Lung-gut axis of microbiome alterations following co-exposure to ultrafine carbon black and ozone

Abstract

Background: Microbial dysbiosis is a potential mediator of air pollution-induced adverse outcomes. However, a systemic comparison of the lung and gut microbiome alterations and lung-gut axis following air pollution exposure is scant. In this study, we exposed male C57BL/6J mice to inhaled air, CB (10 mg/m³), O₃ (2 ppm) or CB + O₃ mixture for 3 h/day for either one day or four consecutive days and were euthanized 24 h post last exposure. The lung and gut microbiome were quantified by 16 s sequencing.

Results: Multiple CB + O₃ exposures induced an increase in the lung inflammatory cells (neutrophils, eosinophils and B lymphocytes), reduced absolute bacterial load in the lungs and increased load in the gut. CB + O₃ exposure was more potent as it decreased lung microbiome alpha diversity just after a single exposure. CB + O₃ co-exposure uniquely increased Clostridiaceae and Prevotellaceae in the lungs. Serum short chain fatty acids (SCFA) (acetate and propionate) were increased significantly only after CB + O₃ co-exposure. A significant increase in SCFA producing bacterial families (Ruminococcaceae, Lachnospiraceae, and Eubacterium) were also observed in the gut after multiple exposures. Co-exposure induced significant alterations in the gut derived metabolite receptors/mediator (Gcg, Glp-1r, Cck) mRNA expression. Oxidative stress related mRNA expression in lungs, and oxidant levels in the BALF, serum and gut significantly increased after CB + O₃ exposures.

Conclusion: Our study confirms distinct gut and lung microbiome alterations after CB + O₃ inhalation co-exposure and indicate a potential homeostatic shift in the gut microbiome to counter deleterious impacts of environmental exposures on metabolic system.

Keywords: Co-exposure; EPR; Inflammation; Inhalation; Microbial dysbiosis; Ozone; Ultrafine carbon black.

Fig. 1

Study layout and analysis of lung inflammation. A Layout of animal exposure experiments. B Heat-map showing alterations in immune cell profile. C57BL/6J mice (8–12 weeks) were exposed to air, CB (10 mg/m³), O₃ (2 ppm) or CB + O₃ for 3 h for either one or four exposures and euthanized 24 h post last exposure. Data are presented as mean ± standard error of mean (SEM) and analyzed by Two-way Analysis of Variance (ANOVA) followed by Tukey’s post hoc test. Lung tissues were homogenized, stained and analyzed in LSRFortessa II. n = 3–5. *p ≤ 0.05. *denotes significantly difference from air, # denotes significantly different from O₃ at the same time point, and $ denotes significantly different between single and multiple exposure groups

Fig. 2

Alterations in the lung microbiome. A Unique alterations in OTUs after single exposure B Family level analysis of shared and unique OTUs after multiple exposures C Shannon alpha diversity index. The boxplots show median, quartile, smallest and largest observations. D Bacterial taxonomic profiles at the family level in the lung microbiota after single day CB, O₃ or CB + O₃ exposure. E Bacterial taxonomic profiles at the family level in the lung microbiota after multiple days CB, O₃ or CB + O₃ exposure. F Bacterial taxonomic profiles (family level) after removing top three families (single exposure). G Bacterial taxonomic profiles (family level) after removing top three families (multiple exposures). H Absolute quantification of bacterial load per ng of lung DNA was analyzed using Qiacuity digital PCR. I Correlation between absolute bacterial load in the lungs and inflammatory cells (neutrophils, eosinophils, B and T cells) after multiple exposures. *p ≤ 0.05. *denotes significantly difference compared to air, ^#denotes significantly difference compared between ozone alone and co-exposure. Data are presented as mean ± standard error of mean (SEM) and analyzed by Two-way ANOVA followed by Tukey’s post hoc test. The Kruskal–Wallis test was performed to determine the statistical significance of Shannon diversity index n = 3–5

Fig. 3

Alterations of alpha diversity indices and relative abundance of gut microbial communities after multiple exposures. A Family level analysis of shared and unique microbiota at multiple exposures showing altered unique OTUs compared to air. B Shannon diversity index. The boxplots show median, quartile, smallest and largest observations. C Bacterial taxonomic profiles at the family level in the colon microbiota after multiple days CB, O₃ or CB + O₃ exposure. D Bacterial taxonomic profiles (top three families removed) after multiple exposures. E–G Significant increase in Xylanphilum, Oscillibacter and Anaeroplasma in the colon contents. H Lactobacillus significantly increased after multiple CB + O₃ co-exposure. I Bacteroidetes level in the gut DNA increased significantly compared to control. J Firmicutes level altered in the gut. K Absolute bacterial load per ng of colon content DNA was analyzed using Qiacuity digital PCR. *p ≤ 0.05. *denotes significantly difference compared to air. ^#denotes significantly difference between ozone and co-exposure. The Kruskal–Wallis test was performed to determine the statistical significance of diversity index. n = 5–6

Fig. 4

Alterations in oxidative stress response. A Alterations in Duox2, p22, Gpx1, Gpx2, Gpx3, Gpx4, Nrf2, and Ho-1 gene expression in lungs after single air, CB, O₃ and CB + O₃ exposure. B Significant increase in H₂O₂ levels in the lungs after single co-exposure. C Representative room temperature X-band EPR spectra of CM^• radical in bronchoalveolar lavage fluid after single exposure, and plot of EPR signal intensity D Representative room temperature EPR spectra of CM^• radical in serum after single exposure, and plot of signal intensity. E Representative room temperature EPR spectra of CM^• radical in gut samples after multiple exposures and plot of EPR signal intensity. F Correlation of absolute bacterial load in the lungs to hydrogen peroxide levels after single exposure. G-H Correlation of absolute bacterial load in the lung and redox levels in the lavage and serum, respectively after single inhalation exposure. Gene expression data are presented as log fold changes compared to 18 s (internal control). Data are presented as mean ± standard error of mean (SEM) and analyzed by One-way Analysis of Variance (ANOVA) followed by Tukey’s post hoc test. n = 5–6. *p ≤ 0.05. *denotes significantly difference from air, and ^# denotes significantly different from O₃ at the same time point

Fig. 5

Alterations in short chain fatty acid content and respective microbiome. A Alterations in serum acetate after multiple exposures. B–C Alteration in propionate and butyrate content in serum by GC–MS after multiple exposures. D–G Significant increase in Ruminiclostridium 5, Ruminiclostridium 6, Ruminococcaceae_uncultured and Lacnospiraceae_uncultured in the colon content after multiple CB + O₃ exposure. Data are presented as mean ± standard error of mean (SEM) and analyzed by One-way Analysis of Variance (ANOVA) followed by Tukey’s post hoc test. n = 5–6. *p ≤ 0.05. *denotes significantly difference from air, ^# denotes significantly different from O₃ at the same time point

Fig. 6

Alterations in gut microbiome produced secondary mediators. A Alteration in the Gcg mRNA expression after multiple exposures in the colon as log fold change, B–D Alteration in the Glp1r, Cck and Pyy mRNA expression after multiple exposures in the colon as log fold change. E, F Correlation of Glp1r and Cck mRNA expression and Lactobacillas abundance after multiple exposures in the colon. Data are presented as mean ± standard error of mean (SEM) and analyzed by One-way Analysis of Variance (ANOVA) followed by Tukey’s post hoc test. n = 5–6. *p ≤ 0.05. *denotes significantly difference from air

Fig. 7

Overview figure representing co-exposure induced pulmonary and intestinal alteration. Neutrophils, eosinophils and B cells were significantly increased in the lungs after multiple CB + O₃ co-exposure. Microbial alpha diversity and total bacterial load were reduced in the lungs. CB + O₃ exposure also significantly induced serum short chain fatty acids (SCFA) and oxidant level (BALF, serum and gut tissue). Gut total bacterial load, Lactobacillus and Bididobacterium significantly increased after multiple CB + O₃ exposure