Interleukin 26 (IL-26) is a new member of the IL-10 family that is highly expressed in rheumatoid arthritis (RA). However, the functions of IL-26 produced by macrophages in RA have not been elucidated. In the present work, we evaluated the effects and the mechanisms of IL-26 on M1 and M2 macrophage differentiation. Human or mouse macrophage cells were treated with lipopolysaccharides (LPS), interferon gamma (IFNγ), or IL-4 alone or concurrently treated with IL-26 to monitor M1 or M2 macrophage subtypes. The expression level of M1 or M2 macrophage genes was evaluated by reverse transcription polymerase chain reaction (RT-PCR) and enzyme-linked immunosorbent assay (ELISA). The molecular mechanisms of downstream signaling activation during differentiation were investigated by immunoblotting assay. Our results found that IL-26 promoted macrophage cells from CD80+ M1 macrophage differentiation, not from the CD206+ M2 phenotype. The messenger RNA of M1-type macrophage markers tumor necrosis factor alpha (TNFα) and inducible nitric oxide synthase (iNOS) was up-regulated in the IL-26-treated group. Also, the M1-related proinflammatory cytokines TNFα and IL-6 were induced after IL-26 stimulation. Interestingly, IL-10, a cytokine marker of M2 macrophage, was also elevated after IL-26 stimulation. Moreover, the M1-like macrophage stimulated by IL-26 underwent cJUN, nuclear factor kappa B (NF-κB), and signal transducer and activator of transcription 1 (STAT1) activation. Our findings suggested the role of IL-26 in synovial macrophages of active rheumatoid arthritis and provided a new insight into IL-26 as a candidate therapeutic target in rheumatoid arthritis.

Rheumatoid arthritis (RA) is an autoimmune disease with chronic inflammation, bone erosion, and joint deformation. Synovial tissue in RA patients is full of proinflammatory cytokines and infiltrated immune cells, such as T help (Th) 9, Th17, macrophages, and osteoclasts. Recent reports emphasized a new member of the interleukin (IL)-10 family, IL-26, an inducer of IL-17A that is overexpressed in RA patients. Our previous works found that IL-26 inhibits osteoclastogenesis and conducts monocyte differentiation toward M1 macrophages. In this study, we aimed to clarify the effect of IL-26 on macrophages linking to Th9 and Th17 in IL-9 and IL-17 expression and downstream signal transduction. Murine and human macrophage cell lines and primary culture cells were used and stimulated by IL26. Cytokines expressions were evaluated by flow cytometry. Signal transduction and transcription factors expression were detected by Western blot and real time-PCR. Our results show that IL-26 and IL-9 colocalized in macrophage in RA synovium. IL-26 directly induces macrophage inflammatory cytokines IL-9 and IL-17A expression. IL-26 increases the IL-9 and IL-17A upstream mechanisms IRF4 and RelB expression. Moreover, the AKT-FoxO1 pathway is also activated by IL-26 in IL-9 and IL-17A expressing macrophage. Blockage of AKT phosphorylation enhances IL-26 stimulating IL-9-producing macrophage cells. In conclusion, our results support that IL-26 promotes IL-9- and IL-17-expressing macrophage and might initiate IL-9- and IL-17-related adaptive immunity in rheumatoid arthritis. Targeting IL-26 may a potential therapeutic strategy for rheumatoid arthritis or other IL-9 plus IL-17 dominant diseases.