Abstract Rationale Type 2 inflammation enhances airway smooth muscle cell (ASM) hypercontractility and AHR to promote bronchocontriction through pro-inflammatory cytokines, IL-4 and IL-13. IL-4 and IL-13 signaling stimulates JAK/Stat6 and PI3K/mTOR signaling pathways. Actin polymerization is a critical step in ASM contraction and is regulated by actin regulatory proteins and ASM interactions with extracellular matrix (ECM) proteins. In human ASM cells, we recently observed that IL-4, but not IL-13, increases contraction and actin polymerization yet the underlying mechanisms remain unknown. In the present study, we examined the effects of pharmacological inhibitors of Stat6, PI3K, and mTOR pathways on IL-4-induced ASM contraction and actin polymerization. Method Primary human ASM cells were isolated from human airways and cultured in growth media. ASM were seeded in 2 mg/mL collagen gels or glass slide, serum straved for 48 h and then treated with media only, IL-4 (10 ng/mL), and in the presence or absence of pharmacological inhibitors for Stat6 (20 nM AS-1517499), PI3K inhibitors (10 uM LY294004 and 250 nM PF-04691502), mTORC1 inhibitor (rapamycin 5 nM), mTORC2 inhibitor (JR-AB2-011 50 uM), actin polymerization inhibitor (0.2 uM cytochalasin D) for 48 or 120 h. After 48 h treatment, cells were stained with fluorescence-conjugated antibodies for polymerized F-actin and monomeric G-actin (F/G ratio). Gel diameter was measured daily for 120 h. Expression of actin and ECM regulatory proteins (SMA, CRACD, cofilin, lysyl oxidase) were measured by qPCR or western blot. Results IL-4 significantly increased the collagen gel contraction by ASM. Treatment with Stat6 inhibitor significantly inhibited by IL-4-induced ASM contraction. Additionally, exposure to PI3K inhibitors and rapamycin, a mTORC1 inhibitor (IC50=0.5 nM), abrogated gel contraction at 5 nM. Meanwhile treatment with a mTORC2 inhibitor (IC50=0.36 uM) also reduced ASM contraction albeit at a greater concentration (50 uM). IL-4 significantly increased F/G ratio, which was significantly inhibited by exposure to PI3K and mTORC1 inhibitors and also Stat6 inhibitor. Treatment with an actin polymerization inhibitor reduced IL-4-induced gel contraction and F/G ratio. Interestingly, IL-4 increased gene expression of actin regulatory proteins, CRACD, LOX, and LOX3, while also reducing cofilin protein expression. These effects of IL-4 were abrogated by PI3K inhibition. Conclusion These novel data suggest that IL-4 induces human ASM cell contraction through Stat6 and PI3K/mTORC1 pathways and involve regulation of actin polymerization and ECM proteins. Collectively, these data reveal new signaling mechanism by which IL-4 promotes ASM actin polymerization and hypercontractility. This abstract is funded by: R01-HL155095(Britt) and R01-HL155095 Diversity Supplement (Ruwanpathirana).
Ruwanpathirana et al. (2026) studied this question.