α-Ketoglutarate-Dependent KDM6 Histone Demethylases and Interferon-Stimulated Gene Expression in Lupus

Abstract

Objective: We aimed to investigate the hypothesis that interferon (IFN)-stimulated gene (ISG) expression in systemic lupus erythematosus (SLE) monocytes is linked to changes in metabolic reprogramming and epigenetic regulation of ISG expression.

Methods: Monocytes from healthy volunteers and patients with SLE at baseline or following IFNα treatment were analyzed by extracellular flux analysis, proteomics, metabolomics, chromatin immunoprecipitation, and gene expression. The histone demethylases KDM6A/B were inhibited using glycogen synthase kinase J4 (GSK-J4). GSK-J4 was tested in pristane and resiquimod (R848) models of IFN-driven SLE.

Results: SLE monocytes had enhanced rates of glycolysis and oxidative phosphorylation compared to healthy control monocytes, as well as increased levels of isocitrate dehydrogenase and its product, α-ketoglutarate (α-KG). Because α-KG is a required cofactor for histone demethylases KDM6A and KDM6B, we hypothesized that IFNα may be driving "trained immune" responses through altering histone methylation. IFNα priming (day 1) resulted in a sustained increase in the expression of ISGs in primed cells (day 5) and enhanced expression on restimulation with IFNα. Importantly, decreased H3K27 trimethylation was observed at the promoters of ISGs following IFNα priming. Finally, GSK-J4 (KDM6A/B inhibitor) resulted in decreased ISG expression in SLE patient monocytes, as well as reduced autoantibody production, ISG expression, and kidney pathology in R848-treated BALB/c mice.

Conclusion: Our study suggests long-term IFNα exposure alters the epigenetic regulation of ISG expression in SLE monocytes via changes in immunometabolism, a mechanism reflecting trained immunity to type I IFN. Importantly, it opens the possibility that targeting histone-modifying enzymes, such as KDM6A/B, may reduce IFN responses in SLE.

Figure 1.. SLE Monocytes show enhanced metabolic activity which can be mimicked by IFNα stimulation in healthy control monocytes.

(A) Oxygen consumption rate (OCR) and (B) extracellular acidification rate (ECAR) was compared between SLE and healthy control (HC) monocytes under basal conditions. Data in A&B is representative of at least 10 individual experiments and is presented as mean±S.D. (C) OCR and ECAR at baseline were compared between paired SLE patients with either high or low baseline levels of IFI27. Data is representative of at least 3 individual experiments and is presented as mean±S.D. (D&E) OCR and ECAR measurements were made in healthy control monocytes treated with IFNα (1000 U/ml) for 24 hours (n=11). Data is representative of individual experiments and is presented as mean±S.D. Data in the right-hand panels represents baseline values of paired samples stimulated with IFNα 24 hours (n=11). Statistical significance was determined using student’s (A-C) unpaired and (D) paired t-test *p<0.05, **p <0.01, ***p <0.001, ****p<0.0001; ns = not significant.

Figure 2.. SLE patients have altered expression of isocitrate dehydrogenase 2 (IDH2) which is driven by IFNα.

(A, B) Targeted proteomic analysis demonstrates significant increase in expression of IDH2 in SLE monocytes (SLE; n=5) compared to monocytes from healthy controls (HC; n=5 (C) Metabolomic analysis demonstrates increased levels of intracellular α-KG in SLE monocytes (n=5) compared to HC monocytes (n=5); (D) Comparative proteomic analysis of IDH2 levels in monocytes from SLE patients with moderate to active disease (AP) (n=7), inactive disease (IAP) (n=5) or healthy controls (n=5); ((E-F) Overnight IFNα treatment of THP1 monocytic cells increased expression of IDH2 mRNA (E) and protein (F) as measured by qPCR and western blotting when compared to non-treated (NT) cells, respectively. Data is representative of >3 individual experiments.; (A, B, C, E, F) Data is presented as mean±S.D. and statistical significance was determined by student’s unpaired t-test and (D) one-way ANOVA. *p<0.05, **p <0.01, ***p <0.001, ****p<0.0001; ns = not significant.

Figure 3.. α-KG and IFNα pretreatment results in enhanced IFN-driven gene expression.

(A) Healthy control monocytes were untreated or pretreated with (A) 2μM cell permeable dimethyl-αKG followed by stimulation overnight with IFNα, as indicated. IFI27 expression was determined by qPCR; (B) THP1 cells were untreated or pretreated with 10μM AGI-6780 followed by stimulation overnight with IFNα, as indicated. IFI27 and ISG15 expression was determined by qPCR; (C) Trained immunity schematic whereby (D) healthy control monocytes are pulsed with PBS or IFNα (1000U/ml) for 24 hours, washed, restimulated with PBS or IFNα (1000U/ml) on day 5 for 24 hours (as indicated) and IFI27 expression determined by qPCR. Data in A, B & D is representative at least n=3 individual experiments and is presented as mean±S.D. Statistical significance was determined using One-Way ANOVA and Tukey’s multiple comparison test. **p <0.01, ***p <0.001, ****p<0.0001, ns = not significant; (E) Gene set enrichment analysis of RNAseq data from THP1 cells pulsed with IFNα for 24hours, washed and then rested for 5 days demonstrate that genes associated with the IFNα response are most highly enriched compared to non-treated control cells (n=2 for each group).

Figure 4.. Epigenetic regulation of ISG expression is altered by IFNα pretreatment and ISG expression can be reversed by inhibiting KDM6A/B in both SLE patient.

(A, B) THP1 cells trained with IFNα (1000U/ml) showed significant (A) decreased H3K27me3 marks and (B) increased H3K4me3 marks at the IFI27 promoter compared to naive THP1 cells; Data shown is from 3 independent experiments and is presented as mean±S.E.M. Statistical significance was determined using One-Way ANOVA and Tukey’s multiple comparison test, ****p<0.0001, ***p<0.001, *p<0.05, ns=not significant. (C) CD14+ Monocytes from healthy control were treated with α-KG (2μM) for 24 hours as indicated, H3K27me3 marks were quantified by Geometric Mean of H3K27me3-Alexa Fluor 488 positive area and are represented as mean±S.D., n=2. Statistical significance was determined using a one-tailed paired Student’s t test *p<0.05. (D-F) Level of H3K27me3 were analyzed in healthy controls (HC, n=4) and SLE (n=6) PBMCs by flow cytometry. (C) UMAP plot showing distribution of monocyte populations and (D) H3K27me3 levels in HC (top panel) and SLE cells (bottom panel). (E) Relative H3K27me3 expression in CD14⁺ positive monocyte population in HC and SLE (third panel).

Figure 5.. ISG expression can be reversed by inhibiting KDM6A/B in both healthy control and SLE monocytes and in pristane-treated C57Bl/6 mice.

(A, B) Monocytes from (A) healthy controls and (B) SLE patients were pretreated with GSK-J4 (25μM) as indicated, followed by overnight treatment with PBS or IFNα (1000U/ml). ISG expression was measured by qPCR. Data is representative of individual experiments (n=5) and is presented as mean±S.D; (C) C57Bl/6 mice (n=5 per group) were treated with pristane (i.p) on day 0, followed by GSK-J4 (10μg/kg) or vehicle control on day 6. Mice were sacrificed on Day 7. qPCR was used to assess ISG expression in monocytes isolated from the peritoneal cavity of pristane-treated mice, treated with or without GSK-J4 (10μg/kg). Data is presented as mean±S.D; Statistical significance was determined using student’s t-test, *p<0.05, ns= not significant. (A-B) Statistical significance was determined using One-Way ANOVA and Tukey’s multiple comparison test or (C) student’s t-test, ****p<0.0001, ***p<0.001, *p<0.05, ns= not significant.

Figure 6.. Inhibition of resiquimod-induced disease progression in Balb/c mice by GSK-J4.

(A) Schematic representation of the 35-day resiquimod/GSK treatment protocol performed on wild-type Balb/c mice (n=10 per group). (B) Measurement of mouse serum creatinine levels in vehicle control, resiquimod, and GSK+resiquimod treated mice. (C) Detection of anti-double stranded DNA (total IgG) through optical density (O.D.) measurement at 450nm in mouse serum from vehicle control, resiquimod, and GSK+resiquimod. (D) Quantification of Ifi27 expression in mouse kidney using qPCR analysis. (E) Immunohistochemical staining for glomerular immune complex and C3 deposits in renal sections from vehicle control, resiquimod, and GSK+resiquimod treated mice groups. PAS, IgG, and C3 staining results for renal sections. Images were taken at 40× (PAS) and 10× (IgG & C3) magnifications. (F) Glomerular size was quantified on at least 40 glomeruli from 3 different mice group. (G) Glomerular cellularity was determined by counting total nuclear cells in each glomerulus using light microscopy. In panels B-D and F-G, data is presented as mean±S.D. Statistical analysis was performed using One-Way ANOVA and Tukey’s multiple comparison test. ****p<0.0001, ***p<0.001, *p<0.05, ns= not significant.