Single-cell and spatial transcriptomics reveal the pathogenesis of chronic granulomatous disease under natural conditions [SPF or CL]

Chronic granulomatous disease (CGD) is a rare and hereditary immunodeficiency disorder attributed to a malfunction in the NADPH oxidase 2 (NOX2) of phagocytes. Owing to the deficiency of NOX2-derived reactive oxygen species, individuals with CGD exhibit increased susceptibility to pathogens, including bacteria and fungi. This defect also contributes to an excessive inflammatory response, leading to tissue damage. In this study, we established a CGD infection model with Ncf2-/- genetic background through controlled environmental exposure. Within Specific Pathogen-Free (SPF) facilities, Ncf2-/- mice showed no signs of infection. However, under CL grade conditions (CL), these mice spontaneously developed fibro-encapsulated necrotizing lung granulomas. Through single-cell RNA sequencing (scRNA-seq) and flow cytometry, we detected a significant expansion of neutrophils and monocyte-derived macrophages (MDMs) in the lung tissues of CGD mice, identifying a NOS2high neutrophil subset characterized by a proinflammatory transcriptional signature and a specific MDMs subset marked by NOS2 and ARG1. In the spatial transcriptomics data, the NOS2high neutrophils were located in the core area of granulomas, promoting the inflammatory response by highly expressing Mif, Il1b, and Il1a, while the specific macrophage subset was situated at the periphery of granuloma lesions, facilitating extracellular matrix remodeling by expressing Fn1, Mmp9, Mmp12, and Mmp14. Pharmacological inhibition of MIF can significantly diminish neutrophil infiltration and granuloma formation via MIF/NLRP3/IL1ß axis. Furthermore, loss of the myeloid-specific pro-survival gene Morrbid resulted in the apoptosis of myeloid cells in CGD and loss of Il1r1 also mitigated diseases, leading to reduction of systemic inflammation in the two compound mutants. In summary, through controlling the environmental factors, we constructed a natural model of CGD infection, elucidated granuloma formation by single-cell and spatial transcriptomics, and developed three interventions. Overall design: We compared changes in lung tissue in WT and Ncf2-/- mice under different environmental conditions (SPF or CL). We performed Single-cell RNA sequencing (SPF WT, SPF NCF2-/-, CL WT, CL NCF2-/-) and spatial transcriptome sequencing (CL WT, CL NCF2-/-) on mouse lung tissue. In addition, we extracted mouse bone marrow cells for Single-cell RNA sequencing (CL WT, CL NCF2-/-) and sorted bone marrow lin cells for bulk RNA sequencing.