RSV bronchiolitis and pneumonia causes significant mortality in young infants every year. The survivors become susceptible to asthma. In our present study, using a mouse model of BALB/C and NKT−/− mice, we examined the pulmonary responses to RSV infection in the very young hosts as opposed to relatively older hosts. We also examined the effects of exogenous osteopontin (OPN) administration on the alveolar cellular constitution by direct microscopy and flowcytometry and the role of NKT cells in this process. Pulmonary damage was assessed by measuring the Lactic Dehydrogenase (LDH) levels in the BALF by ELISA and histopathological examination by microscopy. Microscopy revealed a decreased lymphocyte response in the bronchoalevolar lavage fluid (BALF) of RSV-infected-4-day- old pups as compared to that of -14- and -20-day-old mice; but an enhanced neutrophil response, which was associated with increased LDH levels. Flowcytometry revealed that administration of OPN had downregulatory effects on lung lymphocyte (CD4+ and CD8+) responses in all age groups with proportional upregulation of lung macrophage (CD11bmed/hi, CD11Clo/−) responses. This effect was less pronounced in the older age groups. Additionally, activation of macrophages, as indicated by MHCII expression, was NKT dependent. Moreover, RSV- infection significantly inhibited lung CD8+ T cell activation as indicated by decreased CD44 expression, which was recovered by OPN administration. Understanding the underlying mechanisms of RSV-infection induced differential immune responses and OPN-mediated differential immunomodulation in different age groups may help develop newer therapeutic strategies.

RSV bronchiolitis and pneumonia in infancy is known to be associated with increased susceptibility to asthma. This phenomenon is characterized by a hyper reactive airway with a predominant Th2 response and a skewed dendritic cell (DC) polarization, which can be modulated by exogenous OPN administration. OPN is an immunomodulatory agent inducing a strong Th1 polarization in RSV infected lungs. In our present study, we used a neonatal mouse model to examine the transcriptional regulation of this immunomodulation in RSV-infected pups. RSV-infected pups were intranasally administered with or without different doses of OPN, and messenger RNA (mRNA) expression of GATA-3 and STAT-6 were examined by PCR and RTPCR methods. At day 3 post-infection, GATA-3 expression was significantly higher in the OPN treated lungs compared to that in the PBS treated lungs, contradicting the enhanced Th1 cytokine responses observed after OPN treatment. Interestingly, an enhanced expression of STAT-6 was also observed at the same time point in the OPN treated and RSV infected pups compared to that in the PBS treated pups. Of note, STAT-6 influences IL4 transcription which has a prominent role in Th2 differentiation. On the other hand, IL-4 instructs DC to promote Th1 differentiation which is STAT-6 dependent. Thus, the transcriptional regulation of OPN-induced immunomodulation is not one-dimensional and will need further investigations into the dynamics of other transcriptional factors (e.g. T-bet and Foxp3) to establish OPN as a novel therapeutic in the prevention of RSV-induced susceptibility to asthma.

Survivors of RSV bronchiolitis and pneumonia develop increased susceptibility to asthma. We showed that skewed polarization of dendritic cells (DC) towards plasmacytoid DC (pDC) in the pups in response to RSV is inhibited by exogenous administration of osteopontin (OPN) with an associated Th1 immune response. In the present study, we examined the expression of MHC-II, an indicator of antigen presenting function, on the antigen presenting cells (APC) in the RSV-infected lungs and its dependence on NKT cells. Wild type (WT) and NKT knock out (NKT-/-) pups were infected with RSV and were treated with or without OPN. Expression of MHCII on the lung APCs, including macrophages (MQ) and DCs (pDC; CD11blo/-CD11Clo/-CD45Rhi) and mDC; CD11blo/-CD11ChiCD45Rlo/-), was determined by flowcytometry (FACS). The effect of OPN on MHCII expression in the presence or absence of NKT cells was studied. FACS revealed that expression of MHCII was higher on the pDCs than mDCs in the RSV-infected WT lungs. In the absence of NKT cells, MHCII expression on pDCs was increased. OPN treatment augmented expression of MHCII on MQ and DCs, which was further enhanced in the absence of NKT cells. These data suggest that OPN- induced MHCII expression on the lung APCs is NKT dependent. Further experiments will explore the underlying mechanism of this phenomenon, which may help developing a therapeutic strategy to stop skewed polarization of DC in RSV infected lungs in preventing susceptibility to asthma.

RSV bronchiolitis and pneumonia during early infancy is associated with increased susceptibility to asthma; and is characterized by a hyper-responsiveness airway and a predominant Th2 response. Using a BALB/C neonatal mouse model we have observed a skewed polarization of DCs towards plasmacytoid DCs (pDCs: CD11blo CD11clo CD45R+) over myeloid DC (mDCs: CD11blo CD11chi/med CD45R-), as determined by flow cytometry (FACS). This was associated with increased and decreased production of Th2 (IL-10) and Th1 (IFN-g) cytokines, respectively, in the alveolar spaces, as measured by ELISA. A temporal observation of enhanced NKT (CD3-CD19-CD1d+) cell response was also determined by FACS. In addition, FACS revealed that RSV infection of NKT cell knock out (CD1d-/-) neonatal mouse had lower proportions of pDCs and a higher proportions of mDCs compared to the wild type (WT) mouse of same age. Amount of IL-10 in bronchoalveolar lavage fluid was lower in the CD1d-/- mouse than that in the WT mouse. This data suggests that NKT cells are instrumental in skewed polarization of DC in response to RSV infection, which in turn may influence the T cell responses and hence, development of susceptibility to asthma in later life.

RSV infection in early infancy makes individuals susceptible to develop asthma in later life due to airway hyper-responsiveness (AHR) to innocuous antigens, which is characterized by enhanced Th2 responses. Using a neonatal mouse model, we investigated whether differential NKT cell responses and skewed polarization of DC are involved in the underlying mechanism. Flow cytometry revealed a significant increase in the proportions of NKT cells (CD3-CD19-CD1d+) in RSV-infected 5 days old lungs compared to that of 7 and 34 days old lungs. A comparable increase in the proportions of plasmacytoid DCs (pDCs; CD11blo CD11clo CD45R+) to myeloid DC (mDCs; CD11blo CD11chi/med CD45R-) was also determined by flow cytometry. To note, DC phenotypes involved in antigen presentation influence the outcomes of the immune responses. In support of this notion, RSV infection induced a significantly higher production of Th2 cytokines than Th1 cytokines in the lungs of younglings. In contrast, RSV-infected adult lungs produced significantly higher amount of Th1 cytokines. Furthermore, NK cell response was not enhanced in the young lungs as opposed to that in the adult lungs in response to RSV infection. Thus, enhanced NKT cell and reduced NK cell responses to RSV infection in the neonatal lungs, along with an enhanced Th2 response, may have induced a skewed polarization of DCs to pDCs leading to development of AHR and susceptibility to asthma.