ROLE OF EPITHELIAL LTA4H-LTB4 SIGNALING IN RESOLUTION OF COLONIC MUCOSAL WOUNDS

Abstract Neutrophils (PMN), the first immune cells recruited to site of mucosal injury, are critical for wound healing, but too little or too many neutrophils perturb mucosal repair resulting in chronic, poorly healing wounds, as seen in individuals with chronic inflammatory bowel disease (IBD). Despite the clinical significance of dysregulated PMN intestinal influx, many details underlying temporal regulation of PMN recruitment that maintain the balance between inflammation and repair remain unknown. Leukotriene B4 (LTB4), a potent chemoattractant binding the high-affinity receptor BLT1 on PMNs, is a well-established regulator of neutrophil recruitment. We recently demonstrated that BLT1 is also expressed on colonic epithelium, where epithelial BLT1-LTB4 signaling is critical for resolution of colonic mucosal wounds. While is it known that LTB4 is derived from arachidonic acid (AA) by the successive action of the enzyme 5-lipoxygenase (5-LO), and the terminal enzyme LTA4 hydrolase (LTA4H), the precise source of LTA4H and its role in mucosal wound repair is unknown. Using RNAscope in situ hybridization assay, we found that intestinal epithelial cells (IEC) adjoining mucosal wounds significantly upregulate Lta4h as early as 1hour post-injury. Single-cell RNA sequencing confirmed upregulation of Lta4h in murine colonic wounds, and colonoids from ulcerative colitis (UC) patients showed higher LTA4H expression than healthy controls, indicating that both injury and inflammation enhance epithelial LTA4H. To understand the role of epithelial LTA4H in resolution of mucosal injury, we generated mice selectively lacking LTA4H in IEC (Lta4hΔIEC). In vivo pinch biopsy-based colonoscopy revealed a significantly impaired wound healing in Lta4hΔIEC mice compared to controls. We are now investigating how epithelial LTA4H modulates PMN and monocyte recruitment to the wounds. In order to recapitulate a physiologically relevant microenvironment for our studies, we performed experiments under hypoxic conditions as observed in the gut, especially during inflammation. We observed that injury mediated hypoxia in mucosal wounds induce Lta4h expression in IECs via hypoxia-inducible factor (HIF) signaling. Our in-vitro data indicates that Lta4h and hypoxia inducible factors (Hif-1α and Hif-2α) are significantly upregulated under hypoxic conditions in primary mouse colonoids. Finally, we find that hypoxia driven LTA4H expression in IECs leads to increased LTB4 production. Overall, these findings demonstrate that upon mucosal injury, hypoxia mediated stabilization of HIFs in IEC regulate epithelial LTA4H expression and thereby LTB4 production to promote resolution of colonic mucosal injury. Our findings highlight a novel pro-repair function of LTA4H-LTB4 signaling during mucosal wound healing that’s perturbed in pathological conditions such as ulcerative colitis.