Rationale. Emerging findings link asthma and allergy to gut microbiome alterations, however causality remains largely unproven. Asthma onset can be promoted by environmental airborne particles, but mechanisms are poorly understood. At the same time, pulmonary particle exposure was shown to alter gut microbiome. Among the particles microplastics are a ‘new’ type and are increasingly found in the organism of humans; accumulating evidence implies health risks, but often no link to disease is seen making causality hard to establish. Our mouse model integrates these factors for a mechanistic study of asthma origin: prenatal exposure to environmental particles concentrated from urban air (CAP), diesel exhaust particles (DEP) or microplastic particles (MP) predisposes neonates to asthma. We hypothesized that particle-altered gut flora mediates asthma predisposition, and aimed to identify causative microbiome components and their metabolic activity operative in modulating immune signaling. Methods. Airway exposure of pregnant dams to CAP, DEP or MP triggers predisposition to asthma in newborns, so that they develop asthma after an intentionally low-dose of allergen that is innocuous in control pups. Here we performed gut microbiome transplant (GMT) from these asthma-predisposed neonates (without any allergen exposure) to intact recipients: asthma onset was determined as airway hyperresponsiveness, eosinophilia in bronchoalveolar lavage, lung tissue infiltration and elevation of IL-4, IL-5 and IL-13. Microbiome and metabolite changes were tested by metagenomic sequencing and metabolomics. Results. GMT conferred asthma predisposition to naïve recipients in all three particle groups. To test whether live microbes are required for the effect we radiation sterilized the GMT material, which ablated its ability to confer disease. To narrow down the effect we co-treated the GMT with antibacterials which also abrogated the GMT effect. Metagenomics revealed that more abundant in the CAP and DEP samples were Acetatifactor, Butyribacter, Kineothrix, Coproplasma and Lachnospiraceae bacteria (MP samples are work-in-progress). Functional analysis suggested a potential mechanistic explanation: these ‘suspect’ species are linked to short-chain fatty acids (acetate, butyrate) production known to affect dendritic cells (DCs). When tested, we found acetate and butyrate levels altered and DC function skewed toward allergy responses. Conclusions. Prenatal exposure to CAP, DEP or MP particles changes the gut microbiome of newborns making it causative of increased asthma predisposition. Live, but not dead, microbiome transplant confers the effect to recipients. Metagenomics identified potential bacteria ‘suspects’, informing future species-specific studies. The microbiome interaction with host immunity involves bacteria-derived fatty acids that modulate DC function leading to enhanced allergen presentation.

Despite broad knowledge of the pathogenesis, our understanding of the origin of allergy and asthma remains poor, preventing etiotropic treatments. The gut microbiome is seen to be altered in asthmatics; however, proof of causality of the microbiome alterations is lacking. We report on gut microbiome transplantation (GMT) from mice predisposed to asthma by maternal exposure to pro-allergy environmental particles into naive recipients. This GMT confers asthma predisposition, and the effect is abrogated by gamma sterilization of the transplant material or by co-administration of antibacterials, indicating that viable bacteria are mediating the effect. Metagenomics identifies key changes in the "pro-asthma" microbiome, and metabolomics links the identified species to altered production of butyrate known to act on immune cells and epigenetic mechanisms. We further show that transplant recipients develop DNA methylation alterations in dendritic cells. Finally, dendritic cells with an altered methylome present allergen to T cells, and this effect is abrogated by an epigenetically acting drug in vitro.

Targeted, promoter-specific removal of DNA methylation marks is emerging as a promising strategy for experimental and therapeutic regulation of gene expression. Research to date has relied largely on the expression of transgene-encoded epigenetic editor constructs in target cells. While effective for in vitro demonstrations, alternative approaches are needed for greater translatability to humans. Here, we describe the design of recombinant, gene-targeted epigenetic editor proteins that are directly taken up by mouse lung cells following administration in vivo without transgenesis, vectors, or packaging tools. Proteins are targeted to their intended promoter using either dCas9 or artificial zinc finger domains, and thymine-DNA-glycosylase (TDG) and ten-eleven translocation proteins (Tet) catalytic domains mediate specific demethylation. Results demonstrate intranuclear arrival of epigenetic editors in vitro and in mice, local DNA demethylation, and resulting highly gene-specific derepression of the transcriptional response, which confers sensitivity to interferon (IFN) stimulation. Therefore, this study provides proof of principle for vector-free, targeted promoter demethylation in vivo.

Titanium dioxide (TiO2), a particle with widespread presence in manufacturing industries, is used as a control in pollution studies. Of importance, TiO2 exposure has been shown to cause pulmonary inflammation and immune dysfunction. During pregnancy, increased cardiovascular stress occurs, inducing or aggravating conditions such as high blood pressure, diabetes, and preeclampsia. While a relation between particle exposure and cardiovascular disease has been observed, its role during pregnancy requires further understanding. Herein, we investigated the effect of TiO2 exposure on cardiac tissue in vitro and in vivo. In vitro studies using human cardiomyocytes showed TiO2 increased toxicity and necrotic death associated with enhanced oxidative stress. The use of N-acetylcysteine (NAC), a general antioxidant, rescued the myocytes from TiO2-induced necrotic death. Pregnant (P) or non-pregnant (NP) mice were challenged with TiO2 to assess the effect of pregnancy and exposure in cardiac tissue. Pregnancy alone was shown to alter the cardiac proteome with upregulation of inflammation biomarkers. Upon exposure, both P and NP mice showed notable proteome remodeling with pathways associated with oxidative stress, cardiac function, cell death, and inflammation being altered. Immunoblots confirmed changes in catalase, Complement (C) 5, and glutathione S-transferase mu 1 (GSTM1). These findings demonstrate that TiO2 can cause cardiac damage with characteristics similar to those in myocardial infarction and ischemia. Exposure during pregnancy can exacerbate some of these effects. Preventative measures should be taken to limit exposure and future studies could indicate long-term exposure impacts. ### Competing Interest Statement The authors have declared no competing interest. * (TiO2) : Titanium dioxide (NAC) : N-acetylcysteine (GSTM1) : glutathione S-transferase mu 1 (P) : pregnant (NP) : non-pregnant (C) : complement (PM) : particulate matter (LDH) : Lactate dehydrogenase (PBS) : phosphate buffered saline (SDS) : sodium-dodecyl sulfate (LC-MS/MS) : Liquid chromatography-tandem mass spectrometry (MFI) : mean fluorescent intensity (GO) : gene ontology (TBST) : 5% BSA 0.01% Tween-20 Tris Buffered Saline (ROS) : reactive oxygen species (LFQ) : label-free quantitation (FDR) : false discovery rate (PCA) : principal component analysis (H2O2) : hydrogen peroxide (O2-) : superoxide (O-) : hydroxyl radical National Institute of Environmental Health Sciences, R01ES030227

Introduction:Research has shown epigenetic change via alternation of the methylation profile of human skeletal muscle DNA after Cardio-Pulmonary Bypass (CPB). In this study, we investigated the change in epigenome-wide DNA methylation profiles of porcine myocardium after ischemic insult in the setting of treatment with extracellular vesicle (EV) therapy in normal vs. high-fat diet (HFD) pigs.Methods:Four groups of three pigs underwent ameroid constrictor placement to the left circumflex artery (LCx) and were assigned to the following groups: (1) normal diet saline injection; (2) normal diet EV injection; (3) HFD saline injection; and (4) HFD EV injection. DNA methylation was profiled via reduced-representation bisulfite sequencing (RRBS) and compared using a custom bioinformatic pipeline.Results:After initial analysis, 441 loci had a nominal P value < 0.05 when examining the effect of ischemia vs. normal heart tissue on a normal diet in the absence of treatment. 426 loci at P value threshold < 0.05 were identified when comparing the ischemic vs. normal tissue from high-fat diet animals. When examining the effect of EV treatment in ischemic tissue in subjects on a normal diet, there were 574 loci with nominal P value < 0.05 with two loci Fructosamine 3 kinase related protein [(FN3KRP) (P < 0.001)] and SNTG1 (P = 0.03) significant after Bonferroni correction. When examining the effect of EV treatment in ischemic tissue in HFD, there were 511 loci with nominal P values < 0.05. After Bonferroni correction, two loci had P values less than 0.05, betacellulin [(BTC) (P = 0.008)] and [proprotein convertase subtilisin/kexin type 7 (PCSK7) (P = 0.01)].Conclusions:Alterations in DNA methylation were identified in pig myocardium after ischemic insult, change in diet, and treatment with EVs. Hundreds of differentially methylated loci were detected, but the magnitude of the effects was low. These changes represent significant alterations in DNA methylation and merit further investigation.

Tweetable abstract Exploring uropathogenic E. coli-induced epigenetic changes in uroepithelial cells contributing to recurrent UTIs and potential therapeutic strategies. Understanding these mechanisms could inform novel UTI interventions.

Aberrant DNA hypermethylation at regulatory cis-elements of particular genes is seen in a plethora of pathological conditions including cardiovascular, neurological, immunological, gastrointestinal and renal diseases, as well as in cancer, diabetes and others. Thus, approaches for experimental and therapeutic DNA demethylation have a great potential to demonstrate mechanistic importance, and even causality of epigenetic alterations, and may open novel avenues to epigenetic cures. However, existing methods based on DNA methyltransferase inhibitors that elicit genome-wide demethylation are not suitable for treatment of diseases with specific epimutations and provide a limited experimental value. Therefore, gene-specific epigenetic editing is a critical approach for epigenetic re-activation of silenced genes. Site-specific demethylation can be achieved by utilizing sequence-dependent DNA-binding molecules such as zinc finger protein array (ZFA), transcription activator-like effector (TALE) and clustered regularly interspaced short palindromic repeat-associated dead Cas9 (CRISPR/dCas9). Synthetic proteins, where these DNA-binding domains are fused with the DNA demethylases such as ten-eleven translocation (Tet) and thymine DNA glycosylase (TDG) enzymes, successfully induced or enhanced transcriptional responsiveness at targeted loci. However, a number of challenges, including the dependence on transgenesis for delivery of the fusion constructs, remain issues to be solved. In this review, we detail current and potential approaches to gene-specific DNA demethylation as a novel epigenetic editing-based therapeutic strategy.

Particulate matter in the air exacerbates airway inflammation (AI) in asthma; moreover, prenatal exposure to concentrated urban air particles (CAPs) and diesel exhaust particles (DEPs) predisposes the offspring to asthma and worsens the resolution of AI in response to allergens. We previously tested the hypothesis that such exposure impairs the pathways of specialized proresolving mediators that are critical for resolution and found declined Lipoxin A4 (LxA4) and Resolvin E2 (RvE2) levels in the "at-risk" pups of exposed mothers. Here, we hypothesized that supplementation with synthetic LxA4 or RvE2 via the airway can ameliorate AI after allergen exposure, which has not been tested in models with environmental toxicant triggers. BALB/c newborns with an asthma predisposition resultant from prenatal exposure to CAPs and DEPs were treated once daily for 3 days with 750 ng/mouse of LxA4 or 300 ng/mouse of RvE2 through intranasal instillation, and they were tested with the intentionally low-dose ovalbumin protocol that elicits asthma in the offspring of particle-exposed mothers but not control mothers, mimicking the enigmatic maternal transmission of asthma seen in humans. LxA4 and RvE2 ameliorated the asthma phenotype and improved AI resolution, which was seen as declining airway eosinophilia, lung tissue infiltration, and proallergic cytokine levels.

Introduction/Hypothesis: Hyperproliferative endothelial cells (ECs) play an important role in the pathogenesis of pulmonary arterial hypertension (PAH), by formation of occlusive plexiform lesions. We previously reported that Anoctamin-1 (ANO1), a calcium-activated chloride channel, expression is increased in lung ECs from patients with PAH and is associated with increased EC proliferation. However, the regulatory mechanism underlying ANO1 expression in PAH remains unclear. DNA methylation has been recognized as an important mechanism regulating gene expression. In this study, we test the hypothesis that ANO1 expression in PAH is epigenetically regulated via DNA methylation. Methods/Results: DNA was extracted from lung ECs of patients with idiopathic PAH (n=7) and controls (n=6), followed by Bisulfite Next-Generation Sequencing (EpigenDx, MA) to determine DNA methylation profile in human ANO1 gene (Ensembl Gene ID: ENSG00000131620). We used 19 assays to cover 97 CpG sites in the human ANO1 gene, including regions of 5’-upstream, 5’-UTR, and different introns. Our data show that the methylation of 29 of 97 (30%) CpG sites in human ANO1 gene is significantly altered in lung ECs from PAH patients than that from control subjects. Among the significantly altered 29 CpG sites, the methylation at 22 CpG sites (76%) is significantly decreased. We developed a CRISPR-based approach to target four differentially methylated CpG sites to determine the specific contribution of their methylation in regulation of human ANO1 gene. We demonstrate that demethylation of one of the sites, CpG#-73, in human embryonic kidney cells resulted in an increase in ANO1 mRNA expression compared to control, suggesting that decreased DNA methylation is sufficient to upregulate human ANO1 expression. Conclusions: Our results demonstrate that DNA methylation in human ANO1 gene is significantly altered in PAH and reduction in DNA methylation may serve as a regulatory mechanism for ANO1 upregulation in settings of PAH.

Our results demonstrate that DNA methylation in human ANO1 gene is significantly altered in PAH and reduction in DNA methylation may serve as a regulatory mechanism for ANO1 upregulation in settings of PAH.

Parental asthma or allergy have been linked to higher risk of asthma in a child; this occurs to a variable extent in different study populations. Moreover, it is debated whether maternal more so than paternal asthma history is a stronger predisposing factor: while in some countries/populations the maternal effect was clearly seen over paternal, in others the parental effects were equivalent, and in a few studies paternal effect dominated. Here we aimed to determine parental asthma and allergy effect in the Danish GEneral SUburban population Study (GESUS). This cross-sectional study has involved 21,362 adults aged 20+ years in the suburbs of Copenhagen. We used a combination of questionnaire approach, history of prescribed asthma medications and pulmonary function testing to determine odds ratios for maternal and paternal (and combined) asthma and allergy linked to asthma in the test subjects. We found that the input of maternal vs. paternal asthma effect was approximately equal (age and sex-adjusted OR 2.46, 95% CI: 2.15-2.81 for asthmatic mothers vs. 2.97, 2.58-3.42 for asthmatic fathers), except for the "ever asthma" age and sex-adjusted odds ratios where paternal allergy seems to have conferred a marginally greater effect (age and sex-adj. OR 1.96 for maternal allergy vs. 2.44 for paternal allergy, p = 0.03). Stratifying for gestational tobacco smoking did not affect the maternal results. We conclude that in the GESUS study parental asthma or allergy were strongly linked to higher asthma risk in offspring, without a prominent maternal or paternal effect.

In recent years, microbiome research has expanded from the gastrointestinal tract to other host sites previously thought to be abacterial such as the lungs. Yet, the effects of pregnancy in the lung and gut microbiome remains unclear. Here we examined the changes in the gut and lung microbiome in mice at 14 days of gestation. Lung tissue and stool samples were collected from pregnant and non-pregnant female BALB/c mice, DNA was isolated, amplified, and bacterial specific V4 16S rRNA gene was sequenced. Using an in-house bioinformatic pipeline we assessed the microbial composition of each organ using stool and lung tissue samples. The stool data showed that Lachnospiraceae and Lactobacillaceae were more abundant in the pregnant mice. Likewise, Lactobacillaceae were dominant in the lungs of pregnant mice. However, Streptococcaceae were dominant in the lungs of non-pregnant mice with a low microbial abundance in the pregnant mice. A permutation test showed that pregnancy significantly contributes to the variance in both the lung and stool microbiome. At the same time, we estimate that 49% of the total detected operational taxonomic units were shared between the stool and lung data. After removing common stool-associated bacteria from the lung dataset, no microbial differential abundance was detected between the pregnant and non-pregnant lung microbial community. Thus, pregnancy contributes to variance to the lung and stool microbiome but not in the unique lung microbiota.

The respiratory tract has a resident microbiome with low biomass and limited diversity. This results in difficulties with sample preparation for sequencing due to uneven bacteria-to-host DNA ratio, especially for small tissue samples such as mouse lungs. We compared effectiveness of current procedures used for DNA extraction in microbiome studies. Bronchoalveolar lavage fluid (BALF) and lung tissue samples were collected to test different forms of sample pre-treatment and extraction methods to increase bacterial DNA yield and optimize library preparation. DNA extraction using a pre-treatment method of mechanical lysis (lung tissue) and one-step centrifugation (BALF) increased DNA yield and bacterial content of samples. In contrast, a significant increase of environmental contamination was detected after phenol chloroform isoamyl alcohol (PCI) extraction and nested PCR. While PCI has been a standard procedure used in microbiome studies, our data suggests that it is not efficient for DNA extraction of frozen low biomass samples. Finally, a DNA Enrichment kit was tested and found to improve the 16S copy number of lung tissue with a minor shift in microbial composition. Overall, we present a standardized method to provide high yielding DNA and improve sequencing coverage of low microbial biomass frozen samples with minimal contamination.

PURPOSE OF REVIEW:Increases in ambient levels of air pollutants have been linked to lung inflammation and remodeling, processes that lead to the development and exacerbation of allergic asthma. Conventional research has focused on the role of CD4+ T helper 2 (TH2) cells in the pathogenesis of air pollution-induced asthma. However, much work in the past decade has uncovered an array of air pollution-induced non-TH2 immune mechanisms that contribute to allergic airway inflammation and disease.RECENT FINDINGS:In this article, we review current research demonstrating the connection between common air pollutants and their downstream effects on non-TH2 immune responses emerging as key players in asthma, including PRRs, ILCs, and non-TH2 T cell subsets. We also discuss the proposed mechanisms by which air pollution increases immune-mediated asthma risk, including pre-existing genetic risk, epigenetic alterations in immune cells, and perturbation of the composition and function of the lung and gut microbiomes. Together, these studies reveal the multifaceted impacts of various air pollutants on innate and adaptive immune functions via genetic, epigenetic, and microbiome-based mechanisms that facilitate the induction and worsening of asthma.

Maternal gestational exposures to traffic and urban air pollutant particulates have been linked to increased risk and/or worsening asthma in children; however, mechanisms underlying this vertical transmission are not entirely understood. It was postulated that gestational particle exposure might affect the ability to elicit specialized proresolving mediator (SPM) responses upon allergen encounter in neonates. Lipidomic profiling of 50 SPMs was performed in lungs of neonates born to mice exposed to concentrated urban air particles (CAP), diesel exhaust particles (DEP), or less immunotoxic titanium dioxide particles (TiO2). While asthma-like phenotypes were induced with identical eosinophilia intensity across neonates of all particle-exposed mothers, levels of LXA4, HEPE and HETE isoforms, and HDoHe were only decreased by CAP and DEP only but not by TiO2. However, RvE2 and RvD1 were inhibited by all particles. In contrast, isomers of Maresin1 and Protectin D1 were variably elevated by CAP and DEP, whereas Protectin DX, PGE2, and TxB2 were increased in all groups. Only Protectin D1/DX, MaR1(n-3,DPA), 5(S),15(S)-DiHETE, PGE2, and RvE3 correlated with eosinophilia but the majority of other analytes, elevated or inhibited, showed no marked correlation with inflammation intensity. Evidence indicates that gestational particle exposure leads to both particle-specific and nonspecific effects on the SPM network.

There is mounting evidence that environmental exposures can result in effects on health that can be transmitted across generations, without the need for a direct exposure to the original factor, for example, the effect of grandparental smoking on grandchildren. Hence, an individual's health should be investigated with the knowledge of cross-generational influences. Epigenetic factors are molecular factors or processes that regulate genome activity and may impact cross-generational effects. Epigenetic transgenerational inheritance has been demonstrated in plants and animals, but the presence and extent of this process in humans are currently being investigated. Experimental data in animals support transmission of asthma risk across generations from a single exposure to the deleterious factor and suggest that the nature of this transmission is in part due to changes in DNA methylation, the most studied epigenetic process. The association of father's prepuberty exposure with offspring risk of asthma and lung function deficit may also be mediated by epigenetic processes. Multi-generational birth cohorts are ideal to investigate the presence and impact of transfer of disease susceptibility across generations and underlying mechanisms. However, multi-generational studies require recruitment and assessment of participants over several decades. Investigation of adult multi-generation cohorts is less resource intensive but run the risk of recall bias. Statistical analysis is challenging given varying degrees of longitudinal and hierarchical data but path analyses, structural equation modelling and multilevel modelling can be employed, and directed networks addressing longitudinal effects deserve exploration as an effort to study causal pathways.