The Role of Arp2/3 Complex in Inflammatory Activation and TLR4 Endocytosis

Abstract

The Arp2/3 complex is a major actin filament nucleator, which produces branched filaments in different cells types such as fibroblasts, and macrophages. It has been shown that Arp2/3 depletion in primary bone marrow-derived macrophages results in disrupted integrin function and reduced F-actin production, and an alteration in cell shape and protrusion. However, significant questions remain regarding the molecular mechanism of Arp2/3 complex function in inflammatory activation and haptosensing. Here, we utilize Arpc2-/- macrophages with disrupted Arp2/3 complex function to determine its role in inflammatory regulation including post-translational regulations, gene expression, cytokine secretion and TLR4 endocytosis. TLR4 is a specific immune receptor stimulated by bacterial lipopolysaccharides (LPS) which stimulates robust inflammatory activation. Here we use biochemical techniques, immunofluorescence, Time-Lapse imaging, Traction-Force Microscopy and RNA seq to compare WT and Arpc2-/- macrophage activation during acute and chronic LPS/Interferonɣ stimulation. We find that cell shape changes, altered TLR4 endocytosis and higher expression levels of inflammatory hallmarks such as iNOS, P-NFKb and P-ERK upon loss of Arp2/3 function. Our preliminary results demonstrate that Arpc2-/- macrophages are primed under baseline conditions and hyper-activated after LPS/Interferonɣ stimulation. These preliminary results demonstrate a difference between WT and Arpc2-/- macrophages in inflammatory activation and a de-regulation of the Arpc2-/- cytoskeleton during inflammatory activation. We will utilize these results to further dissect the molecular role of Arp2/3 complex in the inflammatory activation, and will potentially lead us to a better understanding of human inflammatory disorders.