D-079. sPLUNC-1 Is a Sialylated Peptide Secreted Abundantly by Nasopharyngeal Epithelial Cells that Kills Nontypeable Haemophilus influenzae
We have recently demonstrated that the short palate, lung, and nasal epithelium clone protein (sPLUNC-1) kills nontypeable H. influenzae, Moraxella catarrhalis, and Streptococcus pneumoniae, and that this effector of the innate immune system is produced in the middle ear of children. These data suggested that sPLUNC-1 likely plays an important role in protecting the uppermost airway from bacteria that cause otitis media (OM). However, the lack of an established cell culture system, utilizing cells that originate from a respiratory tract tissue important to the OM disease course and that produces native sPLUNC-1, hampered progress in our understanding of the dynamic interactions between sPLUNC-1 and causative agents of OM. The chinchilla is the preferred rodent host used to model human OM, and we have demonstrated that sPLUNC-1 is expressed by cultures of differentiated, pseudostratified, ciliated and mucus-secreting chinchilla nasopharyngeal epithelial cells (CNPEs). We determined that sPLUNC-1 comprised approximately 5% of the total protein secreted from the apical surface of CNPEs, demonstrating that sPLUNC-1 was a predominant constituent of the CNPE secretome. We further showed that sPLUNC-1 produced by polarized CNPEs was post-translationally modified in a manner similar to the cognate peptide found in vivo, as both contained sialic acid linked to galactose as the terminal sugar on the polypeptide. We determined that goblet cells were the primary source of secreted sPLUNC-1 in CNPE cultures, as antibodies directed against this protein labeled these cells. We are currently determining optimal conditions for generating a silencing RNA-mediated knockdown in sPLUNC-1 expression in CNPE cultures. With the developments described herein, we can now begin to: unravel the mechanisms underlying regulation of sPLUNC-1 gene expression, determine the impact of sialylation on the antimicrobial activity of sPLUNC-1, and decipher the role of sPLUNC-1 in influencing bacterial colonization of the mucosal surface of the uppermost airway. This work was supported by K99 DC008966 and R01 DC005847 from the NIDCD/NIH and N01 AI30040 from the NIAID.