IL‐13/IL4Rα Signaling Increases Tension in Human Circular and Longitudinal Esophageal Smooth Muscle Through Distinct Molecular Pathways: Potential Contribution to Reduced Esophageal Distensibility in EoE

Abstract

Introduction Reduced esophageal distensibility (ED) in EoE has been attributed largely to fibrosis, but inflammatory cell infiltration and increased tension in esophageal smooth muscle (ESM) also might contribute to reduced ED. EoE is a type 2 immune disease in which type 2 cytokines IL-4 and IL-13 signal through the IL-4 receptor alpha (IL4Rα), activating Janus kinases (JAKs) that in turn activate STAT6. Dupilumab, a monoclonal antibody against IL4Rα, can improve ED in EoE patients, but it is unclear whether this is due to effects on fibrosis, inflammatory cells, or ESM tension. To explore how IL4Rα signaling might affect ESM tension, we treated ESM cells with IL-13 and selective inhibitors of STAT6 (AS1517499, 600 nM) or JAK1/2 (ruxolitinib, 100 ng/ml). Since ESM tension is induced by phosphorylation of myosin light chains through PKC and ROCK pathways, we also studied effects of selective inhibitors of PKC (calphostin C, 500 nM) and ROCK (Y-27632, 10 µM) on ESM tension. Methods Using 1 circular (C) and 1 longitudinal (L) telomerase-expressing ESM cell lines generated from muscularis propria of a human organ donor, cell contraction was assessed in the attached gel contraction model. Gels containing equal numbers of ESM cells were cultured for 48 hours with and without IL-13 (50 ng/ml), during which ESM tension developed; contraction was initiated by releasing the gel from the sides of the culture plate. Histamine (100 µM), a contraction mediator, was added immediately prior to release in some studies. Contraction was assessed by measuring gel surface area over 24 hours using Image J. Gel contraction studies were repeated in the presence of pathway inhibitors. Results In non-treated cells, gel sizes of CESM were significant smaller at all time points than those of LESM (p<0.001). Compared to non-treated controls, IL-13 significantly reduced gel size in both CESM and LESM (Figures 1&2). Also compared to non-treated controls, IL-13-treatment resulted in smaller gel sizes at all time points following histamine stimulation in both CESM and LESM (p<0.05). In LESM, IL-13-induced reductions in gel size were significantly inhibited by ruxolitinib, calphostin C, and Y-27632, but not by AS1517499 (Figure 2). In CESM, these IL-13-induced reductions were significantly inhibited by ruxolitinib and calphostin C, but not by Y-27632 or AS1517499 (Figure 1). Conclusions CESM generates more baseline tension that LESM. IL-13 increases tension and enhances the contractile response to histamine in both CESM and LESM. In LESM, both the PKC and ROCK pathways contribute to IL-13-mediated increases in tension whereas in CESM, only the PKC pathway contributes to this effect. These findings demonstrate distinct molecular pathways whereby IL-13 contributes to LESM and CESM tension, and suggest that IL-13-induced ESM tension may contribute to the reduced ED of EoE.