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Objective：Macrophages are the main source of inflammatory mediators and play important roles in the pathogenesis of rheumatoid arthritis (RA). Interleukin-21 (IL-21) regulates both innate and adaptive immune responses and exerts major effects on inflammatory responses that promote the development of RA. However, its effect on macrophage polarisation remains unclear. Methods：CD14+ monocytes of the peripheral blood of Human healthy donors (HD) and RA, and macrophages of RA synovial fluid (RA-SF MΦs) were isolated. IL-21 receptor (IL-21R) was detected by flow cytometry. Cytokine production by MΦs from different sources pre-treated with IL-21 and/or LPS was measured by real-time polymerase chain reaction (RT-PCR) and ELISA. CD14+ monocytes were differentiated into M1-like and M2-like macrophages via stimulation with GM-CSF, interferon-γ (IFN-γ), and LPS or M-CSF, IL-4, and IL-13, respectively. To determine the effect of IL-21 on macrophage polarisation, macrophage phenotypes, gene expression, and cytokine secretion were detected by flow cytometry, RT-PCR, and ELISA. TLR4 and ERK1/2 were determined by western blotting. Results：IL-21 exerted different effects on LPS-mediated inflammatory responses in various derived MΦs, and inhibited macrophages polarisation to M1-like macrophages and promote their polarisation to M2-like macrophages in HD and RA. Moreover, IL-21 inhibited LPS-mediated secretion of inflammatory cytokines, probably by downregulating the ERK1/2, in RA-SF MΦs. Conclusion：For the first time, we indicated that IL-21 inhibits LPS-mediated cytokine production in RA-SF MΦs, and impairs pro-inflammatory activity of M1-like macrophages, hereby exerting anti-inflammatory effects on RA. Thus, IL-21 might not be an appropriate therapeutic target for RA. [ABSTRACT FROM AUTHOR]

Interleukin‐21 (IL‐21) is a type I cytokine produced by activated T cells that promotes cytokine production in monocytes. Monocytes are activated by Toll‐like receptors (TLRs) to produce pro‐inflammatory mediators. However, little is known about the regulatory effect of IL‐21 on TLR‐mediated inflammation in human monocytes. This study investigated the potential association between IL‐21 and TLR2/4‐mediated inflammation in human monocytic THP‐1 cells. First, the expression of the IL‐21 receptor (IL‐21R) in human monocytic THP‐1 cells was examined by flow cytometry. Then, THP‐1 cells were treated with either the TLR2 ligand peptidoglycan (PGN) or the TLR4 ligand lipopolysaccharide (LPS) with or without IL‐21. Then, the production of several cytokines (IL‐6, IL‐8, TNF‐α, IFN‐γ and IL‐10), expression of TLR2/4, and activation of the downstream signaling pathways of mitogen‐activated protein kinase (MAPK), Janus kinase (JAK)/signal transducer and activator of transcription 3 (STAT3), phosphatidylinositol 3‐kinase (PI3K)/protein kinase B (Akt), and nuclear factor‐kappa B (NF‐κB) were determined. We found that IL‐21R was highly expressed in human monocytic THP‐1 cells and that IL‐21 induced TLR2 and TLR4 expression and further enhanced PGN/LPS‐mediated TLR2/4 expression. In addition, IL‐21 also upregulated the expression of IL‐6, IL‐8, TNF‐α, IFN‐γ and IL‐10 and enhanced TLR2/4‐mediated cytokine production in THP‐1 cells via phosphorylation of the STAT3, Akt and p38 MAPK signalling pathways. Our study suggests, for the first time that IL‐21 enhances TLR2/4‐mediated cytokine production in human monocytic THP‐1 cells by activating the STAT3, PI3K/Akt and p38 MAPK signalling pathways. [ABSTRACT FROM AUTHOR]