Roflumilast Reduced the IL-18-Induced Inflammatory Response in Fibroblast-Like Synoviocytes (FLS)

Abstract

Pro-inflammatory cytokines, such as the IL-18-induced inflammatory response and associated damage in fibroblast-like synoviocytes (FLS), play an important role in the pathogenesis of rheumatoid arthritis (RA). Roflumilast, an inhibitor of phosphodiesterase-4 (PDE-4), has been licensed for the treatment of chronic obstructive pulmonary disease (COPD). However, it is unknown whether roflumilast possesses a protective effect against the IL-18-induced inflammatory response in FLS. We found that roflumilast attenuated IL-18-induced oxidative stress by reducing the production of reactive oxygen species and malondialdehyde (MDA) in MH7A fibroblast-like synoviocytes (FLS). Additionally, roflumilast prevented IL-18-induced expressions and secretions of pro-inflammatory cytokines such as IL-6, IL-8, and TNF-α. Importantly, we found that roflumilast inhibited IL-18-induced expressions of chemokines such as CCL5, CXCL9, and CXCL10. Further, roflumilast inhibited the expression of extracellular matrix degradative enzymes, such as matrix metalloproteinase-3 (MMP-3) and MMP-13. Mechanistically, we found that roflumilast suppressed the activation of the transcriptional factor AP-1 and NF-κB. Our results suggest that roflumilast might be a potential therapeutic agent for the treatment of RA.

Figure 1

Effects of roflumilast in cell viability of MH7A fibroblast-like synoviocytes (FLS). (A) Molecular structure of roflumilast; (B) MH7A cells were stimulated with roflumilast at concentrations of 0, 0.5, 1, 5, 10, 50, and 100 μM for 24 h. Cell viability was measured using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) assay (^#P < 0.05, ^##P < 0.01 vs vehicle group).

Figure 2

Roflumilast reduced IL-18-induced oxidative stress in MH7A FLS. Cells were stimulated with IL-18 (10 ng/mL) in the presence or absence of 5 and 10 μM roflumilast. (A) Intracellular ROS was measured using dihydroethidium (DHE) staining; scale bar, 200 μm. (B) Production of MDA (^####P < 0.0001 vs vehicle group; **P < 0.01, ***P < 0.001 vs IL-18 group).

Figure 3

Roflumilast reduced IL-18-induced expressions and secretions of pro-inflammatory cytokines IL-6, IL-8, and TNF-α in MH7A FLS. Cells were stimulated with IL-18 (10 ng/mL) in the presence or absence of 5 and 10 μM roflumilast. (A–C) mRNA of IL-6, IL-8, and TNF-α; (D–F) secretions of IL-6, IL-8, and TNF-α (^####P < 0.0001 vs vehicle group; **P < 0.01, ***P < 0.001 vs IL-18 group).

Figure 4

Roflumilast reduced IL-18 induced expression and secretion of pro-inflammatory chemokines CCL5, CXCL9, and CXCL10 in MH7A FLS. Cells were stimulated with IL-18 (10 ng/mL) in the presence or absence of 5 and 10 μM roflumilast. (A–C) mRNA of CCL5, CXCL9, and CXCL10; (D–F) secretions of CCL5, CXCL9, and CXCL10 (^####P < 0.0001 vs vehicle group; **P < 0.01, ***P < 0.001 vs IL-18 group).

Figure 5

Roflumilast reduced IL-18-induced expressions and secretions of MMP-3 and MMP-13 in MH7A FLS. Cells were stimulated with IL-18 (10 ng/mL) in the presence or absence of 5 and 10 μM roflumilast. mRNA of MMP-3 (A) and MMP-13 (B); protein levels of MMP-3 (C) and MMP-13 (D) (^####P < 0.0001 vs vehicle group; **P < 0.01, ***P < 0.001 vs IL-18 group).

Figure 6

Roflumilast reduced IL-18-induced activation of AP-1 in MH7A FLS. Cells were stimulated with IL-18 (10 ng/mL) in the presence or absence of 5 and 10 μM roflumilast. (A) Protein expression of c-Jun and c-Fos and (B) luciferase activity of AP-1 (^####P < 0.0001 vs vehicle group; **P < 0.01, ***P < 0.001 vs IL-18 group).

Figure 7

Roflumilast reduced IL-18-induced activation of NF-κB in MH7A RA-FLS. Cells were stimulated with IL-18 (10 ng/mL) in the presence or absence of 5 and 10 μM roflumilast. (A) Nuclear levels of NF-κB p65; (B) luciferase activity of NF-κB (^####P < 0.0001 vs vehicle group; **P < 0.01, ***P < 0.001 vs IL-18 group).