Introduction: EoE is characterized by Th2 inflammation of the esophagus. In esophageal epithelial cells, the Th2 cytokines IL-4 and IL-13 both signal through IL-4 receptor-α (IL4Rα) to activate Janus kinase 1 (JAK1), leading to increased expression of eotaxin-3, a chemoattractant that draws eosinophils into the EoE esophagus. In EoE patients, treatment with weekly subcutaneous injections of dupilumab, a monoclonal antibody to IL4Rα, decreases esophageal mucosal eosinophil counts and improves dysphagia. Dysphagia in EoE might result from cytokine and eosinophil effects on esophageal smooth muscle (ESM) as well as on mucosa and, in earlier studies, we found that IL-4 and IL-13 signal through IL4Rα/JAK1 in ESM cells to increase expression of eotaxin-3 and CPI-17, a myosin phosphatase inhibitor that increases muscle tension. Upadacitinib, a JAK1 inhibitor, is an oral agent used to treat several inflammatory diseases including atopic dermatitis, ulcerative colitis, and Crohn’s disease. Since dupilumab works upstream of JAK1, we hypothesized that a JAK1 inhibitor like upadacitinib also should be an effective EoE treatment. In this study, we aimed to explore upadacitinib effects on Th2 cytokine-stimulated expression of eotaxin-3 and CPI-17 in esophageal epithelial and ESM cells, and on ESM tension.
Methods: We used 2 telomerase-expressing esophageal epithelial cell lines from EoE patients and 2 telomerase-expressing circular ESM cell lines from human organ donors. Cells were treated with IL-13 (50 ng/ml) or IL-4 (10 ng/ml), with and without upadacitinib (10-500 nM). We used ELISA to measure eotaxin-3 protein. For gel contraction studies, gels with equal numbers of ESM cells were treated with IL-13 or IL-4, with or without upadacitinib (100 nM) for 48 hours, during which tension developed; contraction was initiated by releasing the gel from the sides of the culture plate, and gel surface area was measured 1 hour later by Image J; qPCR was used to measure CPI-17 mRNA.
Results: Th2 cytokines significantly increased eotaxin-3 protein in esophageal epithelial cells and in ESM cells, an effect blocked by all doses of upadacitinib (Figure 1). In ESM cells, Th2 cytokines increased CPI-17 mRNA levels significantly, an effect also blocked by upadacitinib (Figure 2). In ESM cells, Th2 cytokine treatment increased the slope of gel contraction (an index of muscle tension) significantly, and upadacitinib significantly reduced that slope (Figure 2).
Conclusions: In both esophageal epithelial cells and circular ESM cells, blockade of JAK1 signaling with upadacitinib decreases Th2 cytokine-stimulated eotaxin-3 production. In ESM cells, upadacitinib also blocks Th2 cytokine-stimulated increases in CPI-17 mRNA expression and in muscle tension. These findings suggest that upadacitinib could be a useful oral agent for EoE patients, especially those who have ESM involvement.

<b>Figure 1.</b> Upadacitinib (Upa) reduces Th2 cytokine-stimulated increases in eotaxin-3 secretion in esophageal epithelial cells from EoE patients (A,B) and in circular esophageal smooth muscle (ESM-C) cells from organ donors (C,D). Bar graphs depict mean ± SEM from 3 biological replicates. ***p≤ 0.001 compared with untreated controls (C); +++p≤ 0.001 compared with corresponding IL-13 or IL-4 treatment.

Figure 1. Upadacitinib (Upa) reduces Th2 cytokine-stimulated increases in eotaxin-3 secretion in esophageal epithelial cells from EoE patients (A,B) and in circular esophageal smooth muscle (ESM-C) cells from organ donors (C,D). Bar graphs depict mean ± SEM from 3 biological replicates. ***p≤ 0.001 compared with untreated controls (C); +++p≤ 0.001 compared with corresponding IL-13 or IL-4 treatment.

<b>Figure 2.</b> Upadacitinib (Upa) reduces Th2 cytokine-stimulated increases in CPI-17 mRNA expression and tension in circular esophageal smooth muscle cells (ESM-C) from organ donors. (A,B) CPI-17 mRNA expression and (C,D) slope of contraction induced by Th2 cytokines in cells treated with and without upadacitinib (100 nM). Bar graphs depict mean ± SEM from 3 biological replicates. ***p≤ 0.001 compared with untreated controls (C); +++p≤ 0.001 compared with corresponding IL-13 or IL-4 treatment.

Figure 2. Upadacitinib (Upa) reduces Th2 cytokine-stimulated increases in CPI-17 mRNA expression and tension in circular esophageal smooth muscle cells (ESM-C) from organ donors. (A,B) CPI-17 mRNA expression and (C,D) slope of contraction induced by Th2 cytokines in cells treated with and without upadacitinib (100 nM). Bar graphs depict mean ± SEM from 3 biological replicates. ***p≤ 0.001 compared with untreated controls (C); +++p≤ 0.001 compared with corresponding IL-13 or IL-4 treatment.

Melissa Nelson

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