Preconceptional smoking alters spermatozoal miRNAs of murine fathers and affects offspring's body weight.

BACKGROUND: Active smoking has been reported among 7% of teenagers worldwide, with ages ranging from 13 to 15 years. An epidemiological study suggested that preconceptional paternal smoking is associated with adolescent obesity in boys. We developed a murine adolescent smoking model before conception to investigate the paternal molecular causes of changes in offspring's phenotype. METHOD: Male and female C57BL/6J mice were exposed to increasing doses of mainstream cigarette smoke (CS) from onset of puberty for 6 weeks and mated with room air (RA) controls. RESULTS: Thirteen miRNAs were upregulated and 32 downregulated in the spermatozoa of CS-exposed fathers, while there were no significant differences in the count and morphological integrity of spermatozoa, as well as the proliferation of spermatogonia between CS- and RA-exposed fathers. Offspring from preconceptional CS-exposed mothers had lower body weights (p = 0.007). Moreover, data from offspring from CS-exposed fathers suggested a potential increase in body weight (p = 0.062). CONCLUSION: We showed that preconceptional paternal CS exposure regulates spermatozoal miRNAs, and possibly influences the body weight of F1 progeny in early life. The regulated miRNAs may modulate transmittable epigenetic changes to offspring, thus influence the development of respiratory- and metabolic-related diseases such as obesity, a mechanism that warrants further studies for elaborate explanations.

Cigarette Smoke Affects Dendritic Cell Populations, Epithelial Barrier Function, and the Immune Response to Viral Infection With H1N1.

Smokers with apparently "healthy" lungs suffer from more severe and frequent viral respiratory infections, but the mechanisms underlying this observation are still unclear. Epithelial cells and dendritic cells (DC) form the first line of defense against inhaled noxes such as smoke or viruses. We therefore aimed to obtain insight into how cigarette smoke affects DCs and epithelial cells and how this influences the response to viral infection. Female C57BL/6J mice were exposed to cigarette smoke (CS) for 1 h daily for 24 days and then challenged i.n. with the viral mimic and Toll-like receptor 3 (TLR3) ligand poly (I:C) after the last exposure. DC subpopulations were analyzed 24 h later in whole lung homogenates by flow cytometry. Calu-3 cells or human precision-cut lung slices (PCLS) cultured at air-liquid interface were exposed to CS or air and subsequently inoculated with influenza H1N1. At 48 h post infection cytokines were analyzed by multiplex technology. Cytotoxic effects were measured by release of lactate dehydrogenase (LDH) and confocal imaging. In Calu-3 cells the trans-epithelial electrical resistance (TEER) was assessed. Smoke exposure of mice increased numbers of inflammatory and plasmacytoid DCs in lung tissue. Additional poly (I:C) challenge further increased the population of inflammatory DCs and conventional DCs, especially CD11b+ cDCs. Smoke exposure led to a loss of the barrier function in Calu-3 cells, which was further exaggerated by additional influenza H1N1 infection. Influenza H1N1-induced secretion of antiviral cytokines (IFN-α2a, IFN-λ, interferon-γ-induced protein 10 [IP-10]), pro-inflammatory cytokine IL-6, as well as T cell-associated cytokines (e.g., I-TAC) were completely suppressed in both Calu-3 cells and human PCLS after smoke exposure. In summary, cigarette smoke exposure increased the number of inflammatory DCs in the lung and disrupted epithelial barrier functions, both of which was further enhanced by viral stimulation. Additionally, the antiviral immune response to influenza H1N1 was strongly suppressed by smoke. These data suggest that smoke impairs protective innate mechanisms in the lung, which could be responsible for the increased susceptibility to viral infections in "healthy" smokers.

The need for clean air: The way air pollution and climate change affect allergic rhinitis and asthma.

Air pollution and climate change have a significant impact on human health and well-being and contribute to the onset and aggravation of allergic rhinitis and asthma among other chronic respiratory diseases. In Westernized countries, households have experienced a process of increasing insulation and individuals tend to spend most of their time indoors. These sequelae implicate a high exposure to indoor allergens (house dust mites, pets, molds, etc), tobacco smoke, and other pollutants, which have an impact on respiratory health. Outdoor air pollution derived from traffic and other human activities not only has a direct negative effect on human health but also enhances the allergenicity of some plants and contributes to global warming. Climate change modifies the availability and distribution of plant- and fungal-derived allergens and increases the frequency of extreme climate events. This review summarizes the effects of indoor air pollution, outdoor air pollution, and subsequent climate change on asthma and allergic rhinitis in children and adults and addresses the policy adjustments and lifestyle changes required to mitigate their deleterious effects.

Human airway epithelial extracellular vesicle miRNA signature is altered upon asthma development.

BACKGROUND: miRNAs are master regulators of signaling pathways critically involved in asthma and are transferred between cells in extracellular vesicles (EV). We aimed to investigate whether the miRNA content of EV secreted by primary normal human bronchial epithelial cells (NHBE) is altered upon asthma development. METHODS: NHBE cells were cultured at air-liquid interface and treated with interleukin (IL)-13 to induce an asthma-like phenotype. EV isolations by precipitation from basal culture medium or apical surface wash were characterized by nanoparticle tracking analysis, transmission electron microscopy, and Western blot, and EV-associated miRNAs were identified by a RT-qPCR-based profiling. Significant candidates were confirmed in EVs isolated by size-exclusion chromatography from nasal lavages of children with mild-to-moderate (n = 8) or severe asthma (n = 9), and healthy controls (n = 9). RESULTS: NHBE cells secrete EVs to the apical and basal side. 47 miRNAs were expressed in EVs and 16 thereof were significantly altered in basal EV upon IL-13 treatment. Expression of miRNAs could be confirmed in EVs from human nasal lavages. Of note, levels of miR-92b, miR-210, and miR-34a significantly correlated with lung function parameters in children (FEV1 FVC%pred and FEF25-75%pred ), thus lower sEV-miRNA levels in nasal lavages associated with airway obstruction. Subsequent ingenuity pathway analysis predicted the miRNAs to regulate Th2 polarization and dendritic cell maturation. CONCLUSION: Our data indicate that secretion of miRNAs in EVs from the airway epithelium, in particular miR-34a, miR-92b, and miR-210, might be involved in the early development of a Th2 response in the airways and asthma.

In utero exposure to cigarette smoke and effects across generations: A conference of animals on asthma.

BACKGROUND: The prevalence of asthma and chronic obstructive pulmonary disease (COPD) has risen markedly over the last decades and is reaching epidemic proportions. However, underlying molecular mechanisms are not fully understood, hampering the urgently needed development of approaches to prevent these diseases. It is well established from epidemiological studies that prenatal exposure to cigarette smoke is one of the main risk factors for aberrant lung function development or reduced fetal growth, but also for the development of asthma and possibly COPD later in life. Of note, recent evidence suggests that the disease risk can be transferred across generations, that is, from grandparents to their grandchildren. While initial studies in mouse models on in utero smoke exposure have provided important mechanistic insights, there are still knowledge gaps that need to be filled. OBJECTIVE: Thus, in this review, we summarize current knowledge on this topic derived from mouse models, while also introducing two other relevant animal models: the fruit fly Drosophila melanogaster and the zebrafish Danio rerio. METHODS: This review is based on an intensive review of PubMed-listed transgenerational animal studies from 1902 to 2018 and focuses in detail on selected literature due to space limitations. RESULTS: This review gives a comprehensive overview of mechanistic insights obtained in studies with the three species, while highlighting the remaining knowledge gaps. We will further discuss potential (dis)advantages of all three animal models. CONCLUSION/CLINICAL RELEVANCE: Many studies have already addressed transgenerational inheritance of disease risk in mouse, zebrafish or fly models. We here propose a novel strategy for how these three model organisms can be synergistically combined to achieve a more detailed understanding of in utero cigarette smoke-induced transgenerational inheritance of disease risk.

miR-142-3p is associated with aberrant WNT signaling during airway remodeling in asthma.

Asthma is characterized by a chronic inflammation and remodeling of the airways. Although inflammation can be controlled, therapeutic options to revert remodeling do not exist. Thus, there is a large and unmet need to understand the underlying molecular mechanisms to develop novel therapies. We previously identified a pivotal role for miR-142-3p in regulating airway smooth muscle (ASM) precursor cell proliferation during lung development by fine-tuning the Wingless/Integrase I (WNT) signaling. Thus, we here aimed to investigate the relevance of this interaction in asthma. We performed quantitative RT-PCR and immune staining in a murine model for ovalbumin-induced allergic airway inflammation and in bronchial biopsies from patients with asthma and isolated primary fibroblasts thereof. miR-142-3p was increased in hyperproliferative regions of lung in murine and human asthma, whereas this microRNA (miRNA) was excluded from regions with differentiated ASM cells. Increases in miR-142-3p were associated with a decrease of its known target Adenomatous polyposis coli. Furthermore, we observed a differential expression of miR-142-3p in bronchial biopsies from patients with early or late onset severe asthma, which coincided with a differential WNT signature. Our data suggest that miR-142-3p is involved in regulating the balance between proliferation and differentiation of ASM cells in asthma, possibly via controlling WNT signaling. Thus, this miRNA might be an interesting target to prevent ASM hyperproliferation in asthma.

Intrauterine smoke exposure deregulates lung function, pulmonary transcriptomes, and in particular insulin-like growth factor (IGF)-1 in a sex-specific manner.

Prenatal exposure to tobacco smoke is a significant risk-factor for airway disease development. Furthermore, the high prevalence of pregnant smoking women requires the establishment of strategies for offspring lung protection. Therefore, we here aimed to understand the molecular mechanism of how prenatal smoke exposure affects fetal lung development. We used a mouse model recapitulating clinical findings of prenatally exposed children, where pregnant mice were exposed to smoke until c-section or spontaneous delivery, and offspring weight development and lung function was monitored. Additionally, we investigated pulmonary transcriptome changes in fetal lungs (GD18.5) by mRNA/miRNA arrays, network analyses and qPCR. The results demonstrated that prenatally exposed mice showed intrauterine and postnatal growth retardation, and impaired lung function. 1340 genes and 133 miRNAs were found to be significantly dysregulated by in utero smoke exposure, and we identified Insulin-like growth factor 1 (Igf1) as a top hierarchical node in a network analysis. Moreover, Igf1 mRNA was increased in female murine offspring and in prenatally exposed children. These findings suggest that prenatal smoking is associated with a dysregulation of several genes, including Igf1 in a sex-specific manner. Thus, our results could represent a novel link between smoke exposure, abberant lung development and impaired lung function.

The potential for targeted rewriting of epigenetic marks in COPD as a new therapeutic approach.

Chronic obstructive pulmonary disease (COPD) is an age and smoking related progressive, pulmonary disorder presenting with poorly reversible airflow limitation as a result of chronic bronchitis and emphysema. The prevalence, disease burden for the individual, and mortality of COPD continues to increase, whereas no effective treatment strategies are available. For many years now, a combination of bronchodilators and anti-inflammatory corticosteroids has been most widely used for therapeutic management of patients with persistent COPD. However, this approach has had disappointing results as a large number of COPD patients are corticosteroid resistant. In patients with COPD, there is emerging evidence showing aberrant expression of epigenetic marks such as DNA methylation, histone modifications and microRNAs in blood, sputum and lung tissue. Therefore, novel therapeutic approaches may exist using epigenetic therapy. This review aims to describe and summarize current knowledge of aberrant expression of epigenetic marks in COPD. In addition, tools available for restoration of epigenetic marks are described, as well as delivery mechanisms of epigenetic editors to cells. Targeting epigenetic marks might be a very promising tool for treatment and lung regeneration in COPD in the future.

Early Career Members at the ERS LSC 2017: mechanistic overlap between chronic lung injury and cancer.

.@EarlyCareerERS looks back on #LSC2017 http://ow.ly/I3M730fkn5X.

Pulmonary microRNA profiles identify involvement of Creb1 and Sec14l3 in bronchial epithelial changes in allergic asthma.

Asthma is highly prevalent, but current therapies cannot influence the chronic course of the disease. It is thus important to understand underlying early molecular events. In this study, we aimed to use microRNAs (miRNAs) - which are critical regulators of signaling cascades - to identify so far uncharacterized asthma pathogenesis pathways. Therefore, deregulation of miRNAs was assessed in whole lungs from mice with ovalbumin (OVA)-induced allergic airway inflammation (AAI). In silico predicted target genes were confirmed in reporter assays and in house-dust-mite (HDM) induced AAI and primary human bronchial epithelial cells (NHBE) cultured at the air-liquid interface. We identified and validated the transcription factor cAMP-responsive element binding protein (Creb1) and its transcriptional co-activators (Crtc1-3) as targets of miR-17, miR-144, and miR-21. Sec14-like 3 (Sec14l3) - a putative target of Creb1 - was down-regulated in both asthma models and in NHBE cells upon IL13 treatment, while it's expression correlated with ciliated cell development and decreased along with increasing goblet cell metaplasia. Finally, we propose that Creb1/Crtc1-3 and Sec14l3 could be important for early responses of the bronchial epithelium to Th2-stimuli. This study shows that miRNA profiles can be used to identify novel targets that would be overlooked in mRNA based strategies.

Maternal Pre-Pregnancy Obesity Is Associated with Altered Placental Transcriptome.

Maternal obesity has a major impact on pregnancy outcomes. There is growing evidence that maternal obesity has a negative influence on placental development and function, thereby adversely influencing offspring programming and health outcomes. However, the molecular mechanisms underlying these processes are poorly understood. We analysed ten term placenta's whole transcriptomes in obese (n = 5) and normal weight women (n = 5), using the Affymetrix microarray platform. Analyses of expression data were carried out using non-parametric methods. Hierarchical clustering and principal component analysis showed a clear distinction in placental transcriptome between obese and normal weight women. We identified 72 differentially regulated genes, with most being down-regulated in obesity (n = 61). Functional analyses of the targets using DAVID and IPA confirm the dysregulation of previously identified processes and pathways in the placenta from obese women, including inflammation and immune responses, lipid metabolism, cancer pathways, and angiogenesis. In addition, we detected new molecular aspects of obesity-derived effects on the placenta, involving the glucocorticoid receptor signalling pathway and dysregulation of several genes including CCL2, FSTL3, IGFBP1, MMP12, PRG2, PRL, QSOX1, SERPINE2 and TAC3. Our global gene expression profiling approach demonstrates that maternal obesity creates a unique in utero environment that impairs the placental transcriptome.

D-tryptophan from probiotic bacteria influences the gut microbiome and allergic airway disease.

BACKGROUND: Chronic immune diseases, such as asthma, are highly prevalent. Currently available pharmaceuticals improve symptoms but cannot cure the disease. This prompted demands for alternatives to pharmaceuticals, such as probiotics, for the prevention of allergic disease. However, clinical trials have produced inconsistent results. This is at least partly explained by the highly complex crosstalk among probiotic bacteria, the host's microbiota, and immune cells. The identification of a bioactive substance from probiotic bacteria could circumvent this difficulty. OBJECTIVE: We sought to identify and characterize a bioactive probiotic metabolite for potential prevention of allergic airway disease. METHODS: Probiotic supernatants were screened for their ability to concordantly decrease the constitutive CCL17 secretion of a human Hodgkin lymphoma cell line and prevent upregulation of costimulatory molecules of LPS-stimulated human dendritic cells. RESULTS: Supernatants from 13 of 37 tested probiotic strains showed immunoactivity. Bioassay-guided chromatographic fractionation of 2 supernatants according to polarity, followed by total ion chromatography and mass spectrometry, yielded C11H12N2O2 as the molecular formula of a bioactive substance. Proton nuclear magnetic resonance and enantiomeric separation identified D-tryptophan. In contrast, L-tryptophan and 11 other D-amino acids were inactive. Feeding D-tryptophan to mice before experimental asthma induction increased numbers of lung and gut regulatory T cells, decreased lung TH2 responses, and ameliorated allergic airway inflammation and hyperresponsiveness. Allergic airway inflammation reduced gut microbial diversity, which was increased by D-tryptophan. CONCLUSIONS: D-tryptophan is a newly identified product from probiotic bacteria. Our findings support the concept that defined bacterial products can be exploited in novel preventative strategies for chronic immune diseases.

(-)-Adrenaline elicits positive inotropic, lusitropic, and biochemical effects through beta2 -adrenoceptors in human atrial myocardium from nonfailing and failing hearts, consistent with Gs coupling but not with Gi coupling.

Activation of either coexisting beta1- or beta2 -adrenoceptors with noradrenaline or adrenaline, respectively, causes maximum increases of contractility of human atrial myocardium. Previous biochemical work with the beta2 -selective agonist zinterol is consistent with activation of the cascade beta2 -adrenoceptors-->Gsalpha-protein-->adenylyl cyclase-->cAMP-->protein kinase (PKA)-->phosphorylation of phospholamban, troponin I, and C-protein-->hastened relaxation of human atria from nonfailing hearts. However, in feline and rodent myocardium, catecholamines and zinterol usually do not hasten relaxation through activation of beta2 -adrenoceptors, presumably because of coupling of the receptors to Gi protein. It is unknown whether the endogenously occurring beta2 -adrenoceptor agonist adrenaline acts through the above cascade in human atrium and whether its mode of action could be changed in heart failure. We assessed the effects of (-)-adrenaline, mediated through beta2 -adrenoceptors (in the presence of CGP 20712A 300 nM to block beta1 -adrenoceptors), on contractility and relaxation of right atrial trabecula obtained from nonfailing and failing human hearts. Cyclic AMP levels were measured as well as phosphorylation of phospholamban, troponin I, and protein C with Western blots and the back-phosphorylation procedure. For comparison, beta1 -adrenoceptor-mediated effects of (-)-noradrenaline were investigated in the presence of ICI 118,551 (50 nM to block beta2 -adrenoceptors). The positive inotropic effects of both (-)-noradrenaline and (-)-adrenaline were accompanied by reductions in time to peak force and time to reach 50% relaxation. (-)-Adrenaline caused similar positive inotropic and lusitropic effects in atrial trabeculae from failing hearts. However, the inotropic potency, but not the lusitropic potency, of (-)-noradrenaline was reduced fourfold in atrial trabeculae from heart failure patients. Both (-)-adrenaline and (-)-noradrenaline enhanced cyclic AMP levels and produced phosphorylation of phospholamban, troponin I, and C-protein to a similar extent in atrial trabeculae from nonfailing hearts. The hastening of relaxation caused by (-)-adrenaline together with the PKA-catalyzed phosphorylation of the three proteins involved in relaxation, indicate coupling of beta2 -adrenoceptors to Gs protein. The phosphorylation of phospholamban at serine16 and threonine17 evoked by (-)-adrenaline through beta2 -adrenoceptors and by (-)-noradrenaline through beta1 -adrenoceptors was not different in atria from nonfailing and failing hearts. Activation of beta2 -adrenoceptors caused an increase in phosphorylase a activity in atrium from failing hearts further emphasizing the presence of the beta2 -adrenoceptor-Gsalpha-protein pathway in human heart. The positive inotropic and lusitropic potencies of (-)-adrenaline were conserved across Arg16Gly- and Gln27Glu-beta2 -adrenoceptor polymorphisms in the right atrium from patients undergoing coronary artery bypass surgery, chronically treated with beta1 -selective blockers. The persistent relaxant and biochemical effects of (-)-adrenaline through beta2 -adrenoceptors and of (-)-noradrenaline through beta1 -adrenoceptors in heart failure are inconsistent with an important role of coupling of beta2 -adrenoceptors with Gialpha-protein in human atrial myocardium.

New insights into beta2-adrenoceptor signaling in the adult rat heart.

OBJECTIVE: The role of cAMP in beta(2)-adrenoceptor signaling and its functional relevance in adult rat heart has been the subject of considerable controversy. Therefore, we investigated the beta(2)-adrenoceptor pathways in both adult cardiomyocytes and in the intact hearts of Wistar rats with respect to protein kinase A (at Ser16)-, the key event in shortening of relaxation time, and CaM kinase II (at Thr17)-dependent phospholamban phosphorylation. METHODS: Contractile and cellular beta(1)/beta(2)-adrenergic responses were studied in parallel on the same perfused rat heart. (-)Isoproterenol and the beta(2)-adrenergic agonists zinterol and procaterol were used to discriminate the beta-adrenoceptor subtype-related actions. RESULTS: Beta(2)-adrenoceptor stimulation induces protein kinase A-dependent phospholamban phosphorylation in both adult cardiomyocytes and in adult hearts of rats. The beta(2)-adrenoceptor-mediated shortening of relaxation time in the heart correlates with Ser16 phosphorylation. Adenosine elicited antiadrenergic action on both beta(1)- and beta(2)-adrenergic signaling cascades by reducing the phosphorylation status of phospholamban. Only beta(1)-adrenoceptor stimulation produced significant CaM kinase II-related Thr17 phosphorylation, troponin I phosphorylation and activation of phosphorylase a. CONCLUSIONS: Our findings clearly show that beta(2)-adrenoceptor signaling is coupled to phospholamban phosphorylation and shortening of relaxation time in the adult rat heart.

Expression of human angiotensinogen-renin in rat: effects on transcription and heart function.

In double transgenic rats (dTGR) harboring the human angiotensinogen (hAOGEN) and human renin (hREN) genes, we studied cardiac transcript levels of hypertrophy-related, Ca(2+) regulatory, and beta-adrenoceptor-associated proteins. The contractile properties and the cellular signaling of isolated hearts exposed to (-)isoproterenol and/or angiotensin (Ang) I were evaluated. dTGR developed hypertension of 174.1+/- 7.6 versus 109.6 +/- 2.0 mm Hg (P<0.05) in Sprague-Dawley rats and heart hypertrophy. In hearts of dTGR, the transcript levels of ANP, beta-MHC, and alpha-MHC were altered (percentage versus Sprague-Dawley rats, 100%) by 304%, 178%, and 78%, respectively. Transcript levels of L-type Ca(2+) channel, Ca(2+) release channel, SERCA2a, phospholamban, G(i)- and G(s)-proteins were unchanged. Isolated hearts of dTGR indicated higher baseline contractility versus Sprague-Dawley rats. (-)Isoproterenol-modified contractility occurred in both groups; however, the extent (predrug value, 100%) was less in hearts of dTGR versus Sprague-Dawley rats (+dP/dt, 310 +/- 42% versus 534 +/- 63%; P<0.05). Interestingly, (-)isoproterenol shortened the relaxation time by equivalent to 25% in both groups. This finding was reflected by a protein kinase A-related phospholamban phosphorylation. Ang I depressed the heart contractility but did not interact with the protein kinase A pathway. In conclusion, we have found that expression of the hAOGEN-hREN complex in dTGR elicited specific effects on transcripts of ANP and myofibrillar proteins. Although the beta-adrenergically mediated relaxation was not impaired in the hypertrophied hearts, the extent of beta-adrenergic inotropic responsiveness was reduced.