Lack of protein prenylation promotes NLRP3 inflammasome assembly in human monocytes
Authors: Oliver P. Skinner, Julie Jurczyluk, Paul J. Baker, Seth L. Masters, Alicia G. Rios Wilks, Misaki S. Clearwater, Avril A.B. Robertson, Kate Schroder, Sam Mehr, Marcia A. Munoz, Michael J. Rogers
Journal: Journal of Allergy and Clinical Immunology (2019)

Mevalonate kinase deficiency (MKD; also known as hyper-IgD syndrome) is a rare autoinflammatory disorder caused by recessive hypomorphic mutations in the MVK gene.1Stoffels M. Jongekrijg J. Remijn T. Kok N. van der Meer J.W. Simon A. TLR2/TLR4-dependent exaggerated cytokine production in hyperimmunoglobulinaemia D and periodic fever syndrome.Rheumatology (Oxford). 2015; 54: 363-368Crossref PubMed Scopus (32) Google Scholar Impaired activity of mevalonate kinase, a crucial component of the mevalonate-cholesterol biosynthesis pathway, leads to decreased production of isoprenoid lipids, defective posttranslational protein prenylation, and cytosolic accumulation of unprenylated Rab proteins and other small GTPases in cells from patients with MKD.2Munoz M.A. Jurczyluk J. Mehr S. Chai R.C. Arts R.J.W. Sheu A. et al.Defective protein prenylation is a diagnostic biomarker of mevalonate kinase deficiency.J Allergy Clin Immunol. 2017; 140: 873-875Abstract Full Text Full Text PDF PubMed Scopus (20) Google Scholar It has been proposed that excessive production of IL-1β from monocytes of patients with MKD is caused by loss of protein prenylation, specifically geranylgeranylation.3Mandey S.H. Kuijk L.M. Frenkel J. Waterham H.R. A role for geranylgeranylation in interleukin-1beta secretion.Arthritis Rheum. 2006; 54: 3690-3695Crossref PubMed Scopus (150) Google Scholar In this setting defective protein prenylation promotes inflammasome activation and cleavage of pro–IL-1β, thereby triggering systemic inflammatory flares. However, the precise underlying mechanisms, including the type of inflammasome that is activated, remain unclear. Recently, 2 studies suggested that loss of protein prenylation in macrophages leads to activation of the pyrin inflammasome, either through mechanisms involving loss of prenylated K-Ras4Akula M.K. Shi M. Jiang Z. Foster C.E. Miao D. Li A.S. et al.Control of the innate immune response by the mevalonate pathway.Nat Immunol. 2016; 17: 922-929Crossref PubMed Scopus (133) Google Scholar or loss of prenylated Rho GTPase.5Park Y.H. Wood G. Kastner D.L. Chae J.J. Pyrin inflammasome activation and RhoA signaling in the autoinflammatory diseases FMF and HIDS.Nat Immunol. 2016; 17: 914-921Crossref PubMed Scopus (324) Google Scholar However, other inflammasomes could feasibly also play a role because cholesterol homeostasis is linked to NLRP3 inflammasome activation.6Guo C. Chi Z. Jiang D. Xu T. Yu W. Wang Z. et al.Cholesterol homeostatic regulator SCAP-SREBP2 integrates NLRP3 inflammasome activation and cholesterol biosynthetic signaling in macrophages.Immunity. 2018; 49: 842-856.e7Abstract Full Text Full Text PDF PubMed Scopus (119) Google Scholar To further clarify this issue, we pharmacologically inhibited the mevalonate pathway in human THP-1 monocytic cells with simvastatin (SIM) and then stimulated the cells with bacterial LPS, an archetypal inflammatory stimulus that acts through Toll-like receptor (TLR) 4. In THP-1 cells and human PBMCs, blocking protein prenylation with statins or other pharmacologic agents, such as GGTI-298, before LPS stimulation mimics the excessive release of IL-1β from PBMCs of patients with MKD.3Mandey S.H. Kuijk L.M. Frenkel J. Waterham H.R. A role for geranylgeranylation in interleukin-1beta secretion.Arthritis Rheum. 2006; 54: 3690-3695Crossref PubMed Scopus (150) Google Scholar, 7Jurczyluk J. Munoz M.A. Skinner O.P. Chai R.C. Ali N. Palendira U. et al.Mevalonate kinase deficiency leads to decreased prenylation of Rab GTPases.Immunol Cell Biol. 2016; 94: 994-999Crossref PubMed Scopus (12) Google Scholar First, we used an in vitro prenylation assay and Western blotting to identify a concentration of SIM that profoundly inhibited Rab and Rap1A prenylation in THP-1 cells without causing loss of cell viability (see Fig E1, A and B, in this article's Online Repository at www.jacionline.org). Pretreatment with 5 μmol/L SIM for 24 hours was used thereafter, and this substantially increased IL-1β release after LPS stimulation (Fig 1, A). Cells with an inflammasome "speck" containing ASC (apoptosis-associated speck-like protein containing C-terminal caspase recruitment domain) were virtually absent in THP-1 cells in the absence or presence of SIM alone (0.6% and 1.6% of cells, respectively). However, SIM pretreatment significantly increased the proportion of speck-containing cells after 4 hours of LPS stimulation, from 30% to 45% (Fig 1, B). Similarly, SIM boosted LPS-induced caspase-1 activity and pyroptotic cell death (Fig 1, C, and see Fig E1, C). This increase in inflammasome formation, caspase-1 activation, and pyroptosis was accompanied by a quadrupling of LPS-induced IL-1β (and a doubling of IL-18) release into the extracellular medium from SIM-pretreated cells (Fig 1, D). SIM prevents protein prenylation by blocking synthesis of essential isoprenoid lipids, particularly geranylgeranyl diphosphate. This can be overcome by addition of exogenous geranylgeraniol (GGOH) to allow normal protein geranylgeranylation. Supplementing THP-1 cells with 50 μmol/L GGOH completely abolished the synergistic effect of SIM on LPS-induced inflammasome formation, caspase-1 activation, pyroptosis, and IL-1β and IL-18 release (Fig 1, B-D, and see Fig E1, C). We confirmed that GGOH prevented accumulation of unprenylated Rab and Rap1A proteins in SIM-treated THP-1 cells (Fig 1, E), supporting the concept that SIM exacerbates inflammasome activation as a result of loss of protein prenylation rather than through any off-target effects. To determine the requirement for NLRP3 or pyrin in SIM-enhanced inflammasome formation, we used THP-1 cells in which the NLRP3, MEFV, or CASP1 genes were disabled by means of doxycycline-inducible CRISPR/Cas9 gene editing (Fig 1, F).8Baker P.J. Boucher D. Bierschenk D. Tebartz C. Whitney P.G. D'Silva D.B. et al.NLRP3 inflammasome activation downstream of cytoplasmic LPS recognition by both caspase-4 and caspase-5.Eur J Immunol. 2015; 45: 2918-2926Crossref PubMed Scopus (99) Google Scholar NLRP3 or caspase-1 deficiency abolished LPS-induced IL-1β release and completely prevented the synergistic effect of SIM pretreatment, but pyrin deficiency did not affect IL-1β production under any conditions (Fig 1, G). Furthermore, NLRP3 deficiency in THP-1 cells also ablated LPS-induced ASC speck formation and caspase-1 activity with and without SIM pretreatment (see Fig E1, D and E). Unlike caspase-1 deficiency (Fig 1, G), lack of caspase-4/5 had no effect on IL-1β release (see Fig E1, F), indicating that SIM did not promote a noncanonical inflammasome pathway. Consistent with a requirement for the NLRP3 inflammasome, we found that preventing K+ efflux by using high extracellular [K+] or neutralizing reactive oxygen species completely blocked the synergistic effect of SIM (see Fig E1, G and H). Together, these observations clearly demonstrate that loss of prenylation in human THP-1 monocytes enhances the LPS-induced formation of an NLRP3-dependent and pyrin-independent inflammasome. We next examined whether pharmacologic inhibition of NLRP3 could prevent the excessive inflammasome activation caused by defective protein prenylation in THP-1 cells. Treatment with the small-molecule NLRP3 inhibitor MCC9509Coll R.C. Robertson A.A. Chae J.J. Higgins S.C. Munoz-Planillo R. Inserra M.C. et al.A small-molecule inhibitor of the NLRP3 inflammasome for the treatment of inflammatory diseases.Nat Med. 2015; 21: 248-255Crossref PubMed Scopus (1527) Google Scholar completely blocked LPS-induced caspase-1 activation (Fig 1, H), IL-1β release (Fig 1, I) and pyroptosis (see Fig E1, C) in the presence or absence of SIM. Similarly, MCC950 significantly inhibited the increased IL-1β release from THP-1 cells pretreated with GGTI-298 (see Fig E1, I). Finally, we tested the effect of MCC950 on PBMCs isolated from a patient with a mild form of MKD/hyper-IgD syndrome (compound heterozygous MVKV377I/H20N mutations) and from both parents (heterozygous for either MVKV377I/+ or MVKH20N/+). We have previously shown that protein prenylation is defective in PBMCs from this patient but not in either parent.2Munoz M.A. Jurczyluk J. Mehr S. Chai R.C. Arts R.J.W. Sheu A. et al.Defective protein prenylation is a diagnostic biomarker of mevalonate kinase deficiency.J Allergy Clin Immunol. 2017; 140: 873-875Abstract Full Text Full Text PDF PubMed Scopus (20) Google Scholar Stimulation with LPS and the NLRP3 activator nigericin elicited much greater IL-1β release in cells from the patient with MKD than from either parent, and this response was abolished by MCC950 (Fig 1, J). Our observations clearly demonstrate that lack of prenylation in monocytic cells (using statin treatment, a pharmacologic model of MKD, or using authentic MKD cells from a patient) leads to enhanced formation of an NLRP3-dependent and pyrin-independent inflammasome upon LPS stimulation. Although loss of prenylation has been suggested to activate the pyrin inflammasome in mouse bone marrow–derived macrophages,4Akula M.K. Shi M. Jiang Z. Foster C.E. Miao D. Li A.S. et al.Control of the innate immune response by the mevalonate pathway.Nat Immunol. 2016; 17: 922-929Crossref PubMed Scopus (133) Google Scholar, 5Park Y.H. Wood G. Kastner D.L. Chae J.J. Pyrin inflammasome activation and RhoA signaling in the autoinflammatory diseases FMF and HIDS.Nat Immunol. 2016; 17: 914-921Crossref PubMed Scopus (324) Google Scholar it is possible that defective prenylation promotes activity of distinct inflammasomes in different cell types. In monocytes, loss of protein prenylation caused by SIM treatment was alone insufficient for inducing NLRP3 inflammasome formation or caspase-1 activation (Fig 1, B and C) but substantially boosted LPS-induced inflammasome activity (speck formation, caspase-1 activation, IL-1β and IL-18 release, and pyroptosis) on stimulation with LPS (Fig 1, B-D, and see Fig E1, C). The underlying mechanism remains to be clarified but did not involve an enhancement of LPS-induced changes in mRNA levels (see Fig E2, A, in this article's Online Repository at www.jacionline.org) or changes in protein levels (see Fig E2, B) of pro–IL-1β, pro–IL-18, NLRP3, ASC, or pro–caspase-1. Enhanced inflammasome formation in prenylation-deficient THP-1 cells also occurred after stimulation with the TLR2 agonist Pam3CSK4 (Fig 1, K), which is consistent with the enhanced sensitivity of PBMCs from patients with MKD to both TLR2 and TLR4 agonists.1Stoffels M. Jongekrijg J. Remijn T. Kok N. van der Meer J.W. Simon A. TLR2/TLR4-dependent exaggerated cytokine production in hyperimmunoglobulinaemia D and periodic fever syndrome.Rheumatology (Oxford). 2015; 54: 363-368Crossref PubMed Scopus (32) Google Scholar These findings are of clinical relevance because they demonstrate that targeting the pyrin inflammasome in isolation might not be sufficient to resolve all the pathology associated with MKD. Rather, approaches to overcome the metabolic defect in the mevalonate pathway and restore normal protein prenylation could be more effective at preventing broader inflammasome activation. We thank Professor Kirill Alexandrov and Dr Zakir Tnimov (University of Queensland) for providing reagents for the Rab prenylation assay and Dr Ryan Chai (Garvan Institute) and Dr Rebecca Coll (University of Queensland) for technical advice. THP-1 cells (obtained from ATCC [Manassas, Va] and confirmed mycoplasma free throughout the studies) were seeded into 25-cm3 flasks (5-8 × 105 cells/mL in RPMI/10% heat-inactivated FCS) and cultured with or without 5 μmol/L SIM (Sigma-Aldrich, St Louis, Mo) or 10 μmol/L GGTI-298 (Sigma-Aldrich) with or without 50 μmol/L GGOH (Sigma-Aldrich) for 24 hours. Cells were then replated into 96-well plates in triplicate wells (1 × 106 cells/mL in 200 μL) and stimulated with 200 ng/mL LPS (Escherichia coli O111:B4; Sigma-Aldrich) for 4 hours with or without 10 μmol/L MCC950, 50 mmol/L KCl, or 100 μmol/L butylated hydroxyanisole (Sigma-Aldrich) or stimulated for 4 hours with 1 μg/mL Pam3CSK4 (R&D Systems, Minneapolis, Minn). Conditioned media were then collected for measurement of human IL-1β and IL-18 (Instant ELISA; eBioscience, San Diego, Calif). Unless stated otherwise, values shown of IL-1β or IL-18 release are from 1 experiment and representative of at least 3 independent experiments (IL-1β) or 2 experiments (IL-18). For time-course studies, THP-1 cells were seeded into 6-well plates (106 cells in 1 mL) and pretreated with 5 μmol/L SIM for 24 hours, followed by addition of 200 ng/mL LPS. Samples of conditioned media for IL-1β ELISA were then taken at 1-hour intervals up to 6 hours, and cells were collected for quantitative PCR analysis and Western blotting. IL-1β values are means ± SDs from 3 independent experiments. Fresh blood samples from a patient with MKD and both unaffected parents were obtained after informed consent. PBMCs were isolated from buffy coat preparations and seeded in duplicate at 3 × 106 cells/well (96 well-plate) with or without 50 μmol/L MCC950 and then stimulated with 1000 ng/mL LPS for 3 hours and then 10 μmol/L nigericin for 30 minutes. IL-1β release into conditioned media was assayed by means of ELISA (Instant ELISA; eBioscience). Values shown are means ± SDs from 1 experiment and representative of 2 independent experiments. RNA was extracted from cell pellets for quantitative RT-PCR analysis, as described previously.E1Jurczyluk J. Munoz M.A. Skinner O.P. Chai R.C. Ali N. Palendira U. et al.Mevalonate kinase deficiency leads to decreased prenylation of Rab GTPases.Immunol Cell Biol. 2016; 94: 994-999Crossref PubMed Scopus (24) Google Scholar mRNA expression of the following genes was assessed by using TaqMan probes: IL1B, IL18, NLRP3, MEFV, ASC, CASP1, and β2-microglobulin (B2M; TaqMan probes from Thermo Fisher Scientific, Waltham, Mass; Hs01555410_m1, Hs01038788_m1, Hs00918082_m1, Hs00925524_m1, Hs01547324_gH, Hs00354836_m1, and Hs99999907_m1 respectively). Values are expressed relative to B2M as a control and are means ± SDs from 3 independent experiments. THP-1 cells expressing Cas9 that had been lentivirally transduced with doxycycline-inducible sgRNA to NLRP3, MEFV, CASP1, and CASP4 and CASP5 (or untransduced control cells)E2Baker P.J. Boucher D. Bierschenk D. Tebartz C. Whitney P.G. D'Silva D.B. et al.NLRP3 inflammasome activation downstream of cytoplasmic LPS recognition by both caspase-4 and caspase-5.Eur J Immunol. 2015; 45: 2918-2926Crossref PubMed Scopus (207) Google Scholar were cultured with 1 μg/mL doxycycline for 72 hours and then without doxycycline for a further 24 hours. Loss of protein expression was confirmed by means of Western blotting (analysis of cleaved caspase-1 [p10] was performed in LPS-treated cells). After doxycycline induction, cells were treated with SIM for 24 hours before 4 hours of LPS stimulation, as described above. Unless stated otherwise, values of IL-1β release are means ± SDs from 3 independent experiments. Cell pellets were extracted by using 66 mmol/L Tris-Cl (pH 7.4), 2% SDS, 1× EDTA-free Complete Protease Inhibitor (Roche, Mannheim, Germany), and 30 μg of lysate were analyzed by means of Western blotting for pro–IL-1β (AF-201-NA, 1:000; R&D Systems), caspase-1 (D7F10, 1:000; Cell Signaling, Danvers, Mass), NLRP3 (Cryo-2, 1:1000; AdipoGen Life Sciences, San Diego, Calif), pyrin (AL196, 1:000; AdipoGen Life Sciences), caspase-4 (M029-3, 1:1000; MBL, Woburn, Mass), and β-actin (A1978, 1:1000; Sigma-Aldrich) by using peroxidase-labeled secondary antibodies and chemiluminescence detection by using SuperSignal West Pico and Femto reagent (Thermo Fisher Scientific, Waltham, Mass). Unprenylated Rap1A or unprenylated Rab proteins were detected in 30 μg of cell lysate by using Western blotting for unprenylated Rap1A or by using an in vitro prenylation assay to detect unprenylated Rab GTPases, as described previously.E1Jurczyluk J. Munoz M.A. Skinner O.P. Chai R.C. Ali N. Palendira U. et al.Mevalonate kinase deficiency leads to decreased prenylation of Rab GTPases.Immunol Cell Biol. 2016; 94: 994-999Crossref PubMed Scopus (24) Google Scholar All blots shown are representative of at least 3 separate experiments. THP-1 cells were cultured for 24 hours with or without 5 μmol/L SIM before seeding at 1 × 106 cells/mL in 96-well plates. The caspase-1 inhibitor VX-765 (50 μmol/L; Merck, Whitehouse Station, NJ) was added before 4 hours of stimulation with 200 ng/mL LPS or 1 μg/mL Pam3CSK4. Cells were cytospun onto glass slides, fixed with 10% formalin, and immunostained for ASC (anti-ASC antibody N-15, sc-22514-R, Santa Cruz Biotechnology, Dallas, Tex; Alexa Fluor 647 secondary antibody and 4′-6-diamidino-2-phenylindole dihydrochloride nuclear counterstain) before imaging with a Leica DM 5500 fluorescence microscope (Leica, Wetzlar, Germany). Quantification of ASC specks was performed by using ImageJ image analysis software (National Institutes of Health, Bethesda, Md) with 6 fields of view at ×20 magnification per sample. Each field of view had 100 to 600 cells. Unless stated otherwise, percentages of cells with ASC specks are means ± SDs from 3 independent experiments. THP-1 cells were cultured for 24 hours with or without 5 μmol/L SIM and reseeded in white, clear-bottom, 96-well plates, as described above, before 1 hour of stimulation with 200 ng/mL LPS. As a positive control, cells were treated with 10 μmol/L nigericin (Sigma-Aldrich) for 45 minutes. One hundred microliters of Caspase-Glo 1 reagent (Promega, Madison, Wis) was added to wells at room temperature before measuring luminescence after 1 hour on a CLARIOstar (BMG Labtech, Cary, NC) plate reader. Values were expressed as percentages of caspase-1 activity in control cells and, unless stated otherwise, are means ± SDs from 3 independent experiments. Cell viability was assessed by using Trypan Blue exclusion on THP-1 cultures treated with various concentrations of SIM (1, 2, 5, 10, and 20 μmol/L) for 24 hours. Pyroptotic cell death was measured based on propidium iodide uptake (50 μg/mL; Sigma-Aldrich) and flow cytometric analysis with a CytoFLEX (Beckman Coulter, Fullerton, Calif) flow cytometer. One-way ANOVA with the Tukey multiple comparisons post hoc test was performed with GraphPad Prism software (v7; GraphPad Software, La Jolla, Calif).Fig E2Pretreatment of THP-1 cells with SIM did not enhance mRNA or protein expression of inflammasome components/substrates. A, Quantitative PCR analysis of mRNA expression of genes encoding IL-1β, IL-18, NLRP3, pyrin (MEFV), ASC, and caspase-1 in control cells (black lines) or SIM-pretreated cells (red lines) for up to 6 hours after LPS stimulation. Values are means ± SDs from 3 independent experiments. B, Western blots showing protein levels of pro–IL-1β, NLRP3, pyrin, and pro–caspase-1 in control or SIM-pretreated cells for up to 4 hours after LPS stimulation. Zero hours represents control or SIM pretreatment before LPS stimulation; β-actin was used as a loading control. Blots are representative of 3 independent experiments.View Large Image Figure ViewerDownload Hi-res image Download (PPT)