MALVINA PIZZUTO ¹, LAURA HURTADO NAVARRO¹, MARÍA CRISTINA MOLINA LÓPEZ¹, Jean-Marie Ruysschaert², PABLO PELEGRIN VIVANCOS¹

(1) CIRUGÍA DIGESTIVA, ENDOCRINA Y TRASPLANTE DE ÓRGANOS ABDOMINALES, IMIB-Arrixaca, España
(2) Université Libre de Bruxelles, Bélgica

Toll-like receptors (TLRs) are proteins that act like the sentinels of our cells. They detect bacteria and viruses through the recognition of pathogen motifs such as LPS, a lipid of Gram-negative bacterial membrane, among others. These motifs are called Pathogen-Associated Molecular Patterns (PAMPs). When a TLR recognizes a PAMP, it activates the transcription factor NF-κB and induces the secretion of inflammatory proteins called cytokines, such as TNF-α, which alert the immune system to fight the pathogens. The NF-κB activation also produces proteins called NLRP3 and pro-IL1β. NLRP3 is activated by a decrease of intracellular concentration of K+, induced among others by ATP or toxins such as nigericin. Activated NLRP3 binds ASC and caspase-1 proteins to form the NLRP3-inflammasome. Then caspase-1 cleaves and activates IL-1β, a potent inflammatory cytokine. Caspase-1 also cleaves gasdermin D (GSDMD) to induce pore formation in the cell membrane, through which IL-1β is secreted, and water gathered, eventually breaking up the membrane, causing the cell death. Thus the cell is sacrificed. This kind of cell dead is called pyroptosis and is accompanied by the release of intracellular content such as the enzyme LDH and inflammatory molecules that amplify the inflammatory response. Since NLRP3 and pro-IL1β are produced by NF-κB activation, to activate the NLRP3-inflammasome, the NLRP3 stimuli have to be combined with a TLR activator. For instance nigericin induces IL1β secretion only after LPS treatment. Yet, we have found a molecule able to activate both TLR and NLRP3: the Ornithine Lipid (OL), a component of bacteria such as Vibrio Cholerae grown in the absence of phosphate.

Bone marrow-derived macrophages (BMDM) from C57BL/6 wild type mice, or Nlrp3-/-, Pycard-/- or Casp1/11-/- mice were treated for 4h or 18h with LPS, OL or LPS and nigericin. Supernatants were assayed for TNF-α and IL-1β secretion by ELISA, and pyroptotic cell death by LDH assay. Cell lysates were assayed for pro-IL-1beta and NLRP3 expression by qPCR and Western blot. HEK-Blue-mTLR4 cells were treated 24h with LPS or OL. TLR4-dependent NF-κB activation was measured by SEAP assay.

In HEKBlue-mTLR4 cells, OL was able to activate TLR4. OL induced TNF-α secretion, and the expression of pro-IL-1β and NLRP3 mRNA and protein in macrophages from wild type mice. OL also induce IL1β secretion dependent on cellular K+ efflux and NLRP3/caspase-1 inflammasome, as it was blocked by specific inhibitors and on Nlrp3-/-, Pycard-/- or Casp1/11-/- macrophages. In line, OL induced NLRP3 dependent pyroptosis, that was absent in Casp1/11-/- macrophages.

OL induces the secretion of IL-1β and pyroptosis though NLRP3 inflammasome activation. In contrast with the majority of NLRP3 triggers, OL does not require a two-step activation protocol, as OL is able to activate NF-κB, probably by engaging TLR4. However, given the similarity of OL with TLR2 agonists, we could expect also TLR2 activation by OL.