Follistatin-like 1 regulates inflammatory cytokine release from lung fibroblasts and airway smooth muscle cells

Rationale Chronic obstructive pulmonary disease (COPD) is an inflammatory disease of the lung, with cigarette smoking as the main risk factor. Patients with COPD exhibit an increased level of the neutrophil chemoattractant interleukin-1β (IL-1β). One of the IL-1β-inducible proteins, Follistatin-like 1 (FSTL1) has been demonstrated to be implicated in inflammatory diseases, such as arthritis. It has been demonstrated that FSTL1 contributes to the release of inflammatory cytokines, including interleukin-8 (IL-8). However, the regulatory role of FSTL1 in inflammation in COPD is currently unknown. Methods This study aims to unravel the role of FSTL1 in inflammatory cytokine release. Lung tissue from COPD patients was stained using FSTL1 antibody. MRC-5 human lung fibroblasts and immortalized airway smooth muscle cells were treated with either cigarette smoke extract (CSE) or IL-1β. FSTL1 and IL-8 mRNA expression were measured using qPCR. Gain-of-function studies were performed using FLAG-tagged FSTL1, CFP served as control. Loss-of-function studies were performed using FSTL1 siRNA, non-targeting siRNA served as control. Subsequently, transfected cells were treated with or without CSE or IL-1β for 24h. The release of IL-8 in the supernatant was measured by ELISA. Results We found that FSTL1 is expressed in lung tissue from COPD patients, particularly in structural cells and inflammatory cells. In vitro, a dose-dependent downregulation of FSTL1 mRNA expression was observed in lung fibroblasts and airway smooth muscle cells in response to CSE, whereas IL-1β dose dependently induced FSTL1 mRNA expression. The mRNA expression of IL-8 was markedly increased by 15% CSE (∼8-fold) and IL-1β (∼3000-fold) in non-transfected lung fibroblasts and by CSE (∼2.5-fold) and IL-1β (∼50-fold) in airway smooth muscle cells. Interestingly, in FSTL1 overexpressing cells, CSE- and IL-1β-induced IL-8 release were augmented 2-fold and 1.5-fold, respectively. In contrast, silencing of FSTL1 attenuated CSE- and IL-1β-induced IL-8 release by about 50% and 20%, respectively, without affecting basal IL-8 release. Similar effects of FSTL1 overexpression and silencing on IL-8 release were observed in airway smooth muscle cells, demonstrating the importance of FSTL1 in CSE- and IL-1β-induced IL-8 release from lung fibroblasts and airway smooth muscle cells. Conclusions FSTL1 contributes to CSE- and IL-1β-induced IL-8 release from lung fibroblasts and airway smooth muscle cells, indicating that FSTL1 may contribute to airway inflammation in COPD.