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Induces Persistent DNA Methylation and -Specific Immune Changes

This information is current as Andrew R. DiNardo, Tomoki Nishiguchi, Emily M. Mace, of October 5, 2021. Kimal Rajapakshe, Godwin Mtetwa, Alexander Kay, Gugu Maphalala, W. Evan Secor, Rojelio Mejia, Jordan S. Orange, Cristian Coarfa, Kapil N. Bhalla, Edward A. Graviss, Anna M. Mandalakas and George Makedonas J Immunol 2018; 201:124-133; Prepublished online 11 May 2018; Downloaded from doi: 10.4049/jimmunol.1800101 http://www.jimmunol.org/content/201/1/124 http://www.jimmunol.org/ Supplementary http://www.jimmunol.org/content/suppl/2018/05/10/jimmunol.180010 Material 1.DCSupplemental References This article cites 76 articles, 18 of which you can access for free at: http://www.jimmunol.org/content/201/1/124.full#ref-list-1

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The Journal of Immunology is published twice each month by The American Association of Immunologists, Inc., 1451 Rockville Pike, Suite 650, Rockville, MD 20852 Copyright © 2018 by The American Association of Immunologists, Inc. All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. The Journal of Immunology

Schistosomiasis Induces Persistent DNA Methylation and Tuberculosis-Specific Immune Changes

Andrew R. DiNardo,* Tomoki Nishiguchi,* Emily M. Mace,†,‡ Kimal Rajapakshe,x Godwin Mtetwa,{ Alexander Kay,{ Gugu Maphalala,‖ W. Evan Secor,# Rojelio Mejia,** Jordan S. Orange,†,‡ Cristian Coarfa,x Kapil N. Bhalla,†† Edward A. Graviss,‡‡ Anna M. Mandalakas,*,1 and George Makedonas†,‡,1

Epigenetic mechanisms, such as DNA methylation, determine immune cell phenotype. To understand the epigenetic alterations induced by helminth coinfections, we evaluated the longitudinal effect of and schistosomiasis on CD4+ T cell DNA methylation and the downstream tuberculosis (TB)–specific and bacillus Calmette–Gue´rin–induced immune pheno- type. All experiments were performed on human primary immune cells from a longitudinal cohort of recently TB-exposed children. Compared with age-matched uninfected controls, children with active haematobium and lumbri- Downloaded from coides infection had 751 differentially DNA-methylated , with 72% hypermethylated. ontology pathway analysis identified inhibition of IFN-g signaling, cellular proliferation, and the Th1 pathway. Targeted real-time quantitative PCR after methyl-specific endonuclease digestion confirmed DNA hypermethylation of the transcription factors BATF3, ID2, STAT5A, IRF5, PPARg, RUNX2, IRF4, and NFATC1 and cytokines or cytokine receptors IFNGR1, TNFS11, RELT (TNF receptor), IL12RB2, and IL12B (p < 0.001; Sidak–Bonferroni). Functional blockage of the IFN-g signaling pathway was confirmed, with helminth-infected individuals having decreased upregulation of IFN-g–inducible genes (Mann–Whitney p < 0.05). Hypomethylation of the IL-4 pathway http://www.jimmunol.org/ and DNA hypermethylation of the Th1 pathway was confirmed by Ag-specific multidimensional flow cytometry demonstrating decreased TB-specific IFN-g and TNF and increased IL-4 production by CD4+ T cells (Wilcoxon signed-rank p <0.05).In S. haematobium–infected individuals, these DNA methylation and immune phenotypic changes persisted at least 6 mo after successful deworming. This work demonstrates that helminth infection induces DNA methylation and immune perturbations that inhibit TB- specific immune control and that the duration of these changes are helminth specific. The Journal of Immunology, 2018, 201: 124–133.

he immune system has multiple mechanisms to remain subsets (Th1, Th2, Th9, Th17, T regulatory cells [Tregs], Th22, and T responsive to changing environments. It is now clear that follicular helper cells) (4–6). To differentiate into a Th1 CD4+ T cell, by guest on October 5, 2021 T both the innate and adaptive immune response use epi- the IFNG promoter is demethylated and the IL4 locus undergoes genetic mechanisms of gene regulation to maintain long-term repressive histone modifications (7–9). Although previously thought phenotypes. For example, monocytes “trained” with b-glucan or to be immutable, epigenetic mechanisms have recently been de- bacillus Calmette–Gue´rin (BCG) undergo epigenetic chromatin al- scribed that allow T cells to shift between Th1 and Th2 phenotypes terations that alter TNF and IL-6 production and improve Staphylo- depending on environmental stimuli (10). Understanding the duration coccus, Candida, and mycobacterial killing (1, 2). Long-term and mechanisms of epigenetic-mediated immune polarization is phenotypic changes in adaptive immunity are similarly epigenetically critical to predicting and reversing detrimental immune perturbations. mediated. The RAG-mediated TCR rearrangement requires hypo- Helminths are a heterogeneous group of parasites estimated to methylation of RAG and other critical genes (3). After TCR rear- affect a quarter of the world’s population (11, 12). Although most rangement, T cells remain responsive to changing environments via are subclinical, they can induce growth retardation, epigenetic modifications that polarize CD4+ T cells into phenotypic nutritional deficiencies, and profoundly perturb immunogenicity

*The Global Tuberculosis Program, Immigrant and , Department of This work was supported by a Burroughs Wellcome Fund/American Society of Pediatrics, Texas Children’s Hospital, Baylor College of Medicine, Houston, TX and Postdoctoral Fellowship in Tropical Infectious Dis- 77030; †Department of , Baylor College of Medicine, Houston, TX eases (early career grant), the Thrasher Research Fund (early career grant), and 77030; ‡Texas Children’s Hospital Center for Human Immunobiology, Department National Institutes of Health Grant T-32 AI 55413–11 (to A.R.D.). of Pediatrics, Texas Children’s Hospital Center for Human Immunobiology, Baylor x The findings and conclusions in this report are those of the authors and do not College of Medicine, Houston, TX 77030; Dan L Duncan Comprehensive Cancer { necessarily represent the views of the Centers for Disease Control and Prevention. Center, Baylor College of Medicine, Houston, TX 77030; Baylor–Swaziland Children’s Foundation, Mbabane H100, Swaziland; ‖Ministry of Health, Mbabane Address correspondence and reprint requests to Dr. Andrew R. DiNardo, Baylor H100, Swaziland; #Parasitic Diseases Branch, Division of Parasitic Diseases and College of Medicine, Feigin Center, Suite 630, 1102 Bates Avenue, Houston, TX , Centers for Disease Control and Prevention, Atlanta, GA 30333; 77030. E-mail address: [email protected] **Department of Pediatrics, National School of Tropical Medicine, Texas The online version of this article contains supplemental material. Children’s Hospital Center for Human Immunobiology, Houston, TX 77030; ††Department of Leukemia, MD Anderson Cancer Center, Houston, TX 77030; Abbreviations used in this article: BCG, bacillus Calmette–Gue´rin; gDNA, genomic and ‡‡Department of Pathology and Genomic Medicine, Houston Methodist DNA; GO, ; IQR, interquartile range; MF, macrophage; MDMF, Hospital Research Institute, Houston, TX 77030 monocyte-derived MF; MSMD, Mendelian susceptibility to mycobacterial disease; O&P, ova and parasite microscopy; qPCR, quantitative PCR; SEA, S. mansoni– 1A.M.M. and G.M. are colast authors and contributed equally to this work. soluble egg Ag; TB, tuberculosis; Treg, T regulatory cell. ORCIDs: 0000-0003-2172-1491 (G. Mtetwa); 0000-0002-3635-8405 (G. Maphalala); 0000- 0003-0584-5961 (W.E.S.); 0000-0003-4134-4558 (R.M.); 0000-0003-1024-5813 (E.A.G.). Copyright Ó 2018 by The American Association of Immunologists, Inc. 0022-1767/18/$35.00 Received for publication January 23, 2018. Accepted for publication April 9, 2018. www.jimmunol.org/cgi/doi/10.4049/jimmunol.1800101 The Journal of Immunology 125

(12–14). The helminth-induced immune response is characterized real-time PCR were performed. Schistosomiasis serology was performed by tolerance and a type 2 immune polarization (15, 16) with de- by standard ELISA. In brief, 96-well ELISA plates were coated with 2 mg/ml creased IFN-g, IL-12, and IL-17 (17) and increased Tregs (18), S. mansoni–soluble egg Ag (SEA) in 0.1 M sodium bicarbonate buffer (pH 9.6) for 2 h, followed by washing with PBS plus 0.3% Tween 20 and blocking IL-4, IL-5, IL-10 (19), and TGF-b (20). This profound type 2 and with PBS/Tween containing 5% dry milk powder. Plates were again washed immune-tolerant phenotype undermines the type 1 immune re- and incubated with a 1:50 dilution of participant plasma in PBS/Tween/milk sponse to , Salmonella, tetanus, and BCG (21–25) and is for 30 min. Plates were again washed and incubated with a 1:50,000 dilution the suspected rationale for increased TB disease progression of HRP-conjugated mouse anti-human IgG (SouthernBiotech, Birmingham, AL) for 30 min. After a final , SureBlue TMB (Kirkegaard & Perry among helminth-infected individuals (11, 26–28). In contrast to Laboratories, Gaithersburg, MD) substrate was added to plates for 5 min and the type 2 immune response required to expel helminths, Myco- the reaction was stopped using 1 N H2SO4. Plates were read at 450 nm on a bacterium tuberculosis infection requires a type 1 polarized CD4– Molecular Devices (Sunnyvale, CA) microplate reader, and patient results macrophage (MF) immune axis for effective host response. were compared with a standard curve consisting of sera from confirmed Individuals with IL-12–IFN-g axis defects due to Mendelian sus- schistosomiasis patients that was included on each plate using SoftMax Pro software (Molecular Devices). ceptibility to mycobacterial diseases (MSMD), HIV, and TNF inhibi- Stool microscopy was performed using the McMaster protocol (52). Two tors are prone to disseminated BCG, tuberculosis (TB) disease grams of stool were added to 28 ml of NaCl and sugar flotation solution progression, and infection with other intracellular (29, 30). and homogenized by aggressive hand mixing followed by straining and Stimulation of MFs with TNF and IFN-g induces intracellular killing viewing on a McMaster chamber slide. Urine microscopy was performed after 400 3 g centrifugation. via phagolysosome maturation and NO production, whereas IL-4 in- Multiparallel helminth quantitative PCR (qPCR) was performed as duces a diametrically opposed reaction to direct MFsawayfromNO previously described (53). DNA was extracted from 50 mg of stool using and toward collagen synthesis (31). Therefore, when MFsarepolar- the MP FastDNA SPIN Kit for Soil. For urine DNA extraction, the con- ized with IL-4, they exhibit decreased phagolysosome maturation, di- centrated pellet was heated at 90˚C for 10 min followed by DNA isolation Downloaded from minished NO production, and reduced M. tuberculosis killing (32–35). using the MP FastDNA SPIN Kit. From stool, helminth-specific PCR was performed for A. lumbricoides, , Ancylostoma duo- In the meˆle´e of host– interactions, pathogens induce denale, , and Strongyloides stercoralis using primers and epigenetic changes to subvert host immunity. HIV induces DNA FAM-labeled minor groove binding probes (Supplemental Table I). Sim- methyltransferases expression that hypermethylates IFNG and IL2. ilarly, qPCR specific for S. haematobium was performed from the HIV also hypomethylates PD1, resulting in decreased IFN-g and DNA isolated from urine with organism-specific primers and probes

(Supplemental Table I). The qPCR reactions were performed in duplicate http://www.jimmunol.org/ IL-2 while increasing PD-1 (36–39). Similar to other intracellular with 2 ml of stool- or urine-derived template DNA, 900 nM primers, pathogens (40–42), M. tuberculosis induces epigenetic changes that 250 nM probe, and 3.5 ml of TaqMan Fast MasterMix and acquired on a benefit mycobacterial survival by altering MF Ag-presentation and QuantStudio 3 in Mbabane, Swaziland, after 40 cycles. Quantification was inflammasome function (43–48). CD4+ T cell cytokine production performed using a standard curve consisting of 10-fold serial dilutions of is critical for MF polarization and M. tuberculosis control (49, 50). purified plasmid DNA containing the target sequences. Composite helminth status was defined according to serology, microscopy, In murine studies, schistosomiasis induces epigenetic changes and PCR status. Active helminth infection was defined as microscopy and/or that induce alternative activated MFs(7)andaTh2CD4+ Tcell PCR positive. Remote infection was classified as serology positive but mi- phenotype characterized by DNA hypermethylation of the IFNG croscopy and PCR negative at both baseline and the 2- and 6-mo follow-up. promoter (8). Therefore, to elucidate the longitudinal epige- Uninfected controls were microscopy, PCR, and serology negative. by guest on October 5, 2021 netic–immune effects of helminth coinfection, we evaluated the DNA methylation effect of and on + + To evaluate CD4 T cell DNA methylation, CD14-positive selection was CD4 T cell DNA methylation and TB-specific immune phenotype. followed by CD4-negative selection (Stemcell Technologies) to obtain These studies indicate that Schistosoma and Ascaris induce global .93% CD4+ T cell population, determined by flow cytometry. Cellular changes in host DNA methylation and immune phenotypic changes DNA was then isolated using the DNeasy lymphocyte protocol (Qiagen). in CD4+ Tcells.ForS. haematobium, these host DNA methylation To perform the DNA Methylation EPIC array, genomic DNA (gDNA) from CD4+ T cells was quantified using a Qubit 3.0 Fluorometer (Thermo Fisher and immune phenotypic perturbations are relatively stable and Scientific, Waltham, MA). gDNA quality was analyzed using Agilent 4200 persist at least 6 mo after successful antihelminthic therapy. TapeStation System (Agilent Technologies, Sugar Land, TX) for integrity check and NanoDrop ND1000 Spectrophotometer for purity check. gDNA Materials and Methods (250–750 ng) was treated with sodium bisulfite using the Zymo EZ DNA Methylation Kit (Zymo Research, Irvine, CA). We followed the Illumina Cohort recommended incubation conditions during bisulfite conversion to maximize Asymptomatic children 6 mo to 15 y of age with recent intense TB exposure DNA conversion rate. Illumina Infinium HumanMethylation 850 and Human [as quantified by a standardized TB exposure score (51)] were enrolled after Methylation EPIC BeadChip (Illumina, San Diego, CA) were run on an Illu- obtaining assent and written informed consent of their parent or caregiver. mina iScan System (Illumina) using the manufacturer’s standard protocol. As per national guidelines, participants were empirically dewormed with Validation of the Infinium DNA Methylation EPIC data were performed and treated with isoniazid preventative therapy if they were using real-time PCR after isolated DNA underwent methylation-sensitive ,5 y of age or HIV infected. Individuals with HIV infection were excluded or -dependent digestion with an EpiTect II DNA Methylation from this analysis. All individuals were BCG vaccinated as confirmed by a Kit. The relative percentage of methylated and unmethylated DNA BCG scar and/or card. Participants completed clinical evaluation at 2 was determined relative to mock and double-digested DNA as previously and 6 mo for TB symptoms and provided stool and urine for helminth described (54). evaluation. was collected for helminth serology and PBMC collection at each visit. Stool and urine were collected for parasite microscopy and PCR Multidimensional immune profiling at each visit. The protocol was reviewed and approved by the Baylor College To identify TB-specific and BCG-induced immunity, PBMCs were stimulated of Medicine Children’s Foundation–Swaziland, Baylor College of Medicine, with 2.5 mg each of ESAT-6 and CFP-10 overlapping peptide pools or 5 mg and the Swaziland Scientific and Ethics Committee. The protocol was also of BCG sonicate, respectively. BCG- and TB-specific immune responses reviewed at the Centers for Disease Control and Prevention, which issued a were compared with vehicular control (dimethyl sulfoxide) stimulation, and nonengaged determination. All methods were performed in compliance with staphylococcal enterotoxin B was used as a positive control. Cell stimulations institutional and international guidelines for the Protection of Human Sub- occurred in the presence of brefeldin A (1 mg/ml), monensin (0.7 mg/ml), and jects in concordance with the Declaration of Helsinki. CD28/CD49 (2.5 mg/ml). Following overnight (16 h) stimulation, cells were Helminth status stained with an amine reactive viability dye (Ghost Dye), surface Abs (CD4, CD8, CD56, and PD-1), and intracellular Abs (CD3, IFN-g, TNF, IL-4, IL-10, To comprehensively describe a child’s helminth status, serology, ova and IL-13, perforin, T-bet, GATA-3, and Ki-67) and acquired on a BD LSR II parasite microscopy (O&P), and helminth-specific multiparallel quantitative Fortessa (Supplemental Table I). A minimum of 70% viability was the 126 HELMINTHS INDUCE DNA HYPERMETHYLATION AND IMMUNE ALTERATION

Table I. Demographics and TB exposure of clinical cohort

TB Exposure Score Sex Age

n Microscopy or PCR Serology Mean Median p Value (% Male) Median p Value IQ1 IQ3 Helminth uninfected 18 Negative Negative 7.25 8 N/A 42.00 4.5 N/A 2.4 8.8 Active helminth 8 Positive Positive/negative 7 6 0.7 33.30 4.9 0.9 2.1 9.3 infection Past schistosomiasis 6 Negative SEA positive 6.8 7 0.6 33.30 8.5 0.4 2.8 9 Past ascariasis 6 Negative Ascaris positive 6.6 6 0.3 40.00 7.5 0.2 6.6 10.5 IQ1, first quartile; IQ3, third quartile; N/A, not applicable. threshold for credible data. All reported TB-specific or BCG-induced values discovery rate ,1%), of which 72% were hypermethylated. were subtracted from the background nonstimulated immune response. Gene ontology (GO) analysis identified significant DNA Gene expression methylation perturbation in the immune system in the IL-4 and IFN-g response, regulation of cytokine production, cell prolif- PBMCs were stimulated overnight with IFN-g (50 ng/ml) or media control, and 16 h later, CD4+ T cells were isolated via magnetic bead negative eration, mTOR signaling, and glycolysis (Fig. 1). selection as described above. From 100 ng of RNA, the Human Immu- To validate these results, targeted DNA methylation of specific nology v2 probe set was performed using the NanoString nCounter. genes was evaluated by PCR after methyl-specific endonuclease Background noise was normalized using eight negative control probes and digestion. Compared with helminth-uninfected healthy controls, Downloaded from the geometric mean of positive controls and 15 housekeeping genes. helminth-infected individuals had increased DNA methylation Monocyte-derived MF immune polarization for the transcription factors IRF4 (5.7%, IQR 4.7–7.0% versus 0.6%, IQR 0–1.1%), IRF5 (34.8%, IQR 27.5–38.4% versus Adherent monocytes from healthy control PBMCs were cultured for 7 d at 37˚C with 10% human AB serum and 10 ng/ml M-CSF and GM-CSF. 0.9%, IQR 0–1.4%), BATF3 (50%, IQR 50–50% versus 2.2%, After a 7-d conversion to monocyte-derived MFs (MDMF), the cells IQR 1.6–2.7%), ID2 (48.4%, IQR 50–50% versus 1.9%, IQR 2 were polarized with IL-4 20 ng/ml or IFN-g 50 ng/ml for 48 h and then 0.2–1.3%), RUNX2 (33.3%, IQR 26.2–37.4% versus 1.2%, IQR http://www.jimmunol.org/ brought to the biosafety level three. The MDMFs from three healthy 0–1.2%), PPARg (46.3%, IQR 46.2–50.0% versus 2.3%, IQR 1.5– controls were incubated with live H37Rv M. tuberculosis at a multiplicity of infection of 3:1 overnight, nonphagocytosed extracellular bacilli were 3.0%), and NFATC1 (60.9%, IQR 60.4–71.4% versus 3.0%, IQR removed by washing, MDMF were lysed with ice-cold deionized water, 0–4.4%) (p , 0.001, Sidak–Bonferroni; Fig. 2A, Table II). Sim- and mycobacteria were cultured on 7H11 plates at 1:5 and 1:25 dilutions. ilarly, compared with uninfected controls, helminth-infected children Twenty-one days later, CFUs were manually counted. had increased CD4+ DNA methylation of cytokine or cytokine re- Statistical analysis ceptors IFNGR1 (20.1%, IQR 18.4–20.2% versus 0.6%, IQR 0–0.6%), TNFS11 (5.8%, IQR 4.9–7.7% versus 0.6%, IQR 0.3– Flow cytometry data analysis was performed in FlowJo (Tree Star, Ashland, 0.7%), RELT (TNF receptor) (12.1%, IQR 7.6–16.2% versus 0.9%, OR), GraphPad 6.0 (GraphPad Software), and SPICE (available at http://exon. by guest on October 5, 2021 niaid.nih.gov/spice). The nonparametric Mann–Whitney U test was used to IQR 0.5–1.5%), IL12RB2 (34.1%, IQR 28.7–34.9% versus 2.2%, compare datasets. For multiple comparisons, the Sidak–Bonferroni test was IQR 0–3.4%), and IL12B (14.3%, IQR 8.4–15.6% versus 0.7%, IQR performed. Statistically significant differentially methylated genes were visu- 0–1.0%) (p , 0.001, Sidak–Bonferroni; Fig. 2A, Table II). By alized using Ingenuity Pathway Analysis (Qiagen Bioinformatics). The .idat contrast, MAP1LC3, a gene essential in autophagy, was hypo- files from methylation data were imported into the R statistical system using the Bioconductor (http://www.bioconductor.org) minfi package (55). Pre- methylated 9% in active helminth infection and remained hypo- processed and normalized data from minfi were used by the Bioconductor methylated in remote schistosomiasis (negative by stool and urine limma package to identify the significantly differentiated probes with a p value O&P and PCR for helminth infection, but serology-positive) infection ,0.05 and log2-fold change .2or,0.5 (55). Statistically significant differ- (p , 0.001, Sidak–Bonferroni; Fig. 2A). entially methylated genes were visualized using Ingenuity Pathway Analysis Children with remote Ascaris infection had a CD4+ DNA (Qiagen Bioinformatics). Further gene enrichment analysis was performed by employing the gene set collections compiled by the Molecular Signature methylation signature statistically similar to the healthy control Database (http://www.broadinstitute.org/gsea/msigdb/) using the hypergeometric group (Fig. 2A). By contrast, children with remote schistoso- distribution (statistical significance achieved at p , 0.05) (56). miasis had CD4+ DNA methylation signatures statistically similar to the active helminth infection group (Fig. 2A). To Results further evaluate if schistosomiasis induces persistent DNA Participant demographics methylation alterations in CD4+ T cells, we repeated the DNA Among the children enrolled who had exposure to a microbiologically methylation evaluation 6 mo after enrollment with negative confirmed pulmonary TB case in the past 6 mo, 59% of children were microscopyandPCRat2and6motoconfirmsuccessful female and the median age was 6.1 (interquartile range [IQR] 2.7– deworming. Six months after successful deworming, the DNA + 10.5). The median and mean TB exposure scores were 7 (Table I). methylation of CD4 T cells remained elevated and statistically similar to actively infected individuals (Fig. 2A). Comparison Helminth infection induces CD4 DNA methylation changes between DNA methylation and respective helminth status is that persist after schistosomiasis shownintheheatmapinFig.2B.Pathwayanalysisofthese We evaluated how helminth status altered CD4+ T cell DNA methyl- results identified helminth-induced inhibition of the Th1 and ation. Individuals with current Ascaris or schistosome infec- IFN-g signaling pathway (Fig. 3 p =2.97e233 and p =6.01e217, tion (n = 8), confirmed by O&P or by multiparallel PCR, have respectively). unique CD4+ T cell DNA methylation signatures compared with uninfected, TB-exposed children (n = 18). Using the DNA Helminth infection mutes upregulation of IFN-g–inducible Methylation EPIC array to analyze ∼850,000 CpG probes, gene expression helminth-infected individuals had 751 genes with differentially To validate the effect of helminth-induced DNA hyper- methylated probes compared with uninfected controls (false methylation of the IFN-g signaling pathway, CD4+ T cells The Journal of Immunology 127 Downloaded from

FIGURE 1. Global CD4+ T cell DNA methylation changes during helminth infection. The Illumina DNA Methylation EPIC array was performed on CD4+ T cells from helminth-infected and uninfected individuals (n = 5 per group). (A) Volcano plot showing the b values for hypomethylation (blue dots) and hypermethylation (red dots) in helminth-infected individuals compared with uninfected controls. (B) GO pathways enriched in genes with DNA hypomethylation after helminth infection. (C) GO pathways enriched in genes with DNA hypermethylation after helminth http://www.jimmunol.org/ infection. were stimulated overnight with 50 ng/ml of IFN-g. IDO, (25.5, IQR 16.6–38.7 versus 3.8, IQR 0.8–6.7; p , 0.05 by Mann– SERPING1, STAT1, IRF1, FCGR1A/B, GBP1, ICAM1, HLA- Whitney U test; Fig. 4B). DRA, HLA-DRB3, and HLA-DQA1, the IFN-g–inducible genes specific to CD4+ T cells (57), were evaluated, demonstrating that Helminth infection results in persistent immune IDO upregulation was muted by helminth infection (IDO fold phenotypic perturbations 2 upregulation 12.24, IQR 9.4–16.0 versus 0.02, IQR 0.3–0.4; p = DNA methylation decreases transcription factor binding affinity, by guest on October 5, 2021 0.001, Sidak–Bonferroni; Fig. 4A). Using a previously described thereby repressing gene expression. To validate the func- CD4+ TcellIFN-g–inducible gene signature (57), helminths tional impact of helminth infection–induced DNA methylation induced a 6.7-fold decrease in IFN-g–stimulated gene upregulation changes in CD4+ T cells, we performed flow cytometry–based

FIGURE 2. Targeted DNA methylation of transcription factors, cytokines, and surface receptors depending on helminth infection. Methyl-specific endonuclease digestion followed by real-time qPCR was performed on isolated DNA from CD4+ T cells from Ascaris-infected, schistosome-infected, or uninfected children. Successful deworming was confirmed by negative PCR and microscopy 2 and 6 mo after antihelminth therapy (n = 6 per group). (A) Bar graphs depict the median and range of the percentage of DNA methylation depending on helminth status. (B) Heatmap of the CD4+ T cell DNA methylation signature depending on helminth status. S.h, S. haematobium. 128 HELMINTHS INDUCE DNA HYPERMETHYLATION AND IMMUNE ALTERATION

Table II. Median and IQR of DNA methylation differences in helminth-infected and uninfected individuals

Helminth Uninfected Controls

Median (%) IQ1–IQ3 (%) Median (%) IQ1–IQ3 (%) IRF4 5.70 4.7–7.0 0.60 0–1.1 IRF5 34.50 27.5–38.4 0.90 0–1.4 BATF3 50 50–50 2.20 1.6–2.7 ID2 48.40 50–50 1.90 0.2–1.3 RUNX2 33.30 26.2–37.4 1.20 0–1.2 PPARg 46.30 46.2–50 2.30 1.5–3.0 NFATC1 60.90 60.4–71.4 3.00 0–4.4 IFNGR1 20.10 18.4–20.2 0.60 0–0.6 TNFS11 5.80 4.9–7.7 0.60 0.3–0.7 RELT 12.10 7.6–16.2 0.90 0.5–1.5 IL12RB2 34.40 28.7–34.9 0.20 0–3.4 IL12B 14.30 8.4–15.6 0.70 0–1.0 IQ1, first quartile; IQ3, third quartile. multidimensional immune profiling after ex vivo stimulation with signed-rank test p , 0.05; Fig. 5). The increased frequency of IL-

TB-specific peptides or BCG sonicate. The characteristic immune 4–producing subsets was associated with a decrease in TB-specific Downloaded from alteration induced by helminth infections is an increase in IL-4 CD4+ T cells producing IFN-g and TNF (median 0.0%, IQR 0.0– production by CD4+ T cells as well as by other lymphocytes, mast 0.0 versus 0.4%, IQR 0.16–0.5%; Wilcoxon signed-rank test cells, eosinophils, and basophils (58, 59). We found that PBMCs p , 0.05). DNA methylation alterations in individuals with active from recently TB-exposed individuals with active helminth in- helminth infection were associated with a decrease in TB-specific fection had an increased frequency of TB-specific (ESAT-6 and proliferation. Similarly, TB-specific upregulation of Ki-67 was +

CFP-10 stimulation) CD4 T cells upregulating IL-4 (median decreased in helminth-infected individuals compared with http://www.jimmunol.org/ 3.0%, IQR 3.0–4.5% versus 0.3%, IQR 0.07–0.37; Wilcoxon uninfected controls (median 0.1%, IQR 0.03–0.18% versus 0.08% by guest on October 5, 2021

FIGURE 3. Pathway analysis identified inhibition of the Th1 and IFN-g signaling pathway by helminth infection. Methyl-specific endonuclease digestion followed by real-time qPCR was performed on isolated DNA from CD4+ T cells from helminth-infected or uninfected children. Genes outlined in pink expressed increased DNA methylation in helminth-infected children compared with uninfected age-matched controls (n = 6 per group). (A) Th1 signaling pathway. (B) IFN-g signaling pathway. The Journal of Immunology 129

FIGURE 4. Helminths decrease IFN-g–inducible gene upregulation. PBMCs from recently TB-exposed children were stimulated overnight with IFN-g (50 ng/ml) followed by CD4+ T cell selection and RNA isolation. Fold upregulation was determined by comparing nonstimulated gene expression to IFN-g Downloaded from gene expression (n = 8 helminths and 4 controls). (A) IFN-g–inducible gene expression of specific genes. #p , 0.001, Sidak–Bonferroni test. (B) IFN-g– inducible gene expression depending on helminth status. Lines represent median and minimum to maximum range. p , 0.05, Mann–Whitney U test.

IQR 0.05–0.1%; Wilcoxon signed-rank test p , 0.05). Further- demonstrated an increased percentage of BCG-induced CD4+ more, helminth-infected individuals had a decrease in the quantity T cells upregulating IL-4 (median 1.2%, IQR 0.8–1.8% versus 0.7, of TB-specific proliferating CD4+ T cells (an increase in Ki-67 IQR 0.3–0.8%; Wilcoxon signed-rank test p , 0.05; Supplemental http://www.jimmunol.org/ above baseline) producing TNF and IFN-g (median 0.0, IQR Fig. 2). 0.00–0.00 versus 0.02%, IQR 0.01–0.05%; Wilcoxon signed-rank We next evaluated the persistence of helminth-induced immune test p , 0.05; Supplemental Fig. 1). Because all individuals were perturbations on TB-specific and BCG-induced immune responses BCG vaccinated, the BCG-induced immune response was also 6 mo after antihelminth therapy. Successful antihelminth therapy evaluated. Notably, individuals with active helminth infection was documented with negative repeat stool and urine O&P and by guest on October 5, 2021

FIGURE 5. Helminths decrease the M. tuberculosis–specific Th1 CD4+ T cell function, with associated increases in Th2. PBMCs from recently TB- exposed children were stimulated overnight with M. tuberculosis–specific peptides (ESAT-6 and CFP-10) peptide pools (n = 18 helminth uninfected and 8 helminth infected). (A) Flow cytometry gating strategy. (B) Representative dot plots of nonstimulated, ESAT-6– and CFP-10–stimulated, or BCG-stimulated CD4+ T cells. (C) The overall M. tuberculosis–specific CD4+ T cell response is illustrated as a pie graph with TNF-a (yellow arcs), IFN-g (red arcs), and IL-4 (green arcs). Each pie slice corresponds to a discreet functional subpopulation, outlined in (D). The Th1/Th2 ratio was determined by the ratio of IFN-g and TNF-a compared with IL-4. (D) The bar graph represents the absolute frequency of CD4+ T cells producing IFN-g, TNF-a, or IL-4. Data represent 18 helminth-uninfected and 8 active helminth-infected individuals. #p , 0.05 between helminth-uninfected and -infected groups, Wilcoxon test. 130 HELMINTHS INDUCE DNA HYPERMETHYLATION AND IMMUNE ALTERATION Downloaded from

FIGURE 6. Persistent Th2/Th1 perturbations after schistosomiasis. Children with or without helminth infection had PBMCs collected at baseline and http://www.jimmunol.org/ 6 mo later. Children with Ascaris or S. haematobium were treated with albendazole or , respectively. Successful deworming was confirmed by microscopy and PCR at 2 and 6 mo. The overall M. tuberculosis–specific CD4+ T cell response is illustrated as a pie graph with TNF-a (yellow arcs), IFN-g (red arcs), and IL-4 (green arcs). The Th1/Th2 ratio was determined by the ratio of IFN-g and TNF-a compared with IL-4. Data represent six individuals per helminth group and 18 uninfected controls. helminth PCR at 2 and 6 mo after study enrollment. For schis- immune changes has been previously evaluated with mixed re- tosomiasis, but not ascariasis, 6 mo after successful antihelminthic sults. After leaving helminth-endemic areas, refugees experienced therapy, the CD4+ T cell immune phenotype demonstrated an decreased , IgE, and HLA-DR on CD3+ T cells 6–12 by guest on October 5, 2021 increased percentage of IL-4 and a decreased percentage of TNF mo after deworming (61). However, other studies have shown that and IFN-g Ag-specific producing CD4+ T cells compared with remote ascariasis (as determined by elevated Ascaris-specific IgE) uninfected controls (median 0.05%, IQR 0.01–0.05% versus 0.4%, is inversely associated with tuberculin skin test reactivity (62). IQR 0.16–0.5%; Wilcoxon signed-rank test p , 0.05; Fig. 6). Further, 14 mo after in utero schistosomiasis exposure, children Previous reports have established that the supernatant of CD4+ still had a 25-fold decrease in purified derivative–induced T cells incubated with SEA was less effective at forming a mature IFN-g production (21). The mixed results from previous studies phagolysosome with intracellular M. tuberculosis (32). Consis- are likely due to variability of helminths evaluated and variability tent with this, MDMF treated with IL-4 instead of IFN-g had a of the immune assessments performed. The cohort in the current 3.8-fold decreased ability to kill intracellular M. tuberculosis study consisted of Ascaris and S. haematobium–infected children (Wilcoxon signed-rank test p = 0.039; Supplemental Fig. 3). who were recently household contacts of individuals with mi- crobiologically confirmed TB. Their helminth status was docu- Discussion mented by urine and stool PCR, microscopy, and serology. To remain responsive to changing environments, the immune Participants were dewormed and reevaluated 2 and 6 mo later to system undergoes epigenetic modifications to refine cell pheno- confirm conversion from active to remote helminth status. These types (10, 60). CD4+ T cells in particular have an epigenetically studies demonstrate that schistosomiasis has persistent DNA methyl- mediated spectrum of phenotypes that respond to intracellular ation and TB-specific and BCG-induced immune perturbations pathogens (Th1), extracellular parasites (Th2 and Th9), and bac- that remain shifted away from Th1 immunity 6 mo after suc- teria (Th17 and Th22) and limit autoimmunity (Tregs) (60). After cessful S. haematobium deworming. encountering a pathogen, both the innate and adaptive immune There is a paucity of data describing the duration of epigenetic responses retain immunologic memory (2). In this article, we show control of immune responses after a host–pathogen interaction. that recently TB-exposed asymptomatic children with helminth In vitro, memory Th1 and Th2 cells retain posttranslational epi- infection have CD4+ T cell DNA methylation signatures that inhibit genetic modifications (H3K4Ac of IL4 and IFNG promoter) for the TB-specific and BCG-induced Th1 immune phenotype. Of in- .20 cell divisions (63). In a sepsis model, IL-12 remained epi- terest, these CD4+ T cell DNA methylation and immune phenotype genetically inhibited for at least 6 wk (64). In vivo, epigenetic perturbations persist for at least 6 mo in children previously infected alterations persisted 3 mo after BCG vaccination (2), and CD8+ with S. haematobium, but not in children previously infected with lymphocytes retain permissive PD1 DNA hypomethylation marks A. lumbricoides. 2 y after successful antiretroviral therapy (38). In this study, Helminth infection induces a Th2 and immune-tolerant response we show that the schistosomiasis-induced CD4+ T cells DNA that subverts cell-mediated immunity to B virus, BCG, methylation signatures persist at least 6 mo after successful and cholera (21, 58, 61). The duration of helminth-induced deworming for schistosomiasis, but not ascariasis. The continued The Journal of Immunology 131 effect of schistosome infections on the host immune system after (Figs. 5, 6). Further, by increasing the DNA methylation of the treatment is consistent with observations that the pathology as- downstream IFN-g signaling pathway, IFN-g was less capable of sociated with schistosomiasis endures beyond infection (65, 66). upregulating the IFN-g–inducible genes (Fig. 4). These experi- The difference in the duration of DNA methylation and immune ments focused on the DNA methylation and immune phenotype of perturbations is likely related to the different life cycles of these CD4+ T cells. A critical role of IFN-g is to induce phenotypic organisms. Whereas Ascaris is an intraluminal infection and is changes in MFs, specifically to upregulate genes necessary for passed out of the body intact following treatment, S. haematobium phagolysosome maturation, generation of lysosomal acidification, live in the venous plexus, and successful chemotherapy and reactive oxygen species and NO synthase production (57). eliminates the worms; however, the eggs remain imbedded in the Future studies should evaluate if DNA methylation changes in the tissue, causing inflammation that can persist for years after suc- IFN-g signaling pathway block MF phagolysosome maturation, cessful treatment (66). For at least some period of time, the eggs reactive oxygen species and NO synthase, or the generation of continue to induce a type 2 immune response that is diametrically lysosomal acidification with resultant decreased intracellular opposed to the type 1 immune response directed at intracellular M. tuberculosis killing. coinfections, such as M. tuberculosis (11, 12, 26, 27, 61). Helminths perturb the immune system and increase the risk of Deworming combined with BCG revaccination was successful at TB progression. Therefore, they are an excellent model to elucidate restoring Ag-specific cell-mediated immunity in a previous the immunity necessary to control M. tuberculosis infection. In this study of Ascaris- and -infected individuals (22). This study, we confirm that ex vivo IFN-g improves M. tuberculosis simple strategy should be evaluated in a schistosomiasis- killing but that helminths induce epigenetic DNA methylation infected population. changes in CD4+ T cells that hamper both IFN-g production and After schistosome infection, STAT-6– and JMJD3-mediated the downstream intracellular IFN-g signaling pathway. These Downloaded from alterations in histone H3 lysine 27 (H3K27) trimethylation in- findings highlight the importance of persistent DNA methylation duce an alternatively activated MF phenotype (7, 67). Similarly, perturbations in immune function and provide plausible explana- murine studies have shown that schistosomiasis induces CD4+ tions for increased rate of TB progression in the setting of hel- T cells to shift to a Th2 phenotype associated with DNA hypo- minth coinfection. Future studies should evaluate means to reverse methylation of the IL4 and Gata3 genes (68). Whereas IL4 and helminth-induced detrimental DNA methylation changes to Gata3 were not hypomethylated in this cohort, the GO pathway restore mycobacterial immunity. Similarly, longitudinal cohort http://www.jimmunol.org/ regulating IL-4 was hypomethylated (Fig. 1), with associated in- studies need to be undertaken to evaluate if DNA methylation creased production of IL-4 (Fig. 4). This supports the previous signatures described in this article correlate with increased rates of evidence that the Th2 pathway is epigenetically selected and not a TB progression. default pathway (69). There are numerous potential nonexclusive mechanisms through Disclosures which helminth may alter DNA methylation. 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