Synthetic double-stranded RNA enhances airway inflammation and remodelling in a rat model of asthma

Abstract

Respiratory viral infections are frequently associated with exacerbations of asthma. Double-stranded RNA (dsRNA) produced during viral infections may be one of the stimuli for exacerbation. We aimed to assess the potential effect of dsRNA on certain aspects of chronic asthma through the administration of polyinosine-polycytidylic acid (poly I:C), synthetic dsRNA, to a rat model of asthma. Brown Norway rats were sensitized to ovalbumin and challenged three times to evoke airway remodelling. The effect of poly I:C on the ovalbumin-induced airway inflammation and structural changes was assessed from bronchoalveolar lavage fluid and histological findings. The expression of cytokines and chemokines was evaluated by real-time quantitative reverse transcription PCR and ELISA. Ovalbumin-challenged animals showed an increased number of total cells and eosinophils in bronchoalveolar lavage fluid compared with PBS-challenged controls. Ovalbumin-challenged animals treated with poly I:C showed an increased number of total cells and neutrophils in bronchoalveolar lavage fluid compared with those without poly I:C treatment. Ovalbumin-challenged animals showed goblet cell hyperplasia, increased airway smooth muscle mass, and proliferation of both airway epithelial cells and airway smooth muscle cells. Treatment with poly I:C enhanced these structural changes. Among the cytokines and chemokines examined, the expression of interleukins 12 and 17 and of transforming growth factor-β(1) in ovalbumin-challenged animals treated with poly I:C was significantly increased compared with those of the other groups. Double-stranded RNA enhanced airway inflammation and remodelling in a rat model of bronchial asthma. These observations suggest that viral infections may promote airway remodelling.

Figure 1

Protocol for our rat model of asthma (a). Ovalbumin (OVA) sensitization was performed by subcutaneous injection of 1 mg OVA. On days 15, 20 and 25, the rats were challenged by aerosol with 5% OVA solution or with PBS. Rats were divided into four groups (b). Two groups of rats received synthetic dsRNA, polyinosine-polycytidylic acid (poly I:C). The other two groups received PBS.

Figure 2

Analyses of total cells and differential cell counts in bronchoalveolar lavage fluid. Total cells (a). Differential cell counts of eosinophils (b), neutrophils (c), macrophages (d) and lymphocytes (e). Data are presented as means (SEM). *P < 0·05.

Figure 3

Representative haematoxylin & eosin-stained lung sections from PBS-treated PBS-challenged animals (a), polyinosine-polycytidylic acid (poly I:C) -treated PBS-challenged animals (b), PBS-treated ovalbumin (OVA) -challenged animals (c), and poly I:C-treated OVA-challenged animals (d).

Figure 4

Representative periodic acid-Schiff (PAS) staining of lung sections from PBS-treated PBS-challenged animals (a), polyinosine-polycytidylic acid (poly I:C) -treated PBS-challenged animals (b), PBS-treated ovalbumin (OVA) -challenged animals (c), and poly I:C-treated OVA-challenged animals (d). Goblet cell hyperplasia is determined by counting the number of PAS-positive cells per airway and is normalized for airway size by dividing by the perimeter of the basement membrane (P_BM) (e). Data are presented as means (SEM). *P < 0·05.

Figure 5

Representative airway smooth muscle-specific α-actin staining (red) of lung sections from PBS-treated PBS-challenged animals (a), polyinosine-polycytidylic acid (poly I:C) -treated PBS-challenged animals (b), PBS-treated ovalbumin (OVA) -challenged animals (c), and poly I:C-treated OVA-challenged animals (d). Smooth muscle-specific α-actin staining area is normalized for airway size by dividing by the square of the perimeter of the basement membrane () (e). Data are presented as means (SEM). *P < 0·05.

Figure 6

Representative airways stained by both proliferating cell nuclear antigen (PCNA, purple) and airway smooth muscle-specific α-actin (red) from PBS-treated PBS-challenged animals (a), polyinosine-polycytidylic acid (poly I:C) -treated PBS-challenged animals (b), PBS-treated ovalbumin (OVA) -challenged animals (c), and poly I:C-treated OVA-challenged animals (d). Quantification of PCNA-positive epithelial cells was performed by counting the number of PCNA-positive epithelial cells and normalizing for airway size was performed by dividing by the perimeter of the basement membrane (P_BM) (e). Data are presented as means (SEM). *P < 0·05.

Figure 7

Representative airway smooth muscle (ASM) stained by both proliferating cell nuclear antigen (PCNA, purple) and ASM-specific α-actin (red) from PBS-treated PBS-challenged animals (a), polyinosine-polycytidylic acid (poly I:C) -treated PBS-challenged animals (b), PBS-treated ovalbumin (OVA) -challenged animals (c), and poly I:C-treated OVA-challenged animals (d). Arrows indicate PCNA-positive ASM cells. PCNA-positive ASM cells were counted and normalized for airway size by dividing by the square of the perimeter of the basement membrane () (e). Data are presented as means (SEM). *P < 0·05.

Figure 8

Quantitative RT-PCR of cytokine and chemokine mRNA in lung homogenates (a–i) and interleukin-17 (IL-17) and transforming growth factor-β₁ (TGF-β₁) in bronchoalveolar lavage fluid measured by ELISA (j, k) from PBS-treated PBS-challenged animals (PBS/PBS), polyinosine-polycytidylic acid-treated PBS-challenged animals (poly I:C/PBS), PBS-treated ovalbumin (OVA) -challenged animals (PBS/OVA), and poly I:C-treated OVA-challenged animals (poly I:C/OVA). Data are presented as means (SEM). *P < 0·05.