Asthma is triggered or worsened by environmental exposures and is associated with epigenetic changes in humans and animal models. Microbial dysbiosis in the gut and the lung is increasingly being associated with the incidence and severity of asthma, however causality studies are lacking. We have adapted a mouse model that focuses on the ONSET of allergic asthma early in life after an in utero exposure to environmental particles to study how microbiome may lead to the asthma onset. In this model, we have shown that maternal exposures (to allergen or particulate matter, e.g. concentrated urban air particles (CAP), diesel exhaust particles (DEP) and titanium dioxide particles (TiO2), trigger increased asthma risk in several generations of the offspring. Humans are widely exposed to these particulates, especially in urban and industrial settings, where the incidence of asthma is also higher. We found that the increased ‘preparedness’ for asthma in these neonates is associated with DNA methylation changes in key immune cells – dendritic cells (DC) that are essential in asthma origin. Important unanswered questions are why these epigenetic changes occur, and whether there is a causative link to the aberrant microbiome seen in asthma. We hypothesize that in utero exposures to particles alter the microbiome of the pregnant mice and their offspring, which then signals to the immune cells in a way that predisposes the offspring to allergy. In Specific Aim 1, we will test what happens to the maternal microbiome (gut, lung and vaginal) after the gestational particle exposure, as it is the maternal flora that largely seeds the neonate’s microbiome. Longitudinal profiling will employ a multifaceted approach, including 16S/ITS taxonomic profiling, metagenomic sequencing and targeted metabolomics, for the comprehensive analysis of the composition and metabolism of the microbes. In Specific Aim 2, we will examine the neonatal gut microbiome via similar longitudinal profiling, including their response to allergen and establishment of the asthma phenotype. Importantly, we will perform causality experiments by transferring the hypothetically aberrant flora from the “asthma-at-risk” donor pups (born to the dams treated with particles) to normal recipients, and vice versa: fecal microbiota transplant (FMT). Finally, we will test the effect of the FMT on the recipient’s DC epigenome. In Specific Aim 3, we will similarly profile neonatal lung microbiome and will test the effect of antibiotic- based alteration of the aberrant lung microflora on asthma preparedness. Significance: Here we postulate two, potentially interconnected, mechanisms in asthma onset: epigenetics and the microbiome. Both the epigenetic alterations in immune cells and the dysbiosis in the gut and lung have been linked to asthma in humans and mouse models but causality studies are lacking. The proposed research addresses this gap in knowledge in a study designed to test basic mechanisms of relativ...