent findings should be further investigated. Therefore, we do not completely rule out the possibility that IL-1β contributes to TLR4-mediated pathogenesis in antibody-induced arthritis. Consistent with this suggestion, Ji et al. demonstrated that joint IL-1β expression levels were significantly increased three to six days after K/BxN serum transfer and suggested that IL-1 and TNF-β play critical roles in antibody-induced arthritis . Furthermore, our experiments demonstrated that recombinant IL-1β restored joint inflammation in TLR4-/- mice, indicating that IL-1β promotes antibody-mediated joint inflammation, depending on TLR4-mediated immune responses.
Our data indicate that monocytes from HCV patients are activated in vivo. This interferes with their differentiation into DC, leading to deficient TLR4 signaling in these cells that are enable to induce a Th1 response. This specific defect is linked to the activation of the MEK/ERK pathwayTLR4 is expressed not only in joint-infiltrating immune cells, but also in non-hematopoietic joint tissues, and regulates joint inflammation by mediating the production of various cytokines [27, 28]. Several studies have reported that macrophages, mast cells, NKT cells and Gr-1+ cells play crucial roles in antibody-induced arthritis, and express TLR4 on the cell surface [16, 17, 19, 20, 29]. Our experiments demonstrated that adoptive transfer of WT mast cells or macrophages fully restored joint inflammation in macrophage- and mast cell-depleted WT mice, respectively, indicating that TLR4-expressing macrophages and mast cells, rather than non-hematopoietic joint cells, are crucial to antibody-induced arthritis. However, adoptive transfer of Gr-1+ cell-depleted spleen cells only partially restored joint inflammation in TLR4-/- mice, indicating that Gr-1+ cells contribute less significantly to the promotion of antibody-induced arthritis via TLR4-mediated signals than macrophages and mast cells, although Gr-1+ cells play a crucial role in the development of joint inflammation. With respect to NKT cells, our recent study clearly demonstrated that invariant NKT cells express TLR4, which promotes antibody-induced arthritis , although the expression patterns of TLR4 in NKT cells are controversial [31–33]. Therefore, macrophages, mast cells, Gr-1+ cells and invariant NKT cells promote antibody-induced arthritis by expressing TLR4. Furthermore, levels of TLR4, which was constitutively expressed in the joints, gradually increased, peaked, and then gradually decreased in our current experiments. Consistent with the TLR4 expression pattern in the joints, levels of the endogenous TLR4 ligands HSP60, HMGB1 and fibronectin were also increased in the joint tissues of WT mice during antibody-induced arthritis. Moreover, antibody-induced arthritis was developed in WT, but not in TLR4-/- mice in the absence of exogenous TLR4 ligand, indicating that TLR4 endogenous ligands contribute to developing antibody-induced arthritis. Therefore, TLR4 on immune cells may be engaged by endogenous or exogenous ligands, which induce TLR4-mediated downstream immunological events. Consistent with our results, levels of endogenous TLR4 ligands, including HMGB-1, s100 proteins and hyaluronic acid were found to be elevated in the synovial fluid or serum of RA patients , and concentrations were correlated with clinical scores in RA patients .
For therapeutic purposes, it would be beneficial to inhibit TLR4 signals, IL-12 production, and the effects of IL-12 on IL-1β and IFN-γ production in antibody-induced joint inflammation. Several studies have demonstrated that anti-IL-12 mAbs ameliorate CIA in mice [36, 37], suggesting that a blockade of IL-12 with a neutralizing mAb may be a useful therapeutic strategy for rheumatoid arthritis. Alternatively, strategies to block the functional activity of TLR4-expressing effector cells may also be helpful in treating rheumatoid arthritis.