miR–9-5p regulates immunometabolic and epigenetic pathways in β-glucan–trained immunity via IDH3α

Trained immunity, induced by β-glucan in monocytes, is mediated by activating metabolic pathways that result in epigenetic rewiring of cellular functional programs; however, molecular mechanisms underlying these changes remain unclear. Here, we report a key immunometabolic and epigenetic pathway mediated by the miR–9-5p-isocitrate dehydrogenase 3α (IDH3α) axis in trained immunity. We found that β-glucan–trained miR–9-5p–/– monocytes showed decreased IL-1β, IL-6, and TNF-α production after LPS stimulation. Trained miR–9-5p–/– mice produced decreased levels of proinflammatory cytokines upon rechallenge in vivo and had worse protection against Candida albicans infection. miR–9-5p targeted IDH3α and reduced α-ketoglutarate (α-KG) levels to stabilize HIF-1α, which promoted glycolysis. Accumulating succinate and fumarate via miR–9-5p action integrated immunometabolic circuits to induce histone modifications by inhibiting KDM5 demethylases. β-Glucan–trained monocytes exhibited low IDH3α levels, and IDH3α overexpression blocked the induction of trained immunity by monocytes. Monocytes with IDH3α variants from autosomal recessive retinitis pigmentosa patients showed a trained immunity phenotype at immunometabolic and epigenetic levels. These findings suggest that miR–9-5p and IDH3α act as critical metabolic and epigenetic switches in trained immunity.


Introduction
Trained immunity, the nonspecific memory of the innate immune system, has been present by a wide variety of recent -but also older -studies in plants, invertebrates, and mammals (1,2).Innate immune cells were previously challenged in vitro with microbial components such as β-glucan of the Candida albicans (C.albicans) cell wall or the Bacillus Calmette-Guérin (BCG) vaccine and restimulated with the same or different microbial insult a week after training, resulting in increased capacity of the challenged cells to produce cytokines compared with nontrained cells (3,4).Additionally, when mice were trained in vivo with β-glucan, BCG, or a low dose of C. albicans, they showed lower mortality after lethal C. albicans reinfections (5).Trained monocytes have high glucose consumption, lactate production, and NAD + /NADH ratio, displaying a shift from oxidative phosphorylation to aerobic glycolysis (Warburg effect), depending on the activation of mTOR through the Dectin-1/Akt/HIF-1α pathway (6).Of note, other metabolites from the tricarboxylic acid (TCA) cycle also play key roles in monocyte remodeling.Fumarate promotes the stabilization of HIF-1α, which are responsible for mounting trained immunity; in contrast, α-KG leads to the hydroxylation of HIF-1α for degradation and impaired monocyte induction of trained immunity (7).Causal to the enhanced inflammatory response in trained immunity is the increased deposition of histone marks that are positively correlated with transcription at the promoters of key immune genes.The promoters of trained immune genes are enriched for H3K4me3 (8).Accumulation of fumarate contributes to trained immunity phenotype by inhibiting KDM5 histone demethylases (responsible for H3K4me3 demethylation) and, thus, influencing epigenetic reprogramming, whereas α-ketoglutarate (α-KG) counteracts this effect (7).However, molecular mechanisms underlying cellular metabolism reprogramming, with a shift from oxidative phosphorylation to aerobic glycolysis and the accumulation of metabolites from the TCA cycle, have not been fully deciphered, thereby impairing understanding of trained immunity.
Trained immunity, induced by β-glucan in monocytes, is mediated by activating metabolic pathways that result in epigenetic rewiring of cellular functional programs; however, molecular mechanisms underlying these changes remain unclear.Here, we report a key immunometabolic and epigenetic pathway mediated by the miR-9-5p-isocitrate dehydrogenase 3α (IDH3α) axis in trained immunity.We found that β-glucan-trained miR-9-5p -/-monocytes showed decreased IL-1β, IL-6, and TNF-α production after LPS stimulation.Trained miR-9-5p -/-mice produced decreased levels of proinflammatory cytokines upon rechallenge in vivo and had worse protection against Candida albicans infection.miR-9-5p targeted IDH3α and reduced α-ketoglutarate (α-KG) levels to stabilize HIF-1α, which promoted glycolysis.Accumulating succinate and fumarate via miR-9-5p action integrated immunometabolic circuits to induce histone modifications by inhibiting KDM5 demethylases.β-Glucan-trained monocytes exhibited low IDH3α levels, and IDH3α overexpression blocked the induction of trained immunity by monocytes.Monocytes with IDH3α variants from autosomal recessive retinitis pigmentosa patients showed a trained immunity phenotype at immunometabolic and epigenetic levels.These findings suggest that miR-9-5p and IDH3α act as critical metabolic and epigenetic switches in trained immunity.

R E S E A R C H A R T I C L E
JCI Insight 2021;6(9):e144260 https://doi.org/10.1172/jci.insight.144260 Isocitrate dehydrogenases (IDHs) are a group of enzymes that catalyze the oxidative decarboxylation of isocitrate (ICT) to α-KG.Three IDH paralogs, localized in different subcellular compartments, show different enzymatic characteristics.IDH1 and IDH2 are homodimeric nicotinamide adenine dinucleotide phosphate-dependent (NADP-dependent) enzymes that reversibly convert ICT to α-KG (9).Previously, IDH1 and IDH2 inhibition was shown to reduce α-KG and NADPH levels and inhibit the α-KG-dependent function of Jumonji-C domain-containing histone lysine residue demethylases, resulting in a global hypermethylation phenotype (10).IDH3 is a nicotinamide adenine dinucleotide-dependent (NAD-dependent) heterotetrameric enzyme that irreversibly catalyzes the conversion of ICT to α-KG in the TCA cycle.Diminished IDH3 activity reduced the ratio of α-KG to succinate and fumarate, which in return, promotes hypoxia-inducible factor 1α-dependent (HIF-1α-dependent) upregulation of glycolytic enzymes and dampens oxidative phosphorylation (10).Addressing whether and how IDH activity is regulated in monocytes trained by β-glucan and whether this regulation involves metabolic and epigenetic rewiring of trained immunity is fundamental.
miRNAs are short, single-stranded RNAs that regulate posttranscriptional mRNA expression by binding to complementary mRNA sequences, resulting in translational repression and gene silencing (11).These noncoding RNAs play critical roles in various physiological processes, such as cell differentiation, cellular rewiring, metabolism reprogramming, and epigenetic modification (12).However, our understanding of the role of miRNAs in modulating trained immunity is limited (13).Here, our aim was to unveil the early transcriptional and metabolic events following monocyte exposed to β-glucan and how the resulting epigenetic changes determine the function of trained monocytes.We showed that miR-9-5p was abundantly expressed in β-glucan-trained monocytes via the Dectin-1/Akt/mTOR/GSK3β pathway and that it contributed to trained immunity via IDH3α.

R E S E A R C H A R T I C L E
JCI Insight 2021;6(9):e144260 https://doi.org/10.1172/jci.insight.144260miR-9-5p triggers the switch to glycolysis in trained immunity.To determine the molecular mechanism that miR-9-5p uses to induce trained immunity, we performed an unbiased assessment of whole-genome mRNA expression patterns after training WT and miR-9-5p -/-monocytes with β-glucan.Monocytes were trained as described above, and after 5 days of rest (before restimulation) (Figure 2E), monocyte-derived-macrophages were harvested for RNA sequencing (RNA-seq) analysis (Supplemental Table 2).Heatmap analysis revealed distinct expression patterns of some mRNAs when β-glucan-trained miR-9-5p -/-monocytes were compared with control samples (Figure 4A).Pathway analysis of these differentially regulated genes in β-glucan-trained monocytes between WT and miR-9-5p -/-mice showed that several pathways were involved in trained immunity, such as Akt/mTOR signaling, HIF-1α signaling, and inflammatory cytokine signaling (Figure 4B and Supplemental Figure 5A).Furthermore, the decreased glycolysis induction and increased oxidative phosphorylation were observed in miR-9-5p -/-monocytes compared with that of naive cells (Figure 4C and Supplemental Figure 5A).Monocytes trained with β-glucan were shown to switch from oxidative metabolism to glycolysis in an Akt/mTOR pathway-dependent manner (6).Thus, we hypothesized that miR-9-5p was involved in aerobic glycolysis reprogramming during β-glucan-induced trained immunity.Regarding the molecular pathway, Akt and mTOR were phosphorylated in response to β-glucan training in WT monocytes, being consistent with previous results (Supplemental Figure 2, A and C) but comparable between WT and miR-9-5p -/-monocytes (Figure 4D and Supplemental Figure 5B).In an analysis of glycolysis key proteins, hexokinase 2 (HK2) and glucose transporter-1 (GLUT1) were decreased upon β-glucan treatment in miR-9-5p -/-monocytes (Figure 4D), consistent with the qPCR results, in which HK2 and GLUT1 displayed similar reduction (Supplemental Figure 5C).Additionally, glucose consumption, lactate production, and the NAD + /NADH ratio were significantly reduced in miR-9-5p -/-monocytes induced with β-glucan compared with that in WT cells (Figure 4, E and F).Furthermore, we examined the extracellular acidification rate (ECAR) by glycolysis stress test in β-glucantrained monocytes before LPS stimulation (Figure 4G).Training with β-glucan for 6 days increased ECAR in WT monocytes due to a metabolic shift; however, it decreased ECAR in miR-9-5p -/-monocytes (Figure 4G), as reflected by inhibited basal and maximal glycolysis, together with a lower glycolytic reserve (Figure 4H).In contrast, basal oxygen consumption after β-glucan training was increased in miR-9-5p -/-monocytes by approximately 50% compared with that in control monocytes (Supplemental Figure 5D).These data demonstrate that miR-9-5p switches the energy metabolism from oxidative phosphorylation to glycolysis in β-glucan-triggered trained immunity.
β-Glucan training of monocytes resulted in decreased biological activity of KDM5 demethylases, which corresponds to the time point with the increasing fumarate concentration (7).α-KG is a known cofactor of the KDM5, which can actively remove lysine trimethylation of H3K4me3 (20).KDM5 activity could be inhibited by fumarate but could be restored by the addition of α-KG (Supplemental Figure 6A).Due to the elevated ratio of α-KG/succinate and fumarate, we assessed whether miR-9-5p loss affected the KDM5 activity in β-glucan-trained monocytes.We found that KDM5 activity was significantly increased in β-glucan training of miR-9-5p -/-monocytes compared with that in WT cells (Figure 5K).Addition of fumarate to miR-9-5p -/-monocytes reduced the KDM5 activity to the level of WT cells (Supplemental Figure 6, B  and C).H3K4me3 modification at the promoters of proinflammatory cytokine and aerobic glycolysis genes was specifically induced by β-glucan training (7).Epigenetic changes in H3K4me3 at the promoter sites of TNFA, IL-6, HK2, and GLUT1 were abolished in β-glucan training of miR-9-5p -/-monocytes (Figure 5L).Fumarate addition restored the H3K4me3 levels at TNFA promoters in miR-9-5p -/-monocytes trained by
miR-9-5p regulates DNA hypomethylation.IDH3α loss of function affects DNA methylation (10).Having identified IDH3α as a target of miR-9-5p, we evaluated whether and to what extent differential methylation in human monocytes transfected with miR-9-5p and a scrambled control.RNA-seq analysis identified 3057 differentially expressed genes (Supplemental Figure 9A).The higher number of hypomethylated relative to hypermethylated CpGs revealed an overall global increase in DNA hypomethylation in miR-9-5p-overexpressing monocytes (Supplemental Figure 9B).Integrative analysis of gene expression and methylation data show that expression of 1189 genes (out of 3057) correlated with their CpG hypomethylation status (Supplemental Figure 9C).Pathway enrichment analysis of genes with correlation between gene expression and demethylation identified cellular metabolic pathway and inflammatory response as key pathways regulated through hypomethylation-driven expression changes upon miR-9-5p overexpression (Supplemental Figure 9D).Correlation for CpG sites was mainly located within island and shores, compared with shelf and open sea (Supplemental Figure 9E).The promoter regions of HK2 and TNFA showed hypomethylation, while IDH3A methylated status changed little (Supplemental Figure 9F).
Additionally, knocking down miR-9-5p partially resulted in DNA hypomethylation of the IDH3A promoter under training conditions (Supplemental Figure 9, G and H).Together, these results indicate that the miR-9-5p-controlled immunometabolic change in β-glucan-trained monocytes may occur through hypomethylation of trained immune genes.
IDH3α impairs the metabolic and epigenetic changes in β-glucan-trained immunity.To examine whether IDH3α affected the induction of trained immunity via β-glucan, we generated IDH3α-overexpressing monocytes (Figure 7A).We found that IDH3α gain of function inhibited IL-1β production, glucose consumption, and lactate production in β-glucan training of monocytes (Figure 7B).Downregulation of oxygen consumption upon exposure to β-glucan was partially restored in IDH3α-overexpressing monocytes (Figure 7, C and  D).The α-KG level was increased approximately 2-fold in IDH3α-overexpressing monocytes trained with β-glucan; in contrast, fumarate level was decreased (Figure 7E).KDM5 activity was also greatly increased in monocytes overexpressing IDH3α (Figure 7F), together with the decrease of H3K4me3 level at TNFA promoter (Figure 7G).Of note, β-glucan-trained monocytes with an IDH3α mutation showed increased production of IL-1β, lactate, and fumarate compared with that in IDH3α-overexpressing cells (Figure 7, H and I).Moreover, IDH3α-mutant monocytes showed the decreased KDM5 activity and the increased H3K4me3 enrichment at the TNFA promoter (Figure 7, J and K).Thus, IDH3α dampened the energy metabolism and epigenetic changes in monocytes trained with β-glucan.Monocytes with an IDH3α mutation (p.Ala-175-Val) from patients with autosomal recessive retinitis pigmentosa (ArRP) show a trained immunity phenotype.IDH3α variants were identified as crucial causes of typical ArRP (22).Without stimulation, ArRP patients suffer sustained inflammation attacks, characterized by increased macrophage activity and increased chemokine and cytokine levels (23).However, the molecular mechanism of this increased inflammatory state is still unclear.We hypothesized that this is due to a trainedlike phenotype of ArRP monocyte-derived macrophages with IDH3α mutants, which likely induces the critical glycolytic switch and the epigenetic change.We analyzed monocytes from 3 adult patients with ArRP that had an IDH3α mutation (p.Ala-175-Val).In line with our hypothesis, monocytes from these patients produced significantly more TNF-α, IL-6, and IL-1β upon stimulation with several ligands compared with healthy controls (Figure 8A).RNA-seq analysis revealed that IDH3α (p.Ala-175-Val) variant monocytes exposed to media or LPS for 24 hours, or left unstimulated (Supplemental Table 3), showed differential expression of multiple genes (Figure 8B).Pathway analysis of the these expression data is displayed in Figure 8C and shows upregulated inflammatory pathways, such as NF-κB signaling, supporting the hypothesis of a hyperinflammatory state.Although the expression profiles of Akt-mTOR pathway showed little change (Supplemental Figure 10A), we observed increased expression of glycolysis and cytokine genes in ArRP patient monocytes after 24 hours of media treatment, as well as decreased oxidative phosphorylation pathway activity (Figure 8B and Supplemental Figure 10A).Glucose consumption and lactate production were increased in monocytes with an IDH3α mutation (p.Ala-175-Val), compared with that in healthy donor cells, whereas the ratio of α-KG to fumarate was decreased (Supplemental Figure 10,  B and C).Moreover, KDM5 activity was decreased in IDH3α-mutated monocytes (Supplemental Figure 10D).H3K4me3 levels at TNFA, IL-6, HK2, and GLUT1 promoters were higher in ArRP monocytes with an IDH3α mutation than that in normal cells (Figure 8D).These results demonstrate that IDH3α loss of function may be one of the main contributors to the inflammatory signatures in ArRP.

Discussion
Trained immunity describes the ability of innate immune cells to form immunological memories of prior encounters with pathogens.Recollection of these memories during a secondary encounter manifests a broadly enhanced inflammatory response characterized by the increased transcription of innate immune genes (3).Despite this phenomenon having been described over a decade ago, our understanding of the molecular mechanisms responsible for this phenotype is still incomplete.Herein, β-glucan-trained miR-9-5p -/-monocytes showed decreased IL-1β, IL-6, and TNF-α production after LPS stimulation.Trained miR-9-5p -/-mice produced decreased proinflammatory cytokines upon rechallenge in vivo and had worse protection against C. albicans infection.We highlight the mechanistic role of miR-9-5p in the establishment and maintenance of discrete, long-lasting epigenetic modifications that are causal to the trained immune response.The discovery of the central role of miR-9-5p function in the establishment of trained immunity has revealed a class of therapeutic targets for potentially controlling inflammatory responses in a discrete manner.Furthermore, as the catalog of functionally investigated microRNAs transcripts grow, we foresee that more microRNAs that are important for the regulation of trained immunity and inflammation will be revealed.
Trained immunity undergoes an oxygen-independent metabolic switch from oxidative phosphorylation to aerobic glycolysis (24).However, it is not clear what regulates this metabolic reprogramming.Previously, the Akt/mTOR pathway was shown to participate in the immunometabolic changes in trained immunity (6).In this context, we found that pharmacological inhibition or downregulation of the Akt/mTOR pathway reduced miR-9-5p expression.To further elucidate the mechanisms through which Akt/mTOR regulates miR-9-5p level, we turned to GSK3β, which binds directly to the microprocessor complex and facilitates microRNA biogenesis (25).Additionally, Akt/mTOR inhibition reduced GSK3β protein levels, and the increase in microRNA biogenesis associated with Akt/mTOR activation was reversed by GSK3β inhibition (14), suggesting that Akt/mTOR regulates microRNA biogenesis via GSK3β.In this context, we found that total GSK3β level was increased in monocytes trained with β-glucan.Also loss of function of Akt/mTOR led to reduction of GSK3β.GSK3β inhibition also decreased miR-9-5p level in β-glucan-trained monocytes.These results suggest that β-glucan-trained immunity could induce miR-9-5p expression via activation of the Akt/mTOR/GSK3β pathway.miR-9-5p -/-monocytes trained with β-glucan did not alter the phosphorylation of Akt and mTOR, and they supported miR-9-5p acting potentially downstream to it.Upon β-glucan training, HIF-1α plays an important role in mounting glycolysis via Akt/mTOR activation (26).Expression of HIF-1α at protein levels was significantly reduced in β-glucan-trained miR-9-5p -/-monocytes compared with WT cells.Actually, miR-9-5p deficiency promoted the PHD2-mediated hydroxylation of HIF-1α, indicating that miR-9-5p stabilized HIF-1α in trained immunity.We also found that, in miR-9-5p -/-monocytes, lactate production and glucose consumption were decreased, whereas oxygen consumption was increased, demonstrating that induction of miR-9-5p directly promoted the switch of glucose metabolism from oxidative phosphorylation to glycolysis.Our results identify miR-9-5p as a critical molecular switch promoting aerobic glycolysis.
Genome-wide changes in histone modifications have been shown to underlie trained immunity in monocytes (27).Trained immunity induces the accumulation of key metabolites in the TCA cycle, such as fumarate and succinate, and decreased the level of α-KG (28,29).The metabolites act as cofactors for epigenetic enzymes, resulting in the induction of histone modifications, such as H3K4me3, by regulating KDM5 activity (8).However, the molecular mechanisms underlying the metabolites accumulation in  B, D, E, F, and G); *P < 0.05 by 1-way ANOVA/Tukey's multiple comparisons test (I, J, and K).Unprocessed original scans of blots are shown in Supplemental Figure 11.trained immunity have not been deciphered.Here, miR-9-5p deficiency increased the level of effective α-KG by promoting the ratio of α-KG to fumarate and succinate.α-KG acts as a cofactor to enhance KDM5 activity for H3K4 demethylation, whereas fumarate, having a similar molecular structure to α-KG, can act as an antagonizing factor (7).We found that KDM5 activity was increased in β-glucan-trained miR-9-5p -/-monocytes, and the trimethylation of H3K4 was reduced at the promoters of TNFA, IL-6, HK2, and GLUT1 due to the increase in the amount of α-KG.This indicated that the metabolic and epigenetic changes were intertwined and highly dependent on miR-9-5p expression in trained immunity.Diminished IDH3 activity results in the metabolic switch from oxidative phosphorylation to glycolysis (30).IDH3α inactivation reduced the ratio of α-KG to fumarate and succinate (10).In this context, IDH3α inhibited the metabolic switch from oxidative phosphorylation to glycolysis and increased the α-KG/fumarate ratio, impairing epigenetic changes in trained monocytes by increasing KDM5 activity.IDH3α gain of function reduced the enrichment of H3K4me3 at the promoters of TNFA in monocytes upon β-glucan training.Actually, miR-9-5p directly targeted IDH3α in the processes of trained immunity.Recently, IDH3α was found to interact with cytosolic serine hydroxymethyltransferase (cSHMT) to enhance S-adenosyl methionine (SAM) generation (10), which involves depositing the repressive H3K9me3 mark in trained immunity (31).Gain or loss of IDH3α function regulates the expression of the methyl group donor SAM, which should be explored in β-glucan-trained immunity.It is likely that IDH3α downregulation led to the immunometabolic programs, contributing to the induction of β-glucan-trained immunity.Thus, our study revealed that the miR-9-5p/IDH3α axis is one of the main contributors to the immunometabolic and epigenetic remodeling in trained immunity.
ArRP is an inherited form of retinal dystrophy caused by inherited or acquired mutations in over 50 different genes, including IDH3A (22,32).Loss-of-function and missense mutations in IDH3α are implicated in families exhibiting ArRP (22).In this disease, there is increased macrophage activity and levels of chemokines and cytokines (IL-1β) in patients and rodent models (23,33).However, it is unclear whether this increased inflammatory state is the cause or a consequence of this currently untreatable disease.We found that an IDH3α mutation led to the increased production of proinflammatory cytokines and accumulation of TCA cycle metabolites in β-glucan-induced trained immunity.In addition to the beneficial effects of trained immunity as a host defense mechanism, trained immunity plays a deleterious role in the induction and/or persistence of inflammatory diseases if inappropriately activated (34,35).Thus, we hypothesized that induction of the trained immunity phenotype, characterized by exaggerated cytokine production from monocytes due to an IDH3α mutation, may play a role in the pathophysiology of ArRP.This hypothesis was validated via the demonstration of excessive cytokine production in ArRP monocytes with an IDH3α mutation (p.Ala-175-Val), accompanied with transcriptional and epigenetic changes.In previous studies, NLRP3 expression was significantly upregulated in the early-onset model up to 7-16 weeks, and IL-1β expression was still upregulated in the late-onset model from 16 weeks (33,36), suggesting that, in addition to the mechanism dependent on NLRP3 inflammasome activation, an IDH3α mutation may play a causative role in the development of the hyperinflammatory state observed in ArRP.However, the study was limited by the small sample size, and more samples from different populations are needed to confirm this hypothesis.Because the loss of function of IDH3A in mice is lethal, further studies are required to investigate the role of IDH3α in trained immunity via the generation of IDH3A homozygous mutation in mice to reveal the link between the inflammatory state induced by IDH3α and ArRP.We have provided an insight into understanding the hyperinflammatory state of patients with ArRP regarding trained immunity.
In conclusion, miR-9-5p promoted the rewiring of cellular metabolism that modulates the epigenetic programming of metabolic genes in β-glucan training of monocyte.As a direct target of miR-9-5p, IDH3α impaired the key metabolite changes (a switch from oxidative phosphorylation to aerobic glycolysis and the accumulation of fumarate and succinate).Moreover, monocytes from patients with ArRP, having an IDH3α mutation, may acquire a trained immunity phenotype, characterized by the increased expression of cytokines and genes involved in the glycolysis pathway.We have identified the miR-9-5p/IDH3α axis as a crucial mechanism linking the stimulation of innate immune pathways with the induction of epigenetic and metabolic changes in trained immunity.Thus, therapeutic strategies targeting the miR-9-5p/IDH3α axis may represent a promising approach for prevention of inflammation.
Buffy coats from healthy volunteers and ArRP patients obtained from the the Sixth Affiliated Hospital of Guangzhou Medical University were ethically approved by the Institutional Human Ethics Committee.
Trained immunity in vitro models.Blood mononuclear cells were isolated from WT and miR-9-5p -/-mice using Ficoll-Paque (GE Healthcare) as previously described (7).Briefly, 1 × 10 7 to 2 × 10 7 PBMCs were layered on top of a hyperosmotic Percoll solution (48.5% Percoll [Sigma-Aldrich], 41.5% sterile H 2 O, and 0.16 M filter-sterilized NaCl) and centrifuged for 15 minutes at 600g at room temperature.The interphase layer was isolated, and cells were washed with cold PBS.Human monocytes were purified by MACS

Figure 8 .
Figure 8. Monocytes with an IDH3α mutation (p.Ala-175-Val) from patients with autosomal recessive retinitis pigmentosa (ArRP) show a trained immunity phenotype.(A) Monocytes from patients with autosomal recessive retinitis pigmentosa (ArRP), who had the IDH3α (p.Ala-175-Val) mutation, and matching healthy volunteers were exposed ex vivo to several ligands for 24 hours.Cytokine expression levels were measured in the supernatants (n = 5 independent experiments).(B) Heatmap of individual donors for genes that show LPS responses in controls and patients with ArRP who had IDH3α (p.Ala-175-Val) mutation.Monocytes were either untreated or stimulated for 24 hours with media or LPS.RNA-seq was performed once in triplicate (n = 3 versus 3).(C) Pathway associated with genes that show higher expression in ArRP patients monocytes exposed to 24-hour RPMI compared with controls (n = 3 versus 3).(D) H3K4me3 levels were determined at the promoter sites of TNFA, IL-6, HK2, and GLUT1 in ArRP patient monocytes exposed to 24-hour RPMI compared with controls (n = 4 independent experiments).Data are shown as means ± SEM, *P < 0.05, **P < 0.01, or ***P < 0.001 by 2-tailed Student's t test (A and D).