Thymic stromal lymphopoietin induces migration in human airway smooth muscle cells

Abstract

Airway remodeling due to increased airway smooth muscle (ASM) mass, likely due to enhanced migration and proliferation, has been shown to be highly associated with decline in lung function in asthma. Thymic stromal lymphopoietin (TSLP) is an IL-7-like, pro-allergic cytokine that has been shown to be necessary and sufficient for the development of allergic asthma. Human ASM (HASM) cells express TSLP receptor (TSLPR), the activation of which leads to enhanced release of proinflammatory mediators such as IL-6, CCL11/eotaxin-1, and CXCL8/IL-8. We show here that TSLP induces HASM cell migration through STAT3 activation since lentiviral-shRNA inhibition of STAT3 abrogated the TSLP-induced cell migration. Moreover, TSLP induced multiple cytoskeleton changes in HASM cells such as actin polymerization, cell polarization, and activation of small GTPase Rac1. Collectively, our data suggest a pro-migratory function of TSLP in ASM remodeling and provides better rationale for targeting TSLP/TSLPR pathway for therapeutic approaches in allergic asthma.

Figure 1. TSLP induces Human airway smooth muscle (HASM) cell migration.

(A) HASM cells seeded in Boyden upper chamber were stimulated with TSLP (1, 10 ng/ml) and PDGF (10 ng/ml) in lower chamber, and migrated cells were analyzed by light microscope (magnification, x200). Migrated cells were quantitated and presented in (B) ***p < 0.001 vs medium control (One-way ANOVA followed by Newman-Keuls Multiple Comparison Test).

Figure 2. STAT3 mediates TSLP-induced HASM cell migration.

(A) Lentiviral-shRNA transduction silenced the STAT3 expression in HASM cells as shown by Western blotting. Scramble- (B) and STAT3-shRNA (C) silenced HASM cells were analyzed for migration. (D) TSLP-induced HASM cell migration was quantified and presented as fold increase relative to unstimulated control. *p < 0.05, ***p < 0.001, One-way ANOVA followed by Newman-Keuls Multiple Comparison Test.

Figure 3. TSLP induces cytoskeleton changes in HASM.

(A) Polymerized actin in TSLP or PDGF-stimulated HASM cells was analyzed by phalloidin staining, quantified as percent polarized cells. **p < 0.01, ***p < 0.001 vs unstimulated control; One-way ANOVA and Newman-Keuls Multiple Comparison Test.

Figure 4. TSLP and PDGF induce Rac1 activation in HASM cells.

GTPase Rac1 activity in TSLP-stimulated HASM cells was measured by G-LISA Rac1 activation assay kit. The Rac1 activity is presented as relative luminescence units (RLU) over unstimulated control.