Blocking protein prenylation in cell culture models of HIDS leads to the formation of NLRP3 inflammasomes by a mechanism dependent on extracellular ATP — ASN Events
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  3. Blocking protein prenylation in cell culture models of HIDS leads to the formation of NLRP3 inflammasomes by a mechanism dependent on extracellular ATP

Blocking protein prenylation in cell culture models of HIDS leads to the formation of NLRP3 inflammasomes by a mechanism dependent on extracellular ATP (#374)

Oliver P Skinner 1 , Alicia G Rios Wilks 1 , Julie Jurczyluk 1 , Kristen Perry 1 , Ryan C Chai 1 , Paul J Baker 2 , Seth L Masters 2 , Avril Robertson 3 , Kate Schroder 3 , Marcia A Munoz 1 , Michael J Rogers 1
  1. Bone Biology Division, Garvan Institute of Medical Research, Sydney, NSW, Australia
  2. Division of Inflammation, Walter and Eliza Hall Institute of Medical Research, Melbourne, Victoria, Australia
  3. Cell Biology & Molecular Medicine Division, Institute for Molecular Bioscience, Brisbane, Queensland, Australia

Protein prenylation is an essential process dependent on isoprenoid lipids from the mevalonate pathway. Mutations in mevalonate kinase lead to the autoinflammatory disease HIDS. We recently demonstrated that HIDS PBMCs are defective in prenylation; it has been proposed that lack of prenylation causes activation of the pyrin inflammasome and caspase-1-mediated IL-1β release. We used cell culture models, in which prenylation was inhibited by statin treatment, to clarify the mechanism of inflammasome activation.

Protein prenylation was inhibited in THP-1 monocytes by treatment for 24hr with 5μM simvastatin (SIM). SIM pre-treatment caused a 5-fold increase in IL-1β release following LPS stimulation, without affecting mRNA or protein levels of proIL-1β, NLRP3 or pyrin compared to LPS alone. This effect was completely inhibited in the presence of 1-10μM MCC950, a specific inhibitor of NLRP3 inflammasomes. Furthermore, deletion of NLRP3 or CASP1 using CRISPR/Cas9 abolished the release of IL-1β whereas deletion of MEFV (pyrin) had no effect. 500μM oxo-ATP (a P2X7 antagonist) also abolished SIM-stimulated IL-1β release, consistent with the requirement for extracellular ATP/P2X7 to fully activate NLRP3 inflammasomes in monocytes.

To examine whether inhibition of prenylation was sufficient to trigger inflammasome formation we used immortalized bone marrow macrophages expressing mCherry-ASC. In these cells, LPS-induced IL-1β release is dependent on extracellular ATP/P2X7. Live cell imaging, and ASC immunostaining, revealed that 15-20% of cells contained an inflammasome 24hr after 2μM SIM treatment but this was absent in untreated cells. SIM pre-treated cells also released IL-1β following stimulation with LPS alone; this was completely prevented by adding oxo-ATP or MCC950, or by restoring prenylation with exogenous lipid.

These observations demonstrate that blocking prenylation in cell culture models of HIDS leads to the formation of NLRP3 inflammasomes by a mechanism dependent on extracellular ATP, challenging the view that pyrin inflammasomes are involved in the inflammatory pathology of HIDS.

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