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. 2020 Apr 21;31(3):107548.
doi: 10.1016/j.celrep.2020.107548.

The Set7 Lysine Methyltransferase Regulates Plasticity in Oxidative Phosphorylation Necessary for Trained Immunity Induced by β-Glucan

Samuel T Keating  1 , Laszlo Groh  1 , Charlotte D C C van der Heijden  1 , Hanah Rodriguez  2 , Jéssica C Dos Santos  1 , Stephanie Fanucchi  3 , Jun Okabe  2 , Harikrishnan Kaipananickal  4 , Jelmer H van Puffelen  5 , Leonie Helder  1 , Marlies P Noz  1 , Vasiliki Matzaraki  1 , Yang Li  6 , L Charlotte J de Bree  7 , Valerie A C M Koeken  1 , Simone J C F M Moorlag  1 , Vera P Mourits  1 , Jorge Domínguez-Andrés  1 , Marije Oosting  1 , Elianne P Bulthuis  8 , Werner J H Koopman  8 , Musa Mhlanga  9 , Assam El-Osta  10 , Leo A B Joosten  11 , Mihai G Netea  12 , Niels P Riksen  13
Affiliations

The Set7 Lysine Methyltransferase Regulates Plasticity in Oxidative Phosphorylation Necessary for Trained Immunity Induced by β-Glucan

Samuel T Keating et al. Cell Rep. .

Abstract

Trained immunity confers a sustained augmented response of innate immune cells to a secondary challenge, via a process dependent on metabolic and transcriptional reprogramming. Because of its previous associations with metabolic and transcriptional memory, as well as the importance of H3 histone lysine 4 monomethylation (H3K4me1) to innate immune memory, we hypothesize that the Set7 methyltransferase has an important role in trained immunity induced by β-glucan. Using pharmacological studies of human primary monocytes, we identify trained immunity-specific immunometabolic pathways regulated by Set7, including a previously unreported H3K4me1-dependent plasticity in the induction of oxidative phosphorylation. Recapitulation of β-glucan training in vivo additionally identifies Set7-dependent changes in gene expression previously associated with the modulation of myelopoiesis progenitors in trained immunity. By revealing Set7 as a key regulator of trained immunity, these findings provide mechanistic insight into sustained metabolic changes and underscore the importance of characterizing regulatory circuits of innate immune memory.

Keywords: Set7; immunometabolism; inflammation; macrophage; methylation; monocyte; oxidative phosphorylation; trained immunity; β-glucan.

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Conflict of interest statement

Declaration of Interests W.J.H.K. is a scientific advisor of Khondrion (Nijmegen, the Netherlands) and of Fortify Therapeutics. These subject matter experts had no involvement in the data collection, analysis and interpretation, writing of the manuscript, and the decision to submit the manuscript for publication.

Figures

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Graphical abstract
Figure 1
Figure 1
Set7 Is Associated with Trained Immunity Induced by β-Glucan (A) Graphical outline of in vitro training methods. Adherent monocytes (Mo) were stimulated with 1 μg/mL β-glucan or standard culture medium (RPMI) for 24 h (first stimulus), allowed to differentiate to macrophages (Mϕ) for 5 days, and restimulated for 24 h with LPS or RPMI on day 6. (B) Production of pro-inflammatory cytokines TNFα and IL-6 by trained macrophages following restimulation (n = 6 healthy volunteers per group). (C) Expression of SETD7 mRNA prior to restimulation (6 d) (n = 7 healthy volunteers per group). Representative western blot analysis of differentiated macrophages performed on day 6, prior to restimulation. Set7 encoded by the SETD7 gene; Rpl29k5me2 as a marker for Set7 activity; β-actin was used as loading control. (D) Single-nucleotide polymorphisms (SNPs) within SETD7 suggestively associated with trained responses to β-glucan in peripheral blood mononuclear cells (n = 267). Age- and sex-corrected TNFα and IL-6 changes are shown as boxplots for rs7680948 and rs56183115, respectively. (E) SNPs near SETD7 suggestively associated with trained responses to the bacillus Calmette-Guérin vaccine in peripheral blood mononuclear cells. Age- and sex-corrected TNFα and IL-6 changes are shown as boxplots for rs795971 (n = 213) and rs6816973 (n = 248), respectively. (F) SNPs near SETD7 suggestively associated with trained responses to the oxidized low-density lipoprotein in peripheral blood mononuclear cells. Age- and sex-corrected TNFα and IL-6 changes are shown as boxplots for rs6536295 (n = 197) and rs10020166 (n = 225), respectively. Data are represented as mean ± SEM. ∗p < 0.05, Wilcoxon signed-rank test.
Figure 2
Figure 2
Pharmacological Inhibition of Set7 Dose-Dependently Attenuates the Pro-inflammatory Cytokine Response of Trained Immunity In Vitro (A) Western blot analysis of β-glucan-trained macrophages incubated with cyproheptadine (CPH) for 24 h. HSP90 was used as a loading control. (B) Graphical overview of in vitro training methods. Adherent monocytes (Mo) were stimulated with β-glucan or RPMI culture medium for 24 h in the presence of CPH or DMSO vehicle control, allowed to differentiate to macrophages (Mϕ), and restimulated for 24 h with LPS, Pam3Cys, or RPMI on day 6. (C and D) Production of (C) TNFα and (D) IL-6 by β-glucan-trained macrophages incubated with CPH or 5′-methylthioadenosine (MTA) for the first 24 h of in vitro training and restimulated with LPS (n = 7 healthy volunteers). (E) Production of TNFα by β-glucan-trained macrophages incubated with CPH for the first 24 h of in vitro training and restimulated with Pam3Cys (n = 3 healthy volunteers). (F) Expression of SETD7 mRNA on day 6 by cells trained with β-glucan in the presence of 100 μM CPH (n = 6 healthy volunteers; open bar represents DMSO vehicle control). (G) Expression of RPL29 mRNA at 24 h by cells trained with β-glucan in the presence of 100 μM CPH (n = 3 healthy volunteers; open bar represents DMSO vehicle control). (H) Analysis of lactate dehydrogenase (LDH) as a measure of cytotoxicity in cells incubated with 100 μM CPH for 24 h (n = 3 healthy volunteers). (I) Analysis of viability and apoptosis with Annexin V and PI staining in cells incubated with 100 μM CPH versus DMSO vehicle controls for 24 h (n = 3 healthy volunteers). Fold change difference between CPH and vehicle controls. (J and K) Production of (J) TNFα and (K) IL-6 by β-glucan-trained macrophages incubated with sinefungin for the first 24 h of in vitro training and restimulated with LPS (n = 6 healthy volunteers). (L) Production of TNFα and IL-6 by bacillus Calmette-Guérin (BCG)-trained macrophages incubated with 100 μM CPH for the first 24 h of in vitro training and restimulated with LPS (n = 6 healthy volunteers). (M) Production of TNFα and IL-6 by laminarin-trained macrophages incubated with 100 μM CPH for the first 24 h of in vitro training and restimulated with LPS (n = 6 healthy volunteers). Data are represented as mean ± SEM. ∗p < 0.05, Wilcoxon signed-rank test or t test where appropriate. See also Figures S1 and S2.
Figure 3
Figure 3
Set7 Regulates Trained Immunity Induced by β-Glucan In Vivo (A) Representative western blot of Set7 protein expression in the bone marrow of wild-type (WT) and Setd7 KO mice. β-Actin was used as loading control. Expression of Setd7 mRNA in the bone marrow of WT and Setd7 KO mice (n = 7 mice per group). (B) Schematic overview of in vivo induction of trained immunity by β-glucan. (C) Plasma levels of TNFα, IL-6, and IL-1β in WT and Setd7 KO mice trained with PBS or β-glucan on day 1 and administered LPS on day 6 (n = 6–9 mice per group). (D) Day 6 analysis of Setd7 mRNA expression in the bone marrow of WT mice administered PBS or β-glucan (n = 7 mice per group). (E) Bone marrow mRNA expression of Csf2, Il1b, and Cd34 in WT and Setd7 KO mice trained with PBS or β-glucan on day 1 and administered LPS on day 6 (n = 6–7 mice per group). Data are represented as mean ± SEM. ∗p < 0.05, ∗∗p < 0.01, Mann-Whitney test.
Figure 4
Figure 4
Plasticity in the Induction of Oxidative Phosphorylation Is Important for the Pro-inflammatory Cytokine Production by Cells Trained by β-Glucan (A) Lactate production by β-glucan-trained macrophages incubated with CPH for the first 24 h of in vitro training (n = 6 healthy volunteers). (B) Oxygen consumption analysis (seahorse) of macrophages 5 days after incubation with β-glucan and co-incubation with CPH. Basal and maximum oxygen consumption rates (OCR) are indicated (n = 5 healthy volunteers; open bars represent DMSO vehicle controls). (C and D) Heatmap of the p values of association between SNPs mapped to genes involved in oxidative phosphorylation (OXPHOS) and the tricarboxylic acid (TCA) cycle and the magnitude of cytokine production capacity by PBMCs trained with β-glucan in vitro isolated from (C) 300BCG (cohort 1) and (D) 200FG (cohort 2). The color legend for the heatmap indicates the range of p values from QTL mapping. Boxplots show the genotype-stratified cytokine levels for the OXPHOS and TCA cycle loci (cohort 1, n = 238 healthy volunteers for TNFα, n = 251 healthy volunteers for IL-6; cohort 2, n = 119 healthy individuals). (E) Production of IL-6 by β-glucan-trained macrophages incubated with 1 μM oligomycin for the first 24 h of in vitro training and restimulated with LPS (n = 8 healthy volunteers). Data are represented as mean ± SEM. ∗p < 0.05, ∗∗p < 0.01, Wilcoxon signed-rank test or Mann-Whitney test where appropriate. See also Figure S3.
Figure 5
Figure 5
Inhibition of Set7 Regulates Metabolic Changes in Macrophages Trained with β-Glucan (A) Key TCA cycle metabolite concentrations in primary human monocytes/macrophages measured 24 h and 5 days after incubation with β-glucan and co-incubation with CPH (n = 6 healthy volunteers; open bars represent DMSO vehicle controls). (B) Schematic overview of the TCA cycle and succinate dehydrogenase (SDH) complex. Enzymes analyzed for gene expression in (C) and (D) are indicated. (C) Expression analysis of genes encoding enzymes involved in the TCA cycle by primary human monocytes/macrophages measured 24 h and 5 days after incubation with β-glucan and co-incubation with 100 μM CPH (n = 6–8 healthy volunteers; open bars represent DMSO vehicle controls). (D) Expression analysis of genes encoding SDH subunits in primary human monocytes/macrophages measured 24 h and 5 days after incubation with β-glucan and co-incubation with 100 μM CPH (n = 6–8 healthy volunteers; open bars represent DMSO vehicle controls). (E) Bone marrow mRNA expression of Mdh2, Sdhb, Fh1, and Suclg1 in WT and Setd7 KO mice trained with PBS or β-glucan on day 1 and administered LPS on day 6 (n = 6–7 mice per group). Data are represented as mean ± SEM. ∗p < 0.05, ∗∗p < 0.01, Wilcoxon signed-rank test or Mann-Whitney test where appropriate.
Figure 6
Figure 6
H3K4me1 Changes at Distal Enhancers Regulating MDH2 and SDHB Support Metabolic Reprogramming of Macrophages Trained with β-Glucan (A) Gene-enhancer interactions within the topologically associating domain (TAD) surrounding MDH2 derived from ChIA-PET interactions in K562 cells (upper panel). ChIP-seq-derived H3K4me1 maps of these enhancer regions in monocytes stimulated with β-glucan for 24 h (lower panels). (B) Gene-enhancer interactions within the TAD surrounding SDHB derived from ChIA-PET interactions in K562 cells (upper panel). ChIP-seq-derived H3K4me1 maps of these enhancer regions in monocytes stimulated with β-glucan for 24 h (lower panels). (C) Levels of H3K4me1 at enhancer sites associated with the transcriptional regulation of MDH2 in primary human macrophages measured 5 days after incubation with β-glucan and co-incubation with 100 μM CPH (n = 6 healthy volunteers; open bars represent DMSO vehicle controls). (D) Levels of H3K4me1 at enhancer sites associated with the transcriptional regulation of SDHB in primary human macrophages measured 5 days after incubation with β-glucan and co-incubation with 100 μM CPH (n = 6 healthy volunteers; open bars represent DMSO vehicle controls). Data are represented as mean ± SEM. ∗p < 0.05, ∗∗p < 0.01, Wilcoxon signed-rank test. See also Figures S4, S5, and S6.
Figure 7
Figure 7
Set7 Regulates Metabolic Changes in Cells Trained with β-Glucan β-Glucan stimulation activates Set7 to write the H3K4me1 modification to distal enhancers associated with sustained MDH2 and SDHB gene expression leading to increases in TCA cycle metabolites and increased oxidative phosphorylation important for enhanced cytokine production in trained immunity.
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