Elsevier

Cytokine

Volume 63, Issue 2, August 2013, Pages 194-200
Cytokine

Analysis of bronchoalveolar lavage fluid in a mouse model of bronchial asthma and H1N1 2009 infection

https://doi.org/10.1016/j.cyto.2013.04.035Get rights and content

Highlights

  • Mice with bronchial asthma are sensitized to infection with H1N1 2009.

  • IL-6 and TNF-α levels were higher in infected asthmatic mice than in the controls.

  • Increased viral replication may contribute to worsening respiratory symptoms.

  • TGF-β1 pro-inflammatory effects may exacerbate bronchitis in this model.

Abstract

Background

Bronchial asthma is known as a risk factor of admission to the intensive care unit. However, the mechanism by which pandemic 2009 H1N1 (A(H1N1)pdm09) infection increases the severity of symptoms in patients with bronchial asthma is unknown; therefore, we aimed at determining this mechanism.

Methods

Inflammatory cell levels in the bronchoalveolar lavage (BAL) fluid from the non-asthma/mock, non-asthma/A(H1N1)pdm09, asthma/mock, and asthma/A(H1N1)pdm09 groups were determined using BALB/c mice. Cell infiltration levels, cytokine levels, and viral titers were compared among the groups.

Results

Neutrophil, monocyte, interleukin (IL)-5, IL-6, IL-10, IL-13, and tumor necrosis factor (TNF)-α levels were significantly higher in the BAL fluid from the non-asthma/A(H1N1)pdm09 and asthma/A(H1N1)pdm09 groups than in the mock groups (p < 0.05 for neutrophils and monocytes; p < 0.01 for the rest). The number of eosinophils and CD8+ lymphocytes and the level of transforming growth factor beta 1 (TGF-β1) in BAL fluid in the asthma/A(H1N1)pdm09 group were significantly higher among all groups (p < 0.05 for eosinophils and CD8+ lymphocytes; p < 0.01 for TGF-β1). The levels of IL-6, IL-10, IL-13, and TNF-α were significantly higher in the asthma/A(H1N1)pdm09 group than in the non-asthma/A(H1N1)pdm09 group (p < 0.05 for IL-6 and IL-10; p < 0.01 for IL-13 and TNF-α). The level of IFN-γ in the asthma/A(H1N1)pdm09 group was significantly lower than that in the non-asthma/A(H1N1)pdm09 group (p < 0.05). The viral titers in the BAL fluids were higher in the asthma/A(H1N1)pdm09 group than in the non-asthma/A(H1N1)pdm09 group (p < 0.05). Histopathological examination showed more severe infiltration of inflammatory cells and destruction of lung tissue in the asthma/A(H1N1)pdm09 group than in the non-asthma/A(H1N1)pdm09 group.

Conclusions

Severe pulmonary inflammation induced by elevated levels of cytokines, combined with increased viral replication due to decreased IFN-γ levels, may contribute to worsening respiratory symptoms in patients with bronchial asthma and A(H1N1)pdm09 infection.

Introduction

The 2009 pandemic H1N1 (A(H1N1)pdm09) influenza virus originated in pigs and emerged among humans in Mexico during the spring of 2009 before spreading globally [1]. Most cases of A(H1N1)pdm09 infection involved mild symptoms, but some patients had severe respiratory problems such as severe pneumonia and acute respiratory distress syndrome (ARDS); these were particularly common in children and young adults [1], [2], [3], [4]. In children, bronchial asthma increases the risk of admission to the hospital and intensive care unit [4], [5], [6], [7]. A cytokine-mediated inflammatory response has been well documented in cases of pneumonia and ARDS [8], [9], [10], but to our knowledge, there are no published findings regarding the cytokine profile of bronchoalveolar lavage (BAL) fluid from patients who have bronchial asthma and are also infected by A(H1N1)pdm09. Thus, the mechanism by which A(H1N1)pdm09 infection increases the severity of symptoms in patients with bronchial asthma remains unclear.

In this study, we investigated the levels of cytokines and viral titers in BAL fluid in a mouse model of bronchial asthma with A(H1N1)pdm09 infection to determine the mechanism by which A(H1N1)pdm09 infection increases the severity of symptoms in mice with bronchial asthma.

Section snippets

Sensitization and allergen challenge of mice

BALB/c mice aged 6–8 weeks (Chiyoda Kaihatsu Co., Ltd., Tokyo, Japan) were sensitized and challenged with grade II ovalbumin (OVA; Sigma Chemical Co., St. Louis, MO, USA) or phosphate-buffered saline (PBS). For sensitization, mice were injected subcutaneously on days 0 and 14 with 50 μg OVA, which was dissolved in 300 μl PBS containing 2 mg aluminum hydroxide (alum; Wako Pure Chemical Industries, Ltd., Osaka, Japan). On days 22, 24, 26, and 28, the mice inhaled 1% (w/v) OVA in PBS for 30 min via a

Analysis of the cells in the BAL fluid

The number of inflammatory cells in the BAL fluid from the different groups is illustrated in Fig. 2. The number of neutrophils and monocytes in the non-asthma/A(H1N1)pdm09 and asthma/A(H1N1)pdm09 groups was significantly higher than that in the mock groups (all p < 0.05). The number of eosinophils in the asthma/mock group was significantly higher than that in the non-asthma/mock and non-asthma/A(H1N1)pdm09 groups (both p < 0.05), and the number of eosinophils in the asthma/A(H1N1)pdm09 groups was

Discussion

To our knowledge, this is the first report of BAL fluid analysis in A(H1N1)pdm09-infected mice with bronchial asthma. The results show that A(H1N1)pdm09 infection induces high levels of pro-inflammatory cytokines such as IL-6 and TNF-α in BAL fluid and that this increase is enhanced by the presence of bronchial asthma.

IL-6 and TNF-α are known as pro-inflammatory cytokines [11], [12], [13]. The levels of these cytokines are elevated in the sputum and BAL fluid of patients with asthma and are

Acknowledgements

This study was supported by Grants from the Ministry of Health, Labor and Welfare (H21-Shink-Ippan-010), Japan.

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