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Bioinformatics analysis and verification of key candidate genes influencing the pathogenesis of chronic rhinosinusitis with nasal polyps

ABSTRACT: Objectives: Chronic rhinosinusitis (CRS) with nasal polyps (CRSwNP) is a prominent public health issue. Furthermore, the prognosis of eosinophilic CRSwNP is poor, with a high recurrence rate. The underlying molecular mechanisms of eosinophilic CRSwNP remain unclear. Therefore, in this study, we sought to determine the crucial genes underlying eosinophil infiltration in eosinophilic CRSwNP pathogenesis. Methods: We used the Gene Expression Omnibus database (GEO) (GSE36830 and GSE23552 datasets) to mine gene expression profiles of CRSwNP patients and normal subjects. Differentially expressed genes (DEGs) between normal and CRSwNP tissues were identified and subjected to the Kyoto Encyclopedia of Genes and Genomes (KEGG) and Gene Ontology (GO) enrichment analyses. Co-expression networks were established using a weighted gene co-expression network analysis (WGCNA) and single-sample gene set enrichment analysis (GSEA). Protein-protein interaction networks were developed to detect functional protein modules. Based on the common DEGs, candidate miRNAs and related lncRNAs were predicted using the mirTarBase and StarBase databases. Finally, we generated immune cell subtypes of CRSwNP. Results: A total of 146 DEGs were identified. Of these, 131 genes were upregulated, whereas 15 were downregulated. GO analysis indicated that DEGs primarily participated in leukocyte chemotaxis and migration as well as cell chemotaxis. KEGG pathway analysis suggested that DEGs participated in the interactions between cytokines and viral proteins, osteoclast differentiation, and cytokine-cytokine receptor interactions. Real-time quantitative polymerase chain reaction analysis showed that Complement C5a Receptor 1 (C5AR1), C-C Motif Chemokine Receptor 3 (CCR3), Complement C3a Receptor 1 (C3AR1), and C-C Motif Chemokine Ligand 13 (CCL13) expression levels were significantly upregulated in nasal polyps, whereas C-C Motif Chemokine Ligand 4 (CCL4) expression levels were significantly downregulated. Conclusions: The candidate genes identified in this study may influence the activation and accumulation of eosinophils, cell chemotaxis, and inflammatory responses, thereby potentially representing molecular targets for future studies of CRSwNP.

SUBMITTER: Chen G

PROVIDER: S-EPMC10006810 | biostudies-literature | 2023 Jan

REPOSITORIES: biostudies-literature

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Project description:BACKGROUND:Chronic rhinosinusitis with nasal polyps (CRSwNP) is characterized by an alteration in airway epithelial cell functions including barrier function, wound repair mechanisms, mucociliary clearance. The mechanisms leading to epithelial cell dysfunction in nasal polyps (NPs) remain poorly understood. Our hypothesis was that among the inflammatory cytokines involved in NPs, IL-6 could alter epithelial repair mechanisms and mucociliary clearance. The aim of this study was to evaluate the in vitro effects of IL-6 on epithelial repair mechanisms in a wound repair model and on ciliary beating in primary cultures of Human Nasal Epithelial Cells (HNEC). METHODS:Primary cultures of HNEC taken from 38 patients during surgical procedures for CRSwNP were used in an in vitro model of wound healing. Effects of increasing concentrations of IL-6 (1 ng/mL, 10 ng/mL, and 100 ng/mL) and other ILs (IL-5, IL-9, IL-10) on wound closure kinetics were compared to cultures without IL-modulation. After wound closure, the differentiation process was characterized under basal conditions and after IL supplementation using cytokeratin-14, MUC5AC, and ?IV tubulin as immunomarkers of basal, mucus, and ciliated cells, respectively. The ciliated edges of primary cultures were analyzed on IL-6 modulation by digital high-speed video-microscopy to measure: ciliary beating frequency (CBF), ciliary length, relative ciliary density, metachronal wavelength and the ciliary beating efficiency index. RESULTS:Our results showed that: (i) IL-6 accelerated airway wound repair in vitro, with a dose-response effect whereas no effect was observed after other ILs-stimulation. After 24 h, 79% of wounded wells with IL6-100 were fully repaired, vs 46% in the IL6-10 group, 28% in the IL6-1 group and 15% in the control group; (ii) specific migration analyses of closed wound at late repair stage (Day 12) showed IL-6 had the highest migration compared with other ILs (iii) The study of the IL-6 effect on ciliary function showed that CBF and metachronal wave increased but without significant modifications of ciliary density, length of cilia and efficiency index. CONCLUSION:The up-regulated epithelial cell proliferation observed in polyps could be induced by IL-6 in the case of prior epithelial damage. IL-6 could be a major cytokine in NP physiopathology.

Project description:BackgroundMolecular signatures of chronic rhinosinusitis with nasal polyps (CRSwNP) related to macrophages remain unclear. This study aimed to develop a macrophage-associated diagnostic signature for CRSwNP.MethodsTranscriptome data from 54 patients with CRSwNP and 37 healthy controls across GSE136825, GSE36830, and GSE72713 were used to identify differentially expressed genes (DEGs) between two groups. Gene Set Enrichment Analysis and Weighted Gene Co-Expression Network Analysis pinpointed crucial pathways and gene clusters. A diagnostic model was created from these analyses and receiver operating characteristic curve (ROC), and further validated in our transcriptome data from 29 samples. Immune cell infiltration analysis was performed and linked those diagnostic genes to macrophages and verified by single-cell RNA sequencing data. Immunofluorescence co-staining of CD163 and HMOX1 was performed in nasal tissues. Mouse bone marrow-derived macrophage (BMDMs) cultures were used in functional experiments. Correlations between the expression of HMOX1 and eotaxin genes were investigated.ResultsDEGs of CRSwNP versus control group were enriched in the INTERLEUKIN_4_AND_13_SIGNALING pathways. A four-gene diagnostic model (HMOX1, ALOX5, F13A1 and ITGB2) was developed and demonstrated high diagnostic precision with an area under ROC curve of 0.980 for training dataset and 0.895 for test dataset. M2 macrophage presence and HMOX1 expression significantly correlated with CRSwNP (p < 0.001). Single-cell RNA sequencing data underscored the altered cellular composition in CRSwNP, with HMOX1 notably expressed in M2 macrophages. Immunofluorescence staining highlighted the increased infiltration of CD163+ M2 macrophages in nasal mucosa samples of eosinophilic CRSwNP, which correlated with HMOX1 protein levels (p < 0.05). The HMOX1 inhibitor zinc protoporphyrin reduced the ratio of CD163 + HMOX1 + M2 macrophages in mouse BMDM cultures (p < 0.05). HMOX1 expression showed a strong positive correlation with the expression of eotaxin genes (CCL11, CCL24, and CCL26; p < 0.05 respectively).ConclusionM2 macrophage-derived HMOX1 can be used as an innovative diagnostic signature for CRSwNP, which might be a potential regulator of eosinophilic inflammation.

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Bioinformatics analysis and verification of key candidate genes influencing the pathogenesis of chronic rhinosinusitis with nasal polyps

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