Differential expression of cytokine transcripts in neonatal and adult ovine alveolar macrophages in response to respiratory syncytial virus or toll-like receptor ligation.

Alveolar macrophages (AMvarphis) secrete regulatory molecules that are believed to be critical in maintaining normal lung homeostasis. However, in response to activating signals, AMvarphis have been shown to become highly phagocytic cells capable of secreting significant levels of pro-inflammatory cytokines. There is evidence to suggest that susceptibility of Mvarphi subpopulations to viral infection, and their subsequent cytokine/chemokine response, is dependent on age of the host. In the present study, we compared bovine respiratory syncytial virus (BRSV) replication and induction of cytokine responses in neonatal ovine AMvarphis to those cells isolated from adult animals. While neonatal AMvarphis could be infected with BRSV, viral replication was limited as previously shown for AMvarphis from mature animals. Interestingly, following BRSV infection, peak mRNA levels of IL-1beta and IL-8 in neonatal AMvarphi were several fold higher than levels induced in adult AMvarphis. In addition, peak mRNA expression for the cytokines examined occurred at earlier time points in neonatal AMvarphis compared to adult AMvarphis. However, the data indicated that viral replication was not required for the induction of specific cytokines in either neonatal or adult AMvarphis. TLR3 and TLR4 agonists induced significantly higher levels of cytokine transcripts than BRSV in both neonatal and adult AMvarphis. It was recently proposed that immaturity of the neonatal immune system extends from production of pro-inflammatory cytokines to regulation of such responses. Differential regulation of cytokines in neonatal AMvarphis compared to adult AMvarphis in response to RSV could be a contributory factor to more severe clinical episodes seen in neonates.

Neonatal ovine pulmonary dendritic cells support bovine respiratory syncytial virus replication with enhanced interleukin (IL)-4 And IL-10 gene transcripts.

The lung microenvironment is constantly exposed to microorganisms and particulate matter. Lung dendritic cells (DCs) play a crucial role in the uptake and processing of antigens found within the respiratory tract. Respiratory syncytial virus (RSV) is a common respiratory tract pathogen in children that induces an influx of DCs to the mucosal surfaces of the lung. Using a neonatal lamb model, we examined the in vivo permissiveness of DCs to RSV infection, as well as overall cell surface changes and cytokine responses of isolated lung DCs after bovine RSV (BRSV) infection. We report that isolated lung DCs and alveolar macrophages support BRSV replication. Isolated lung DCs were determined to be susceptible to BRSV infection as demonstrated by quantification of BRSV non-structural protein 2 mRNA. BRSV infection induced an initial upregulation of CD14 expression on lung DCs, but by 5 d postinfection expression was similar to that on control cells. No significant changes in CD80/86 or MHC class I expression were seen on lung DCs after BRSV infection. Low to moderate expression of MHC class II and DEC-205 was detected by day 5 postinfection. Initially, on day 3 postinfection, lung DCs from BRSV-infected lambs had decreased endocytosis of fluorescein isothiocyanate (FITC)-ovalbumin (OVA). The amount of FITC-OVA endocytosed by lung DCs isolated on day 5 postinfection was similar to that of controls. The most interesting observation was the induction of immunomodulatory interleukin (IL)-4 and IL-10 cytokine gene transcription in lung DCs and alveolar macrophages after in vivo infection with BRSV. Overall, these findings are the first to demonstrate that neonatal lung DCs support in vivo BRSV replication and produce type II cytokines after viral infection.

Pulmonary dendritic cells isolated from neonatal and adult ovine lung tissue.

Lung dendritic cells (DCs) are potent antigen presenting cells (APCs) that initiate and modulate the adaptive immune response upon microbial infection within the pulmonary environment. For the first time, neonatal and adult lung DCs in a large animal model were compared in these studies. Here, we isolated and identified lung DCs in both neonatal and adult sheep, a valuable experimental animal utilized in pulmonary studies of naturally occurring respiratory diseases. Neonatal lung DCs exhibited characteristic dendrites and morphology when observed by transmission electron microscopy and expressed low to moderate DEC-205, CD80/86, MHC class II and CD 14. Regardless of age, lung DCs were functionally able to endocytose FITC conjugated ovalbumin but to a lesser degree than monocyte-derived DCs. In addition, neonatal lung DCs were demonstrated to be potent stimulators of allogeneic T cell proliferation. Together, these results demonstrate that neonatal and adult lung DCs are functionally similar. It is apparent from the data presented that neonatal pulmonary DCs do not exhibit an intrinsic functional defect that would impair their ability to take up antigen and stimulate naïve T cells. These data support growing evidence that neonatal immune responses may differ from adults due to different microenvironmental influences rather than differences in dendritic cell maturation states.

Reduced clearance of respiratory syncytial virus infection in a preterm lamb model.

Respiratory syncytial virus (RSV) causes significant respiratory disease in children worldwide. For the study of severe RSV disease seen in preterm infants, a suitable animal model is lacking. The novel hypothesis of this study was that preterm lambs are susceptible to bovine RSV (bRSV) infection, an analogous pneumovirus with ruminant host specificity, and that there would be age-dependent differences in select RSV disease parameters. During RSV infection, preterm lambs had elevated temperatures and respiration rates with mild anorexia and cough compared to controls. Gross lesions included multifocal consolidation and atelectasis with foci of hyperinflation. Microscopic lesions included multifocal alveolar septal thickening and bronchiolitis. Immunohistochemistry localized the RSV antigen to all layers of bronchiolar epithelium from a few basal cells to numerous sloughing epithelia. A few mononuclear cells were also immunoreactive. To assess for age-dependent differences in RSV infection, neonatal lambs were infected similarly to the preterm lambs or with a high-titer viral inoculum. Using morphometry at day 7 of infection, preterm lambs had significantly more cellular immunoreactivity for RSV antigen (P <0.05) and syncytial cell formation (P <0.05) than either group of neonatal lambs. This work suggests that perinatal RSV clearance is age-dependent, which may explain the severity of RSV infection in preterm infants. The preterm lamb model is useful for assessing age-dependent mechanisms of severe RSV infection.