IL-17 intensifies IFN-γ-induced NOS2 upregulation in RAW 264.7 cells by further activating STAT1 and NF-κB

Interleukin-17 (IL-17) is a signature cytokine of Th17 cells. Previous research has indicated that IL-17 plays a proinflammatory role by exacerbating interferon-γ (IFN-γ)-induced inflammation. However, prior to this study, it was not known whether inducible nitric oxide synthase (iNOS or NOS2), a signature molecule of inflammation, could be intensified by IL-17 when combined with IFN-γ. Thus, we explored the roles and underlying mechanisms of IL-17 and IFN-γ in the regulation of NOS2 expression in RAW 264.7 cells using qPCR, western blot analysis, colorimetric analysis, ChIP assay and statistical analysis. Although IL-17 alone did not induce NOS2 expression or nitric oxide (NO) production, as shown by western blot analysis and colorimetric analysis, it intensified IFN-γ-induced NOS2 upregulation and NO production in RAW 264.7 cells. The alteration of relevant transcription factors demonstrated that a combination of IFN-γ and IL-17 enhanced Tyr701-phosphorylated signal transducer and activator of transcription 1 [p-STAT1(Y701)] and nuclear factor-κB (NF-κB) activation, nuclear translocations and their binding to the NOS2 promoter, compared with IFN-γ alone, as illustrated by the results of the western blot analysis and ChIP assay. Also, using the corresponding inhibitors of STAT1 and NF-κB, we noted downregulation of the expression of NOS2 induced by IFN-γ alone or in combination with IL-17, respectively. In addition, IFN-γ increased phosphorylated (p-)p38 mitogen-activated protein kinase (MAPK), and accelerated the activation of the NF-κB pathway and the expression of NOS2, but phosphorylated extracellular signal-regulated kinase 1/2 (p-ERK1/2) was reduced by treatment with IFN-γ and IL-17. IL-17 intensified the activation of the NF-κB pathway and NOS2 upregulation induced by IFN-γ by increasing the phosphorylation of p38 MAPK and limiting the phosphorylation of ERK1/2. Taken together, these results suggest that IL-17 intensified IFN-γ-induced NOS2 upregulation and NO production by increasing the transcription activity of p-STAT1(Y701) and NF-κB in RAW 264.7 cells. Further activation of the NF-κB pathway induced by IL-17 relied on enhanced phosphorylation of p38 MAPK and decreased phosphorylation of ERK1/2. The mechanism suggested in this study provides novel information which may be used for anti-inflammatory therapy with IL-17.