Single-cell Signatures Reveal Immune Dysregulation in Nontuberculous Mycobacterial Pulmonary Disease

Background Nontuberculous mycobacterial (NTM) pulmonary disease is characterized by chronic infection and dysregulated immunity. However, cell type-specific transcriptional changes driving disease progression remain poorly defined. Methods We performed single-cell RNA sequencing (scRNA-seq) on bronchoalveolar lavage fluid (BALF) samples from individuals with NTM pulmonary disease and integrated these data with external control BALF single-cell datasets. Cell clustering, reference-based annotation, differential expression analysis, and KEGG pathway enrichment were performed, and selected transcriptional signatures were validated using in vitro NTM-infected cellular models. Results Distinct transcriptional profiles were identified in alveolar epithelial and innate immune cells. Alveolar type II cells showed increased expression of KRT7, MUC1, LAMP3, and ABCA3, with reduced ATP1A1 and ATP1B1. Monocytes exhibited higher CCR2, SELL, and TLR2, but lower LYZ and CX3CR1. Macrophages displayed decreased CD74 and SIGLEC1, among other immune-regulatory genes. These alterations were reproduced in in vitro NTM-infected alveolar type II cells, monocytes, and macrophages, confirming disease-relevant expression signatures. Conclusions Our integrated single-cell and in vitro transcriptomic analyses reveal consistent dysregulation of immune and epithelial cells in NTM pulmonary disease. These findings suggest impaired antigen presentation and reduced immune activation, particularly in macrophage and monocyte populations. The concordance between in vivo and in vitro data highlights the robustness of these transcriptional signatures and identifies potential targets for immunomodulatory therapy in NTM infection.