Lysine methyltransferase G9a is an important modulator of trained immunity

Abstract

Objectives: Histone methyltransferase G9a, also known as Euchromatic Histone Lysine Methyltransferase 2 (EHMT2), mediates H3K9 methylation which is associated with transcriptional repression. It possesses immunomodulatory effects and is overexpressed in multiple types of cancer. In this study, we investigated the role of G9a in the induction of trained immunity, a de facto innate immune memory, and its effects in non-muscle-invasive bladder cancer (NMIBC) patients treated with intravesical Bacillus Calmette-Guérin (BCG).

Methods: EHMT2 expression was assessed upon induction of trained immunity by RNA sequencing and Western blotting. G9a inhibitor BIX-01294 was used to investigate the effect on trained immunity responses in vitro. Subsequent cytokine production was measured by ELISA, epigenetic modifications were measured by ChIP-qPCR, Seahorse technology was used to measure metabolic changes, and a luminescence assay was used to measure ROS release. RNA sequencing was performed on BIX-01294-treated monocytes ex vivo.

Results: The expression of EHMT2 mRNA and protein decreased in monocytes during induction of trained immunity. G9a inhibition by BIX-01294 induced trained immunity and amplified trained immunity responses evoked by various microbial ligands in vitro. This was accompanied by decreased H3K9me2 at the promoters of pro-inflammatory genes. G9a inhibition was also associated with amplified ex vivo trained immunity responses in circulating monocytes of NMIBC patients. Additionally, altered RNA expression of inflammatory genes in monocytes of NMIBC patients was observed upon ex vivo G9a inhibition. Furthermore, intravesical BCG therapy decreased H3K9me2 at the promoter of pro-inflammatory genes.

Conclusion: Inhibition of G9a is important in the induction of trained immunity, and G9a may represent a novel therapeutic target in NMIBC patients.

Keywords: BCG; EHMT2; G9a; inflammation; non‐muscle‐invasive bladder cancer; trained immunity.

Figure 1

Euchromatic Histone Lysine Methyltransferase 2 (EHMT2) expression is decreased upon induction of trained immunity. (a) Outline of in vitro training method: isolated monocytes were stimulated or left in culture medium alone (RPMI) for 24 h, rested for 5 days, and restimulated with non‐specific stimuli for 24 h. (b) Percoll‐isolated monocytes were stimulated for 24 h with 1 μg mL⁻¹ β‐glucan or 10 ng mL⁻¹ LPS. Cells were analysed by performing RNA sequencing at baseline (prior to stimulation), after 24 h (upon stimulation, n = 2 for LPS and n = 7 for RPMI and β‐glucan) and 6 days (before restimulation, n = 10 for RPMI and β‐glucan). EHMT2 mRNA expression is shown as average reads per kilobase million (RPKM; 2 independent experiments, mean ± SEM, n.s., Wilcoxon signed‐rank test). (c) Relative G9a expression by Western blot of day 6 β‐glucan (2 μg mL⁻¹)‐trained monocytes (n = 7, each line represents one individual) normalised for actin loading control (3 independent experiments, mean ± SEM, *P < 0.05, Wilcoxon signed‐rank test). (d) Healthy individuals received endotoxin, other healthy individuals were administered with BCG, and EHMT2 mRNA expression before, and respectively 4 h, and 1 month later, in monocytes was assessed. (e) EHMT2 mRNA expression in RPKM in monocytes by performing RNA‐seq before and 4 h after 2 ng kg⁻¹ endotoxin administration (n = 12, each line represents one individual, 1 experiment, **P < 0.01, Wilcoxon signed‐rank test). (f) EHMT2 mRNA expression in RPKM in monocytes by performing RNA‐seq, before and 1 month after BCG vaccination in healthy individuals (n = 6, 1 experiment, each line represents one individual, Wilcoxon signed‐rank test, n.s.).

Figure 2

G9a inhibition is accompanied by decreased H3K9me2 at gene promoters involved in trained immunity (a) qRT‐PCR Euchromatic Histone Lysine Methyltransferase 2 (EHMT2) mRNA expression of monocytes after 4h, 1 day, 6 days, upon 24‐h stimulation ± 1 µm BIX‐01294 adjusted for housekeeping gene. Fold change compared to t = 0 (n = 7, mean ± SEM, three independent experiments, Mann–Whitney U‐test, n.s.). (b) G9a protein expression by Western blot of day 6 monocytes incubated for 24 h ± 1 µm BIX‐01294, normalised for actin loading control (n = 7, each line represents one individual, three independent experiments, Wilcoxon signed‐rank test, n.s.). (c) ChIP‐qPCR H3K9me2 levels of day 6 2 μg mL⁻¹ β‐glucan‐trained monocytes ± 1 µm BIX‐01294, at promoters of TNFA, IL1B, IL6 and MTOR (n = 7 RPMI, n = 9 β‐glucan and β‐glucan/RPMI + BIX‐01294, three independent experiments, *P < 0.05, **P < 0.01, Wilcoxon signed‐rank test, mean ± SEM).

Figure 3

G9a inhibition by BIX‐01294 results in increased trained immunity responses in vitro.(a) Cytokine assessment in supernatant of monocytes incubated for 24 h with 2 μg mL⁻¹ β‐glucan, 5 μg mL⁻¹ BCG, 10 μg mL⁻¹ oxLDL, 1 ng mL⁻¹ LPS, and non‐stimulated (RPMI), ±1 µm BIX‐01294, and restimulated with 10 ng mL⁻¹ LPS for 24 h at day 6 (RPMI n = 27, β‐glucan n = 27, LPS n = 11, BCG n = 15, oxLDL n = 8, each line represents one individual, total of 10 independent experiments). (b) Basal extracellular acidification rate (ECAR) and oxidative consumption rate (OCR) of day 6 2 μg mL⁻¹ β‐glucan‐trained monocytes ± 1 µm BIX‐01294, measured using Seahorse (n = 7, three independent experiments). *P < 0.05, **P < 0.01, ***P < 0.001, Wilcoxon signed‐rank test, mean ± SEM.

Figure 4

G9a inhibition in BCG‐induced trained immunity increases, but is not dependent on, ROS production. (a) ROS luminescence production after Zymosan (1 mg mL⁻¹) stimulation of day 6 BCG‐trained monocytes ±1 µm BIX‐01294 (n = 7, mean ± SEM, three independent experiments, *P < 0.05, Wilcoxon signed‐rank test). (b) IL‐6 and TNF‐α fold changes of monocytes incubated for 24 h with 2 μg mL⁻¹ β‐glucan, 5 μg mL⁻¹ BCG, ± 1 µm BIX‐01294, ±1 mm NAC, which were restimulated after 6 days with 10 ng mL⁻¹ LPS (n = 9 for RPMI and BCG in three independent experiments, n = 6 for β‐glucan in two independent experiments, mean ± SEM, *P < 0.05, Wilcoxon signed‐rank test). (c) IL‐6 and TNF‐α fold changes of monocytes from chronic granulomatous disease (CGD) patients and healthy individuals incubated for 24 h with 2 μg mL⁻¹ β‐glucan ± 1 µm BIX‐01294 which were restimulated after 6 days with 10 ng mL⁻¹ LPS (n = 3 HC, n = 5 CGD, mean ± SEM, 1 experiment, each colour represents one individual, Mann–Whitney U‐test, n.s.).

Figure 5

G9a inhibition is associated with amplified trained immunity responses in NMIBC patients, and BCG immunotherapy results in decreased H3K9me2 marks. (a) Fold changes of cytokine production compared to baseline (RPMI without BIX‐01294) in supernatant of circulating monocytes from NMIBC patients incubated for 24 h with 2 μg mL⁻¹ β‐glucan, 5 μg mL⁻¹ BCG, and non‐stimulated (RPMI), ± 1 µm BIX‐01294, washed and restimulated with 10 ng mL⁻¹ LPS for 24 h at day 6 (n = 5, for each donor and visit an independent experiment). Each colour represents one patient. (b) ChIP‐qPCR H3K9me2 levels of isolated circulating monocytes from NMIBC patients after BCG treatment (i.e. approximately 6 weeks after the 6^th weekly BCG instillation) compared to baseline, at promoters of TNFA, IL1B, IL6 (n = 6 (one outlier was excluded, Grubb's test, P < 0.05), three independent experiments, Wilcoxon signed‐rank test). Each colour represents one patient.

Figure 6

G9a inhibition in bladder cancer patients results in differentially expressed inflammatory‐related genes. (a) CD14⁺ monocytes were isolated from 4 healthy individuals and 4 bladder cancer patients, and supplemented with 1 µm BIX‐01294 for 24 h, subsequently RNA was isolated for RNA sequencing (four independent experiments). (b, c) Heatmap of significantly upregulated (b) and downregulated (c) genes by RNA sequencing of monocytes from bladder cancer patients ± BIX‐01294 treatment for 24 h [n = 4 control (CTRL), n = 4 BIX‐01294 treatment (BIX)]. (d) Individual plots of differentially expressed genes TNFRSF11A, DNMT3A, IL18BP, IL1B, expressed in log₂ RPKM (n = 4 for each group, each colour represents one donor). BC, bladder cancer patient; HC, healthy control. Unadjusted P‐values derived from DEseq are displayed.