Neutrophil influx into the intestinal lumen is critical in the response to infectious agents, but is also associated with intestinal damage observed in idiopathic inflammatory bowel disease (IBD). Our prior studies have shown that this influx is mediated by the chemoattractant actions of hepoxilin A3 (HXA3) secreted from the luminal surface of epithelial cells by the efflux pump MRP2. Preliminary studies herein provide evidence that epithelial cells export another class of bioactive lipids from their apical surface, N-acyl ethanolamine class endocannabinoids via the multidrug resistance transporter P- glycoprotein (P-gp), which counteract this inflammatory response. Loss of endocannabinoid signaling through CB2, the peripheral cannabinoid receptor, leads to increased pathology and neutrophil influx during acute intestinal inflammation, consistent with events of IBD. Such preliminary data underscore a key role for epithelial cells in balancing the secretion of pro- and anti-inflammatory lipids via surface efflux pumps in order to control neutrophil infiltration and maintain homeostasis in the healthy intestine. Thus, we have identified a new layer of regulation of neutrophil function in the intestine. Unlike other anti- inflammatory lipids, such as the specialized pro-resolving mediators that are produced by immune cells, we have discovered that epithelial cells constitutively produce and apically secrete endocannabinoids, which we refer to as AMEND (activity modulating epithelial-neutrophil discourse), to locally establish an anti-inflammatory environment to maintain intestinal homeostasis. Thus, it appears that there are two systems, an anti-inflammatory P-gp secretion of endocannabinoids pathway and a pro-inflammatory MRP2 secretion of HxA3 pathway, that function in a balanced and coordinated manner that can be used by epithelial cells to integrate signals from the local environment to maintain exquisite control over the initiation of intestinal inflammation. We seek to further understand the interaction between HXA3 and neutrophils, and to investigate the mechanisms by which AMEND regulates HXA3 activity during homeostasis and disease. Bridging this gap could help explain how commensal bacteria can stabilize a state of tolerance and how genetic variations in specific elements of either pathway might predispose individuals to development of IBD. To address these questions, we will determine whether P-gp/AMEND and MRP2/HXA3 pathways modulate the balance between microbial tolerance and inflammatory responses, and that disturbance of this balance leads to pathological inflammation. Thus, Aim 1 is designed to assess the imbalance between the Pgp/EC and MRP2/HXA3 pathways in patients who suffer from ulcerative colitis, and Aim 2 will examine whether dysbiotic vs. healthy human stool differentially modulates the P-gp and MRP pathways in the intestine.
A broad body of research has established that the resident microbiota in the intestine play a critical role in host physiology, metabolism, and immunity, and that perturbations in the gut microbiota are associated with a wide variety of disease states. Because the resident microbiota are known to contribute to tolerance and homeostasis in the healthy intestine, this proposal will test the central hypothesis that the normal microbiota actively drives the endocannabinoid efflux through the apical transporter P-glycoprotein to prevent unnecessary inflammation in patients who suffer from ulcerative colitis. Unlike other anti-inflammatory lipids, such as the specialized pro-resolving mediators that are produced by immune cells, our studies support the novel concept that epithelial cells themselves, as a result of a ?healthy microbiota?, constitutively produce and apically secrete endocannabinoids to locally establish an anti-inflammatory environment and maintain intestinal homeostasis.
