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.

Single-Cell Profiling of Premature Neonate Airways Reveals a Continuum of Myeloid Differentiation.

Single-cell genomic technologies hold great potential to advance our understanding of lung development and disease. A major limitation lies in accessing intact cells from primary lung tissues for profiling human airway health. Sampling methods such as endotracheal aspiration that are compatible with clinical interventions could enable longitudinal studies, the enrollment of large cohorts, and the development of novel diagnostics. To explore single-cell RNA sequencing profiling of the cell types present at birth in the airway lumen of extremely premature neonates (<28 wk gestation), we isolated cells from endotracheal aspirates collected from intubated neonates within the first hour after birth. We generated data on 10 subjects, providing a rich view of airway luminal biology at a critical developmental period. Our results show that cells present in the airways of premature neonates primarily represent a continuum of myeloid differentiation, including fetal monocytes (25% of total), intermediate myeloid populations (48%), and macrophages (2.6%). Applying trajectory analysis to the myeloid populations, we identified two trajectories consistent with the developmental stages of interstitial and alveolar macrophages, as well as a third trajectory presenting an alternative pathway bridging the distinct macrophage precursors. The three trajectories share many dynamic genes (N = 5,451), but also have distinct transcriptional changes (259 alveolar-specific, 666 interstitial-specific, and 285 bridging-specific). Overall, our results define cells isolated within the so-called "golden hour of birth" in extremely premature neonate airways, representing complex lung biology, and can be used in studies of human development and disease.