The immune response to influenza virus is exquisitely sensitive to suppression by 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD); however, the cellular mechanisms underlying the suppressive effects of TCDD are unknown. Mice exposed to TCDD exhibited a dose-responsive increase in mortality following an otherwise non-lethal influenza virus infection. Given that cytotoxic T lymphocytes (CTL) are generally thought to resolve primary infections in the lung, we tested the hypothesis that exposure to TCDD suppresses T-cell responsiveness, leading to decreased CTL in the lung. After infection with influenza virus, naive CD8+ lymphocytes are activated and differentiate in the mediastinal lymph node (MLN). In mice exposed to TCDD and infected with influenza virus, the number of CD8+ MLN cells was reduced 60% compared to vehicle-treated mice. Moreover, MLN cells from TCDD-treated mice failed to develop cytolytic activity, and the production of interleukin (IL)-2 and interferon (IFN)-gamma was suppressed. Exposure to TCDD also altered the production of virus-specific antibodies, decreased the recruitment of CD8+ cells to the lung, reduced the percentage and number of bronchoalveolar lavage cells bearing a CTL phenotype (CD8+CD44hiCD62L(l) degrees ), and suppressed IL-12 levels in the lung. Despite our findings that exposure to TCDD suppressed T cell-dependent functions, the cytolytic activity of lung lavage cells from TCDD and vehicle treated mice was equivalent, and IFN gamma levels in the lungs of mice treated with TCDD were enhanced 10-fold. Thus, while exposure to TCDD suppressed a number of responses associated with the development of adaptive immunity to influenza virus, a direct link between these effects and enhanced susceptibility to influenza remains unclear.

Rationale: Lungs of adult mice exposed to hyperoxia as newborns are simplified and exhibit reduced function much like that observed in people who had bronchopulmonary dysplasia (BPD) as infants. Because survivors of BPD also show increased risk for symptomatic respiratory infections, we investigated how neonatal hyperoxia affected the response of adult mice infected with influenza A virus infection. Objectives: To determine whether neonatal hyperoxia increased the severity of influenza A virus infection in adult mice. Methods: Adult female mice exposed to room air or hyperoxia between Postnatal Days 1 and 4 were infected with a sublethal dose of influenza A virus. Measurements and Main Results: The number of macrophages, neutrophils, and lymphocytes observed in airways of infected mice that had been exposed to hyperoxia as neonates was significantly greater than in infected siblings that had been exposed to room air. Enhanced inflammation correlated with increased levels of monocyte chemotactic protein-1 (CCL2) in lavage fluid, whereas infectionassociated changes in IFN-g, IL-1b, IL-6, tumor necrosis factor-a, KC, granulocyte-macrophage colony-stimulating factor, and macrophage inflammatory protein-1a, and production of virus-specific antibodies, were largely unaffected. Increased mortality of mice exposed to neonatal hyperoxia occurred by Day 14 of infection, and was associated with persistent inflammation and fibrosis. Conclusions: These data suggest that the disruptive effect of hyperoxia on neonatal lung development also reprograms key innate immunoregulatory pathways in the lung, which may contribute to exacerbated pathology and poorer resistance to respiratory viral infections typically seen in people who had BPD.

The Developmental Origins of Health and Disease (DOHaD) paradigm is one of the most rapidly expanding areas of biomedical research. Environmental stressors that can impact on DOHaD encompass a variety of environmental and occupational hazards as well as deficiency and oversupply of nutrients and energy. They can disrupt early developmental processes and lead to increased susceptibility to disease/dysfunctions later in life. Presentations at the fourth Conference on Prenatal Programming and Toxicity in Boston, in October 2014, provided important insights and led to new recommendations for research and public health action. The conference highlighted vulnerable exposure windows that can occur as early as the preconception period and epigenetics as a major mechanism than can lead to disadvantageous "reprogramming" of the genome, thereby potentially resulting in transgenerational effects. Stem cells can also be targets of environmental stressors, thus paving another way for effects that may last a lifetime. Current testing paradigms do not allow proper characterization of risk factors and their interactions. Thus, relevant exposure levels and combinations for testing must be identified from human exposure situations and outcome assessments. Testing of potential underpinning mechanisms and biomarker development require laboratory animal models and in vitro approaches. Only few large-scale birth cohorts exist, and collaboration between birth cohorts on a global scale should be facilitated. DOHaD-based research has a crucial role in establishing factors leading to detrimental outcomes and developing early preventative/remediation strategies to combat these risks.

Many ligands for the aryl hydrocarbon receptor (AhR) are considered endocrine disruptors and carcinogens, and assessment of adverse health effects in humans exposed to such chemicals has often focused on malignancies, including breast cancer. Mammary tissue contains the AhR, and inappropriate activation of the AhR during fetal development causes defects in mammary development that persist into adulthood. However, it is not known whether the extensive differentiation of mammary tissue that occurs during pregnancy is also sensitive to disruption by AhR activation. To examine this, we exposed pregnant C57Bl/6 mice to 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) on days 0, 7, and 14 of pregnancy. Examination of mammary glands on days 9, 12, and 17 of pregnancy and on the day of parturition showed severe defects in development, including stunted growth, decreased branching, and poor formation of lobular alveolar structures. This impaired differentiation was biologically significant, as expression of whey acidic protein in the gland was suppressed, and all pups born to TCDD-treated dams died within 24 h of birth. Analysis of circulating progesterone, prolactin, and estradiol suggest that hormone production was slightly impaired by inappropriate activation of the AhR. However, hormone levels were affected only very late in pregnancy. Given that the observed defects in gland development preceded these hormonal effects, altered hormone levels are an unlikely mechanistic explanation for impaired mammary development. This novel finding that AhR activation during pregnancy disrupts mammary gland differentiation raises questions about the susceptibility of mammary tissue to direct injury by endocrine disrupting agents and the potential for AhR-mediated signaling to adversely affect lactation and breast tissue development in human populations.

Unlike their role in bacterial infection, less is known about the role of neutrophils during pulmonary viral infection. Exposure to pollutant 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD, dioxin) results in excess neutrophils in the lungs of mice infected with influenza A virus. TCDD is the most potent agonist for the aryl hydrocarbon receptor (AhR), and exposure to AhR ligands has been correlated with exacerbated inflammatory lung diseases. However, knowledge of the effects of AhR agonists on neutrophils is limited. Likewise, the factors regulating neutrophil responses during respiratory viral infections are not well characterized. To address these knowledge gaps, we determined the in vivo levels of KC, MIP-1alpha, MIP-2, LIX, IL-6, and C5a in infected mouse lungs. Our data show that these neutrophil chemoattractants are generally produced transiently in the lung within 12-24 h of infection. We also report that expression of CD11a, CD11b, CD49d, CD31, and CD38 is increased on pulmonary neutrophils in response to influenza virus. Using AhR-deficient mice, we demonstrate that excess neutrophilia in the lung is mediated by activation of the AhR and that this enhanced neutrophilia correlates directly with decreased survival in TCDD-exposed mice. Although AhR activation results in more neutrophils in the lungs, we show that this is not mediated by deregulation in levels of common neutrophil chemoattractants, expression of adhesion molecules on pulmonary neutrophils, or delayed death of neutrophils. Likewise, exposure to TCDD did not enhance pulmonary neutrophil function. This study provides an important first step in elucidating the mechanisms by which AhR agonists exacerbate pulmonary inflammatory responses.

Bisphenol A (BPA) is a high-production volume chemical classified as an environmental estrogen and used primarily in the plastics industry. BPA's increased usage correlates with rising BPA levels in people and a corresponding increase in the incidence of asthma. Due to limited studies, the contribution of maternal BPA exposure to allergic asthma pathogenesis is unclear. Using two established mouse models of allergic asthma, we examined whether developmental exposure to BPA alters hallmarks of allergic lung inflammation in adult offspring. Pregnant C57BL/6 dams were gavaged with 0, 0.5, 5, 50, or 500 μg BPA/kg/day from gestational day 6 until postnatal day 21. To induce allergic inflammation, adult offspring were mucosally sensitized with inhaled ovalbumin containing low-dose lipopolysaccharide or ip sensitized using ovalbumin with alum followed by ovalbumin aerosol challenge. In the mucosal sensitization model, female offspring that were maternally exposed to ≥ 50 μg BPA/kg/day displayed enhanced airway lymphocytic and lung inflammation, compared with offspring of control dams. Peritoneally sensitized, female offspring exposed to ≤ 50 μg BPA/kg/day presented dampened lung eosinophilia, compared with vehicle controls. Male offspring did not exhibit these differences in either sensitization model. Our data demonstrate that maternal exposure to BPA has subtle and qualitatively different effects on allergic inflammation, which are critically dependent upon route of allergen sensitization and sex. However, these subtle, yet persistent changes due to developmental exposure to BPA did not lead to significant differences in overall airway responsiveness, suggesting that early life exposure to BPA does not exacerbate allergic inflammation into adulthood.

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The response of CD8+ T cells to influenza virus is very sensitive to modulation by aryl hydrocarbon receptor (AhR) agonists; however, the mechanism underlying AhR-mediated alterations in CD8+ T cell function remains unclear. Moreover, very little is known regarding how AhR activation affects anamnestic CD8+ T cell responses. In this study, we analyzed how AhR activation by the pollutant 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) alters the in vivo distribution and frequency of CD8+ T cells specific for three different influenza A virus epitopes during and after the resolution of a primary infection. We then determined the effects of TCDD on the expansion of virus-specific memory CD8+ T cells during recall challenge. Adoptive transfer of AhR-null CD8+ T cells into congenic AhR+/+ recipients, and the generation of CD45.2AhR−/−→CD45.1AhR+/+ chimeric mice demonstrate that AhR-regulated events within hemopoietic cells, but not directly within CD8+ T cells, underlie suppressed expansion of virus-specific CD8+ T cells during primary infection. Using a dual-adoptive transfer approach, we directly compared the responsiveness of virus-specific memory CD8+ T cells created in the presence or absence of TCDD, which revealed that despite profound suppression of the primary response to influenza virus, the recall response of virus-specific CD8+ T cells that form in the presence of TCDD is only mildly impaired. Thus, the delayed kinetics of the recall response in TCDD-treated mice reflects the fact that there are fewer memory cells at the time of reinfection rather than an inherent defect in the responsive capacity of virus-specific memory CD8+ cells.