Role of Macrophage Mechanobiology in Diabetic Gastroenteropathy
The long list of complications associated with diabetes mellitus includes gastroenteropathies such as gastroparesis, a syndrome defined by delayed emptying of gastric contents with no mechanical obstruction. Few treatments are available for gastroparesis, which can be a disabling disorder due to not only the symptoms of nausea, vomiting, early satiety, bloating and pain but also the adverse impact of altered gastric emptying on glycemic control. It has become clear that several cell types that regulate gastrointestinal motility are important for gastroparesis' pathophysiology. Still, the most robust link is between delayed gastric emptying and changes in macrophage polarization, which associates with the loss of pacemaker cells (interstitial cells of Cajal, ICC) in the gastric muscularis propria. Since gastroparesis is also associated with stiffer tissues, and forces may activate macrophages, the impact of forces on macrophage function may be especially relevant for macrophages in gastroparesis. This proposal's overall hypothesis is that in gastroparesis, pro-inflammatory Piezo1 macrophage release IL6, and TNFa in response to mechanical force, induce loss of ICC and promote gastric dysfunction. We generated exciting preliminary data for this application: 1) single-cell transcriptomics of CD45+ cells from the gastric muscularis propria of mice identifies a population of gastric macrophages expressing Piezo1, 2) Piezo1 macrophages are enriched in pro-inflammatory genes, such as IL6 and TNFa in comparison to non Piezo1 macrophages that have a resident, non-inflammatory phenotype 3) We developed a novel in-vitro primary culture of isolated macrophages that retain Piezo1 expression. We will test the hypothesis in 2 not overlapping AIMs.To test the first AIM to determine how macrophages respond to mechanical force through Piezo1 we will use a novel macrophage primary culture to determine the calcium response and the cytokine release in the presence of a Piezo1 specific agonist. To test the second AIM to determine the location of Piezo1 macrophages and the effect of their depletion on gastric function in diabetic mice, we will generate a diabetic Piezo1floxflox/Lyz2cre mouse model to study the effect of Piezo1 macrophages depletion on ICC and gastric emptying. This study is poised to provide the mechanistic basis for understanding how mechanical cues lead to macrophage activation in gastroparesis. Completion of the project will identify new directions for testing novel options to treat both gastroparesis and other complications of diabetes.

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