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

Zhihua WangDepartment of Respiratory and Critical Care Medicine, National Clinical Research Center of Respiratory Disease, Tongji Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan 430030, People’s Republic of China, [email protected]

Junling ZhaoDepartment of Respiratory and Critical Care Medicine, National Clinical Research Center of Respiratory Disease, Tongji Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan 430030, People’s Republic of China, [email protected]

Ting WangDepartment of Respiratory and Critical Care Medicine, National Clinical Research Center of Respiratory Disease, Tongji Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan 430030, People’s Republic of China, [email protected]

Xiaohui DuDepartment of Respiratory and Critical Care Medicine, National Clinical Research Center of Respiratory Disease, Tongji Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan 430030, People’s Republic of China, [email protected]

Jungang XieDepartment of Respiratory and Critical Care Medicine, National Clinical Research Center of Respiratory Disease, Tongji Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan 430030, People’s Republic of China, [email protected]Correspondence[email protected]

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:

Supplementary materials

Figure S1 PM_2.5 aggravated smoking-induced inflammatory cell infiltration in lungs of mice.

Notes: (A–L) Representative double immunofluorescence–stained lung-tissue sections from control, PM_2.5, smoking, and PM_2.5 + smoking groups, labeled for Ly6G⁺ neutrophils (red, A–D), CD3⁺ T lymphocytes (green, E–H), and composite images (I–L; original magnification 400×, bar 50 μm), using DAPI nuclear counterstain (blue).

Abbreviation: PM_2.5, particulate matter ≤2.5 μm.

Figure S1 PM2.5 aggravated smoking-induced inflammatory cell infiltration in lungs of mice.Notes: (A–L) Representative double immunofluorescence–stained lung-tissue sections from control, PM2.5, smoking, and PM2.5 + smoking groups, labeled for Ly6G+ neutrophils (red, A–D), CD3+ T lymphocytes (green, E–H), and composite images (I–L; original magnification 400×, bar 50 μm), using DAPI nuclear counterstain (blue).Abbreviation: PM2.5, particulate matter ≤2.5 μm.

Figure S2 PM_2.5 aggravated smoking-induced hyperplasia of alveolar epithelial cells and small-airway epithelia in lungs of mice.

Note: (A–D) Representative immunohistochemistry-stained lung sections from control, PM_2.5, smoking, and PM_2.5 + smoking group, labeled for PCNA (brown, original magnification 200×, bar 50 μm).

Abbreviation: PM_2.5, particulate matter ≤2.5 μm.

Figure S2 PM2.5 aggravated smoking-induced hyperplasia of alveolar epithelial cells and small-airway epithelia in lungs of mice.Note: (A–D) Representative immunohistochemistry-stained lung sections from control, PM2.5, smoking, and PM2.5 + smoking group, labeled for PCNA (brown, original magnification 200×, bar 50 μm).Abbreviation: PM2.5, particulate matter ≤2.5 μm.

Figure S3 Correlation between levels of Wnt5a and inflammatory factors (IL6 and IL8) in mice and 16HBE cells.

Notes: (A, B) Correlation between levels of Wnt5a and IL6/IL8 in mice (n=5 mice/group); (C, D) correlation between levels of Wnt5a and IL6/IL8 in 16HBE cells. Data represent the relative mRNA expressions of Wnt5a and inflammatory factors in mice or cells. Pearson or Spearman analysis was used to calculate correlation (R)- and P-values.

Abbreviations: PM_2.5, particulate matter ≤2.5 μm; 16HBE, 16 human bronchial epithelial cells.

Figure S3 Correlation between levels of Wnt5a and inflammatory factors (IL6 and IL8) in mice and 16HBE cells.Notes: (A, B) Correlation between levels of Wnt5a and IL6/IL8 in mice (n=5 mice/group); (C, D) correlation between levels of Wnt5a and IL6/IL8 in 16HBE cells. Data represent the relative mRNA expressions of Wnt5a and inflammatory factors in mice or cells. Pearson or Spearman analysis was used to calculate correlation (R)- and P-values.Abbreviations: PM2.5, particulate matter ≤2.5 μm; 16HBE, 16 human bronchial epithelial cells.

Figure S4 Expression levels of Wnt5a analyzed by Western blot.

Notes: (A, B) Cells preincubated with BOX5 (200 μM) or vehicle (PBS) for 1 hour, then exposed or unexposed to PM_2.5 (100 μg/mL) + CSE (10%) for 24 hours (qualitative and quantitative Western blot, β-actin as loading control). Data expressed as mean ± SD. *P<0.05; **P<0.01.

Abbreviations: PM_2.5, particulate matter ≤2.5 μm; CSE, cigarette-smoke extract.

Figure S4 Expression levels of Wnt5a analyzed by Western blot.Notes: (A, B) Cells preincubated with BOX5 (200 μM) or vehicle (PBS) for 1 hour, then exposed or unexposed to PM2.5 (100 μg/mL) + CSE (10%) for 24 hours (qualitative and quantitative Western blot, β-actin as loading control). Data expressed as mean ± SD. *P<0.05; **P<0.01.Abbreviations: PM2.5, particulate matter ≤2.5 μm; CSE, cigarette-smoke extract.

Figure S5 Wnt5a in regulation of airway inflammation induced by PM_2.5 and smoking/CSE.

Abbreviations: PM_2.5, particulate matter ≤2.5 μm; CSE, cigarette-smoke extract.

Acknowledgments

This study was supported by the National Natural Science Foundation of China (81570033, 81570047, 81370145, and 81370156), National Key Basic Research and Development Program (973 Program, 20l5CB553403), Chinese Medical Association Research Project (2013BAI09B00), National Key Technologies R&D Program (2016YFC1303900 and 2016YFC1304700), and National Key Research and Development Program in China (2016YFC0903600).

Disclosure

The authors report no conflicts of interest in this work.