Pulsating Fluid Shear Stress Induces an Altered Macrophage Mechanotype with a Moderate Pro-inflammatory Profile and Reduced Phagocytosis

Physical activity exposes immune cells in skeletal muscle to dynamic mechanical cues, but whether shear stress directly controls macrophage mechanotype, activation, and function remains unclear. In this research, we tested the effects of 1 h pulsating fluid shear stress (PFSS; 0.7 Pa, 1 Hz) on RAW 264.7 and mouse bone marrow-derived macrophages (BMDMs) and compared responses with lipopolysaccharide (LPS; 100 ng/mL). Live imaging and nanoindentation were used to quantify deformation and cortical stiffness, immunocytochemistry assessed morphology and F-actin organization, Griess assays measured nitric oxide (NO), RT-qPCR and multiplex assays profiled inflammatory mediators, Western blotting assessed ERK1/2 activation, and pHrodo bioparticles assessed phagocytosis. PFSS rapidly reduced cell apex height during pulsatile loading and increased projected cell area and Young's modulus after treatment, indicating deformation with cortical stiffening. PFSS caused acute NO release and ERK1/2 phosphorylation, both of which were abolished by EGTA. PFSS transiently increased Il1b and Tnf expression, and elevated TNF-alpha secretion at 6 and 24 h, but did not induce the broad inflammatory transcriptional response observed after LPS. PFSS and LPS both reduced phagocytosis. These PFSS-induced responses, including rapid NO release and selective pro-inflammatory gene expression, were reproduced in primary BMDMs. These findings show that short-term PFSS drives a Ca ²⁺ -dependent, mechanically dominated macrophage response characterized by altered mechanotype, moderate pro-inflammatory activation, and impaired phagocytic function. Fluid shear stress may therefore tune macrophage behavior independently of full classical inflammatory activation, with implications for understanding immune regulation during exercise-mediated skeletal muscle repair.