1. Metabolic Induction of Trained Immunity through the Mevalonate Pathway
Siroon Bekkering, Rob J.W. Arts, Boris Novakovic, Ioannis Kourtzelis, Charlotte D.C.C. van der Heijden, Yang Li, Calin D. Popa, Rob ter Horst, Julia van Tuijl, Romana T. Netea-Maier, Frank L. van de Veerdonk, Triantafyllos Chavakis, Leo A.B. Joosten, Jos W.M. van der Meer, Henk Stunnenberg, Niels P. Riksen, Mihai G. Netea 
Cell 
Volume 172, Issue 1, Pages e9 (January 2018)
DOI: /j.cell
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3. Figure 1 The Cholesterol Synthesis Pathway in Trained Immunity
(A) Transcriptional expression of macrophages 5 days after β-glucan exposure. Red arrow, significant upregulation. Overview of the cholesterol synthesis pathway and the used inhibitors are shown.
(B) Monocytes were trained with β-glucan ± inhibitors of the cholesterol synthesis pathway for 24 hr. Five days after β-glucan exposure, lactate concentrations were measured in the supernatants and macrophages were restimulated for 24 hr with LPS, and TNF-α concentrations were measured in the supernatant (n = 6, mean ± SEM).
(C) Mouse blood mononuclear cells were trained with β-glucan ± fluvastatin on day 1. At day 6, cells were stimulated for 24 hr, and TNF-α was determined in the supernatants (n = 6, mean ± SEM).
(D and E) Monocytes were trained with oxLDL (D) or BCG (E) +/− fluvastatin for 24 hr. Five days later, epigenetic (H3K4me3) changes at the TNFA promoter and lactate concentrations in the supernatant were measured. Macrophages were also restimulated for 24 hr with LPS to determine TNF-α concentrations in the supernatant (n = 6, mean ± SEM).
(F) Monocytes were trained with oxLDL ± fluvastatin for 24 hr. Five days later, foam cell formation was induced by exposure to high concentration of oxLDL. After 24 hr, cells were stained with oil red O. ∗p < 0.05.
(G) Monocytes were trained with oxLDL ± fluvastatin for 24 hr. After 5 days rest in culture medium, RNA was isolated and expression of scavenger receptor CD36 and SR-A, and the cholesterol efflux transporters ABCA1 and ABCG1 were determined (n = 6, mean ± SEM). ∗p < 0.05.
See also Figure S1 and Table S4.
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4. Figure 2 Mevalonate Induces Trained Immunity
(A) Adding mevalonate (500 μM) during the first 24 hr can rescue the inhibition of oxLDL/β-glucan-induced training by fluvastatin (n = 6, mean ± SEM).
(B and C) Human (B) and mouse (C) monocytes were trained with mevalonate for 24 hr and restimulated with LPS at day 6. TNF-α concentration was measured in the supernatants (n = 6, mean ± SEM).
(D) When 6-fluoromevalonate was added during the first 24 hr, training with mevalonate could be enhanced (n = 6, mean ± SEM).
(E) Lactate production of RPMI or LPS restimulated mevalonate-trained macrophages at day 6 (n = 10, mean ± SEM).
(F) Adding a methyltransferase inhibitor (MTA) inhibits the induction of trained immunity (n = 6, mean ± SEM).
(G) H3K4me3 at promoters of TNFA and IL6 was assessed at day 5 of mevalonate-trained macrophages (n = 6, mean ± SEM). ∗p < 0.05, ∗∗p < 0.01.
See also Table S5.
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5. Figure 3 Signaling in Mevalonate-Trained Monocytes
(A) When 2-deoxyglucose (2-DG) or rapamycin were added during the first 24 hr, mevalonate (500 μM, left panel) and β-glucan (5 μg/mL, right panel)-induced training could be inhibited (n = 6, mean ± SEM).
(B) Western blot showing increased p-mTOR, p-S6K, and p-4EBP1 upon mevalonate training. Treatment with IGF1-R inhibitor (IGFR-i) inhibited these effects.
(C) Inhibition of the IGF1-R during resting of β-glucan-induced training inhibits the induction of trained immunity.
(D) When an IGF1-R inhibitor or an IGF1-R antibody were added to mevalonate training, the induction of trained immunity was diminished (n = 6, mean ± SEM).
(E) Blocking the IGF1-R inhibits the epigenetic changes induced by mevalonate training (n = 6, mean ± SEM).
(F) Monocytes were incubated for 4 or 24 hr with 500 μM or 5 μg/mL β-glucan. After incubation, IGF-R expression on monocytes was determined.
(G) Monocytes were trained with IGF1 during 24 hr. Five days after exposure, concentrations were measured in the supernatant after LPS/Pam3Cys restimulation (n = 6, mean ± SEM).
(H) Monocytes of 200 subjects were in vitro trained by β-glucan or BCG and restimulated with LPS. In supernatants TNF-α, IL-6 and lactate (LAC) were determined. From all subjects, SNP information for the IGF1R was extracted from a genome-wide Illumina Infinium SNP array. SNP analysis revealed four SNPs in the IGF1R with a significant association with LPS-induced TNF-α production in monocytes trained with β-glucan and BCG.
(I) Proposed working mechanism of the amplification of trained immunity through mevalonate interaction with IGF1-R at the level of the cell membrane. ∗p < 0.05.
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6. Figure 4 Epigenetic Changes Induced by Mevalonate
(A) Experimental setup for mevalonate-induced trained macrophages.
(B) Summary of H3K27ac peaks that show higher or lower deposition in mevalonate-trained cells relative to control macrophages.
(C) Boxplots of H3K27ac peaks.
(D) Heatmap of H3K27ac intensity at peaks that show increased and decreased H3K27ac in mevalonate-trained macrophages. Data are plotted over an area of ±12 kb from peak center.
(E) Pathway analysis of H3K27ac differentially regulated genes (n = 2).
See also Table S1.
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7. Figure 5 HIDS Patients Display a Trained Immunity-like Phenotype
(A) Monocytes from HIDS patients and matched healthy volunteers were exposed ex vivo to several ligands for 24 hr. Cytokine were measured in the supernatants (n = 4, mean ± SEM).
(B) Heatmap showing expression data of monocytes of HIDS patients versus healthy volunteers either unstimulated or 24 hr stimulated with RPMI or LPS (n = 3 versus n = 3).
(C) Top pathways associated with genes that show higher expression in HIDS patients exposed to 24 hr RPMI compared to controls (n = 3 versus n = 3).
(D) Monocytes of 3 HIDS patients and 3 healthy volunteers were exposed to RPMI or LPS for 24 hr or left unstimulated (unstim). Expression of genes in the glycolysis pathway, mTOR pathway, or cytokines were determined. Fold differences in expression of these genes between healthy volunteers and HIDS patients were calculated by dividing expression of HIDS patients by healthy volunteers (n = 3 versus n = 3). ∗p < 0.05.
See also Figures S1 and S2 and Tables S2 and S3.
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8. Figure S1 Additional In Vitro Experiments, Related to Figure 1B
(A) Monocytes were trained with β-glucan (left) or oxLDL (right) +/− different concentrations of fluvastatin during 24h. Five days after exposure macrophages were restimulated for 24h with LPS and TNFα concentrations were measured in the supernatant. (N = 6, mean ± SEM).
(B) Related to Figure 2G. Monocytes were trained with mevalonate (500 μM) for 24h. At day 6 DNA was isolated and H3K9me3 was determined at the promoters of IL6 and TNFA. (N = 6, mean ± SEM).
(C) Monocytes were pre-incubated with RPMI as negative control or 500 μM R-mevalonate. After 4h, the cells were stimulated with either RPMI as negative control or 50 ng/ml IGF1 for 15 minutes and Akt phosphorylation was determined by western blot.
(D) Related to Figure 3H. Example. Boxplot presentation of LPS-restimulated BCG-trained monocytes. TNFα production based on the rs polymorphism in IGF1R.
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9. Figure S2 HIDS Expression Analysis, Related to Figure 5
Heatmap of individual donors for genes that show LPS responses in controls and HIDS patients. Monocytes were either unstimulated (= baseline) or stimulated for 24h with RPMI or LPS.
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10. Figure S3
Comparison of H3K27ac Peaks in HIDS Monocytes versus Mevalonate-Trained Macrophages, Related to Figure 5
At the left, graphs display the median and mean read counts of the H3K27ac peaks that were significantly up or down in HIDS patient monocytes. At the right, graphs display median and mean read counts at the same H3K27ac peaks.
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