Innate Immune Response to LPS in Airway Epithelium Is Dependent on Chronological Age and Antecedent Exposures

The immune mechanisms for neonatal susceptibility to respiratory pathogens are poorly understood. Given that mucosal surfaces serve as a first line of host defense, we hypothesized that the innate immune response to infectious agents may be developmentally regulated in airway epithelium. To test this hypothesis, we determined whether the expression of IL-8 and IL-6 in airway epithelium after LPS exposure is dependent on chronological age. Tracheas from infant, juvenile, and adult rhesus monkeys were first exposed to LPS ex vivo, and then processed for air–liquid interface primary airway epithelial cell cultures and secondary LPS treatment in vitro. Compared with adult cultures, infant and juvenile cultures expressed significantly reduced concentrations of IL-8 after LPS treatment. IL-8 protein in cultures increased with animal age, whereas LPS-induced IL-6 protein was predominantly associated with juvenile cultures. Toll-like receptor (TLR) pathway RT-PCR arrays showed differential expressions of multiple mRNAs in infant cultures relative to adult cultures, including IL-1α, TLR10, and the peptidoglycan recognition protein PGLYRP2. To determine whether the age-dependent cytokine response to LPS is reflective of antecedent exposures, we assessed primary airway epithelial cell cultures established from juvenile monkeys housed in filtered air since birth. Filtered air–housed animal cultures exhibited LPS-induced IL-8 and IL-6 expression that was discordant with age-matched ambient air–housed animals. A single LPS aerosol in vivo also affected this cytokine profile. Cumulatively, our findings demonstrate that the innate immune response to LPS in airway epithelium is variable with age, and may be modulated by previous environmental exposures

Early life ozone exposure results in dysregulated innate immune function and altered microRNA expression in airway epithelium.

Exposure to ozone has been associated with increased incidence of respiratory morbidity in humans; however the mechanism(s) behind the enhancement of susceptibility are unclear. We have previously reported that exposure to episodic ozone during postnatal development results in an attenuated peripheral blood cytokine response to lipopolysaccharide (LPS) that persists with maturity. As the lung is closely interfaced with the external environment, we hypothesized that the conducting airway epithelium of neonates may also be a target of immunomodulation by ozone. To test this hypothesis, we evaluated primary airway epithelial cell cultures derived from juvenile rhesus macaque monkeys with a prior history of episodic postnatal ozone exposure. Innate immune function was measured by expression of the proinflammatory cytokines IL-6 and IL-8 in primary cultures established following in vivo LPS challenge or, in response to in vitro LPS treatment. Postnatal ozone exposure resulted in significantly attenuated IL-6 mRNA and protein expression in primary cultures from juvenile animals; IL-8 mRNA was also significantly reduced. The effect of antecedent ozone exposure was modulated by in vivo LPS challenge, as primary cultures exhibited enhanced cytokine expression upon secondary in vitro LPS treatment. Assessment of potential IL-6-targeting microRNAs miR-149, miR-202, and miR-410 showed differential expression in primary cultures based upon animal exposure history. Functional assays revealed that miR-149 is capable of binding to the IL-6 3' UTR and decreasing IL-6 protein synthesis in airway epithelial cell lines. Cumulatively, our findings suggest that episodic ozone during early life contributes to the molecular programming of airway epithelium, such that memory from prior exposures is retained in the form of a dysregulated IL-6 and IL-8 response to LPS; differentially expressed microRNAs such as miR-149 may play a role in the persistent modulation of the epithelial innate immune response towards microbes in the mature lung

Early Life Ozone Exposure Results in Dysregulated Innate Immune Function and Altered microRNA Expression in Airway Epithelium

<div><p>Exposure to ozone has been associated with increased incidence of respiratory morbidity in humans; however the mechanism(s) behind the enhancement of susceptibility are unclear. We have previously reported that exposure to episodic ozone during postnatal development results in an attenuated peripheral blood cytokine response to lipopolysaccharide (LPS) that persists with maturity. As the lung is closely interfaced with the external environment, we hypothesized that the conducting airway epithelium of neonates may also be a target of immunomodulation by ozone. To test this hypothesis, we evaluated primary airway epithelial cell cultures derived from juvenile rhesus macaque monkeys with a prior history of episodic postnatal ozone exposure. Innate immune function was measured by expression of the proinflammatory cytokines IL-6 and IL-8 in primary cultures established following <i>in vivo</i> LPS challenge or, in response to <i>in vitro</i> LPS treatment. Postnatal ozone exposure resulted in significantly attenuated IL-6 mRNA and protein expression in primary cultures from juvenile animals; IL-8 mRNA was also significantly reduced. The effect of antecedent ozone exposure was modulated by <i>in vivo</i> LPS challenge, as primary cultures exhibited enhanced cytokine expression upon secondary <i>in vitro</i> LPS treatment. Assessment of potential IL-6-targeting microRNAs miR-149, miR-202, and miR-410 showed differential expression in primary cultures based upon animal exposure history. Functional assays revealed that miR-149 is capable of binding to the IL-6 3′ UTR and decreasing IL-6 protein synthesis in airway epithelial cell lines. Cumulatively, our findings suggest that episodic ozone during early life contributes to the molecular programming of airway epithelium, such that memory from prior exposures is retained in the form of a dysregulated IL-6 and IL-8 response to LPS; differentially expressed microRNAs such as miR-149 may play a role in the persistent modulation of the epithelial innate immune response towards microbes in the mature lung.</p></div

Discharged patients and prognoses of caregivers after two years - Part III of the Berlin deinstitutionalisation study

<p>Airway epithelial cells harvested from one-year-old juvenile rhesus monkeys with prior ozone and/or LPS exposure, as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0090401#pone-0090401-g001" target="_blank">Figure 1</a> were cultured under air-liquid interface conditions and subsequently treated with increasing doses of LPS <i>in vitro</i>. Cultures were evaluated for IL-8 mRNA (A, C) and protein (B, D) expression at 24 h post-treatment. Results show the average +/− SE. *p<0.05, **p<0.01 by two-way ANOVA comparing <i>in vivo</i> exposure and <i>in vitro</i> LPS concentration (n = 4 for each group except filtered air controls n = 5).</p

Effect of postnatal ozone exposure on miR-149, miR-202, miR-410 and miR-let7a expression in juvenile monkey airway epithelial cell cultures.

<p>Comparison of constitutive (0 µg/ml) and LPS-induced (1 µg/ml, 6 h post-treatment) microRNA expression in primary airway epithelial cell cultures derived from one-year-old juvenile rhesus monkeys with prior ozone and/or LPS exposure as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0090401#pone-0090401-g001" target="_blank">Figure 1</a>. (A) miRNA-149, (B) miRNA-202, (C) miRNA-410, (D) miRNA-let7a. Graphs show the average +/− SE of the 2?-(delta CT) where the delta CT =  (microRNA of interest – endogenous control microRNA (RNU-6b)). microRNAs were extracted from n = 3–5 animals per group. Results from Student's t-tests comparing microRNA expression at baseline and post- <i>in vitro</i> LPS treatment are shown with horizontal bars. *p<0.05, **<i>p</i><0.005, ***<i>p</i><0.0003.</p

Assessment of microRNA regulation of IL-6 in human airway epithelial cell cultures.

<p>To evaluate the ability of the putative IL-6-targeting microRNAs to regulate expression of IL-6, the BEAS-2B cell line was transfected with a negative control (NegCO) microRNA or mimics for miR-149, miR-202 or miR-410. (A) Expression of microRNAs in BEAS-2B 24 h post-transfection with a NegCO microRNA or mimics for miR-149, miR-202, or miR-410. Values are reported as the average +/− SE of the 2?-(delta CT) relative to the endogenous control microRNA RNU-6b. (B) IL-6 mRNA and (C) IL-6 protein expression was evaluated in transfected BEAS-2B cells after 24 h. Data are reported as the fold-change in IL-6 mRNA or IL-6 protein as compared to no mimic controls for each experiment. (D) Binding of microRNA mimics to IL-6 mRNA was tested in the HBE1 cell line with the IL-6 3′UTR cloned into a firefly/renilla luciferase plasmid reporter system. For transfection normalization across samples, the relative luciferase units are reported for firefly versus renilla luciferase. Data are from 4 separate experiments. *p<0.05, **p<0.005 by Student's t-test.</p

IL-6 immunofluorescence staining in juvenile monkey trachea following postnatal ozone and/or <i>in vivo</i> LPS.

<p>Trachea cryosections from a representative animal in each exposure group were stained with FITC-conjugated anti-human IL-6 mouse monoclonal antibody (B, D, F, and H). FITC-conjugated mouse IgG1 isotype controls are included for comparison (A, C, E, and G). Images were collected at 20× magnification. Scale bar =  100 µm. White arrows indicate IL-6 staining.</p

Small-Magnitude Effect Sizes in Epigenetic End Points are Important in Children's Environmental Health Studies: The Children's Environmental Health and Disease Prevention Research Center's Epigenetics Working Group.

BackgroundCharacterization of the epigenome is a primary interest for children's environmental health researchers studying the environmental influences on human populations, particularly those studying the role of pregnancy and early-life exposures on later-in-life health outcomes.ObjectivesOur objective was to consider the state of the science in environmental epigenetics research and to focus on DNA methylation and the collective observations of many studies being conducted within the Children's Environmental Health and Disease Prevention Research Centers, as they relate to the Developmental Origins of Health and Disease (DOHaD) hypothesis.MethodsWe address the current laboratory and statistical tools available for epigenetic analyses, discuss methods for validation and interpretation of findings, particularly when magnitudes of effect are small, question the functional relevance of findings, and discuss the future for environmental epigenetics research.DiscussionA common finding in environmental epigenetic studies is the small-magnitude epigenetic effect sizes that result from such exposures. Although it is reasonable and necessary that we question the relevance of such small effects, we present examples in which small effects persist and have been replicated across populations and across time. We encourage a critical discourse on the interpretation of such small changes and further research on their functional relevance for children's health.ConclusionThe dynamic nature of the epigenome will require an emphasis on future longitudinal studies in which the epigenome is profiled over time, over changing environmental exposures, and over generations to better understand the multiple ways in which the epigenome may respond to environmental stimuli

Small-Magnitude Effect Sizes in Epigenetic End Points are Important in Children’s Environmental Health Studies: The Children’s Environmental Health and Disease Prevention Research Center’s Epigenetics Working Group

BACKGROUND: Characterization of the epigenome is a primary interest for children’s environmental health researchers studying the environmental influences on human populations, particularly those studying the role of pregnancy and early-life exposures on later-in-life health outcomes. OBJECTIVES: Our objective was to consider the state of the science in environmental epigenetics research and to focus on DNA methylation and the collective observations of many studies being conducted within the Children’s Environmental Health and Disease Prevention Research Centers, as they relate to the Developmental Origins of Health and Disease (DOHaD) hypothesis. METHODS: We address the current laboratory and statistical tools available for epigenetic analyses, discuss methods for validation and interpretation of findings, particularly when magnitudes of effect are small, question the functional relevance of findings, and discuss the future for environmental epigenetics research. DISCUSSION: A common finding in environmental epigenetic studies is the small-magnitude epigenetic effect sizes that result from such exposures. Although it is reasonable and necessary that we question the relevance of such small effects, we present examples in which small effects persist and have been replicated across populations and across time. We encourage a critical discourse on the interpretation of such small changes and further research on their functional relevance for children’s health. CONCLUSION: The dynamic nature of the epigenome will require an emphasis on future longitudinal studies in which the epigenome is profiled over time, over changing environmental exposures, and over generations to better understand the multiple ways in which the epigenome may respond to environmental stimuli. CITATION: Breton CV, Marsit CJ, Faustman E, Nadeau K, Goodrich JM, Dolinoy DC, Herbstman J, Holland N, LaSalle JM, Schmidt R, Yousefi P, Perera F, Joubert BR, Wiemels J, Taylor M, Yang IV, Chen R, Hew KM, Freeland DM, Miller R, Murphy SK. 2017. Small-magnitude effect sizes in epigenetic end points are important in children’s environmental health studies: the Children’s Environmental Health and Disease Prevention Research Center’s Epigenetics Working Group. Environ Health Perspect 125:–526; http://dx.doi.org/10.1289/EHP59