Vesicular and extravesicular protein analyses from the airspaces of ozone-exposed mice revealed signatures associated with mucoinflammatory lung disease

Kshitiz Paudel,Sonika Patial,Yogesh Saini,Xue Wen,Mehmet Kesimer,Thao Vo,Richa Gupta,Ishita Choudhary

doi:10.1038/s41598-021-02256-5

Kshitiz Paudel, Sonika Patial + Show 6 more

Open Access

https://doi.org/10.1038/s41598-021-02256-5

Copy DOI

Abstract

Lung epithelial lining fluid (ELF) harbors a variety of proteins that influence homeostatic and stress responses in the airspaces. Exosomes, nano-sized extracellular vesicles, contain many proteins that vary in abundance and composition based on the prevailing conditions. Ozone causes inflammatory responses in the airspaces of experimental animals and humans. However, the exosomal protein signatures contained within the ELF from ozone-exposed lung airspaces remain poorly characterized. To explore this, we hypothesized that ozone triggers the release of exosome-bound inflammatory proteins from various cells that reflect mucoobstructive lung disease. Accordingly, we repetitively exposed adult male and female C57BL/6 mice to HEPA-filtered air (air) or 0.8 ppm ozone (4 h per day) for 14 days (five consecutive days of exposure, 2 days of rest, five consecutive days of exposure, 2 days of rest, four consecutive days of exposure). Exosome-bound proteomic signatures, as well as the levels of soluble inflammatory mediators in the bronchoalveolar lavage fluid (BALF), were determined 12–16 h after the last exposure. Principal component analyses of the exosome-bound proteome revealed a clear distinction between air-exposed and ozone-exposed mice, as well as between ozone-exposed males and ozone-exposed females. In addition to 575 proteins that were enriched in both sexes upon ozone exposure, 243 and 326 proteins were enriched uniquely in ozone-exposed males and females, respectively. Ingenuity pathway analyses on enriched proteins between ozone- and air-exposed mice revealed enrichment of pro-inflammatory pathways. More specifically, macrophage activation-related proteins were enriched in exosomes from ozone-exposed mice. Cytokine analyses on the BALF revealed elevated levels of G-CSF, KC, IP-10, IL-6, and IL-5 in ozone-exposed mice. Finally, the histopathological assessment revealed significantly enhanced intracellular localization of mucoinflammatory proteins including MUC5B and FIZZ1 in ozone-exposed mice in a cell-specific manner indicating the cellular sources of the proteins that are ferried in the exosomes upon ozone-induced lung injury. Collectively, this study identified exosomal, secretory, and cell-specific proteins and biological pathways following repetitive exposure of mice to ozone.

Highlights

Lung epithelial lining fluid (ELF) harbors a variety of proteins that influence homeostatic and stress responses in the airspaces
The epithelial lining fluid (ELF), a thin liquid layer covering the epithelial cells in the airway and alveolar spaces, contains exosomes that are released from the resident and/or recruited cells[14]
While air-exposed males and females had basal or undetectable levels of all these five cytokines, ozone exposure resulted in a significant elevation in the concentration of these five cytokines, i.e., granulocyte-colony stimulating factor (G-CSF), KC, IP-10, IL-6, and IL-5 in both the sexes (Fig. 1B–F)

Summary

Introduction

Lung epithelial lining fluid (ELF) harbors a variety of proteins that influence homeostatic and stress responses in the airspaces. Exosomes are nano-sized extracellular vesicles (EV) that originate from the endosomal compartment of the cells and are known to contain biomolecules including proteins, lipids, RNA, DNA, and m etabolites[1,2,3] The composition of these biomolecules in bodily fluids such as plasma[4,5], epithelial lining fluid (ELF)6,7, saliva8, milk[8], and urine[8,9] may yield valuable information about their cellular origins, physiological roles, prevailing pathological stresses, and, clinically, may have diagnostic and prognostic values[10,11,12]. We hypothesized that repetitive ozone exposure triggers the release of exosome-bound inflammatory proteins from various cells that reflect the mucoobstructive lung disease. Our second hypothesis was that exosomes from ozone-exposed females possess unique protein signatures that cause exaggerated inflammatory responses To test these hypotheses, we asked a series of questions including, (1) Which proteins are enriched in the airspace-derived exosomes from healthy lungs (homeostasis)? This study revealed several interesting findings related to ozone- and sex-specific protein signatures within the pulmonary airspaces

Methods

Results

Conclusion