Abstract 572: Netrin-1 Induces Cytoskeletal-mediated Rewiring Of Mitochondrial Metabolism And Immunity

Abstract

Background: Cardiometabolic diseases are characterized by maladaptive immune infiltration in metabolic tissues. This results in unresolved inflammation, mediated by macrophages (M∅). Netrin-1 (Ntn1) causes macrophage retention by inhibiting cytoskeletal activity and induces a pro-inflammatory phenotype through receptor Unc5B. Emerging evidence suggests that cellular mechanics and metabolism are reciprocally regulated, but whether this plays a role in macrophage immunometabolism has not been explored. We hypothesized that Ntn1 stimulates a cytoskeletal-mediated rewiring of cellular metabolism toward a pro-inflammatory phenotype in macrophages. Methods/Results: We report that mechanical stress stimulates Ntn1 and Unc5b expression in macrophages along with proinflammatory genes known to promote atherosclerosis. RNA-sequencing analysis comparing control (M∅ WT ) with Ntn1 -silenced macrophages (M∅ Δ Ntn1 ) under high mechanical stress revealed a transcriptional signature associated with decreased biomechanical sensing and increased energy metabolism. This was confirmed by extracellular flux assays showing higher mitochondrial respiration in M∅ Δ Ntn1 . In addition, flow cytometry and live-cell imaging showed cytoskeletal remodelling and improved mitochondrial functionality in M∅ Δ Ntn1 as measured by higher mitochondrial potential, membrane integrity and network complexity. Importantly, incubation with recombinant Ntn1 reversed the effects of genetic loss of Ntn1 , but not that of Unc5b , suggesting receptor dependency. Furthermore, as Ntn1 is tied to cytoskeletal reorganization via Rho-associated kinase (ROCK) inhibition, we tested the effects of ROCK signaling on mitochondrial metabolism. Small molecule-mediated ROCK pathway activation raised mitochondrial membrane potential in M∅ WT , mimicking loss of Ntn1 , whereas ROCK inhibition restored mitochondrial membrane potential in M∅ Δ Ntn1 to wild type level. Conclusion: Taken together, our data shows that Ntn1, through Unc5B, decreases mitochondrial function through ROCK-mediated cytoskeletal reorganization. This suggests that Ntn1 perpetuates inflammation in cardiometabolic diseases through a cytoskeletal regulation of immunometabolism in macrophages.