BackgroundPoor interferon gamma (IFNγ) production during respiratory syncytial virus (RSV) is associated with prolonged viral clearance and increased disease severity in neonatal mice and humans. We previously showed that intra-nasal delivery of IFNγ significantly enhances RSV clearance from neonatal lungs prior to observed T-lymphocyte recruitment or activation, suggesting an innate immune mechanism of viral clearance. We further showed that alveolar macrophages dominate the RSV-infected neonatal airways relative to adults, consistent with human neonatal autopsy data. Therefore, the goal of this work was to determine the role of neonatal alveolar macrophages in IFNγ-mediated RSV clearance.MethodsClodronate liposomes, flow cytometry, viral plaque assays, and histology were used to examine the role of alveolar macrophages (AMs) and the effects of intra-nasal IFNγ in RSV infected neonatal Balb/c mice. The functional outcomes of AM depletion were determined quantitatively by viral titers using plaque assay. Illness was assessed by measuring reduced weight gain.ResultsAM activation during RSV infection was age-dependent and correlated tightly with IFNγ exposure. Higher doses of IFNγ more efficiently stimulated AM activation and expedited RSV clearance without significantly affecting weight gain. The presence of AMs were independently associated with improved RSV clearance, whereas AM depletion but not IFNγ exposure, significantly impaired weight gain in RSV-infected neonates.ConclusionWe show here for the first time, that IFNγ is critical for neonatal RSV clearance and that it depends, in part, on alveolar macrophages (AMs) for efficient viral clearing effects. Early reductions in viral burden are likely to have profound short- and long-term immune effects in the vulnerable post-natally developing lung environment. Studies are ongoing to elucidate the pathologic effects associated with early versus delayed RSV clearance in developing neonatal airways.

Respiratory syncytial virus (RSV) is the leading cause of lower respiratory tract infections in young children and is further associated with increased healthcare utilization and cost of care in the first years of life. Severe RSV disease during infancy has also been linked to the later development of allergic asthma, yet there remains no licensed RSV vaccine or treatment. Pre-clinical and clinical studies have shown that disease severity and development of allergic asthma are associated with differences in cytokine production. As a result, stimulation of the innate host immune response with immune potentiators is gaining attention for their prospective application in populations with limited immune responses to antigenic stimuli or against pathogens for which vaccines do not exist. Specifically, macrophage-activating cytokines such as interferon gamma (IFNγ) and granulocyte colony-stimulating factor (GM-CSF) are commercially available immune potentiators used to prevent infections in patients with chronic granulomatous disease and febrile neutropenia, respectively. Moreover, an increasing number of reports describe the protective function of IFNγ and GM-CSF as vaccine adjuvants. Although a positive correlation between cytokine production and age has previously been reported, little is known about age-dependent cytokine metabolism or immune activating responses in infant compared to adult lungs. Here we use a non-compartmental pharmacokinetic model in naïve and RSV-infected infant and adult BALB/c mice to determine the effect of age on IFNγ and GM-CSF elimination and innate cell activation following intranasal delivery.

Respiratory syncytial virus (RSV) is one of the leading causes of respiratory tract infections in children. Nearly every child will be infected by the age of two, yet there is currently no vaccine or effective treatment for RSV infection. Previous studies have shown that immunotherapy, such as recombinant GM-CSF (rGM-CSF), expedites viral clearance, likely due to increased activation and accumulation of pulmonary antigen-presenting cells (APCs). This study examines the pharmacokinetics and pharmacodynamics of a weight-based, inhaled rGM-CSF dose in infant compared to adult mice. Using a Luminex assay, the concentration of rGM-CSF in digested lung tissue and blood was analyzed over 24 hours. In lung digest, rGM-CSF was rapidly detected in both ages with levels peaking at 1 hour and decreasing to endogenous levels by 8 hours. rGM-CSF levels in the blood peaked at 0.5 hours and decreased by 4 hours in pups, while adult levels peaked at 1 hour and decreased by 8 hours. Whole lung was analyzed by flow cytometry for macrophage activation at 4, 8, 24, and 48 hours post dose. In pups and adults, CD11c+/CD11b+ expression increased, suggesting activation or infiltration of APCs. CD86 and MHC class II expression increased significantly in adults, with a more marked MHCII increase at 48 hours. Using a non-compartmental pharmacokinetic modeling approach, we predict that increasing the dose from 50 to 170ng/g will achieve similar biological outcomes in pup compared to adult mice.