Maternal Pregravid Obesity Remodels the DNA Methylation Landscape of Cord Blood Monocytes Disrupting Their Inflammatory Program This information is current as of September 26, 2021. Suhas Sureshchandra, Randall M. Wilson, Maham Rais, Nicole E. Marshall, Jonathan Q. Purnell, Kent L. Thornburg and Ilhem Messaoudi J Immunol published online 8 September 2017 http://www.jimmunol.org/content/early/2017/09/07/jimmun Downloaded from ol.1700434

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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 © 2017 by The American Association of Immunologists, Inc. All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. Published September 8, 2017, doi:10.4049/jimmunol.1700434 The Journal of Immunology

Maternal Pregravid Obesity Remodels the DNA Methylation Landscape of Cord Blood Monocytes Disrupting Their Inflammatory Program

Suhas Sureshchandra,* Randall M. Wilson,† Maham Rais,† Nicole E. Marshall,‡ Jonathan Q. Purnell,x Kent L. Thornburg,x and Ilhem Messaoudi*

Prepregnancy maternal obesity is associated with adverse outcomes for the offspring, including increased incidence of neonatal bacterial sepsis and necrotizing enterocolitis. We recently reported that umbilical cord blood (UCB) monocytes from babies born to obese mothers generate a reduced IL-6/TNF-a response to TLR 1/2 and 4 ligands compared to those collected from lean mothers. These observations suggest altered development of the offspring’s immune system, which in turn results in dysregulated

function. We therefore investigated transcriptional and epigenetic differences within UCB monocytes stratified by prepregnancy Downloaded from maternal body mass index. We show that UCB monocytes from babies born to obese mothers generate a dampened response to LPS stimulation compared with those born to lean mothers, at the level of secreted immune mediators and transcription. Because expression profiles of resting UCB monocytes from both groups were comparable, we next investigated the role of epigenetic differences. Indeed, we detected stark differences in methylation levels within promoters and regulatory regions of involved in TLR signaling in resting UCB monocytes. Interestingly, the DNA methylation status of resting cells was highly predictive of

transcriptional changes post-LPS stimulation, suggesting that cytosine methylation is one of the dominant mechanisms driving http://www.jimmunol.org/ functional inadequacy in UCB monocytes obtained from babies born to obese mothers. These data highlight a potentially critical role of maternal pregravid obesity-associated epigenetic changes in influencing the function of an offspring’s monocytes at birth. These findings further our understanding of mechanisms that explain the increased risk of infection in neonates born to mothers with high prepregnancy body mass index. The Journal of Immunology, 2017, 199: 000–000.

lmost 37% of women of childbearing age are categorized obesity include increased rates of preeclampsia, gestational hyper- as obese, making obesity one of the most common tension, gestational diabetes, placental abruption, preterm delivery, comorbidities during pregnancy (1). It is well estab- and cesarean delivery (1). For the fetus, complications include A by guest on September 26, 2021 lished that a high prepregnancy (pregravid) body mass index increased risk of stillbirth, abnormal growth, and cardiac or neural (BMI) is associated with detrimental health outcomes for both tube defects (1, 4). Moreover, neonates born to obese mothers are mother and child (2–4). Maternal complications of pregravid at increased risk of bacterial sepsis and necrotizing enterocolitis, requiring admission to the neonatal intensive care unit (5, 6). *Department of Molecular Biology and Biochemistry, School of Biological Sciences, Some adverse health outcomes for the offspring persist into University of California Irvine, Irvine, CA 92697; †Division of Biomedical Sciences, Uni- adulthood, including increased susceptibility to respiratory in- versity of California Riverside, Riverside, CA 92521; ‡Maternal-Fetal Medicine, x fections such as respiratory syncytial virus (7, 8), asthma (9), Oregon Health and Science University, Portland, OR 97239; and Department of Medicine, The Knight Cardiovascular Institute, Oregon Health and Science Uni- wheezing (10), cancer (11), type 2 diabetes (12), and cardiovas- versity, Portland, OR 97239 cular disease (13), culminating in an increased risk for all-cause ORCIDs: 0000-0003-2555-2111 (M.R.); 0000-0003-3105-555X (N.E.M.). offspring mortality (14). Received for publication March 24, 2017. Accepted for publication August 7, 2017. The aforementioned observations strongly suggest that pregravid This work was supported by National Institutes of Health Grants KL2TR000152 (to obesity disrupts the development and maturation of the offspring’s N.E.M.) and R03AI112808 (to I.M.) and the National Center for Advancing Trans- immune system in utero. This hypothesis is supported by studies lational Sciences of the National Institutes of Health under Award UL1TR000128. in murine models that have shown detrimental effects on the and methylation data have been submitted to the National Center for Biotechnology Information Sequence Read Archive (http://www.ncbi.nlm.nih.gov/ immune system of pups born to obese compared with lean dams sra) under accession number PRJNA398556. (15, 16). Specifically, pups born to obese dams generated disparate S.S., N.E.M., J.Q.P., K.L.T., and I.M. conceived and designed the experiments; S.S., IgG (smaller) and IgE (larger) Ab responses following vaccination R.M.W., and M.R. performed the experiments; S.S. and R.M.W. analyzed the data; with OVA (15). If these results are true in humans, they could S.S. and I.M. wrote the paper with input from N.E.M., J.Q.P., and K.L.T. explain the increased incidence of asthma (9) and wheezing (10) Address correspondence and reprint requests to Dr. Ilhem Messaoudi, 2400 Biolog- ical Sciences III, University of California Irvine, Irvine, CA 92697-3900. E-mail in children born to obese mothers. Additional rodent studies address: [email protected] reported greater morbidity and mortality following bacterial The online version of this article contains supplemental material. infection (Escherichia coli sepsis and methicillin-resistant Staphy- Abbreviations used in this article: BMI, body mass index; DEG, differentially expressed lococcus aureus infection) and greater susceptibility to auto- gene; DMC, differentially methylated cytosine; DMR, differentially methylated region; immune encephalitis in pups born to obese dams (16). Lastly, ex eDMR, empirical DMR; FC, fold change; FDR, false discovery rate; GO, ; RPKM, read per kilobase of transcript per million mapped reads; TF, tran- vivo LPS stimulation of colonic lamina propria lymphocytes scription factor; TSS, transcription start site; UCB, umbilical cord blood; UCBMC, resulted in increased secretion of inflammatory cytokines IL-6, UCB mononuclear cell. IL-1b, and IL-17, whereas LPS stimulation of splenocytes resulted Copyright Ó 2017 by The American Association of Immunologists, Inc. 0022-1767/17/$35.00 in decreased levels of TNF-a and IL-6 in pups born to obese dams

www.jimmunol.org/cgi/doi/10.4049/jimmunol.1700434 2 EPIGENETIC IMPACT OF MATERNAL OBESITY ON CORD BLOOD MONOCYTE

(16). Collectively, these data strongly suggest dysregulated im- Abs conjugated to magnetic microbeads per the manufacturer’s recom- munity in pups born to obese dams where aberrant responses (such mendations (Miltenyi Biotec, San Diego, CA). Magnetically bound UCB as to auto-antigens) are exaggerated, whereas protective anti- monocytes were washed and eluted for collection. Positive selection of UCB monocytes was chosen to ensure high purity of the population, microbial responses are reduced. Similarly, a baboon study which was assessed using flow cytometry and was $90% for all samples reported significant changes in the expression of genes involved in analyzed. Ag presentation, complement and coagulation cascade, leukocyte LPS stimulation and detection of soluble mediators migration, and BCR signaling in PBMCs collected from infants born to obese compared with lean dams (17). Purified UCB monocytes were plated at 2 3 105 per well and stimulated However, only a few studies have explored the impact of ma- with 100 ng (1 mg/ml) of LPS (TLR4 ligand, E. coli 055:B5; InvivoGen, ternal pregravid obesity on neonatal immune function in humans. San Diego, CA) or left untreated for 16 h at 37˚C and 5% CO2. The above concentration of LPS was previously standardized for robust detection of One study reported that children born to obese mothers have a 16- gene expression in primary human monocytes (22, 23). Following the 16 h fold higher risk of having detectable levels of C reactive incubation, cells were spun down. Supernatants were collected for analysis after adjusting for BMI, Tanner stages, and gender compared with using the human ProcartaPlex multiplex immunoassay (eBioscience/ children born to lean mothers, suggesting that high pregravid Affymetrix), which simultaneously measures the concentration of 45 cy- tokines (TNF-a, TNF-b, IFNG, IL-1a, IL-1b, IL-6, IL-13, IL-15, IL-18, maternal BMI results in dysregulated inflammatory responses in IL-22, IL-23, and IL-27, IL-1RA, IL-4, and IL-10), chemokines (MIP1a, the offspring (18). More recently, we have demonstrated that pre- MIP1b, MCP-1, IL-8, IP10, RANTES, and Eotaxin), and growth factors gravid maternal BMI is associated with a reduced number of (VEGF, SDF1a, EGF, PDGF, GMCSF, HGF, and FGF2) (http://www. umbilical cord blood (UCB) CD4+ T cells, IL-4–secreting CD4+ ebioscience.com/human-cytokine-chemokine-growth-factor-1-45-plex- procartaplex-multiplex-kit.htm). Values below the limit of detection were T cells, and a diminished ability of monocytes and myeloid den- Downloaded from designated as half of the lowest limit. dritic cells to respond to ex vivo stimulation with LPS (TLR4 Principal component analysis of cytokine, chemokine, and growth ligand) and HKLM/Pam3CSK4 (TLR1/2 ligands) (19). However, factors was performed using the prcomp function and visualized using the the mechanisms that drive these dysregulated processes are ggbiplot package in R. Differences in protein levels between the two groups unknown. before and after stimulation were statistically assessed using two-way ANOVA and corrected for multiple comparisons using Sidak multiple Therefore, in this study, we sought to investigate the mechanisms comparison test. Differences in protein levels following stimulation after underlying the suppressed responses of UCB monocytes collected corrections for resting levels was statistically assessed using an unpaired http://www.jimmunol.org/ from babies born to obese mothers following LPS stimulation. We t test (GraphPad, San Diego, CA). chose to focus on monocytes in light of the critical role they play in Cell migration assay host defense during the early neonatal period (20). In addition, monocytes are some of the earliest phagocytic cells in the fetus, Migratory potential of PBMCs was measured using CytoSelect 96-well Cell 3 5 appearing as early as week 3 of gestation (21), making them ex- Migration Assay (Cell Biolabs, San Diego, CA). Briefly, 2 10 adult PBMCs were incubated in serum-free media in the upper wells of the quisitely sensitive to reprogramming by the maternal metabolic migration plate, whereas supernatants collected following LPS stimulation environment. We carried out a combination of functional and of UCB monocytes were placed in lower wells. Migration plates were genomics analyses to uncover the impact of pregravid obesity on incubated at 37˚C and 5% CO2 for 5 h. The number of cells that migrated purified UCB monocyte response to LPS. First, we measured into the lower wells was quantified using CyQuant cell proliferation assay by guest on September 26, 2021 differences in cytokine, chemokine, and growth factor production per the manufacturer’s instructions. Absolute numbers of migrated cells were calculated using a standard curve for CyQuant assay with a linear between UCB monocytes collected from babies born to obese and range of fluorescence limited from 50 to 50,000 cells. Supernatant from lean mothers. We then determined differences in transcriptional LPS-stimulated purified adult monocytes (n = 2) was used as a positive activity between these two groups, and finally, we assessed dif- control, whereas media served as negative control. ferences in DNA methylation. Our analyses revealed significant RNA-sequencing dampening of immune mediator production coupled with a re- pressed transcriptional program and changes in levels of methyl- RNA from stimulated and unstimulated monocyte cell pellets was extracted using Zymo Research Direct-zol RNA mini-prep (Zymo Research, San ation in genomic loci that overlap regulatory regions of genes Diego, CA) per the manufacturer’s instructions. RNA concentration and critical for mediating a proinflammatory response in UCB mono- integrity was determined using Agilent 2100 Bioanalyzer. Following rRNA cytes of babies born to obese mothers. depletion using Ribo-Gone rRNA removal kit (Clontech, Mountain View, CA), libraries were constructed using SMARTer Stranded RNA-Seq kit (Clontech). Briefly, rRNA-depleted RNA was fragmented, converted to ds Materials and Methods cDNA and ligated to adapters. The roughly 300 bp-long fragments were Subjects then amplified by PCR and selected by size exclusion. Each library was prepared with unique index facilitating multiplexing of several samples for This research project was approved by the Institutional Ethics Review sequencing. Following quality control for size, quality, and concentrations, Boards of Oregon Health and Science University and University of Cal- libraries were multiplexed and sequenced to single-end 100 bp sequencing ifornia Riverside. All subjects provided signed consent before enrolling in using the Illumina HiSeq2500 platform. the study. The characteristics of this cohort were described earlier (19). For the studies described in this manuscript, a total of 18 UCB mononuclear RNA-sequencing bioinformatics cell (UCBMC) samples collected from nonsmoking women without ges- tational diabetes who had an uncomplicated, singleton gestation were used: Raw reads were assessed for quality using FASTQC (www.bioinformatics. 8 women with a mean age of 31.25 6 4.9 y and a prepregnancy BMI of babraham.ac.uk/projects/fastqc) and trimmed using TrimGalore (www. 21.8 6 1.9 kg/m2 (lean); and 10 women with a mean age of 30.5 6 5.6 and bioinformatics.babraham.ac.uk/projects/trim_galore/). To conform to a prepregnancy BMI of 36.6 6 4.5 kg/m2 (obese). The racial distribution Clontech library prep protocol, five bases from the leading end and three was as follows: 15 Caucasian, 1 Asian American/Pacific Islander, 1 bases at the trailing end were trimmed, with minimum base quality of 30 American Indian/Alaskan Native, and 1 unknown. ensuring reads of a minimum length of 50 bases. Reads were then aligned to the using TopHat2 (https://ccb.jhu.edu/software/tophat/ Cell separation and purification index.shtml). Reads mapping uniquely to exonic regions were counted gene-wise using GenomicRanges package in R. Differentially expressed UCBMCs were obtained by standard density gradient centrifugation over genes (DEGs) were determined using edgeR (https://bioconductor.org/ Ficoll (BD Biosciences, San Jose, CA), resuspended in 10% DMSO/FBS, packages/release/bioc/html/edgeR.html) and defined as those with fold frozen using Mr. Frosty Freezing Containers (Thermo Fisher Scientific, change (FC) $2 and a false discovery rate (FDR) corrected p value #0.05. Waltham, MA), and stored in liquid nitrogen until analysis. Frozen Functional enrichment of DEGs and pathway over-representation was UCBMCs were thawed and UCB monocytes were purified using CD14 performed using InnateDB (http://www.innatedb.com/redirect.do?go=batchGo) The Journal of Immunology 3 and confirmed independently using DAVID (https://david.ncifcrf.gov/summary. ciation regions were extended 5KB upstream of transcription start site jsp). Transcriptional regulation of DEGs was performed using cisRed (http:// (TSS) and 5KB downstream of transcription termination site with the reads www.cisred.org/). All plots were generated in R using gplots. Gene networks per kilobase of transcript per million mapped reads (RPKM) of the gene were generated using Metacore (Thomson Reuters, Washington, DC). assigned as the expression value for the sample (Supplemental Fig. 4). For methylation scores, we considered only cytosines with measured values Methyl-sequencing across all samples. Median b levels within each region for a sample were assigned as the methylation level for the particular sample. b values were DNA cytosine methylation levels in purified resting UCB monocytes were measured taking into consideration differing coverage at different genomic measured at single base resolution using a targeted bisulfite sequencing locations allowing us to perform a weighted correlation analysis. Next, for approach (SureSelectXT Human Methyl-Seq enrichment system; Agilent, each gene, sample-wise weighted correlation between b values and RPKM Santa Clara, CA), focusing on regions where methylation is known to was performed using the rcorr function from Hmisc package (http://biostat. impact gene regulation [cancer tissue–specific differentially methylated mc.vanderbilt.edu/wiki/Main/Hmisc) in R, and corrected for multiple hy- 6 regions (DMRs), GENCODE promoters, CpG islands, shores and shelves pothesis testing generating false discovery rates for each association. 4 kb, DNase I hypersensitive sites, and RefGenes]. Restricting methylation measurements to a relevant portion of the genome increases the statistical power for detecting subtle alterations in gene regulatory regions. Briefly, Results 200 ng genomic DNA was isolated from resting 2–5 3 105 CD14+ UCB monocytes from babies born to obese mothers respond monocytes using Quick-gDNA MiniPrep (Zymo Research, Irvine, CA), poorly to LPS sheared to 100–200 bp using a Covaris Ultrasonicator (Covaris, Woburn, MA), and verified using Agilent 2100 Bioanalyzer. The ends of sheared Our previous studies showed a reduced ability of monocytes from DNA were repaired, 39 adenylated, and ligated with methylated adapters. babies born to obese mothers to respond to LPS (TLR4 agonist) as These DNA fragments were then hybridized with 120 nt biotinylated RNA well as HKLM/Pam3CSK (TLR1/2 ligands) (19). However, these library fragments that recognize methylated DNA regions and isolated Downloaded from using streptavidin beads followed by bisulfite conversion using Zymo EZ experiments were carried out using total UCBMC and only DNA Methylation-Gold Kit (Zymo), which converts unmethylated cyto- measured intracellular levels of TNF-a and IL-6. To specifically sines to uracils. Libraries were then PCR amplified, ligated with unique assess the impact of pregravid BMI on the functional ability of indices, and sequenced on an Illumina NextSeq500 platform to generate UCB monocytes to respond to the TLR4 ligand LPS, CD14+ 60 million 50 bp paired-end reads per sample. Efficiency of bisulfite monocytes were purified from UCBMC collected from babies conversion was measured using 20 pg of unmethylated phage l DNA spiked in with each sample before DNA fragmentation. The bisulfite born to lean (lean group) and obese (obese group) mothers, then nonconversion rate was calculated as the percentage of cytosines se- cultured overnight in the presence or absence of LPS (Fig. 1A). http://www.jimmunol.org/ quenced at cytosine reference positions in the l genome. Six lean and three Production of cytokines, chemokines, and growth factors was obese samples were used in these experiments, and among them three lean determined in the supernatant using a human multiplex immu- and three obese samples were also included in the stimulation studies, allowing us to carry out a pairwise correlation analysis between gene noassay. Principal component analysis clearly indicates that al- expression and DNA methylation patterns. though unstimulated and stimulated UCB monocytes isolated from the lean group formed distinct immune mediator production Methyl-sequencing bioinformatics profiles, those from the obese group had overlapping profiles in- Raw reads were assessed for quality and trimmed to ensure bases with dicative of the lack of response to LPS (Fig. 1B). quality scores ,30 and reads shorter than 50 bases were eliminated. In particular, we observed dampened responses across several Quality-passed reads were aligned to the human genome hg19 using mediators secreted following LPS stimulation including proin- by guest on September 26, 2021 Bismark (http://www.bioinformatics.babraham.ac.uk/projects/bismark/). flammatory mediators IL-1a and IL-1b (Fig. 1C), anti- To measure bisulfite conversion rates, reads were mapped to the phage l genome that was spiked into each library. PCR duplicates in the alignment inflammatory cytokines IL-10 and IL-1RA (Fig. 1D), and che- files were filtered using picard tools (https://broadinstitute.github.io/picard/) mokines CCL4, CCL5, CXCL1, and CXCL10 (Fig. 1E). Except and file conversions were performed using SAMtools (http://samtools. IL-4, we observed no differences in analyte levels produced by sourceforge.net/). Single base resolution methylation calls were made using resting cells from both groups (Fig. 1D). These differences in the the R package methylKit (https://github.com/al2na/methylKit). Differences in levels of secreted immune mediators were observed in the absence methylation between lean and obese groups were measured using a logistic + regression model built in methylKit, allowing us to identify differentially of any variations in the frequencies of either total or CD16 UCB methylated cytosines (DMCs) with at least 25% difference in methylation monocytes from the lean or obese group (19). Moreover, expres- levels and an FDR corrected p value of at least 0.05. DMCs overlapping sion of cell-surface receptors CD14, CD16, and TLR4 was com- X, Y, and mitochondrial genome were eliminated from sub- parable between lean and obese groups (Supplemental Fig. 1A). sequent analyses. We performed optimized region analysis of methylation using empirical DMRs (eDMRs) (https://github.com/ShengLi/edmr), which To determine whether the reduced production of soluble mediators uses a bimodal distribution to identify accurate boundaries of regions or loci by UCB monocytes from the obese group was functionally rele- harboring significant epigenetic changes without a priori assignment of DMR vant, we measured the ability of adult PBMCs to migrate in re- length. DMC and DMR locations were annotated using HOMER (http:// sponse to the supernatant collected from LPS-stimulated UCB homer.salk.edu). DMRs overlapping genic and intergenic regions were enriched using InnateDB, DAVID, and GREAT (http://bejerano.stanford. monocytes from lean and obese groups. In line with the reduced edu/great/public/html/). concentration of several chemokines, we detected a significant reduction in the ability of adult PBMCs to migrate in response to Correlation between DNA methylation and gene expression the LPS-supernatant from the obese group compared with the lean For context-specific association between DNA methylation changes and group (Fig. 1F). gene expression, we first compared DMR annotations with gene expression changes between stimulated cells from both groups after correcting basal UCB monocytes from lean and obese groups are transcription levels in resting cells. For each DMR, the methylation dif- transcriptionally distinct following LPS stimulation ference scores were considered. For the expression of genes associated with We next investigated if the dampened response of neonatal the genomic context, the FC in the obese group relative to the lean group following stimulation was considered after correcting the transcriptional monocytes from the obese group stems from reduced transcrip- levels in corresponding unstimulated cells. Only DEGs with an FDR #10% tional activation following LPS stimulation using RNA-sequencing were considered. (Fig. 1A). As described for the soluble mediators above, the We then used a second approach for genome-wide correlations between transcriptional profiles looked indistinguishable prior to LPS methylation and gene expression. Using an extension of the established statistical framework, sample-specific correlations were evaluated (24) stimulation (Fig. 2A, Supplemental Fig. 1B). However, despite using three lean and three obese samples for which both RNA-sequencing comparable surface and gene expression of TLR4 (Supplemental and methyl-sequencing data were available. Briefly, for each gene, asso- Fig. 1A, 1C), large gene expression changes were observed in the 4 EPIGENETIC IMPACT OF MATERNAL OBESITY ON CORD BLOOD MONOCYTE Downloaded from http://www.jimmunol.org/ by guest on September 26, 2021 FIGURE 1. UCB monocytes from babies born to obese mothers (obese group) generate dampened responses following ex vivo LPS stimulation. (A) Experimental design interrogating the influence of maternal pregravid obesity on transcriptional, epigenetic, and functional responses of cord blood monocytes. (B) Principal Component Analysis of immune mediators released by monocytes from lean and obese groups in the absence and presence of LPS as measured by multiplexed ELISA assay. (C–E) Secreted levels of (C) proinflammatory mediators, (D) regulatory cytokines, and (E) chemokines following LPS stimulation. (F) Bar graphs representing migration of adult PBMC toward the media of LPS-stimulated UCB monocytes from lean and obese groups. *p , 0.05, **p , 0.01, ***p , 0.001, ****p , 0.0001. lean (Fig. 2B, Supplemental Fig. 1D) but not the obese group pression of classic stress responders such as mitochondrial (Fig. 2B, Supplemental Fig. 1E) following LPS stimulation. More encoded SOD2 and zinc transporter SLC39A8 (Fig. 2E). These specifically, whereas 825 upregulated and 890 downregulated observations suggest an overall suppression of the LPS-induced genes were identified in UCB monocytes from the lean group transcriptional program in UCB monocytes from the obese following LPS stimulation (FDR #5%, FC $2) (Fig. 2B), mono- group. Not surprisingly, the number of genes trans-activated by cytes from the obese group show very limited gene-expression transcription factors (TFs) regulated by the metabolic status of changes following stimulation (93 up- and 3 downregulated) monocytes (LXRA/RXRA, PPARA, PPARG, and VDR) was (Fig. 2B). dramatically reduced in the obese group (Fig. 2F). Only 48 genes were upregulated in both the lean and obese groups in response to LPS, with 777 and 45 genes upregulated UCB monocytes from the obese group fail to transcriptionally exclusively in the lean and obese groups respectively (Fig. 2C). activate and suppress key regulatory processes involved in The48sharedDEGsenrichedtopathwaysassociatedwithLPS cellular response to LPS response (Fig. 2D), and comprised genes important for the in- Functional enrichment of the 777 genes upregulated exclusively in flammatory response including cytokines and chemokines UCB monocytes from the lean group following LPS stimulation (IL1B, IL6, IL2RA, IL10,andCXCL1), regulators of T cell ac- revealed significant over-representation of inflammatory innate tivation (CD80, CD274,andSLAMF1), and cell adhesion immune processes, such as JUN kinase activity and MyD88- (MUCL1, LIMK2,andITGB8) (Fig. 2D). However, the magni- dependent TLR signaling pathway (Fig. 3A). Some of the nota- tudes of FC of these shared upregulated genes were lower in the ble DEGs within these processes include genes involved in toll- obese group compared with the lean group (Fig. 2E). Addi- like engagement (TLR1, TLR2, TLR8, PTX3, TNIP1, and tionally, expression of TNFRSF1, the major receptor for TNFA ACOD1), proinflammatory responses (TNF, IL1A, IL15, TNFAIP, in myeloid cells and TNIP3, a negative regulator of NF-kB– and IRGM), cell migration (CCR7, CCL20, CCL19, CXCL3, and induced gene expression program, was reduced in the obese CXCL8), and pathogen pattern recognition (FPR2, NOD1, TLRs 1, group (Fig. 2E). Similar trends were also observed in the ex- 2, and 8) (Fig. 3B). The Journal of Immunology 5

FIGURE 2. LPS stimulation induces a blunted transcriptional response in UCB monocytes from the obese group. (A) Principal Component Analysis Downloaded from of transcriptional profiles of unstimulated UCB monocytes from the lean and obese group. Sample circled in red (obese group) was identified as an outlier and removed from subsequent analysis. (B) Volcano plots representing overall gene expression changes observed in lean (left) and obese (right)

group following LPS stimulation. Genes displaying http://www.jimmunol.org/ statistically significant differences in expression following LPS stimulation compared with resting state are marked in pink. The numbers of DEGs that are either upregulated (Up) or downregulated (Down) are indicated. (C) Venn diagram identifying overlap of upregulated DEGs in lean and obese groups following LPS stimulation. (D) Functional enrichment (biological processes) of the 48 DEGs upregulated in both obese and lean groups carried out using InnateDB. Numbers within the bars in- by guest on September 26, 2021 dicate the number of genes that mapped to the GO term. (E) Scatter plot of FCs and regression line based on best fit of the 48 DEGs upregulated in both groups revealed lower magnitudes of upregu- lation in the obese group. (F) Heatmap of the per- centages of genes regulated by LPS inducible and metabolically sensitive TFs in lean and obese groups. The percentages were calculated based on total number of predicted genes regulated by each TF as determined by cisRed.

Some of the 45 genes upregulated exclusively in UCB mono- include FLI1, a negative regulator of matrix metalloproteases and cytes from the obese group played a role in signaling (CACNA1F, the anti-inflammatory cytokine IL-10 (25), and the long non- MECOM, PPP2R2C, and CSF2) and metabolism (EPHX2, coding RNA oncogene LINC00511, which plays a role in chro- NUBP2, and CHRM3) (Table I). Notable genes within this group matin remodeling (26) (Table I). 6 EPIGENETIC IMPACT OF MATERNAL OBESITY ON CORD BLOOD MONOCYTE Downloaded from

FIGURE 3. UCB monocytes from the lean group display a robust LPS inducible inflammatory tran- scriptional program. (A) Functional enrichment of the 777 genes upregulated exclusively in the lean group carried out using InnateDB. Numbers within the bars indicate the number of genes that mapped to each of http://www.jimmunol.org/ the GO terms. (B) Heatmap displaying expression levels (RPKM) of the 31 genes that mapped to the GO term “inflammatory response.” (C) Functional enrich- ment of 890 genes upregulated only in the lean group following LPS stimulation as reported by InnateDB. Numbers within the bars indicate the number of DEGs that mapped to each of the GO terms. (D) Heatmap of immune-related genes downregulated exclusively in the lean group. by guest on September 26, 2021

A large number of genes were downregulated exclusively in downregulated DEGs directly interact with one another and are UCB monocytes from the lean group. These DEGs enriched to regulated by key TFs AP-1, SMAD3, CREB1, and IRF8 regulatory processes such as mRNA catabolic processes (e.g., (Supplemental Fig. 2B). L ribosomal ), translation initiation, and termination (Fig. 3C, Supplemental Fig. 2A). We also observed downregula- Maternal obesity is associated with global hypomethylation in tion of genes involved in Ag presentation (HLADMA, HLADPA1, UCB monocytes HLADPB1, HLADQA1, HLADQB1, and HLADRA), T cell co- Given the large functional and transcriptional differences between stimulation (CD4, CD86, PAK1, SPN, ICOSLG, and MAP3K14) lean and obese groups, we next wanted to identify the epigenetic [innate immune response, and response to wounding (Fig. 3C, mechanisms by which maternal obesity dysregulates UCB 3D)]. Additionally, network analysis revealed that several of the monocyte response to LPS stimulation. Given previous reports of The Journal of Immunology 7

Table I. Genes exclusively upregulated by LPS in obese group

Gene ID FC Gene Symbol Gene Name ENSG00000166069 12.27 TMCO5A Transmembrane and coiled-coil membrane 5A ENSG00000273478 11.06 N/A Uncharacterized ENSG00000184156 10.31 KCNQ3 Potassium voltage-gated channel subfamily Q member 3 ENSG00000241106 10.25 HLA-DOB MHC, class II, DO b ENSG00000257931 10.23 N/A Uncharacterized ENSG00000215871 10.12 N/A Uncharacterized ENSG00000138028 10.03 CGREF1 Cell growth regulator with EF-hand domain 1 ENSG00000222036 9.79 POTEM POTE ankyrin domain family member M ENSG00000213344 9.63 PCNPP3 PEST containing nuclear protein pseudogene 3 ENSG00000250658 9.61 N/A Uncharacterized ENSG00000224607 9.61 IGKV1D-27 Ig k variable 1D-27 (pseudogene) ENSG00000234320 9.30 N/A Uncharacterized ENSG00000144452 9.15 ABCA12 ATP binding cassette subfamily A member 12 ENSG00000234335 8.87 RPS4XP11 Ribosomal protein S43 pseudogene 11 ENSG00000164400 8.85 CSF2 Colony stimulating factor 2 ENSG00000198307 8.78 N/A Uncharacterized ENSG00000227107 8.76 N/A Uncharacterized ENSG00000123243 8.24 ITIH5 Intera-trypsin inhibitor H chain family member 5 ENSG00000203721 8.19 LINC00862 Long intergenic nonprotein coding RNA 862 Downloaded from ENSG00000102001 8.18 CACNA1F Calcium voltage-gated channel subunit alpha1 F ENSG00000266664 7.78 N/A Uncharacterized ENSG00000112742 7.77 TTK TTK protein kinase ENSG00000259315 7.69 ACTG1P17 Actin g 1 pseudogene 17 ENSG00000157429 7.64 ZNF19 Zinc finger protein 19 ENSG00000268355 7.63 N/A Uncharacterized ENSG00000150672 7.59 DLG2 Discs large MAGUK scaffold protein 2 http://www.jimmunol.org/ ENSG00000065618 7.30 COL17A1 Collagen type XVII a 1 ENSG00000171811 7.29 CFAP46 Cilia and flagella associated protein 46 ENSG00000227036 7.08 LINC00511 Long intergenic nonprotein coding RNA 511 ENSG00000229727 7.06 N/A Uncharacterized ENSG00000163121 7.05 NEURL3 Neuralized E3 ubiquitin protein 3 ENSG00000185834 6.72 RPL12P4 Ribosomal protein L12 pseudogene 4 ENSG00000224424 6.52 PRKAR2A-AS1 PRKAR2A antisense RNA 1 ENSG00000120915 6.22 EPHX2 epoxide 2 ENSG00000085276 5.38 MECOM MDS1 and EVI1 complex ENSG00000147130 5.37 ZMYM3 Zinc finger MYM-type containing 3

ENSG00000133019 5.36 CHRM3 Cholinergic receptor muscarinic 3 by guest on September 26, 2021 ENSG00000074211 5.33 PPP2R2C Protein phosphatase 2 regulatory subunit B g ENSG00000100726 4.74 TELO2 Telomere maintenance 2 ENSG00000239899 4.74 RN7SL674P RNA, 7SL, cytoplasmic 674, pseudogene ENSG00000185803 4.32 SLC52A2 Solute carrier family 52 member 2 ENSG00000171045 3.69 TSNARE1 T-SNARE domain containing 1 ENSG00000095906 3.43 NUBP2 Nucleotide binding protein 2 ENSG00000134326 3.18 CMPK2 Cytidine/uridine monophosphate kinase 2 ENSG00000151702 2.99 FLI1 Fli-1 proto-oncogene, ETS TF List of the 45 DEGs upregulated only in UCB monocytes from the obese group following LPS stimulation. maternal obesity-associated DNA methylation changes in select Functional enrichment of genes differentially methylated in 59 loci in offspring PBMC, and its role in predicting cellular re- regulatory and promoter regions using DAVID revealed over- sponses to biological stimuli like LPS (27), we investigated representation of gene ontology (GO) terms such as immune re- pregravid obesity-induced changes in the methylome of UCB sponse (HLAG, CCR3, CCR6, CCR9, NCR2, ITK, and LSP1) and monocytes. We measured global changes in methylation levels inflammatory response (NFKB1, NFKBIZ, NLRC4, S100A12, of 2,278,000–3,392,222 cytosines at single nucleotide resolution, IL37, IL23A, IL36G, CLEC7A, CXCR6, and CXCL10) (Fig. 4D). using a targeted approach. After filtering methylation changes Moreover, differential methylation of cytosines within CpG is- that were not measured across all samples and those located on X lands was associated with genes involved in regulating IL4 syn- and Y chromosomes, a total of 1,769,148 DMCs were included thesis (ICOSLG, IRF4, and ZFPM1), myeloid cell differentiation in our analysis. Overall, maternal obesity was associated with (CBFA2T3, FAM20C, NFATC1, and ZFPM1), and transcriptional more hypomethylation (8887 cytosines, Db $25%, q # 0.05) repression (HDAC4, NCOR2, PRDM16, CTBP2, and ZFPM1)as than hypermethylation (4838 cytosines, Db #25%, q # 0.05) in determined using GREAT (Fig. 4E). UCB monocytes (Fig. 4A, Supplemental Fig. 3A). These trends were consistent across all genomic contexts (Fig. 4B) and were Maternal obesity is associated with DNA methylation changes uniformly distributed across all chromosomes (Supplemental in genomic regions that regulate expression of key immune Fig. 3B). Methylation changes were more pronounced in 59 genes in UCB monocytes regulatory and promoter regions (Fig. 4B). The global trends in Having established the general pattern of methylation changes in methylation changes agree with methylation patterns in long promoter regions, we next asked if resolution of these DMCs into interspersed nuclear elements, which have been previously DMRs would allow us to identify changes in specific genes with reported as a bona fide measure of methylation levels in the higher confidence and biological relevance. Without a priori as- genome (Fig. 4C) (28). signment of region lengths, eDMR (https://github.com/ShengLi/ 8 EPIGENETIC IMPACT OF MATERNAL OBESITY ON CORD BLOOD MONOCYTE Downloaded from http://www.jimmunol.org/ by guest on September 26, 2021

FIGURE 4. Maternal pregravid obesity is associated with global hypomethylation in UCB monocytes as measured by targeted bisulfite sequencing. (A) Distribution of overall single base resolution methylation changes in UCB monocytes from obese group relative to lean group. (B) Bar graph representing context-specific changes in methylation following correction for lengths of genomic regions indicates particular enrichment at the 59 regulatory and promoter regions. The pie charts above each bar represent the proportion of raw number of hypo- and hypermethylated cytosines in each context after including corrections for genomic lengths. (C) Profile of frequencies of DMCs overlapping long interspersed nuclear elements and CpG islands. (D) Genes with DMCs in 59 regulatory regions preferentially enrich to immune system processes. Numbers within the bars indicate the number of genes that mapped to the GO term. (E) Functional enrichment of genes regulated by DMCs in CpG islands using cis-regulatory model described by GREAT shows over- representation of inflammatory processes. Numbers within the bars indicate the number of genes that mapped to the GO term. edmr) identified 1506 DMRs with 1020 DMRs containing at least DEFB1) (Fig. 5C). Finally, we report hypomethylated DMRs two DMCs (Fig. 5A): 375 hypermethylated (Db $20%, q # 0.05) overlapping first exons of CLECL1 and NLRC3, two genes critical and 645 hypomethylated (Db #20%, q # 0.05) DMRs (Fig. 5B). for the ability of innate cells to activate T cells (Fig. 5C). We Although the lengths of these regions were highly variable observed very limited hypermethylation overlapping metabolic (Supplemental Fig. 3C), trends of over-representation in 59 regu- genes (RHOH, MGAT4A), but large hypermethylated changes oc- latory regions were maintained in both hypomethylated and curring at 39 and promoter regions of immune genes (CD101, ETV3, hypermethylated DMRs (Supplemental Fig. 3D). Importantly, our S100B, HLAE, HLAC, ITK, RHOH, ZFP36L1,andTNFRSF25), analysis revealed hypomethylation in promoters and 59 regulatory and stress-response genes (TXNIP and DDIT4). regions of genes involved in metabolism (CAMK1, PPARG, PAQR8, FOLR2, LDLRAD4, ENO1, and ICA1) (Fig. 5C). Addi- Intergenic DNA methylation changes overlap cis elements tionally, some DMRs overlapped genes important in cell migration regulating defense response and immune development and adhesion in myeloid cells (FGR, ITGAX, CDH5, and LAMB3), Because a large number of DMRs overlapped intergenic regions and defense response (DOK3, TRIM40, TREM1, CD59, IRF5, and (Supplemental Fig. 3D), we asked if these changes overlap cis- The Journal of Immunology 9 Downloaded from http://www.jimmunol.org/

FIGURE 5. Pregravid obesity-associated changes in methyl- ation in UCB monocytes overlap genes critical in metabolism, cell adhesion, and migration. (A) Distribution of the number of cytosines residing in DMRs determined by eDMR. (B) Bar graph representing the number of hyper- and hypomethylated DMRs. (C) Volcano plot of immune genes with DMRs over- D

lapping 39 and 59 regulatory regions. ( ) Functional enrichment by guest on September 26, 2021 of cis-regulatory associations of intergenic DMRs as predicted by GREAT indicates impact on immune system response, de- velopment, and apoptosis. 10 EPIGENETIC IMPACT OF MATERNAL OBESITY ON CORD BLOOD MONOCYTE regulatory regions with possible associations with coding regions. unable to capture subtle differences in sample-specific methyl- We addressed this question using GREAT, with a proximal regu- ation and gene expression. latory assignment of 5 kb upstream and 1 kb downstream of TSS, We therefore used a second, more robust, and sensitive method to and a distal regulatory assignment of 100 kb on either side of TSS. measure sample-wise association between DNA methylation levels This analysis revealed significant methylation changes in in- and gene expression levels, allowing us to measure association tergenic regions regulating defense response (HLA-DRA, HLA- before and after stimulation separately (Supplemental Fig. 4). This DRB5, IFNAR1, IFNGR2, IRF2, CD180, and CXCL1), hemato- approach not only accounts for contributions of intergenic DNA poietic development (, CD28, CEBPA, VEGFA, and EPHA2) methylation changes, but also captures heterogeneity in methyl- and apoptosis (BCL6, KDM2B, PSMA6, FLT4, DAD1, and ation and expression changes among individual samples before ERCC3) (Fig. 5D). and after LPS stimulation. This approach revealed no statistically Given recent reports of potential cross-talk between DNA significant associations in unstimulated cells, but 54 positive and methylation and methylation in transcriptional silencing of negative associations were detected in post-LPS stimulation ex- genes during endotoxin tolerance of in vitro cultured monocytes pression profiles, suggesting that maternal obesity–associated (29), we compared maternal obesity–associated DMRs to histone DNA methylation changes are more predictive of gene expression data available from Human ENCODE and BLUEPRINT consortia changes following ex vivo stimulation, as previously reported in for CD14+CD162 monocytes (http://epigenomesportal.ca/ihec/) adult PBMCs (27) (Table III) (Fig. 7A). because the overwhelming majority of UCB monocytes are Significant associations between gene expression and DNA CD162 (19). This analysis showed that DMRs with large mag- methylation was detected in several genes that regulate monocyte nitudes of methylation differences (both hyper- and hypomethyl- activation and polarization (Fig. 7B). The most striking example Downloaded from ation) overlapped inactive promoters and open chromatin (Fig. of a robust association was the correlation between expression 6A). Because DNA methylation has been previously shown to and methylation of PPARG (Fig. 7C), a critical metabolically modulate TF occupancy in a context-specific fashion (30), we sensitive TF that regulates LPS-inducible gene expression. In explored preferential TF binding sites in different contexts of particular, PPARG promotes monocyte/macrophage differenti- DMRs. Promoter DMRs had preferential binding sites for PU.1/ ation and regulates uptake of oxidized low density lipoprotein

SP1 TF, whereas intergenic DMRs had preferential cJun/AP1 (38, 39). Negative associations were also significant for http://www.jimmunol.org/ binding sites, and open chromatin regions had increased CEBPB NFATC1, a TF that mediates cell differentiation events in re- binding sites (Fig. 6B). Importantly, all of these TFs are critical for sponse to TNF (40), and ZNF516, a methylation target in my- the LPS-inducible transcriptional program in monocytes and eloid leukemia (41). We also detected a positive association for macrophages (31). In comparison, we observed modest changes IRF5, a transcriptional repressor that polarizes macrophages to in both active and inactive enhancers (Fig. 6A). We next de- an inflammatory phenotype and supports a Th1-Th17 response termined whether these modest changes in inducible enhancer (42), and FOXP1, a TF that regulates monocyte differentiation regions could affect levels of LPS-inducible enhancer RNAs via its role in integrin engagement (43, 44). following stimulation. Therefore, we measured expression Because we detected significant associations in genes important changes of 257 long noncoding and enhancer RNA previously for monocyte activation and differentiation, we sought to com- by guest on September 26, 2021 reported in human THP1 cells following a 4 h LPS stimulation putationally analyze the expression level of genes previously (32). This analysis revealed dampened expression of LPS- classified as transcriptional markers of M1 and M2 macrophages inducible enhancers (Fig. 6C), including IL6 and IL10RB en- (45). Because LPS polarizes macrophages to an M1 phenotype, hancer RNA (Fig. 6D) in UCB monocytes from the obese group, we focused on transcriptional levels of M1 and M2 markers in supporting the general theme of dampened transcriptional ac- resting monocytes only. Several cytokine, chemokine, and recep- tivation in these cells. tor markers of M1 macrophages (CCL19, IL12B, IL2RA, CCL20, IL6, and CCR7) showed higher expression in UCB monocytes High pregravid maternal BMI-associated DNA methylation from the lean group compared with monocytes from the obese patterns correlate with altered gene expression in group. However, these cells had greater expression of apoptotic UCB monocytes genes (FAS, XAF1, and BIRC3) and solute carriers (SLC31A2 and Next, we integrated the expression and methylation datasets to SLC7A5) compared with the lean counterparts (Fig. 7D). In con- identify correlations between maternal pregravid obesity-induced trast, M2 cytokine and receptor markers (MSR1, CCL13, CD302, DNA methylation changes in resting UCB monocytes and corre- CLEC7A, P2RY5, and MS4A6A) had higher expression in the sponding gene expression changes following LPS stimulation. We obese group (Fig. 7E), whereas and solute carriers that used two approaches to address this question. Our first method mediate the M2 phenotype were more upregulated in the lean measured gene expression differences between stimulated mono- group. These gene expression profiles strongly suggest that UCB cytes from both groups after correcting for expression levels in monocytes from the lean group are more poised toward differ- resting cells and compared it with methylation changes in those entiating into an M1 phenotype, whereas those from the obese genes in a context specific manner. This analysis revealed that only group are poised toward a regulatory phenotype. 30 DMRs overlapped gene bodies with significant gene expression changes (Table II). Some of these genes included: ADA (adenosine Discussion deaminase), which plays a critical role in monocyte/macrophage In utero exposure to environmental factors can influence cellular maturation (33); ZFP36L1, critical for monocyte/macrophage developmental processes and long-term health outcomes in the differentiation (34); and KLF2 (FC = 2.87), which regulates offspring (46). Of particular importance, maternal nutrition proinflammatory activation of monocytes (35). Additionally, this serves as a critical early environmental signal that can perturb list included two TLR4 interacting genes: CD180, a TLR4 ac- the gestational milieu to influence metabolic plasticity during cessory protein that negatively regulates TLR4 signaling (36), and fetal development (47, 48). This phenomenon has been dem- LY86, which cooperates with TLR4 and CD180 to mediate the onstrated in animal models of nutritional constraint (48–50), response to LPS (37) (Table II). This approach, however, com- intrauterine growth restriction (51–53), and epidemiological pares overall changes in methylation with gene expression and is studies (54, 55). Findings from these studies demonstrate that The Journal of Immunology 11 Downloaded from

FIGURE 6. Pregravid obesity-associated DMRs within intergenic regions are more prevalent in inactive promoters. (A) Violin plots describing methylation changes in DMRs overlapping known monocyte promoter and enhancer profiles predicted by ENSEMBL’s Regulatory Segmentation tool. The http://www.jimmunol.org/ violins represent symmetric distribution of methylation dif- ferences in each region, with violin height representing the range of methylation difference. Inactive promoters and open chromatin regions displayed the broadest range of changes. (B) Enriched motifs for DMRs overlapping different genomic contexts predicted by HOMER. (C) Venn diagram of statisti- cally significant enhancer and long noncoding RNA differen- tially expressed following LPS stimulation in lean and obese groups. (D) Pre- and poststimulation levels of IL-6 and IL- 10RB enhancer RNAs in lean and obese groups indicate sig- by guest on September 26, 2021 nificant changes in the lean group only. **p , 0.01, ****p , 0.0001.

reprogramming of gene expression and rewiring of epigenetic Therefore, in this study, we investigated how the maternal obese circuitry mediate early alteration of offspring cellular and de- environment reprograms the neonatal immune system. velopmental function. However, although pregravid obesity is Initial studies from our laboratory demonstrated that UCB widespread and has been linked to several adverse outcomes for monocytes collected from babies born to obese mothers fail to both mother and child, the mechanisms remain under studied. respond vigorously to TLR1/2 and TLR4 ligands (19). TLRs play a 12 EPIGENETIC IMPACT OF MATERNAL OBESITY ON CORD BLOOD MONOCYTE

Table II. Direct context-specific association between gene expression study that documented reduced IL-6 and TNF-a production in and DNA methylation response to LPS by splenocytes isolated from pups born to obese dams fed a high-fat Western diet during gestation (16). Interest- Gene Methylation Difference Expression FC ingly, we observed reduced IL4 levels in resting cells from the 39 regulatory region obese group. IL4 suppresses LPS-induced production of IL-12 ADA 25.58 2.29 and IL-10 in human peripheral blood monocytes (62) and in- FAN46A 229.05 22.49 hibits adipogenesis by downregulating expression of PPARG and TCF7L1 223.81 4.58 ZFP36L1 26.74 21.79 CEBPA in adipocytes (63). All four of these genes were down- 59 regulatory region regulated in UCB monocytes from the obese group relative to the ANKRD22 229.07 5.17 lean group. Transcriptional analysis using RNA-sequencing con- APMAP 225.09 22.09 firmed the failure of UCB monocytes from the obese group to 2 2 CECR2 21.25 2.67 fully activate the LPS-inducible inflammatory gene expression FAM65B 25.26 1.76 MIR3945HG 227.06 3.57 program. Even genes activated by LPS stimulation in both groups PDCD1LG2 235.49 3.05 showed relatively lower magnitudes of upregulation in the obese TMEM177 21.52 3.79 group compared with the lean group. These transcriptional dif- UBASH3B 225.14 21.75 ferences were seen despite comparable levels of CD14 and TLR4 ZNF385A 225.55 22.32 expression as well as frequencies of nonclassical monocytes Introns + + BHLHE40-AS1 222.77 7.74 (CD14 CD16 ) in unstimulated UCB monocytes, suggesting CD180 226.39 22.86 the involvement of transcriptional/posttranscriptional regulatory Downloaded from FOXN3 224.01 21.63 mechanisms. 2 2 GPR157 28.74 2.53 Several mechanisms can regulate gene expression. Previous KIAA0930 221.57 22.30 KIAA0930 221.93 22.30 studies have shown that maternal pregravid obesity-associated high LINC00862 230.47 7.95 concentrations of glucose, insulin, and fatty acids, as well as LY86 21.61 23.37 hormones (e.g., insulin and leptin), and inflammatory mediators MAFA 22.25 3.05 (e.g., IL-6 and TNF-a) cross the placenta and influence neuroen- http://www.jimmunol.org/ MEGF6 225.46 3.05 MEIKIN 24.45 23.47 docrine and brain development (64). These changes are mediated, RAPGRP1 24.46 2.17 in part, by epigenetic mechanisms, including DNA methyl- RGS2 221.81 22.92 ation, which affect pathways including lipid peroxidation and RPS6KA2 226.85 22.91 corticosteroid-receptor expression (65). Similarly, previous clini- 2 SH3PXD2B 29.64 2.34 cal studies have demonstrated that maternal BMI is associated SLC25A37 224.23 2.33 SORT1 224.42 22.43 with hypomethylation of an offspring’s peripheral blood cells at TBC1D1 222.87 2.80 genes involved in inflammatory and metabolic pathways that can TIMM13 231.52 22.15 persist for years (66, 67). However, no studies to date have in- Exons vestigated the relationship between pregravid maternal obesity- by guest on September 26, 2021 AKR1A1 223.10 22.10 CMYA5 24.93 4.85 induced methylation changes and immune function in the off- FAM46A 229.05 22.49 spring. Moreover, DNA methylation is highly cell specific, and FAM78A 230.60 21.89 measurements made in heterogeneous populations of cells such as GBP5 223.07 2.56 PBMCs do not necessarily allow us to infer contributions of cell KLF2 23.66 2.87 subsets. Therefore, we interrogated methylation changes at a LGALS12 221.56 24.23 SNORD17 222.49 2.977 single cytosine resolution in UCB monocytes and its association TCF7L1 223.81 4.58 with LPS-induced gene expression changes. ZFP36L1 26.74 21.79 As previously shown in total blood leukocytes (66, 67), our ZFP36L1 23.12 21.79 analysis revealed global hypomethylation in UCB monocytes from Summary of direct context-specific changes in genes with statistically significant babies born to obese mothers relative to their lean counterparts. expression and methylation changes. The gene names have been categorized by Profiling these changes in intergenic and gene regulatory regions specific contexts in which methylation changes were observed. Columns two and three represent changes in methylation and gene expression, respectively. revealed significant over-representation of critical innate immune genes with potential functional relevance to both the metabolic status of neonatal monocytes and its ability to participate in critical role in the recognition of pathogens that are relevant to host defense and inflammation. Additional analysis of intergenic neonates, including ones recognized by TLR2 [group B Strepto- regions and regulatory regions showed statistically significant coccus (56), Listeria monocytogenes, Mycoplasma hominis (57), methylation changes in genomic regions critical for monocyte Candida albicans hyphae, and CMV (58)] and TLR4 [Entero- ability to respond to LPS (AP1, CEPBP) and differentiation bacteriaceae, C. albicans blastoconidia (58), and respiratory (PU.1). These TFs dictate both early and late transcriptional syncytial virus (59)]. Dysregulated TLR signaling has been at- responses to LPS and mediate cytokine responses and cell tributed to development of several early diseases in neonates such differentiation events in monocytes, suggesting that maternal as necrotizing enterocolitis (60). Given that babies born to obese obesity-associated changes in DNA methylation levels in UCB mothers are at increased risk of neonatal infections such as necro- monocytes predispose the cells to respond differently following tizing enterocolitis and bacterial sepsis (5, 6, 61), we investigated secondary stimulation such as with LPS. the impact of pregravid obesity on functional, transcriptional, Recent studies in human immune cells have shown that although and epigenetic profiles of UCB monocytes in response to LPS DNA methylation is stable when cells are presented with acute stimulation. biological stimulus like LPS (68), it is predictive of ex vivo In agreement with our previous study (19), we report dampened stimulation responses of PBMCs (27). We therefore used this ra- UCB monocyte cytokine and chemokine responses in the obese tionale to determine if maternal obesity-associated methylation group following LPS. Furthermore, our data agree with a previous changes in resting UCB monocytes were predictive of its tran- The Journal of Immunology 13

Table III. Weighted pairwise association between gene expression and DNA methylation

Gene Chr Start Stop Spearman SBNO2 chr19 1102569 1102675 1 SBNO2 chr19 1177517 1177605 1 PPARG chr3 12392758 12392795 1 ARHGAP26 chr5 142431271 142431331 1 ZNF516 chr18 74201176 74201349 21 AC007278.3 chr2 103052681 103052768 21 PTRH1 chr9 130474243 130474298 21 IRF5 chr7 128579512 128580582 1 NFATC1 chr18 77269145 77269329 21 IGF2BP2 chr3 185429286 185429418 1 MIR3945 chr4 185776752 185776854 1 PPARG chr3 12391954 12392194 1 JAK1 chr1 65363869 65363974 1 ZNF516 chr18 74198756 74198855 21 RP5-968J1.1 chr20 1798583 1798776 21 WDR59 chr16 75034758 75034983 21 ANKRD13A chr12 110450642 110450723 1 PIK3R5 chr17 8867288 8867387 21 CTD-2319I12.1 chr17 58160030 58160123 21 Downloaded from IL1R1 chr2 102680182 102680254 21 MOAP1 chr14 93652953 93653040 1 METTL9 chr16 21663891 21663937 1 RASA3 chr13 114900020 114900137 21 UXS1 chr2 106712973 106713176 1 TIMM13 chr19 2423123 2423287 21 SEP9 chr17 75385143 75385269 21 http://www.jimmunol.org/ RGCC chr13 42037300 42037548 21 AC003104.1 chr17 40425651 40425728 1 TBC1D7 chr6 13303043 13303065 1 TRAJ20 chr14 22993035 22993190 1 FOXP1 chr3 71542816 71542914 1 TRAK1 chr3 42258087 42258157 21 SNAP23 chr15 42803175 42803323 1 ERGIC1 chr5 172314050 172314166 21 MXRA7 chr17 74709515 74709775 1 RP11-589N15.1 chr8 11754669 11755204 1

BIN3 chr8 22503551 22503623 1 by guest on September 26, 2021 AEN chr15 89168304 89168351 1 GPR97 chr16 57701448 57701619 1 ZC3H12A chr1 37937355 37937438 21 ENDOD1 chr11 94841383 94841420 21 TREM1 chr6 41254310 41254471 1 ZNF710 chr15 90547691 90548203 1 STX5 chr11 62573843 62574403 1 RP11-426C22.5 chr16 29188779 29189157 21 TRAJ22 chr14 22993035 22993190 1 ADAM9 chr8 38855985 38856736 1 MBNL1 chr3 152046667 152046751 21 PARN chr16 14530501 14530619 1 SF1 chr11 64539603 64539824 21 FCGR2C chr1 161574652 161575205 1 TMEM80 chr11 692948 693088 1 HSPA7 chr1 161574652 161575205 1 RP11-25K21.6 chr1 161574652 161575205 1 Summary of genes with significant association between DNA methylation and expression. All associations: FDR p value ,0.01. Each gene is provided with genomic location for which a significant association with gene expression is reported.

scriptional response to an ex vivo stimulation. As recently de- levels of M1 genes in lean groups and M2 genes in obese groups, scribed for adult PBMC (27), sample-wise association between suggesting that UCB monocytes from the obese group may be transcriptional and methylation levels suggest that methylation poised toward a regulatory phenotype. levels of genes in resting cells are more predictive of gene- The small number of associations detected suggests that in the expression patterns following LPS stimulation than in resting context of maternal obesity, DNA methylation is not the sole UCB monocytes. The association between methylation and post- regulatory mechanism for gene expression. Alternative regulatory LPS gene expression profiles also suggest differences in polari- mechanisms include histone posttranslational modifications that zation potential of UCB monocytes. Particularly, TFs such as could result in active chromatin remodeling. Indeed, maternal high- PPARG (38, 39), IRF5 (42), and FOXP1 (44) have established fat diet–induced epigenetic changes in inhibitory histone marks in critical roles in regulating the differentiation of monocytes into offspring monocytes such as H3K9 trimethylation have been proinflammatory (M1) or regulatory (M2) macrophages. Indeed, reported in rodent models (69, 70). These changes have direct transcriptional analysis of resting UCB monocytes indicate higher consequences on monocyte function by altering expression of 14 EPIGENETIC IMPACT OF MATERNAL OBESITY ON CORD BLOOD MONOCYTE Downloaded from http://www.jimmunol.org/ by guest on September 26, 2021

FIGURE 7. Pregravid obesity-associated alterations in cytosine methylation within resting UCB monocytes are predictive of LPS-inducible transcrip- tional responses. (A) Bar graph representing number of significant associations reported by pairwise weighted integration of DNA methylation and gene expression profiles following corrections for multiple testing. (B) Assignment of biological functions to 54 genes with significant associations between expression profile and methylation status 5KB surrounding gene body. (C) DNA methylation changes within two DMRs overlapping PPARG gene (top). Gene expression changes in lean (left bottom) and obese (right bottom) groups following stimulation. (D) Scatter plot comparing normalized transcript levels of transcriptional markers of M1 and (E) M2 macrophages in resting cells from lean and obese groups. The x- and y-axes represent RPKMs from lean and obese groups respectively. Only genes outside the 95% confidence interval of the regression line are annotated. genes such as TLR4 and LBP (16). However, the absence of ex- vestigate global changes in histone H3 methylation footprint, pression differences in LPS receptors CD14 and TLR4 in resting combined with expression changes of enhancer and long non- UCB monocytes from obese and lean groups in our study suggests coding RNA using unbiased genome-wide approaches. the involvement of other mechanisms that regulate transcription. Another possibility is that increased in utero exposure to a high- One example of such a mechanism is chromatin remodeling of fat diet resulted in altered TLR4 as well as TLR1/2 signaling due to cis-regulatory regions of LPS-inducible genes mediated by histone activation by fatty acids (71, 72). Alternatively, maternal obesity methylation changes and regulatory RNA. Future studies will in- may lead to an enhanced inflammatory environment within the The Journal of Immunology 15 placenta, which promotes immune tolerance. Indeed, in nonhuman 16. Myles, I. A., N. M. Fontecilla, B. M. Janelsins, P. J. Vithayathil, J. A. Segre, and primates and humans, maternal obesity resulted in the accumu- S. K. Datta. 2013. Parental dietary fat intake alters offspring microbiome and immunity. J. Immunol. 191: 3200–3209. lation of macrophages within the placenta (73), and increased 17. Farley, D., M. E. Tejero, A. G. Comuzzie, P. B. Higgins, L. Cox, S. L. Werner, expression of inflammatory molecules IL-1, TNFA, and MCP-1 S. L. Jenkins, C. Li, J. Choi, E. J. Dick, Jr., et al. 2009. Feto-placental adaptations (74). The contribution of an altered maternal gut microbiome as a to maternal obesity in the baboon. Placenta 30: 752–760. 18. Leibowitz, K. L., R. H. Moore, R. S. Ahima, A. J. Stunkard, V. A. Stallings, consequence of obesity cannot be ruled out either (75). 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