Fine-particulate matter aggravates cigarette smoke extract-induced airway inflammation via Wnt5a-ERK pathway in COPD

Abstract

Background: Exposure to environmental particulate matter (PM) ≤2.5 μm in diameter (PM_2.5) and smoking are common contributors to COPD, and pertinent research implicates both factors in pulmonary inflammation. Using in vivo mouse and in vitro human cellular models, we investigated the joint impact of PM_2.5 pollution, and cigarette smoke (CS) in mice or cigarette-smoke extract (CSE) in cells on COPD inflammation, and explored potential mechanisms.

Methods: Tissue changes in lungs of C57BL/6 mice exposed to PM_2.5 and CS were studied by light microscopy, H&E, immunochemistry, and immunofluorescence-stained sections. Levels of inflammatory factors induced by PM_2.5/CS in mice and PM_2.5/CSE in 16HBE cells were also monitored by quantitative reverse-transcription (qRT)-PCR and ELISA. Expression of genes related to the Wnt5a-signaling pathway was assessed at transcriptional and protein levels using immunofluorescence, qRT-PCR, and Western blotting.

Results: Inflammatory response to combined exposure of PM_2.5 and CS or CSE in mouse and 16HBE cells surpassed responses incited separately. Although separate PM_2.5 and CS/CSE exposure upregulated the expression of Wnt5a (a member of the Wnt-secreted glycoprotein family), combined PM_2.5 and CS/CSE exposure produced a steeper rise in Wnt5a levels. Use of a Wnt5a antagonist (BOX5) successfully blocked related inflammatory effects. ERK phosphorylation appeared to mediate the effects of Wnt5a in the COPD model, promoting PM_2.5 aggravation of CS/CSE-induced airway inflammation.

Conclusion: Our findings suggest that combined PM_2.5 and CS/CSE exposure induce airway inflammation and Wnt5a expression in vivo in mice and in vitro in 16HBE cells. Furthermore, PM_2.5 seems to aggravate CS/CSE-induced inflammation via the Wnt5a-ERK pathway in the context of COPD.

Keywords: COPD; PM2.5; Wnt5a; airway inflammation.

Figure 1

PM_2.5 aggravated smoking-induced histological changes and inflammation in lungs of mice. Notes: (A–D) H&E-stained sections of lung from control, PM_2.5, smoking, and PM_2.5 + smoking groups (original magnification 200×, bar 100 μm); (E, F) IL6 and IL8 mRNA expression in lungs of mice (normalized to β-actin level, n=5 mice/group). Data expressed as mean ± SD. *P<0.05; ***P<0.001. Abbreviation: PM_2.5, particulate matter ≤2.5 μm.

Figure 2

PM_2.5 or CSE exposure increased production of cytokines in 16HBE cells. Notes: (A) Cells left unexposed or variably exposed to CSE (2.5%, 5%, 10%, or 20%) for 24 hours; (B) cells left unexposed or variably exposed to 10% CSE (6, 12, 24, or 48 hours); (C) cells left unexposed or variably exposed to PM_2.5 (25 μg/mL, 50 μg/mL, 100 μg/mL, or 200 μg/mL) for 24 hours; (D) cells left unexposed or variably exposed to 100 μg/mL PM_2.5 (6, 12, 24, or 48 hours). Levels of IL6 and IL8 determined in culture supernatants (ELISA). Data expressed as mean ± SD. *P<0.05; **P<0.01; ***P<0.001. Abbreviations: PM_2.5, particulate matter ≤2.5 μm; CSE, cigarette-smoke extract.

Figure 3

PM_2.5 aggravated CSE-induced inflammation in 16HBE cells. Notes: (A) Cell viability assessed after exposure to PM_2.5 (25–200 μg/mL) in combination with 10% CSE for 24 hours; (B–E) cells left unexposed or exposed to PM_2.5 (100 μg/mL), CSE (10%), or PM_2.5 (100 μg/mL) + CSE (10%) for 24 hours. Levels of IL6 and IL8 determined in culture supernatants (ELISA), as well as levels of IL6 and IL8 mRNA expression (quantitative reverse-transcription PCR, normalized to β-actin level). Data expressed as mean ± SD. *P<0.05; **P<0.01; ***P<0.001. 25 PS, 25 μg/mL PM_2.5+10% CSE; 50 PS, 50 μg/mL PM_2.5+10% CSE; 100 PS, 100 μg/mL PM_2.5+10% CSE; 200 PS, 200 μg/mL PM_2.5+10% CSE. Abbreviations: PM_2.5, particulate matter ≤2.5 μm; CSE, cigarette-smoke extract.

Figure 4

PM_2.5 and smoking/CSE exposure upregulated expression of Wnt5a in lungs of mice and 16HBE cells. Notes: (A–D) Representative immunofluorescence-stained lung sections from control, PM_2.5, smoking, and PM_2.5 + smoking mouse groups, labeled for Wnt5a (red, original magnification 400×, bar 50 μm), using DAPI nuclear counterstain (blue); (E) levels of Wnt5a mRNA expressed in mouse lungs, shown by group (normalized to β-actin level, n=5 mice/group); (F–H) cells left unexposed or variably exposed to PM_2.5, CSE, or PM_2.5 + CSE for 24 hours: Wnt5a transcription (quantitative reverse-transcription PCR; normalized to β-actin level) and protein levels (qualitative and quantitative Western blot, β-actin as loading control). Data expressed as mean ± SD. *P<0.05; **P<0.01; ***P<0.001. Abbreviations: PM_2.5, particulate matter ≤2.5 μm; CSE, cigarette-smoke extract.

Figure 5

Wnt5a antagonist downregulated levels of proinflammatory cytokines and Wnt5a. Notes: (A) Cell viability measured after exposure to BOX5 at various concentrations (100–300 μM). (B–E) Cells pretreated with BOX5 (200 μM) for 1 hour prior to PM_2.5 (100 μg/mL) or 10% CSE exposure for 24 hours: levels of IL6 and IL8 protein in culture supernatants (ELISA), as well as levels of IL6 and IL8 mRNA expression in 16HBE cells (quantitative reverse-transcription PCR; normalized to β-actin level). (F, G) Representative band of Wnt5a on Western blot (β-actin as loading control); (H, I) Quantitative Western blot analysis of Wnt5a. Data expressed as mean ± SD. *P<0.05; **P<0.01; ***P<0.001. Abbreviation: PM_2.5, particulate matter ≤2.5 μm.

Figure 6

Expression levels of P-ERK1/2 and T-ERK1/2 analyzed by Western blot. Notes: (A) Mice left unexposed or variably exposed to PM_2.5, smoking, or PM_2.5 + smoking for 10 months (n=5 mice/group); (B) cells left unexposed or variably exposed to PM_2.5 (100 μg/mL), CSE (10%), or PM_2.5 (100 μg/mL) + CSE (10%) for 24 hours; (C, D) cells preincubated with BOX5 (200 μM) or vehicle (PBS) for 1 hour, then exposed or unexposed to PM_2.5/CSE for 24 hours. Data expressed as mean ± SD. *P<0.05; **P<0.01; ***P<0.001. Abbreviations: PM_2.5, particulate matter ≤2.5 μm; CSE, cigarette-smoke extract.