Exposure of neonates to respiratory syncytial virus is critical in determining subsequent airway response in adults

Abstract

Background: Respiratory syncytial virus (RSV) is the most common cause of acute bronchiolitis in infants and the elderly. Furthermore, epidemiological data suggest that RSV infection during infancy is a potent trigger of subsequent wheeze and asthma development. However, the mechanism by which RSV contributes to asthma is complex and remains largely unknown. A recent study indicates that the age of initial RSV infection is a key factor in determining airway response to RSV rechallenge. We hypothesized that severe RSV infection during neonatal development significantly alters lung structure and the pulmonary immune micro-environment; and thus, neonatal RSV infection is crucial in the development of or predisposition to allergic inflammatory diseases such as asthma.

Methods: To investigate this hypothesis the present study was conducted in a neonatal mouse model of RSV-induced pulmonary inflammation and airway dysfunction. Seven-day-old mice were infected with RSV (2 x 10(5) TCID50/g body weight) and allowed to mature to adulthood. To determine if neonatal RSV infection predisposed adult animals to enhanced pathophysiological responses to allergens, these mice were then sensitized and challenged with ovalbumin. Various endpoints including lung function, histopathology, cytokine production, and cellularity in bronchoalveolar lavage were examined.

Results: RSV infection in neonates alone led to inflammatory airway disease characterized by airway hyperreactivity, peribronchial and perivascular inflammation, and subepithelial fibrosis in adults. If early RSV infection was followed by allergen exposure, this pulmonary phenotype was exacerbated. The initial response to neonatal RSV infection resulted in increased TNF-alpha levels in bronchoalveolar lavage. Interestingly, increased levels of IL-13 and mucus hyperproduction were observed almost three months after the initial infection with RSV.

Conclusion: Neonatal RSV exposure results in long term pulmonary inflammation and exacerbates allergic airways disease. The early increase in TNF-alpha in the bronchoalveolar lavage implicates this inflammatory cytokine in orchestrating these events. Finally, the data presented emphasize IL-13 and TNF-alpha as potential therapeutic targets for treating RSV induced-asthma.

Figure 1

Schematic of study protocol and exposure groups. Neonatal mice (7 days of age) were infected with RSV (2 × 10⁵ TCID50/g body weight; RSS, ROO groups). On protocol days 41 and 55, mice were injected i.p. with ovalbumin complexed to Imject Alum (OVA and ROO groups) or with isotonic saline (SAL and RSS groups). These mice were then exposed to aerosolized ovalbumin or saline for 20 minutes on protocol days 65, 66, and 67. n = 8/group.

Figure 2

Airway hyperreactivity in RSV and/or Ova exposed mice. Mice were infected with RSV as neonates (RSS) and exposed to Ova 5 wks later (ROO). Controls were sham infected and exposed to Saline (SAL) or Ova (OVA). A. Lung resistance of each group is plotted as a function of increasing doses of inhaled MeCh, using single compartment model. B. Penh is plotted as a function of increasing dose of inhaled MeCh (0 to 50 mg/ml). Data were collected on protocol days 69 (A) or 96 (B), normalized to saline airway responses as described in methods, and expressed as mean ± SEM. n = 4–5/group. ***p < 0.001; **p < 0.01.

Figure 3

BAL cellularity in RSV and/or Ova exposed mice. Bronchoalveolar lavage fluid was isolated on protocol day 69. RSV and/or Ova induced significant increase in total BAL cellularity. In mice exposed to RSV as neonates (RSS), this increase correlated with elevated levels of macrophages; whereas, in mice exposed to Ova (ROO and Ova), this increase correlated to elevation in eosinophil numbers. Data are expressed as means ± SEM, n = 3/group. ***p < 0.001

Figure 4

Cytokine levels in the BAL fluid of mice exposed to RSV and/or Ova. Bronchoalveolar lavage fluid was isolated 5 hr post-infection with RSV (A) or on protocol day 69 (B). A. Elevated levels of TNF-α, IL-5, IL-4, and IL-2 were observed as early as 5 hrs post-infection, although significance over SAL controls was observed only for TNF-α. IL-2, IL-4 and IL-5 were below the limit of detection in the SAL mice. B. IL-13 was significantly elevated in RSS mice; while TNF-α, IL-5, and IL-13 were significantly enhanced in mice exposed to RSV and Ova (ROO). IL-13 was below the limit of detection in control animals (SAL). Data are expressed as means ± SEM, n = 3/group. ***p < 0.001, **p < 0.01, and *p < 0.05.

Figure 5

Lung histopathology of mice exposed to RSV and/or Ova. Lung tissue sections were obtained from mice on protocol day 69. Tissue sections were stained with H&E (A), PAS (B), and MT (C). A. Lung inflammation, B. mucus (purple) hyperproduction, and C. collagen (blue) deposition were observed in mice exposed to RSV and/or Ova. D. Scores were assigned to these histological endpoints by two independent observers and were recorded on a scale of 0–4. Increased deposition of peribronchial and perivascular collagen was observed in the subepithelial reticular layer of the airways in RSS, OVA and ROO mice. In all cases, neonatal RSV infection (RSS) induced persistent lung pathologies including increased peribronchial inflammation, mucus production, and subepithelial fibrosis were exacerbated by subsequent exposure to allergen (ROO). n = 3/group. ***p < 0.001, *p < 0.05.