Environmental particulate matter induces murine intestinal inflammatory responses and alters the gut microbiome

Abstract

Background: Particulate matter (PM) is a key pollutant in ambient air that has been associated with negative health conditions in urban environments. The aim of this study was to examine the effects of orally administered PM on the gut microbiome and immune function under normal and inflammatory conditions.

Methods: Wild-type 129/SvEv mice were gavaged with Ottawa urban PM10 (EHC-93) for 7-14 days and mucosal gene expression analyzed using Ingenuity Pathways software. Intestinal permeability was measured by lactulose/mannitol excretion in urine. At sacrifice, segments of small and large intestine were cultured and cytokine secretion measured. Splenocytes were isolated and incubated with PM10 for measurement of proliferation. Long-term effects of exposure (35 days) on intestinal cytokine expression were measured in wild-type and IL-10 deficient (IL-10(-/-)) mice. Microbial composition of stool samples was assessed using terminal restriction fragment length polymorphism. Short chain fatty acids were measured in caecum.

Results: Short-term treatment of wild-type mice with PM10 altered immune gene expression, enhanced pro-inflammatory cytokine secretion in the small intestine, increased gut permeability, and induced hyporesponsiveness in splenocytes. Long-term treatment of wild-type and IL-10(-/-) mice increased pro-inflammatory cytokine expression in the colon and altered short chain fatty acid concentrations and microbial composition. IL-10(-/-) mice had increased disease as evidenced by enhanced histological damage.

Conclusions: Ingestion of airborne particulate matter alters the gut microbiome and induces acute and chronic inflammatory responses in the intestine.

Figure 1. Ingenuity Pathway gene networks.

The most highly significant gene networks identified in the Ingenuity Pathway analysis of the gene expression data in response to PM₁₀ are shown for small intestine at days 7(A) and 14(B) and colon at days 7(C) and (D). The networks are displayed graphically as nodes (genes/gene products) and edges (the biological relationships between the nodes). The intensity of the node color indicates the degree of up (red) or down (green) regulation in gene expression. Nodes are displayed using shapes that represent the functional class of the gene product. Edges are displayed as a direct interaction (solid line).

Figure 2. Cytokine secretion from isolated small intestine (SI) and colon taken from WT mice treated with PM₁₀ for 7 (D7) or 14 (D14) days.

A significant increase in CXCL1 (A), IL-1β (B) and IL-10 (E) was seen in the small intestine at day 7. Bars are mean ± SEM n = 6 for all groups ^a p<0.05 compared with control at day 7; ^bp<0.05 compared with control at day 14.

Figure 3. PM₁₀ increased small intestinal permeability and induced hyporesponsiveness in splenocytes.

A) Lactulose/mannitol excretion in urine was measured weekly. B) Statistical analysis of areas under the curve of mice treated with PM₁₀ or vehicle. Small intestinal permeability from the PM₁₀ treated group was significantly higher compared with vehicle treated group. C) Proliferation of isolated splenocytes from mice treated for 14 days under basal conditions and in response to PM₁₀ (0.5 mg/ml). Response to αCD3 was used as a positive control and results are displayed as a ratio to αCD3 proliferation. Splenocytes isolated from mice that had been treated with PM₁₀ for 14 days had less proliferation in response to PM₁₀ compared with control mice. ^ap<0.05 compared with control mice, n = 8; ^bp<0.05 compared with control in response to PM_10. N = 6.

Figure 4. Cytokine expression in small intestine (SI) and colon taken from WT and IL-10^−/− mice treated with PM₁₀ for 35 days.

A significant increase in IL-17 (A), IL-1β (c), TNFα (D), IL-12 (E) and IL-13 (F) was seen in the colon of IL-10^−/− mice treated with PM₁₀. PM₁₀ induced increased IL-17 and IL-13 in the colons of WT mice. Bars are mean ± SEM n = 6–9 for all groups ^a p<0.05 compared with WT control colon; ^bp<0.05 compared with IL-10^−/− control colon; ^cp<0.05 compared with IL-10^−/− control SI.

Figure 5. Microbiota composition in stool samples from WT and IL-10^−/− mice after 35 days of treatment with PM₁₀.

Stool was analyzed using T-RFLP. (A) Relative abundance of phyla. Table provides mean ± SEM for each phylum as the percentage of total sequences. (B) Bacteria communities were clustered using partial least squares discriminant analysis (PLS-DA). WT mice clustered independently from IL-10 mice, and PM₁₀ treatment shifted the microbiota in both WT and IL-10^−/− groups. WT control: Black squares (n = 9); IL-10^−/− control: Blue triangles (n = 6); WT+PM₁₀: Red circles (n = 3); IL-10^−/−+PM₁₀: green crosses (n = 3).

Figure 6. Relative abundance of phyla in stool samples from WT and IL-10^−/− mice at days 0 and 35.

In IL-10^−/− mice, PM₁₀ decreased percentages of Bacteroidetes and increased Firmicutes compared with day 0 (Figure 6). PM₁₀ increased amounts of Verrucomicrobia in both WT and IL-10^−/− mice. Bars are mean ± SEM n = 6–9 for all groups ^*p<0.05 compared with day 0.

Figure 7. Short chain fatty acids in cecal contents.

IL-10^−/− mice exposed to PM₁₀ showed a significant increase in isovaleric (F) and isobutyric (C) and a decrease in butyric acid (D). WT mice had a significant decrease in butyric acid (D) and valeric acid (F). Bars are mean ± SEM n = 6 for all groups ^a p<0.05 compared with WT control mice; ^bp<0.05 compared with IL-10^−/− control mice.