Background/Purpose: Toll-like receptors (TLRs) have been shown to contribute to the inflammatory response in rheumatoid arthritis (RA). A number of TLRs have been found to be upregulated in synovial tissue (TLR2, TLR3, TLR4), synovial monocytes (TLR4) as well as macrophages (TLR2, TLR4) in RA versus osteoarthritis (OA). Based on phenotypic and functional differences macrophages can be divided into different subsets. At present there is limited knowledge on the functional role of macrophage subsets in rheumatoid arthritis. Here we investigated the impact of TLR2, TLR3 and TLR4 signaling on monocytes (M0) and monocyte derived M1 and M2 macrophages. We compared inflammatory and anti-inflammatory cytokine release, cell surface marker and TLR gene expression as well as viability of M0, M1 and M2 macrophages.

Methods: Monocytes were isolated from buffy coats of 4-6 healthy donors by CD14 microbead separation and differentiated into M1 and M2 by culturing them in the presence of 50ng/ml GM-CSF and M-CSF, respectively, for 6 to 8 days. For M0, CD14+ cells were kept in standard medium for 3 days. Subsequently, cells were stimulated for 24 hours with Pam3, LPS or PolyIC. Cytokine release was measured by ELISA, gene expression by qRT-PCR and viability by WST-1 assay. Cells were stained with fluorescently labeled antibodies for CD markers and analyzed by FACS.

Results: We demonstrate that stimulation of M2 macrophages with the TLR2-ligand Pam3 but not with the TLR4-ligand LPS leads to a decrease in the IL-10/IL-6 and IL-10/IL-8 ratio. TLR2 and TLR3 basal gene expression levels were similar between the cell types, whereas TLR4 expression was 3.7 fold higher in M2 than M0 or M1. TLR4 was strongly downregulated by LPS and Pam3 in M1 and M2, but not in M0. In contrast, TLR2 was upregulated by Pam3 and LPS. CD14 and CD163 expression was not affected by TLR ligands. CD86 levels were increased in M1 by all TLR ligands tested and in M0 by Pam3 but decreased in M2 by LPS and Pam3. CD80 was increased by Pam3 and LPS in M1 and by LPS in M2. Stimulation with TLR2 ligands was found to significantly increase M2 viability.

Conclusion: We demonstrate that stimulation of in vitro differentiated M2 macrophages with TLR2 ligands leads to increased proinflammatory cytokine production, suggesting that the antiinflammatory activity of M2 macrophages is reduced in an inflammatory milieu with abundant TLR2 ligands as can be found in RA. In line with these observations, Pam3 upregulated TLR2 mRNA expression in M2 macrophages in an autocrine fashion and increased M2 viability, thereby enhancing TLR2-induced proinflammatory effects.

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ACR Meeting Abstracts - https://acrabstracts.org/abstract/impact-of-tlr-stimulation-on-cytokine-production-in-macrophage-subsets-tlr2-stimulation-impairs-anti-inflammatory-activity-of-m2-macrophages/