Abstract 1027: Mechanical Stress Is A Non-canonical Regulator Of Inflammatory Metabolism In Immune Cells Via Netrin-1

Atherosclerotic cardiovascular disease (CVD) is characterized by chronic inflammation of the artery wall triggered by lipoprotein deposition. Maladaptive immune responses, primarily mediated by macrophages (Mø), promote disease progression including extracellular matrix (ECM) remodeling. Emerging evidence suggests a relationship between cellular mechanics and metabolic programming, yet the role of ECM-induced mechanical stress on Mø immunometabolism remains unexplored. We hypothesized that mechanical stress induces cytoskeletal-mediated rewiring of cellular metabolism that fuels inflammation in plaque macrophages. To investigate the role of matrix-induced mechanical stress on Mø, we used an in vitro system that reproduces the elasticity parameters of the atherosclerotic plaque environment. Compared to stiff matrices, Mø cultured on soft matrices showed marked morphological changes, accompanied by increased inflammation and decreased mitochondrial function as assessed by RNA sequencing and flow cytometry analyses. Notably, cytoskeletal changes were associated with profound mitochondrial network reorganization and increased ROS generation. RNA-seq and ligand-receptor pairing analysis identified the neuroimmune guidance molecule netrin-1 ( Ntn1 ) and its receptor Unc5b as most upregulated in reponse to mechanical stress. Loss-of-function studies showed that netrin-1-deficiency protects from mechanical stress-induced metabolic changes observed in WT Mø, as measured by mitochondrial network remodelling and higher mitochondrial respiration. Importantly, incubation with recombinant netrin-1 reversed the effects of genetic loss of Ntn1 , but not Unc5b , suggesting receptor dependency. Mechanistically, we showed that netrin-1 controls cytoskeletal reorganization via the Rho-associated kinase (ROCK) pathway to alter mitochondrial function, and that ROCK inhibition recapitulated the effect of Ntn1 silencing on mitochondrial potential. Collectively, our data link Ntn1-Uncb mediated changes in cytoskeletal rearrangement to alterations in mitochondrial metabolism that dictate Mø inflammation, and implicate the ECM as a non-canonical regulator of immunometabolism that perpetuates inflammation in CVD.