Objective The mechanisms that contribute to the persistent activation of macrophages in rheumatoid arthritis (RA) are incompletely understood. This study was performed to determine the contribution of endogenous gp96 in TLR-mediated macrophage activation in RA. Methods RA synovial fluids (SFs) were employed to activate macrophages and HEK-TLR2 and HEK-TLR4 cells. Neutralizing antibodies to TLR2, TLR4 and gp96 were employed to inhibit activation. RA SF macrophages were isolated by CD14 negative selection. Cell activation was measured by the expression of TNFα or IL-8 mRNA. Arthritis was induced employing the K/BxN serum transfer model, gp96 expression determined by Immunoblot analysis, ELISA and immunohistochemistry. The arthritis was treated with neutralizing anit-gp96 or control serum. Results RA SF induced the activation of macrophages and HEK-TLR2 and HEK-TLR4 cells. RA SF-induced macrophage and HEK-TLR2 activation was suppressed by neutralizing anti-gp96 antibody only when high (>800 ng/ml), but not low (<400 ng/ml), concentrations of gp96 were present. Neutralization of RA SF macrophage cell surface gp96 inhibited the constitutive expression of TNFα. Supporting its role in RA, joint tissue gp96 expression was induced in the K/BxN serum transfer model of RA, and neutralizing antibodies to gp96, ameliorated joint inflammation on clinical and histologic examination. Conclusions These observations support the role of gp96 as an endogenous TLR2 ligand in RA and identify the TLR2 pathway as a therapeutic target.

Objective A non-apoptotic role for Fas signaling has been implicated in the regulation of inflammation and innate immunity. These studies were performed to elucidate the role of Fas signaling on macrophages in the development of arthritis. Methods K/BxN serum transfer arthritis was induced in a mouse line with Fas conditionally deleted in the myeloid lineage (Fasf/f, LyzMcre). The arthritis was assessed clinically and histologically. IL-1β, CXCL5, IL-10, IL-6 and gp96 expression was determined by ELISA. Bone marrow derived macrophages were activated by IL-1β and gp96. Cells were analyzed for phenotype and apoptosis by flow cytometry. Results The onset of arthritis in Fasf/f, LyzMcre mice was comparable to that observed in control mice, however, resolution was accelerated during the chronic phase. The attenuated arthritis was associated with reduced articular expression of the endogenous TLR2 ligand gp96 and the neutrophil chemokine CXCL5, and the enhanced expression of IL-10. Activation with IL-1β or gp96 induced increased IL-10 in Fas deficient, compared with control, macrophages. IL-10 suppressed IL-1β plus gp96 induced IL-6 and CXCL5. IL-1β-mediated activation of ERK, which regulates IL-10 expression, was increased in Fas-deficient macrophages. Conclusions Together, our observations suggest that impaired Fas signaling results in the enhanced expression of anti-inflammatory IL-10 and reduced gp96, which are associated with accelerated resolution of inflammation in the chronic phase of arthritis. These observations suggest that strategies to reduce endogenous TLR ligands and increase IL-10 may be beneficial in patients with RA.

IntroductionOur objectives were to examine mononuclear cell gene expression profiles in patients with systemic lupus erythematosus (SLE) and healthy controls and to compare subsets with and without atherosclerosis to determine which genes’ expression is related to atherosclerosis in SLE.MethodsMonocytes were obtained from 20 patients with SLE and 16 healthy controls and were in vitro-differentiated into macrophages. Subjects also underwent laboratory and imaging studies to evaluate for subclinical atherosclerosis. Whole-genome RNA expression microarray was performed, and gene expression was examined.ResultsGene expression profiling was used to identify gene signatures that differentiated patients from controls and individuals with and without atherosclerosis. In monocytes, 9 out of 20 patients with SLE had an interferon-inducible signature compared with 2 out of 16 controls. By looking at gene expression during monocyte-to-macrophage differentiation, we identified pathways which were differentially regulated between SLE and controls and identified signatures based on relevant intracellular signaling molecules which could differentiate SLE patients with atherosclerosis from controls. Among patients with SLE, we used a previously defined 344-gene atherosclerosis signature in monocyte-to-macrophage differentiation to identify patient subgroups with and without atherosclerosis. Interestingly, this signature further classified patients on the basis of the presence of SLE disease activity and cardiovascular risk factors.ConclusionsMany genes were differentially regulated during monocyte-to-macrophage differentiation in SLE patients compared with controls. The expression of these genes in mononuclear cells is important in the pathogenesis of SLE, and molecular profiling using gene expression can help stratify SLE patients who may be at risk for development of atherosclerosis.

RA is a chronic inflammatory disease characterized by the persistent expression of inflammatory cytokines from macrophages, which may be mediated, in part, through TLR2 signaling. Earlier studies demonstrate a role for TLR2 signaling in dampening the arthritis in IL-1Ra-/- mice, which was mediated through T cells. This study was performed to determine whether TLR2 signaling plays a role in the pathogenesis of T cell-independent arthritis triggered by transferring serum from K/BxN mice. We documented more severe arthritis in Tlr2-/- mice compared with WT controls. The Tlr2-/- mice also demonstrated increased inflammation, erosion, pannus formation, and osteoclastogenesis, as well as increased IL-1β and decreased IL-10 within the joints. In vitro bone marrow-differentiated macrophages expressed comparable levels of activating and inhibitory FcγRs, however when stimulated with immune complexes, the Tlr2-/- macrophages expressed decreased IL-10 and reduced activation of Akt and ERK. Our findings indicate that Tlr2-/- promotes the effector phase of arthritis through decreased IL-10 by macrophages, which is important, not only as an anti-inflammatory cytokine but also in restraining the differentiation and activation of osteoclasts.