Ornithine Lipid Activates Both TLR4 and the non-canonical NLRP3 Inflammasome

Abstract

AbstractToll-like receptors (TLRs) are proteins that act as the sentinels of mammalian cells by detecting bacteria and viruses motifs such as the phospholipid lipopolysaccharides (LPS) from Gram-negative bacterial membrane, among others. Bacteria like Vibrio cholerae, when grow in phosphate-depleted medium are unable to produce LPS and other phospholipids and therefore increase the synthesis of ornithine lipids (OLs) to keep membrane integrity and survival. We found that, although the huge structural differences between OL and LPS, our immune system is still able to detect OL and trigger immune response through TLR4 and the non-canonical Nucleotide-binding domain and leucine-rich repeat-containing pyrin protein 3 (NLRP3) inflammasome. Similar to LPS, OL induced TLR4-dependent tumor necrosis factor (TNF)-α secretion and Nuclear Factor (NF)-κB activation and therefore also elicited the priming of the NLRP3 inflammasome. Moreover, incubation of macrophages with OL causes caspase-dependent cleavage of gasdermin D (GSDMD) and consequent K+ efflux-dependent NLRP3 activation and interleukin (IL)-1β secretion.Results abstract (if needed)In bone-marrow derived murine macrophages (BMDM) from wild type mice, OLs induced hallmarks of NF-κB activation such as TNF-α secretion and the expression of pro-IL-1β and NLRP3. OL induced NF-κB activation dependent on TLR4 as demonstrated by the NF-κB activation measured in HEK293 reporting cells expressing uniquely TLR4 and by the decrease of TNF-α secretion in macrophages caused by antibodies blocking TLR4. OLs also induced IL-1β secretion dependent on cellular K+ efflux and NLRP3/caspase-1 inflammasome, as it was blocked by specific inhibitors and absent on Nlrp3−/−, Pycard−/− or Casp1/11−/− macrophages.