The objectives of this study were to define the role of IFN-γ in the induction of protective responses against the development of altered airway function after reinfection with RSV. Because recurrent RSV infection is frequent in humans, we hypothesized that the consequences of reinfection with RSV may be determined at the time of initial infection, where IFN-γ could play a key regulator of the response. In an earlier study (11
), neonatal RSV infection was shown to predispose mice to develop more severe airway disease on reinfection. This amplified and altered response to reinfection was characterized by the development of significant AHR associated with a marked airway eosinophilia and mucus hyperproduction. By contrast, primary infection at later age (e.g., at weaning) elicited a protective airway response to reinfection characterized by an exuberant lymphocytic inflammatory response, but without development of AHR or airway eosinophilia and mucus hyperproduction. Interestingly, the most distinctive difference observed between the two ages in terms of response to primary RSV infection was lower IFN-γ production in the lungs of infected newborn mice. The present study determined that the outcome of reinfection with RSV (development of, or protection against, altered airway responses) is dependent on IFN-γ production during primary RSV infection.
The results of this study demonstrate that IFN-γ is not essential to the development of AHR during primary RSV infection, because IFN-γ−/−
mice developed AHR to the same extent as did WT mice after primary RSV infection. This finding contradicts an early report, which suggested that IFN-γ might contribute to RSV-mediated AHR in mice (25
); however, AHR was not documented in this study (e.g., by assessment of airway responsiveness to MCh) nor was the outcome of reinfection examined in IFN-γ−/−
mice. Durbin and colleagues compared the responses of IFN-γ−/−
and WT mice to primary RSV infection and found no differences in lung pathology, cytokine levels, or viral replication and rate of clearance between the two strains of mice even after inoculation with high doses of RSV (i.e., 107
PFUs [plaque-forming units]) (26
). In contrast, mice lacking STAT1, a common signaling pathway shared by IFN-γ and IFN-α/β, developed marked lung pathology characterized by a prominent eosinophilia, a Th2-biased cytokine response, but no significant alteration in rate of viral clearance after primary RSV infection. Surprisingly, all these strains of mice including IFN-γ−/−
mice appeared to be protected and did not develop signs of illness as defined by weight loss after reinfection with RSV. Nonetheless, it was concluded that STAT1 activation by both type I (α/β) and type II (γ) IFNs plays an important role in establishing a protective Th1-mediated immune response to RSV infection. In the present study, no significant differences were seen between IFN-γ−/−
mice and WT mice in AHR or airway inflammation after primary RSV infection, which is not inconsistent with the findings reported by Durbin and colleagues (26
). However, our data clearly show that IFN-γ−/−
mice are not protected from, but rather predisposed to, developing altered airway responses (AHR, eosinophilia, and mucus hyperproduction) on reinfection, further establishing a critical role for IFN-γ in determining the outcome of reinfection with RSV. Interestingly, treatment of IFN-γ−/−
mice by administration of rIFN-γ during the primary RSV infection prevented the subsequent development of altered airway responses on reinfection of these mice; treatment during reinfection was without effect. Likewise, adoptive transfer of IFN-γ–sufficient T cells into IFN-γ−/−
mice during the primary infection but not during reinfection also prevented the development of altered airway responses on reinfection in IFN-γ−/−
mice. These data support our hypothesis that deficient (or absence of) IFN-γ production during initial RSV infection predisposes to the development of altered airway responses on subsequent reinfection with this virus. This is further revealed in neonatal mice in which deficient IFN-γ production during initial RSV infection appears to be the basis for the subsequent development of altered and Th2-biased airway responses (enhanced AHR, airway eosinophilia, and mucus hyperproduction) on reinfection with RSV.
IFN-γ, a cytokine mainly produced by NK cells and activated CD4+
T cells, promotes cell-mediated immune responses to intracellular pathogens such as viruses and has well-characterized antiviral activity (27
). The role of IFN-γ in RSV-mediated disease is not well understood, but there is an apparent association between severity of RSV bronchiolitis and IFN-γ production. Earlier studies reported that infants hospitalized for severe lower respiratory tract illness due to RSV infection and requiring ventilation had lower IFN-γ production by blood mononuclear cells compared with those with a milder illness (15
). Moreover, the levels of IFN-γ measured in nasopharyngeal aspirates were lower in infants hospitalized for severe RSV bronchiolitis compared with those exhibiting milder disease. In one study, deficient IFN-γ production by blood mononuclear cells at the time of RSV bronchiolitis was found to be an indicator of lower pulmonary function and increased airway responsiveness to histamine 5 months later, and appeared to predict the development of asthma in infants hospitalized for severe RSV bronchiolitis (14
). A prospective study found that infants who developed lower respiratory tract disease (bronchiolitis) had lower levels of IFN-γ in nasal lavage fluids compared with those who developed upper respiratory tract disease alone after RSV infection (17
). Recent findings from a study of hospitalized infants suggested that decreased IFN-γ production is more a characteristic of lower respiratory tract illness related to RSV than other respiratory viruses (29
). Taken together, these human studies suggest an important role for initial IFN-γ production in determining the outcome of RSV-mediated airway disease.
IFN-γ production usually identifies a Th1-type response, which is often accompanied by a vigorous CD8+
cytolytic T lymphocyte (CTL) response to viral infection in animal models (30
). By analogy, a weak IFN-γ and/or CD8+
CTL response could result in delayed virus clearance, leading in turn to an increased inflammatory response and more severe illness. Indeed, infants with a severe clinical course of RSV disease exhibit lower CD8+
T-cell and CD8+
T-cell counts during the acute phase of illness (13
). This is in accord with previous findings of RSV-specific cellular cytotoxic immune responses in infants with mild illness but not in those with the most severe disease (31
), and with the findings of reduced numbers of CD8+
T cells in the peripheral blood of infants with severe disease, compared with infants with milder forms of illness caused by RSV (32
There are several potential mechanisms by which impaired or lower IFN-γ production during primary RSV infection might be associated with enhanced AHR and airway inflammation after reinfection with RSV. Impaired IFN-γ at birth may facilitate skewing of T-cell differentiation toward a Th2 phenotype, thereby predisposing to subsequent development of atopic wheezing and asthma in children (33
). However, deficient IFN-γ may also reflect either decreased maturation of CD8+
T cells or decreased numbers of NK cells or Th1 cells. Previous in vitro
studies have shown that addition of IFN-γ to activated T cells leads to acquisition of cytotoxic activities by CD8+
but not CD4+
T cells (35
). In our study, the elevated viral load seen in mice lacking IFN-γ may reflect decreased cytolytic activity by CD8+
T cells, and the ability of IFN-γ–sufficient T cells to restore protection suggested that a T-cell source is sufficient for this protection. There is also evidence to suggest that both development and maturation of primary CD8+
T-cell responses are critically dependent on help from CD4+
T cells during priming (36
). Because both CD4+
T cells are a source of IFN-γ during RSV infection, interaction between both T-cell subsets is possibly required for mediating full protection. The nature of CD4 T-cell–mediated help is not clear but may involve production of IL-2 to facilitate CD8+
T-cell expansion and IFN-γ to promote the acquisition of cytotoxic activity by these cells. IFN-γ also regulates the production of IgG2a
, a major class of complement-fixing and virus-neutralizing antibodies in mice; thus, a deficiency in IgG2a
antibody production may also contribute to increased viral load in the lung. However, based on our findings, AHR appeared to correlate with lung immunopathology, not viral load.
Human RSV is known to elicit an adaptive (virus-specific) T-cell–mediated immune response in mice despite a limited viral replication in the lung of these animals, which may be explained by species-related differences in host permissiveness to the pathogen. Usually, no apparent clinical signs of disease are observed in mice after infection with doses up to 106
PFUs of human RSV. However, clinical signs of disease (e.g., ruffled fur and significant weight loss) are typically observed on Days 3–4 after infection of mice with higher doses (107
PFUs) of human RSV (38
). In humans, there is a lack of information as to what extent RSV can replicate in vivo
in the lower airways and it is unclear whether severity of RSV disease (bronchiolitis) can directly be related to the rate of viral replication in the lower respiratory tract of children. Previous studies have described marked differences between human primary nasal epithelial cells, bronchial epithelial cells, and alveolar macrophages in permissiveness to RSV infection and viral replication in vitro
). Although both nasal and bronchial epithelial cells were equally permissive to RSV infection in vitro
, nasal epithelial cells produced much more virus than bronchial epithelial cells (10-fold at 0.1 multiplicity of infection [MOI], 3-fold at 1 MOI). Alveolar macrophages were much less permissive and restricted viral replication. These observations suggest that RSV replication might also be limited (restricted) in human lower airways, the major site of RSV-induced bronchiolitis and altered airway function (wheezing).
Analysis of cells recovered by lavage from the airways of children with acute bronchiolitis revealed predominant airway neutrophilia in some studies (40
), whereas lymphocytes appear to be more prominent in tissue (41
). However, the role of neutrophils in RSV-mediated disease remains unclear. In one study, repeated BAL samples were obtained from both term and preterm infants hospitalized for 7 days for severe RSV bronchiolitis requiring ventilation (42
). Although neutrophils predominated during the first 2 to 3 days after intubation, the numbers of these cells declined progressively, reaching baseline control values by Day 4. By contrast, despite the severity of RSV bronchiolitis, preterm infants did not exhibit such a prominent BAL neutrophilia (no difference compared with nonbronchiolitic control infant group). In the mouse model, neutrophils never predominate in the BAL fluid at the peak of RSV-induced lung inflammation (i.e., Days 6–7 postinfection). However, a transient BAL neutrophilia can be observed early (Days 1–3) after RSV infection in mice (unpublished observations). We are currently investigating this neutrophilic response in the mouse model to define its role in RSV-induced airway inflammation and altered airway function.
The exact mechanisms of AHR are not really clear and there is not a single common pathway that leads to this alteration, which can be triggered by allergen, virus, or pollution. Studies of murine models of allergic airway responsiveness have established a key role for IL-13 as a downstream mediator that regulates AHR and mucus hyperproduction and contributes to airway eosinophilia (reviewed in Reference 43
). This pathophysiology is believed to be initiated during sensitization by an IL-4–dependent mechanism that drives the development of Th2 response. We have previously shown that IL-13 is not required for the development of AHR after primary RSV infection (44
). However, similar to allergen-mediated AHR and lung histopathology, the pathophysiology (AHR, airway eosinophilia, and mucus hyperproduction) that develops on reinfection of IFN-γ–deficient mice (adult IFN-γ−/−
or newborn mice) with RSV is likely driven by a Th2 (IL-13) response that develops due to the deficient (or the absence of) IFN-γ production during initial RSV infection. The fact that rIFN-γ did not alter the responses when administered during reinfection suggests that IFN-γ has no direct effect on AHR and cannot reverse established Th2 responses and lung histopathology. On the other hand, the effectiveness of IFN-γ treatment during the primary RSV infection in preventing the subsequent Th2-biased, altered airway responses that develop on reinfection further emphasizes the important role IFN-γ plays during initial infection in determining the outcome of reinfection with RSV. IFN-γ enhances Th1 responses and is an important counterregulator of Th2 development, particularly during priming (45
). Conceivably, deficiency or lack of IFN-γ production during initial RSV infection (priming) may result in the unopposed differentiation of a Th2-biased response, leading to development of eosinophilia, mucus hyperproduction, and AHR on subsequent reinfection with this virus.
In conclusion, the results of this study demonstrate that IFN-γ production at the time of initial infection is a critical factor that determines the outcome of reinfection with RSV in mice. In conjunction with several clinical reports, these findings are important to our understanding of the pathogenesis of post-RSV wheezing and asthma and may provide novel insights for the development of immunotherapeutic approaches for the prevention of RSV-mediated long-term sequelae in infants with severe RSV bronchiolitis.