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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.

Background: Respiratory syncytial virus (RSV) is a leading cause of bronchiolitis and pneumonia in young children worldwide, and no vaccine is currently available. Inactivated RSV vaccines tested in the 1960's led to vaccine-enhanced disease upon viral challenge, which has undermined RSV vaccine development. RSV infection is increasingly being recognized as an important pathogen in the elderly, as well as other individuals with compromised pulmonary immunity. A safe and effective inactivated RSV vaccine would be of tremendous therapeutic benefit to many of these populations., Principal Findings: In these preclinical studies, a mouse model was utilized to assess the efficacy of a novel, nanoemulsion-adjuvanted, inactivated mucosal RSV vaccine. Our results demonstrate that NE-RSV immunization induced durable, RSV-specific humoral responses, both systemically and in the lungs. Vaccinated mice exhibited increased protection against subsequent live viral challenge, which was associated with an enhanced Th1/Th17 response. In these studies, NE-RSV vaccinated mice displayed no evidence of Th2 mediated immunopotentiation, as has been previously described for other inactivated RSV vaccines., Conclusions: These studies indicate that nanoemulsion-based inactivated RSV vaccination can augment viral-specific immunity, decrease mucus production and increase viral clearance, without evidence of Th2 immune mediated pathology.

Development of bronchus-associated lymphoid tissue has been suggested to enhance local antiviral immune responses; however, ectopic lymph node formation often corresponds to chronic inflammatory diseases. These studies investigated the role of ectopic pulmonary lymph nodes upon respiratory syncytial virus (RSV) infection using CCR7-deficient mice, which develop bronchus-associated lymphoid tissue early in life. CCR7(-/-) mice exhibited impaired secondary lymph node formation, enhanced effector T cell responses and pathogenic mucus production in the lung after RSV infection. IL-17 production from CD4 T cells in CCR7(-/-) mice was most remarkably enhanced. Wild-type animals reconstituted with CCR7(-/-) bone marrow recapitulated the pathogenic lung phenotype in CCR7(-/-) mice, whereas CCR7(-/-) animals reconstituted with wild-type bone marrow had normal lymph node development, diminished IL-17 production and reduced lung pathology. Mixed bone marrow chimeras revealed an alteration of immune responses only in CCR7(-/-) T cells, suggesting that impaired trafficking promotes local effector cell generation. Lymphotoxin-α-deficient mice infected with RSV were used to further examine locally induced immune responses and demonstrated increased mucus production and amplified cytokine responses in the lung, especially IL-17. Neutralization of IL-17 in CCR7(-/-) or in lymphotoxin-α-deficient animals specifically inhibited mucus hypersecretion and reduced IL-13. Thus, immune cell trafficking to secondary lymph nodes is necessary for appropriate cytokine responses to RSV as well as modulation of the local environment.

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.

CC chemokine receptor 1 (CCR1) is found on a variety of cells in the immune system and has been shown to play an important role in the host response to pathogens. These studies used a murine model of virus-induced exacerbation of allergic airway disease to examine the role of CCR1 on T cells associated with immune responses taking place in the lung. Lungs of virally exacerbated allergic animals contained elevated levels of interferon-gamma and interleukin-13 and increased levels of CCR1 ligands CCL3 and CCL5. CCR1 expression on T cells was increased in virally exacerbated allergic animals over the level observed in mice sensitized to allergen or exposed to viral infection alone. Using mice deficient for CCR1, we observed decreased airway hyperreactivity and Th2 cytokine production from CD4(+) T cells when this receptor was absent. Transfer studies demonstrated that neither CD4(+) nor CD8(+) T cells from CCR1(-/-) mice migrated to the lymph node as efficiently as wild-type T cells. Intracellular cytokine staining in wild-type mice revealed that CCR1(+) CD4(+) and CD8(+) T cells are associated with interleukin-13 production. Thus, these studies identify CCR1 as a potential target for alleviating T-cell accumulation during exacerbation of asthmatic disease.

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.