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

Distinct type 2-high inflammation associated molecular signatures of chronic rhinosinusitis with... 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 enrich- ment 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 activ- ity 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. *Correspondence: dr.luozhang@139.com; wangcs830@126.com Luo Zhang, Chengshuo Wang contributed equally to the study Department of Otolaryngology, Head and Neck Surgery, Beijing TongRen Hospital, Capital Medical University, No. 1, DongJiaoMinXiang, DongCheng District, Beijing 100730, China Full list of author information is available at the end of the article © The Author(s) 2020. This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creat iveco mmons .org/licen ses/by/4.0/. The Creative Commons Public Domain Dedication waiver (http://creat iveco mmons .org/publi cdoma in/ zero/1.0/) applies to the data made available in this article, unless otherwise stated in a credit line to the data. Wang et al. Clin Transl Allergy (2020) 10:26 Page 2 of 14 Conclusions: Patients with CRSwNP + AS have distinct type 2-high inflammation-associated molecular signatures in nasal tissues compared to patients with CRSwNP-alone. Keywords: Chronic rhinosinusitis with nasal polyps, Asthma, Type 2 inflammation, Molecular endotype, Transcriptome sequencing Background associated transcriptome signatures, indicated by cod- Chronic rhinosinusitis (CRS), a disease characterized by ing mRNAs and long non-coding RNAs (lncRNAs), chronic inflammation of the sinonasal tissue, affects 5.5– were found in patients with CRSwNP + AS compared to 28% of the general population [1]. CRS with nasal polyps patients with CRSwNP-alone. (CRSwNP) accounts for approximately 20% of all CRS and has greater severity of clinical disease [2]. Asthma Materials and methods is one of the most common chronic inflammatory disor - Subjects ders of the lower airway worldwide with increasing mor- A total of 195 subjects, including 65 CRSwNP patients bidity. Studies have reported that up to 60% of CRSwNP with comorbid asthma (CRSwNP + AS), 99 patients patients have comorbid asthma (CRSwNP + AS), which with CRSwNP-alone and 31 healthy control subjects is one of the most challenging CRS subtypes to treat [3]. were enrolled in series from June 2017 to March 2018 Patients with CRSwNP + AS have greater disease sever- in the Rhinology Department of Beijing TongRen Hos- ity, higher recurrence rates of nasal polyps after surgery, pital. Patients with CRSwNP were diagnosed accord- poorer asthma control and higher costs [4–6]. ing to the European Position Paper on Rhinosinusitis There is evidence that eosinophilic CRSwNP tends to and Nasal Polyps 2020 guidelines [1]. The diagnosis of have comorbid asthma more frequently [7]. Formation comorbid asthma was based on the Global Initiative for of IgE, which is independent of the presence of allergy Asthma 2019 guidelines. The diagnosis of allergic rhini - in nasal polyp tissue, is also associated with asthmatic tis was according to Allergic Rhinitis and Its Impact on condition in patients with CRSwNP [8]. However, there Asthma 2016 guidelines. Atopy was confirmed based is still no clear explanation for the association between on positive test for serum antigen-specific IgE (cut-off CRSwNP and asthma, and likewise the pathogenic mech- value, 0.35kUA/L), measured by Immuno-CAP 100 sys- anisms leading to CRSwNP and asthma are uncertain. tem (Pharmacia, Uppsala, Sweden). Patients undergoing The united airway concept suggests that the upper and septoplasty because of anatomic variations and without lower airway inflammation share common pathogenic other sinonasal diseases were recruited as control sub- mechanisms and influence each other [9, 10]. There is jects. All subjects were aged 18 to 70 years. Subjects with evidence that several features of inflammatory pattern, immunodeficiency, fungal sinusitis, coagulation disorder, disrupted epithelial barrier and airway remodelling are neoplasia, pregnancy and aspirin-exacerbated respiratory similar in CRSwNP and asthma [11–13]. Thus, under - disease (AERD) were excluded. None of the patients had standing the molecular relationship between CRSwNP been treated with corticosteroids, antibiotics or biologics and comorbid asthma may help to reveal the mechanisms within the 4-week period before surgery, and no patient that underlie airway chronic inflammation. had symptoms of infection at the time of sampling, apart CRSwNP is a heterogeneous inflammatory condi - from symptoms of chronic rhinosinusitis or asthma. tion with different endotypes [14]. The majority of white Recurrence was defined as the presence of nasal polyps patients with CRSwNP in western countries have a type observed under nasal endoscopy, together with at least 2 pattern of inflammation characterized by pronounced one symptom (nasal obstruction, rhinorrhea, headache/ eosinophilia and high levels of interleukin-4 (IL-4), IL-5 facial pain, reduction or loss of smell, sleep disturbance/ and IL-13 cytokines [15]. In contrast, Chinese patients fatigue) lasting at least 1 week, despite appropriate intra- with CRSwNP have lower type 2 inflammation and nasal corticosteroid treatment. The postoperative follow- show higher degree of type 1/type 3 inflammation [16, up period was 18  months for assessment of recurrence/ 17]. Furthermore, evidence from previous studies on non-recurrence of nasal polyps [1, 21]. All patients were epidemiology and clinical characteristics, suggests that followed-up after weeks 1, 2, 4, and 12, and then once CRSwNP + AS may be considered a subtype of CRSwNP every 3 months for up to 18 months by the same surgeon, [18–20]. Thus, this study aimed to investigate the charac - who was blinded to all laboratory data [21]. Nasal tissue teristics of type 2 inflammation and molecular endotypes samples were collected from the inferior turbinate of con- associated with CRSwNP + AS by whole-transcriptome trol subjects and the nasal polyps of CRSwNP patients sequencing. Distinct type 2-high inflammation and its during surgery. The tissue samples were processed for W ang et al. Clin Transl Allergy (2020) 10:26 Page 3 of 14 staining with haematoxylin and eosin, RNA sequencing paired-end reads were generated by Novogene Bioinfor- and ELISA as previously described [22].The Ethics Com - matics Technology Cooperation (Beijing, China). mittee of Beijing Tongren Hospital approved this study, and all subjects signed informed consent forms prior to RNA sequencing data analysis enrolment in the study. Adapters and low-quality tail were trimmed from reads prior to read alignment. Clean sequence reads were aligned to the human genome with Hisat2 (v2.0.5). Cuf- Histological evaluation of polyp tissue flinks (v2.2.1) was used to assemble transcripts, estimate Nasal polyp tissues were immediately formalin fixed after the abundance of these transcripts, and detect differen - surgery, and then dehydrated and embedded in paraf- tial expression among samples. For mRNA analyses, the fin. Paraffin sections were stained with haematoxylin reference genome build GRCh37 was chosen as the anno- and eosin (H&E) and processed for histological evalua- tation references. For lncRNA analyses, the GENCODE tion. All sections were examined by optical microscopy v19 database was chosen as the annotation references. at × 400 magnification. The absolute numbers and per - Fragments per kilo-base of exon per million fragments centages of infiltrating inflammatory cells; including mapped (FPKM) of both lncRNAs and mRNAs in each eosinophils, neutrophils, plasma cells, and lymphocytes; sample was calculated based on the length of the frag- were recorded as mean of six non-overlapping regions in ments and reads count mapped to this fragment. Dif- each section by two independent pathologists, who were ferential expression analysis was performed using blinded to the study design and clinical background of Cuffdiff software (v2.2.1). An adjusted P < 0.05 plus fold the patients. change > 2 was used as the cut-off for significantly differ - entially expressed (DE) mRNAs (DE-mRNAs) and lncR- Assessment of total IgE in nasal tissues NAs (DE-lncRNAs). Concentrations of total IgE in nasal tissues were assayed using the Human IgE ELISA Kit (Arigo Biolaboratories Pathway analysis of DE‑mRNAs and function prediction Corporation, Taiwan). Briefly, fresh nasal tissues were of DE‑lncRNAs placed into RIPA lysis buffer with 1% protease inhibitor DE-mRNAs were loaded into Enrichr (https ://amp. cocktail (Thermo Fisher Scientific) and homogenized pharm .mssm.edu/Enric hr/) [23] for pathway enrichment using a standard bench-top homogenizer (Qiagen, Valen- analysis, and the top significantly enriched (P < 0.05) cia, CA). The tissue homogenates were centrifuged and KEGG and Biocarta pathways with high Combined supernatants were collected for IgE analysis, according to Score, provided by Enrichr, were determined. the manufacturer’s instructions. All samples were tested In order to predict DE-lncRNA functions, we applied in duplicate. weighted gene coexpression network analysis (WGCNA) [24] to construct a coexpression network between DE- RNA isolation and RNA sequencing lncRNAs and their highly correlated DE-mRNAs based Nasal tissue samples of CRSwNP + AS (n = 10), on the Pearson correlation coefficient between their CRSwNP-alone (n = 10), and control (n = 9) were ran- normalized expression levels. Briefly, network construc - domly selected for whole-transcriptome sequencing. The tion and module detection were performed using the nasal tissue samples collected after surgery were freshly “blockwise Modules” function in the WGCNA pack- preserved in RNAlater solution (Qiagen, Hilden, Ger- age. A coexpression similarity matrix was calculated by many) and processed for extraction and purification of computing Pearson correlations between all gene pairs, total RNA using the RNeasy Kit (Qiagen, Hilden, Ger- and then transformed into an adjacency matrix using a many), according to the manufacturer’s instructions. The soft threshold power (β) equal to 18. A dynamic tree cut quantity and quality of the isolated RNA was determined algorithm was used to detect groups of highly correlated with NanoDrop 2000 Spectrophotometer (Thermo Fis - genes. Modules were defined as the branches cut-off of cher Scientific) and 2100 TapeStation Automated Elec - the tree and each module was labelled in unique col- trophoresis System (Agilent Technologies), and samples ours, of which grey colour contains probes not assigned with an RNA integrity number of greater than 8.0 were to any module. The module eigengenes were utilized to chosen for sequencing. Ribosomal RNA was removed represent each module, which was calculated via the first and sequencing libraries were prepared using the rRNA- principal component. Using the module eigengenes, the depleted RNA by NEBNext UltraTM Directional RNA relationship between module and tissue type (CRSwNP- Library Prep Kit (New England Biolabs, USA), following alone and CRSwNP + AS) was estimated. the manufacturer’s instructions. RNA sequencing was Enrichr was used to perform the functional enrich- performed on the Illumina Hiseq platform and 150  bp ment analysis for each module, and the top significantly Wang et al. Clin Transl Allergy (2020) 10:26 Page 4 of 14 enriched pathways were determined. In interesting mod- from CRSwNP + AS patients were significantly higher ules that related to the disease condition, top hub genes compared to IgE levels in nasal tissues of CRSwNP-alone with high connectivity and edges with weight above a patients and controls (Fig.  1f ). Moreover, Spearman cor- threshold of 0.1 were identified and visualized using the relation test showed that the percentage of local eosino- cytoscape network. phils was positively correlated with the concentration of tissue IgE (Additional file 1: Figure S1). Statistical analysis All data are presented as medians and interquartile range Whole transcriptome profiling of nasal tissues (IQR) except for age, which is presented as mean ± SD. from CRSwNP + AS and CRSwNP‑alone Data analysis was performed using GraphPad Prism Ver- To identify the gene expression profiles of CRSwNP sion 7.0 (GraphPad Software, La Jolla, Calif ). All para- with and without comorbid asthma, RNA sequencing metric variants were analyzed using Student t tests, and was performed on nasal tissue samples from control, nonparametric variants were analyzed by using Mann– CRSwNP-alone and CRSwNP + AS patients. Analysis of Whitney U tests. The χ or Fisher exact test was used for DE-mRNAs and DE-lncRNAs demonstrated that there qualitative data. Relationships between variables were were 5218 DE-mRNAs and 2949 DE-lncRNAs between evaluated using Spearman correlation analysis. Differ - CRSwNP-alone and control, and 2512 DE-mRNAs and ences were considered significant at P value < 0.05. 464 DE-lncRNAs between CRSwNP + AS and con- trol (Additional file  1: Figure S2). Additionally, 968 DE- Results mRNAs and 312 DE-lncRNAs were identified between Demographic and clinical characteristics of the subjects CRSwNP + AS and CRSwNP-alone. Hierarchical cluster- Demographic and clinic characteristics of all partici- ing of top 500 differentially expressed genes was shown in pants enrolled in this study are presented in the Addi- Additional file 1: Figure S3. tional file  1: Table S1. There was no significant difference with regard to age, gender and smoker status between Common dysregulated genes shared by CRSwNP + AS the 3 groups. Peripheral blood eosinophils and total IgE and CRSwNP‑alone were increased in both subtypes of CRSwNP patients A total of 1988 common DE-mRNAs were shared by compared to control subjects. Assessment for the sig- CRSwNP-alone and CRSwNP + AS respectively com- nificance of differences between CRSwNP + AS group pared to control (Fig.  2a and Additional file  1: Table S2). and CRSwNP-alone group indicated that atopy and Assessment of these common DE-mRNAs by pathway recurrence were significantly more frequent in patients enrichment analysis using Enrichr Demonstrated that with CRSwNP + AS than in patients with CRSwNP- the top enriched KEGG pathways were associated with alone (62.5% vs 28.6% and 66.7% vs 26.9%, respectively). cytokine–cytokine receptor interaction, chemokine Patients with CRSwNP + AS also had a significantly signalling pathway, staphylococcus aureus infection, decreased forced exhalation volume in one second asthma, and cell adhesion molecules (Fig.  2b). The top (FEV1)/forced vital capacity (FVC) ratio and an increased enriched BioCarta pathways were related to eicosanoid fractional exhaled nitric oxide (FeNO) compared to metabolism, the co-stimulatory signal during T cell acti- patients with CRSwNP-alone. Similarly, CRSwNP + AS vation and IL-10 anti-inflammatory signalling pathway patients had a significantly higher percentage of eosino - (Fig.  2b). Likewise, gene ontology enrichment analysis phils (6.45%) and total IgE (143.00 kU/l) in peripheral showed that common DE-mRNAs were mainly associ- blood compared to CRSwNP-alone patients (3.00% and ated with regulation of immune system process, immune 53.90 kU/l, respectively; Fig. 1a, b). response, response to stimulus and cell–cell signalling (Additional file 1: Figure S4). Local features of nasal tissue in different patient group Similarly, a total of 176 common DE-lncRNAs were To identify the local inflammatory patterns of nasal tissue shared by CRSwNP + AS versus control and CRSwNP- in CRSwNP patients with and without comorbid asthma, alone versus control (Fig.  2c and Additional file  1: the infiltrating eosinophils, neutrophils, plasma cells, and Table  S2). WGCNA was applied to explore the poten- lymphocytes were assessed. Tissue sections stained with tial functions of common DE-lncRNAs, and construc- H&E demonstrated that patients with CRSwNP + AS had tion of a hierarchical clustering tree demonstrated 9 significantly more eosinophils in nasal polyp tissues than modules, each of which was labelled with a distinct col- patients with CRSwNP-alone (Fig. 1c–e). our (Fig.  2e). The size of these modules ranged from 41 Given the relationship between IgE and Type 2 immune to 539 genes; with the largest turquoise coloured mod- response, we further examined the total IgE levels in ule (Additional file  1: Figure S5) comprising 45 lncR- nasal tissues and found that IgE levels in nasal tissues NAs and 494 mRNAs, which were highly coexpressed. W ang et al. Clin Transl Allergy (2020) 10:26 Page 5 of 14 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 Pathway enrichment analysis showed that the genes receptor signalling pathway, staphylococcus aureus infec- of this turquoise module were mainly associated with tion, chemokine signalling pathway, and Th1, Th2, Th17 cytokine–cytokine receptor interaction, asthma, T cell cell differentiation (Fig. 2d). Wang et al. Clin Transl Allergy (2020) 10:26 Page 6 of 14 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 W ang et al. Clin Transl Allergy (2020) 10:26 Page 7 of 14 Figure  2f shows the top 50 hub genes with high con- Th1 and Th2 cell differentiation (Additional file  1: Figure nectivity and edge weigh identified and visualized by S6). cytoscape network. LINC01146,  the only one lncRNA of these top hub genes, was significantly up-regulated in Distinct transcriptome signatures in nasal tissue both CRSwNP-alone and CRSwNP + AS. Pathway analy- of CRSwNP + AS sis of the coexpressed mRNAs of LINC01146 demon- Overall, 212 mRNAs were down-regulated and 756 strated that LINC01146 was mostly associated with T cell mRNAs up-regulated in nasal tissues of CRSwNP + AS receptor signalling pathway, natural killer cell mediated patients, compared to nasal tissues of CRSwNP-alone cytotoxicity, Fc gamma R-mediated phagocytosis, and patients (Fig.  3a). The 50 most significant DE-mRNAs are shown in Additional file  1: Table S3. Enrichr pathway 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 Wang et al. Clin Transl Allergy (2020) 10:26 Page 8 of 14 analysis showed that arachidonic acid metabolism, ECM- (Fig.  5a). The top 50 significant DE-lncRNAs are shown receptor interaction, IL-17 signalling pathway, GATA3 in Additional file  1: Table  S5. A coexpression network participate in activating Th2 cytokine genes, and fibrinol - constructed based on the expression of DE-lncRNAs ysis pathway were the top significant pathways enriched and DE-mRNAs using WGCNA demonstrated 7 col- by DE-mRNAs (Fig. 3b). our-coded modules underneath the cluster tree (Addi- Detailed examination of the expression of genes tional file  1: Figure S7). Assessment of the relationship involved in arachidonic acid metabolism demonstrated between each module and tissue type (CRSwNP + AS), that 14 of 16 DE-mRNAs related to arachidonic acid estimated using the module eigengenes, demonstrated metabolism; including PTGS1, TBXAS1, ALOX15B, that all 7 modules were significantly correlated with tis - PLA2G4D, LTC4S, GGT1, GGT5, GPX6, PLA2G4B, sue type changes (Fig. 5b). Of these, the blue module was PLA2G6, CYP4F8, JMJD7-PLA2G4B, PTGIS and GPX3; most negatively correlated with the phenotypic traits of −4 were up-regulated in CRSwNP + AS compared to CRSwNP + AS (r = − 0.73, P = 2 × 10 ), and the yel- CRSwNP-alone (Fig. 3c). low module the most positively correlated (r = 0.66, P = 0.001). Severe type 2 inflammation in nasal tissues Overall, there were 31 lncRNAs and 94 mRNAs in the of CRSwNP + AS blue module, and only one lncRNA (HK3-006) and 63 As indicated by above transcriptome data, type 1, type mRNAs in the yellow module. Pathway enrichment anal- 2 and type 3 related signalling pathways might be dif- ysis indicated that the blue module was mainly associated ferentiated between CRSwNP-alone and CRSwNP + AS. with IL-17 signalling pathway and cytokine–cytokine u Th s, we investigated the mRNA expression of critical receptor interaction (Fig. 5c), whereas the yellow module cytokines and their receptors, which indicate the activi- was mainly related to asthma, arachidonic acid metabo- ties of different inflammatory endotypes; namely (1) lism, and signalling pathways such as calcium and JAK- IFNG and IFNG receptor (IFNGR1 and IFNGR2) for type STAT signalling pathway (Fig.  5d). Top 50 hub genes 1 inflammation; (2) IL5, IL4, IL13 and their receptors identified from the blue and yellow modules, respectively, (IL5RA, IL5RB, IL4R, IL13RA1 and IL13RA2) for type are shown in Fig. 5 e, f. 2 inflammation; (3) IL17A and IL17A receptor (IL17RA and IL17RC) for type 3 inflammation; as well as (4) TNF, Discussion IL1B, IL6 and their receptors (TNFRSF1A, TNFRSF1B, Both CRSwNP and asthma are airway inflammatory IL1R1, IL1R2 and IL6R) for proinflammatory activity. disorders, which have serious effects on quality of life. We found that the expression of cytokines IL5 and A great proportion of CRSwNP patients have comor- IL13, and receptors IL5RA and IL5RB, indicating aug- bid asthma, which makes it one of the most challenging mented type 2 inflammation, was significantly enhanced phenotypes to treat [3, 12]. Furthermore, the molecular in both CRSwNP-alone and CRSwNP + AS compared endotypes of CRSwNP + AS are not clear. In this regard, to control (Fig.  3d and Additional file  1: Table  S4). Fur- to our knowledge this is the first study to investigate the thermore, nasal tissue from CRSwNP + AS demonstrated molecular endotypes of CRSwNP + AS compared to significantly higher expression of IL4, IL5 and IL13; and CRSwNP-alone, by whole-transcriptome RNA sequenc- conversely significantly lower expression of IL17A than ing. Our study demonstrated that type 2-high inflamma - nasal tissue of CRSwNP-alone. tion patterns and their associated transcriptome features Assessment of correlations between the significant were distinct in nasal tissues of CRSwNP + AS patients cytokines and the other significant inflammatory indi - compared to CRSwNP-alone patients. cators determined above, demonstrated that the expres- Studies have indicated that CRS is a heterogeneous dis- sion of both IL5 and IL13 was positively correlated with ease with several unclear endotypes, which are mainly the percentage of local eosinophils, concentration of tis- characterized by type 1, 2, and 3 inflammatory patterns sue IgE and the expression of LTC4S, which reflected an [15, 25–27]. Furthermore, some recent studies have imbalanced arachidonic acid metabolism (Fig.  4a–f ). In indicated that the most prevalent endotype in CRSwNP contrast, the expression of IL17A was negatively cor- is characterized by type 2 inflammation [28], and that related with tissue IgE and the expression of LTC4S type 2 inflammation in CRSwNP may be differenti - (Fig. 4g–i). ated into moderate and severe type 2 inflammatory pat - terns according to the intensity of inflammation [29]. In LncRNA signatures in nasal tissue of CRSwNP + AS accordance with these studies, the findings for the differ - We identified 229 up-regulated and 83 down-regulated entially expressed type 1, type 2 and type 3 inflammation- DE-lncRNAs in nasal tissues of CRSwNP + AS patients related genes and pathways in the present study suggest compared to nasal tissues of CRSwNP-alone patients that the CRSwNP + AS endotype is associated with a W ang et al. Clin Transl Allergy (2020) 10:26 Page 9 of 14 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 more severe type 2 inflammation, compared to CRSwNP- of cytokines IL-5, IL-4 and IL-13 [31]. Whilst more alone endotype. Furthermore, in accordance with the than 80% of Western white patients with CRSwNP are findings of Tomassen and colleagues [15] that severe characterized by type 2 inflammation, less than 50% of type 2 CRSwNP increased asthma prevalence, our study CRSwNP cases in East Asian countries show features of has also demonstrated that in addition to more frequent type 2 reactions [32, 33]. Like CRSwNP, asthma is also atopy, more local eosinophil infiltration, and higher level a complex and heterogeneous disease, and two major of tissue IgE, the chance of recurrence of CRSwNP was endotypes of asthma, type 2-high asthma and type 2-low also increased in patients with CRSwNP + AS compared asthma, have been described, based on underlying air- to patients with CRSwNP-alone. Indeed, we have previ- way immune-mediated inflammation [34, 35]. In this ously shown that high proportion of eosinophils in nasal regard approximately 50% to 60% of all patients with tissue act as a reliable prognostic indicator for CRSwNP severe asthma in Europe and the United States account recurrence [30]. Similarly, a more recent study has indi- for type 2-high asthma, compared to 38.5% of the severe cated that asthma in CRS patients was the only fac- asthmatic patients with type 2-high asthma in China tor that increases the chance of recurrence in CRSwNP [36–39]. Thus, whilst it is possible that generally more patients [6]; suggesting that comorbid asthma might also Western patients with CRSwNP + AS are also likely to be a strong indicator for CRSwNP recurrence. be characterised with type 2-high inflammation than Airway type 2 inflammation is mainly mediated by Asian patients CRSwNP + AS, this nevertheless needs to eosinophils, mast cells, Th2 cells, ILC2s and IgE-produc - be confirmed in well-designed multicenter studies in the ing B cells, in combination with increased production future. Wang et al. Clin Transl Allergy (2020) 10:26 Page 10 of 14 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 Our findings for RNA sequencing provides valuable demonstrated that regardless of the subtypes of CRSwNP, information for exploring the general molecular mecha- genes and pathways that most likely contribute to the nisms underlying the pathogenesis of CRSwNP. Similar pathogenesis of CRSwNP, appear to be mainly associ- to the findings of Peng and colleagues [40], our study has ated with cytokine and chemokine signalling pathway, W ang et al. Clin Transl Allergy (2020) 10:26 Page 11 of 14 staphylococcus aureus infection, eicosanoid metabolism inflammation in the nasal tissues of CRSwNP + AS and cell adhesion molecules. In this respect, the well- patients. The increased expression of PTGS1 and PTGIS known genes or biomarkers closely related to CRSwNP; may accelerate the conversion of arachidonic acid to for example CLC, POSTN, CCL18, IL13, TSLP and prostacyclins (PGI2) and prostaglandins (PGD2, PGE2), BPIFA1; were also identified as the top DE-mRNAs in which are also related to an aggravated airway inflam - both CRSwNP-alone and CRSwNP + AS groups. mation and asthmatic conditions [48, 49]. Moreover, However, the transcriptome signatures of the increased expression of ALOX15B in CRSwNP + AS CRSwNP + AS were characterized by distinct groups of might have an active proinflammatory role [50]. Collec - differentially expressed genes and their enriched path - tively, these findings suggest that enhanced activity of ways, compared to CRSwNP-alone. In particular, the pre- arachidonic acid metabolism might help to augment type sent study showed that the CRSwNP + AS endotype was 2 inflammation in CRSwNP + AS [45, 51]. mainly associated with higher activities of arachidonic Th2 and Th17 signalling pathways are two major reg - acid metabolism, Th2 signalling pathway and fibrinolysis ulatory pathways, which are inversely interrelated in pathway, and lower activity of IL-17 signalling pathway. patients with CRSwNP [17]. In accordanc ewith this, gen- Beyond that, the most significant DE-mRNAs between eral observation, our RNA sequencing data also revealed CRSwNP + AS and CRSwNP-alone also provide impor- higher activities of Th2 signalling pathway and lower tant information. In line with the high concentration of activities of IL-17 signalling pathway in CRSwNP + AS tissue IgE in CRSwNP + AS, increased mRNA expres- than in CRSwNP-alone. In this respect IL-4, IL-5 and sion of constant region of heavy chain of IgE (IGHE) was IL-13 are typical Th2 cytokines, which reflect the sever - also confirmed by RNA sequencing. Some recently iden - ity of type 2 inflammation, whereas IL-17 is a typical tified biomarkers of CRSwNP, for example CST1 [41], Th17 cytokine. Indeed, the present study further showed were also found to be differentially expressed between that the expression of IL5 and IL13 was positively cor- CRSwNP + AS and CRSwNP-alone. Importantly, the pre- related with other indicators of type 2 inflammation sent study showed that some of the most significant DE- (i.e. increased local eosinophils and tissue IgE). Simi- mRNAs; including ITLN1, KCNA3 and CCR10; which larly, genes that are related to arachidonic acid metabo- were up-regulated in nasal tissue of CRSwNP + AS com- lism were also correlated with the expression of type 2 pared to nasal tissue of CRSwNP-alone (Additional file  1: cytokines, thus, emphasising the crucial role of type 2 Figure S8), are also expressed in the bronchial tissue and inflammation in CRSwNP + AS patients. contribute to the pathogenesis of asthma [42–44]. This LncRNAs play important roles in various biologi- suggests that signature genes identified in nasal tissue cal processes and are emerging as reliable biomark- of CRSwNP + AS might also play important roles in the ers and potential therapeutic targets of human chronic pathogenesis of asthma. Moreover, consistent with the diseases [52, 53]. However, comparatively few studies united airways concept, it is tempting to speculate that have reported the involvement of lncRNAs in chronic the pattern of gene expression in the upper airway may nasal inflammation. Yue and colleagues [54] found that be influenced by the prevailing conditions in the lower linc00632 was down-regulated in nasal tissues of aller- airway, and as such prevalence of comorbid asthmatic gic rhinitis patients and inhibited IL-13 induced inflam - conditions might exacerbate type 2 inflammation in nasal matory cytokine and mucus production. Wang and tissue of CRSwNP patients. colleagues [55] showed that lncRNA XLOC_010280 It has been well demonstrated that alterations in the might regulate the expression of CCL18 and eosinophilic arachidonic acid pathway play an important role in air- inflammation in eosinophilic CRSwNP. In the present way inflammatory conditions like rhinosinusitis, nasal study, whole-transcriptome sequencing has revealed sev- polyps, allergic rhinitis, and asthma [45]. We found a eral dysregulated lncRNAs in both subtypes of CRSwNP, generally enhanced activity of arachidonic acid metabo- which will provide a useful pool of candidate lncRNAs in lism in CRSwNP + AS, indicated by the up-regulated future investigations of CRSwNP endotypes. expression of PLA2 (PLA2G4A, PLA2G4B, PLA2G4D WGCNA is an effective method of multigene analysis and PLA2G6), which may promotes the release of mem- to construct coexpression network, and has successfully brane-bound arachidonic acid [46]. The imbalanced syn - been applied for studying mRNAs and lncRNAs to dis- thesis of eicosanoids characterized by increased synthesis tinguish dysfunctional regulatory subnetworks, select of cysteinyl leukotrienes (CysLTs) is correlated with the out potentially key genes, and predict lncRNA func- inflammatory pattern and severity of the airway inflam - tions [56, 57]. We applied WGCNA to predict functions mation [45, 47]. Consistent with this, we also showed an of DE-lncRNAs and identify hub genes in the present increased expression of LTC4S, which promotes the bio- study and showed that common dysregulated lncRNAs synthesis of CysLTs and indicates the presence of severe in CRSwNP-alone and CRSwNP + AS have very similar Wang et al. Clin Transl Allergy (2020) 10:26 Page 12 of 14 specific information on the role of different cell types predictive functions to common DE-mRNAs, as indi- as biomarkers of CRSwNP with or without comorbid cated by genes in the maximal module. Furthermore, we asthma. The use of inferior turbinate tissue from controls identified LINC01146 as the only top hub lncRNA, which for comparison with nasal polyp tissues from CRSwNP might play a key role in the pathogenesis of CRSwNP. patients is also as a limitation of the study, particularly Although LINC01146 has also been found to be dysregu- as nasal polyps rarely arise from inferior turbinates in lated in hepatocellular carcinoma, its precise function in healthy control subjects. However, using mucosa tissue this condition is not clear [58]. Further study is needed to from different locations such as middle turbinate and validate the function of common dysregulated lncRNAs ethmoid tissue as controls may avoid regional variations in CRSwNP. in gene expression. The present study also identified HK3-006 as the only top hub lncRNA that was expressed in the module that most positively correlated with phenotypic traits of Conclusions CRSwNP + AS; and may be related to asthma pathway The present study demonstrated that CRSwNP patients and arachidonic acid metabolism as predicted by its with comorbid asthma have distinct type 2-high inflam - highly coexpressed mRNAs. Similarly, several top hub mation-associated transcriptome profiles, as indicated lncRNAs were identified in the module that most nega - by differential expression profiles of key mRNAs and tively correlated with phenotypic traits of CRSwNP + AS. lncRNAs in nasal tissue, compared to patients with Among these lncRNAs, we have identified a new CRSwNP-alone. Differences in molecular mechanisms lncRNA, LINC686, which is most likely to be associated and type 2 inflammation-related molecules in CRSwNP with IL-17 signalling pathway. Although an increasing with comorbid asthma may be useful in better under- number of studies show that lncRNAs are involved in the standing the mechanisms underlying the development regulation of cytokine signalling and inflammation [59, of different endotypes of CRS, as well as development 60], our understanding of the functions of lncRNA is just of potential biomarkers and targeted gene therapies for beginning to develop. CRSwNP in the future. The present study has some limitations. First, this study did not investigate any subgroups of CRSwNP + AS Supplementary information and CRSwNP-alone. This may be of importance as Supplementary information accompanies this paper at https ://doi. both CRSwNP and asthma are heterogeneous diseases, org/10.1186/s1360 1-020-00332 -z. and one recent study has shown that patients with CRSwNP + AS can be grouped into 3 subtypes with dis- Additional file 1: Table S1. Demographic and clinical characteristics of tinct inflammatory status and disease severity [19]. How - study subjects. Table S2. Common DE-mRNAs and DE-lncRNAs shared by ever, the present study was designed to directly compare CRSwNP-alone versus control and CRSwNP+AS versus control. Table S3. CRSwNP + AS with CRSwNP-alone. Furthermore, as a Top 50 DE-mRNAs of CRSwNP+AS versus CRSwNP-alone. Table S4. Expression of key cytokines and their receptors in nasal tissues from substantial number of Asian CRSwNP patients are type control subjects and CRSwNP-alone and CRSwNP+AS patients. Table S5. 1 and/or type 3 inclined, it would be more appropri- Top 50 DE-lncRNAs of CRSwNP+AS versus CRSwNP-alone. Figure S1. ate to compare type 2 CRSwNP endotype with type 2 Correlation of infiltrating eosinophils and total IgE in nasal tissues of patients with CRSwNP. Figure S2. Differentially expressed mRNAs and CRSwNP + AS endotype for a better understanding of differentially expressed lncRNAs in nasal tissues of CRSwNP patients. the mechanisms and pathways operating in either endo- Figure S3. Hierarchical clustering of differentially expressed genes. Figure type. Second, as the sample size used for RNA sequenc- S4. GO biological processes enriched by common dysregulated genes in CRSwNP+AS and CRSwNP-alone. FigureS5. Gene number of modules ing is relatively small, this might decrease the statistical identified by weighted gene co-expression network analysis. Figure S6. power for gene signature profiling. Third, we did not The expression and potential functions of LINC01146. Figure S7. Gene evaluate lower airway inflammation by assessment of modules identified by WGCNA based on expression of DE-mRNAs and DE- lncRNAs in CRSwNP+AS versus CRSwNP-alone. Figure S8. Asthma related inflammatory cells and cytokines in bronchial biopsy genes expressed in nasal tissue of patients with CRSwNP+AS and patients or induced sputum, which might be helpful in under- with CRSwNP-alone. standing the association between CRSwNP and asthma. Fourth, assessment of critical cytokines and their recep- Abbreviations tors was based only on RNA expression and need to be CRS: Chronic rhinosinusitis; CRSwNP: Chronic rhinosinusitis with nasal polyps; CRSwNP + AS: Chronic Rhinosinusitis with Nasal Polyps with Comorbid verified according to the levels of corresponding proteins Asthma; IL: Interleukin; lncRNA: Long non-coding RNA; DE: Differentially in the tissue. Fifth, whole tissue samples with multiple cell expressed; FPKM: Fragments per kilo-base of exon per million fragments types were used for RNA sequencing and no single-cell mapped; WGCNA: Weighted gene coexpression network analysis; KEGG: Kyoto Encyclopedia of Genes and Genomes. RNA sequencing was used. 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Distinct type 2-high inflammation associated molecular signatures of chronic rhinosinusitis with nasal polyps with comorbid asthma

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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 enrich- ment 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 activ- ity 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. *Correspondence: dr.luozhang@139.com; wangcs830@126.com Luo Zhang, Chengshuo Wang contributed equally to the study Department of Otolaryngology, Head and Neck Surgery, Beijing TongRen Hospital, Capital Medical University, No. 1, DongJiaoMinXiang, DongCheng District, Beijing 100730, China Full list of author information is available at the end of the article © The Author(s) 2020. This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creat iveco mmons .org/licen ses/by/4.0/. The Creative Commons Public Domain Dedication waiver (http://creat iveco mmons .org/publi cdoma in/ zero/1.0/) applies to the data made available in this article, unless otherwise stated in a credit line to the data. Wang et al. Clin Transl Allergy (2020) 10:26 Page 2 of 14 Conclusions: Patients with CRSwNP + AS have distinct type 2-high inflammation-associated molecular signatures in nasal tissues compared to patients with CRSwNP-alone. Keywords: Chronic rhinosinusitis with nasal polyps, Asthma, Type 2 inflammation, Molecular endotype, Transcriptome sequencing Background associated transcriptome signatures, indicated by cod- Chronic rhinosinusitis (CRS), a disease characterized by ing mRNAs and long non-coding RNAs (lncRNAs), chronic inflammation of the sinonasal tissue, affects 5.5– were found in patients with CRSwNP + AS compared to 28% of the general population [1]. CRS with nasal polyps patients with CRSwNP-alone. (CRSwNP) accounts for approximately 20% of all CRS and has greater severity of clinical disease [2]. Asthma Materials and methods is one of the most common chronic inflammatory disor - Subjects ders of the lower airway worldwide with increasing mor- A total of 195 subjects, including 65 CRSwNP patients bidity. Studies have reported that up to 60% of CRSwNP with comorbid asthma (CRSwNP + AS), 99 patients patients have comorbid asthma (CRSwNP + AS), which with CRSwNP-alone and 31 healthy control subjects is one of the most challenging CRS subtypes to treat [3]. were enrolled in series from June 2017 to March 2018 Patients with CRSwNP + AS have greater disease sever- in the Rhinology Department of Beijing TongRen Hos- ity, higher recurrence rates of nasal polyps after surgery, pital. Patients with CRSwNP were diagnosed accord- poorer asthma control and higher costs [4–6]. ing to the European Position Paper on Rhinosinusitis There is evidence that eosinophilic CRSwNP tends to and Nasal Polyps 2020 guidelines [1]. The diagnosis of have comorbid asthma more frequently [7]. Formation comorbid asthma was based on the Global Initiative for of IgE, which is independent of the presence of allergy Asthma 2019 guidelines. The diagnosis of allergic rhini - in nasal polyp tissue, is also associated with asthmatic tis was according to Allergic Rhinitis and Its Impact on condition in patients with CRSwNP [8]. However, there Asthma 2016 guidelines. Atopy was confirmed based is still no clear explanation for the association between on positive test for serum antigen-specific IgE (cut-off CRSwNP and asthma, and likewise the pathogenic mech- value, 0.35kUA/L), measured by Immuno-CAP 100 sys- anisms leading to CRSwNP and asthma are uncertain. tem (Pharmacia, Uppsala, Sweden). Patients undergoing The united airway concept suggests that the upper and septoplasty because of anatomic variations and without lower airway inflammation share common pathogenic other sinonasal diseases were recruited as control sub- mechanisms and influence each other [9, 10]. There is jects. All subjects were aged 18 to 70 years. Subjects with evidence that several features of inflammatory pattern, immunodeficiency, fungal sinusitis, coagulation disorder, disrupted epithelial barrier and airway remodelling are neoplasia, pregnancy and aspirin-exacerbated respiratory similar in CRSwNP and asthma [11–13]. Thus, under - disease (AERD) were excluded. None of the patients had standing the molecular relationship between CRSwNP been treated with corticosteroids, antibiotics or biologics and comorbid asthma may help to reveal the mechanisms within the 4-week period before surgery, and no patient that underlie airway chronic inflammation. had symptoms of infection at the time of sampling, apart CRSwNP is a heterogeneous inflammatory condi - from symptoms of chronic rhinosinusitis or asthma. tion with different endotypes [14]. The majority of white Recurrence was defined as the presence of nasal polyps patients with CRSwNP in western countries have a type observed under nasal endoscopy, together with at least 2 pattern of inflammation characterized by pronounced one symptom (nasal obstruction, rhinorrhea, headache/ eosinophilia and high levels of interleukin-4 (IL-4), IL-5 facial pain, reduction or loss of smell, sleep disturbance/ and IL-13 cytokines [15]. In contrast, Chinese patients fatigue) lasting at least 1 week, despite appropriate intra- with CRSwNP have lower type 2 inflammation and nasal corticosteroid treatment. The postoperative follow- show higher degree of type 1/type 3 inflammation [16, up period was 18  months for assessment of recurrence/ 17]. Furthermore, evidence from previous studies on non-recurrence of nasal polyps [1, 21]. All patients were epidemiology and clinical characteristics, suggests that followed-up after weeks 1, 2, 4, and 12, and then once CRSwNP + AS may be considered a subtype of CRSwNP every 3 months for up to 18 months by the same surgeon, [18–20]. Thus, this study aimed to investigate the charac - who was blinded to all laboratory data [21]. Nasal tissue teristics of type 2 inflammation and molecular endotypes samples were collected from the inferior turbinate of con- associated with CRSwNP + AS by whole-transcriptome trol subjects and the nasal polyps of CRSwNP patients sequencing. Distinct type 2-high inflammation and its during surgery. The tissue samples were processed for W ang et al. Clin Transl Allergy (2020) 10:26 Page 3 of 14 staining with haematoxylin and eosin, RNA sequencing paired-end reads were generated by Novogene Bioinfor- and ELISA as previously described [22].The Ethics Com - matics Technology Cooperation (Beijing, China). mittee of Beijing Tongren Hospital approved this study, and all subjects signed informed consent forms prior to RNA sequencing data analysis enrolment in the study. Adapters and low-quality tail were trimmed from reads prior to read alignment. Clean sequence reads were aligned to the human genome with Hisat2 (v2.0.5). Cuf- Histological evaluation of polyp tissue flinks (v2.2.1) was used to assemble transcripts, estimate Nasal polyp tissues were immediately formalin fixed after the abundance of these transcripts, and detect differen - surgery, and then dehydrated and embedded in paraf- tial expression among samples. For mRNA analyses, the fin. Paraffin sections were stained with haematoxylin reference genome build GRCh37 was chosen as the anno- and eosin (H&E) and processed for histological evalua- tation references. For lncRNA analyses, the GENCODE tion. All sections were examined by optical microscopy v19 database was chosen as the annotation references. at × 400 magnification. The absolute numbers and per - Fragments per kilo-base of exon per million fragments centages of infiltrating inflammatory cells; including mapped (FPKM) of both lncRNAs and mRNAs in each eosinophils, neutrophils, plasma cells, and lymphocytes; sample was calculated based on the length of the frag- were recorded as mean of six non-overlapping regions in ments and reads count mapped to this fragment. Dif- each section by two independent pathologists, who were ferential expression analysis was performed using blinded to the study design and clinical background of Cuffdiff software (v2.2.1). An adjusted P < 0.05 plus fold the patients. change > 2 was used as the cut-off for significantly differ - entially expressed (DE) mRNAs (DE-mRNAs) and lncR- Assessment of total IgE in nasal tissues NAs (DE-lncRNAs). Concentrations of total IgE in nasal tissues were assayed using the Human IgE ELISA Kit (Arigo Biolaboratories Pathway analysis of DE‑mRNAs and function prediction Corporation, Taiwan). Briefly, fresh nasal tissues were of DE‑lncRNAs placed into RIPA lysis buffer with 1% protease inhibitor DE-mRNAs were loaded into Enrichr (https ://amp. cocktail (Thermo Fisher Scientific) and homogenized pharm .mssm.edu/Enric hr/) [23] for pathway enrichment using a standard bench-top homogenizer (Qiagen, Valen- analysis, and the top significantly enriched (P < 0.05) cia, CA). The tissue homogenates were centrifuged and KEGG and Biocarta pathways with high Combined supernatants were collected for IgE analysis, according to Score, provided by Enrichr, were determined. the manufacturer’s instructions. All samples were tested In order to predict DE-lncRNA functions, we applied in duplicate. weighted gene coexpression network analysis (WGCNA) [24] to construct a coexpression network between DE- RNA isolation and RNA sequencing lncRNAs and their highly correlated DE-mRNAs based Nasal tissue samples of CRSwNP + AS (n = 10), on the Pearson correlation coefficient between their CRSwNP-alone (n = 10), and control (n = 9) were ran- normalized expression levels. Briefly, network construc - domly selected for whole-transcriptome sequencing. The tion and module detection were performed using the nasal tissue samples collected after surgery were freshly “blockwise Modules” function in the WGCNA pack- preserved in RNAlater solution (Qiagen, Hilden, Ger- age. A coexpression similarity matrix was calculated by many) and processed for extraction and purification of computing Pearson correlations between all gene pairs, total RNA using the RNeasy Kit (Qiagen, Hilden, Ger- and then transformed into an adjacency matrix using a many), according to the manufacturer’s instructions. The soft threshold power (β) equal to 18. A dynamic tree cut quantity and quality of the isolated RNA was determined algorithm was used to detect groups of highly correlated with NanoDrop 2000 Spectrophotometer (Thermo Fis - genes. Modules were defined as the branches cut-off of cher Scientific) and 2100 TapeStation Automated Elec - the tree and each module was labelled in unique col- trophoresis System (Agilent Technologies), and samples ours, of which grey colour contains probes not assigned with an RNA integrity number of greater than 8.0 were to any module. The module eigengenes were utilized to chosen for sequencing. Ribosomal RNA was removed represent each module, which was calculated via the first and sequencing libraries were prepared using the rRNA- principal component. Using the module eigengenes, the depleted RNA by NEBNext UltraTM Directional RNA relationship between module and tissue type (CRSwNP- Library Prep Kit (New England Biolabs, USA), following alone and CRSwNP + AS) was estimated. the manufacturer’s instructions. RNA sequencing was Enrichr was used to perform the functional enrich- performed on the Illumina Hiseq platform and 150  bp ment analysis for each module, and the top significantly Wang et al. Clin Transl Allergy (2020) 10:26 Page 4 of 14 enriched pathways were determined. In interesting mod- from CRSwNP + AS patients were significantly higher ules that related to the disease condition, top hub genes compared to IgE levels in nasal tissues of CRSwNP-alone with high connectivity and edges with weight above a patients and controls (Fig.  1f ). Moreover, Spearman cor- threshold of 0.1 were identified and visualized using the relation test showed that the percentage of local eosino- cytoscape network. phils was positively correlated with the concentration of tissue IgE (Additional file 1: Figure S1). Statistical analysis All data are presented as medians and interquartile range Whole transcriptome profiling of nasal tissues (IQR) except for age, which is presented as mean ± SD. from CRSwNP + AS and CRSwNP‑alone Data analysis was performed using GraphPad Prism Ver- To identify the gene expression profiles of CRSwNP sion 7.0 (GraphPad Software, La Jolla, Calif ). All para- with and without comorbid asthma, RNA sequencing metric variants were analyzed using Student t tests, and was performed on nasal tissue samples from control, nonparametric variants were analyzed by using Mann– CRSwNP-alone and CRSwNP + AS patients. Analysis of Whitney U tests. The χ or Fisher exact test was used for DE-mRNAs and DE-lncRNAs demonstrated that there qualitative data. Relationships between variables were were 5218 DE-mRNAs and 2949 DE-lncRNAs between evaluated using Spearman correlation analysis. Differ - CRSwNP-alone and control, and 2512 DE-mRNAs and ences were considered significant at P value < 0.05. 464 DE-lncRNAs between CRSwNP + AS and con- trol (Additional file  1: Figure S2). Additionally, 968 DE- Results mRNAs and 312 DE-lncRNAs were identified between Demographic and clinical characteristics of the subjects CRSwNP + AS and CRSwNP-alone. Hierarchical cluster- Demographic and clinic characteristics of all partici- ing of top 500 differentially expressed genes was shown in pants enrolled in this study are presented in the Addi- Additional file 1: Figure S3. tional file  1: Table S1. There was no significant difference with regard to age, gender and smoker status between Common dysregulated genes shared by CRSwNP + AS the 3 groups. Peripheral blood eosinophils and total IgE and CRSwNP‑alone were increased in both subtypes of CRSwNP patients A total of 1988 common DE-mRNAs were shared by compared to control subjects. Assessment for the sig- CRSwNP-alone and CRSwNP + AS respectively com- nificance of differences between CRSwNP + AS group pared to control (Fig.  2a and Additional file  1: Table S2). and CRSwNP-alone group indicated that atopy and Assessment of these common DE-mRNAs by pathway recurrence were significantly more frequent in patients enrichment analysis using Enrichr Demonstrated that with CRSwNP + AS than in patients with CRSwNP- the top enriched KEGG pathways were associated with alone (62.5% vs 28.6% and 66.7% vs 26.9%, respectively). cytokine–cytokine receptor interaction, chemokine Patients with CRSwNP + AS also had a significantly signalling pathway, staphylococcus aureus infection, decreased forced exhalation volume in one second asthma, and cell adhesion molecules (Fig.  2b). The top (FEV1)/forced vital capacity (FVC) ratio and an increased enriched BioCarta pathways were related to eicosanoid fractional exhaled nitric oxide (FeNO) compared to metabolism, the co-stimulatory signal during T cell acti- patients with CRSwNP-alone. Similarly, CRSwNP + AS vation and IL-10 anti-inflammatory signalling pathway patients had a significantly higher percentage of eosino - (Fig.  2b). Likewise, gene ontology enrichment analysis phils (6.45%) and total IgE (143.00 kU/l) in peripheral showed that common DE-mRNAs were mainly associ- blood compared to CRSwNP-alone patients (3.00% and ated with regulation of immune system process, immune 53.90 kU/l, respectively; Fig. 1a, b). response, response to stimulus and cell–cell signalling (Additional file 1: Figure S4). Local features of nasal tissue in different patient group Similarly, a total of 176 common DE-lncRNAs were To identify the local inflammatory patterns of nasal tissue shared by CRSwNP + AS versus control and CRSwNP- in CRSwNP patients with and without comorbid asthma, alone versus control (Fig.  2c and Additional file  1: the infiltrating eosinophils, neutrophils, plasma cells, and Table  S2). WGCNA was applied to explore the poten- lymphocytes were assessed. Tissue sections stained with tial functions of common DE-lncRNAs, and construc- H&E demonstrated that patients with CRSwNP + AS had tion of a hierarchical clustering tree demonstrated 9 significantly more eosinophils in nasal polyp tissues than modules, each of which was labelled with a distinct col- patients with CRSwNP-alone (Fig. 1c–e). our (Fig.  2e). The size of these modules ranged from 41 Given the relationship between IgE and Type 2 immune to 539 genes; with the largest turquoise coloured mod- response, we further examined the total IgE levels in ule (Additional file  1: Figure S5) comprising 45 lncR- nasal tissues and found that IgE levels in nasal tissues NAs and 494 mRNAs, which were highly coexpressed. W ang et al. Clin Transl Allergy (2020) 10:26 Page 5 of 14 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 Pathway enrichment analysis showed that the genes receptor signalling pathway, staphylococcus aureus infec- of this turquoise module were mainly associated with tion, chemokine signalling pathway, and Th1, Th2, Th17 cytokine–cytokine receptor interaction, asthma, T cell cell differentiation (Fig. 2d). Wang et al. Clin Transl Allergy (2020) 10:26 Page 6 of 14 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 W ang et al. Clin Transl Allergy (2020) 10:26 Page 7 of 14 Figure  2f shows the top 50 hub genes with high con- Th1 and Th2 cell differentiation (Additional file  1: Figure nectivity and edge weigh identified and visualized by S6). cytoscape network. LINC01146,  the only one lncRNA of these top hub genes, was significantly up-regulated in Distinct transcriptome signatures in nasal tissue both CRSwNP-alone and CRSwNP + AS. Pathway analy- of CRSwNP + AS sis of the coexpressed mRNAs of LINC01146 demon- Overall, 212 mRNAs were down-regulated and 756 strated that LINC01146 was mostly associated with T cell mRNAs up-regulated in nasal tissues of CRSwNP + AS receptor signalling pathway, natural killer cell mediated patients, compared to nasal tissues of CRSwNP-alone cytotoxicity, Fc gamma R-mediated phagocytosis, and patients (Fig.  3a). The 50 most significant DE-mRNAs are shown in Additional file  1: Table S3. Enrichr pathway 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 Wang et al. Clin Transl Allergy (2020) 10:26 Page 8 of 14 analysis showed that arachidonic acid metabolism, ECM- (Fig.  5a). The top 50 significant DE-lncRNAs are shown receptor interaction, IL-17 signalling pathway, GATA3 in Additional file  1: Table  S5. A coexpression network participate in activating Th2 cytokine genes, and fibrinol - constructed based on the expression of DE-lncRNAs ysis pathway were the top significant pathways enriched and DE-mRNAs using WGCNA demonstrated 7 col- by DE-mRNAs (Fig. 3b). our-coded modules underneath the cluster tree (Addi- Detailed examination of the expression of genes tional file  1: Figure S7). Assessment of the relationship involved in arachidonic acid metabolism demonstrated between each module and tissue type (CRSwNP + AS), that 14 of 16 DE-mRNAs related to arachidonic acid estimated using the module eigengenes, demonstrated metabolism; including PTGS1, TBXAS1, ALOX15B, that all 7 modules were significantly correlated with tis - PLA2G4D, LTC4S, GGT1, GGT5, GPX6, PLA2G4B, sue type changes (Fig. 5b). Of these, the blue module was PLA2G6, CYP4F8, JMJD7-PLA2G4B, PTGIS and GPX3; most negatively correlated with the phenotypic traits of −4 were up-regulated in CRSwNP + AS compared to CRSwNP + AS (r = − 0.73, P = 2 × 10 ), and the yel- CRSwNP-alone (Fig. 3c). low module the most positively correlated (r = 0.66, P = 0.001). Severe type 2 inflammation in nasal tissues Overall, there were 31 lncRNAs and 94 mRNAs in the of CRSwNP + AS blue module, and only one lncRNA (HK3-006) and 63 As indicated by above transcriptome data, type 1, type mRNAs in the yellow module. Pathway enrichment anal- 2 and type 3 related signalling pathways might be dif- ysis indicated that the blue module was mainly associated ferentiated between CRSwNP-alone and CRSwNP + AS. with IL-17 signalling pathway and cytokine–cytokine u Th s, we investigated the mRNA expression of critical receptor interaction (Fig. 5c), whereas the yellow module cytokines and their receptors, which indicate the activi- was mainly related to asthma, arachidonic acid metabo- ties of different inflammatory endotypes; namely (1) lism, and signalling pathways such as calcium and JAK- IFNG and IFNG receptor (IFNGR1 and IFNGR2) for type STAT signalling pathway (Fig.  5d). Top 50 hub genes 1 inflammation; (2) IL5, IL4, IL13 and their receptors identified from the blue and yellow modules, respectively, (IL5RA, IL5RB, IL4R, IL13RA1 and IL13RA2) for type are shown in Fig. 5 e, f. 2 inflammation; (3) IL17A and IL17A receptor (IL17RA and IL17RC) for type 3 inflammation; as well as (4) TNF, Discussion IL1B, IL6 and their receptors (TNFRSF1A, TNFRSF1B, Both CRSwNP and asthma are airway inflammatory IL1R1, IL1R2 and IL6R) for proinflammatory activity. disorders, which have serious effects on quality of life. We found that the expression of cytokines IL5 and A great proportion of CRSwNP patients have comor- IL13, and receptors IL5RA and IL5RB, indicating aug- bid asthma, which makes it one of the most challenging mented type 2 inflammation, was significantly enhanced phenotypes to treat [3, 12]. Furthermore, the molecular in both CRSwNP-alone and CRSwNP + AS compared endotypes of CRSwNP + AS are not clear. In this regard, to control (Fig.  3d and Additional file  1: Table  S4). Fur- to our knowledge this is the first study to investigate the thermore, nasal tissue from CRSwNP + AS demonstrated molecular endotypes of CRSwNP + AS compared to significantly higher expression of IL4, IL5 and IL13; and CRSwNP-alone, by whole-transcriptome RNA sequenc- conversely significantly lower expression of IL17A than ing. Our study demonstrated that type 2-high inflamma - nasal tissue of CRSwNP-alone. tion patterns and their associated transcriptome features Assessment of correlations between the significant were distinct in nasal tissues of CRSwNP + AS patients cytokines and the other significant inflammatory indi - compared to CRSwNP-alone patients. cators determined above, demonstrated that the expres- Studies have indicated that CRS is a heterogeneous dis- sion of both IL5 and IL13 was positively correlated with ease with several unclear endotypes, which are mainly the percentage of local eosinophils, concentration of tis- characterized by type 1, 2, and 3 inflammatory patterns sue IgE and the expression of LTC4S, which reflected an [15, 25–27]. Furthermore, some recent studies have imbalanced arachidonic acid metabolism (Fig.  4a–f ). In indicated that the most prevalent endotype in CRSwNP contrast, the expression of IL17A was negatively cor- is characterized by type 2 inflammation [28], and that related with tissue IgE and the expression of LTC4S type 2 inflammation in CRSwNP may be differenti - (Fig. 4g–i). ated into moderate and severe type 2 inflammatory pat - terns according to the intensity of inflammation [29]. In LncRNA signatures in nasal tissue of CRSwNP + AS accordance with these studies, the findings for the differ - We identified 229 up-regulated and 83 down-regulated entially expressed type 1, type 2 and type 3 inflammation- DE-lncRNAs in nasal tissues of CRSwNP + AS patients related genes and pathways in the present study suggest compared to nasal tissues of CRSwNP-alone patients that the CRSwNP + AS endotype is associated with a W ang et al. Clin Transl Allergy (2020) 10:26 Page 9 of 14 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 more severe type 2 inflammation, compared to CRSwNP- of cytokines IL-5, IL-4 and IL-13 [31]. Whilst more alone endotype. Furthermore, in accordance with the than 80% of Western white patients with CRSwNP are findings of Tomassen and colleagues [15] that severe characterized by type 2 inflammation, less than 50% of type 2 CRSwNP increased asthma prevalence, our study CRSwNP cases in East Asian countries show features of has also demonstrated that in addition to more frequent type 2 reactions [32, 33]. Like CRSwNP, asthma is also atopy, more local eosinophil infiltration, and higher level a complex and heterogeneous disease, and two major of tissue IgE, the chance of recurrence of CRSwNP was endotypes of asthma, type 2-high asthma and type 2-low also increased in patients with CRSwNP + AS compared asthma, have been described, based on underlying air- to patients with CRSwNP-alone. Indeed, we have previ- way immune-mediated inflammation [34, 35]. In this ously shown that high proportion of eosinophils in nasal regard approximately 50% to 60% of all patients with tissue act as a reliable prognostic indicator for CRSwNP severe asthma in Europe and the United States account recurrence [30]. Similarly, a more recent study has indi- for type 2-high asthma, compared to 38.5% of the severe cated that asthma in CRS patients was the only fac- asthmatic patients with type 2-high asthma in China tor that increases the chance of recurrence in CRSwNP [36–39]. Thus, whilst it is possible that generally more patients [6]; suggesting that comorbid asthma might also Western patients with CRSwNP + AS are also likely to be a strong indicator for CRSwNP recurrence. be characterised with type 2-high inflammation than Airway type 2 inflammation is mainly mediated by Asian patients CRSwNP + AS, this nevertheless needs to eosinophils, mast cells, Th2 cells, ILC2s and IgE-produc - be confirmed in well-designed multicenter studies in the ing B cells, in combination with increased production future. Wang et al. Clin Transl Allergy (2020) 10:26 Page 10 of 14 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 Our findings for RNA sequencing provides valuable demonstrated that regardless of the subtypes of CRSwNP, information for exploring the general molecular mecha- genes and pathways that most likely contribute to the nisms underlying the pathogenesis of CRSwNP. Similar pathogenesis of CRSwNP, appear to be mainly associ- to the findings of Peng and colleagues [40], our study has ated with cytokine and chemokine signalling pathway, W ang et al. Clin Transl Allergy (2020) 10:26 Page 11 of 14 staphylococcus aureus infection, eicosanoid metabolism inflammation in the nasal tissues of CRSwNP + AS and cell adhesion molecules. In this respect, the well- patients. The increased expression of PTGS1 and PTGIS known genes or biomarkers closely related to CRSwNP; may accelerate the conversion of arachidonic acid to for example CLC, POSTN, CCL18, IL13, TSLP and prostacyclins (PGI2) and prostaglandins (PGD2, PGE2), BPIFA1; were also identified as the top DE-mRNAs in which are also related to an aggravated airway inflam - both CRSwNP-alone and CRSwNP + AS groups. mation and asthmatic conditions [48, 49]. Moreover, However, the transcriptome signatures of the increased expression of ALOX15B in CRSwNP + AS CRSwNP + AS were characterized by distinct groups of might have an active proinflammatory role [50]. Collec - differentially expressed genes and their enriched path - tively, these findings suggest that enhanced activity of ways, compared to CRSwNP-alone. In particular, the pre- arachidonic acid metabolism might help to augment type sent study showed that the CRSwNP + AS endotype was 2 inflammation in CRSwNP + AS [45, 51]. mainly associated with higher activities of arachidonic Th2 and Th17 signalling pathways are two major reg - acid metabolism, Th2 signalling pathway and fibrinolysis ulatory pathways, which are inversely interrelated in pathway, and lower activity of IL-17 signalling pathway. patients with CRSwNP [17]. In accordanc ewith this, gen- Beyond that, the most significant DE-mRNAs between eral observation, our RNA sequencing data also revealed CRSwNP + AS and CRSwNP-alone also provide impor- higher activities of Th2 signalling pathway and lower tant information. In line with the high concentration of activities of IL-17 signalling pathway in CRSwNP + AS tissue IgE in CRSwNP + AS, increased mRNA expres- than in CRSwNP-alone. In this respect IL-4, IL-5 and sion of constant region of heavy chain of IgE (IGHE) was IL-13 are typical Th2 cytokines, which reflect the sever - also confirmed by RNA sequencing. Some recently iden - ity of type 2 inflammation, whereas IL-17 is a typical tified biomarkers of CRSwNP, for example CST1 [41], Th17 cytokine. Indeed, the present study further showed were also found to be differentially expressed between that the expression of IL5 and IL13 was positively cor- CRSwNP + AS and CRSwNP-alone. Importantly, the pre- related with other indicators of type 2 inflammation sent study showed that some of the most significant DE- (i.e. increased local eosinophils and tissue IgE). Simi- mRNAs; including ITLN1, KCNA3 and CCR10; which larly, genes that are related to arachidonic acid metabo- were up-regulated in nasal tissue of CRSwNP + AS com- lism were also correlated with the expression of type 2 pared to nasal tissue of CRSwNP-alone (Additional file  1: cytokines, thus, emphasising the crucial role of type 2 Figure S8), are also expressed in the bronchial tissue and inflammation in CRSwNP + AS patients. contribute to the pathogenesis of asthma [42–44]. This LncRNAs play important roles in various biologi- suggests that signature genes identified in nasal tissue cal processes and are emerging as reliable biomark- of CRSwNP + AS might also play important roles in the ers and potential therapeutic targets of human chronic pathogenesis of asthma. Moreover, consistent with the diseases [52, 53]. However, comparatively few studies united airways concept, it is tempting to speculate that have reported the involvement of lncRNAs in chronic the pattern of gene expression in the upper airway may nasal inflammation. Yue and colleagues [54] found that be influenced by the prevailing conditions in the lower linc00632 was down-regulated in nasal tissues of aller- airway, and as such prevalence of comorbid asthmatic gic rhinitis patients and inhibited IL-13 induced inflam - conditions might exacerbate type 2 inflammation in nasal matory cytokine and mucus production. Wang and tissue of CRSwNP patients. colleagues [55] showed that lncRNA XLOC_010280 It has been well demonstrated that alterations in the might regulate the expression of CCL18 and eosinophilic arachidonic acid pathway play an important role in air- inflammation in eosinophilic CRSwNP. In the present way inflammatory conditions like rhinosinusitis, nasal study, whole-transcriptome sequencing has revealed sev- polyps, allergic rhinitis, and asthma [45]. We found a eral dysregulated lncRNAs in both subtypes of CRSwNP, generally enhanced activity of arachidonic acid metabo- which will provide a useful pool of candidate lncRNAs in lism in CRSwNP + AS, indicated by the up-regulated future investigations of CRSwNP endotypes. expression of PLA2 (PLA2G4A, PLA2G4B, PLA2G4D WGCNA is an effective method of multigene analysis and PLA2G6), which may promotes the release of mem- to construct coexpression network, and has successfully brane-bound arachidonic acid [46]. The imbalanced syn - been applied for studying mRNAs and lncRNAs to dis- thesis of eicosanoids characterized by increased synthesis tinguish dysfunctional regulatory subnetworks, select of cysteinyl leukotrienes (CysLTs) is correlated with the out potentially key genes, and predict lncRNA func- inflammatory pattern and severity of the airway inflam - tions [56, 57]. We applied WGCNA to predict functions mation [45, 47]. Consistent with this, we also showed an of DE-lncRNAs and identify hub genes in the present increased expression of LTC4S, which promotes the bio- study and showed that common dysregulated lncRNAs synthesis of CysLTs and indicates the presence of severe in CRSwNP-alone and CRSwNP + AS have very similar Wang et al. Clin Transl Allergy (2020) 10:26 Page 12 of 14 specific information on the role of different cell types predictive functions to common DE-mRNAs, as indi- as biomarkers of CRSwNP with or without comorbid cated by genes in the maximal module. Furthermore, we asthma. The use of inferior turbinate tissue from controls identified LINC01146 as the only top hub lncRNA, which for comparison with nasal polyp tissues from CRSwNP might play a key role in the pathogenesis of CRSwNP. patients is also as a limitation of the study, particularly Although LINC01146 has also been found to be dysregu- as nasal polyps rarely arise from inferior turbinates in lated in hepatocellular carcinoma, its precise function in healthy control subjects. However, using mucosa tissue this condition is not clear [58]. Further study is needed to from different locations such as middle turbinate and validate the function of common dysregulated lncRNAs ethmoid tissue as controls may avoid regional variations in CRSwNP. in gene expression. The present study also identified HK3-006 as the only top hub lncRNA that was expressed in the module that most positively correlated with phenotypic traits of Conclusions CRSwNP + AS; and may be related to asthma pathway The present study demonstrated that CRSwNP patients and arachidonic acid metabolism as predicted by its with comorbid asthma have distinct type 2-high inflam - highly coexpressed mRNAs. Similarly, several top hub mation-associated transcriptome profiles, as indicated lncRNAs were identified in the module that most nega - by differential expression profiles of key mRNAs and tively correlated with phenotypic traits of CRSwNP + AS. lncRNAs in nasal tissue, compared to patients with Among these lncRNAs, we have identified a new CRSwNP-alone. Differences in molecular mechanisms lncRNA, LINC686, which is most likely to be associated and type 2 inflammation-related molecules in CRSwNP with IL-17 signalling pathway. Although an increasing with comorbid asthma may be useful in better under- number of studies show that lncRNAs are involved in the standing the mechanisms underlying the development regulation of cytokine signalling and inflammation [59, of different endotypes of CRS, as well as development 60], our understanding of the functions of lncRNA is just of potential biomarkers and targeted gene therapies for beginning to develop. CRSwNP in the future. The present study has some limitations. First, this study did not investigate any subgroups of CRSwNP + AS Supplementary information and CRSwNP-alone. This may be of importance as Supplementary information accompanies this paper at https ://doi. both CRSwNP and asthma are heterogeneous diseases, org/10.1186/s1360 1-020-00332 -z. and one recent study has shown that patients with CRSwNP + AS can be grouped into 3 subtypes with dis- Additional file 1: Table S1. Demographic and clinical characteristics of tinct inflammatory status and disease severity [19]. How - study subjects. Table S2. Common DE-mRNAs and DE-lncRNAs shared by ever, the present study was designed to directly compare CRSwNP-alone versus control and CRSwNP+AS versus control. Table S3. CRSwNP + AS with CRSwNP-alone. Furthermore, as a Top 50 DE-mRNAs of CRSwNP+AS versus CRSwNP-alone. Table S4. Expression of key cytokines and their receptors in nasal tissues from substantial number of Asian CRSwNP patients are type control subjects and CRSwNP-alone and CRSwNP+AS patients. Table S5. 1 and/or type 3 inclined, it would be more appropri- Top 50 DE-lncRNAs of CRSwNP+AS versus CRSwNP-alone. Figure S1. ate to compare type 2 CRSwNP endotype with type 2 Correlation of infiltrating eosinophils and total IgE in nasal tissues of patients with CRSwNP. Figure S2. Differentially expressed mRNAs and CRSwNP + AS endotype for a better understanding of differentially expressed lncRNAs in nasal tissues of CRSwNP patients. the mechanisms and pathways operating in either endo- Figure S3. Hierarchical clustering of differentially expressed genes. Figure type. Second, as the sample size used for RNA sequenc- S4. GO biological processes enriched by common dysregulated genes in CRSwNP+AS and CRSwNP-alone. FigureS5. Gene number of modules ing is relatively small, this might decrease the statistical identified by weighted gene co-expression network analysis. Figure S6. power for gene signature profiling. Third, we did not The expression and potential functions of LINC01146. Figure S7. Gene evaluate lower airway inflammation by assessment of modules identified by WGCNA based on expression of DE-mRNAs and DE- lncRNAs in CRSwNP+AS versus CRSwNP-alone. Figure S8. Asthma related inflammatory cells and cytokines in bronchial biopsy genes expressed in nasal tissue of patients with CRSwNP+AS and patients or induced sputum, which might be helpful in under- with CRSwNP-alone. standing the association between CRSwNP and asthma. Fourth, assessment of critical cytokines and their recep- Abbreviations tors was based only on RNA expression and need to be CRS: Chronic rhinosinusitis; CRSwNP: Chronic rhinosinusitis with nasal polyps; CRSwNP + AS: Chronic Rhinosinusitis with Nasal Polyps with Comorbid verified according to the levels of corresponding proteins Asthma; IL: Interleukin; lncRNA: Long non-coding RNA; DE: Differentially in the tissue. Fifth, whole tissue samples with multiple cell expressed; FPKM: Fragments per kilo-base of exon per million fragments types were used for RNA sequencing and no single-cell mapped; WGCNA: Weighted gene coexpression network analysis; KEGG: Kyoto Encyclopedia of Genes and Genomes. RNA sequencing was used. 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Miyata J, Fukunaga K, Kawashima Y, Ohara O, Arita M. Cysteinyl leukot- Publisher’s Note riene metabolism of human eosinophils in allergic disease. Allergol Int. Springer Nature remains neutral with regard to jurisdictional claims in pub- 2020;69(1):28–34. lished maps and institutional affiliations. 48. Ishiura Y, Fujimura M, Nobata K, Oribe Y, Abo M, Myou S. Prostaglandin I2 enhances cough reflex sensitivity to capsaicin in the asthmatic airway. Cough. 2007;3:2. Ready to submit your research ? Choose BMC and benefit from: fast, convenient online submission thorough peer review by experienced researchers in your field rapid publication on acceptance support for research data, including large and complex data types • gold Open Access which fosters wider collaboration and increased citations maximum visibility for your research: over 100M website views per year At BMC, research is always in progress. Learn more biomedcentral.com/submissions

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