Immune consequences of exposure to β-pinene oxidation aerosols: adult versus gestational murine models

Abstract

Background: While studies demonstrating the adverse effects of air pollution on human health are accumulating, studies on secondary organic aerosol (SOA) are scarce. However, SOA accounts for a significant portion of airborne particulate matter. In particular, pinene biogenic SOA contributes predominantly to SOA loading in the outdoor atmosphere of natural and urban areas and are also emitted indoors because of the presence of terpenes in numerous consumer products. Our aim was to study the immune consequences of acute exposure to β-pinene ozonolysis gaseous and SOA products in mice. This reaction was generated in an atmospheric simulation chamber, and the mice were exposed to the particulate and gaseous products, to the gaseous products only, or to synthetic air 2 h per day for 3 days in real time in a whole-body inhalation chamber. Exposures were performed in adulthood or in utero. Since some adverse effects only occur in individuals weakened by existing immune activation, such as low-grade inflammation, the immune response was measured in the steady state or in a state of moderate systemic inflammation induced by lipopolysaccharide administration.

Results: Exposure of healthy adult mice caused minor immunosuppression in the lungs. However, in adult mice weakened by moderate systemic inflammation, the same exposure conditions revealed that mice exposed to the β-pinene ozonolysis particulate and gaseous products presented deficient pulmonary and systemic immune responses, including excessive recruitment of B lymphocytes, CD4⁺ T lymphocytes, CD11b⁺ dendritic cells, inflammatory monocytes and neutrophils in the lungs and defective recruitment of regulatory T cells in the spleen. In offspring exposed to β-pinene ozonolysis products in utero, the LPS-induced upregulation of Ccl2, Cxcl10 and Icam1 mRNA levels in the lungs and the activation of dendritic cells in the spleen were excessive in female mice. The male offspring developed a normal response to moderate systemic inflammation, except for impaired activation of CD4⁺ T cells and increased activation of CD103⁺ dendritic cells in the spleen.

Conclusion: In mice, pulmonary and systemic immune reactions in response to moderate systemic inflammation are dysregulated by exposure to common secondary oxidation products, highlighting interest in the role of these neglected atmospheric compounds in immune disease development and susceptibility to infections.

Keywords: Air pollution; Gestational; Immune response; Inflammation; Lung; Mice; Secondary organic aerosols; Systemic.

Fig. 1

Setup developed to expose mice to β-pinene oxidation products in real time. (A) Experimental scheme of generation and exposure to β-pinene oxidation products overall (pink area) or without SOA (blue area). (B) SOA mass concentrations measured via SMPS analyses before and after the pumping of the atmospheric simulation chamber for 2 h at 2 L.min^− 1. (C) Representative aerosol concentration and size distribution measured by the SPMS

Fig. 2

Pulmonary response to acute direct exposure in adulthood to β-pinene oxidation products. (A) Experimental design. n = 10/group. (B) Inspiratory capacity function. (C) Mouse XL cytokine panel assay in BAL fluid. (D) Total cell counts in the lungs. (E-H) Immune cell frequency in the lungs. Parent population was CD45⁺CD3⁺TCRb⁺ cells for CD4⁺, CD8⁺, Reg T cells and CD45⁺CD5⁺NK1.1⁺ cells for NKT like T cells. (I) mRNA expression levels of cytokines in the lungs. A representative of two independent experiments is shown. The data represent the means ± SEMs. * p < 0.05, ** p < 0.01

Fig. 3

Pulmonary response to moderate systemic inflammation after acute direct exposure in adulthood to β-pinene oxidation products. (A) Experimental design. n = 10/group. (B) Mouse XL cytokine panel assay in BAL fluid. (C-F) Immune cell frequency in the lungs. Parent population was CD45⁺CD3⁺TCRβ⁺ cells for CD4⁺, CD8⁺, Reg T cells and CD45⁺CD5⁺NK1.1⁺ cells for NKT like T cells. (G) mRNA expression levels of cytokines in the lungs. A representative of two independent experiments is shown. The data represent the means ± SEMs. * p < 0.05, ** p < 0.01

Fig. 4

Systemic response to moderate systemic inflammation after acute direct exposure in adulthood to β-pinene oxidation products. (A-C) Immune cell frequency in the spleen. Parent population was CD45⁺CD3⁺TCRβ⁺ cells for CD4⁺, CD8⁺, Reg T cells and CD45⁺CD5⁺NK1.1⁺ cells for NKT like T cells. (D) Mouse XL cytokine panel assay in the liver. (E) mRNA expression levels of cytokines in the liver. A pool of two independent experiments is shown. The data represent the means ± SEMs. * p < 0.05, ** p < 0.01

Scheme 1

Experimental design for gestational exposure to β-pinene oxidation products

Fig. 5

Pulmonary response to moderate systemic inflammation in female offspring exposed to β-pinene oxidation products in utero. (A-D). Immune cell frequency in the lungs. Parent population was CD45⁺CD3⁺TCRβ⁺ cells for CD4⁺, CD8⁺, Reg T cells and CD45⁺CD5⁺NK1.1⁺ cells for NKT like T cells. (E) mRNA expression levels of cytokines in the lungs. A pool of two independent experiments is shown. n = 10/group. The data represent the means ± SEMs. * p < 0.05, ** p < 0.01, *** p < 0.001

Fig. 6

Systemic response to moderate systemic inflammation in female offspring exposed to β-pinene oxidation products in utero. (A-D) Immune cell frequency in the spleen. Parent population was CD45⁺CD3⁺TCRβ⁺ cells for CD4⁺, CD8⁺, Reg T cells and CD45⁺CD5⁺NK1.1⁺ cells for NKT like T cells. (E-F) CD69 and CD25 activation markers on T cells. (G-H) CD86 and Iab activation markers on dendritic cells. (I) mRNA expression levels of cytokines in the liver. A pool of two independent experiments is shown. n = 10/group. The data represent the means ± SEMs. * p < 0.05, ** p < 0.01, *** p < 0.001

Fig. 7

Pulmonary response to moderate systemic inflammation in male offspring exposed to β-pinene oxidation products in utero. (A-D) Frequencies of major immune cells in the lungs. Parent population was CD45⁺CD3⁺TCRβ⁺ cells for CD4⁺, CD8⁺, Reg T cells and CD45⁺CD5⁺NK1.1⁺ cells for NKT like T cells. (E) mRNA expression levels of cytokines in the lungs. A pool of two independent experiments is shown. n = 10/group. The data represent the means ± SEMs. * p < 0.05, ** p < 0.01, *** p < 0.001

Fig. 8

Systemic response to moderate systemic inflammation in male offspring exposed to β-pinene oxidation products in utero. (A-D) Frequencies of major immune cells in the spleen. Parent population was CD45⁺CD3⁺TCRβ⁺ cells for CD4⁺, CD8⁺, Reg T cells and CD45⁺CD5⁺NK1.1⁺ cells for NKT like T cells. (E-F) CD69 and CD25 activation markers on T cells. (G-H) CD86 and Iab activation markers on dendritic cells. (I) mRNA expression levels in the liver. A pool of two independent experiments is shown. n = 10/group. The data represent the means ± SEMs. * p < 0.05, ** p < 0.01, *** p < 0.001