A NOVEL STAT3/6-MEDIATED MITOCHONDRIAL-IMMUNE PATHWAY CONTRIBUTES TO EOE PATHOGENESIS

Introduction: Although the pathobiology of eosinophilic esophagitis (EoE) remains incompletely understood, Th2 cytokines IL-4 and IL-13 are key mediators of the disease. Genetic evidence supports a role for mitochondria in EoE but the mechanisms regulating mitochondrial biology as well as the functional significance of this organelle in EoE remain unknown.
Methods: Mitochondria was assessed in esophageal biopsies and serum of EoE patients or controls subjects with normal esophageal pathology via immunohistochemistry (IHC) for mitochondrial proteins, including MTCO1. Genetic mitochondria depletion was achieved in vivo using K5CreERT2;Tfam^loxp/loxp mice. EoE was induced in mice using MC903/Ovalbumin (OVA) and peeled epithelium was evaluated for mitochondrial DNA (mtDNA) by qPCR and MTCO1 by immunoblotting. Immortalized human esophageal keratinocytes (EPC2-hTERT) were treated with EoE-relevant cytokines IL-4, IL-5, IL-13, IL-1b [each at 10 ng/mL], or TNFα [40 ng/mL] for 7 days. Ruxolitinib [100 ng/mL] inhibited JAK1/2. mtDNA in EPC2-hTERT cells and culture media was measured.
Results: Evidence of increased mitochondria was detected in esophageal epithelium of active EoE patients as compared to controls and inactive EoE patients (Fig. 1A, B). Active EoE patients further displayed increased levels of mtDNA in serum. Increased mtDNA was also detected in esophageal epithelium of mice with MC903/OVA-mediated EoE (Fig. 1C, D). Depletion of mitochondrial transcription factor TFAM limited EoE-associated inflammation (Fig. 1E, F), indicating that mitochondria accumulation promotes EoE pathogenesis. To investigate the signals in the EoE inflammatory milieu that promote mitochondria accumulation in esophageal epithelium, EPC2-hTERT cells and primary esophageal keratinocytes were stimulated with EoE-relevant cytokines. Increased mtDNA level was detected in EPC2-hTERT cells and was increased uniquely in response to IL-13 (Fig. 2A). Evaluation of EPC2-hTERT culture medium revealed increased mtDNA in response to either IL-4 or IL-13 (Fig. 2B). We next examined the role of JAK signaling in mtDNA accumulation in response to IL-4 and IL-13. IL-13-mediated mtDNA accumulation was fully abrogated by JAK inhibitor Ruxolitinib (Fig. 2A, B). We next assessed STAT phosphorylation, which occurs downstream of JAK signaling and directs subcellular localization of STAT3 and STAT6 (Fig. 2C). IL-4 and IL-13 both promoted STAT6 Tyr⁶⁴¹ phosphorylation and STAT3 Ser⁷²⁷ phosphorylation while only IL-13 impacted phosphorylation of STAT6 Tyr⁷⁰⁵ (Fig. 2D). Ruxolitinib suppressed STAT6 Tyr⁶⁴¹ and STAT3 Ser⁷²⁷ phosphorylation mediated by either IL-4 or IL-13. Its effects on STAT6 Ser⁷²⁷ were not as robust (Fig. 2D).
Conclusions: IL-13 and IL-4 in the EoE inflammatory milieu activate STAT3/6 signaling, increasing mitochondria in esophageal epithelial cells, and promoting EoE inflammation.