Diesel Exhaust Particulates Induce Neutrophilic Lung Inflammation by Modulating Endoplasmic Reticulum Stress-Mediated CXCL1/KC Expression in Alveolar Macrophages

Abstract

Diesel exhaust particulates (DEP) have adverse effects on the respiratory system. Endoplasmic reticulum (ER) abnormalities contribute to lung inflammation. However, the relationship between DEP exposure and ER stress in the respiratory immune system and especially the alveolar macrophages (AM) is poorly understood. Here, we examined ER stress and inflammatory responses using both in vivo and in vitro study. For in vivo study, mice were intratracheally instilled with 25, 50, and 100 μg DEP and in vitro AM were stimulated with DEP at 1, 2, and 3 mg/mL. DEP increased lung weight and the number of inflammatory cells, especially neutrophils, and inflammatory cytokines in bronchoalveolar lavage fluid of mice. DEP also increased the number of DEP-pigmented AM and ER stress markers including bound immunoglobulin protein (BiP) and CCAAT/enhancer binding protein-homologous protein (CHOP) were upregulated in the lungs of DEP-treated mice. In an in vitro study, DEP caused cell damage, increased intracellular reactive oxygen species, and upregulated inflammatory genes and ER stress-related BiP, CHOP, splicing X-box binding protein 1, and activating transcription factor 4 expressions in AM. Furthermore, DEP released the C-X-C Motif Chemokine Ligand 1 (CXCL1/KC) in AM. In conclusion, DEP may contribute to neutrophilic lung inflammation pathogenesis by modulating ER stress-mediated CXCL1/KC expression in AM.

Keywords: alveolar macrophages; chemokine CXCL1/KC; diesel exhaust particulate; endoplasmic reticulum stress; neutrophilic lung inflammation; particulate matter 2.5.

Figure 1

Changes in (A) body weight and (B) relative lung weight in mice in response to DEP instillation. Relative lung weights were calculated as follows: relative organ weight = lung weight (g)/final body weight (g) × 100%. Data are means ± SD (n = 5 per group). ^# p < 0.05 or ^## p < 0.01 vs. vehicle control.

Figure 2

(A) Cellular changes in BALF obtained from naïve control (NC), vehicle control (VC), and DEP 25 μg (DEP 25), DEP 50 μg (DEP 50), or DEP 100 μg (DEP 100) mice. Mice were treated 3 times by DEP and sacrificed at day 9. Data are means ± SD (n = 5 per group). ^# p < 0.05 or ^## p < 0.01 vs. vehicle control. (B) Histological changes in lung tissue caused by DEP instillation. Representative H&E-stained sections of lung tissue excised from DEP-induced mice. Red and black arrows indicate black particle-laden alveolar macrophages and inflammatory cells, respectively. Scale bars: 50 μm. Inflammatory cytokine levels including (C) TNF-α, (D) IL-1β, and (E) IL-17 in BALF of mice. Data are means ± SD (n = 4 per group). ^# p < 0.05 vs. vehicle control.

Figure 3

Expression levels of the ER stress markers BiP and CHOP in DEP-induced mice. Representative Western blots and relative densities of (A) BiP and (B) CHOP in lung tissues of DEP-induced mice. Mice were treated three times by DEP and sacrificed at day 9. Data are means ± SD (n = 5 per group). ^# p < 0.05 vs vehicle control.

Figure 4

ER stress-related gene expression levels. (A) BiP, (B) CHOP, (C) sXBP-1, and (D) ATF4 in DEP-stimulated AM. AM were stimulated DEP 1, 2, or 3 mg mL⁻¹ with. RT-qPCR was performed 3 h after DEP stimulation. Data are means ± SD (n = 3 per group). ^#p < 0.05 vs. control.

Figure 5

Expression levels of the ER stress markers BiP and CHOP in DEP-stimulated AM. Representative Western blots and relative densities of (A) BiP and (B) CHOP in DEP-stimulated AM. AM were stimulated with DEP 1, 2, or 3 mg mL⁻¹. Western blotting was performed 3 h after DEP stimulation. Data are means ± SD (n = 3 per group). ^# p < 0.05 or ^### p < 0.001 vs. control.

Figure 6

(A–F) Relative mRNA expression levels for inflammatory factors in DEP-stimulated AM. Data are means ± SD (n = 3 per group). ^# p < 0.05 vs. control.

Figure 7

Representative Western blots and relative densities of (A) TNF-α, (B) IL-1β, (C) TLR4, and (D) CXCL1/KC in DEP-stimulated AM. AM were stimulated with DEP 1, 2, or 3 mg mL⁻¹. Western blotting was performed 3 h after DEP stimulation. Data are means ± SD (n = 3 per group). ^# p < 0.05 or ^### p < 0.001 vs. control.

Figure 8

DEP induces oxidative stress-mediated cytotoxicity in DEP-treated AM. (A) Cytotoxicity assessment by MTT assay. (B) ROS production was measured by DCF-DA staining. AM were subjected to DEP (1, 2, or 3 mg mL⁻¹). DCF-DA staining and the MTT assay were performed 3 h after DEP challenge. (C) NAC pretreatment inhibits oxidative stress in DEP-stimulated AM. ROS production was measured in living cells by DCF-DA staining using flow cytometry. AM were pretreated with H₂O₂ for 30 min and NAC for 2 h and were stimulated with DEP (3 mg mL⁻¹) for 3 h. Data are means ± SD (n = 3 per group). ^#p < 0.05, ^##p< 0.01, ^###p< 0.001 or vs. control; ^***p< 0.001 or vs. DEP group.