Inflammatory expression profiles in monocyte-to-macrophage differentiation in patients with systemic lupus erythematosus and relationship with atherosclerosis.

INTRODUCTION: Our 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. METHODS: Monocytes 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. RESULTS: Gene 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. CONCLUSIONS: Many 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.

Fas signaling in macrophages promotes chronicity in K/BxN serum-induced arthritis.

OBJECTIVE: A nonapoptotic role of Fas signaling has been implicated in the regulation of inflammation and innate immunity. This study was undertaken to elucidate the contribution of Fas signaling in macrophages to the development of arthritis. METHODS: K/BxN serum-transfer arthritis was induced in a mouse line in which Fas was conditionally deleted in the myeloid lineage (Cre(LysM) Fas(flox/flox) mice). The arthritis was assessed clinically and histologically. Expression of interleukin-1ß (IL-1ß), CXCL5, IL-10, IL-6, and gp96 was determined by enzyme-linked immunosorbent assay. Bone marrow-derived macrophages were activated with IL-1ß and gp96. Cell phenotype and apoptosis were analyzed by flow cytometry. RESULTS: Arthritis onset in Cre(LysM) Fas(flox/flox) mice was comparable with 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 Toll-like receptor 2 (TLR-2) ligand gp96 and the neutrophil chemotactic chemokine CXCL5, and enhanced expression of IL-10. Activation with IL-1ß or gp96 induced increased IL-10 expression in Fas-deficient murine macrophages compared with control macrophages. IL-10 suppressed IL-6 and CXCL5 expression induced by IL-1ß plus gp96. IL-1ß-mediated activation of ERK, which regulates IL-10 expression, was increased in Fas-deficient mouse macrophages. CONCLUSION: Taken together, our findings indicate that impaired Fas signaling results in enhanced expression of antiinflammatory IL-10 and reduced expression of gp96, and these effects are associated with accelerated resolution of inflammation during the chronic phase of arthritis. These observations suggest that strategies to reduce endogenous TLR ligands and increase IL-10 may be beneficial in the treatment of rheumatoid arthritis.

TLR2 deletion promotes arthritis through reduction of IL-10.

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-1ß 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.

Glycoprotein 96 perpetuates the persistent inflammation of rheumatoid arthritis.

OBJECTIVE: The mechanisms that contribute to the persistent activation of macrophages in rheumatoid arthritis (RA) are incompletely understood. The aim of this study was to determine the contribution of endogenous gp96 in Toll-like receptor (TLR)-mediated macrophage activation in RA. METHODS: RA synovial fluid was used to activate macrophages and HEK-TLR-2 and HEK-TLR-4 cells. Neutralizing antibodies to TLR-2, TLR-4, and gp96 were used to inhibit activation. RA synovial fluid macrophages were isolated by CD14 negative selection. Cell activation was measured by the expression of tumor necrosis factor α (TNFα) or interleukin-8 messenger RNA. Arthritis was induced in mice by K/BxN serum transfer. The expression of gp96 was determined by immunoblot analysis, enzyme-linked immunosorbent assay, and immunohistochemistry. Arthritis was treated with neutralizing anti-gp96 antiserum or control serum. RESULTS: RA synovial fluid induced the activation of macrophages and HEK-TLR-2 and HEK-TLR-4 cells. RA synovial fluid-induced macrophage and HEK-TLR-2 activation was suppressed by neutralizing anti-gp96 antibodies only in the presence of high (>800 ng/ml) rather than low (<400 ng/ml) concentrations of gp96. Neutralization of RA synovial fluid macrophage cell surface gp96 inhibited the constitutive expression of TNFα. Supporting the role of gp96 in RA, joint tissue gp96 expression was induced in mice with the K/BxN serum-induced arthritis, and neutralizing antibodies to gp96 ameliorated joint inflammation, as determined by clinical and histologic examination. CONCLUSION: These observations support the notion that gp96 plays a role as an endogenous TLR-2 ligand in RA and identify the TLR-2 pathway as a therapeutic target.