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Early life is a critical period for the progressive establishment of immunity in response to environmental stimuli; the impact of airborne challenges on this process is not well defined. In a longitudinal fashion, we determined the effect of episodic house dust mite (HDM) aerosol and ozone inhalation, both separately and combined, on peripheral blood immune cell phenotypes and cytokine expression from 4 to 25weeks of age in an infant rhesus monkey model of childhood development. Immune profiles in peripheral blood were compared with lung lavage at 25weeks of age. Independent of exposure, peripheral blood cell counts fluctuated with chronologic age of animals, while IFNγ and IL-4 mRNA levels increased over time in a linear fashion. At 12weeks of age, total WBC, lymphocyte numbers, FoxP3 mRNA and IL-12 mRNA were dramatically reduced relative to earlier time points, but increased to a steady state with age. Exposure effects were observed for monocyte numbers, as well as CCR3, FoxP3, and IL-12 mRNA levels in peripheral blood. Significant differences in cell surface marker and cytokine expression were detected following in vitro HDM or PMA/ionomycin stimulation of PBMC isolated from animals exposed to either HDM or ozone. Lavage revealed a mixed immune phenotype of FoxP3, IFNγ and eosinophilia in association with combined HDM plus ozone exposure, which was not observed in blood. Collectively, our findings show that airborne challenges during postnatal development elicit measureable cell and cytokine changes in peripheral blood over time, but exposure-induced immune profiles are not mirrored in the lung., (Copyright © 2017 Elsevier Inc. All rights reserved.)

The long-term health effects of wildfire smoke exposure in pediatric populations are not known. The objectives of this study were to determine if early life exposure to wildfire smoke can affect parameters of immunity and airway physiology that are detectable with maturity. We studied a mixed-sex cohort of rhesus macaque monkeys that were exposed as infants to ambient wood smoke from a series of Northern California wildfires in the summer of 2008. Peripheral blood mononuclear cells (PBMCs) and pulmonary function measures were obtained when animals were approximately 3 years of age. PBMCs were cultured with either LPS or flagellin, followed by measurement of secreted IL-8 and IL-6 protein. PBMCs from a subset of female animals were also evaluated by Toll-like receptor (TLR) pathway mRNA analysis. Induction of IL-8 protein synthesis with either LPS or flagellin was significantly reduced in PBMC cultures from wildfire smoke-exposed female monkeys. In contrast, LPS- or flagellin-induced IL-6 protein synthesis was significantly reduced in PBMC cultures from wildfire smoke-exposed male monkeys. Baseline and TLR ligand-induced expression of the transcription factor, RelB, was globally modulated in PBMCs from wildfire smoke-exposed monkeys, with additional TLR pathway genes affected in a ligand-dependent manner. Wildfire smoke-exposed monkeys displayed significantly reduced inspiratory capacity, residual volume, vital capacity, functional residual capacity, and total lung capacity per unit of body weight relative to control animals. Our findings suggest that ambient wildfire smoke exposure during infancy results in sex-dependent attenuation of systemic TLR responses and reduced lung volume in adolescence.

Respiratory tract infections are a leading cause of morbidity and mortality in children under 5 years of age. Increased susceptibility to infection is associated with deficiencies in immunity during early childhood. Airway epithelium represents the first line of mucosal defense against inhaled pathogens. However, little is known about epithelial immune mechanisms in the maturing lung. IL-22 and its receptor IL-22R1 are important in host defense and repair of epithelial barriers. The objective of this study was to determine whether a quantitative difference in IL-22R1 exists between infant and adult airways using the rhesus macaque monkey as a model of childhood lung development. Immunofluorescence staining of tracheal tissue revealed minimal expression of IL-22R1 in epithelium at 1 month of age, with a progressive increase in fluorescence-positive basal cells through 1 year of age. Western blot analysis of tracheal lysates confirmed significant age-dependent differences in IL-22R1 protein content. Further, primary tracheobronchial epithelial cell cultures established from infant and adult monkeys showed differential IL-22R1 mRNA and protein expression in vitro. To begin to assess the regulation of age-dependent IL-22R1 expression in airway epithelium, the effect of histone deacetylase and DNA methyltransferase inhibitors was evaluated. IL-22R1 mRNA in adult cultures was not altered by 5-aza-2'-deoxycytidine or trichostatin A. IL-22R1 mRNA in infant cultures showed no change with 5-aza-2'-deoxycytidine but was significantly increased after trichostatin A treatment; however, IL-22R1 protein did not increase concurrently. These data suggest that IL-22R1 in airway epithelium is regulated, in part, by epigenetic mechanisms that are dependent on chronologic age.

Unlabelled: Influenza is the cause of significant morbidity and mortality in pediatric populations. The contribution of pulmonary host defense mechanisms to viral respiratory infection susceptibility in very young children is poorly understood. As a surrogate to compare mucosal immune responses of infant and adult lungs, rhesus monkey primary airway epithelial cell cultures were infected with pandemic influenza A/H1N1 virus in vitro. Virus replication, cytokine secretion, cell viability, and type I interferon (IFN) pathway PCR array profiles were evaluated for both infant and adult cultures. In comparison with adult cultures, infant cultures showed significantly increased levels of H1N1 replication, reduced alpha interferon (IFN-α) protein synthesis, and no difference in cell death following infection. Age-dependent differences in expression levels of multiple genes associated with the type I IFN pathway were observed in H1N1-infected cultures. To investigate the pulmonary and systemic responses to H1N1 infection in early life, infant monkeys were inoculated with H1N1 by upper airway administration. Animals were monitored for virus and parameters of inflammation over a 14-day period. High H1N1 titers were recovered from airways at day 1, with viral RNA remaining detectable until day 9 postinfection. Despite viral clearance, bronchiolitis and alveolitis persisted at day 14 postinfection; histopathological analysis revealed alveolar septal thickening and intermittent type II pneumocyte hyperplasia. Our overall findings are consistent with the known susceptibility of pediatric populations to respiratory virus infection and suggest that intrinsic developmental differences in airway epithelial cell immune function may contribute to the limited efficacy of host defense during early childhood., Importance: To the best of our knowledge, this study represents the first report of intrinsic developmental differences in infant airway epithelial cells that may contribute to the increased susceptibility of the host to respiratory virus infections. Despite the global burden of influenza, there are currently no vaccine formulations approved for children <6 months of age. Given the challenges of conducting experimental studies involving pediatric patients, rhesus monkeys are an ideal laboratory animal model to investigate the maturation of pulmonary mucosal immune mechanisms during early life because they are most similar to those of humans with regard to postnatal maturation of the lung structure and the immune system. Thus, our findings are highly relevant to translational medicine, and these data may ultimately lead to novel approaches that enhance airway immunity in very young children., (Copyright © 2014, American Society for Microbiology. All Rights Reserved.)

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.

Epidemiology supports a causal link between air pollutant exposure and childhood asthma, but the mechanisms are unknown. We have previously reported that ozone exposure can alter the anatomic distribution of CD25+ lymphocytes in airways of allergen-sensitized infant rhesus monkeys. Here, we hypothesized that ozone may also affect eosinophil trafficking to allergen-sensitized infant airways. To test this hypothesis, we measured blood, lavage, and airway mucosa eosinophils in 3-month old monkeys following cyclical ozone and house dust mite (HDM) aerosol exposures. We also determined if eotaxin family members (CCL11, CCL24, CCL26) are associated with eosinophil location in response to exposures. In lavage, eosinophil numbers increased in animals exposed to ozone and/or HDM. Ozone+HDM animals showed significantly increased CCL24 and CCL26 protein in lavage, but the concentration of CCL11, CCL24, and CCL26 was independent of eosinophil number for all exposure groups. In airway mucosa, eosinophils increased with exposure to HDM alone; comparatively, ozone and ozone+HDM resulted in reduced eosinophils. CCL26 mRNA and immunofluorescence staining increased in airway mucosa of HDM alone animals and correlated with eosinophil volume. In ozone+HDM animal groups, CCL24 mRNA and immunofluorescence increased along with CCR3 mRNA, but did not correlate with airway mucosa eosinophils. Cumulatively, our data indicate that ozone exposure results in a profile of airway eosinophil migration that is distinct from HDM mediated pathways. CCL24 was found to be induced only by combined ozone and HDM exposure, however expression was not associated with the presence of eosinophils within the airway mucosa., (Copyright © 2011 Elsevier Inc. All rights reserved.)

The epidemiologic link between air pollutant exposure and asthma has been supported by experimental findings, but the mechanisms are not understood. In this study, we evaluated the impact of combined ozone and house dust mite (HDM) exposure on the immunophenotype of peripheral blood and airway lymphocytes from rhesus macaque monkeys during the postnatal period of development. Starting at 30 days of age, monkeys were exposed to 11 cycles of filtered air, ozone, HDM aerosol, or ozone+HDM aerosol. Each cycle consisted of ozone delivered at 0.5 ppm for 5 days (8 h/day), followed by 9 days of filtered air; animals received HDM aerosol during the last 3 days of each ozone exposure period. Between 2-3 months of age, animals co-exposed to ozone+HDM exhibited a decline in total circulating leukocyte numbers and increased total circulating lymphocyte frequency. At 3 months of age, blood CD4+/CD25+ lymphocytes were increased with ozone+HDM. At 6 months of age, CD4+/CD25+ and CD8+/CD25+ lymphocyte populations increased in both blood and lavage of ozone+HDM animals. Overall volume of CD25+ cells within airway mucosa increased with HDM exposure. Ozone did not have an additive effect on volume of mucosal CD25+ cells in HDM-exposed animals, but did alter the anatomical distribution of this cell type throughout the proximal and distal airways. We conclude that a window of postnatal development is sensitive to air pollutant and allergen exposure, resulting in immunomodulation of peripheral blood and airway lymphocyte frequency and trafficking.

The use of appropriate extraction and amplification controls for acellular specimens is not standardized in the clinical laboratory community. Extraction controls and checks for inhibitors of amplification in cellular specimens are most often accomplished by amplification of an internal human genomic target. This approach is not feasible for acellular specimens, which may contain little or no amplifiable genomic material. Other specimen types, such as stool, frequently contain amplification inhibitors. Failure to test for these inhibitors can result in the reporting of false-negative results. The goal of this study was to evaluate the use of a T4 bacteriophage as an extraction and amplification control for acellular specimens. The T4 bacteriophage assay was evaluated for use as a control in 290 specimens, including cerebrospinal fluid, serum, and filtered stool. Extraction procedures on two automated instruments were assessed, including the Roche MagNAPure Compact (Roche Diagnostics, Indianapolis, IN) and the QIAGEN BioRobot M48 (QIAGEN, Valencia, CA), along with the manual QIAGEN extraction method. The T4 bacteriophage can be extracted reliably and reproducibly from cerebral spinal fluid, serum, and filtered stool and, therefore, is useful as both an extraction control and inhibitor check for these specimen sources.

Background: The effect of chronic environmental aeroallergen exposure on the immune system and airways has not been experimentally defined in very young children., Objective: The purpose of this study was to determine the immunophenotype of peripheral blood and airway leucocytes in the newborn rhesus macaque monkey, following recurrent aerosol exposure to house dust mite (HDM) (Dermatophagoides farinae)., Methods: A regimen of HDM aerosolization was initiated for 2 h per day, three times per week, starting when rhesus macaque monkeys were 1 week of age. All monkeys were inoculated with diptheria, tetanus, and acellular pertussis vaccine at 5 weeks of age to simulate human infant vaccination schedules., Results: Following 8 weeks of HDM aeroallergen exposure, infant monkeys exhibited a significant reduction in the total peripheral blood lymphocyte numbers and a decreased frequency of peripheral blood CD4+ T lymphocytes with a CD45RA-'memory' immunophenotype. Lavage CD4+ T lymphocytes from HDM-exposed monkeys showed elevated expression of CD25, as well as an increase in CD45RA-/CD62L-/CD11ahigh immunophenotype. Eosinophils were more abundant within airways of HDM-exposed monkeys, accumulating maximally within the trachea., Conclusion: These data demonstrate the development of immunological responses following chronic inhalation of a common environmental allergen during postnatal maturation in the non-human primate.

Twenty-four infant rhesus monkeys (30 days old) were exposed to 11 episodes of filtered air (FA), house dust mite allergen aerosol (HDMA), ozone (O3), or HDMA + O3 (5 days each followed by 9 days of FA). Ozone was delivered for 8 h/day at 0.5 ppm. Twelve of the monkeys were sensitized to house dust mite allergen (Dermatophagoides farinae) at ages 14 and 28 days by subcutaneous inoculation (SQ) of HDMA in alum and intraperitoneal injection of heat-killed Bordetella pertussis cells. Sensitized monkeys were exposed to HDMA aerosol for 2 h/day on days 3-5 of either FA (n = 6) or O3 (n = 6) exposure. Nonsensitized monkeys were exposed to either FA (n = 6) or O3 (n = 6). During the exposure regimen, parameters of allergy (i.e., serum IgE, histamine, and eosinophilia), airways resistance, reactivity, and structural remodeling were evaluated. Eleven repeated 5-day cycles of inhaling 0.5 ppm ozone over a 6-month period had only mild effects on the airways of nonsensitized infant rhesus monkeys. Similarly, the repeated inhalation of HDMA by HDMA-sensitized infant monkeys resulted in only mild airway effects, with the exception of a marked increase in proximal airway and terminal bronchiole content of eosinophils. In contrast, the combined cyclic inhalation of ozone and HDMA by HDMA sensitized infants monkeys resulted in a marked increase in serum IgE, serum histamine, and airways eosinophilia. Furthermore, combined cyclic inhalation of ozone and HDMA resulted in even greater alterations in airway structure and content that were associated with a significant elevation in baseline airways resistance and reactivity. These results suggest that ozone can amplify the allergic and structural remodeling effects of HDMA sensitization and inhalation.

Rats were intratracheally instilled with bleomycin or with silica (quartz) dust to induce lung fibrosis. Several weeks later, purified collagen chains (or collagen digests) were isolated from the lungs of these animals and from age-matched controls instilled intratracheally with saline solution, and the ratios of hydroxylysine to lysine and of the dysfunctional cross-links DHLNL to HLNL were quantified. Collagen from fibrotic lungs had significantly higher ratios of DHLNL:HLNL than did control lungs, 15.5 +/- 4.8 and 17.1 +/- 4.8 vs. 2.3 +/- 0.5 for the silica-instilled and the bleomycin-instilled animals, respectively. The hydroxylysine:lysine ratio was significantly increased for the alpha 1(I) chain, to a value 170% of that of lung collagen from control animals, and for several of its constituent CNBr peptides. Lung tissue was exhaustively digested with collagenase and specific cross-linked peptides were isolated and characterized. The cross-linked alpha 1(I) x alpha 1(I) peptide linked by the residues 87 x 16C, with a ratio of DHLNL:HLNL of 17:1, demonstrated that the increased hydroxylation of the dysfunctional cross-links in fibrotic lung collagen could be accounted for in part by increased hydroxylation of the lysine residue at position 16C of the C-terminal telopeptide of the collagen alpha 1(I) chain. It proved impossible to locate the corresponding N-terminal cross-linked fragment from alpha 1(I) x alpha 1(I) chains, 9N x 930, possibly due to further reactions of this material to form the material referred to as poly(CB6). Isolated poly (CB6) accounted for more than half of the total alpha 1(I)CB6 peptide expected in lung collagen, and had a hydroxylysine:lysine content 2.8 times greater in bleomycin-treated animals than in their age-matched controls. Evidence was also found for a cross-linked alpha 1(III) x alpha 1(I) peptide linking residue 87 from the alpha 1(III) chain with residue 16C from the alpha 1(I) chain; it also had an increased ratio of DHLNL:HLNL. We conclude that the increased hydroxylation of lysine observed in two different animal models of lung fibrosis occurs preferentially at the N- and C-terminal nonhelical extension peptides of the alpha 1(I) collagen chains, and that this apparent specificity of overhydroxylation of fibrotic collagen may have important structural and pathological consequences.

This study was designed to investigate whether the changes in lysine hydroxylation known to occur in hypertrophic tendon occur randomly or at specific lysine residues in the type I collagen molecule. Peptides corresponding to the two known major cross-linking sites of type I collagen (a lysine (or hydroxylysine) at position 9N cross-linked to a hydroxylysine at 930 and a lysine (or hydroxylysine) at position 16C cross-linked to a hydroxylysine at position 87) were prepared by collagenase digestion, size fractionation, and separation by high performance liquid chromatography from normal chicken tendon and from chicken tendon subjected to increased tensile load as a result of muscle hypertrophy. The ratio of the difunctional cross-links dihydroxylysinonorleucine to hydroxylysinonorleucine in normal tendon is 0.75:1; this ratio is increased to 6:1 in hypertrophic tendon. The dihydroxylysinonorleucine to hydroxylysinonorleucine ratio is increased to the same extent in samples of the purified cross-linked peptides derived from both the N-terminal and C-terminal lysine aldehyde residues. On the other hand, the relative hydroxylysine content of preparations of the pooled larger helical peptides obtained by cyanogen bromide digestion of normal and hypertrophic tendons was essentially identical. These results demonstrate that there is a specific increase in hydroxylation of only the N- and C-terminal non-helical lysine residues that participate in the formation of the reducible difunctional cross-links of type I collagen in hypertrophic tendon, while the extent of hydroxylation of lysine residues in the helical regions is not affected. The specific mechanism by which the enzyme lysyl hydroxylase acting on its substrate can distinguish between lysine residues destined to be in non-helical versus helical regions in a nascent collagenous peptide that has not yet attained its final secondary structure remains to be defined.

Rat alveolar macrophages were exposed to silica dust (quartz) suspended in culture medium (SiO2, dry particle size less than 5 microns in diameter) and fluctuation in their cytosolic free calcium content ([Ca2+]i) was detected in cell monolayers with a fluorescent calcium probe (Indo-1AM). Cytosolic free calcium content was correlated with lactate dehydrogenase (LDH) release, an index of cell damage. SiO2 induced a concentration- and time-dependent increase of cytosolic free Ca2+ ion concentration and LDH release. [Ca2+]i was increased about fivefold when cells were exposed to 200 micrograms of SiO2 per milliliter (3 ml per dish) for 2 hr. [Ca2+]i changed within 15 min of SiO2 treatment, whereas LDH release was measurably increased only after 30 min. Chelation of extracellular Ca2+ by 2 mM ethylene glycol bis(beta-aminoethyl ether) N,N'-tetraacetate did not prevent SiO2-induced fluctuation of macrophage [Ca2+]i, but did partially prevent the SiO2-induced increase in LDH release (p less than 0.01). We conclude that a very early event in SiO2-induced damage of alveolar macrophages involves mobilization of intracellular calcium pools to increase [Ca2+]i. These results suggest that SiO2-induced macrophage damage, a key event in the development of silicosis, may involve perturbation of intracellular calcium homeostasis.

Collagen synthesized by tissue minces from lungs of rats administered 1 unit of bleomycin by intratracheal instillation 1 or 2 wk earlier contained relatively more hydroxylysine than did collagen made by lungs from saline-instilled control animals. Most, if not all, of the relative increase in lysine hydroxylation could be localized to the alpha 1 (I) chain of type I collagen. Lung homogenates from bleomycin-treated rats showed increased activity of lysyl hydroxylase (EC 1.14.11.4), the enzyme catalyzing the conversion of collagen-bound lysine to hydroxylysine. Thus, the increased hydroxylation of lysine and of lysine-derived cross-links previously observed in collagen of diseased human lungs and in animal models of lung fibrosis is reflected in an in vitro system.

A hitherto unexpected synergism between the oxidant air pollutants ozone or nitrogen dioxide and a respirable-sized aerosol of ammonium sulfate was observed during controlled exposures of rats to these substances. Response of rat lungs to these pollutants was quantitated by measurement of apparent collagen synthesis rates in vitro by lung minces from exposed animals. Dose-response curves to either O3 or NO2 were altered in the presence of 5 mg/m3 of (NH4)2SO4 aerosol. Morphometric and histologic observations of lungs from rats exposed to high levels of ozone, with and without concurrent exposure to the (NH4)2SO4 particles, confirmed such synergistic effects. In a separate set of experiments, rats were exposed at near ambient levels to mixtures of ozone and sulfuric acid aerosol (submicron-sized aerosol). Potentiation of ozone effects on lung collagen synthesis rates was also observed in these experiments. These observations may have broad implications for the appropriate evaluation of laboratory data in the setting of ambient air quality standards and/or threshold limit values for occupational safety.

The effects of vitamin B-6 deficiency and ozone exposure on selected features of connective tissue metabolism in lung were investigated in groups of weanling male rats fed one of three diets: B-6-supplemented, fed ad lib; B-6-deficient, fed ad lib; or B-6-supplemented, restricted to the food intake of deficient rats for 5 weeks. Also, perinatal rat pups were studied that were nursed from dams fed one of the 3 diets from parturition to day 15 of lactation. During the final week of each experiment, half of the rats in each of the groups were exposed to 0.64 ppm of ozone (23.5 h per day). The collagen and elastin content, collagen synthesis rate, total protein synthesis rate, and lysyloxidase activity of lungs were measured. Perinatal pups rendered vitamin B-6-deficient were particularly sensitive to ozone exposure (65% died as compared to fewer than 5% of the ad lib or food-restricted controls). When L-proline incorporation into collagen and total protein was investigated using lung minces, food restriction and B-6-deficiency resulted in about one-half the incorporation normally observed. Total lung lysyl oxidase activity was also decreased in B-6-deficient and food-restricted rats compared to B-6-supplemented rats fed ad lib. Exposure to ozone resulted in increased lysyl oxidase activity and collagen synthesis in lungs from B-6-supplemented rats, but such responses were not observed in B-6-deficient or food-restricted (FR) rats exposed to ozone.

Bleomycin is an antineoplastic agent that causes a dose-related lung fibrosis that limits its therapeutic effectiveness. It has been proposed that the cellular toxicity and antitumor effects of bleomycin occur by formation of O2-Fe(II)-bleomycin complexes that degrade DNA and release O2- and OH radicals that attack other cellular components. Twice daily injections of the iron chelator deferoxamine were utilized in an attempt to ameliorate bleomycin-induced lung fibrosis. They failed to diminish bleomycin-induced lung inflammation and fibrosis in rats.

Two groups of weanling or young adult rats were fed ad lib casein-based diets containing 4 or 16% protein. Food was restricted in a third group (fed the 16% protein diet) to the amount consumed daily by rats (adult or weanlings) fed the 4% diet. After 3 weeks (weanlings) or 1, 3 or 5 weeks (adults), one-half of the rats in each group were exposed to 0.64 ppm (1.28 mg/m3) of ozone for 7 days (23.5 h each day). Several parameters were then evaluated related to lung connective tissue metabolism including: (1) total lung hydroxyproline, (2) total lung elastin, (3) apparent rates for lung collagen synthesis and elastin accumulation and (4) lung and body weights. In general, the response to protein deficiency and food restriction was more pronounced than to ozone exposure. Protein deficiency and food restriction resulted in decreased lung size and collagen content. However, the ability of lung to respond to ozone (in relative terms) was not altered by changes in diet as assessed by changes in lung weight or the collagen synthetic rate.