Characterizing the Macrophage Population in Patients With Idiopathic Subglottic Stenosis

Abstract

ObjectivesIdiopathic subglottic stenosis (iSGS) is characterized by progressive fibrosis and subglottic luminal narrowing. Currently, immune characterization has focused on T‐cells; however, macrophages remain largely unexplored. The goals of this study are to characterize the transcriptome of iSGS macrophages and the fibrogenic nature of identifed biomarkers.Study DesignBioinformatics and in vitro.MethodsHuman tracheal biopsies from iSGS scar (n = 4), and matched non‐scar (n = 4) regions were analyzed using single‐cell RNA‐seq (scRNA‐seq). Immunofluorescence (IF) was performed on rapidly processed autopsies (RPA) and iSGS tracheal resections (n = 4) to co‐localize S100A8/9 and CD11b. Collagen gene/protein expression was assessed in iSGS fibroblasts (n = 4) treated with protein S100A8/9 (1000 ng/ml). Macrophages were subclustered to identify distinct subpopulations.ResultsscRNA‐seq analysis revealed S100A8/S100A9 (fold change (FC) = 4.1/1.88, p < 0.001) as top differentially expressed genes in iSGS macrophages. IF exhibited increased CD11b+/S100A8/9+ cells in tracheal samples of iSGS versus RPA (26.75% ± 7.08 vs. 0.594% ± 0.974, n = 4, p = 0.029). iSGS fibroblasts treated with S100A8/9 demonstrated increased gene expression of COL1A1 (FC = 2.30 ± 0.45, p = 0.03, n = 4) and COL3A1 (FC = 2.44 ± 0.40, p = 0.03, n = 4). COL1A1 protein assays revealed an increase in the experimental group, albeit not significant, (p = 0.12, n = 4). Finally, macrophage sub clustering revealed one subpopulation as a predominant source of S100A8/S100A9 expression (FC = 7.94/5.47, p < 0.001).ConclusionsS100A8/9 is a key biomarker in iSGS macrophages. Although S100A8/9 demonstrates profibrotic nature in vitro, the role of S100A8/9+ macrophages in vivo warrants further investigation.Level of EvidenceNA Laryngoscope, 133:2308–2316, 2023

Murine Model of Airway Fibrosis has Anatomic, Physiologic, and Molecular Congruency to Human iSGS

“…The role of IL‐17A implicates the involvement of myeloid cells, such as monocytes, in the iSGS inflammatory/immune response. Ospino et al demonstrate an increase in cells that are CD11b+ (macrophages) that also express the proinflammatory protein S100A8/A9 in human iSGS tracheal samples when compared to healthy controls 18 . Similarly a study using the murine SGS model found increased levels of dysregulated macrophages, specifically M2 macrophages, in the murine stenotic tracheal samples.…”

“…that are CD11b+ (macrophages) that also express the proinflammatory protein S100A8/A9 in human iSGS tracheal samples when compared to healthy controls. 18 Similarly a study using the murine SGS model found increased levels of dysregulated macrophages, specifically M2 macrophages, in the murine stenotic tracheal samples. This was demonstrated by increased gene expression of the M2 marker Arg1 in SGS mice compared to normal controls.…”

Transcriptional profiling sheds light on the fibrotic aspects of idiopathic subglottic tracheal stenosis

“…The application of newly developed methods such as single cell RNA sequencing (scRNAseq) provides important insights into the prevailing cellular and transcriptional situation of multiple pathologies (12). Using this method, research groups have decoded the pathologic airway epithelium of ISGS and identified S100A8/A9 as a key biomarker of ISGS macrophages (12)(13)(14). Fibrotic diseases are typically driven by multiple different cell types, with fibroblasts assuming a crucial role as the primary producers of the ECM in most cases (15).…”