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

Abstract

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.

Figure 1

Nitric oxide synthase 2 (NOS2) expression and nitric oxide (NO) production induced by interferon-γ (IFN-γ) and/or interleukin-17 (IL-17) in macrophages. (A-a, B and C-a) Representative western blot analysis of NOS2 in RAW 264.7 cells treated with indicated cytokines for 24 h. (A-b and C-b) Average quantification obtained by densitometric analysis for western blot analysis. Data are expressed as the density ratio of target protein to its non-treated level in arbitrary units. (D) Expression of NOS2 at the mRNA level in RAW 264.7 cells treated with indicated cytokines for 12 h. β-actin was selected as a housekeeping gene. (E) Nitrite concentration in the supernatant of cultured RAW 264.7 cells treated with indicated cytokines for 24 or 48 h. (F-a) Representative western blot analysis and (F-b) quantification of NOS2 in peritoneal macrophages treated with indicated cytokines for 24 h. β-actin was used as a loading control. Data are presented as the means ± SD from three independent experiments. ^*p<0.05 and ^**p<0.01.

Figure 2

Signal transducer and activator of transcription 1 (STAT1) pathway and nitric oxide synthase 2 (NOS2) expression induced by interferon-γ (IFN-γ) and/or interleukin-17 (IL-17) in RAW 264.7 cells. (A-a, B and C) Representative western blot analysis of p-STAT1(Y701) or p-STAT1(S727) in RAW 264.7 cells treated with indicated cytokines for 5 min or specified periods of time. (D) Representative western blot analysis of STAT1 in RAW 264.7 cells treated with STAT1 inhibitor fludarabine (Flu) for 24 h. (E and F-a) Representative western blot analysis of p-STAT1(Y701) or NOS2 in RAW 264.7 cells pretreated with Flu for 24 h prior to cytokine exposure for 5 min or 24 h. (G) Representative western blot analysis of NOS2 in RAW 264.7 cells pretreated with Janus kinase (JAK) inhibitor AG-490 for 30 min prior to cytokine exposure for 24 h. β-actin was used as a loading control. (A-b and F-b) Average quantification obtained by densitometric analysis for western blot analysis. Data are expressed as the density ratio of the target protein to its non-treated level in arbitrary units. Data are presented as the means ± SD from three independent experiments. ^*p<0.05 and ^**p<0.01.

Figure 3

Nuclear translocation and nitric oxide synthase 2 (NOS2) promoter binding of Tyr701-phosphorylated signal transducer and activator of transcription 1 [p-STAT1(Y701)] by interferon-γ (IFN-γ) and/or interleukin-17 (IL-17) in RAW 264.7 cells. (A-a) Representative western blot analysis of nuclear translocation of p-STAT1(Y701) in RAW 264.7 cells treated with the indicated cytokines for 1 h. β-actin was used as a cytoplasmic loading control, and histone H3 was used as a nuclear loading control. (A-b) Average quantification of nuclear and cytoplasmic p-STAT1(Y701) obtained by densitometric analysis for western blot analysis. Data are expressed as the density ratio of target protein to its non-treated level in arbitrary units. (A-c) p-STAT1(Y701) ratio of nucleus to cytoplasm. (B) Chromatin immunoprecipitation (ChIP) analysis of p-STAT1(Y701) occupancy on the proximal interferon-gamma-activated site (GAS) of NOS2 promoter in RAW 264.7 cells treated with indicated cytokines for 1 h. Antibodies: R, anti-RNA polymerase II antibody; I, IgG; S, anti-p-STAT1(Y701) antibody. Data are presented as the means ± SD from three independent experiments. ^*p<0.05 and ^**p<0.01.

Figure 4

NF-κB pathway and nitric oxide synthase 2 (NOS2) expression induced by interferon-γ (IFN-γ) and/or interleukin-17 (IL-17) in RAW 264.7 cells. (A-a and B-a) Representative western blot analysis of IκBα degradation in RAW 264.7 cells treated with the indicated cytokines for 4 h. (C-a) Representative western blot analysis of NOS2 in RAW 264.7 cells pretreated with the NF-κB inhibitor SN50 for 1 h prior to cytokine exposure for 24 h. β-actin was used as a loading control. (A-b, B-b and C-b) Average quantification obtained by densitometric analysis for western blot analysis. Data are expressed as the density ratio of target protein to its non-treated level in arbitrary units. Data are presented as the means ± SD from three independent experiments. ^*p<0.05 and ^**p<0.01.

Figure 5

Nuclear translocation and nitric oxide synthase 2 (NOS2) promoter binding of p65 by interferon-γ (IFN-γ) and/or interleukin-17 (IL-17) in RAW 264.7 cells. (A-a) Representative western blot analysis of nuclear translocation of p65 in RAW 264.7 cells treated with indicated cytokines for 1 h. β-actin was used as a cytoplasmic loading control, and histone H3 was used as a nuclear loading control. (A-b) Average quantification of nuclear and cytoplasmic p65 obtained by densitometric analysis for western blot analysis. Data are expressed as the density ratio of target protein to its non-treated level in arbitrary units. (B) Chromatin immunoprecipitation (ChIP) analysis of p65 occupancy on the proximal NF-κB element of the NOS2 promoter in RAW 264.7 cells treated with indicated cytokines for 1 h. Antibodies: R, anti-RNA Polymerase II antibody; I, IgG; p, anti-p65 antibody. Data are presented as the means ± SD from three independent experiments. ^*p<0.05 and ^**p<0.01.

Figure 6

p38 mitogen-activated protein kinase (MAPK) pathway and nitric oxide synthase 2 (NOS2) expression induced by interferon-γ (IFN-γ) and/or interleukin-17 (IL-17) in RAW 264.7 cells. (A-a) Representative western blot analysis of p-p38 in RAW 264.7 cells treated with indicated cytokines for 5 min. (B-a and C-a) Representative western blot analysis of NOS2 and IκBα degradation in RAW 264.7 cells pretreated with p-p38 inhibitor SB203580 for 30 min prior to cytokine exposure for 24 or 4 h. (D-a and E) Representative western blot analysis of phosphorylated extracellular signal-regulated kinase 1/2 (p-ERK1/2) or Tyr701-phosphorylated signal transducer and activator of transcription 1 [p-STAT1(Y701)] in RAW 264.7 cells pretreated with SB203580 for 30 min prior to cytokine exposure for 30 or 5 min. β-actin was used as a loading control. (A-b, B-b, C-b and D-b) Average quantification obtained by densitometric analysis for western blot analysis. Data are expressed as the density ratio of the target protein to its non-treated level in arbitrary units. Data are presented as the means ± SD from three independent experiments. ^**p<0.01.

Figure 7

Extracellular signal-regulated kinase 1/2 (ERK1/2) pathway and nitric oxide synthase 2 (NOS2) expression induced by interferon-γ (IFN-γ) and/or interleukin-17 (IL-17) in RAW 264.7 cells. (A-a) Representative western blot analysis of p-ERK1/2 in RAW 264.7 cells treated with indicated cytokines for 30 min. (B-a, C-a and D-a) Representative western blot analysis of NOS2, IκBα degradation and p-p38 in RAW 264.7 cells pretreated with p-ERK1/2 inhibitor PD98059 for 30 min prior to cytokine exposure for 24 h, 4 h or 5 min. β-actin was used as a loading control. (A-b, B-b, C-b and D-b) Average quantification obtained by densitometric analysis for western blot analysis. Data are expressed as the density ratio of target protein to its non-treated level in arbitrary units. Data are presented as the means ± SD from three independent experiments. ^**p<0.01.

Figure 8

Schematic diagram of the molecular mechanisms by which interleukin-17 (IL-17) intensified interferon-γ (IFN-γ)-induced nitric oxide synthase 2, inducible (NOS2) upregulation in RAW 264.7 cells. IFNGR1/2, interferon gamma receptor 1/2; p-ERK1/2, phosphorylated extracellular signal-regulated kinase 1/2; JAK1/2, Janus kinase 1/2.