Peritoneal and bone marrow macrophages were obtained from vimentin knockout and wild-type controls. Cells were analyzed for bacterial phagocytosis, bacterial killing and production of reactive oxygen species by chemiluminescence, H2O2 and NADPH production measurements. Redox was measured by high-performance liquid chromatography. Immunopreceipitation and Western blot analysis were performed to show the interaction between the p47 phox subunit of the NADPH oxidase complex with vimentin at the plasma membrane. Co-localization of p47phox and vimentin at the plasma membrane was confirmed via confocal fluorescence microscopy analysis.
Here we report that peritoneal and bone marrow-derived macrophages of vimentin knockout (Vim KO) mice generated higher levels ROS upon activation compared with wild-type (WT) controls leading to reduced intracellular redox potential. This enhanced production of ROS by Vim KO phagocytes correlated with an improved capacity to mediate in vitro bacterial killing. Lack of vimentin accelerated the p47phox subunit of the NADPH oxidase complex from the cytosol to the plasma membrane via its interaction with the trans-located p47phox subunit at the plasma membrane. Challenging Vim KO and WT mice with a lethal dose of Escherichia coli (E. coli) demonstrated that lack of vimentin improved bacterial clearance and prolonged mouse survival.
These findings suggest that vimentin impedes production of ROS by interfering with the assembly and function of the NADPH oxidase complex. Remarkably, the absence of vimentin conferred significant resistance to death by bacterial septicemia. Based on our results, we propose that vimentin modulates the intensity of the innate immune response by attenuating ROS production, a surprising role for this highly abundant but poorly understood intermediate filament protein that has been recently implicated in the pathogenicity of arthritis.
Disclosure: N. Mor-Vaknin, None; M. Legendre, None; Y. Yu, None; C. H. C. Serezani, None; S. Garg, None; A. Jatzek, None; M. D. Swanson, None; S. Teitz-Tennenbaum, None; A. Punturieri, None; N. C. Engleberg, None; R. Banerjee, None; M. Peters-Golden, None; D. Markovitz, None.