Abstract 21312: Lactate Dehydrogenase A Affects Neutrophil Transcellular Extravasation by Upregulating Invadopodia Signaling

Abstract

During inflammation, such as ischemia reperfusion injury or atherosclerosis, excessive neutrophil infiltration drives pathogenesis. Yet, the relationship between neutrophil transmigration and inflammation is ill-defined. Neutrophils migrate through endothelial cells (EC) via para (between EC) and transcellular (through an EC, TCM) mechanisms. During TCM, neutrophils interact with EC via invadopodia-like structures to form a ‘pore’ into EC membrane, thus facilitating neutrophil migration through EC body. We recently reported that deficiency in Rap1b, a member of RasGTPase superfamily, enhanced neutrophil recruitment to inflamed lungs and susceptibility to endotoxin shock. Rap1b-/- neutrophils exhibit increased TCM, invadopodia-like formation and metalloproteinase (MMP) release, suggesting mode of migration may influence inflammation outcome. Here, we used Rap1b-/- neutrophils to further investigate the link between neutrophil migration and inflammation. Using mass spectrometry, we found 2-fold increase in glycolytic enzymes hexokinase1, LDHA and PGK1, in Rap1b-/- protrusions compared to WT. Immunofluorescent staining and western blotting confirmed this observation. Inhibition of LDHA with a specific inhibitor FX11, inhibited Rap1b-/- invadopodia protrusions, MMP release and TCM. We then tested effects of LDHA inhibition on neutrophil transendothelial migration in vivo. Rap1b-/- and WT neutrophils were tagged with cell tracker dyes and transferred to recipient mice, treated with FX11 or DMSO control. Ear microvasculature was stimulated with FMLP and labeled with PECAM antibody to visualize EC junctions. More Rap1b-/- cells migrated out of vessels compared to WT cells at the same vascular environment. FX11 treatment significantly abrogated this increase. This suggests that LDHA and enhanced glycolytic metabolism play critical roles during transmigration in vivo perhaps by changing neutrophil migratory behavior and inflammation outcome. Since lactate, the glycolytic byproduct, induces milieu acidification and acidity play a major role in ischemic damage to the heart, our findings may be important for our understanding of cardiovascular injury and for the development of approaches for effective treatment.