Although Piwi proteins and Piwi-interacting RNAs (piRNAs) genetically repress transposable elements (TEs), it is unclear how the highly diverse piRNA populations direct Piwi proteins to silence TE targets without silencing the entire transcriptome. To determine the capacity of piRNA-mediated silencing, we introduced reporter genes into Drosophila OSS cells, which express microRNAs (miRNAs) and piRNAs, and compared the Piwi pathway to the Argonaute pathway in gene regulation. Reporter constructs containing several target sites that were robustly silenced by miRNAs were not silenced to the same degrees by piRNAs. However, another set of reporters we designed to enable a large number of both TE-directed and genic piRNAs to bind were robustly silenced by the PIWI/piRNA complex in OSS cells. These reporters show that a bulk of piRNAs are required to pair to the reporter's transcripts and not the reporter's DNA sequence to engage PIWI-mediated silencing. Following our genome-wide study of PIWI-regulated targets in OSS cells, we assessed candidate gene elements with our reporter platform. These results suggest TE sequences are the most direct of PIWI regulatory targets while coding genes are less directly affected by PIWI targeting. Finally, our study suggests that the PIWI transcriptional silencing mechanism triggers robust chromatin changes on targets with sufficient piRNA binding, and preferentially regulates TE transcripts because proteincoding transcripts lack a threshold of targeting by piRNA populations. This reporter platform will facilitate future dissections of the PIWI-targeting mechanism.

Successfully fighting infection requires a properly tuned immune system. Recent epidemiological studies link exposure to pollutants that bind the aryl hydrocarbon receptor (AHR) during development with poorer immune responses later in life. Yet, how developmental triggering of AHR durably alters immune cell function remains unknown. Using a mouse model, we show that developmental activation of AHR leads to long-lasting reduction in the response of CD8+ T cells during influenza virus infection, cells critical for resolving primary infection. Combining genome-wide approaches, we demonstrate that developmental activation alters DNA methylation and gene expression patterns in isolated CD8+ T cells prior to and during infection. Altered transcriptional profiles in CD8+ T cells from developmentally exposed mice reflect changes in pathways involved in proliferation and immunoregulation, with an overall pattern that bears hallmarks of T cell exhaustion. Developmental exposure also changed DNA methylation across the genome, but differences were most pronounced following infection, where we observed inverse correlation between promoter methylation and gene expression. This points to altered regulation of DNA methylation as one mechanism by which AHR causes durable changes in T cell function. Discovering that distinct gene sets and pathways were differentially changed in developmentally exposed mice prior to and after infection further reveals that the process of CD8+ T cell activation is rendered fundamentally different by early life AHR signaling. These findings reveal a novel role for AHR in the developing immune system: regulating DNA methylation and gene expression as T cells respond to infection later in life.

SummaryRecent studies have linked health fates of children to environmental exposures of their great grandparents. However, few studies have considered whether ancestral exposures influence immune function across generations. Here, we report transgenerational inheritance of altered T cell responses resulting from maternal (F0) exposure to the aryl hydrocarbon receptor ligand 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD). Since F0 exposure to TCDD has been linked to transgenerational transmission of reproductive problems, we asked whether maternal TCDD exposure also caused transgenerational changes in immune function. F0 exposure caused transgenerational effects on the CD8+ T cell response to influenza virus infection in females but not in males. Outcrosses showed changes were passed through both parental lineages. These data demonstrate that F0 exposure to an aryl hydrocarbon receptor (AHR) agonist causes durable changes to immune responses that can affect subsequent generations. This has broad implications for understanding how the environment of prior generations shapes susceptibility to pathogens and antiviral immunity in later generations.

Early life environmental exposures drive lasting changes to the function of the immune system and can contribute to disease later in life. One of the ways environmental factors act is through cellular receptors. The aryl hydrocarbon receptor (AHR) is expressed by immune cells and binds numerous xenobiotics. Early life exposure to chemicals that bind the AHR impairs CD4 + T cell responses to influenza A virus (IAV) infection in adulthood. However, the cellular mechanisms that underlie these durable changes remain poorly defined. Transcriptomic profiling of sorted CD4 + T cells identified changes in genes involved in proliferation, differentiation, and metabolic pathways were associated with triggering AHR during development. Functional bioassays confirmed that CD4 + T cells from infected developmentally exposed offspring exhibit reduced proliferation, differentiation, and cellular metabolism. Thus, developmental AHR activation shapes T cell responsive capacity later in life by affecting integrated cellular pathways, which collectively alter responses later in life. Given that coordinated shifts in T cell metabolism are essential for T cell responses to numerous challenges, and that humans are constantly exposed to many different types of AHR ligands, this has far-reaching implications for how AHR signaling, particularly during development, durably influences T cell mediated immune responses across the lifespan.

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 CD4 + T cells. Yet, the underlying affected cellular pathways via which activating the AhR early in life causes the responses of CD4 + T cells to remain affected into adulthood remain unclear. OBJECTIVE: Our goal was to identify cellular mechanisms that drive impaired CD4 + 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 CD4 + 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 methylationaltering drug therapies on the response of CD4 + T cells from developmentally exposed mice to infection. RESULTS: Gene and protein expression showed that developmental AhR activation reduced CD4 + 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 CD4 + T-cell genome. Treatment of developmentally exposed offspring with DNA methylation-altering drugs alleviated some, but not all, of the impaired CD4 + 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 CD4 + T-cell functional responsiveness.