Control of the innate immune response by the mevalonate pathway

Abstract

Deficiency in mevalonate kinase (MVK) causes systemic inflammation. However, the molecular mechanisms linking the mevalonate pathway to inflammation remain obscure. Geranylgeranyl pyrophosphate, a non-sterol intermediate of the mevalonate pathway, is the substrate for protein geranylgeranylation, a protein post-translational modification that is catalyzed by protein geranylgeranyl transferase I (GGTase I). Pyrin is an innate immune sensor that forms an active inflammasome in response to bacterial toxins. Mutations in MEFV (encoding human PYRIN) result in autoinflammatory familial Mediterranean fever syndrome. We found that protein geranylgeranylation enabled Toll-like receptor (TLR)-induced activation of phosphatidylinositol-3-OH kinase (PI(3)K) by promoting the interaction between the small GTPase Kras and the PI(3)K catalytic subunit p110δ. Macrophages that were deficient in GGTase I or p110δ exhibited constitutive release of interleukin 1β that was dependent on MEFV but independent of the NLRP3, AIM2 and NLRC4 inflammasomes. In the absence of protein geranylgeranylation, compromised PI(3)K activity allows an unchecked TLR-induced inflammatory responses and constitutive activation of the Pyrin inflammasome.

Figure 1. Pggt1b deficiency augments proinflammatory cytokine production while suppressing IFN-β and IL-10 production

Quantitation of IL-1β (a), TNF (b), IL-6 (c), IL-12 (d), IFN-β (e), IL-10 (f) and RANTES (g) in the supernatant of BMDMs by ELISA eight hours after stimulation with TLR ligands. Data (mean±SD) are representative of three independent experiments with similar results.

Figure 2. Neutrophilia and enhanced susceptibility to endotoxic shock in Pggt1b^fl/flLyz2-Cre mice

(a) Representative contour-plot of CD11b⁺, Ly6G⁺ granulocytes in the peripheral blood of Pggt1b^fl/+Lyz2-Cre and Pggt1b^fl/flLyz2-Cre mice. (b) Granulocyte percentage from sex- and age-matched control Pggt1b^fl/+Lyz2-Cre (n=7) and Pggt1b^fl/flLyz2-Cre (n=7) mice. (c) Survival of Pggt1b ^fl/+Lyz2-Cre (dotted line) (n=8) and Pggt1b^fl/flLyz2-Cre (solid line) (n=8) mice injected with a lethal dose of LPS. ELISA quantitation of IL-1β (d), TNF (e) and IL-6 (f) in the serum from Pggt1b ^fl/+Lyz2-Cre and Pggt1b^fl/flLyz2-Cre mice two hours post LPS injection. *P=.0.011, **P=0.0061, ***P=0.0006, ****P<0.0001 (Two Way ANOVA). Data are representative of two (a, b) or three (c–f) independent experiments.

Figure 3. Compromised LPS-induced Akt, GSK3β and mTOR Phosphorylation in the Absence of Pggt1b

(a) Immunoblots of phospho-Ser473 and Thr308 of Akt in the cell lysate of BMDMs stimulated with LPS at 10ng/ml. (b) Immunoblots of phospho-Gsk3β Ser9, phospho-RelA Ser536 and phospho-c-Jun Thr239 in the cell lysate of BMDMs stimulated with LPS at 10ng/ml. (c) Immunoblots of phospho-p70S6K Thr389 in the cell lysate of -BMDMs stimulated with LPS at 10ng/ml. Data in (a), (b) and (c) are from the same experiment which is representative of three independent experiments with similar results.

Figure 4. Protein Geranylgeranylation controls TLR-induced cytokine production and inflammasome activation through the PI(3)Kinase-p110δ

Production of IL-1β (a), IL-12(p40) (b), TNF(c), IL-6 (d) and IFN-β (e) by wild-type and Pik3cd^−/− (p110δ) BMDMs stimulated with LPS; (f) Production of IL-1β, TNF, IL-12(p40) or IL-10 in supernatants of wild type (WT) or Pggt1b deficient BMDMs reconstituted with empty vector (EV) or that harboring cDNAs encoding wild type p110δ, wild type Pggt1b or a constitutively active form of p110δ (E1211K). *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001 (Two Way ANOVA). Data (mean±SD) are representative of 3 independent experiments with similar results.

Figure 5. Protein geranylgeranylation controls the Kras-p110δ interaction that licenses TLR-induced p110δ activation

(a) Autoradiography of in vivo labeling of Ras proteins immunoprecipitated from wild type or Pggt1b deficient BMDMs. (b) Transcript abundance of Hras, Kras, and Nras in mouse BMDMs 48 hours after transfection with control or siRNA against H, K, Nras genes; (c) Immunoblot of phospho-Akt S473 after LPS stimulation of BMDMs 48 hours after transfection with control or siRNA against Hras, Kras, or Nras as described in (b); (d) Image J quantitation of phospho-Akt S473 in LPS-stimulated samples described in (c), the ratios between the phospho-Akt S473 band and the corresponding pan-Akt band were calculated. The ratio in siRNA scramble control was set as 100%. (e) Immunoblot of lysate or anti-p110δ immunoprecipitates of wild type immortalized macrophages stably infected with EV or pMSCV constructs containing cDNAs encoding wild type or the geranylgeranylated form (CAAL) of Hras, Kras, and Nras proteins; (f) Immunoblot of anti-p110α, β and δ immunoprecipitate or lysate of wild type or Pggt1b deficient BMDMs stimulated with LPS. Data are representatives of two (a) or three (b–f) independent experiments with similar results.

Figure 6. Spontaneous IL-1β secretion in Pggt1b and Pik3cd^−/− macrophages is mediated by pyrin inflammasome

(a)Immunoblots of the culture supernatant or the cell lysate of LPS or LPS plus nigericin-stimulated wild-type and Pggt1b deficient BMDMs; (b) Relative abundance of transcripts of Nlrc4, Aim2 and Mefv in primary BMDMs 48 hours after transfection with control or siRNA against Aim2, Nlrc4 or Mefv; (c)Immunoblot of supernatant or cell lysate of Pggt1b deficient BMDMs pre-transfected with siRNA as described in (b) and stimulated with LPS; (d) Immunoblot of supernatants or cell lysate of wild type, Akt1^−/−and Pik3cd^−/− BMDMs stimulated with LPS; (e) Immunoblot of supernatant or cell lysate of of Pggt1b deficient BMDMs reconstituted with EV or that harboring cDNAs encoding wild type p110δ, wild type Pggt1b or a constitutively active form of p110δ (E1211K); (f) Immunoblot of Pik3cd^−/−BMDMs stimulated with LPS that was pre-transfected with siRNA against Aim2, Nlrc4 and Mefv (g) & (h) Relative transcript abundance of Mefv in Pggt1b deficient(g) or Pik3cd^−/− (h) BMDMs upon LPS stimulation; (i) Immunoblot of Pyrin in cell lysate of LPS-stimulated Pggt1b deficient and control BMDMs. Data are representative of 3 independent experiments with similar results.

Figure 7. Enhanced cytokine production and inflammasome activation in PBMCs from HIDs patients treated with LPS and simvastatin

(a) Immunoblot of cleaved IL-1β from supernatant of LPS or LPS plus simvastatin stimulated PBMCs from healthy control or HIDs patients. (b) NanoString analysis of gene expression of proinflammatory cytokines from healthy control PBMCs stimulated with LPS or LPS plus simvastatin. (c) Immunoblot of pan-Akt and phospho-Akt-Ser473 of cell lysates of human PBMCs stimulated with LPS pre-treated overnight with vehicle or simvastatin (5μM). Data are representative of two experiments on the same patient samples with similar results.