Fine Particulate Matter-Induced Exacerbation of Allergic Asthma via Activation of T-cell Immunoglobulin and Mucin Domain 1

Abstract

Background: Fine particulate matter (PM_2.5) exacerbates airway inflammation and hyperreactivity in patients with asthma, but the mechanism remains unclear. The aim of this study was to observe the effects of prolonged exposure to high concentrations of PM_2.5on the pathology and airway hyperresponsiveness (AHR) of BALB/c mice undergoing sensitization and challenge with ovalbumin (OVA) and to observe the effects of apoptosis and T-cell immunoglobulin and mucin domain 1 (TIM-1) in this process.

Methods: Forty female BALB/c mice were divided into four groups: control group, OVA group, OVA/PM group, and PM group (n = 10 in each group). Mice in the control group were exposed to filtered clean air. Mice in the OVA group were sensitized and challenged with OVA. Mice in the OVA/PM group were sensitized and challenged as in the OVA group and then exposed to PM_2.5for 4 h per day and 5 days per week for a total of 8 weeks using a nose-only "PM_2.5online enrichment system" in The Second Hospital of Hebei Medical University. Mice in the PM group were exposed to the PM_2.5 online enrichment system only. AHR was detected. Bronchoalveolar lavage fluid (BALF) was collected for cell classification. The levels of interleukin-4 (IL-4), IL-5, and IL-33 in BALF were measured using enzyme-linked immunosorbent assay. Changes in histological structures were examined by light microscopy, and changes in ultramicrostructures were detected by electron microscopy. Apoptosis was determined by terminal deoxynucleotidyl transferase-mediated dUTP nick end-labeling (TUNEL) assay in the lung tissues. Western blotting and immunohistochemistry were utilized to analyze the expression of Bcl-2, Bax, and TIM-1 in the lungs.

Results: The results showed that AHR in the OVA/PM group was significantly more severe than that in the OVA and PM groups (P < 0.05). AHR in the PM group was also considerably more severe than that in the control group (P < 0.05). The BALF of OVA/PM group (28.00 ± 6.08 vs. 12.33 ± 4.51, t = 4.631, P = 0.002) and PM group (29.00 ± 3.00 vs. 12.33 ± 4.51, t = 4.927, P = 0.001) had more lymphocytes than the BALF of the control group. The number of neutrophils in the BALF of the OVA/PM group (6.67 ± 1.53 vs. 3.33 ± 1.53, t = 2.886, P = 0.020) and PM group (6.67 ± 1.53 vs. 3.33 ± 1.53, t = 2.886, P = 0.020) was much higher than those in the BALF of OVA group (P < 0.05). TUNEL assays showed that the number of apoptotic cells in the OVA/PM group was significantly higher than that in the OVA group (Tunel immunohistochemical scores [IHS%], 1.20 ± 0.18 vs. 0.51 ± 0.03, t = 8.094, P < 0.001) and PM group (Tunel IHS%, 1.20 ± 0.18 vs. 0.51 ± 0.09, t = 8.094, P < 0.001), and that the number of apoptotic cells in the PM group was significantly higher than that in the control group (Tunel IHS%, 0.51 ± 0.09 vs. 0.26 ± 0.03, t = 2.894, P = 0.020). The concentrations of IL-4 (77.44 ± 11.19 vs. 48.02 ± 10.02 pg/ml, t = 4.595, P = 0.002) and IL-5 (15.65 ± 1.19 vs. 12.35 ± 0.95 pg/ml, t = 3.806, P = 0.005) and the Bax/Bcl-2 ratio (1.51 ± 0.18 vs. 0.48 ± 0.10, t = 9.654, P < 0.001) and TIM-1/β-actin ratio (0.78 ± 0.11 vs. 0.40 ± 0.06, t = 6.818, P < 0.001) in the OVA/PM group were increased compared to those in the OVA group. The concentrations of IL-4 (77.44 ± 11.19 vs. 41.47 ± 3.40 pg/ml, t = 5.617, P = 0.001) and IL-5 (15.65 ± 1.19 vs. 10.99 ± 1.40 pg/ml, t = 5.374, P = 0.001) and the Bax/Bcl-2 ratio (1.51 ± 0.18 vs. 0.97 ± 0.16, t = 5.000, P = 0.001) and TIM-1/β-actin ratio (0.78 ± 0.11 vs. 0.31 ± 0.06, t = 8.545, P < 0.001) in the OVA/PM group were increased compared to those in the PM group. The concentration of IL-4 (41.47 ± 3.40 vs. 25.46 ± 2.98 pg/ml, t = 2.501, P = 0.037) and the Bax/Bcl-2 ratio (0.97 ± 0.16 vs. 0.18 ± 0.03, t = 7.439, P < 0.001) and TIM-1/β-actin ratio (0.31 ± 0.06 vs. 0.02 ± 0.01, t = 5.109, P = 0.001) in the PM group were also higher than those in the control group.

Conclusions: Exacerbated AHR associated with allergic asthma caused by PM_2.5is related to increased apoptosis and TIM-1 activation. These data might provide insights into therapeutic targets for the treatment of acute exacerbations of asthma induced by PM_2.5.

Keywords: Apoptosis; Asthma; Fine Particulate Matter; T-cell Immunoglobulin and Mucin Domain 1.

Figure 1

The mean concentration of PM_2.5 in the exposure chamber from February 14 to April 11, 2017, in Shijiazhuang, Hebei, China. PM_2.5: Fine particulate matter.

Figure 2

Airway responsiveness in different groups. (a) Rrs in the control, OVA, OVA/PM, and PM groups at different concentrations of aerosolized Mch. (b) R_N in the four groups. (c) G in the four groups. (d) H in the four groups. Values are represented as the mean ± SD. Based on one-way ANOVA, followed by LSD multiple range tests, comparing with control group, a significant difference is indicated by *P < 0.001, ^†P < 0.01, and ^‡P < 0.05. Comparing with PM group, a significant difference is indicated by ^§P < 0.01 and ^||P < 0.05. Comparing with OVA group, a significant difference is indicated by ^¶P < 0.01 and **P < 0.05 (n = 6 per group). Rrs: Respiratory system resistance; R_N: Airway resistance; G: Tissue damping; H: Tissue elasticity; Mch: Methacholine; PM: Particulate matter; OVA: Ovalbumin; SD: Standard deviation; ANOVA: Analysis of variance; LSD: Least significant difference.

Figure 3

Cell classification in BALF: number of eosinophils, lymphocytes, neutrophils, and macrophages based on a total of 200 cells in the BALF of the control, OVA, OVA/PM, and PM groups. Values are mean ± SD. Using one-way ANOVA, followed by LSD multiple range test, comparing with control group, a significant difference is indicated by *P < 0.001, ^†P < 0.01, and ^‡P < 0.05. Comparing with PM group, significant difference is indicated by ^§P < 0.001, ^||P < 0.01, and ^¶P < 0.05. Comparing with OVA group, significant difference is indicated by **P < 0.05 (n = 6 per group). BALF: Bronchoalveolar lavage fluid; PM: Particulate matter; OVA: Ovalbumin; SD: Standard deviation; ANOVA: Analysis of variance; LSD: Least significant difference.

Figure 4

Cytokine analysis in BALF with ELISA: IL-4, IL-5, and IL-33 levels in the BALF of the control, OVA, OVA/PM, and PM groups. Values are mean ± SD. Using one-way ANOVA, followed by LSD multiple range test, comparing with control group, significant difference is indicated by *P < 0.001, ^†P < 0.01, and ^‡P < 0.05. Comparing with PM group, a significant difference is indicated by ^§P < 0.01 and ^||P < 0.05. Comparing with OVA group, a significant difference is indicated by ^¶P < 0.01 (n = 6 per group). ELISA: Enzyme-linked immunosorbent assay; IL-4: Interleukin-4; IL-5: Interleukin-5; IL-33: Interleukin-33; PM: Particulate matter; OVA: Ovalbumin; SD: Standard deviation; ANOVA: Analysis of variance; LSD: Least significant difference.

Figure 5

Comparison of the changes in lung tissues of mice in different groups (hematoxylin and eosin, ×200). The control group had intact terminal bronchioles and alveolar epithelia and showed no inflammation. The OVA group showed hyperplasia of smooth muscles of the small bronchi (black arrow), hyperplasia of lymphatic follicles (star), and infiltration of eosinophils (white arrow). The OVA/PM group displayed mild loss of tracheal epithelial cells, widening of the alveolar septa (black arrows), infiltration of inflammatory cells, and hyperplasia of smooth muscles of the small bronchi (stars). The PM group displayed the presence of inflammatory cells in peribronchiolar regions (black arrow) and substantial alveolar epithelial hyperplasia (stars). PM: Particulate matter; OVA: Ovalbumin.

Figure 6

Comparison of the ultrastructural changes in lung tissues of mice in different groups. The OVA group (both ×15,000) displayed eosinophilic hyperplasia. In the OVA/PM group, black arrow indicates interstitial fibrosis (upper ×8000) and white arrow indicates type II alveolar epithelium with abnormal mitochondria and slight fusion and deterioration of the nuclear membrane and mitochondrial cristae (lower ×5000). In the PM group, black arrows indicate type II alveolar epithelium with abnormal mitochondria and slight fusion and deterioration of the nuclear membrane and mitochondrial cristae (upper ×6000 and lower ×15,000). PM: Particulate matter; OVA: Ovalbumin.

Figure 7

Apoptotic cells were determined by TUNEL staining in the lung tissues of mice in the control, OVA, OVA/PM, and PM groups (×200). TUNEL-positive cells were found in the bronchia and lung cells. Values are mean ± SD. Using one-way ANOVA, followed by LSD multiple range test, comparing with control group, a significant difference is indicated by *P < 0.001 and ^†P < 0.05. Comparing with PM group, a significant difference is indicated by ^‡P < 0.001. Comparing with OVA group, a significant difference is indicated by ^§P < 0.001 (n = 6 per group). TUNEL: Terminal deoxynucleotidyl transferase-mediated dUTP nick end-labeling; IHS: Immunohistochemical scores; PM: Particulate matter; OVA: Ovalbumin; SD: Standard deviation; ANOVA: Analysis of variance; LSD: Least significant difference.

Figure 8

Effects of PM_2.5 on the expression of apoptosis-regulatory proteins and TIM-1 in mice in different groups determined by Western blotting. (a) Images of Western blotting for Bax and Bcl-2. (b) Comparisons of the Bax/Bcl-2 ratio. (c) Images of Western blotting for TIM-1. (d) Comparisons of the expression of TIM-1. Values are mean ± SD. Using one-way ANOVA, followed by LSD multiple range test, comparing with control group, comparing with control group, significant difference is indicated by *P < 0.001, ^†P < 0.01, and ^‡P < 0.05. Comparing with PM group, significant difference is indicated by ^§P < 0.001 and ^||P < 0.01. Comparing with OVA group, significant difference is indicated by ^¶P < 0.001 (n = 6 per group). TIM-1: T-cell Immunoglobulin and Mucin Domain 1; PM: Particulate matter; OVA: Ovalbumin; SD: Standard deviation; ANOVA: Analysis of variance; LSD: Least significant difference.

Figure 9

Effects of PM_2.5 on the expression of apoptotic regulatory proteins and TIM-1 in mice in different groups with immunohistochemistry (×200). (a-d) Images of immunohistochemistry of Bax in control, OVA, OVA/PM, and PM groups; (e) IOD of Bax; (f-i) images of immunohistochemistry of Bcl-2 in four groups; (j) IOD of Bcl-2; (k-n) images of immunohistochemistry of TIM-1in four groups; (o) IHS% of TIM-1. Values are mean ± SD. Using one-way ANOVA, followed by LSD multiple range test, comparing with control group, significant difference is indicated by *P < 0.001, ^†P < 0.01, and ^‡P < 0.05. Comparing with PM group, significant difference is indicated by ^§P < 0.001 and ^||P < 0.01. Comparing with OVA group, significant difference is indicated by ^¶P < 0.001, **P < 0.01, and ^††P < 0.05 (n = 6 per group). IOD: Integral optical density; IHS: Immunohistochemical scores; PM: Particulate matter; OVA: Ovalbumin; SD: Standard deviation; ANOVA: Analysis of variance; LSD: Least significant difference.