There is increasing evidence indicating that exposure to air pollutants may be associated with the onset of several respiratory diseases such as allergic airway disease and chronic obstructive pulmonary disorder (COPD). Many lung diseases demonstrate an outgrowth of pathogenic bacteria belonging to the Proteobacteria phylum, and the incidence of occurrence of these diseases is higher in heavily polluted regions. Within the human body, the lungs are among the first to be exposed to the harmful effects of inhaled pollutants and microbes. Research in the past few decades have expounded on the air-pollution-induced local and systemic inflammatory responses, but the involvement of the lung microbial communities has not yet been well-characterized. Lungs were historically considered to be sterile, but recent advances have demonstrated that the lower respiratory tract is replete with a wide variety of microorganisms - both in health and disease. Recent studies show that these lung microbes may play a significant role in modulating the immune environment by inducing IgA and mucus production.
Air pollutants have previously been shown to alter intestinal bacterial populations that increase susceptibility to inflammatory diseases; however, to date, the effects of traffic-generated air pollutants on the resident microbial communities on the lungs have not been explored. The microbiome is influenced by several factors, including diet and environmental exposures. A large percentage of the Western world population consumes a high-fat (HF) diet which has resulted in the epidemic of obesity. Consumption of an HF diet has been shown to alter the intestinal microflora and increase baseline inflammation. We aimed to understand whether diet might also contribute to the alteration of the commensal lung microbiome, either alone or related to exposure. Thus, we investigated the hypothesis that exposure to air pollutants can alter the commensal lung microbiota, thereby promoting alterations in the lung's immune and inflammatory responses; in addition to determining whether these outcomes are exacerbated by a high fat-diet.
We performed two studies with exposures to different components of air pollutant mixtures on C57Bl/6 mice placed on either a control (LF) diet or a high-fat (HF) diet. Our first exposure study was performed on C57Bl/6 mice with a mixture of gasoline and diesel engine emissions (ME: 30 µg PM/m3 gasoline engine emissions + 70 µg PM/m3 diesel engine emissions) or filtered air (FA) for 6h/d, 7 d/wk for 30 days. The ME study investigated the alterations in immunoglobulin A (IgA), IgG and IgM, and lung microbiota abundance and diversity. Our results revealed ME exposures alongside the HF diet causes a decrease in IgA and IgG when compared to FA controls, thereby decreasing airway barrier protection. This was accompanied by the expansion of bacteria within the Proteobacteria phylum and a decrease in the overall bacterial diversity and richness in the exposed vs. control groups.
In our second study, we exposed C57Bl/6 mice to only the diesel exhaust particle component (35µg DEP, suspended in 35µl 0.9% sterile saline) or sterile saline only (control) twice a week for 30 days. We investigated immunoglobulin profiles by ELISA that revealed a significant increase in IgA and IgG in response to DEP. We also observed an increase in inflammatory tumor necrosis factor (TNF) - α, Interleukin (IL) -10, Toll-like receptors (TLR) - 2,4, nuclear factor kappa B (NF-κB) histologically and by RT-qPCR. Mucus production and collagen deposition within the lungs were also significantly elevated with DEP exposures. Microbial abundance determined quantitatively from the bronchoalveolar lavage fluid (BALF) by qPCR revealed an expansion of bacteria belonging to the Proteobacteria phylum in the DEP exposed groups on the HF diet. We also observed an increase in reactive oxygen and nitrogen species (ROS-RNS) products (nitrates), within the groups that revealed an expansion of Proteobacteria. These observations are most likely due to the unique metabolic capabilities of Proteobacteria to proliferate in inflammatory environments with excess nitrates. We assessed if treatments with probiotics could attenuate the DEP-induced inflammation by supplementing a separate group of study animals on the HF diet with 0.3 g/day of Winclove Ecologic® Barrier probiotics in their drinking water throughout the study. With probiotic treatments, we observed a significant decrease in ROS-RNS that was accompanied by complete elimination of Proteobacteria suggesting that in the absence of nitrates, the expansion of Proteobacteria is curbed effectively. We also observed a decrease in proinflammatory TNF-α and collagen deposition with probiotic treatments, and an increase in IgA levels within the BALF, suggesting that probiotics aid in balancing proinflammatory responses and enhance beneficial immune responses to efficiently mediate the DEP-induced inflammation.
Both studies showed that air pollutants alter the immune defenses and contribute to lung microbial alterations with an expansion of Proteobacteria. The immunoglobulin profiles discordant between the two studies can be explained by the route and/or duration and composition of air pollutant exposure. Collectively these studies suggest that exposure to air pollutants alter immune responses and/or increase the availability of inflammatory by-products within the lungs that can enable the selective outgrowth of pathogenic bacteria. The observed detrimental outcomes are further exacerbated when coupled with the consumption of an HF diet. Importantly, these results may shed light on the missing link between air pollution-induced inflammation and bacterial expansion and also point to therapeutic alternatives to curb bacterial outgrowth in lung disease exacerbations observed in patient populations living and/or working in heavily polluted regions.
