Distinct type 2-high inflammation associated molecular signatures of chronic rhinosinusitis with nasal polyps with comorbid asthma

Abstract

Background: Patients with chronic rhinosinusitis with nasal polyps (CRSwNP) and comorbid asthma have more severe disease and are difficult to treat. However, the molecular endotypes associated with CRSwNP with comorbid asthma (CRSwNP + AS) are not clear. This study aimed to investigate the characteristics of type 2 inflammation and the molecular signatures associated with CRSwNP + AS.

Methods: A total of 195 subjects; including 65 CRSwNP + AS patients, 99 CRSwNP-alone patients, and 31 healthy control subjects; were enrolled in the study. Nasal tissues from patients with CRSwNP + AS, CRSwNP-alone and control subjects were assessed for infiltration of inflammatory cells and concentrations of total IgE. Whole-transcriptome sequencing was performed and differentially expressed (DE) mRNAs and long non-coding RNAs (lncRNAs) and their associated pathways were analyzed. The correlations between type 2 cytokines and local eosinophils, tissue IgE, and transcriptome signatures were evaluated.

Results: Significantly higher local eosinophil infiltration and higher levels of total IgE were found in nasal tissues from CRSwNP + AS patients than in nasal tissues from CRSwNP-alone patients. Furthermore, atopy and recurrence were significantly more frequent in patients with CRSwNP + AS than in patients with CRSwNP-alone (62.5% vs 28.6% and 66.7% vs 26.9%, respectively). RNA sequencing analysis identified 1988 common DE-mRNAs, and 176 common DE-lncRNAs shared by CRSwNP + AS versus control and CRSwNP-alone versus control. Weighted gene coexpression network analysis (WGCNA) identified LINC01146 as hub lncRNA dysregulated in both subtypes of CRSwNP. Overall, 968 DE-mRNAs and 312 DE-lncRNAs were identified between CRSwNP + AS and CRSwNP-alone. Both pathway enrichment analysis and WGCNA indicated that the phenotypic traits of CRSwNP + AS were mainly associated with higher activities of arachidonic acid metabolism, type 2 cytokines related pathway and fibrinolysis pathway, and lower activity of IL-17 signalling pathway. Furthermore, the expression of type 2 cytokines; IL5 and IL13, was positively correlated with local eosinophil infiltration, tissue IgE level, and the expression of DE-mRNAs that related to arachidonic acid metabolism. Moreover, WGCNA identified HK3-006 as hub lncRNA in yellow module that most positively correlated with phenotypic traits of CRSwNP + AS.

Conclusions: Patients with CRSwNP + AS have distinct type 2-high inflammation-associated molecular signatures in nasal tissues compared to patients with CRSwNP-alone.

Keywords: Asthma; Chronic rhinosinusitis with nasal polyps; Molecular endotype; Transcriptome sequencing; Type 2 inflammation.

Fig. 1

Detection of eosinophils and total IgE in blood and nasal tissues from patients with CRSwNP. a, b Blood eosinophils and total IgE was detected in CRSwNP patients with asthma (CRSwNP + AS), CRSwNP-alone and control (Ctrl) subjects. c, d Representative images of haematoxylin and eosin-stained nasal polyp tissues from patients with CRSwNP-alone and CRSwNP + AS. Red arrows point out eosinophils. e The percentage of infiltrating eosinophils, neutrophils, plasma cells and lymphocytes were assessed in nasal tissues of CRSwNP-alone (n = 24) and CRSwNP + AS (n = 25). f Concentrations of total IgE in nasal tissues of Ctrl (n = 10), CRSwNP-alone (n = 14) and CRSwNP + AS (n = 17) were assayed using Human IgE ELISA Kit. Data are presented as medians and interquartile range (IQR). Data shown in a adjusted for smoking, and in b and f adjusted for atopy. *P < 0.05, **P < 0.01, Mann–Whitney U tests. CRSwNP chronic rhinosinusitis with nasal polyps, AS asthma

Fig. 2

Common dysregulated genes shared by CRSwNP + AS and CRSwNP-alone. Nasal tissue samples of CRSwNP + AS (n = 10), CRSwNP-alone (n = 10), and control (n = 9) were analysed by whole-transcriptome sequencing. a Venn diagrams depicting DE-mRNAs of CRSwNP + AS versus control and CRSwNP-alone versus control. The number of DE-mRNAs is marked in the corresponding areas. b The 1988 common DE-mRNAs shared by CRSwNP + AS versus control and CRSwNP-alone versus control were assessed by pathway enrichment analyses using Enrichr. Top 15 significantly enriched KEGG pathways (blue columns) and top 5 significantly enriched BioCarta pathways (turquoise columns) are depicted. P < 0.05 were considered statistically significant. c Venn diagrams depicting DE-lncRNAs of CRSwNP + AS versus control and CRSwNP-alone versus control. d, e The 176 common DE-lncRNAs shared by CRSwNP + AS versus control and CRSwNP-alone versus control were assessed for expression based modules identified by weighted gene coexpression network analysis (WGCNA) and for their potential functions, based on a coexpression network. e Branches of the dendrogram obtained by hierarchical clustering of adjacency based similarity show 9 modules, labelled with a distinct colour, and d Top 15 significantly enriched KEGG pathways (blue column) and top 5 significantly enriched BioCarta pathways (turquoise column) by genes in the largest turquoise module. f Top 50 hub genes of turquoise module visualized by cytoscape network. mRNAs or lncRNAs with high connectivity and edges with weight above a threshold of 0.1 were identified as hub genes. The red nodes denote lncRNAs, and the green nodes denote mRNAs. CRSwNP chronic rhinosinusitis with nasal polyps, AS asthma, lncRNA long non-coding RNA, DE differentially expressed, KEGG Kyoto Encyclopedia of Genes and Genomes

Fig. 3

Differentially expressed genes and pathways between CRSwNP + AS and CRSwNP-alone. a Volcano plots illustrating DE-mRNAs of CRSwNP + AS versus CRSwNP-alone identified by RNA sequencing. b Top 15 KEGG pathways (blue column) and top 5 BioCarta pathways (turquoise column) significantly enriched by DE-mRNAs. c The expression of arachidonic acid metabolism-related DE-mRNAs between CRSwNP + AS and CRSwNP-alone. The colour coding of heat maps represents the gene expression level normalized to Control group, calculated based on fragments per kilo-base of exon per million fragments mapped (FPKM). Yellow box indicates the up-regulated genes in CRSwNP + AS group. d The expression of critical cytokines and their receptors that indicated the activity of different inflammatory endotypes. Yellow stars represent significantly differentially expressed genes between CRSwNP + AS and CRSwNP-alone. P < 0.05 were considered statistically significant. CRSwNP chronic rhinosinusitis with nasal polyps, AS asthma, DE differentially expressed, KEGG Kyoto Encyclopedia of Genes and Genomes

Fig. 4

Correlations between cytokines and inflammatory indicators. Spearman correlation analysis was performed between expression of cytokines IL-5, IL-13, and IL-17A and percentage of local eosinophils, concentration of tissue IgE and LTC4S expression. N = 20 for each group. FPKM fragments per kilo-base of exon per million fragments mapped, IL interleukin-4

Fig. 5

Differentially expressed lncRNAs and pathway analysis between CRSwNP + AS and CRSwNP-alone. a Volcano plots illustrating DE-lncRNAs of CRSwNP + AS versus CRSwNP-alone identified by RNA sequencing. b The correlation between modules and phenotype of CRSwNP + AS. Seven modules were identified by WGCNA based on expression of DE-mRNAs and DE-lncRNAs of CRSwNP + AS versus CRSwNP-alone. Pearson’s correlation coefficient between each module and phenotype of CRSwNP + AS and their associated P values are shown in the corresponding modules. The red and green colours show a strong positive and negative correlation, respectively. c, d All or top 10 KEGG pathways significantly enriched by genes in blue module c and yellow module (d). e, f Top 50 hub genes in blue module (e) and yellow module (f) visualized by cytoscape network. mRNAs or lncRNAs with high connectivity and edges with weight above a threshold of 0.1 were identified as hub genes. The red nodes denote lncRNAs, and the green nodes denote mRNAs. P < 0.05 were considered statistically significant. CRSwNP chronic rhinosinusitis with nasal polyps; AS: asthma, lncRNA long non-coding RNA, DE differentially expressed, KEGG Kyoto Encyclopedia of Genes and Genomes