Glycolipid Metabolite ß-Glucosylceramide Is a Neutrophil Extracellular Trap-Inducing Ligand of Mincle Released during Bacterial Infection and Inflammation.

ABSTRACT

Neutrophil extracellular traps (NETs) are implicated in host defense and inflammatory pathologies alike. A wide range of pathogen- and host-derived factors are known to induce NETs, yet the knowledge about specific receptor-ligand interactions in this response is limited. We previously reported that macrophage-inducible C-type lectin (Mincle) regulates NET formation. In this article, we identify glycosphingolipid ß-glucosylceramide (ß-GlcCer) as a specific NET-inducing ligand of Mincle. We found that purified ß-GlcCer induced NETs in mouse primary neutrophils in vitro and in vivo, and this effect was abrogated in Mincle deficiency. Cell-free ß-GlcCer accumulated in the lungs of pneumonic mice, which correlated with pulmonary NET formation in wild-type, but not in Mincle-/-, mice infected intranasally with Klebsiella pneumoniae Although leukocyte infiltration by ß-GlcCer administration in vivo did not require Mincle, NETs induced by this sphingolipid were important for bacterial clearance during Klebsiella infection. Mechanistically, ß-GlcCer did not activate reactive oxygen species formation in neutrophils but required autophagy and glycolysis for NET formation, because ATG4 inhibitor NSC185058, as well as glycolysis inhibitor 2-deoxy-d-glucose, abrogated ß-GlcCer-induced NETs. Forced autophagy activation by tamoxifen could overcome the inhibitory effect of glycolysis blockage on ß-GlcCer-mediated NET formation, suggesting that autophagy activation is sufficient to induce NETs in response to this metabolite in the absence of glycolysis. Finally, ß-GlcCer accumulated in the plasma of patients with systemic inflammatory response syndrome, and its levels correlated with the extent of systemic NET formation in these patients. Overall, our results posit ß-GlcCer as a potent NET-inducing ligand of Mincle with diagnostic and therapeutic potential in inflammatory disease settings.