Whole Blood Transcriptomics Characterize Molecular Phenotypes of Pulmonary Mycobacterium Avium Complex Disease

Abstract Background: Pulmonary mycobacterium avium complex (MAC) disease shows a range of clinical phenotypes and responses to treatment. While Th1 immunity and neutrophils have been implicated in its pathogenesis, the precise mechanisms remain unclear. Furthermore, comprehensive gene expression analysis in this context are scarce. This study aimed to elucidate the heterogeneity of pulmonary MAC disease by gene expression profiling of whole blood cells. Methods: We enrolled 100 patients with pulmonary MAC disease requiring treatment according to standard guidelines. Whole blood cells were collected for RNA-sequencing before (0M) and one month after the start of treatment (1M). Unsupervised cluster analysis was performed on gene expression data at 0M. Differentially expressed gene (DEG) analysis was performed between each cluster, adjusting for blood cell composition. Results: The 100 patients were stratified into three clusters, Cluster 1 (C1; 58 patients) with mild disease, Cluster 2 (C2; 34 patients) with moderate disease, and Cluster 3 (C3; 8 patients) with severe disease. C3 had higher peripheral blood neutrophil counts compared to C1 and C2. The numbers of DEGs identified was C3 vs C1; 1357 genes, C2 vs C1; 125 genes, and C3 vs C2; 408 genes (|fold change| >1.5, false discovery rate <0.05). Ingenuity Pathway Analysis (IPA) revealed that “Neutrophil degranulation” pathway (96 genes) and “Interferon alpha/beta signaling” pathway (30 genes) were significantly enriched and activated in C3 vs. C1. We further evaluated these 126 genes, focusing on neutrophil-related genes. By cross-referencing with a previous single-cell RNA sequencing study on neutrophils (PMID: 32719519), 24 common genes were identified. Among these, genes related to canonical mature activated neutrophils (G5a; S100A8, S100A9, TIMP2) showed higher expression in C3 compared to C1 and C2. In addition, genes associated with mature interferon dominant activated neutrophils (G5b; IFIT1, ISG15, RSAD2) were expressed in C1, C2 and C3. Previously reported gene expression data for pulmonary nontuberculous mycobacteria disease (GSE97298) supported a trend towards higher expression of these 24 genes in the mortality group. Conclusion: Gene expression analysis of whole blood cells identified three distinct clusters in patients with pulmonary MAC disease. Cluster 3, comprising severe cases, exhibited higher expression of genes related to activated neutrophils compared to the other groups.