Respiratory syncytial virus increases eosinophil extracellular traps in a murine model of asthma - PubMed (original) (raw)

Respiratory syncytial virus increases eosinophil extracellular traps in a murine model of asthma

Josiane Silva Silveira et al. Asia Pac Allergy. 2019.

Abstract

Background: Respiratory viral infections are the leading cause of asthma exacerbations. Eosinophil activation results in the formation of eosinophil extracellular traps (EETs), which release web-like structures of DNA and proteins that bind, disarm and extracellularly kill pathogens.

Objective: We investigated whether the respiratory syncytial virus (RSV) in vitro could induce EETs in bronchoalveolar lavage fluid eosinophils in a murine model of asthma.

Methods: BALB/cJ mice (6-8 weeks old) were sensitized with 2 subcutaneous injections of ovalbumin (20 μg) on days 0 and 7, followed by three intranasal challenges with ovalbumin (100 μg) on days 14, 15, and 16 of the protocol. The control group received Dulbecco's phosphate-buffered saline. Bronchoalveolar lavage fluid eosinophils of ovalbumin group or control group were stimulated with RSV (103 PFU/mL) in vitro for 3 hours. After that, culture supernatant was collected to perform the analyses proposed in this study.

Results: We verified an increase in extracellular DNA concentration in bronchoalveolar lavage fluid eosinophils from ovalbumin group stimulated with RSV (103 PFU/mL) in vitro, which was confirmed by confocal microscopy. We demonstrated that most cells are negative for annexin V and propidium iodide in all groups evaluated. Also, RSV in vitro decreased interferon-ɣ in culture supernatant when compared to the ovalbumin group.

Conclusion: In this study, we demonstrated for the first time that RSV in vitro induces EETs formation in eosinophils from asthmatic mice.

Keywords: Asthma; Eosinophil peroxidase; Extracellular traps; Inflammation; Respiratory syncytial viruses.

Copyright © 2019. Asia Pacific Association of Allergy, Asthma and Clinical Immunology.

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Conflict of interest statement

Conflict of Interest: The authors have no financial conflicts of interest.

Figures

Fig. 1

Fig. 1. Ovalbumin-induced airway eosinophilic inflammation. (A) Total cell counts in BALF. (B-E) Differential cell counts in BALF (eosinophils, macrophages, neutrophils, and lymphocytes. (F) Representative illustration of BALF cells stained with H&E (×400, arrows indicate eosinophils). Results are expressed as mean ± standard deviation, for 7–10 animals in each group, of 3 independent experiments, **p < 0.01, ***p < 0.001. BALF, bronchoalveolar lavage fluid; OVA, ovalbumin.

Fig. 2

Fig. 2. RSV in vitro induces EETs colocalized with EPO in BALF cells from asthmatic mice. (A) Effect of different concentrations of RSV (103–106 PFU/mL) in vitro in extracellular DNA concentration. (B) Extracellular DNA concentration in BALF cells from asthmatic and control mice stimulated with RSV (103 PFU/mL) in vitro or unstimulated. (C) EETs release in BALF cells from asthmatic and control mice stimulated with RSV (103 PFU/mL) in vitro or unstimulated (×630, arrows indicate EETs formation). (D) Analysis of annexin V binding and PI uptake in BALF cells from all groups. Results are expressed as mean ± standard deviation, for 7–10 animals in each group, of 3 independent experiments, *p < 0.05, **p < 0.01, ***p < 0.001. BALF, bronchoalveolar lavage fluid; DPBS, Dulbecco's phosphate-buffered saline; EPO, eosinophil peroxidase; OVA, ovalbumin; PFU, plaque-forming unit; PI, propidium iodide; RSV, respiratory syncytial virus; FITC, fluorescein isothiocyanate.

Fig. 3

Fig. 3. Effect of RSV in BALF cells from asthmatic mice in cytokines levels and EPO activity. (A) IL-4, (B) IFN-ɣ, and (C) EPO activity in from BALF cells (2 × 105 /mL) from asthmatic mice stimulated with RSV (103 PFU/mL) in vitro or unstimulated. Results are expressed as mean ± standard deviation, for 5 animals in each group, of 3 independent experiments, **p < 0.01. BALF, bronchoalveolar lavage fluid; EPO, eosinophil peroxidase; DPBS, Dulbecco's phosphate-buffered saline; IL-4, interleukin-4; IFN-ɣ, interferon-ɣ; OVA, ovalbumin; RSV, respiratory syncytial virus.

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