Background Cigarette smoking is a recognised environmental risk factor not only for the development of rheumatoid arthritis (RA), but also for the severity of established RA. Specifically, smoking is associated with increased disease activity and progression. Sirtuins (SIRTs) are recently discovered regulators of inflammation. In RA, SIRT1 was shown to contribute to apoptosis resistance and pro-inflammatory cytokine production.
Objectives To investigate the impact of cigarette smoke on the expression of SIRT1 in RA synovial fibroblasts (RASF) and to elucidate the function of SIRT1 in the inflammatory response induced by cigarette smoke extract (CSE) and tumor necrosis factor alpha (TNFα).
Methods Synovial tissues were obtained from RA patients undergoing joint replacement surgery. RASF (n=6) were stimulated with 5% CSE or 10 ng/ml TNFα for 24 hours. Protein levels of SIRT1 were detected by immunoblot in cell lysates. The expression of SIRT1 mRNA was measured with SYBR green Real-time PCR. Secreted levels of interleukins (ILs) were detected in cell culture supernatants by ELISA. For overexpression of SIRT1, RASF (n=7) were transfected with the expression vector encoding wild-type SIRT1 or control vector for 72h and stimulated with 5% CSE or 10 ng/ml TNFα for 24h.
Results Western blot analysis revealed reduced expression of SIRT1 upon stimulation of RASF with CSE, but increased SIRT1 protein levels after treatment with TNFα. Since the mRNA levels of SIRT1 were not altered by the treatment of RASF with CSE, this indicated that SIRT1 is regulated posttranscriptionally. Stimulation of RASF with CSE increasedthe secretion of the pro-inflammatory cytokine IL8 (by 1.8-fold±0.1, p<0.02), but did not alter the release of IL6. To investigate the function of SIRT1, SIRT1 was overexpressed in RASF stimulated with CSE. Successful overexpression of SIRT1 was confirmed by western blot. Overexpression of SIRT1 specifically increased the release of IL8 in CSE stimulated RASF (control vector: 1.5-fold±0.2, SIRT1 vector: 2.3-fold±0.5, p=0.03), indicating that SIRT1 promotes IL8 secretion under CSE stimulation. Treatment of RASF with TNFα also enhanced the expression of IL8. However, the overexpression of SIRT1 under TNFα stimulation did not affect the production of IL8. Instead, the secretion of IL6 was increased by overexpression of SIRT1 in TNFα stimulated RASF (control vector: 34.9-fold±9.8, SIRT1 vector: 47.8-fold±11.7, p=0.01). Interestingly, overexpression of SIRT1 in unstimulated cells specifically increased the basalprotein production of IL8 (by 1.5-fold±0.1, p=0.03), but did not affect the expression of IL6.
Conclusions In the current study we found that SIRT1 specifically increases IL8 secretion under CSE treatment, but does not affect TNFα-induced IL8 expression. Instead, SIRT1 enhances IL6 release under TNFα stimulation. Therefore, we conclude that the pro-inflammatory functions of SIRT1 in RASF depend on the inflammatory stimulus. The investigation of mechanisms involved in the regulation of SIRT1 activity under different stimuli will provide insights into the pro-inflammatory properties of SIRT1 and clarify the role of SIRT1 inhibitors in the treatment of inflammatory disorders.
Acknowledgements This work was supported by ZIHP, IAR, IMI-BTCure
Disclosure of Interest None Declared