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. 2021 Jun;21(6):662.
doi: 10.3892/etm.2021.10094. Epub 2021 Apr 21.

Influence of fine particulate matter and its pure particulate fractions on pulmonary immune cells and cytokines in mice

Zhouguang Jiao  1 , Zhanbo Wen  1 , Wenhui Yang  1 , Lingfei Hu  1 , Jinsong Li  1
Affiliations

Influence of fine particulate matter and its pure particulate fractions on pulmonary immune cells and cytokines in mice

Zhouguang Jiao et al. Exp Ther Med. 2021 Jun.

Abstract

Particulate matter with a diameter ≤2.5 µm (PM2.5) has a complex composition and has been associated with the incidence of cardiopulmonary disease and premature death in humans. However, whether pure particulate fractions of PM2.5 (PPP2.5), which are composed primarily of carbon, are responsible for the toxicity caused by ambient particulate matter (original PM2.5 particles, OPP2.5) is currently unclear. The present study assessed the acute toxic effects of OPP2.5 sampled in Beijing, China and of its PPP2.5 fraction in male BALB/c mice. The mice were intratracheally instilled with a single dose of aerosolized OPP2.5 or PPP2.5. Blood, lungs and bronchoalveolar lavage fluid were collected after 24 h for histopathology, flow cytometry and the measurement of pro-inflammatory cytokines/chemokines and other biochemical factors. Both OPP2.5 and PPP2.5 caused acute toxicity, particularly inflammatory responses, including an increase in the levels of pro-inflammatory cytokines and an accumulation of numerous immune cells in the lungs. OPP2.5 induced a stronger inflammatory response than PPP2.5. The complex components adsorbed into the solid core granules of OPP2.5 and the granules themselves contributed to the toxic effects.

Keywords: acute toxicity; histopathology; immune system; inflammation; particulate matter.

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Conflict of interest statement

The authors declare that they have no competing interests.

Figures

Figure 1
Figure 1
Changes in mouse body weight following intratracheal instillation with aerosolized OPP2.5 particles (80 µg), PPP2.5 particles (80 µg) or vehicle (50 µl sterile saline). Data are presented as the mean ± standard error of the mean (n=8/group). **P<0.01 vs. control. OPP2.5, original atmospheric particulate matter with a diameter ≤2.5 µm; PPP2.5, pure atmospheric particulate matter with a diameter ≤2.5 µm.
Figure 2
Figure 2
Levels of biochemical factors in blood and serum following exposure to OPP2.5 particles (80 µg), PPP2.5 particles (80 µg) or vehicle (50 µl sterile saline). (A) Hematocrit level. Relative activities of (B) ALT, (C) ALB, (D) LDH and (E) ALP. (F) Relative content of MCP1. Results were normalized to hematocrit levels. Data are presented as mean ± standard error of the mean (n=8/group). *P<0.05 and **P<0.01 vs. control. #P<0.05 vs. OPP2.5 group. OPP2.5, original atmospheric particulate matter with a diameter ≤2.5 µm; PPP2.5, pure atmospheric particulate matter with a diameter ≤2.5 µm; ALT, alanine aminotransferase; ALB, albumin; LDH, lactate dehydrogenase; ALP, alkaline phosphatase; MCP1, monocyte chemoattractant protein.
Figure 3
Figure 3
Stained sections of lung tissues following exposure to OPP2.5 particles (80 µg), PPP2.5 particles (80 µg) or vehicle (50 µl sterile saline). Widening alveolar septa (black arrows) and notable inflammatory cell infiltration were induced by OPP2.5 and PPP2.5. Magnification, x40 (n=3/group). OPP2.5, original atmospheric particulate matter with a diameter ≤2.5 µm; PPP2.5, pure atmospheric particulate matter with a diameter ≤2.5 µm.
Figure 4
Figure 4
Comparison of pulmonary immune cell numbers obtained by flow cytometry following exposure to OPP2.5 particles (80 µg), PPP2.5 particles (80 µg) or vehicle (50 µl sterile saline). (A) Fluorescence-activated cell sorting gating strategy used to identify pulmonary major immune cells. Live CD45+ cells were gated and myeloid cells were gated on CD11c vs. CD11b. Total myeloid cells were then plotted as MerTK vs. CD64 and the MerTK+ CD64+ macrophage gate was plotted as CD11c vs. MHC II to illustrate the AMs and IM1, IM2 and IM3. A macrophage-deficient gate was plotted with CD11c and MHC II to illustrate DCs, which were plotted as CD11c vs. CD11b to identify Batf3+ and Irf4+ DCs. A macrophage/DC-deficient gate was plotted as Ly6G vs. CD11b to identify neutrophils (CD11b+ Ly6G+) and a macrophage/DC/neutrophil-deficient gate was plotted as SSC vs. F4/80 to identify eosinophils and monocytes. (B) The proportion of CD45+ cells in living cells and the absolute number of CD45+ cells in each sample from treated and control groups. The proportion of (C) AMs, (D) IM1s, (E) neutrophils, (F) DCs, (G) Batf3+, (H) Irf4+ and (I) monocytes in CD45+ cells and their absolute number. The circles represent the corresponding data of each mouse in the control group, the squares represent the corresponding data of each mouse in the OPP2.5 group, and the triangles represent the corresponding data of each mouse in the PPP2.5 group. Data are presented as mean ± standard error of the mean (n=4/group). *P<0.05 and **P<0.01 vs. control. #P<0.05 and ##P<0.01 vs. OPP2.5 group. OPP2.5, original atmospheric particulate matter with a diameter ≤2.5 µm; PPP2.5, pure atmospheric particulate matter with a diameter ≤2.5 µm; CD, cluster of differentiation; MHC, major histocompatibility complex; AMs, alveolar macrophages; IM, interstitial macrophage; DC, dendritic cell; SSC, side scatter.
Figure 5
Figure 5
Levels of biochemical factors (A) AST, (B) LDH, (C) eotaxin, (D) MCP-1, (E) MIP-1α, (F) MIP-2, (G) IL-1β, (H) TNF- α, (I) IL-6, (J) IL-17, (K) LIF, (L) VEGF and (M) CRP in BALF from mice treated with OPP2.5 particles (80 µg), PPP2.5 particles (80 µg) or vehicle (50 µl sterile saline). Data are presented as mean ± standard error of mean (n=8/group). *P<0.05 and **P<0.01 vs. control. #P<0.05 and ##P<0.01 vs. OPP2.5 group. AST, aspartate transaminase; LDH, lactate dehydrogenase; MCP, monocyte chemoattractant protein; MIP, macrophage inflammatory protein; IL, interleukin, TNF, tumor necrosis factor; LIF, leukemia inhibitory factor; VEGF, vascular endothelial growth factor; CRP, C-reactive protein; BALF, bronchoalveolar lavage fluid; OPP2.5, original atmospheric particulate matter with a diameter ≤2.5 µm; PPP2.5, pure atmospheric particulate matter with a diameter ≤2.5 µm.
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Grants and funding

Funding: The present study was supported by the National Science Foundation of China (grant no. 41205102).