ScRNA-seq Expression of APOC2 and IFI27 Identifies Four Families of Alveolar Macrophage Superclusters in Cystic Fibrosis and Healthy BALF

Rationale: Alveolar macrophages (AMs) reside on the luminal surface of the airways and alveoli, ensuring proper gas exchange by ingesting cellular debris and pathogens, and regulating inflammatory responses. Recent studies have highlighted the heterogeneity of airspace macrophages from healthy bronchoalveolar lavage fluid and cystic fibrosis sputum. Understanding the heterogeneity and diverse roles played by AMs and recruited monocytes is critical for treating CF and other airway diseases. Objectives: To identify and compare the cellular composition and functional diversity between CF and healthy airspace macrophages and monocytes. Methods: We performed single-cell RNA sequencing (scRNA-seq) on 113,213 BAL cells from four healthy and three mild CF subjects. Transcriptional data and TotalSeq technology were used to confirm cell surface markers that distinguish resident AMs from recruited monocytes. Measurements and Main Results: Unbiased clustering identified four AM superclusters based on the expression of APOC2 and IFI27 genes. Each supercluster contains four shared subclusters named after their differentially expressed gene(s): IGF1.AMs, CCL18.AMs, CXCL5.AMs, and Cholesterol.AMs. AM Supercluster 4 contained two additional subclusters. Beyond the superclusters, several additional AM clusters were identified: Chemokine, Metallothionein, Interferon, and Cycling AMs. Interestingly, the Chemokine cluster further divides into four subclusters, each selectively expressing a unique combination of chemokines. Lastly, minimal differences were observed between mild CF and healthy individuals in airspace cellular composition. Conclusions: ScRNA-seq identified four AM superclusters. Several fundamental questions require further investigation, including these clusters locations, functions, transcription factors that regulate their distinct programming, and whether similar macrophage superclusters can be found in other organs. ### Competing Interest Statement The authors have declared no competing interest.