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Air pollution poses a significant threat to public health. It is a mixture of particulate matter, gaseous substances, and other secondary substances. Despite epidemiological research indicating the interactive effects of various components of air pollution, existing studies and regulatory limits often focus on single-exposure scenarios. The emerging understanding of the microbiome’s role in regulating immune response, metabolism, and pathophysiology of various diseases points towards interactions between environmental exposures and microbial dysbiosis. In this dissertation, mice were exposed to relevant deposited doses of carbon black (CB) and ozone (O3), and lung inflammation as well as the lung-gut axis of the microbiome alterations were studied. We also studied the role of nucleotide-biding oligomerization domainlike receptor X1 (NLRX1) in mechanistically modulating immune response and microbial balance. Our project involved inhalation exposures, a unique mouse model (NLRX1 knock out), flow cytometric analysis of immune cells, 16s rRNA sequencing, and absolute bacterial load analysis using QIAcuity digital PCR. We demonstrated significant induction of pulmonary inflammatory response after acute CB+O3 co-exposure coupled with reduced diversity indices and a pathogenic bacterial enrichment in the lung microbiome. In contrast, a significant increase in gut bacterial load, abundance of beneficial bacteria, and induction of microbiome-derived secondary metabolites in the systemic circulation indicated a homeostatic shift. Sub-chronic exposure to CB+O3 did not show significant alterations in the inflammatory response and microbiome balance. However, aggravated inflammatory response and microbiome alterations were observed when lungs were challenged with bleomycin post-exposure. A significant role of genetics, exposure, and sex was observed that changed dynamically at different post-exposure time points. In summary, these studies improved the current understanding of particle and gas mixed exposures in terms of their impacts on immune cell profiling, oxidative stress, and microbiome regulation. Furthermore, our project highlighted the role of microbiome alterations in the air pollution-induced exacerbation of lung inflammation and injury identifying a potential role of the lung-gut axis.

Air pollution is a mixture of particulate matter, gaseous substances, and other contaminant, yet most of the studies have addressed single exposure outcomes. The effect of environmental toxicants in lung adverse outcomes can be evaluated by studying the co-exposure scenario. The impact of co-exposures is not well addressed in susceptible populations like patients suffering from acute lung injury, asthma, and fibrosis. Without such knowledge it would be difficult to establish a safe exposure limit for mitigating environmental burden in patients and public. The main goal of this study was to understand the effect of ultrafine particle and ozone co-exposure in lung inflammation and lung function decline. We further studied the effect of nucleotide-binding oligomerization domain-like receptor X1 (NLRX1) in mechanistically modulating these responses. NLRX1 has been implicated in inflammation, oxidative stress and mitochondrial dysfunction in various diseases like inflammatory bowel disease and cancer. However, its role in environmental disease burden is not clear. Therefore, we hypothesized that co-exposure to ultrafine carbon black and ozone induce greater toxicity compared to individual exposure and NLRX1 causes more sensitivity in co-exposure induced lung toxicity. We aimed at understanding the mechanistic impact of ultrafine carbon black and ozone co-exposure in induction of inflammation and lung toxicity. We demonstrated significant induction in lung inflammatory responses and airway hyperresponsiveness via induction of the oxidant-epithelial-alarmin pathway after inhalation co-exposure. Furthermore, we demonstrated that ozone reacted carbon black particles caused endothelial cell dysfunction (increased monolayer permeability and impaired migration) via CXC motif chemokine receptor 3 (CXCR3) mediated pathway which occurs via inflammatory mediators release by macrophages. Our studies also indicate that repeated co-exposure to ultrafine carbon black and ozone can cause mitochondrial dysfunction leading to decreased ATP production. Moreover, we demonstrated that co-exposure induced aggravation of inflammation is significantly higher in mice lacking NLRX1 compared to wild-type mice. We also demonstrated that co-exposure resulted in increased inflammatory responses and lung function decline in acute lung injury model. Our project involved the state-of-the-art inhalation co-exposure at West Virginia University iTox Core Facility, unique mouse model (NLRX1 knock out), mitochondrial function studies and lung function studies with Flexivent™. This project advance in understanding the role of NLRX1 in biological outcomes (inflammation, injury, oxidative stress, mitochondrial function) in response to mixed exposure. These exposures are known to induce lung toxicity, pulmonary inflammation and lung function decline. Moreover, the findings will help to delineate the effect of environmental co-exposures in pulmonary toxicity and develop preventative efforts to mitigate the burden of environmental pollution. This project identified the mechanistic impact of co-exposure in inducing lung toxicity compared to individual gaseous and particulate constituent and can aid in reducing the burden of environmental toxicant in the long run