Th1 cytokines TNF- (cid:2) and IFN- (cid:3) promote corticosteroid resistance in developing human airway smooth muscle

Th1 cytokines TNF- (cid:2) and IFN- (cid:3) promote corticosteroid resistance in developing human airway smooth muscle. Am J Physiol Mol Physiol L71–L81, (CSs) are commonly used to manage wheezing and asthma in pediatric populations. Although corticosteroids are effective in alleviating air- way diseases, some children with more moderate-severe asthma phenotypes show CS resistance and exhibit signiﬁcant airﬂow ob- struction, persistent inﬂammation, and more frequent exacerbations. Previous studies have demonstrated that Th1 cytokines, such as TNF- (cid:2) and IFN- (cid:3) , promote CS resistance in adult human airway smooth muscle (ASM). In the present study, using a human fetal ASM cell model, we tested the hypothesis that TNF- (cid:2) /IFN- (cid:3) induces CS resistance. In contrast to TNF- (cid:2) or IFN- (cid:3) alone, the combination of TNF- (cid:2) /IFN- (cid:3) blunted the ability of ﬂuticasone propionate (FP) to reduce expression of the chemokines CCL5 and CXCL10 despite expression of key anti-inﬂammatory glucocorticoid receptor target genes being largely unaffected by TNF- (cid:2) /IFN- (cid:3) . Expression of the NF- (cid:4) B subunit p65 and phosphorylation of Stat1 were elevated in cells treated with TNF- (cid:2) /IFN- (cid:3) , an effect that remained in the presence of FP. siRNA knockdown studies demonstrated the effects of TNF- (cid:2) /IFN- (cid:3) on increased p65 are mediated by Stat1, a transcription factor activated by IFN- (cid:3) . Expression of TNFAIP3, a negative regulator of NF- (cid:4) B activity, was not altered by TNF- (cid:2) /IFN- (cid:3) . However, the effects of TNF- (cid:2) /IFN- (cid:3) were partially reduced by overexpression of TNFAIP3 but did not inﬂuence p65 expression. Together, these data suggest that IFN- (cid:3) augments the effects of TNF- (cid:2) on chemokines by enhancing expression of key inﬂammatory pathways in the presence of CS. Interactions between TNF- (cid:2) - and IFN- (cid:3) -mediated pathways may promote inﬂammation in asthmatic children resistant to CSs. GR- (cid:2) (ab3580), GR- (cid:5) (ab3581), TNFAIP3 (ab13597), and TATA binding protein (ab63766); and Cell Signaling Technology (Danvers, MA): GAPDH (2118), P-GR Ser211 (4161), and total GR (12041). Nontargeting siRNA (D-001810-01- 05), ON-TARGET plus SMART pool human TNFAIP3 (L-009919-00-0005), and Stat1 siRNA (L-003543-00-0005) were purchased from (Dharmacon, Lafayette, CO). The adenoviral vectors Ad-GFP and Ad-TNFAIP3 were obtained from Welgen (Worchester, MA). Cell culture. Human fetal ASM cells were isolated from de-identiﬁed fetal tracheobronchial tissue (18–22 wk of gestation) as previously described (10, 21). Cells were for in vitro use only and considered exempt by Mayo Institutional Review Board. Cells were obtained in collaboration with Dr. Hitesh Pandya (University of Leicester, UK), Novogenix (Los Angeles, CA), or StemCell Express (Folsom, CA). In prior studies, we have demonstrated that human fetal ASM express smooth muscle markers and Ca 2 (cid:6) regulatory proteins and respond to bronchoconstrictor agonists, all characteristics of primary human ASM (9, 21).