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

Article The Set7 Lysine Methyltransferase Regulates Plasticity in Oxidative Phosphorylation Necessary for Trained Immunity Induced by b-Glucan Graphical Abstract Highlights dSet7 regulates enhanced cytokine production in trained immunity in vitro dSet7 knockout mice are unable to mount trained immunity against endotoxin challenge dSet7 modulates cellular respiration in b-glucan-trained macrophages dSet7-dependent histone methylation regulates MDH2 and SDHB in trained cells Authors Samuel T. Keating, Laszlo Groh, Charlotte D.C.C. van der Heijden, ..., Leo A.B. Joosten, Mihai G. Netea, Niels P. Riksen Correspondence [email protected] In Brief Using a combination of pharmacological and genetic approaches, Keating et al. show that the Set7 methyltransferase is a regulator of trained immunity induced by b-glucan. Activation of Set7 increases oxidative phosphorylation in trained cells via histone lysine methylation at gene enhancers of key enzymes of the TCA cycle. Keating et al., 2020, Cell Reports 31, 107548 April 21, 2020 ª2020 The Author(s). https://doi.org/10.1016/j.celrep.2020.107548 Cell Reports Article The Set7 Lysine Methyltransferase Regulates Plasticity in Oxidative Phosphorylation Necessary for Trained Immunity Induced by b-Glucan Samuel T. Keating, 1 Laszlo Groh, 1 Charlotte D.C.C. van der Heijden, 1 Hanah Rodriguez, 2 Je ´ssica C. dos Santos, 1 Stephanie Fanucchi, 3,4 Jun Okabe, 2 Harikrishnan Kaipananickal, 2,5 Jelmer H. van Puffelen, 1,6 Leonie Helder, 1 Marlies P. Noz, 1 Vasiliki Matzaraki, 1 Yang Li, 1,7 L. Charlotte J. de Bree, 1,8,9 Valerie A.C.M. Koeken, 1 Simone J.C.F.M. Moorlag, 1 Vera P. Mourits, 1 Jorge Domı ´nguez-Andre ´s, 1 Marije Oosting, 1 Elianne P. Bulthuis, 10 Werner J.H. Koopman, 10 Musa Mhlanga, 3 Assam El-Osta, 2,5,11 Leo A.B. Joosten, 1,12 Mihai G. Netea, 1,13 and Niels P. Riksen 1,14, * 1 Department of Internal Medicine and Radboud Institute of Molecular Life Sciences (RIMLS), Radboud University Medical Center, Nijmegen, the Netherlands 2 Epigenetics in Human Health and Disease, Department of Diabetes, Monash University, Melbourne, VIC, Australia 3 Division of Chemical, Systems and Synthetic Biology, Department of Integrative Biomedical Sciences, Faculty of Health Sciences, Institute of Infectious Disease and Molecular Medicine, University of Cape Town, Cape Town, South Africa 4 Gene Expression and Biophysics Group, CSIR Biosciences, Pretoria, South Africa 5 Department of Clinical Pathology, The University of Melbourne, Melbourne, VIC, Australia 6 Department for Health Evidence, Radboud University Medical Center, Nijmegen, the Netherlands 7 Department of Computational Biology for Individualised Infection Medicine, Centre for Individualised Infection Medicine, Helmholtz Centre for Infection Research, Hannover Medical School, 30625 Hannover, Germany 8 Research Center for Vitamins and Vaccines, Bandim Health Project, Statens Serum Institut, Copenhagen, Denmark 9 Odense Patient Data Explorative Network, University of Southern Denmark/Odense University Hospital, Odense, Denmark 10 Department of Biochemistry, Radboud Institute of Molecular Life Sciences (RIMLS), Radboud University Medical Center, Nijmegen, the Netherlands 11 Prince of Wales Hospital, The Chinese University of Hong Kong, Hong Kong City, Hong Kong SAR 12 Department of Medical Genetics, Iuliu Hatieganu University of Medicine and Pharmacy, Cluj-Napoca, Romania 13 Department for Genomics and Immunoregulation, Life and Medical Sciences Institute (LIMES), University of Bonn, Bonn, Germany 14 Lead Contact *Correspondence: [email protected] https://doi.org/10.1016/j.celrep.2020.107548 SUMMARY 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 b-glucan. Using pharmacological studies of human primary monocytes, we identify trained immunity-specific immunometabolic pathways regulated by Set7, including a previously unreported H3K4me1dependent plasticity in the induction of oxidative phosphorylation. Recapitulation of b-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. INTRODUCTION A series of recent discoveries has uncovered how cells of the innate immune system such as monocytes and macrophages undergo functional reprogramming to mount a de facto immune memory of an infectious or inflammatory injury by a process called trained immunity, which facilitates augmented responses to subsequent pathogenic encounters (Netea et al., 2020). In the context of infections or vaccination, trained immunity provides beneficial heterologous effects by the enhanced cytokine response to stimulation with non-related pathogens. Prototypical stimuli of trained immunity include the fungal cell wall component b-glucan (Quintin et al., 2012) and the bacillus CalmetteGue ´rin (BCG) vaccine (Kleinnijenhuis et al., 2012). Recent attention has also turned to endogenous drivers of inflammation as inducers of trained immunity (Bekkering et al., 2014; van der Valk et al., 2016). These stimuli shape innate immunological memories by reprogramming metabolic and transcriptional profiles (Arts et al., 2016a; Cheng et al., 2014). Posttranslational methylation of proteins conveys information to cellular pathways, including those that regulate gene Cell Reports 31, 107548, April 21, 2020 ª2020 The Author(s). 1 This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/). Figure 1. Set7 Is Associated with Trained Immunity Induced by b-Glucan (A) Graphical outline of in vitro training methods. Adherent monocytes (Mo) were stimulated with 1 mg/mL b-glucan or standard culture medium (RPMI) for 24 h (first stimulus), allowed to differentiate to macrophages (M4) for 5 days, and restimulated for 24 h with LPS or RPMI on day 6. (legend continued on next page) 2Cell Reports 31, 107548, April 21, 2020 expression. In vitro experiments with pan-methyltransferase inhibitors revealed the pivotal importance of this chemical modification for trained macrophages (Cheng et al., 2014; Quintin et al., 2012). Changes in histone lysine methyl modifications (H3 histones monomethylated [H3K4me1] or trimethylated [H3K4me3] at lysine 4) underlie b-glucan-induced trained immunity (Novakovic et al., 2016). Signaling factors derived from local tissue environments play key roles in determining macrophage fate, and evidence points to the role of enhancer elements in shaping specialized macrophage populations (Denisenko et al., 2017; Gosselin et al., 2014). H3K4me1 is a chromatin signature of enhancers (Heintzman et al., 2007). This modification was shown to persist at decommissioned distal elements, indicating that H3K4me1 provides a mechanism for epigenetic memory in trained immunity in macrophages (Saeed et al., 2014). Despite this, the mechanisms linking immunological signals induced by microbial stimuli or vaccines to chromatin-dependent changes in trained immunity are unclear. Moreover, the identities of the chromatin-modifying enzymes critical to these processes remain obscure. One enzyme that writes the H3K4me1 modification to transcriptionally activating or poised genomic regions is the Set7 lysine methyltransferase (Wang et al., 2001)(also called Set9 [Nishioka et al., 2002], Set7/9 [Tamura et al., 2018], or KMT7 [Allis et al., 2007], and encoded by SETD7). Set7 writes a persistent H3K4me1 signature pertaining to vascular endothelial inflammatory signaling (Brasacchio et al., 2009). The importance of Set7 for mediating H3K4me1 signatures at enhancers associated with endothelial gene expression was demonstrated using an unbiased epigenome-wide approach (Keating et al., 2014). Although Set7 has not been studied in the specific context of trained immunity, our previous analysis of macrophages trained with b-glucan identified elevated levels of SETD7 expression (Quintin et al., 2012). The current study explored the role of Set7 in b-glucaninduced trained immunity. Using genetic and pharmacological approaches, we demonstrate that Set7 is critical for the induction of trained immunity in vitro and in vivo. We identify a role for Set7 in immunometabolic pathways, including the induction of oxidative phosphorylation (OXPHOS). Characterization of epigenetic networks is key to a deeper understanding of regulatory mechanisms supporting trained immunity, and could identify strategies to modulate pro-inflammatory circuits of the innate immune system. RESULTS Set7 Expression and Activity Are Increased in Human Primary Monocytes and Macrophages Stimulated with b-Glucan To investigate the role of Set7, we adopted a previously described in vitro model of trained immunity using the fungal cell wall component b-glucan (Cheng et al., 2014). Adherent human primary monocytes were incubated with culture medium or b-glucan (1 mg/mL) for 24 h. Cells were washed and incubated in normal culture conditions for a further 5 days, during which time they differentiated into macrophages. On day 6, the cells were restimulated with the Toll-like receptor 4 ligand lipopolysaccharide (LPS) (10 ng/mL) for 24 h and pro-inflammatory cytokine production was measured (Figure 1A). Tumor necrosis factor alpha (TNFa) and IL-6 were measured as functional readouts of trained immunity (Arts et al., 2016a; Cheng et al., 2014). Cells stimulated with b-glucan exhibited enhanced TNFaand IL-6 production following LPS restimulation (Figure 1B). We validated our previous transcriptome data showing that SETD7 mRNA expression was significantly increased on day 6 of the in vitro training protocol in macrophages trained with b-glucan. Currently, there is no gold standard test for Set7 activity in primary cells. However, a recent study identified the ribosomal protein Rpl29, a component of the 60S ribosomal subunit, as a non-histone methylation substrate of Set7 (Hamidi et al., 2018), and dimethylated Rpl29 (Rpl29k5me2) was shown to serve as a reliable biomarker for Set7 activity in cancer cells. We observed that Rpl29k5me2 levels correlated with Set7 protein expression in macrophages trained with b-glucan (Figure 1C). To further investigate the association of Set7 to trained immunity, we conducted a genetic study of peripheral blood mononuclear cells (PBMCs) isolated from 267 healthy volunteers of Western European ancestry. Adherent PBMCs from all volunteers were incubated with trained with culture medium or b-glucan (1 mg/mL) for 24 h as described above. We tested for associations among common single-nucleotide polymorphisms (SNPs) and variation in the magnitude of b-glucan-trained cytokine responses of individual subjects and identified two SNPs suggestively associated (p < 9.99 310 3 ) with adaptive changes in cytokine production mapped within SETD7. Intronic variants rs7680948 (located in intron 4 of SETD7 [p < 0.004]) and rs56183115 (located in intron 3 of SETD7 [p = 0.004]) were associated with the potentiation of TNFaand IL-6 production, respectively, upon induction of trained immunity by b-glucan (Figure 1D). To investigate Set7 in trained immunity induced by (B) Production of pro-inflammatory cytokines TNFaand 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; b-actin was used as loading control. (D) Single-nucleotide polymorphisms (SNPs) within SETD7 suggestively associated with trained responses to b-glucan in peripheral blood mononuclear cells (n = 267). Ageand sex-corrected TNFaand 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-Gue ´rin vaccine in peripheral blood mononuclear cells. Ageand sex-corrected TNFaand 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. Ageand sexcorrected TNFaand 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. Cell Reports 31, 107548, April 21, 2020 3 (legend on next page) 4Cell Reports 31, 107548, April 21, 2020 other stimuli, we tested for associations among SNPs near SETD7 and variation in the magnitude of cytokine responses of individuals trained with BCG and oxidized low-density lipoprotein (oxLDL). We identified numerous SNPs suggestively associated (p < 9.99 310 3 ) with adaptive changes in pro-inflammatory cytokine production mapped within 250 kb of SETD7. Variants rs795971 (located approximately 250 kb upstream of SETD7 [p = 0.007]) and rs6816973 (located approximately 160 kb upstream of SETD7 [p < 0.003]) were associated with the potentiation of TNFaand IL-6 production, respectively, upon induction of trained immunity by BCG (Figure 1E). In addition, we identified that variants rs6536295 (located approximately 3 kb upstream of SETD7 [p < 0.007]) and rs10020166 (located approximately 15 kb upstream of SETD7 [p = 0.01]) were associated with the production of TNFaand IL-6, respectively, upon induction of trained immunity by oxLDL (Figure 1F). Pharmacological Inhibition of Set7 Attenuates b-Glucan-Induced Trained Immunity In Vitro Recent studies demonstrated specific inhibition of Set7 activity by cyproheptadine (CPH) in human breast cancer cells (Takemoto et al., 2016). To test whether CPH similarly inhibits Set7 in primary cells, we measured Rpl29K5me2 levels of macrophages trained with b-glucan and observed a strong reduction of this posttranslational modification following 24-h incubation with 100 mM CPH (Figure 2A). To characterize the effects of CPH on trained immunity, we conducted in vitro training experiments on monocytes exposed to a range of CPH concentrations (Figure 2B). When co-incubated with b-glucan for only the first 24 h of the training protocol, CPH dose-dependently attenuated the heightened responsiveness of trained cells to restimulation with LPS (Figures 2C and 2D). This effect was most pronounced at the highest concentration of CPH tested (100 mM); however, a significant reduction in TNFaproduction was observed after coincubation with 25 mM CPH. Similar inhibition of b-glucan-induced trained immunity by CPH (100 mM) was observed when the cells were restimulated with the Toll-like receptor 2 agonist Pam3Cys (10 mg/mL) (Figure 2E). Exposure to this concentration of CPH did not alter the mRNA expression of SETD7 (measured on day 6, prior to restimulation), indicating that the inhibitory effect on trained immunity occurred at the level of Set7 activity (Figure 2F, open bar represents DMSO vehicle controls). Similarly, RPL29 mRNA expression was unaffected by 100 mM CPH (Figure 2G, open bar represents DMSO vehicle controls). The attenuating effect of CPH on cytokine production was not due to cytotoxicity of the compound (Figure 2H) or induction of apoptosis (Figure 2I). We excluded the possibility that CPH inhibits trained immunity via its antihistamine properties by performing experiments using an alternative antihistamine inhibitor, diphenhydramide, which did not alter trained immunity induced by b-glucan (Figure S1). To validate the observation of Set7-dependent regulation of cytokine production, we also tested the Set7 inhibitor sinefungin (Sasaki et al., 2016). Sinefungin dose-dependently inhibited the heightened production of TNFaand IL-6 by trained cells following restimulation with LPS (Figures 2J and 2K). To test the broader role of Set7 as a key regulator of trained immunity, we assessed the effects of CPH on trained cytokine production by other compounds previously shown to induce trained immunity. Indeed, incubation with 100 mM CPH also inhibited the induction of trained immunity by BCG (Figure 2L). Similarly, the dectin-1 ligand laminarin was previously demonstrated to induce trained immunity (Petit et al., 2019). Here, we show that the augmented TNFaproduction exhibited by macrophages trained with laminarin was inhibited by CPH. A similar trend was observed for IL-6 production, with no difference observed between cells that were incubated with laminarin and CPH, or laminarin alone (Figure 2M). To understand the mechanism of Set7-dependent cytokine production in trained immunity, we assessed the TNF and IL6 Figure 2. Pharmacological Inhibition of Set7 Dose-Dependently Attenuates the Pro-inflammatory Cytokine Response of Trained Immunity In Vitro (A) Western blot analysis of b-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 b-glucan or RPMI culture medium for 24 h in the presence of CPH or DMSO vehicle control, allowed to differentiate to macrophages (M4), and restimulated for 24 h with LPS, Pam3Cys, or RPMI on day 6. (C and D) Production of (C) TNFaand (D) IL-6 by b-glucan-trained macrophages incubated with CPH or 50-methylthioadenosine (MTA) for the first 24 h of in vitro training and restimulated with LPS (n = 7 healthy volunteers). (E) Production of TNFaby b-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 b-glucan in the presence of 100 mM CPH (n = 6 healthy volunteers; open bar represents DMSO vehicle control). (G) Expression of RPL29 mRNA at 24 h by cells trained with b-glucan in the presence of 100 mM 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 mM 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 mM 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) TNFaand (K) IL-6 by b-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 TNFaand IL-6 by bacillus Calmette-Gue ´rin (BCG)-trained macrophages incubated with 100 mM CPH for the first 24 h of in vitro training and restimulated with LPS (n = 6 healthy volunteers). (M) Production of TNFaand IL-6 by laminarin-trained macrophages incubated with 100 mM 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. Cell Reports 31, 107548, April 21, 2020 5 promoters by chromatin immunoprecipitation (ChIP) on day 6. We did not observe significant increases in H3K4me1 enrichment at either promoter in cells trained with b-glucan. A similar pattern of H3K4me1 enrichment was observed for cells trained in the presence of 100 mM CPH (Figure S2), suggesting that Set7 does not directly regulate TNF and IL6 expression via promoter histone methylation. Together, these findings identify an important role for Set7 in trained immunity in vitro. Set7 Regulates Trained Immunity In Vivo Using murine models, we and others (Cheng et al., 2014; GarciaValtanen et al., 2017) have described the specific contribution of b-glucan to the activation of trained immunity in vivo. Wild-type mice that received b-glucan injections exhibited enhanced cytokine production by innate immune cells in response to a secondary challenge or infection (Arts et al., 2016a). We adopted a similar approach to test the role of Set7 in trained immunity in vivo. We generated a Set7 constitutive knockout (Setd7 KO) mouse model by the deletion of Setd7 exon 2 region (unpublished data). Deletion of exon 2, which encodes the first MORN (membrane occupation and recognition nexus) repeat, results in a frameshift and inactivation of Set7. Western blot and gene expression analyses confirmed that Set7 was absent in bone marrow (BM) of the homozygous Setd7 KO mice (Figure 3A). Wild-type and Setd7 KO mice were systemically administered a single intraperitoneal 1-mg dose of b-glucan as described previously (Cheng et al., 2014). Control mice were injected with endotoxin-free phosphate-buffered saline (PBS). Five days after b-glucan administration, the mice were challenged with intraperitoneal injections of 10 mg of LPS, and after 3 h the serum levels of cytokines were quantified (Figure 3B). Contrasting the augmented pro-inflammatory cytokine production in wild-type mice administered b-glucan, mice lacking functional Set7 were unable to mount trained immunity against endotoxin challenge with regard to TNFaand IL-1bproduction. However, the effects of Set7 deletion on the trained production of IL-6 in vivo are less clear, because IL-6 production was already at high levels in wildtype mice that received PBS (Figure 3C). The modulation of myeloid progenitors is an integral component of trained immunity (Christ et al., 2018; Kaufmann et al., 2018; Mitroulis et al., 2018), which can explain the sustained activation of the innate immune system beyond the short life span of circulating myeloid cells. A recent study showed that administration of b-glucan to mice resulted in the expansion and polarization of hematopoietic stem and progenitor cells (HSPCs) toward myelopoiesis, which was associated with elevated signaling by innate immune mediators such as IL-1band granulocyte-macrophage colony-stimulating factor (GM-CSF), as well as changes in lipid and glucose metabolism (Mitroulis et al., 2018). To investigate the role of Set7 in these adaptations, we analyzed Setd7 mRNA in the BM of wild-type mice and observed a trend to increase expression in mice administered b-glucan (Figure 3D). We replicated key findings of the previous study (Mitroulis et al., 2018), albeit in whole bone marrow, and found that b-glucan-dependent transcriptional induction of Csf2 (GMCSF) and Il1b was significantly reduced in Setd7 KO mice. We also observed a reduction in the expression of the surrogate marker for HSPCs Cd34 in Set7 null mice; however, the expression of this gene remained elevated relative to Set7-null mice that received PBS injections (Figure 3E). These data demonstrate that Set7 regulates the in vivo pro-inflammatory cytokine response to induction of trained immunity by b-glucan and indicate an important role in hematopoietic adaptations that support the sustained phenotype. Set7 Regulates Key Metabolic Changes in Macrophages Trained with b-Glucan Trained immunity induced by b-glucan or BCG is characterized by metabolic reprogramming including increased glycolysis and intracellular accumulation of fumarate and mevalonate (Arts et al., 2016a; Bekkering et al., 2018). To explore the potential role of Set7 in the hallmark glycolytic metabolism of trained immunity, we measured extracellular lactate levels in day 6 macrophages trained with b-glucan. We observed a significant increase in these levels in b-glucan-trained cells that was abolished by coincubation with 100 mM CPH for the first 24 h of in vitro training (Figure 4A). In addition to changes in glycolysis, previous studies found that b-glucan training is accompanied by the repression of OXPHOS. In contrast to those findings (Cheng et al., 2014), we observed increased oxygen consumption at day 6 by cells trained with 1 mg/mL b-glucan (Figure 4B). By performing parallel respirometry experiments with the Seahorse XF Extracellular Flux Analyzer and the Oxygraph-2k from Oroboros (the instrument used for measuring oxygen consumption by Cheng et al., 2014), we found that the stimulatory dose of b-glucan can explain the discrepancy between the current (increased oxygen consumption) and previously reported findings (decreased oxygen consumption) (Cheng et al., 2014). Specifically, we observed, using the Oxygraph-2k, training with 1 mg/mL b-glucan stimulated oxygen consumption measured on day 6. On the other hand, the stimulatory dose of 10 mg/mL described by Cheng et al. led to an overall reduction of oxygen consumption by trained macrophages (Figures S3A and S3B). To confirm these observations and to rule out donor-specific variation as the cause, we trained cells from the same set of donors with 1 mg/mL b-glucan or 10 mg/mL b-glucan and analyzed them in parallel using the Seahorse system. While both stimulatory doses showed a trend to increase the extracellular acidification rate, training with 1 mg/mL b-glucan increased oxygen consumption, whereas training with 10 mg/mL b-glucan reduced oxygen consumption for each donor (Figures S3C and S3D). Next, we sought to determine whether Set7 was mechanistically involved in the upregulation of OXPHOS by cells trained with b-glucan. Paralleling its attenuating effect on cytokine production, CPH blunted the increase in oxygen consumption induced by b-glucan (Figure 4B; open bars represent DMSO vehicle controls). To understand the significance of this change in oxygen consumption for the induction of trained immunity, we investigated the effect of genetic variation on individual responses to b-glucan. Drawing from our genetic study of PBMCs isolated from 267 healthy volunteers (cohort 1; 300BCG), we tested for associations among common SNPs (minor allele frequency >5%) and variation in the magnitude of b-glucan-trained TNFa and IL-6 responses of individual subjects. Although genomewide significant cytokine quantitative trait loci (cQTLs) were not 6Cell Reports 31, 107548, April 21, 2020 observed, we identified numerous SNPs suggestively associated (p < 9.99 310 3 ) with adaptive changes in pro-inflammatory cytokine production mapped within 250 kb of genes related to OXPHOS as well as genes encoding key tricarboxylic acid (TCA) cycle enzymes. Variation in genes encoding isocitrate dehydrogenase enzymes was associated with the potentiation of TNFaproduction upon training with b-glucan. Similarly, variation in genes that encode subunits of the succinate dehydrogenase Figure 3. Set7 Regulates Trained Immunity Induced by b-Glucan In Vivo (A) Representative western blot of Set7 protein expression in the bone marrow of wild-type (WT) and Setd7 KO mice. b-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 b-glucan. (C) Plasma levels of TNFa, IL-6, and IL-1bin WT and Setd7 KO mice trained with PBS or b-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 b-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 b-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. Cell Reports 31, 107548, April 21, 2020 7 (legend on next page) 8Cell Reports 31, 107548, April 21, 2020 S.F., J.O., K.H., J.H.v.P., L.H., M.P.N., L.C.J.d.B., V.A.C.M.K., S.J.C.F.M.M., V.P.M., J.D.-A., M.O., E.P.B., and W.J.H.K.; Writing—Original Draft, S.T.K.; Writing—Review and Editing, S.T.K., L.G., C.D.C.C.v.d.H., J.C.d.S., S.F., J.O., J.H.v.P., M.P.N., V.M., L.C.J.d.B., V.A.C.M.K., S.J.C.F.M.M., V.P.M., J.D.-A., M.O., E.P.B., W.J.H.K., L.A.B.J., M.G.N., and N.P.R.; Funding Acquisition, L.A.B.J., M.G.N., and N.P.R.; Supervision, J.O., M.M., A.E.-O., L.A.B.J., M.G.N., and N.P.R. 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. 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Rep. 6, 21718. 16 Cell Reports 31, 107548, April 21, 2020 STAR+METHODS KEY RESOURCES TABLE REAGENT or RESOURCE SOURCE IDENTIFIER Antibodies Rabbit monoclonal anti-mono-methyl-Histone H3 (Lys4) Cell Signaling Technology Cat#5326; RRID: AB_10695148 Rabbit monoclonal anti-di-methyl-Rpl29 (Lys5) Cell Signaling Technology Cat#19495; RRID: AB_2798819 Rabbit polyclonal anti-Set7 Cell Signaling Technology Cat#2813; RRID: AB_823636 Rabbit polyclonal anti-HSP90 Cell Signaling Technology Cat#4874; RRID: AB_2121214 Rabbit polyclonal anti-b-actin Sigma-Aldrich Cat#A2066; RRID: AB_476693 Mouse monoclonal anti-b-actin Cell Signaling Technology Cat#3700; RRID: AB_2242334 swine-anti-rabbit Ig polyclonal secondary HRP antibody Dako Cat#P0217; RRID: AB_2728719 Chemicals, Peptides, and Recombinant Proteins b-glucan (b1,3-(D)-glucan Professor David Williams, College of Medicine, Johnson City, USA N/A Lipopolysaccharide Sigma-Aldrich Cat#L2880 From E.coli serotype 055:B5 Percoll Sigma-Aldrich Cat#P1644 Ficoll-Paque GE Healthcare Cat#17-1440-03 Roswell Park Memorial Institute medium (RPMI) Invitrogen Cat#22406031 iScript reverse transcriptase Bio-Rad Cat#1708840 TRIzol reagent Life Technologies Cat#15596018 SYBR Green Applied Biosciences Cat#4368708 16% Formaldehyde Fisher Scientific Cat#28908 Cyproheptadine Selleckchem Cat#S2044 Sinefungin Sigma-Aldrich Cat#S8559 Bacillus Calmette–Gue ´rin vaccine Statens Serum Institut, Copenhagen, Denmark N/A Laminarin Sigma-Aldrich Cat#L9634 Diphenhydramide hydrochloride Sigma-Aldrich Cat#D3630 Critical Commercial Assays Pierce BCA protein assay kit ThermoFisher Scientific Cat#23225 Human TNFaDuoSet ELISA R&D systems Cat#DY210 Human IL-6 DuoSet ELISA R&D systems Cat#DY206 Mouse TNFaQuantikine ELISA R&D systems Cat#MTA00B Mouse IL-6 Quantikine ELISA R&D systems Cat#M6000B Mouse IL-1bQuantikine ELISA R&D systems Cat#MLB00C Lactate Fluorometric Assay kit Biovision Cat#K607 MinElute PCR purification column QIAGEN Cat#28006 iScript cDNA synthesis kit Bio-Rad Cat#1708891 Cytox 96 assay Promega Cat#G1780 Succinate colorimetric assay kit Sigma-Aldrich Cat#MAK184 Fumarate colorimetric assay kit Sigma-Aldrich Cat#MAK060 Malate colorimetric assay kit Sigma-Aldrich Cat#MAK067 Oxaloacetate colorimetric assay kit Sigma-Aldrich Cat#MAK070 Citrate colorimetric assay kit Sigma-Aldrich Cat#MAK057 Experimental Models: Organisms/Strains 300BCG cohort (Human Functional Genomics Project) N/A https://www.humanfunctionalgenomics.org 200FG cohort (Human Functional Genomics Project) N/A https://www.humanfunctionalgenomics.org (Continued on next page) Cell Reports 31, 107548, April 21, 2020 e1 RESOURCE AVAILABILITY Lead Contact Further information and requests for resources and reagents should be directed to and will be fulfilled by the Lead Contact, Niels P. Riksen ([email protected]). Materials Availability This study did not generate new unique reagents. Data and Code Availability This study did not generate any unique datasets or code. EXPERIMENTAL MODEL AND SUBJECT DETAILS Human subjects With regard to the in vitro studies, buffy coats from male and female healthy donors were obtained after written informed consent (Sanquin blood bank, Nijmegen, the Netherlands). Cells were isolated and experiments conducted on the same day. Human cohorts The 300BCG cohort consists of 267 healthy males and females of Western European ancestry. The second cohort consists of 119 healthy individuals of Western European ancestry from the 200 Functional Genomics cohort (2011/399) of the Human Functional Genomics Project. The 300BCG cohort and 200FG cohort studies were approved by the local ethics committee (CMO regio ArnhemNijmegen, number NL58553.091.16 and number 2011-399, respectively). Inclusion of volunteers and experiments were conducted according to the principles expressed in the Declaration of Helsinki. All volunteers gave written informed consent before any material was taken. Mice The Setd7 knockout (KO) mice were generated by genOway (Lyon, France). The Setd7 targeting vector was designed within the first MORN domain of exon 2 containing two loxP sites (unpublished data). Insertion of loxP sites was introduced 1.6kb upstream of exon 2 by the integration of a loxP-flanked neomycine cassette. A targeting vector containing loxP sites flanking exon 2 of the Setd7 gene was integrated by homologous recombination in mouse embryonic stem cells. Recombinant clones were injected into mouse C57BL/6J strain blastocysts and implanted into pseudopregnant females. Once the construct was integrated into C57BL/6J background mice, it was crossed with a CMV promoter-driven Cre recombinase mouse to create a constitutive Setd7 KO mouse. Wildtype and Setd7 KO mice housed under specific pathogen-free conditions were used at the age of 9-11 weeks. Food and water was provided ad libitum. All animal studies were approved by the Alfred Medical Research and Education Precinct (AMREP) Animal Ethics Committee under guidelines laid down by the National Health and Medical Research Council (NHMRC) of Australia. METHOD DETAILS Cells and reagents Human peripheral blood mononuclear cells (PBMCs) were isolated from healthy volunteers by density-gradient centrifugation over Ficoll-Paque (GE Healthcare). Percoll isolation of monocytes was performed as previously described (Arts et al., 2016a). Cells were cultured in RPMI 1640 Dutch-modified culture medium (RPMI medium, Invitrogen) supplemented with 10 mg/mL gentamicin (Centraform), 2 mM Glutamax (Invitrogen), 1 mM pyruvate (Invitrogen), and 10% pooled human serum. Stimuli and inhibitors used were Escherichia coli lipopolysaccharide (LPS; serotype 055:B5, Sigma-Aldrich, 10 ng/mL), and Pam3Cys (EMC microcollections, Continued REAGENT or RESOURCE SOURCE IDENTIFIER Setd7 knockout mouse Professor Assam El-Osta, Monash University, Melbourne N/A Oligonucleotides See Table S1 This paper N/A Software and Algorithms GraphPad Prism 8.12 Graphpad software https://www.graphpad.com R statistical programming N/A RRID:SCR_001905 e2 Cell Reports 31, 107548, April 21, 2020 L2000, 10 mg/mL), Laminarin (Sigma), BCG (Statens Serum Institut, Copenhagen, Denmark), cyproheptadine (Selleckchem), sinefungin (Sigma), and diphenhydramide (Sigma). In vitro training and pharmacological inhibition b-1,3-(D)-glucan (b-glucan) was kindly provided by Professor David Williams (College of Medicine, Johnson City, USA). For isolation of cell wall b-glucans C. albicans was cultivated in 25 mL of YPD (1% yeast extract, 2% dextrose, 2% peptone) for 48 hours at 30C. The cells were harvested by centrifugation at 5,000x g for 5 minutes and pellet washed once with dH 2 O. The washed cell pellets were then frozen at 20C overnight. Prior to extracting the cell wall b-glucans, the cell pellets were subjected to repeated freeze-thaw cycles (3X) to lyse the cells. Cell pellets were then extracted with a base/acid isolation approach. The supernatant contained the water soluble mannans. Glucans are water insoluble and were harvested by centrifugation and washing in dH 2 O prior to lyophilization. The structure and purity of the b-glucans was determined by solution, high field one and two-dimensional Nuclear Magnetic Resonance Spectroscopy (1 and 2-D NMR). Adherent monocytes were trained as described previously (Bekkering et al., 2014). Cells were incubated with b-glucan (1 mg/mL), Laminarin (1 mg/mL), or BCG (5 mg/mL) for 24 hours, washed with warm phosphate buffered saline (PBS) and incubated in normal culture medium at 37C, 5% CO 2 . For pharmacological inhibition experiments, cells were pre-incubated with cyproheptadine (25100 mM), sinefungin (1, 10, 50, and 100 mg/mL), or diphenhydramide (10, 50, 100 mg/mlL), for 1 hour prior to stimulation. Following 5 days in culture, cells were restimulated with medium alone, 10 ng/mL LPS, or 10 mg/mL Pam3Cys for 24 hours at 37C, 5% CO 2 . Cytokine production was measured in supernatants by enzyme-linked immunosorbent assay (ELISA) according to the manufacturer’s instructions (R&D Systems). Cytokine measurement Cytokine production in supernatants and plasmas was determined using commercial enzyme-linked immunosorbent assay kits for TNFa, IL-6, IL-1b(R&D Systems, MN, USA), IL-10 (Sanquin) according to the instructions of the manufacturers. Annexin V/PI staining and lactate dehydrogenase (LDH) measurements for cell viability Apoptosis and cell viability of monocytes after 24h exposure to 100 mM CPH or vehicle control was evaluated with Annexin V-FITC (Biovision) and Propidium Iodide ECD (Biovision) fluorescence with cytoFLEX flow cytometer (Beckman Coulter) and analyzed with Kaluza 2.1 (Beckman Coulter). Analysis of LDH as a measure of cytotoxicity in cells incubated with CPH for 24 hours was assessed in the supernatants by using a Cytotox 96 kit (Promega). Quantitative RT-PCR Total RNA was isolated from human primary macrophages and total bone marrow of mice using TRIzol reagent according to the manufacturer’s instructions. 0.5-1 mg of total RNA was used to synthesize cDNA with the SuperScript III First-Strand Synthesis System (Thermo Fisher Scientific) according to the manufacturer’s protocol. Quantitative RT-PCR was performed using an Applied Biosciences StepOne PLUS qRT-PCR machine using SYBR Green (Invitrogen). All reactions were performed for at least 6 biological replicates and the values expressed as fold increase in mRNA levels relative to those in non-trained cells. 18s (human) or H3f3a (mouse) was used as a housekeeping gene. qRT-PCR primers are listed in Table S1. Western blot analysis of primary human cells For protein expression analysis of primary human cells, approximately 1 310 6 monocytes exposed for 24 hours to 100 mM CPH and 1310 6 macrophages were lysed with 100 mL of lysis buffer (1M Tris pH 7.4), 5M NaCl, 0.5M EDTA, 10% NP-40, 0.5M NaF, 2.5% sodium deoxycholate, PhosSTOP (Roche), cOmplete (Roche)) prior to stimulation on day 6. The homogenate was frozen, then thawed and centrifuged at 4C for 10 min at 15,000 x g, and the supernatant was taken for analysis. The western blot was performed using a Trans Turbo Blot System (Bio-Rad) according to the manufacturer’s instructions. Protein was loaded and separated on SDSPAGE using 4%–15% gradient precast gels and transferred to nitrocellulose membranes using the semi-dry method (Bio-Rad). Rabbit polyclonal primary antibodies were used for both Set7 (1:1000, 2813, Cell Signaling Technology) and Rpl29k5me2 (1:1000, 19495, Cell Signaling Techonolgy. Swine-anti-rabbit polyclonal secondary HRP antibody (1:5000, P0217, Dako) was used to detect Set7 and Rpl29k5me2 protein expressions. b-actin was detected on the blots using rabbit polyclonal primary antibody (1:1000, A2066, SigmaAldrich) and swine-anti-rabbit polyclonal secondary HRP antibody (1:5000, P0217, Dako). HSP90 was detected on the blots using rabbit polyclonal primary antibody (1:1000, 4874, Cell Signaling Technology) and swine-anti-rabbit polyclonal secondary HRP antibody (1:5000, P0217, Dako). Blots were developed with ECL (GE Healthcare) according to the manufacturer’s instructions. Mouse experiments For the in vivo study of trained immunity, mice were injected intraperitoneally with 1 mg of b-glucan in 200 mL of endotoxin-free phosphate-buffered saline (PBS). Intraperitoneal injections of PBS were performed as control. Five days after b-glucan administration, the mice were injected intraperitoneally with 10 mg of LPS from E. coli 055:B5 (Sigma) as a secondary challenge. Mice were euthanized at 3 hours after the LPS challenge. Cell Reports 31, 107548, April 21, 2020 e3 To isolate bone marrow cells (BMCs), mouse femurs and tibias were collected, trimmed and flushed with DPBS (GIBCO) using a 20 mL syringe with a 25 gauge needle to release BMCs. Bone marrow suspensions were gently harvested on 40 mm nylon mesh strainer (Falcon) in 50 mL conical tubes. After centrifugation (5 min, 350 x g,4 C), the cells were suspended with RBC lysis buffer (155 mM NH 4 Cl, 10 mM KHCO 3 , 0.1mM EDTA) to remove erythrocytes. Protein analyses were performed as described previously (Okabe et al., 2012). Briefly, approximately 5 310 6 BMCs were lysed with 250 mL of buffer C (20 mM HEPES-KOH (pH 7.5), 25% Glycerol, 520 mM KCl, 5 mM MgCl 2 , 0.1 mM EDTA, 1mM DTT, 0.5 mM PMSF, 0.2% NP-40 and proteinase inhibitor cocktail) for 15 min at 4C, then centrifuged for 15 min at 15,000 x g,4 C. The supernatant was collected for analysis. Primary antibodies were used for both Set7 (1:2,000, 2813, Cell Signaling Technology) and b-actin (1:10,000, 3700, Cell Signaling Technology). IRDye 800CW Donkey anti-mouse IgG and IRDye 680RD Donkey anti-rabbit IgG secondary antibodies (1:10,000 each, LI-COR) were used to detect Set7 and b-actin protein signals simultaneously. Metabolic analysis Approximately 1 310 7 monocytes were trained with b-glucan (1 mg/mL) in 10 cm Petri dishes (Greiner) in 10 mL medium volumes for 24 hours, washed with warm PBS and incubated in normal culture medium at 37C, 5% CO 2 . Following 5 days in culture, cells were detached with versene solution (ThermoFisher Scientific) and 1 310 5 cells were plated to overnight-calibrated cartridges in assay medium (RPMI with 0.6 mM glutamine, 5 mM glucose and 1 mM pyruvate [pH adjusted to 7.4]) and incubated for 1 hour in a non-CO 2 - corrected incubator at 37C. Oxygen consumption rate (OCR) was measured using a Cell Mito Stress Kit (for OCR) or a glycolysis stress test kit in an XFp Analyzer (Seahorse Bioscience), with final concentrations of 1 mM oligomycin, 1 mM FCCP, and 0.5 mM rotenone/antimycin A. Oxygen consumption measurement Culture medium was collected from cells treated with either RPMI or b-glucan (1 mg/mL or 10 mg/mL). After stimulation, the cells were trypsinized, washed, and resuspended in the collected culture medium. Cell suspensions containing 1 310 6 cells were then used for cellular O 2 consumption analysis. Oxygen consumption was measured at 37C using polarographic oxygen sensors in a two-chamber Oxygraph (OROBOROS Instruments, Innsbruck, Austria). DatLab software (Oroboros) was used for data acquisition (2 s time interval) and analysis (Gnaiger, 2001). First, basal respiration (baseline oxygen consumption) was measured. Next, leak respiration was determined by addition of 2.5 mM of the specific complex V inhibitor oligomycin A (OLI). Then, maximal electron transport chain complex (ETC) capacity (maximum oxygen consumption) was quantified by applying increasing concentrations of the mitochondrial uncoupler FCCP (0.25 to 20 mM final maximal concentration). Finally, minimal (non-mitochondrial) respiration was assessed by addition of the specific complex I inhibitor rotenone (ROT; 100 nM) and the complex III inhibitor antimycin A (AA; 2.5 mM). Metabolite measurements Cells were trained with b-glucan (1 mg/mL) with and without CPH (100 mM) as described above, using DMSO as a vehicle control. Metabolite concentrations measured from at least 1 310 6 trained monocytes for succinate, fumarate, malate, oxaloacetate, and citrate were determined using a commercial colorimetric assay kit (Sigma) according to the manufacturer’s instructions. Genetic analysis We conducted in vitro b-glucan, BCG and oxLDL training of adherent PBMCs from 267 healthy individuals of Western European ancestry from the 300BCG cohort (NL58553.091.16). DNA samples of these individuals were genotyped using the commercially available SNP chip, Infinium Global Screening Array MD v1.0 from Illumina. Genotype information on approximately 4 million single-nucleotide polymorphisms (SNPs) was obtained upon imputation (MAF > 5% and R 2 > 0.3 for imputation quality). First, raw cytokine levels were log-transformed and the ratio between trained and non-trained cytokine levels were taken as the change of cytokine levels. The cytokine changes were mapped to genotype data using a linear regression model with age and sex as covariates. Genetic outliers (n = 15) and samples stimulated with low b-glucan (< 1 mg/mL) were removed before QTL mapping. We also conducted in vitro b-glucan training of adherent PBMCs in a second cohort of 119 healthy individuals of Western European ancestry from the 200 Functional Genomics cohort (2011/399) of the Human Functional Genomics Project (www. humanfunctionalgenomics.org). Genotype information on approximately 4 million single-nucleotide polymorphisms (SNPs) was obtained using Illumina HumanOmniExpressExome SNP chip upon imputation. Only SNPs with a minor allele frequency of R5% that passed standard quality filters were included in the analysis. Raw cytokine levels were log-transformed and the ratio between trained and non-trained cytokine levels was used to quantify the trained immunity response. They were subsequently mapped to genotype data using a linear regression model with age and sex as co-variates (Li et al., 2016). ChIP-seq and ChIA-PET analysis This study makes use of H3K4me1 ChIP-seq datasets generated by the Blueprint Consortium (Adams et al., 2012). This study makes use of ChIA-PET data (accession number GSM970213). In the graphical display of the ChIA-PET data, the paired end tags (PETs) or chromosomal interactions are represented by two blocks for each end of the contact, connected by a horizontal line. The number of PETs in a cluster reflects the strength of the chromosomal interaction. The pre-processed datasets were visualized using the UCSC genome browser with the GRCh37/hg19 assembly (Kent et al., 2002). Enhancers were identified from the GeneHancer database of e4 Cell Reports 31, 107548, April 21, 2020 human regulatory elements (Fishilevich et al., 2017). Enhancers with the highest annotation-derived confidence score were selected. The study makes use of Hi-C data from the K562 cell line (accession GSE63525). Hi-C maps were generated at 5kb resolution using the 3D genome browser with the GRCh37/hg19 assembly (Wang et al., 2018). Chromatin immunoprecipitation Trained monocytes on day 6 were cross-linked in methanol free 1% formaldehyde, followed by sonication and immunoprecipitation using antibodies against H3K4me1 (Cell Signaling Technology). Immunoprecipitated chromatin was processed further for qRT-PCR analysis using the MiniElute DNA purification kit (QIAGEN). Primers used in the reaction are listed in Table S1. Samples were analyzed with a comparative Ct method on the StepOne PLUS qPCR machine (Applied Biosystems) using SYBR green (Invitrogen) in accordance with the manufacturer’s instructions. QUANTIFICATION AND STATISTICAL ANALYSIS Statistical parameters including the exact value of n, the definition of center, dispersion and precision measures (mean ±SEM), and statistical significance are reported in the figures and figure legends. Statistical analysis was performed using GraphPad Prism 8.12 (GraphPad Inc.). Analysis of human qPCR, ELISA and cellular assays was performed using Wilcoxon signed-rank test, t test or nonparametric Mann-Whitney tests, as appropriate. Analysis of mouse data used Mann-Whitney tests for comparisons between groups. R-package Matrix-eQTL was used for cytokine QTL mapping. A p value < 0.05 (*) was considered statistically significant, (**) p < 0.01. Data are shown as mean ±SEM. Cell Reports 31, 107548, April 21, 2020 e5