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In the present studies, we have established that RSV can elicit a more pathogenic environment dependent on improper DC-associated sensitization. Our initial studies demonstrated that RSV, but not influenza, infection during an allergen exposure into the airway induced a more severe allergen response. The RSV-induced exacerbation included an increased Th2 cytokine response and pathophysiology as monitored by AHR and mucus overproduction. DCs played a central role in the allergen-induced responses, as instilling RSV-infected BMDC into the airway could recapitulate a live virus challenge. With the use of CCR6-/- mice that have a primary defect in the recruitment of mDC subsets, reduced exacerbation of disease was observed when RSV was administered along with allergen. Furthermore, sensitization of mice with RSV-infected BMDC into the airway produced a more severe immune response to a live virus challenge. Subsequently, using RSV-infected BMDC from CCR7-/- mice (that do not migrate efficiently to LNs) to sensitize the exacerbated response demonstrated that the response was dependent on DC migration to the LN. Finally, the ability of RSV-infected DCs to elicit an exacerbated, allergen-induced pathogenic response could be maintained for as long as 3 weeks, suggesting that RSV-infected DCs themselves created an altered immune environment that impacts off-target mucosal responses that could have prolonged effects.

Chemokines are important mediators of the immune response to pathogens, but can also promote chronic inflammatory states. Chemokine receptor 6 (CCR6) is found on immature DC and effector/memory T cells, and binds a single ligand, CCL20, with high affinity. Here, we investigated the role of CCL20 and CCR6 in a pulmonary viral infection caused by RSV, a ubiquitous virus that can cause severe pulmonary complications. Neutralization of CCL20 during RSV infection significantly reduced lung pathology and favored a Th1 effector response. CCR6-deficient animals recapitulated this phenotype, and additionally showed enhanced viral clearance when compared with WT mice. No differences were observed in migration of T cells to the lungs of CCR6(-/-) animals; however, a significant reduction was observed in numbers of conventional DC (cDC), but not plasmacytoid DC, in CCR6(-/-) mice. A pathogenic phenotype could be reconstituted in CCR6(-/-) mice by supplying cDC into the airway, indicating that mere number of cDC dictates the adverse response. Our data suggest that blockade of the CCL20/CCR6 pathway provides an environment whereby the attenuated recruitment of cDC alters the balance of innate immune cells and mediates the efficient antiviral response to RSV.

Recent data have indicated that an important instructive class of signals regulating the immune response is Notch ligand-mediated activation. Using quantitative polymerase chain reaction, we observed that only Delta-like 4 (dll4) was up-regulated on bone marrow-derived dendritic cells after respiratory syncytial virus (RSV) infection, and that it was dependent on MyD88-mediated pathways. Using a polyclonal antibody specific for dll4, the development of RSV-induced disease was examined. Animals treated with anti-dll4 had substantially increased airway hyperresponsiveness compared with control antibody-treated animals. When the lymphocytic lung infiltrate was examined, a significant increase in total CD4+ T cells and activated (perforin+) CD8+ T cells was observed. Isolated lung CD4+ T cells demonstrated significant increases in Th2-type cytokines and a decrease in interferon gamma, demonstrating an association with increased disease pathogenesis. Parallel in vitro studies examining the integrated role of dll4 with interleukin-12 demonstrated that, together, both of these instructive signals direct the immune response toward a more competent, less pathogenic antiviral response. These data demonstrate that dll4-mediated Notch activation is one regulator of antiviral immunity.