Metabolic Remodeling of Neutrophils at Inflammatory Site Drives Invadosome Formation Favoring Transcellular Migration

Neutrophils traffic in and out of underlying vascular bed during hematopoiesis and immunosurveillance. However, during inflammatory conditions such as ischemia reperfusion injury or atherosclerosis, excessive neutrophil infiltration into tissue drives disease pathogenesis. Yet, the relationship between neutrophil transmigration and inflammation is ill-defined. Neutrophil extravasation can occur either between two endothelial cells (paracellular) or directly through an endothelial cell body (transcellular). During transcellular migration, neutrophils interact with underlying endothelial cells (EC) via invadosomal structures, which forms a 'pore' into endothelial cell membrane, thus facilitating neutrophil migration through EC body. We have recently reported that deficiency in Rap1b, a member of Ras superfamily of GTPase, enhanced neutrophil transcellular migration, invadosomal structures and metalloproteinase (MMP) release (Kumar et al, JEM, 2014), in a manner dependent on high Akt activity. Further, Rap1-deficiency increased neutrophil recruitment to inflamed lungs and enhanced susceptibility to endotoxin shock, suggesting mode of neutrophil migration may influence inflammatory outcome. Here, to further understand which factors drive neutrophil transcellular migration, we analyzed protein content of Rap1b-/- invadosomal structures during transcellular diapedesis. For this, neutrophils were stimulated in transwell filters of 1µM pore size, with FMLP placed in the lower chamber, allowing only invadosomal protrusions into the pores. After removing the cell body from top of the filter, mass spectrometric analysis was performed on the invadosomal fraction. About 680 proteins were identified in protrusions isolated from WT or Rap1b-/- neutrophils. As expected, majority of them were cytoskeleton and adhesion proteins. Interestingly, Rap1b-/- invadosomal structures contained more enzymes of glycolytic pathways, including HK1, Lactate dehydrogenase A (LDHA) and phosphoglycerate kinase1 (PGK1). Immunofluorescent staining and western blotting confirmed this observation. Importantly, glycolytic enzymes were present at the tip of the protrusions in colocalization with F-actin suggesting site specific glycolytic activity, raising the hypothesis that metabolic remodeling may influence the route of neutrophil migration. LDHA converts pyruvate to lactate and subsequent milieu acidification, which can then cause MMP activation. Consistently, Rap1b-/- neutrophils exhibited increased uptake of glucose analogue (2-NBDG) and concurrent intracellular acidification, as detected by pH sensitive dye. To investigate the importance of LDHA activity during transcellular migration, Rap1b-/- neutrophils were treated with a specific pharmacological inhibitor of LDHA, namely FX11. In vitro, FX11 treatment significantly decreased transcellular migration of Rap1b-/- neutrophils. It also reduced invadosome formation of Rap1b-/- neutrophils within transwell pores, as well as neutrophil acidity and MMP activity. Furthermore, during neutrophil-endothelial cell interactions in vitro, Rap1b-/- neutrophils caused F-actin depolymerization in EC, likely facilitating transcellular passage; this was inhibited by FX11. To examine its effect in vivo, under same inflammatory microenvironment, 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. Rap1b-/- neutrophils migrated out of vessels at higher frequency than WT cells, which was abrogated by FX11 treatment. Moreover, treatment with FX11 reduced the number of Rap1b-/- neutrophils located away from EC junction (transcellular route), in vivo. These results suggest enhanced local glycolytic metabolism and LDHA activity could act as critical regulators of transcellular migration. Increase in extracellular acidification mediated by LDHA activity, could affect endothelial permeability and alter neutrophil migratory behavior affecting outcome of inflammation. Since milieu acidification plays a major role in ischemic damage to the heart, these findings may be clinically important for our understanding of hyperinflammatory disorders.