CC16 drives VLA-2-dependent SPLUNC1 expression

Rationale: CC16 (Club Cell Secretory Protein) is a protein produced by club cells and other non-ciliated epithelial cells within the lungs. CC16 has been shown to protect against the development of obstructive lung diseases and attenuate pulmonary pathogen burden. Despite recent advances in understanding CC16 effects in circulation, the biological mechanisms of CC16 in pulmonary epithelial responses have not been elucidated. Objectives: We sought to determine if CC16 deficiency impairs epithelial-driven host responses and identify novel receptors expressed within the pulmonary epithelium through which CC16 imparts activity. Methods: We utilized mass spectrometry and quantitative proteomics to investigate how CC16 deficiency impacts apically secreted pulmonary epithelial proteins. Mouse tracheal epithelial cells (MTECS), human nasal epithelial cells (HNECs) and mice were studied in naïve conditions and after Mp challenge. Measurements and main results: We identified 8 antimicrobial proteins significantly decreased by CC16^-/- MTECS, 6 of which were validated by mRNA expression in Severe Asthma Research Program (SARP) cohorts. Short Palate Lung and Nasal Epithelial Clone 1 (SPLUNC1) was the most differentially expressed protein (66-fold) and was the focus of this study. Using a combination of MTECs and HNECs, we found that CC16 enhances pulmonary epithelial-driven SPLUNC1 expression via signaling through the receptor complex Very Late Antigen-2 (VLA-2) and that rCC16 given to mice enhances pulmonary SPLUNC1 production and decreases Mycoplasma pneumoniae (Mp) burden. Likewise, rSPLUNC1 results in decreased Mp burden in mice lacking CC16 mice. The VLA-2 integrin binding site within rCC16 is necessary for induction of SPLUNC1 and the reduction in Mp burden. Conclusion: Our findings demonstrate a novel role for CC16 in epithelial-driven host defense by up-regulating antimicrobials and define a novel epithelial receptor for CC16, VLA-2, through which signaling is necessary for enhanced SPLUNC1 production.