Lung RNA Research Articles

Measuring fetal brain and lung transcripts in amniotic fluid supernatant: a comparison of digital PCR and RT-qPCR methods

Purpose: Amniotic fluid (AF) cell-free RNA is a promising source of information regarding fetal physiology. Digital PCR (dPCR) is a direct approach to nucleic acid detection that reports absolute transcript copy number. The aim of this study was to compare quantification of cell-free fetal brain and lung RNA transcripts in AF by reverse transcription-qPCR (RT-qPCR) and dPCR.Material and methods: Prospective hospital-based study was performed in 2016–2017. Pulmonary genes were quantified in term AF samples collected at elective cesarean birth; neurodevelopmental genes were measured in preterm samples (<34 weeks) obtained from women undergoing clinically-indicated amniocentesis.Results: All 11 women in the term cohort had three lung transcripts and a reference gene successfully amplified from their AF supernatant using RT-qPCR and dPCR. SFTPC was the most abundant lung transcript, present in higher concentrations than the reference gene in seven of the eleven samples. Neurodevelopmental gene transcripts in 12 preterm pregnancies were less reliably detected by both methods and were present in low copy numbers (<10 copies/μl). We observed significant positive correlations between transcript quantification by RT-qPCR and dPCR.Conclusion: This study confirms the presence of several potential mRNA markers of lung and brain development with dPCR and RT-qPCR, and a high correlation between the two methods. Transcripts of presumed fetal brain origin are present in very low copy numbers, which presents challenges to their feasibility as biomarkers of neurodevelopment.

Abstract 682: Novel Mesenchyme Homeobox2-target transcription axes are involved in cancer-drug resistance, overall survival and therapy prognosis in lung cancer patients: a functional epigenome wide study

Abstract Lung cancer remains the most progressive malignant disease strongly resistant to oncological therapies including platinum-derived cancer drugs and Epidermal Growth Factor Receptor (EGFR)-Tyrosine Kinases Inhibitors (TKIs). Homeobox-related gene (HOX) transcription factors as Mesenchyme HOX-2 (MEOX2) have previously been associated with cancer-drug resistance, progression and/or clinical prognosis in lung cancer patients. However, transcriptional mechanisms epigenetically modulated have not totally been elucidated in lung cancer therapy resistance. Here an epigenomic strategy was conducted to identify a novel MEOX2 gene sequence-promoter targets profile, associated or involved in therapy resistance mechanisms in human lung cancer. For that chromatin from human non-small cell lung carcinomas (NSCLC), MEOX2 versus RNA Pol II immunoprecipitation and hybridization assays using gene promoter tiling-arrays and bioinformatics analyses were performed, while a set of quantitative and functional assays with clinical-outcome prognosis validation predictions analyses, were assessed. Stringent bioinformatics results identified a common profile of 13 gene promoter sequences, which included in others Sonic Hedgehog-GLI-1, ALDH1A2, MMP24, RUFY3 and ZEB1 (FDR≤0.1) in NSCLC patients with different clinical outcome data. GLI-1 gene promoter-sequences upstream -2,192 to -109 quantitatively validated, were occupied by MEOX2 and RNA Pol II in both NSCLC cell lines and NSCLC patients, as well as consistently enriched with the histone activation marks H3K27Ac and H3K4me3, in addition, confirmed by the ENCODE database bioinformatics analyses. Furthermore, a set of genetic silencing functional assays validated a novel transcriptional MEOX2-GLI1 axis in a cisplatinum dose-dependent manner, involved in cellular migration, invasion, and proliferation capacity. Finally, MEOX2-GLI1 axis expression was clinically validated and analyzed using Kaplan-Maier survival analyses on an independent cohort of 90 NSCLC patients, identifying a significant MEOX2-dependent GLI-1 overexpression statistically associated with clinical poorer overall survival prognosis and treatment response into cisplatinum-based first-line therapy and/or second-line EGFR-TKIs target therapy protocols. In conclusion, a chromatin-immunoprecipitation and epigenome-wide analysis based on the MEOX2-transcriptional occupation study, identified novel MEOX2-transcriptional gene promoter axes involved in embryonic development, oxidoreductase activity, matrix metalloprotease, cellular polarity, epithelial-mesenchyme phenotype, cancer cell migration and invasion pathways. Promoting cisplatinum-based resistance mechanisms and EGFR-TKIs based therapy response prognosis in human lung cancer. Note: This abstract was not presented at the meeting. Citation Format: Federico Avila Moreno, Leonel Armas lopez, Patricia Piña-Sánchez, Oscar Arrieta, Enrique Gúzman de Alba, Abril Marcela Herrera-Solorio, Blanca Ortiz-Quintero, Patricio Santillán-Doherty, David C Christiani, Joaquín Zúñiga. Novel Mesenchyme Homeobox2-target transcription axes are involved in cancer-drug resistance, overall survival and therapy prognosis in lung cancer patients: a functional epigenome wide study [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 682. doi:10.1158/1538-7445.AM2017-682

Pulmonary fibrosis requires cell-autonomous mesenchymal fibroblast growth factor (FGF) signaling

Idiopathic pulmonary fibrosis (IPF) is characterized by progressive pulmonary scarring, decline in lung function, and often results in death within 3-5 five years after diagnosis. Fibroblast growth factor (FGF) signaling has been implicated in the pathogenesis of IPF; however, the mechanism through which FGF signaling contributes to pulmonary fibrosis remains unclear. We hypothesized that FGF receptor (FGFR) signaling in fibroblasts is required for the fibrotic response to bleomycin. To test this, mice with mesenchyme-specific tamoxifen-inducible inactivation of FGF receptors 1, 2, and 3 (Col1α2-CreER; TCKO mice) were lineage labeled and administered intratracheal bleomycin. Lungs were collected for histologic analysis, whole lung RNA and protein, and dissociated for flow cytometry and FACS. Bleomycin-treated Col1α2-CreER; TCKO mice have decreased pulmonary fibrosis, collagen production, and fewer α-smooth muscle actin-positive (αSMA+) myofibroblasts compared with controls. Freshly isolated Col1α2-CreER; TCKO mesenchymal cells from bleomycin-treated mice have decreased collagen expression compared with wild type mesenchymal cells. Furthermore, lineage labeled FGFR-deficient fibroblasts have decreased enrichment in fibrotic areas and decreased proliferation. These data identify a cell autonomous requirement for mesenchymal FGFR signaling in the development of pulmonary fibrosis, and for the enrichment of the Col1α2-CreER-positive (Col1α2+) mesenchymal lineage in fibrotic tissue following bleomycin exposure. We conclude that mesenchymal FGF signaling is required for the development of pulmonary fibrosis, and that therapeutic strategies aimed directly at mesenchymal FGF signaling could be beneficial in the treatment of IPF.

Loss of IL-4Rα signaling is partially able to restore anti-Mycoplasma pneumoniae IgG titers during allergic airway disease

Abstract Rationale Mycoplasma pneumoniae (Mpn) is the leading bacterial agent of pediatric community acquired pneumonia. Epidemiologic evidence has suggested increased susceptibility to Mpn infection among asthmatics. We hypothesized that allergic airway sensitization would impair host immune responses to Mpn infection, but that loss of Th2 cytokine signals (IL-4, IL-13) would overcome this impairment in a murine model. Methods Wild type (Wt) and IL-4Rα−/− BALB/cJ mice were sensitized and challenged with ovalbumin (OVA) to induce allergic airway inflammation, and then infected with Mpn. Immune parameters were studied by analysis of cellular profiles in bronchoalveolar lavage fluid (BALF), serum IgG and IgE antibody levels to a whole bacterial antigen preparation, recombinant Mpn P1 adhesin (rP1), and OVA. Total lung RNA was used to examine cytokine transcript levels for Th1, Th2 and Th17 biasing transcripts. Results Anti-Mpn and P1-specific total IgG responses were decreased in allergen-sensitized animals compared to unsensitized controls (p &amp;lt; 0.01). Decreased titers of IgG1, IgG2a, IgG2b and IgG3 were present in rP1-specific, but not OVA-specific IgG subclass levels. Loss of IL-4Rα signaling partially restores total and subclass-specific responses. Th2 cytokine mRNA levels were increased in Wt, but not IL-4Rα−/− OVA sensitized groups compared to controls. IL-4Rα−/− animals had increased Interferon-γ(IFN-γ) and IL-17A mRNA levels compared to Wt animals. Conclusions Anti-Mpn IgG antibody titers were decreased in Wt allergic animals compared to unsensitized controls and the impairment in immune response to the pathogen was partially restored by inhibition of Th2 signaling in IL-4Rα−/− animals.

118 - The R213G Polymorphism in SOD3 Protects Early Bleomycin-Induced Pulmonary Inflammation and Attenuates Induction of Genes Involved in Agranulocyte/granulocyte Adhesion and Diapedesis

Extracellular superoxide dismutase (SOD3), a key lung antioxidant enzyme, protects against lung and vascular inflammation. A single nucleotide polymorphism (SNP) substituting arginine to glycine at the 213 amino acid position (R213G) in SOD3 decreases tissue binding, thereby increasing SOD3 in the extracellular fluids such as epithelial lining fluid and plasma. We tested the hypothesis that the R213G SNP modulates key inflammatory signaling pathways in bleomycin induced lung injury to protect against progression to lung fibrosis and pulmonary hypertension (PH). In wild type (WT) mice, bleomycin increased multiple proinflammatory cytokines, measured with V-plex (Meso Scale Discovery) over 21 days. In R213G mice, inflammation increased 3 days post-bleomycin, however resolved by day 21 (n=6-8). Unlike the WT mice, R213G mice did not progress to lung fibrosis or PH, shown by physiologic, morphometric and biochemical measures. We performed high throughput RNAseq and bioinformatics analyses in lung RNA to evaluate gene pathways 7 days post-bleomycin in WT and R213G mice. The top enriched pathways, determined by ingenuity pathway analysis, were agranulocyte and granulocyte adhesion and diapedesis (2.51E-11 and 5.75E-06; p-value, respectively). These innate immune responses have important roles in the recruitment, adhesion and extravasation of leukocytes. Genes involved in adhesion (ITGAM and ITGB2), superoxide generation via NADPH oxidase (CYBB and NCF), and hydrolyzation of extracellular matrix (MMP12 and TIMP1) were preferentially upregulated in the WT mice compared to R213G mice. These findings were validated by qPCR. Bioinformatic analysis identifies potential pathways responsible for the protective effects of the R213G SNP and provides a foundation to better understand the role of redox regulation in inflammatory lung and pulmonary vascular diseases.

Abstract 5135: Detection of IFNγ induced PDL1 expression by combined in situ RNA analysis and protein profiling from a single FFPE slide

Abstract PD1 ligands (PDL1) are often upregulated on the cell surface of many different tumors. The primary role of PDL1 in cancer is to inhibit T-cell mediated immune response. Two general mechanisms for PDL1 expression on tumor cells have been proposed. Innate immune resistance, in which PDL1 expression is induced by the constitutive oncogenic signaling, and adaptive immune resistance, in which PDL1 expression is induced by T-cells releasing interferon-γ (IFNγ) and activating the STAT signaling pathway. In order to differentiate between these two mechanisms, IFNγ mRNA expression is measured as an effective alternative to detecting IFNγ protein. Detection of cytokines by IHC is challenging as secreted proteins are widely diffused and the associated staining pattern appears to lack cellular specificity. RNAscope RNA in situ hybridization (ISH) assay is utilized to measure Interferon-γ (IFNγ) mRNA expression, and MultiOmyxTM multiplexed assay (demonstrated to stain up to 60 protein biomarkers) is utilized to measure CD3, CD4, CD8, CD56, CD68, PD1, and PDL1 protein expression. In this study, combined MO and RNAscope ISH assays, enabled identification of individual cells with characteristic mRNA and protein expression profile. The MultiOmyx assay utilizes a pair of directly conjugated Cyanine dye-labeled (Cy3, Cy5) antibodies per round of staining. Each Cy-dye conjugated antibody recognizes different target proteins. Each round of staining is imaged and followed by novel dye inactivation chemistry, enabling repeated rounds of staining. RNAscope is a novel RNA ISH assay capable of single-molecule detection in individual cells, utilizing hybridization mediated signal amplification. The assay utilizes a pair of RNA target specific oligonucleotide probes, which sequentially hybridize to preamplifier, amplifier, and fluorophore label probes. Utilizing MultiOmyx and RNAscope assays, this study proposed to profile both RNA and protein expression in lung, breast, melanoma, colorectal, esophageal, and prostate cancer samples. Differentiating PDL1 expression induced in response to inflammatory signals produced by an activated T-cell, from PDL1 expression induced by constitutive oncogenic signaling, has potential implications in effectiveness of PD1 blockade therapy. According to the proposed mechanisms, PD1 blockade as a mono therapy may only benefit individuals with strong endogenous immune response. In individuals with weak endogenous immune response, combinational therapies consisting of both immune activation and PD1-pathway blockade may be more effective than either mono therapy alone. Citation Format: Qingyan Au, Kathy Nguyen, Michael S. Lazare, Edward J. Moler, Nicholas Hoe. Detection of IFNγ induced PDL1 expression by combined in situ RNA analysis and protein profiling from a single FFPE slide. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 5135.

Abstract LB-037: Upregulation of Rab, a member of the Ras gene family, is associated with accelerated lung tumor progression in mice exposed in utero to secondhand smoke

Abstract Rationale: While second-hand smoke (SHS) exposure is responsible for more than 7,000 lung cancer deaths annually among US nonsmokers, it is unclear whether there is an association between in utero SHS and lung cancer. We have reported that lungs of mice exposed in utero to SHS and as adults to ovalbumin exhibited up-regulation of miR-155-5p, miR-21-3p and miR-18a-5p and down-regulation of 16 tumor suppressor genes, all predicted targets of these 3 miRNAs, which have been characterized as oncogenic miRNAs. In this pilot study, we investigated whether in utero SHS exposure promotes an oncogenic milieu in lungs of adult mice. Methods: Pregnant Balb/c mice were exposed from gestational days 6 - 19 to 10 mg/m3 of SHS or filtered air. From 16-19 weeks of age, male offspring were treated with 4 weekly intraperitoneal injections of urethane (1 g/kg) or saline. At 44 weeks of age, mice were sacrificed. Broncho-alveolar lavage fluids (BALF) were collected for assessment of inflammatory responses and oxidative stress. Tumors were analyzed for number, size, volume, and histopathological classification. Lung RNA sequencing was also performed. Results: In utero SHS-adult urethane (SU) treatment significantly (p &amp;lt; 0.05) increased the number of tumors larger than 2 mm, and their volumes, compared to their respective air-urethane (AU)-treated controls. Lungs of SU mice also showed significant increases in percentages of BALF lymphocytes and levels of 8-isoprostane, a biomarker of oxidative stress. Bronchial-alveolar carcinomas were present in all SU mice (mean = 3.8 carcinomas/mouse) compared to 85% of AU mice (mean = 2.2 carcinomas/mouse). Moreover, 5 intrapulmonary metastases were observed in the SU group vs. 1 in the AU group. A total of 24 genes were differently expressed in lungs of SU vs. AU mice. These dysregulated genes clustered mainly into 3 functional groups: regulation of cellular processes, protein binding and intracellular signaling cascade. Of these genes, 13 were up-regulated, including the Ras-associated binding GTPase (Rab), a member of the Ras oncogene family-like 2A, which showed a 5-fold increase in SU mice. Conclusion: In this lung cancer model, in utero SHS exposure accelerated tumor growth, created chronic inflammation, increased oxidative stress levels, and promoted the development of carcinomas and intrapulmonary metastases. Our results also suggest that the mechanisms by which in utero SHS exposure accelerates lung tumor progression and malignant transformation are associated with 3 functional clusters of genes that include the over-expression of Rab. Overall, our data indicate that in utero SHS exposure aggravates adult responses to carcinogenic stimuli by promoting an oncogenic milieu in lungs of male mice. Citation Format: Alexandra Noel, Rui Xiao, Zakia Perveen, Viviana Le Donne, Daniel B. Paulsen, Arthur L. Penn. Upregulation of Rab, a member of the Ras gene family, is associated with accelerated lung tumor progression in mice exposed in utero to secondhand smoke. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr LB-037.

Differential regulation of human and mouse IL33 expression in the lungs of human IL33 BAC transgenic mice.

Abstract Interleukin-33 (IL33) is a member of the IL-1 cytokine family. IL33 polymorphisms are strongly associated with asthma susceptibility and IL33 deletion attenuates asthma in mice. However, the use of mouse models to elucidate the role of IL-33 in asthma is hampered by notably different tissue-specific patterns of IL33 expression in humans and mice. To bypass this limitation, we generated mice carrying a 157 kB human BAC transgene (TG) that includes IL33 and its flanking sequences on chromosome 9. This TG contains protective alleles for a number of asthma-associated polymorphisms. Five founders with 1, 2, or 6 BAC copies were obtained. All were healthy, fertile, and transmitted the transgene at the expected Mendelian ratio. To compare expression of transgenic human (h) and endogenous mouse (m)IL33, mice were challenged intranasally with Alternaria, which induces rapid release of mIL-33 in the lungs. Bronchoalveolar lavage fluid analysis showed that hIL-33 was also induced by Alternaria. Analysis of lung RNA by real-time PCR showed that Alternaria-induced hIL33 transcription was TG copy number-dependent and comparable with that of mIL33. IL33 is known to be expressed by human but not mouse endothelial cells. Indeed, we found high levels of hIL33 RNA in endothelial cell-enriched mouse CD31+ lung cells while mIL33 was barely detectable. These results suggest that tissue-specific hIL33 expression is controlled by cis-acting element(s) within the IL33 locus. More generally, our BAC TG appears to include all cis-regulatory elements necessary for faithful tissue-specific and copy number-dependent regulation of hIL33 expression, suggesting our mice are an ideal tool to study IL33 and its role in asthma.

MiRNA-146a, miRNA-203a, and miRNA-223 Modulate Inflammatory Response in LPS- Acute Lung Injury in Sickle Cell Transgenic Mice

Introduction: Mouse models have allowed a wider investigation of the inflammatory process in Sickle cell disease (SCD). Previous studies have demonstrated that neutrophil recruitment inresponse to LPS is upregulated in Berkeley sickle mice and the development of acute LPS-induced airway inflammation is associated with enhanced expression of cytokines, chemokines and MMPs expression in lungs. MicroRNAs (miRNAs) belong to a regulatory class of RNAs with great importance in several biological processes, including the development and fate of immune cells, and in both innate and adaptive immune responses, whereby strong evidence links miRNAs expression to signalling pathways and receptors. Some miRNAs are positive regulators of inflammation and others are involved in the negative-feedback regulation of inflammatory processes. A growing amount of evidence indicates that these molecules are involved in various lung diseases. However, the expression profile of miRNAs in lung injury in SCD have not been investigated. Since pulmonary complications are an important cause of morbidity and mortality in SCD, the aim of this study was to determine whether miRNAs implicated in LPS-mediated inflammation are involved in neutrophil recruitment and inflammation in lung injury in SCD using the sickle mice model.Methods: Acute lung inflammation and injury was induced in wild- type (WT, C57BL/6, n=5) and Berkeley SCD mice (n=5) by intranasal administration of LPS (50 ml of 250 mg/ml). The vehicle group received a similar volume of sterile PBS. BAL was performed 4h after LPS challenge. qPCR analysis was used to examine gene expression and ELISA was used to determine protein production. For miRNAs expression analysis, extraction of lung RNA was performed using mirVanaTM miRNA Isolation kit and TaqManTM MicroRNA Reverse Transcription.The expression level of miRNAs was determined using the Taqman miRNA expression assay by qPCR with U6 snRNA as an endogenous control.Results: Levels of miR-15a-5p, miR-16-5p, miR-26a-5p and miR-98-5p in lung tissue increased almost twofold at 4h after LPS challenge in SS compared with SS PBS group (2.9, 3.7; 2.3; 2.9-fold change, respectively). The expressions of miR-106a-5p, miR-146a-5p, miR-146b-5p, and miR-181a-5p were not altered (2.0; 1.2; 1.55; 1.4-fold change, respectively). However, the expressions of miR-203a-3p and miR-223-3p were significantly up-regulated compared to animals from the PBS group (4.3 and 8.8 fold change, respectively, p<0.05). These increases correlated with the increased expressions of Tnf- α, Il-1 β, Il-6, Cxcl1 and Mmp-9 mRNA in the lungs (LPS: 0.27±0.05; 0.24±0.04; 0.18±0.007; 0.44±0.09, 0.46±0.09 vs PBS: 0.02±0.001; 0.03±0.02; 0.007±0.18; 0.052±0.03; 0.07±0.03, respectively) and increased BAL levels of Tnf-α, Cxcl1 and Mmp-9 (4250±636.4; 2491±454.3; 187.5±16.7 vs PBS: 20.8±8.9; 186.6±35.4, 5.4±2.9, pg/ml, respectively). LPS challenge also led to a higher expression of miRNAs in the lungs of WT animals; however only expression levels of miR-146a-5p and miR-223-3p were significantly up-regulated (2.6 and 3.2-fold change, respectively, p<0.05).Conclusion: We have shown that theLPS-induced inflammatory response in SCD transgenic mice is associated with increases in miR-203a-3p and miR-223-3p expression in the mouse lung; suggesting a role of the miRNAs in the regulation of inflammatory mediator production, since these augmentations are associated with the increases in cytokines and chemokines. Furthermore, miR-146a-5p, a negative regulator of inflammatory gene expression, is not upregulated in SS mice and could contribute to the exaggerated inflammation observed in these animals. These data indicate that alterations in the miRNA expression may be associated with the inflammatory state in SCD. Financial Support: FAPESP DisclosuresNo relevant conflicts of interest to declare.

Incomplete lung recovery following sub-acute inhalation of combustion-derived ultrafine particles in mice.

BackgroundParticulate matter (PM) is one of the six criteria pollutant classes for which National Ambient Air Quality Standards have been set by the United States Environmental Protection Agency. Exposures to PM have been correlated with increased cardio-pulmonary morbidity and mortality. Butadiene soot (BDS), generated from the incomplete combustion of 1,3-butadiene (BD), is both a model PM mixture and a real-life example of a petrochemical product of incomplete combustion. There are numerous events, including wildfires, accidents at refineries and tank car explosions that result in sub-acute exposure to high levels of airborne particles, with the people exposed facing serious health problems. These real-life events highlight the need to investigate the health effects induced by short-term exposure to elevated levels of PM, as well as to assess whether, and if so, how well these adverse effects are resolved over time. In the present study, we investigated the extent of recovery of mouse lungs 10 days after inhalation exposures to environmentally-relevant levels of BDS aerosols had ended.MethodsFemale BALB/c mice exposed to either HEPA-filtered air or to BDS (5 mg/m3 in HEPA filtered air, 4 h/day, 21 consecutive days) were sacrificed immediately, or 10 days after the final BDS exposure. Bronchoalveolar lavage fluid (BALF) was collected for cytology and cytokine analysis. Lung proteins and RNA were extracted for protein and gene expression analysis. Lung histopathology evaluation also was performed.ResultsSub-acute exposures of mice to hydrocarbon-rich ultrafine particles induced: (1) BALF neutrophil elevation; (2) lung mucosal inflammation, and (3) increased BALF IL-1β concentration; with all three outcomes returning to baseline levels 10 days post-exposure. In contrast, (4) lung connective tissue inflammation persisted 10 days post-exposure; (5) we detected time-dependent up-regulation of biotransformation and oxidative stress genes, with incomplete return to baseline levels; and (6) we observed persistent particle alveolar load following 10 days of recovery.ConclusionThese data show that 10 days after a 21-day exposure to 5 mg/m3 of BDS has ended, incomplete lung recovery promotes a pro-biotransformation, pro-oxidant, and pro-inflammatory milieu, which may be a starting point for potential long-term cardio-pulmonary effects.

Identification of microRNAs associated with allergic airway disease using a genetically diverse mouse population.

BackgroundAllergic airway diseases (AADs) such as asthma are characterized in part by granulocytic airway inflammation. The gene regulatory networks that govern granulocyte recruitment are poorly understood, but evidence is accruing that microRNAs (miRNAs) play an important role. To identify miRNAs that may underlie AADs, we used two complementary approaches that leveraged the genotypic and phenotypic diversity of the Collaborative Cross (CC) mouse population. In the first approach, we sought to identify miRNA expression quantitative trait loci (eQTL) that overlap QTL for AAD-related phenotypes. Specifically, CC founder strains and incipient lines of the CC were sensitized and challenged with house dust mite allergen followed by measurement of granulocyte recruitment to the lung. Total lung RNA was isolated and miRNA was measured using arrays for CC founders and qRT-PCR for incipient CC lines.ResultsAmong CC founders, 92 miRNAs were differentially expressed. We measured the expression of 40 of the most highly expressed of these 92 miRNAs in the incipient lines of the CC and identified 18 eQTL corresponding to 14 different miRNAs. Surprisingly, half of these eQTL were distal to the corresponding miRNAs, and even on different chromosomes. One of the largest-effect local miRNA eQTL was for miR-342-3p, for which we identified putative causal variants by bioinformatic analysis of the effects of single nucleotide polymorphisms on RNA structure. None of the miRNA eQTL co-localized with QTL for eosinophil or neutrophil recruitment. In the second approach, we constructed putative miRNA/mRNA regulatory networks and identified three miRNAs (miR-497, miR-351 and miR-31) as candidate master regulators of genes associated with neutrophil recruitment. Analysis of a dataset from human keratinocytes transfected with a miR-31 inhibitor revealed two target genes in common with miR-31 targets correlated with neutrophils, namely Oxsr1 and Nsf.ConclusionsmiRNA expression in the allergically inflamed murine lung is regulated by genetic loci that are smaller in effect size compared to mRNA eQTL and often act in trans. Thus our results indicate that the genetic architecture of miRNA expression is different from mRNA expression. We identified three miRNAs, miR-497, miR-351 and miR-31, that are candidate master regulators of genes associated with neutrophil recruitment. Because miR-31 is expressed in airway epithelia and is predicted to target genes with known links to neutrophilic inflammation, we suggest that miR-31 is a potentially novel regulator of airway inflammation.Electronic supplementary materialThe online version of this article (doi:10.1186/s12864-015-1732-9) contains supplementary material, which is available to authorized users.

Therapeutic doses of irradiation activate viral transcription and induce apoptosis in HIV-1 infected cells

The highly active antiretroviral therapy reduces HIV-1 RNA in plasma to undetectable levels. However, the virus continues to persist in the long-lived resting CD4+ T cells, macrophages and astrocytes which form a viral reservoir in infected individuals. Reactivation of viral transcription is critical since the host immune response in combination with antiretroviral therapy may eradicate the virus. Using the chronically HIV-1 infected T lymphoblastoid and monocytic cell lines, primary quiescent CD4+ T cells and humanized mice infected with dual-tropic HIV-1 89.6, we examined the effect of various X-ray irradiation (IR) doses (used for HIV-related lymphoma treatment and lower doses) on HIV-1 transcription and viability of infected cells. Treatment of both T cells and monocytes with IR, a well-defined stress signal, led to increase of HIV-1 transcription, as evidenced by the presence of RNA polymerase II and reduction of HDAC1 and methyl transferase SUV39H1 on the HIV-1 promoter. This correlated with the increased GFP signal and elevated level of intracellular HIV-1 RNA in the IR-treated quiescent CD4+ T cells infected with GFP-encoding HIV-1. Exposition of latently HIV-1infected monocytes treated with PKC agonist bryostatin 1 to IR enhanced transcription activation effect of this latency-reversing agent. Increased HIV-1 replication after IR correlated with higher cell death: the level of phosphorylated Ser46 in p53, responsible for apoptosis induction, was markedly higher in the HIV-1 infected cells following IR treatment. Exposure of HIV-1 infected humanized mice with undetectable viral RNA level to IR resulted in a significant increase of HIV-1 RNA in plasma, lung and brain tissues. Collectively, these data point to the use of low to moderate dose of IR alone or in combination with HIV-1 transcription activators as a potential application for the “Shock and Kill” strategy for latently HIV-1 infected cells.

Effects of Intravenous Benzo[a]Pyrene Dose Administration on Levels of Exposure Biomarkers, DNA Adducts, and Gene Expression in Rats

The effects of benzo[a]pyrene (BaP) administration on biomarkers of exposure and early effects were studied in male Sprague-Dawley rats intravenously injected with doses of 0.4, 4, 10, or 40 μmol BaP/kg . Blood, tissues, and excreta were collected 8 and 24 h posttreatment. BaP and several of its metabolites were simultaneously measured in blood, tissues and excreta by ultra-high-performance liquid chromatography (UHPLC)/fluorescence. DNA adducts of BaP diol epoxide (BaPDE) in lungs were quantified using an ultrasensitive immunoassay with chemiluminescence detection. Expression of selected genes in lungs of treated rats (lung RNA) compared to control rats was also assessed by quantitative real-time polymerase chain reaction. There was a dose-dependent increase in blood, tissue, and excreted levels of BaP metabolites. At 8 and 24 h postinjection, BaP and hydroxyBaP were found in higher concentrations in blood and tissues compared to other analytes. However, diolBaP were excreted in greater amounts in urine and apparently more rapidly than hydroxyBaP. Mean percentages (± SD) of injected dose excreted in urine as 4,5-diolBaP during the 0–8 h and 0–24 h period posttreatment were 0.16 ± 0.027% and 0.14 ± 0.083%, respectively. Corresponding values for 3-OHBaP were 0.0045 ± 0.0009% and 0.026 ± 0.014%. BaP-diones were not detectable in blood, tissues, and excreta; 7,8-diolBaP and BaPtetrol were found to be minor metabolites. There was also a dose-dependent increase in DNA adduct formation in lung. Analysis of gene expression further showed a modulation of Cyp1a1, Cyp1b1, Nqo1, Nrf2, Fos, and Ahr expression at 10- and 40-μmol/kg doses, but not at the lower doses. This study provided a better assessment of the influence of absorbed BaP doses on biological levels of diolBaP and OHBaP exposure biomarkers and association of the latter with early biological alterations, such as DNA adducts and gene expression.

Co-circulation of soricid- and talpid-borne hantaviruses in Poland

Previously, we reported the discovery of a genetically distinct hantavirus, designated Boginia virus (BOGV), in the Eurasian water shrew (Neomys fodiens), as well as the detection of Seewis virus (SWSV) in the Eurasian common shrew (Sorex araneus), in central Poland. In this expanded study of 133 shrews and 69moles captured during 2010–2013 in central and southeastern Poland, we demonstrate the co-circulation of BOGV in the Eurasian water shrew and SWSV in the Eurasian common shrew, Eurasian pygmy shrew (Sorex minutus) and Mediterranean water shrew (Neomys anomalus). In addition, we found high prevalence of Nova virus (NVAV) infection in the European mole (Talpa europaea), with evidence of NVAV RNA in heart, lung, liver, kidney, spleen and intestine. The nucleotide and amino acid sequence variation of the L segment among the SWSV strains was 0–18.8% and 0–5.4%, respectively. And for the 38 NVAV strains from European moles captured in Huta Dłutowska, the L-segment genetic similarity ranged from 94.1%–100% at the nucleotide level and 96.3%–100% at the amino acid level. Phylogenetic analyses showed geographic-specific lineages of SWSV and NVAV in Poland, not unlike that of rodent-borne hantaviruses, suggesting long-standing host-specific adaptation. The co-circulation and distribution of BOGV, SWSV and NVAV in Poland parallels findings of multiple hantavirus species co-existing in their respective rodent reservoir species elsewhere in Europe. Also, the detection of SWSV in three syntopic shrew species resembles spill over events observed among some rodent-borne hantaviruses.

Cigarette smoke affects dendritic cell maturation in the small airways of patients with chronic obstructive pulmonary disease.

The aim of the present study was to characterize and quantify the numbers and expression levels of cells markers associated with dendritic cell (DC) maturation in small airways in current smokers and non-smokers with or without chronic obstructive pulmonary disease (COPD). Lung tissues from the following 32 patients were obtained during resection for lung cancer: Eight smokers with COPD, eight non-smokers with COPD, eight current smokers without COPD and eight non-smokers without COPD, serving as a control. The tissue sections were immunostained for cluster of differentiation (CD)83+ and CD1a+ to delineate mature and immature DCs, and chemokine receptor type 7 (CCR7+) to detect DC migratory ability. Myeloid DCs were collected from the lung tissues, and subsequently the CD83+ and CCR7+ expression levels in the lung myeloid DCs were detected using flow cytometry. The expression levels of CD83+, CD1a+ and CCR7+ mRNA in total lung RNA were evaluated by reverse transcription quantitative polymerase chain reaction (RT-qPCR). Evident chronic bronchitis and emphysema pathological changes were observed in the lung tissues of patients with COPD. The results revealed that the numbers of CD83+ and CCR7+ DCs were reduced but the numbers of CD1a+ DCs were significantly increased in the COPD group as compared with the control group (P<0.05, respectively). Using RT-qPCR, the expression levels of CCR7+ and CD83+ mRNA were found to be reduced in the smokers with COPD as compared with the non-smokers without COPD group (P<0.05, respectively). Excessive local adaptive immune responses are key elements in the pathogenesis of COPD. Cigarette smoke may stimulate immune responses by impairing the homing of airway DCs to the lymph nodes and reduce the migratory potential of DCs. The present study revealed that COPD is associated with reduced numbers of mature CD83+ DCs and lower CCR7+ expression levels in small airways.

156: IFN-γ and IL-17A drive distinct airway changes in a murine model of steroid-unresponsive severe asthma

Asthma is an inflammatory disease of the airways characterized by airway obstruction due to inappropriate immune responses against some environmental moieties like house dust mite (HDM) and pollens. Depending on the frequency and intensity of exacerbations experienced, asthma can be categorized as mild-moderate asthma (MMA) that is corticosteroid (CS)- responsive and severe asthma (SA) that is poorly managed by CS. SA constitutes only 5–10% of all asthmatics but accounts for 50% of the healthcare costs and morbidity associated with the disease. The presence of neutrophils in asthma has been correlated with refractoriness to CS, fatal exacerbations and severity. To date, our understanding of the pathological mechanisms central to neutrophil-prominent SA is minimal primarily because of the lack of a suitable animal model, limiting the development and testing of new therapeutics. Studies in our lab have shown that unlike patients with milder disease, SA patients consistently have a mixed Th1/Th2/Th17 immune phenotype in their airways despite high dose CS treatment. With the association of viral and bacterial infections with severity and frequency of asthma exacerbations, we hypothesized that the combination of an allergen, HDM, with agents that mimic infection would induce a SA phenotype in mice. As in human severe asthmatics, the immune response in this mouse model elicits mixed granulocytic airway inflammation and airway hyperresponsiveness that are poorly responsive to CS. IFN-γ-deficient and IL-17A-deficient mice subjected to the SA model highlight the differential role played by each cytokine in modulating hallmark features of asthma such as airway inflammation, airway hyperreactivity and steroid responsiveness. To identify additional biomarkers for characterization of SA, total lung RNA from mice subjected to the SA model was analyzed by RNA-Seq. Ingenuity Pathway Analysis of the RNA-Seq data revealed potential intermediates (e.g., lipoproteins, proteases, etc.) in the IFN-γ and IL-17A signaling pathways that can be targeted for developing novel therapies for SA and potentially other steroid-refractory diseases.

Identification of a novel nidovirus in an outbreak of fatal respiratory disease in ball pythons (Python regius).

BackgroundRespiratory infections are important causes of morbidity and mortality in reptiles; however, the causative agents are only infrequently identified.FindingsPneumonia, tracheitis and esophagitis were reported in a collection of ball pythons (Python regius). Eight of 12 snakes had evidence of bacterial pneumonia. High-throughput sequencing of total extracted nucleic acids from lung, esophagus and spleen revealed a novel nidovirus. PCR indicated the presence of viral RNA in lung, trachea, esophagus, liver, and spleen. In situ hybridization confirmed the presence of intracellular, intracytoplasmic viral nucleic acids in the lungs of infected snakes. Phylogenetic analysis based on a 1,136 amino acid segment of the polyprotein suggests that this virus may represent a new species in the subfamily Torovirinae.ConclusionsThis report of a novel nidovirus in ball pythons may provide insight into the pathogenesis of respiratory disease in this species and enhances our knowledge of the diversity of nidoviruses.

Genomic profiling of collaborative cross founder mice infected with respiratory viruses reveals novel transcripts and infection-related strain-specific gene and isoform expression.

Genetic variation between diverse mouse species is well-characterized, yet existing knowledge of the mouse transcriptome comes largely from one mouse strain (C57BL/6J). As such, it is unlikely to reflect the transcriptional complexity of the mouse species. Gene transcription is dynamic and condition-specific; therefore, to better understand the mouse transcriptional response to respiratory virus infection, we infected the eight founder strains of the Collaborative Cross with either influenza A virus or severe acute respiratory syndrome coronavirus and sequenced lung RNA samples at 2 and 4 days after infection. We found numerous instances of transcripts that were not present in the C57BL/6J reference annotation, indicating that a nontrivial proportion of the mouse genome is transcribed but poorly annotated. Of these novel transcripts, 2150 could be aligned to human or rat genomes, but not to existing mouse genomes, suggesting functionally conserved sequences not yet recorded in mouse genomes. We also found that respiratory virus infection induced differential expression of 4287 splicing junctions, resulting in strain-specific isoform expression. Of these, 59 were influenced by strain-specific mutations within 2 base pairs of key intron–exon boundaries, suggesting cis-regulated expression. Our results reveal the complexity of the transcriptional response to viral infection, previously undocumented genomic elements, and extensive diversity in the response across mouse strains. These findings identify hitherto unexplored transcriptional patterns and undocumented transcripts in genetically diverse mice. Host genetic variation drives the complexity and diversity of the host response by eliciting starkly different transcriptional profiles in response to a viral infection.

Effects of peroxisome proliferator-activated receptor-β/δ on sepsis induced acute lung injury

Background Acute lung injury (ALI) and acute respiratory distress syndrome (ARDS) are the first steps in the development of multiple organ failure induced by sepsis. A systemic excessive inflammatory reaction is currently the accepted mechanism of the pathogenesis of sepsis. Several studies have suggested a protective role of the peroxisome proliferator activated receptor-β/δ (PPAR-β/δ) in related inflammatory diseases. But the role of PPARβ/δ in ALI remains uncertain. The aim of this study was to investigate the role and possible mechanism of PPARβ/δ in ALI induced by sepsis. Methods Cecal ligation and puncture (CLP) was used as a sepsis model. Rats were randomly divided into four groups, the control group (CON, n=6), sham-operation group (SHAM, n=12), cecal ligation and puncture group (CLP, n=30), GW501516 group (CLP+GW, n=25), which underwent CLP and were subcutaneously injected with the PPAR-β/δ agonist GW501516 (0.05 mg/100 g body weight). Survival was monitored to 24 hours after operation. Blood pressure, serum creatinine, blood urea nitrogen, aspartate aminotrasferase and alanine aminotrasferase were measured after CLP. Concentrations of tumor necrosis factor α (TNF-α) and interleukin (IL)-1β in serum were detected by enzyme linked immunosorbent assay (ELISA) kits. Lung tissue samples were stained with H&amp;E and scored according to the degree of inflammation. Bacterial colonies were counted in the peritoneal fluid. Alveolar macrophages were cultured and incubated with GW501516 (0.15 μmol/L) and PPARβ/δ adenovirus and then treated with Lipopolysaccharide (2 μg/ml) for 2 hours. The TNF-α, IL-1β and IL-6 RNA in lung and alveolar macrophages were determined by real-time PCR. Phosphorylation of signal transducer and activator of transcription 3 (STAT3) in lung and alveolar macrophages was detected by Western blotting. Results GW501516 significantly increased the survival of septic rats, decreased histological damage of the lungs, reduced inflammatory cytokines in serum and lung tissues of septic rats and did not increase counts of peritoneal bacteria. In vitro, GW501516 and over-expression of PPARβ/δ attenuated gene expression of TNF-α, IL-1β and IL-6 in alveolar macrophages. Both in vivo and in vitro, PPARβ/δ inhibited the phosphorylation of STAT3. Conclusion PPARβ/δ plays a protective role in sepsis induced ALI via suppressing excessive inflammation.

The role of CD4+ T cell subsets in the clearance of Pneumocystis murina pneumonia in mice (MPF6P.734)

Abstract Pneumocystis (PC) Pneumonia remains a serious complication in the immuno-compromised host. The host defense against PC is critically dependent upon CD4+ T cells. The role of specific T-cell subsets that mediate host resistance against PC infection remains unclear. We use STAT transcription factor knockout (KO) mice to determine the role of Th1, Th2, and Th17 cells in PC infection. After 28 days infection with PC, serum, lung tissue, and mediastinal lymph node (LN) were collected. LN and lung tissue cells were measured for cytokines by ELISpot and intracellular staining. Total lung RNA was measured for PC burden by RT-qPCR. Cytokines were assayed from cell culture supernatant. Anti-PC antibody was measured by ELISA.Stat4, Stat6 single KO, and Stat4/6 double KO mice all were resistant to PC compared to the Rag2/gc KO mice. Stat4 was required for a Th1 response and Stat6 was required for a Th2 response. STAT3fl/fl/CD4Cre+ mice had partial susceptibility of PC. Stat3 expression was required in CD4+ T cells for PC specific Th17 responses. STAT4/6 double KO crossed to STAT3fl/fl/CD4Cre+ (Triple KO) mice were highly susceptible to PC infection compare to single and double KO mice. Individual Stat4 and 6 and double mutants clear PC. STAT3fl/fl/CD4Cre+ conferred partial susceptibility to PC. Triple KO were as susceptible for PC as Rag2/gc KO mice. These data support the hypothesis that PC is a true opportunist and that various T-cell subsets can confer protection against infection.