Several studies have suggested that severe RSV lower respiratory tract infection in infancy may induce later development of asthma [3-8]. In 2000, Sigur and colleagues reported that RSV infection in infancy, severe enough to require hospitalization, was associated with asthma and allergy in children up to age 7 [9]. More recently, they reported that the relationship between severe RSV bronchiolitis in infancy and later development of asthma and allergy sensitization are still observed in children up to age 13. [10]. In murine models, numerous studies have been carried out and controversial results have been reported. While some studies propose that previous RSV infection enhances allergen sensitization and exacerbates asthma, others believe that RSV infection in infancy protects against later allergic sensitization [11]. This controversy is now thought to arise from the age of initial RSV infection and the different protocols used for RSV infection and allergen sensitization and challenge. In two independent experiments, researchers suggested that the timing of primary RSV infection is significant for predicting disease outcome. Culley and colleagues reported that if neonatal mice (1 d or 7 d of age) were infected with RSV and rechallenged at 12 wks of age, inflammatory cell recruitment was enhanced (including Th2 cells and eosinophils) [12]. Their data suggested that neonatal exposure to RSV exacerbated subsequent disease upon rechallenge in the adult. [12]. More recently, Dakhama and colleagues infected 1-wk old or 3-wk old mice with RSV, and they found that both primary infections led to increased lung resistance, mucus hyperproduction and inflammation along with increased lymphocytes in the lung. [13]. Furthermore, they demonstrated that the BAL fluid from mice infected with RSV at 1 wk of age had reduced levels of IFN-γ and elevated levels of IL-13 compared to mice initially infected at 3 wks of age. This data indicated that a Th2 polarized response occurred in mice infected with RSV at 1 wk of age, while a Th1-biased response occurred in the mice infected with RSV at 3 wks of age. When rechallenged with RSV 5 weeks later (i.e., infection at 1 wk and reinfection at 6 wks of age), these mice displayed exacerbated disease as indicated by enhanced pulmonary resistance, mucus hyperproduction, eosinophilia, and elevated levels of Th2 cytokines. In contrast, initial infection of mice at 3 wks of age resulted in protection upon RSV rechallenge as indicated by the abolished lung resistance and enhanced viral clearance at rechallenge [13]. These experiments indicate that age at initial RSV infection influences the subsequent immune and physiologic response upon re-exposure to RSV.

In contrast to previously published studies analyzing the effect of RSV infection on allergen exposure in adult mice. [17], the present study investigated the influence of neonatal RSV infection on adult allergen sensitization. Neonatal infection with RSV alone was sufficient to induce long-term pulmonary dysfunction and inflammation. Similarly, neonatal infection of brown Norway rats with parainfluenza type 1 (Sendai) virus also led to increases in pulmonary resistance and hyperresponsiveness to methacholine up to 65d after infection [18]. As in our model, persistent airway dysfunction following neonatal infection with Sendai virus correlated with increased peribronchial fibrosis and pulmonary inflammation. [19]. Furthermore, both models (i.e., neonatal RSV infection and neonatal Sendai virus infection) resulted in significantly increased mRNA [19] and protein levels for TGF-β₁. Although numerous cytokines may contribute to airway remodeling, the elevation of TGF-β₁, a fibrogenic cytokine, in both neonatal viral infection models prior to the development of fibrosis suggests a role this cytokine in the regulation of viral-induced airway remodeling observed in neonates.

More importantly, neonatal infection with RSV predisposed mice to the development of enhanced AHR and inflammation after allergen exposure. In contrast, adult RSV infection prior to allergen exposure seemed to assert a "protective" response as evidenced by significantly decreased allergen induced pulmonary resistance, tissue eosinophilia, and IL-13 levels [20]. Our data presented here and elsewhere [16] extend these findings and, more importantly, demonstrate that the age at initial RSV infection also determines whether RSV infection will exacerbate or prevent subsequent allergic inflammation.

A more recent study using neonatal RSV infection followed by subsequent reinfection of adults with RSV demonstrated that early RSV infection also exacerbates RSV induced diseases in the adult [13]. Interestingly, if the primary RSV infection occurred at 3 wks of age, a protective effect upon secondary infection was observed similar to that reported by Peebles and colleagues [17]. Dakhama and colleagues further established that enhancement of AHR, pulmonary eosinophilia, and mucus hyperproduction during reinfection were dependent on IL-13 [13]. We demonstrated that neonatal RSV infection alone leads to elevated levels of IL-13 in the lung and that exposure to allergen significantly increases IL-13 levels over RSV exposure alone. IL-13 has emerged as a major regulatory molecule involved in mucus hyperproduction and allergen induced AHR [21-23]. It is entirely possible that the long-term AHR, pulmonary inflammation, and mucus production observed in our neonatal RSV model is due to high levels of IL-13. In fact, elevated levels of IL-13 were observed in whole lung homogenates as early as 5 hours post-infection (data not shown) and were again observed in adult lungs on protocol day 69 suggesting that IL-13 is being chronically produced. Although the exact cellular source of IL-13 in this neonatal model of RSV infection is currently unknown, previous studies have demonstrated that epithelial cells and/or macrophages infected by RSV are a significant source of IL-13 and are capable of producing this cytokine for months after the initial infection. [24]. We are currently investigating this possibility.

In our study, TNF-α was significantly elevated in the BALF shortly after infection with RSV. TNF-α is an important cytokine for innate immune responses and a central regulator of inflammatory processes, through binding to distinct membrane receptors, referred to as p55 or TNFR1 and p75 or TNFR2 [25]. TNF-α is likely a central mediator of airway inflammation and AHR in asthma, regulating inflammatory cell infiltration, locally enhancing vascular permeability, and inducing the release of the chemokines. Ultimately, this will lead to chronic inflammation and irreversible airway remodeling. Recently, depletion studies using monoclonal antibody therapy for TNF-α have shown promising effects in viral-specific lung immunopathology. [26], rheumatoid arthritis [27] and inflammatory bowel disease [28]. Moreover, a soluble TNF receptor fusion protein, etanercept, has proven efficacious in treating juvenile rheumatoid arthritis in patients as young as 4 [27]. In viral models, TNF-α depletion reduced recruitment of inflammatory cells, reduced type 1 and type 2 cytokines in BALF, and decreased pulmonary pathology without inhibiting viral clearance [26,29]. Although the precise mechanism by which TNF-α leads to the pathology in lungs after RSV infection remains unknown, our data and these previous studies suggest a key role for TNF-α in chronic inflammation in the lung and subsequent airway remodeling associated with asthma.