Characterization of the Immune Microenvironment and Identification of Biomarkers in Chronic Rhinosinusitis with Nasal Polyps Using Single-Cell RNA Sequencing and Transcriptome Analysis

Abstract

Purpose

Chronic rhinosinusitis is a prevalent condition in the field of otorhinolaryngology; however, its pathogenesis remains to be elucidated. The immunological defense of the nasal mucosa is significantly influenced by dendritic cells (DCs). We identified specific biological indicators linked to DCs and explored their significance in cases of chronic rhinosinusitis with nasal polyps (CRSwNP).

Patients and Methods

We categorized cells using single-cell RNA (scRNA) sequencing, and combined transcriptome sequencing was used to identify potential candidate genes for CRSwNP. We selected three biomarkers based on two algorithms and performed enrichment and immune correlation analyses. Biomarkers were verified using training and validation sets, receiver operating characteristic curves, immunohistochemistry, and quantitative real-time reverse-transcription PCR (qRT-PCR). Variations in biomarker expression were validated using pseudotime analysis. The networks of competing transcription factor (TF)-mRNA and competing endogenous RNA (ceRNA) were established, and the protein drugs associated with these biomarkers were predicted.

Results

Both scRNA-seq and transcriptome data showed that DCs immune infiltration was higher in the CRSwNP group than in the control group. Three DC-related biomarkers (NR4A1, CLEC4G, and CD163) were identified. In CRSwNP, NR4A1 expression decreased, whereas CLEC4G and CD163 expression increased. All biomarkers were shown to be involved in immunological and metabolic pathways by enrichment analysis. These biomarkers were associated with γδ T cells, effector memory CD4 + T cells, regulatory T cells, and immature DCs. According to pseudotime analysis, NR4A1 and CD163 expression decreased from high to low, whereas CLEC4G expression remained low.

Conclusion

We screened and identified potential DC-associated biomarkers of CRSwNP progression by integrating scRNA-seq with whole transcriptome sequencing. We analyzed the biological pathways in which they were involved, explored their molecular regulatory mechanisms and related drugs, and constructed ceRNA, TF-mRNA, and biomarker–drug networks to identify new CRSwNP treatment targets, laying the groundwork for the clinical management of CRSwNP.

Keywords:

• CRSwNP
• dendritic cell
• single-cell sequencing
• transcriptomic sequencing
• biomarker

Abbreviations

AUC, area under the curve; CRS, chronic rhinosinusitis; CRSwNP, chronic rhinosinusitis accompanied by nasal polyps; NP group, nasal polyps; NM group, nasal mucosa tissue samples adjacent to nasal polyps from the CRSwNP group; CM group, nasal mucosa tissue samples from the control group; DCs, Dendritic cells; DCRGs, DC-related genes; ceRNA, endogenous RNA; GEO, Gene Expression Omnibus; GO, Gene Ontology; KEGG, Kyoto Encyclopedia of Genes and Genomes; PCA, principal component analysis; qRT-PCR, quantitative real-time reverse transcription-polymerase chain reaction; scRNA, single cell; SVM–RFE RNA, Support vector machine-recursive feature elimination; TF-mRNA, transcription factor; UMAP, Uniform manifold approximation and projection.

Ethics Approval and Informed Consent

The Beijing Chaoyang Hospital’s Ethics Committee’s Institutional Review Board (No. 2020-3-16-18) gave its approval to this work. All experiments involving human participants adhered to the institutional ethical guidelines and followed the principles of the Declaration of Helsinki. Before participating in the study, all participants involved provided written informed permission.

Data Sharing Statement

The unique inputs included in the study are outlined in the article/supplementary material. Further questions or concerns may be directed to the corresponding author.

Acknowledgments

We appreciate Dr. Lu’s insightful advice and constructive comments regarding our study.

Disclosure

The authors report no conflicts of interest in this work.

Additional information

Funding

This research received funding support from the National Natural Science Foundation of China (82071068).