Integrating Proteomes for Lung Tissues and Lavage Reveals Pathways That Link Responses in Allergen-Challenged Mice.

To capture interplay between biological pathways, we analyzed the proteome from matched lung tissues and bronchoalveolar lavage fluid (BALF) of individual allergen-naïve and house dust mite (HDM)-challenged BALB/c mice, a model of allergic asthma. Unbiased label-free liquid chromatography with tandem mass spectrometry (LC-MS/MS) analysis quantified 2675 proteins from tissues and BALF of allergen-naïve and HDM-exposed mice. In comparing the four datasets, we found significantly greater diversity in proteins between lung tissues and BALF than in the changes induced by HDM challenge. The biological pathways enriched after allergen exposure were compartment-dependent. Lung tissues featured innate immune responses and oxidative stress, while BALF most strongly revealed changes in metabolism. We combined lung tissues and BALF proteomes, which principally highlighted oxidation reduction (redox) pathways, a finding influenced chiefly by the lung tissue dataset. Integrating lung and BALF proteomes also uncovered new proteins and biological pathways that may mediate lung tissue and BALF interactions after allergen challenge, for example, B-cell receptor signaling. We demonstrate that enhanced insight is fostered when different biological compartments from the lung are investigated in parallel. Integration of proteomes from lung tissues and BALF compartments reveals new information about protein networks in response to environmental challenge and interaction between intracellular and extracellular processes.

Prenatal microRNA miR-200b Therapy Improves Nitrofen-induced Pulmonary Hypoplasia Associated With Congenital Diaphragmatic Hernia.

OBJECTIVE: We aimed to evaluate the use of miR-200b as a prenatal transplacental therapy in the nitrofen rat model of abnormal lung development and congenital diaphragmatic hernia (CDH). BACKGROUND: Pulmonary hypoplasia (PH) and pulmonary hypertension determine mortality and morbidity in CDH babies. There is no safe medical prenatal treatment available. We previously discovered that higher miR-200b is associated with better survival in CDH babies. Here, we investigate the role of miR-200b in the nitrofen rat model of PH and CDH and evaluate its use as an in vivo prenatal therapy. METHODS: We profiled miR-200b expression during nitrofen-induced PH using RT-qPCR and in situ hybridization in the nitrofen rat model of PH and CDH. The effects of nitrofen on downstream miR-200b targets were studied in bronchial lung epithelial cells using a SMAD luciferase assay, Western blotting and Immunohistochemistry. We evaluated miR-200b as a lung growth promoting therapy ex vivo and in vivo using lung explant culture and transplacental prenatal therapy in the nitrofen rat model. RESULTS: We show that late lung hypoplasia in CDH is associated with (compensatory) upregulation of miR-200b in less hypoplastic lungs. Increasing miR-200b abundance with mimics early after nitrofen treatment decreases SMAD-driven TGF-ß signaling and rescues lung hypoplasia both in vitro and in vivo. Also, prenatal miR-200b therapy decreases the observed incidence of CDH. CONCLUSIONS: Our data indicate that miR-200b improves PH and decreases the incidence of CDH. Future studies will further exploit this newly discovered prenatal therapy for lung hypoplasia and CDH.

MicroRNA 200b is upregulated in the lungs of fetal rabbits with surgically induced diaphragmatic hernia.

OBJECTIVE: Profiling of miR-200b expression and its targets (transforming growth factor [TGF]-ß2 and ZEB2) in the surgical rabbit congenital diaphragmatic hernia (DH) model before and after tracheal occlusion (TO). METHODS: Thirty-eight timed-pregnant rabbits had left DH creation on gestational day (GD) 23. On GD28, 17 randomly selected fetuses had TO. We harvested fetuses at GD23, GD28, or GD30. We calculated lung-to-body weight ratios, processed lungs for miR-200b in situ hybridization and real-time quantitative polymerase chain reaction, and evaluated effects on downstream targets TGF-ß2 or ZEB2. RESULTS: We obtained 16 DH fetuses (n = 7 GD28 and n = 9 GD30), 13 TO fetuses (GD30), and 38 control fetuses (n = 15 GD23, n = 11 GD28, and n = 12 GD30). Diaphragmatic hernia lungs were hypoplastic, and TO resulted in control lung-to-body weight ratio levels. Term miR-200b-3p levels were significantly upregulated in the hypoplastic compared with control ipsilateral lung (1.906 ± 0.90 vs 0.7429 ± 0.44) (P < .01). Fetal TO ipsilateral lungs displayed a variable miR-200b response on in situ hybridization and polymerase chain reaction, with levels similar to control and congenital DH lungs. The TGF-ß2 was unchanged in hypoplastic and TO lungs, and ZEB2 tended to be reduced in TO compared with DH lungs (1.79 [0.4-2.9] vs 0.73 [0.5-1.4]). CONCLUSIONS: Hypoplastic fetal rabbit lungs display upregulation of miR-200b expression although downstream targets are not different from controls. Following TO, fetal rabbit lungs display a variable miR-200b response.

In utero exposure to gestational diabetes mellitus conditions TLR4 and TLR2 activated IL-1beta responses in spleen cells from rat offspring.

Fetal exposure to gestational diabetes mellitus (GDM) is associated with a higher risk of youth-onset insulin resistance and type 2 diabetes. We have previously shown that the rat offspring of GDM dams are insulin resistant when compared to the offspring of lean dams. Since inflammation influences insulin sensitivity, we examined the impact of fetal exposure to GDM on inflammatory responses in the offspring. In rats, we compared inflammatory activity in newborn pups as well as 16week-old young-adult offspring from lean control dams with offspring from high fat and sucrose diet (HFS)-induced GDM dams. To determine whether there are additive effects of exposure to GDM and post-weaning diets, offspring of lean and GDM dams were fed either low fat or HFS diets until 16weeks of age. Plasma levels of interleukin(IL)-1ß were elevated in the offspring of GDM dams. To determine whether this was related to immune reactivity, spleen cells from both the newborn and 16week-old offspring were isolated and reactivity to the toll-like receptor activators, pam3CSK4 and lipopolysaccharides were measured over a 72h timeframe. Spleen cells of GDM dams exhibited sustained stimulation of interleukin(IL)-1ß and IL-10 production, whereas IL-1ß and IL-10 synthesis diminished over time in spleen cells from the offspring of lean dams. Additive effects of GDM exposure and post-weaning HFS diet were not observed, suggesting the effects of GDM on cytokine production are independent of the post-weaning diet. Thus, we conclude that exposure to GDM in utero may condition the immune reactivity of spleen cells.

Tumor necrosis factor regulates NMDA receptor-mediated airway smooth muscle contractile function and airway responsiveness.

We have shown that N-methyl-d-aspartate receptors (NMDA-Rs) are receptor-operated calcium entry channels in human airway smooth muscle (HASM) during contraction. Tumor necrosis factor (TNF) augments smooth muscle contractility by influencing pathways that regulate intracellular calcium flux and can alter NMDA-R expression and activity in cortical neurons and glial cells. We hypothesized that NMDA-R-mediated Ca(2+) and contractile responses of ASM can be altered by inflammatory mediators, including TNF. In cultured HASM cells, we assessed TNF (10 ng/ml, 48 h) effect on NMDA-R subunit abundance by quantitative PCR, confocal imaging, and immunoblotting. We observed dose- and time-dependent changes in NMDA-R composition: increased obligatory NR1 subunit expression and altered regulatory NR2 and inhibitory NR3 subunits. Measuring intracellular Ca(2+) flux in Fura-2-loaded HASM cultures, we observed that TNF exposure enhanced cytosolic Ca(2+) mobilization and changed the temporal pattern of Ca(2+) flux in individual myocytes induced by NMDA, an NMDA-R selective analog of glutamate. We measured airway responses to NMDA in murine thin-cut lung slices (TCLS) from allergen-naive animals and observed significant airway contraction. However, NMDA acted as a bronchodilator in TCLS from house dust mice-challenged mice and in allergen-naive TCLS subjected to TNF exposure. All contractile or bronchodilator responses were blocked by a selective NMDA-R antagonist, (2R)-amino-5-phosphonopentanoate, and bronchodilator responses were prevented by N(G)-nitro-l-arginine methyl ester (nitric oxide synthase inhibitor) or indomethacin (cyclooxygenase inhibitor). Collectively, we show that TNF augments NMDA-R-mediated Ca(2+) mobilization in HASM cells, whereas in multicellular TCLSs allergic inflammation and TNF exposure leads to NMDA-R-mediated bronchodilation. These findings reveal the unique contribution of ionotrophic NMDA-R to airway hyperreactivity.

The transcriptome of nitrofen-induced pulmonary hypoplasia in the rat model of congenital diaphragmatic hernia.

BACKGROUND: We currently do not know how the herbicide nitrofen induces lung hypoplasia and congenital diaphragmatic hernia in rats. Our aim was to compare the differentially expressed transcriptome of nitrofen-induced hypoplastic lungs to control lungs in embryonic day 13 rat embryos before the development of embryonic diaphragmatic defects. METHODS: Using next-generation sequencing technology, we identified the expression profile of microRNA (miRNA) and mRNA genes. Once the dataset was validated by both RT-qPCR and digital-PCR, we conducted gene ontology, miRNA target analysis, and orthologous miRNA sequence matching for the deregulated miRNAs in silico. RESULTS: Our study identified 186 known mRNA and 100 miRNAs which were differentially expressed in nitrofen-induced hypoplastic lungs. Sixty-four rat miRNAs homologous to known human miRNAs were identified. A subset of these genes may promote lung hypoplasia in rat and/or human, and we discuss their associations. Potential miRNA pathways relevant to nitrofen-induced lung hypoplasia include PI3K, TGF-ß, and cell cycle kinases. CONCLUSION: Nitrofen-induced hypoplastic lungs have an abnormal transcriptome that may lead to impaired development.

NMDA receptors mediate contractile responses in human airway smooth muscle cells.

Human airway smooth muscle (HASM) exhibits enhanced contractility in asthma. Inflammation is associated with airway hypercontractility, but factors that underpin these features are not fully elucidated. Glutamate toxicity associated with increased plasma glutamate concentrations was observed in airway inflammation, suggesting that multisubunit glutamate receptors, N-methyl-d-aspartate receptors (NMDA-R) contribute to airway hyperreactivity. We tested the hypothesis that HASM expresses NMDA-R subunits that can form functional receptors to mediate contractile responses to specific extracellular ligands. In cultured HASM cells, we measured NMDA-R subunit mRNA and protein abundance by quantitative PCR, immunoblotting, flow cytometry, and epifluorescence immunocytochemistry. We measured mRNA for a number of NMDA-R subunits, including the obligatory NR1 subunit, which we confirmed to be present as a protein. In vitro and ex vivo functional NMDA-R activation in HASM cells was measured using intracellular calcium flux (fura-2 AM), collagen gel contraction assays, and murine thin-cut lung slices (TCLS). NMDA, a pharmacological glutamate analog, induced cytosolic calcium mobilization in cultured HASM cells. We detected three different temporal patterns of calcium response, suggesting the presence of heterogeneous myocyte subpopulations. NMDA-R activation also induced airway contraction in murine TCLS and soft collagen gels seeded with HASM cells. Responses in cells, lung slices, and collagen gels were mediated by NMDA-R, as they could be blocked by (2R)-amino-5-phosphonopentanoate, a specific NMDA-R inhibitor. In summary, we reveal the presence of NMDA-R in HASM that mediate contractile responses via glutamatergic mechanisms. These findings suggest that accumulation of glutamate-like ligands for NMDA-R associated with airway inflammation contributes directly to airway hyperreactivity.

New insights into lung development and diseases: the role of microRNAs.

MicroRNAs (miRNAs) are short endogenous noncoding RNA molecules (∼ 22 nucleotides) that can regulate gene expression at the post-transcription level. Research interest in the role of miRNAs in lung biology is emerging. MiRNAs have been implicated in a range of processes such as development, homeostasis, and inflammatory diseases in lung tissues and are capable of inducing differentiation, morphogenesis, and apoptosis. In recent years, several studies have reported that miRNAs are differentially regulated in lung development and lung diseases in response to epigenetic changes, providing new insights for their versatile role in various physiological and pathological processes in the lung. In this review, we discuss the contribution of miRNAs to lung development and diseases and possible future implications in the field of lung biology.

Suppression of influenza A virus replication in human lung epithelial cells by noncytotoxic concentrations bafilomycin A1.

Subcellular trafficking within host cells plays a critical role in viral life cycles, including influenza A virus (IAV). Thus targeting relevant subcellular compartments holds promise for effective intervention to control the impact of influenza infection. Bafilomycin A1 (Baf-A1), when used at relative high concentrations (≥10 nM), inhibits vacuolar ATPase (V-ATPase) and reduces endosome acidification and lysosome number, thus inhibiting IAV replication but promoting host cell cytotoxicity. We tested the hypothesis that much lower doses of Baf-A1 also have anti-IAV activity, but without toxic effects. Thus we assessed the antiviral activity of Baf-A1 at different concentrations (0.1-100 nM) in human alveolar epithelial cells (A549) infected with IAV strain A/PR/8/34 virus (H1N1). Infected and mock-infected cells pre- and cotreated with Baf-A1 were harvested 0-24 h postinfection and analyzed by immunoblotting, immunofluorescence, and confocal and electron microscopy. We found that Baf-A1 had disparate concentration-dependent effects on subcellular organelles and suppressed affected IAV replication. At concentrations ≥10 nM Baf-A1 inhibited acid lysosome formation, which resulted in greatly reduced IAV replication and release. Notably, at a very low concentration of 0.1 nM that is insufficient to reduce lysosome number, Baf-A1 retained the capacity to significantly impair IAV nuclear accumulation as well as IAV replication and release. In contrast to the effects of high concentrations of Baf-A1, very low concentrations did not exhibit cytotoxic effects or induce apoptotic cell death, based on morphological and FACS analyses. In conclusion, our results reveal that low-concentration Baf-A1 is an effective inhibitor of IAV replication, without impacting host cell viability.

Chronic exposure to perfluorinated compounds: Impact on airway hyperresponsiveness and inflammation.

Emerging epidemiological evidence reveals a link between lung disease and exposure to indoor pollutants such as perfluorinated compounds (PFCs). PFC exposure during critical developmental stages may increase asthma susceptibility. Thus, in a murine model, we tested the hypothesis that early life and continued exposure to two ubiquitous household PFCs, perfluorooctanoic acid (PFOA) and perflurooctanesulfonic acid (PFOS), can induce lung dysfunction that exacerbates allergen-induced airway hyperresponsiveness (AHR) and inflammation. Balb/c mice were exposed to PFOA or PFOS (4 mg/kg chow) from gestation day 2 to 12 wk of age by feeding pregnant and nursing dams, and weaned pups. Some pups were also sensitized and challenged with ovalbumin (OVA). We assessed lung function and inflammatory cell and cytokine expression in the lung and examined bronchial goblet cell number. PFOA, but not PFOS, without the OVA sensitization/challenge induced AHR concomitant with a 25-fold increase of lung macrophages. PFOA exposure did not affect OVA-induced lung inflammatory cell number. In contrast, PFOS exposure inhibited OVA-induced lung inflammation, decreasing total cell number in lung lavage by 68.7%. Interferon-γ mRNA in the lung was elevated in all PFC-exposed groups. Despite these effects, neither PFOA nor PFOS affected OVA-induced AHR. Our data do not reveal PFOA or PFOS exposure as a risk factor for more severe allergic asthma-like symptoms, but PFOA alone can induce airway inflammation and alter airway function.