DNA Methylation Patterns in CD4+ T Cells of Naïve and Influenza A Virus-Infected Mice Developmentally Exposed to an Aryl Hydrocarbon Receptor Ligand

Abstract

Background: Early life environmental exposures can have lasting effects on the function of the immune system and contribute to disease later in life. Epidemiological studies have linked early life exposure to xenobiotics that bind the aryl hydrocarbon receptor (AhR) with dysregulated immune responses later in life. Among the immune cells influenced by developmental activation of the AhR are $\text{CD}{4}^{+}$ T cells. Yet, the underlying affected cellular pathways via which activating the AhR early in life causes the responses of $\text{CD}{4}^{+}$ T cells to remain affected into adulthood remain unclear.

Objective: Our goal was to identify cellular mechanisms that drive impaired $\text{CD}{4}^{+}$ T-cell responses later in life following maternal exposure to an exogenous AhR ligand.

Methods: C57BL/6 mice were vertically exposed to the prototype AhR ligand, 2,3,7,8-tetrachlorodibenzo-$p$-dioxin (TCDD), throughout gestation and early postnatal life. The transcriptome and DNA methylation patterns were evaluated in $\text{CD}{4}^{+}$ T cells isolated from naïve and influenza A virus (IAV)-infected adult mice that were developmentally exposed to TCDD or vehicle control. We then assessed the influence of DNA methylation-altering drug therapies on the response of $\text{CD}{4}^{+}$ T cells from developmentally exposed mice to infection.

Results: Gene and protein expression showed that developmental AhR activation reduced $\text{CD}{4}^{+}$ T-cell expansion and effector functions during IAV infection later in life. Furthermore, whole-genome bisulfite sequencing analyses revealed that developmental AhR activation durably programed DNA methylation patterns across the $\text{CD}{4}^{+}$ T-cell genome. Treatment of developmentally exposed offspring with DNA methylation-altering drugs alleviated some, but not all, of the impaired $\text{CD}{4}^{+}$ T-cell responses.

Discussion: Taken together, these results indicate that skewed DNA methylation is one of the mechanisms by which early life exposures can durably change the function of T cells in mice. Furthermore, treatment with DNA methylation-altering drugs after the exposure restored some aspects of $\text{CD}{4}^{+}$ T-cell functional responsiveness. https://doi.org/10.1289/EHP7699.

Figure 1.

Gene expression profiles in $\text{CD}{4}^{+}$ T cells of naïve or influenza A virus (IAV)-infected mice developmentally exposed to vehicle or TCDD. (A) Pregnant mice were administered vehicle or TCDD ($1\mathrm{\mu }\mathrm{g}/\mathrm{kg}$ body weight) on GD0, 7, and 14 and 2 d after parturition. $\text{CD}{4}^{+}$ T cells from lymph nodes of naïve or IAV-infected offspring (8–10 wk of age) were purified, RNA was isolated, and RNA-sequencing (RNA-seq) was performed. For immunologically naïve offspring, peripheral lymph nodes from a single mouse were combined, and there were a total of three replicates per treatment group, with 1 offspring from unique dams used per replicate, and 6 total mice used. For IAV-infected offspring, the mediastinal lymph nodes were used. Mice were sacrificed 9 d after infection, and there were three replicates per treatment group, with 4–6 offspring in each unique pool of cells for a total of 30 total mice used. (B) All of the mice (naïve and infected) were sacrificed on the same day with $\text{CD}{4}^{+}$ T-cell purification, RNA isolation, and RNA-seq performed in parallel. The number of differentially expressed genes (DEGs) was assessed. (C,D) Volcano plots of DEGs. On each plot, the direction of change indicates increased (right; red) or decreased (left; green) gene expression with infection compared with $\text{CD}{4}^{+}$ T cells from immunologically naïve (C) vehicle or (D) TCDD developmentally exposed mice. (E) Venn diagram of the number of DEGs that were up-regulated in both vehicle and TCDD groups, or uniquely up-regulated in only one group. (F) Venn diagram of the number of DEGs that were down-regulated by both vehicle and TCDD exposure, or uniquely down-regulated in only one group. (G,H) Ingenuity Pathway Analysis was used to identify cellular pathways affected by DEGs identified in (C) and (D). T-cell relevant pathways were ranked according to $z$-score, in descending order of highest to lowest $z$-score. Graphs depict pathways from (G) IAV-infected vs. naïve vehicle offspring or (H) IAV-infected vs. naïve TCDD offspring. The $x$-axes denote $–\log$($p$-value) for each pathway. Pathways were plotted according to $z$-score, with highest $z$-scores at the top (red) to lowest at the bottom (green). Gray bars were used when a $z$-score was not predicted. Numerical details are provided in Table S1. Graphic art in (A) was manually created or adapted from Servier Medical Art templates (https://smart.servier.com) and licensed under the Creative Commons Attribution 3.0 Unported License agreement (https://creativecommons.org/licenses/by/3.0/). Note: ATM, ataxia telangiectasia mutated protein; BW, body weight; GD, gestational day; IL, interleukin; iCOS, inducible T-cell costimulator; iCOSL, inducible T-cell costimulator ligand; iNOS, inducible nitric oxide synthase; $\text{NF-}\mathrm{\kappa }\mathrm{B}$, nuclear factor kappa-light-chain-enhancer of activated B cells; PND, postnatal day; TCDD, 2,3,7,8-tetrachlorodibenzo-$p$-dioxin; Th, T helper; TNFR, tumor necrosis factor receptor.

Figure 2.

Exhaustion markers on $\text{CD}{4}^{+}$ T cells during IAV infection following developmental AhR activation via maternal exposure to TCDD or vehicle. Vehicle (7 mice) or TCDD (8 mice) developmentally exposed mice were infected with IAV at maturity (6–8 wk of age). Nine days after infection, mice were euthanized and mediastinal lymph node cells stained for flow cytometry. (A,C,E) Bar graphs show the mean number of $\text{CD}{44}^{\text{hi}}\text{CD}{4}^{+}$ T cells from each exposure group that expressed (A) PD1, (C) CTLA4, or (E) TIM3. (B,D,F) Histograms depict the percentage of $\text{CD}{44}^{\text{hi}}\text{CD}{4}^{+}$ T cells from vehicle (gray solid line) or TCDD (orange dashed line) exposure groups that express (B) PD1, (D) CTLA4, or (F) TIM3. All values are $\text{mean}\pm \text{SEM}$. The numerical information, including $p$-values, are provided in Table S2. All offspring within a treatment group were from separate dams. *, $p\le 0.05$, using Student’s $t$-test. Note: AhR, aryl hydrocarbon receptor; CTLA-4, cytotoxic T-lymphocyte-associated protein 4; IAV, influenza A virus; PD1, programmed cell death protein 1; SEM, standard error of the mean; T, TCDD; TCDD, 2,3,7,8-tetrachlorodibenzo-$p$-dioxin; TIM3, T-cell immunoglobulin and mucin domain-3; V, vehicle.

Figure 3.

Evaluation of senescence-associated characteristics of $\text{CD}{4}^{+}$ T cells from the offspring of vehicle and TCDD-treated dams during IAV infection. Adult (6–8 wk of age) developmentally exposed mice were infected with IAV. Mediastinal lymph nodes (MLNs) were analyzed 9 d after infection. (A,C,E,G) The number of $\text{CD}{4}^{+}$ T cells from vehicle (7 mice) or TCDD (8 mice for KLRG1 and p16; 3 mice for CD107a and perforin) developmentally exposed offspring that express (A) KLRG1, (C) p16, (E) CD107a, or (F) perforin are shown as bar graphs. (B,D,F,H) Scatter plots show the percentage of (B) $\mathrm{p}{16}^{+}$, (D) $\text{KLRG}{1}^{+}$, (F) $\text{CD}107{\mathrm{a}}^{+}$, or (H) ${\text{perforin}}^{+}$ $\text{CD}{4}^{+}$ T cells from infected mice that were developmentally exposed to vehicle (left) or TCDD (right). The numerical information is provided in Table S2. All offspring within a group were from separate dams. Values are $\text{mean}\pm \text{SEM}$. *, $p$ $\le 0.05$, using Student’s $t$-test. Note: CD107a, lysosomal-associated membrane protein 1; IAV, influenza A virus; KLRG1, killer cell lectin-like receptor subfamily G member 1; p16, cyclin-dependent kinase inhibitor 2A; SEM, standard error of the mean; SSC-A, side scatter area; TCDD, 2,3,7,8-tetrachlorodibenzo-$p$-dioxin.

Figure 4.

Assessment of $\text{CD}{4}^{+}$ T cells from developmentally exposed mice during primary and recall responses to IAV. (A) Scheme of experimental set up. Mice were developmentally exposed to vehicle (V) or TCDD (T) and experiment was initiated when the offspring were 6–8 wk of age. (B) The number of virus-specific ${\left(\mathrm{I}\text{-}\mathrm{A}{}^{\mathrm{b}}{\mathrm{NP}}_{311–325}\right)}^{+}$ $\text{CD}{4}^{+}$ T cells in the lymph nodes of naïve mice (8 vehicle; 9 TCDD) was quantified using flow cytometry. (C,D) Offspring of vehicle-treated dams and offspring of TCDD-treated dams were challenged with HKx31 (primary infection). (C) On Day 9 post primary infection, the number of virus-specific $\text{CD}{4}^{+}$ T cells in 8 mice from the vehicle exposure group and 8 mice from the TCDD group was determined using flow cytometry. (D) Mice recovered from primary IAV infection for 79 d. The number of virus-specific $\text{CD}{4}^{+}$ T cells was quantified prior to (7 vehicle; 7 TCDD) and 7 d after PR8 infection (secondary infection; 8 vehicle; 9 TCDD). The mice were 17–19 wk of age at the time of the secondary infection. The mean number of cells and $p$-values are provided in Table S3. At each point in time, offspring within the same exposure group were from separate dams. All values are $\text{mean}\pm \text{SEM}$. *, $p\le 0.05$, using Student’s $t$-test. Note: IAV, influenza A virus; LN, lymph node; MLN, mediastinal lymph node; SEM, standard error of the mean; TCDD, 2,3,7,8-tetrachlorodibenzo-$p$-dioxin; 1^°, primary; 2^°, secondary.

Figure 5.

DNA methylation patterns in $\text{CD}{4}^{+}$ T cells from naïve IAV-infected mice that were developmentally exposed to TCDD or vehicle. Mice were developmentally exposed to vehicle or TCDD, as described in Figure 1. Whole-genome bisulfite sequencing was performed using DNA from purified $\text{CD}{4}^{+}$ T cells from naïve or IAV-infected adult mice (8–10 wk of age). For naïve offspring, peripheral lymph nodes from 1 mouse were pooled, and there was a total of three replicates per treatment group, with 1 offspring from unique dams used per replicate, for a total of 6 mice. For IAV-infected offspring mediastinal lymph nodes were used. Mice were sacrificed 9 d after infection, and there were three replicates per treatment group, with 4–6 offspring in each unique pool of cells for a total of 30 total mice used. All of the mice were sacrificed at the same time. (A) Box plots show global DNA methylation levels. The horizontal line denotes the mean, and boxes depict SEM. Differences between groups were evaluated using a two-way ANOVA with Tukey’s HSD post hoc test, and $p$-values are reported in Table S4. Groups with the same letter were not significantly different from each other. (B) The number of differentially methylated regions (DMRs) that were hypo- (blue; left side) or hyper- (red; right side) methylated are shown. The length of the bar is proportionate to the number of DMRs, and the number of DMRs is denoted on the bar segments. (C–F) Modified Manhattan plots show the distribution of hypomethylated (blue dots; left cluster) and hypermethylated (red dots; right cluster) DMRs across chromosomes in $\text{CD}{4}^{+}$ T cells. (C,D) DMRs in $\text{CD}{4}^{+}$ T cells from naïve (C) or IAV-infected TCDD (D) mice compared with vehicle controls. (E,F) DMRs in $\text{CD}{4}^{+}$ T cells from vehicle (E) or TCDD (F) mice during IAV infection compared with naïve controls. The direction of hyper- and hypomethylation in (B) top two comparisons, (C) and (D) refer to the effect of developmental exposure to TCDD, or refers to the effect of infection in (B) bottom two comparisons, (E) and (F). Global methylation levels were analyzed by ANOVA with a Tukey HSD post hoc test. (G–J) DMRs were mapped to the genome. The total numbers of DMRs are shown. The percent of DMRs in intergenic, intron, TSS, TTS, 3′UTR, exon, noncoding, and 5′UTR regions are shown. The following comparisons were made: (G) DMRs from $\text{CD}{4}^{+}$ T cells from naïve TCDD vs. naïve vehicle mice, (H) DMRs from $\text{CD}{4}^{+}$ T Cells from IAV-infected TCDD vs. IAV-infected vehicle mice, (I) DMRs from $\text{CD}{4}^{+}$ T cells from vehicle IAV-infected vs. vehicle naïve mice, and (J) DMRs from $\text{CD}{4}^{+}$ T cells from TCDD IAV-infected vs. TCDD naïve mice. Note: ANOVA, analysis of variance; HSD, honestly significant difference; IAV, influenza A virus; SEM, standard error of the mean; TCDD, 2,3,7,8-tetrachlorodibenzo-$p$-dioxin; TSS, transcription start site; TTS, transcription termination site; UTR, untranslated region.

Figure 6.

Gene expression and DNA methylation data set integration. The same cells were used for whole-genome bisulfite sequencing (WGBS) and RNA-seq. (A,C) Venn diagrams show the overlap of genes with differentially methylated regions (DMRs), assessed by WGBS, and differentially expressed genes (DEGs), assessed by RNAseq. (A) The DEG and DMR overlap shown for $\text{CD}{4}^{+}$ T cells isolated from vehicle mice prior to and during IAV infection. (C) The DEG and DMR overlap for $\text{CD}{4}^{+}$ T cells isolated from naïve and infected TCDD offspring. (B,D) Ingenuity Pathway Analysis was used to identify and rank T-cell relevant cellular pathways associated with genes with differential expression and DMRs (overlap in Venn diagrams). The pathways impacted by IAV infection in $\text{CD}{4}^{+}$ T cells from (B) vehicle or (D) TCDD developmentally exposed offspring were ranked in descending order according to $p$-value. Pathways were colored according to $z$-score from $–2.324$ (diminished activity, green) to 1.606 (enhanced activity, red). Gray bars were used when a $z$-score was not predicted. The numerical data corresponding to (B) and (D) are in Table 5S. Note: GNRH, gonadotropin-releasing hormone; IAV, influenza A virus; iCOS, inducible T-cell costimulator; iCOSL, inducible T-cell costimulator ligand; IL, interleukin; iNOS, inducible nitric oxide synthase; LPS, lipopolysaccharide; MAPK, mitogen-activated protein kinase; $\text{NF-}\mathrm{\kappa }\mathrm{B}$, nuclear factor kappa-light-chain-enhancer of activated B cells; PEDF, pigment epithelium-derived factor; $\text{PKC}\mathrm{\theta }$, protein kinase C theta; PPARa, peroxisome proliferator-activated receptors; RNA-seq, RNA sequencing; sig, signaling; RXRa, retinoid X receptor; TCDD, 2,3,7,8-tetrachlorodibenzo-$p$-dioxin; TCR, T cell receptor; Th, T helper; TNFR, tumor necrosis factor receptor.

Figure 7.

Effects of treatment with S-adenosyl methionine (SAM) on the $\text{CD}{4}^{+}$ T-cell response to IAV infection in developmentally exposed mice. (A) Mice were developmentally exposed to vehicle or TCDD. At weaning (21 d of age), mice were randomly divided and given normal vivarium water to maintain DNA methylation levels or water containing SAM ($0.5\mathrm{mg}/\mathrm{mL}$) to increase DNA methylation levels. At 6–8 wk of age, offspring of dams given vehicle (V; white bar) and TCDD (T; orange bar) were infected with IAV (HKx31). Mediastinal lymph nodes were harvested 9 d after infection, and cells stained for flow cytometry. (B) The total number of $\text{CD}{4}^{+}$ T cells or (C) virus-specific ${\left(\mathrm{I}\text{-}\mathrm{A}{}^{\mathrm{b}}{\mathrm{NP}}_{311–325}\right)}^{+}$ $\text{CD}{4}^{+}$ T cells were quantified from mice on control (black outline) or SAM (purple outline) water. The percentage of (D) activated ($\text{CD}{44}^{\text{hi}}\text{CD}62{\mathrm{L}}^{-}$) or (E) naïve ($\text{CD}{44}^{\text{lo}}\text{CD}62{\mathrm{L}}^{+}$) $\text{CD}{4}^{+}$ T cells from control or SAM mice. The percentage of (F) Th1 (${\text{Tbet}}^{+}\text{CD}{4}^{+}$) or (G) Tfh ($\text{CXCR}{5}^{+}\text{PD}{1}^{+}\text{CD}{44}^{\text{hi}}\text{CD}{4}^{+}$) isolated from control or SAM mice. The number of mice in each group was as follows: control water vehicle (8), control water TCDD (5), SAM water vehicle (8), and SAM water TCDD (6). Due to a limited number of offspring, individual offspring were defined as the statistical unit, rather than the dam. All data shown are $\text{mean}\pm \text{SEM}$. *, $p\le 0.05$, using a two-way ANOVA with Tukey’s HSD and Student’s $t$-test. #, $p\le 0.05$, using Student’s $t$-test. The numerical data in the graphs, including $p$-values are provided in Table S6. Note: ANOVA, analysis of variance, HSD, honestly significant difference; ${\mathrm{H}}_2\mathrm{O}$, water; IAV, influenza A virus; SEM, standard error of the mean; TCDD, 2,3,7,8-tetrachlorodibenzo-$p$-dioxin; Tfh, T follicular helper; Th, T helper.

Figure 8.

Effects of Zebularine (Zeb) on $\text{CD}{4}^{+}$ T cells from developmentally exposed mice after infection with IAV. (A) Mice were developmentally exposed to vehicle or TCDD. At weaning (21 d of age), mice were randomly selected be given normal vivarium water to maintain DNA methylation levels or water containing Zeb ($0.2\mathrm{mg}/\mathrm{mL}$) to decrease DNA methylation levels. At 6–8 wk of age, mice developmentally exposed to vehicle (white bar) and TCDD (orange bar) were infected with IAV. Mediastinal lymph nodes (MLNs) were harvested 9 d after infection, and cells stained for flow cytometry. (B) The number of $\text{CD}{4}^{+}$ T cells or (C) virus-specific ${\left(\mathrm{I}\text{-}\mathrm{A}{}^{\mathrm{b}}{\mathrm{NP}}_{311–325}\right)}^{+}$ $\text{CD}{4}^{+}$ T cells were quantified from MLNs of mice on control (black outline) or Zeb (green outline) water. The percentage of (D) activated ($\text{CD}{44}^{\text{hi}}\text{CD}62{\mathrm{L}}^{-}$) or (E) naïve ($\text{CD}{44}^{\text{lo}}\text{CD}62{\mathrm{L}}^{+}$) $\text{CD}{4}^{+}$ T cells from control or Zeb mice. The percentage of (F) Th1 (${\text{Tbet}}^{+}\text{CD}{4}^{+}$) or (G) Tfh ($\text{CXCR}{5}^{+}\text{PD}{1}^{+}\text{CD}{44}^{\text{hi}}\text{CD}{4}^{+}$) isolated from control or Zeb mice. For each group, 5–9 developmentally exposed offspring were used: control water vehicle (8), control water TCDD (5), Zeb water vehicle (9), and Zeb water TCDD (6). Due to a limited number of offspring, individual offspring were defined as the statistical unit, rather than the dam. All data shown are $\text{mean}\pm \text{SEM}$. *, $p\le 0.05$, using a two-way ANOVA with Tukey’s HSD and Student’s $t$-test. #, $p\le 0.05$, using Student’s $t$-test. The numerical data in the graphs and $p$-values are provided in Table S7. Note: ANOVA, analysis of variance; HSD, honestly significant difference; ${\mathrm{H}}_2\mathrm{O}$, water; IAV, influenza A virus; SEM, standard error of the mean; T, TCDD; Tbet, T-box transcription factor; TCDD, 2,3,7,8-tetrachlorodibenzo-$p$-dioxin; Tfh, T follicular helper; Th, T helper; V, vehicle.