In the present study, we have shown that neonatal exposure to RSV alone induced long-term airway hyperresponsiveness and pulmonary resistance in mice. This result was correlated with peribronchial inflammation, increased BALF cellularity, increased mucus production, and airway remodeling. Elevations in TNF-α and Th2 cytokines (IL-4 and IL-5) were observed in BALF immediately following neonatal RSV infection. Once these mice had matured (protocol day 69), only IL-13 remained elevated in the BALF. Neonatal RSV infection followed by adult exposure to allergen resulted in significantly higher lung resistance, along with increased total cellularity, eosinophilia, and increased TNF-α and Th2 cytokines (IL-5 and IL-13) in BALF. In addition, TNF-α and IL-13 were significantly higher compared to mice exposed to Ova alone. The most severe lung histopathology was observed in mice exposed to both RSV and Ova, as indicated by severe peribronchial and perivascular inflammation, mucus production, and collagen deposition. Collectively, these data suggest that neonatal RSV infection influences adult immune response to allergen (Ova) and exacerbates allergic pathophysiology in mice long after viral titers are no longer detectable.
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
]. 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
]. 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.
Several studies along with our present data have established the correlation of severe RSV infection followed by allergen exposure and the development of allergic inflammatory disease (i.e., asthma) in mice. Although the mechanism by which the exposure causes asthma and the importance of such exposures in humans need to be further elucidated, our current and previously published data [16
] demonstrate that the initial age of the RSV infection is capable of altering adult pulmonary function and exacerbating pulmonary immunopathology when followed by subsequent allergen exposure. Furthermore, enhanced AHR correlated with chronic pulmonary inflammation, upregulation of the Th2 cytokine, IL-13, and subepithelial fibrosis of the bronchial airways. Increases in TNF-α within 5 hours of RSV infection in our mouse model also suggest a role for this cytokine in the immunopathology of RSV-induced wheeze and asthma development in humans.