Abstract Repeated infections with Streptococcus pneumoniae serotype 19F (Sp19F) alters the surface protein expression and transcriptome of various immune and structural cells including alveolar macrophages and pulmonary epithelial cells, imparting heterotypic immunity against mismatched serotypes of S. pneumoniae. We hypothesized that pulmonary endothelial cells may also undergo remodeling to enhance recruitment of immune cells into the infected experienced lung. To begin testing this hypothesis, C57BL6/J mice were infected with left lobe intratracheal instillations of Sp19F (“experienced”) or saline (“naïve”) at days 0 and 7. At day 35, pulmonary endothelial cells were collected and magnetically enriched based on surface CD31 protein. Flow cytometry with a panel focused on cell adhesion molecules involved in immune cell recruitment was then performed both at baseline and 12 hours post infection with mismatched S. pneumoniae serotype 3 (Sp3). Surface marker expression by total cell counts and median fluorescence intensity was then compared by both manual gating and using opt-SNE, an unbiased toolkit for t-SNE parameter selection. Opt-SNE analyses revealed that endothelial cell clusters were broadly similar, with overlapping populations in the endothelial cells from naïve and experienced mice. Widespread expression of cell adhesion proteins ICAM-1 and ICAM-2 was observed both at baseline and 12 hours post Sp3 infection, comparable in the naïve and experienced groups. However, some small cell populations appeared mildly enriched in naïve or experienced groups. CD34high populations appeared slightly enriched in experienced mice both at baseline and during infection, whereas some VCAM-1high populations appeared slightly enriched in experienced mice only at the infection timepoint. These insights were confirmed in the manual gating. These results suggest that pulmonary endothelial cell adhesion protein expression remains mostly unchanged by a history of pneumococcal pneumonia, contrasting with the extensive surface marker remodeling observed in lung macrophages from experienced mice. Future studies may work to identify other types of surface proteins remodeled on endothelial cells following pneumococcal pneumonia or investigate other measures of cell phenotype potentially altered in experienced lung endothelial cells. This abstract is funded by: NIH, Boston University
Klimek et al. (Fri,) studied this question.