Grujic, Mirjana

Paivandy, Aida

Pejler, Gunnar

Rafati, Nima

English

Publikationer från Uppsala Universitet

DNA demethylation regulates gene expression in IgE-activated mouse mast cells.

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1776  |     LETTERS TO THE EDITORFernando G. Martins1Catarina Ferraz2Luísa G. Vaz2Sofia I. V. Sousa11LEPABE—Laboratory for Process Engineering, Environment, Biotechnology and Energy, Faculty of Engineering, University of Porto, Porto, Portugal2Departamento de Pediatria (UAG-MC), Centro Hospitalar Universitário de São João, Porto, PortugalCorrespondenceSofia I. V. Sousa, LEPABE—Laboratory for Process Engineering, Environment, Biotechnology and Energy, Faculty of Engineering, University of Porto, Rua Dr. Roberto Frias, 4200-465, E221, Porto, Portugal.Email: sofia.sousa@fe.up.ptR E FE R E N C E S 1. Branco PTBS, Nunes RAO, Alvim-Ferraz MCM, et al. Asthma prev-alence and risk factors in early childhood at Northern Portugal. Rev Port Pneumol (English Edition). 2016;22(3):146-150. 2. Sousa SI, Ferraz C, Alvim-Ferraz MC, Vaz LG, Marques AJ, Martins FG. Indoor air pollution on nurseries and primary schools: impact on childhood asthma–study protocol. BMC Public Health. 2012;12:435. 3. GINA.Global Initiativa for Asthma. Global Strategy for Asthma Management and Prevention; 2018. Available from: www.ginas thma.org2018. Accessed June 7, 2018. 4. Oluwole O, Rennie DC, Senthilselvan A, et al. Asthma diag-nosis among children along an urban-rural gradient. J Asthma. 2018;55(11):1242-1252. 5. Beydon N, Davis SD, Lombardi E, et al. An official American Thoracic Society/European Respiratory Society statement: pulmonary function testing in preschool children. Am J Respir Crit Care Med. 2007;175(12):1304-1345. 6. Bjerg-Backlund A, Perzanowski MS, Platts-Mills T, Sandstrom T, Lundback B, Ronmark E. Asthma during the primary school ages–prevalence, remission and the impact of allergic sensitization. Allergy. 2006;61(5):549-555. 7. Aaron SD, Boulet LP, Reddel HK, Gershon AS. Underdiagnosis and Overdiagnosis of Asthma. Am J Respir Crit Care Med. 2018;198(8):1012-1020. 8. Bjerg A, Hedman L, Perzanowski M, Wennergren G, Lundbäck B, Rönmark E. Decreased importance of environmental risk fac-tors for childhood asthma from 1996 to 2006. Clin Exp Allergy. 2015;45(1):146-153. 9. Caminati M, Duric-Filipovic I, Arasi S, Peroni DG, Zivkovic Z, Senna G. Respiratory allergies in childhood: Recent advances and future challenges. Pediatr Allergy Immunol. 2015;26(8):702-710. 10. Beasley R, Clayton T, Crane J, et al. Association between paracetamol use in infancy and childhood, and risk of asthma, rhinoconjunctivitis, and eczema in children aged 6–7 years: analysis from Phase Three of the ISAAC programme. Lancet. 2008;372(9643):1039-1048. 11. Marra F, Marra CA, Richardson K, et al. Antibiotic use in chil-dren is associated with increased risk of asthma. Pediatrics. 2009;123(3):1003-1010. 12. Ni J, Friedman H, Boyd BC, et al. Early antibiotic exposure and de-velopment of asthma and allergic rhinitis in childhood. BMC Pediatr. 2019;19(1):225. 13. Behrens T, Maziak W, Weiland SK, Rzehak P, Siebert E, Keil U. Symptoms of asthma and the home environment. The ISAAC I and III Cross-Sectional Surveys in Münster, Germany. Int Arch Allergy Immunol. 2005;137(1):53-61.SUPPORTING INFORMATIONAdditional supporting information may be found online in the Supporting Information section. DNA demethylation regulates gene expression in IgE-activated mouse mast cellsTo the Editor,Mast cells (MCs) have a major detrimental impact on various allergic conditions. MCs express the high affinity receptor for IgE (FcεRI) and are sensitized by the binding of antigen-specific IgE to such recep-tors. Upon encounter of specific antigen, FcεRI cross-linking occurs, which leads to degranulation and to activated transcription of genes coding for pro-inflammatory cytokines and chemokines.1,2Epigenetics refers to the effects on gene transcription that are unrelated to changes in DNA sequence. Major epigenetic mecha-nisms include DNA methylation, histone modification, and effects of noncoding RNAs.3-6 Generally, DNA methylation of gene promot-ers leads to gene repression whereas DNA demethylation typically leads to activated gene transcription. Considering the major effects of IgE receptor cross-linking on gene transcription in MCs, it appears plausible that epigenetic mechanisms may have an impact on such processes, but this has received relatively little attention.7,8 To pro-vide additional insight into this issue, we here investigated whether IgE-mediated MC activation affects global DNA methylation in MCs and whether modulation of DNA methylation/demethylation affects gene transcription.We used fully mature mouse peritoneal cell-derived MCs with a phenotype similar to that of human mast cells9 (Figure S1A). MCs were sensitized with DNP-specific IgE, followed by IgE receptor cross-linking by the exposure to DNP-conjugated human serum albu-min. This led to substantial MC degranulation (Figure 1A and Figure S1). To assess the effect of IgE receptor cross-linking on global DNA DOI: 10.1111/all.14205      |  1777LETTERS TO THE EDITORmethylation, we used an ELISA based on a monoclonal antibody to 5-methylcytosine. As seen in Figure 1B, IgE receptor cross-linking caused a substantial drop in global DNA methylation, which was most prominent 2 hours after IgE receptor cross-linking. Next, we assessed the effects of DNA methyltransferase (5-azacitidine; inhib-itor of DNMT enzymes) and demethylase (Octyl-ɑ-hydroxyglutarate [2-HG]; inhibitor of Ten-eleven translocation [TET] enzymes) in-hibitors on gene expression in MCs. 2-HG was nontoxic for MCs at concentrations up to 150 µmol/L, and 5-azacitidine was nontoxic up to 10 µmol/L (Figure 1C-D). Next, we tested the effects of 5-azacit-idine and 2-HG (at nontoxic concentrations) on the expression of Il6, Nr4a3, Il3, and Tnf. IgE receptor cross-linking induced an upregulated expression of all these genes. As shown in Figure 1E, the expres-sion of Il6 was profoundly attenuated by the DNA demethylase in-hibitor and, conversely, inhibition of DNA methyltransferase activity potentiated the Il6 expression. Similarly, the expression of Il3 was blunted by DNA demethylase inhibition and potentiated by inhibi-tion of DNA methyltransferase activity (Figure 1F). Neither Tnf nor Nr4a3 expression was significantly repressed by DNA demethylase inhibition (Figure 1G-H). The expression of classical MC markers (Mcpt4, Mcpt6) was not affected by interference with DNA demeth-ylase/ methyltransferase activity (Figure S2).Figure 1 IgE receptor cross-linking causes MC degranulation, decreased global DNA meth-ylation, and effects on gene expression in MCs. Effects on gene ex-pression are modulated by inhibition of DNA demethylase and DNA methyltransferase activity. A, Release of β-hexosaminidase activity from MCs (1.5 × 106 cells) activated by IgE receptor cross-linking. Results are given as mean values ± SEM (n = 6); ***P ≤ .001. B, MCs (2 × 106 cells) were activated by IgE receptor cross-linking. At the time points indicated, cells were recovered followed by purification of DNA and analysis of DNA methylation levels by ELISA. Results are given as mean values ± SEM (n = 6); *P ≤ .05. C, 5-azacitidine (DNMT inhibitor) and (D) 2-HG (TET inhibitor) at the indicated concentra-tions were assessed for effects on cell viability in MCs. E-H, Effect of 5-azacitidine and 2-HG on gene expression in IgE-activated MCs. MCs (1.5 × 106 cells) were activated by IgE receptor cross-linking (IgE + DNP-HSA). As controls, MCs were sensitized with anti-DNP IgE but not subjected to IgE receptor cross-linking (IgE). Inhibitors were added 1h before initiating IgE receptor cross-linking. Two hours after IgE receptor cross-linking, cells were recovered, followed by RNA isolation and qPCR analysis for expression of Il6 (E), Il3 (F), Tnf (G) and Nr4a3 (H). Results are given as mean values ± SEM (n = 3) and are relative to the expression of the housekeeping gene (Hprt). *P ≤ .05; **P ≤ .01; ***P ≤ .001 ****P ≤ .0001To obtain deeper insight into how DNA demethylase inhibition affects gene transcription pathways in MCs, we performed an un-biased transcriptomic analysis, using the AmpliSeq platform. As shown in Figure S3, IgE-mediated MC activation caused upregulated expression of numerous genes. These included several pro-inflam-matory genes, transcription factors, growth factors, and receptors. After interference with DNA demethylation, the expression of a sub-stantial number of IgE-induced genes was blunted (altogether 126 genes; genes with ≥ fourfold repression are highlighted in Figure 2). These included Mrgprx1, S1pr2 (coding for sphingosine-1-phosphate receptor 2), Ccl3, Myc, and Il6. DNA demethylase inhibition also caused an increased expression of several genes in the IgE-activated MCs, many of which having anti-inflammatory properties (eg, Gdf9, Hspa1a (HSP72), Wnt4, Egr1, Tnfaip2, and Shb). DNA demethy-lase inhibition also affected the expression of numerous genes at baseline conditions. Among the genes that were induced by DNA demethylase inhibition under baseline conditions, many were also highly induced under conditions of IgE-mediated MC activation. These included Hspa1a, Hspa1b, Mrgprb8, Cnnm4, Mospd4, Egr1, Klf4, and Ell2 (Figure S4). In addition, several genes were downregu-lated by DNA demethylase inhibition under baseline conditions. Of these, several were also highly downregulated (≥fourfold) by DNA demethylase inhibition in IgE-activated MCs (eg, S1pr2, Lhfpl2, and Nef2).Figure 2 Effect of DNA demethylase blockade on gene ex-pression in IgE-activated primary MCs. MCs (1.5 × 106 cells) were activated for 2 h by IgE receptor cross-linking ± DNA demethylase inhibitor (2-HG), followed by RNA isolation and transcriptome anal-ysis. Differential gene expression analyses were performed using edgeR. A, Log2-normalized expression of genes with significant differential expression between activated MCs (IgE + DNP) and ac-tivated MCs ± DNA demethylase inhibitor (IgE + DNP+2-HG). The side color shows genes clustered by hclust. Among 324 differentially expressed genes, 198 genes were upregulated by 2-HG whereas 126 genes were downregulated. B, List of top genes differentially expressed by |log FC| ≥ 2 (red: upregulated, blue: downregulated)To confirm that findings from the transcriptome analysis were rep-licated with an independent method, we used qPCR analysis where we focused on selected genes: Mrgprx1, Myc, and Ccl3. We also as-sessed the expression of Mrgprx2, a close relative to Mrgprx1. As seen in Figure S5, the qPCR analysis confirmed a profound upregulation of Mrgprx1 in response to IgE-mediated MC activation and also con-firmed that DNA demethylase inhibition suppressed the stimulatory effect of IgE receptor cross-linking on Mrgprx1 expression. A small stimulatory effect of IgE receptor cross-linking on Mrgprx2 expression was also seen, and this effect was blocked by DNA demethylase inhi-bition. The qPCR analysis also confirmed the induction of Ccl3 and Myc by IgE receptor cross-linking, as well as the inhibitory effects of DNA demethylase blockade on the induction of these genes (Figure S5).Altogether, our findings reveal that gene transcription events oc-curring downstream of IgE receptor cross-linking in MCs can be reg-ulated by epigenetic mechanisms. Notably, interference with DNA demethylation blunted only a fraction of those genes that were in-duced by IgE receptor cross-linking. When examining the nature of the genes that were downregulated by DNA demethylase blockade, we found that many of these have pro-inflammatory properties (eg, Myc, S1pr, Il6, and Il3). DNA demethylase inhibition also caused up-regulated expression of numerous genes in the IgE-activated MCs. Intriguingly, many of these latter genes can have anti-inflammatory functions, for example, Hspa1a (HSP72), Wnt4, Egr1, Shb, Crtam, and Tnfaip2. Hence, our findings suggest that interference with DNA demethylation in IgE-activated mouse MCs may dampen se-lected pro-inflammatory pathways and, at the same time, potentiate 1778  |     LETTERS TO THE EDITOR     |  1779LETTERS TO THE EDITORF I G U R E  1   IgE receptor cross-linking causes MC degranulation, decreased global DNA methylation, and effects on gene expression in MCs. Effects on gene expression are modulated by inhibition of DNA demethylase and DNA methyltransferase activity. A, Release of β-hexosaminidase activity from MCs (1.5 × 106 cells) activated by IgE receptor cross-linking. Results are given as mean values ± SEM (n = 6); ***P ≤ .001. B, MCs (2 × 106 cells) were activated by IgE receptor cross-linking. At the time points indicated, cells were recovered followed by purification of DNA and analysis of DNA methylation levels by ELISA. Results are given as mean values ± SEM (n = 6); *P ≤ .05. C, 5-azacitidine (DNMT inhibitor) and (D) 2-HG (TET inhibitor) at the indicated concentrations were assessed for effects on cell viability in MCs. E-H, Effect of 5-azacitidine and 2-HG on gene expression in IgE-activated MCs. MCs (1.5 × 106 cells) were activated by IgE receptor cross-linking (IgE + DNP-HSA). As controls, MCs were sensitized with anti-DNP IgE but not subjected to IgE receptor cross-linking (IgE). Inhibitors were added 1h before initiating IgE receptor cross-linking. Two hours after IgE receptor cross-linking, cells were recovered, followed by RNA isolation and qPCR analysis for expression of Il6 (E), Il3 (F), Tnf (G) and Nr4a3 (H). Results are given as mean values ± SEM (n = 3) and are relative to the expression of the housekeeping gene (Hprt). *P ≤ .05; **P ≤ .01; ***P ≤ .001 ****P ≤ .0001F I G U R E  2   Effect of DNA demethylase blockade on gene expression in IgE-activated primary MCs. MCs (1.5 × 106 cells) were activated for 2 h by IgE receptor cross-linking ± DNA demethylase inhibitor (2-HG), followed by RNA isolation and transcriptome analysis. Differential gene expression analyses were performed using edgeR. A, Log2-normalized expression of genes with significant differential expression between activated MCs (IgE + DNP) and activated MCs ± DNA demethylase inhibitor (IgE + DNP+2-HG). The side color shows genes clustered by hclust. Among 324 differentially expressed genes, 198 genes were upregulated by 2-HG whereas 126 genes were downregulated. B, List of top genes differentially expressed by |log FC| ≥ 2 (red: upregulated, blue: downregulated)1780  |     LETTERS TO THE EDITORvarious anti-inflammatory events. We may therefore propose that interference with epigenetic mechanisms can represent a novel strategy for intervening with pathologies mediated by IgE-activated MCs. However, future studies will be needed to extrapolate the findings to the effects on primary human mast cells. Moreover, such strategies should be adopted with caution, given the possibilities of detrimental side effects associated with global inhibition of DNA demethylation/methylation.KE Y WORDSDNA demethylase, DNA methylation, epigenetics, mast cells, transcriptomeACKNOWLEDG MENTSAmpliSeq analysis was performed at the National Genomics Infrastructure Uppsala (Uppsala Genome Center) of the Science for Life Laboratory, Sweden.CONFLIC T OF INTERE S TThe authors declare no conflict of interest in relation to this work.FUNDING INFORMATIONThis work was supported by grants from The Swedish Heart and Lung Foundation, The Swedish Research Council, The Swedish Cancer Foundation, and The Knut and Alice Wallenberg Foundation.Aida Paivandy1Mirjana Grujic1Nima Rafati2,3Gunnar Pejler1,41Department of Medical Biochemistry and Microbiology, Uppsala University, Uppsala, SwedenEmail: Gunnar.Pejler@imbim.uu.se2Science for Life Laboratory, Department of Medical Biochemistry and Microbiology, Uppsala University, Uppsala, Sweden3Science for Life Laboratory, National Bioinformatics Infrastructure Sweden (NBIS), Uppsala University, Uppsala, Sweden4Department of Anatomy, Physiology and Biochemistry, Swedish University of Agricultural Sciences, Uppsala, SwedenCorrespondenceGunnar Pejler, Department of Medical Biochemistry and Microbiology, Uppsala University, BMC, Box 582, 75123 Uppsala, Sweden.Email: Gunnar.Pejler@imbim.uu.seORCIDGunnar Pejler  https://orcid.org/0000-0002-6779-391X Aida Paivandy and Mirjana Grujic Equal contribution. R E FE R E N C E S 1. Wernersson S, Pejler G. Mast cell granules: armed for battle. Nat Rev Immunol. 2014;14:478-494. 2. Galli SJ, Nakae S, Tsai M. Mast cells in the development of adaptive immune responses. Nat Immunol. 2005;6:135-142. 3. Deaton AM, Bird A. CpG islands and the regulation of transcription. Genes Dev. 2011;25:1010-1022. 4. Jaenisch R, Bird A. Epigenetic regulation of gene expression: how the genome integrates intrinsic and environmental signals. Nat Genet. 2003;33(Suppl):245-254. 5. Schubeler D. Function and information content of DNA methylation. Nature. 2015;517:321-326. 6. Kouzarides T. Chromatin modifications and their function. Cell. 2007;128:693-705. 7. Leoni C, Montagner S, Rinaldi A, et al. Dnmt3a restrains mast cell in-flammatory responses. Proc Natl Acad Sci USA. 2017;114:E1490-E1499. 8. Garcia-Faroldi G, Rönnberg E, Grujic M, Pejler G. Inhibition of BET family epigenetic reader proteins: a novel principle for modulat-ing gene expression in IgE-activated mast cells. Immun Inflamm Dis. 2017;5:141-150. 9. Malbec O, Roget K, Schiffer C, et al. Peritoneal cell-derived mast cells: an in vitro model of mature serosal-type mouse mast cells. J Immunol. 2007;178:6465-6475.SUPPORTING INFORMATIONAdditional supporting information may be found online in the Supporting Information section. Is pollen-food syndrome a frequent comorbidity in adults with irritable bowel syndrome?To the Editor,Irritable bowel syndrome (IBS) affects up to 10% of UK adults, 50% of whom may also have seasonal allergic rhinitis (SAR) and thus an increased risk of developing pollen-food syndrome (PFS) if sensitized to birch tree pollen.1-3 In an exploratory prospective controlled cohort study, we compared the prevalence of PFS in IBS subjects from a secondary care clinic diagnosed using the Rome IV criteria,4 with that of an age and gender-matched control group with DOI: 10.1111/all.14209 

DNA demethylation regulates gene expression in IgE-activated mouse mast cells

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DNA demethylation regulates gene expression in IgE-activated mouse mast cells

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