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Tissue Eosinophilia is Effective for Studying Differential Expression of Genes by RNA sequencing, and may offer Superior Insights into Chronic Rhinosinusitis than Study by Polyp Status

ABSTRACT: RNA sequencing (RNAseq) is being used to study inflammatory pathways in chronic rhinosinusitis (CRS). Our goal was to probe validity of tissue eosinophilia as a metric to study RNAseq in CRS. The study was conducted on prospectively enrolled subjects undergoing sinonasal surgery. Subjects were categorized as control, CRS, CRS with nasal polyps (CRSwNP) and CRS without nasal polyps (CRSsNP). CRS was also categorized by tissue eosinophil levels per high power field (EOS/HPF) as <10 EOS/HPF or ≥10 EOS/HPF. Ethmoidal tissue samples were processed, differentially expressed (DE) genes were calculated and Ingenuity pathway analysis (IPA) performed.Controls separated clearly from CRS by both study criteria (polyp status, EOS/HPF). In both analyses, CRS differentiated into two distinct CRS subgroups. However, heatmaps showed greater homogeneity within each CRS subtype when studied by eosinophilia versus polyp status. Overall, high differential gene expression was found in CRS versus controls, with 736 statistically significant differentially expressed (DE) genes. On comparison between between CRSwNP and CRSsNP, 60 DE genes were found and on analyzing by tissue EOS/HPF, 110DE genes were statistically significant between CRS <10 EOS/HPF and CRS ≥10 EOS/HPF analyses. Tissue eosinophilia as a metric was further validated by finding of IL 17 signalling pathway between <10EOS/HPF (increased) versus ≥10 EOS/HPF samples on pathway analysis. As a metric, tissue eosinophilia is at least as effective as analysis by polyp status for RNA sequencing and may potentially offer superior insights into mechanistic pathways.

ORGANISM(S): Homo sapiens

PROVIDER: GSE198950 | GEO | 2022/03/21

REPOSITORIES: GEO

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Project description:Background: Chronic rhinosinusitis with nasal polyposis (CRSwNP) in western countries is characterized by eosinophilia, IgE production and Th2 cytokine expression. Type 2 innate lymphoid cells (ILC2) from polyps produce IL-5 and IL-13 in response to IL-25 and IL-33 although the relevance of this axis to local mucosal T cell responses is unknown. Objective: To investigate the role of the IL-25/IL-33 axis in local mucosal T cell responses in CRSwNP. Methods: Polyp tissue and blood were obtained from patients undergoing nasal polypectomy. Control nasal biopsies and blood were obtained from healthy volunteers. Tissue was cultured in a short-term explant model. T cell surface phenotype/intracellular cytokines were assessed by flow cytometry. TCR VÎ² analysis was performed with the immunoSEQ assay. Microarrays were performed for gene expression analysis. Results: Using nasal polyp tissue, numerous IL-25 receptor (IL-17RB) positive polarized Th2 cells were identified which were absent in the healthy nasal mucosa and periphery. IL-17RB+CD4+ polyp Th2 cells co-expressed ST2 (IL-33 receptor) and responded to IL-25 and IL-33 with enhanced IL-5 and IL-13 production. Within IL-17RB+CD4+ T cells several identical TCR VÎ² CDR3 sequences were identified in different subjects suggesting clonal expansion driven by a common antigen. Abundant IL-17 producing T cells were observed in healthy nasal mucosal and polyp populations with Th17-related genes the most overexpressed compared to peripheral blood T cells. Conclusion: IL-25 and IL-33 may interact locally with IL-17RB+ST2+ polyp T cells to augment Th2 responses in CRSwNP. A local Th17 response may be the default signature in healthy nasal mucosal immune homeostasis. Three biological replicates. T-helper cells were isolated nasal polyps by explant culture from patients with chronic rhinosinusitis. Cells were then sorted based upon expression of IL17RB by flow cytometric sorting. Resting and activated IL-17RB+ve cells were compared with resting and activated IL-17RB-ve cells.