| BackgroundChronic rhinosinusitis(CRS)is a debilitating inflammatory disease of the nasal mucosa.Nasal polyps(NP)are the most common comorbidity,affecting～30%of patients with CRS,and cause a considerablesocioeconomic burden and impaired quality of life.Clinical management of CRS with NP(CRSwNP)is largely ineffective,partly due to the limited understanding of the underlying pathogenic factors and apaucity of effective therapeutic interventions.Understanding the mechanisms that underlie CRSwNP pathogenesis may help identify targets in critical candidate pathways for therapeutic interventions.RNA sequencing is a powerful means to explore candidate transcripts in an unbiased manner,and to identify cellular and molecular pathways.The known biological processes implicated in CRSwNP are multifaceted,including airway inflammation,tight junction impairment pathogen infection and a defective host defence.The complexity of gene expression patterns in CRSwNP has not been adequately addressed,partly because the reference tissues often used have been suboptimal for biomarker profiling.For instance,differences in gene signatures between NP and control tissues,i.e.inferior turbinate(IT),from healthy subjects might stem from genetic or environmental factors,or both.Additionally,Gene Ontology(GO)analyses have mostly been based on differentially expressed genes(DEGs)identified by microarray assays,but not on gene sets or pathways.Finally,the limited sample sizes have markedly decreased the statistical power for gene signature profiling.So in this study,we conducted genome-wide gene expression analysis to determine candidate pathways and gene networks associated with CRSwNP.MethodsWe performed whole-transcriptomic RNA sequencing on 44 pairs of non-polyp inferior turbinate(CRSwNP-IT)and polyp(CRSwNP-NP)tissues from patients with CRSwNP and 41 inferior turbinate samples from non-CRS controls(CS-IT).To eliminate interindividual differences arising from genetic or environmental factors,we conducted a three-way comparison between the IT in control subjects(CS-IT)versus the paired NP(CRSwNP-NP)and IT(CRSwNP-IT)in patients with CRSwNPHiSeq FASTQ files were mapped to the human genome build hg38 using STAR.Gene counts were obtained with featureCounts using GENCODE version 26 annotations.Principal component analysis(PCA)was carried out in R version 3.3.3 using log2 reads per kilobase million(RPKM)to determine the differences between the samples in each group.DEG analysis was performed using edgeR as pairwise comparisons on genes filtered for log2 RPKM interquartile range>0.5.Venn diagrams were generated using R software.GO enrichment analysis using the Bioconductor package TopGO was performed to identify biological processes and protein functional groups enriched in groups of DEGs.Ingenuity Pathway Analysis was used to define gene sets-determined pathways associated with DEGs.Significance threshold was set to default.Gene Set Enrichment Analysis(GSEA)was conducted using the Bioconductor package fgsea to identify gene sets of enrichment patterns using the Molecular Signatures Database.ResultsPrincipal component-informed analysis at the whole-transcriptome level revealed cilium function and immune regulation as the two main gene ontology(GO)categories differentiating nasal tissues between CRSwNP patients and controls,The first principal component(PC1)comprised genes regulating ciliogenesis and ciliary function(i.e.cilium assembly,ultrastructural protein assembly,cilium motility,intraciliary transport),whereas PC2 mainly consisted of genes modulating inflammatory and immune responses(i.e.chemokine-mediated signalling,innate and adaptive immune response,T-cell proliferation,co-stimulation),and collagen and extracellular matrix(ECM)metabolism(i.e.complex of collagen trimmers,ECM structural constituent and organisation);We detected 6,182 differentially expressed genes(DEGs)between CRSwNP-NP and CS-IT,These DEGs were involved in collagen processing and organisation,inflammatory responses,and O-glycan processing.We detected 915 DEGs between CRSwNP-IT and CS-IT.The top GO sets enriched by these DEGs were involved in the interferon(IFN)signalling pathway and viral responses.Atopy status did not have a major impact on gene expression in various tissues.Ingenuity pathway analysis(IPA)identified significant enrichment of the type 1 interferon signaling and axonal guidance canonical pathways,angiogenesis and collagen and fibrotic changes in CRSwNP(CRSwNP-NP and CRSwNP-IT)tissues compared to control CS-IT.IPA highlights axonal guidance signalling as the common pathway in CRSwNP-NP,CRSwNP-ITand CS-IT.Finally,gene-set enrichment analysis implicated sets of genes co-regulated in processes associated with inflammatory response and aberrant cell differentiation in polyp formation.ConclusionGene signatures involved in defective host-defenses,inflammation and abnormal metabolism of the extracellular matrix are implicated in CRSwNP:(1)The resuluts highlight pronounced differences in gene signatures(particularly cilia-associated and immune regulation genes)in nasal tissues.(2)The resuluts revealed strong DEG signals related to viral responses and airway remodelling in CRSwNP.(3)The findings imply that exaggerated inflammatory responses and epithelial growth may predispose NP formation and that axonal guidance signalling represents a critical target for NP management.(4)The resuluts indicate that gene sets associated with the inflammatory and immune responses are enriched in NP,and support that inflammation and aberrant cell differentiation underlie early-stage NP formation Functional validation of these gene expression patterns will open opportunities for the development of CRSwNP therapeutic interventions such as biologics and immunomodulators.BackgroundNasal polyp(NP)is a common chronic upper airway inflammatory disorder that frequently co-exists with lower airway inflammatory diseases such as asthma.In Asian population,NP is mostly characterized by prominent epithelial remodeling and mixed inflammatory phenotypes.Physiologically,the respiratory cilia maintain proper clearance of pathogens and allergens.We have previously demonstrated the poorly proliferated basal cells and up-regulation of ciliogenesis markers[centrosomal protein 110(CP 110)and fork-head box J1(FOXJ1)]in the aberrantly remodeled epithelium of NP.Furthermore,abnormal ciliary morphology(i.e.,overly dense and lengthened cilia),abnormal expression of dynein axonemal heavy chain 5(DNAH5,the marker crucial to microtubule sliding),and the significantly reduced ciliary beat frequency(CBF)have also been observed in NP.Therefore,abnormal ciliogenesis and/or ultrastructure might be critical drivers of impaired mucociliary clearance(MCC),contributing to the chronic inflammation in NPs.Physiologically,ciliary motility is mainly regulated by the outer dynein arms and radial spoke(RS)complexes.To ensure proper ciliary motility,ciliogenesis is crucial to the formation of axonemes where ciliary ultrastructural proteins are assembled.CP110 reportedly regulated centrosome duplication and centriole conversion to basal bodies.Inflammation-mediated up-regulation of CP110 contributed to defective cilia assembly and decreased motility in CRS.Additionally,increased FOXJ1-positive cell count correlated with longer and denser cilia in NP.Collectively,abnormal expression of CP110 and FOXJ1 may have resulted in disrupted cilia assembly in NP.Currently,most reports focused on isolated ciliogenesis or ciliary ultrastructural marker.The association between ciliogenesis or ciliary ultrastructural marker expression remains understudied.Furthermore,abnormal expression of some ultrastructural markers(i.e.DNAH5)was reportedly present in congenital diseases such as PCD.Because secondary ciliary dyskinesia(SCD)is common among various chronic inflammatory diseases,we hypothesized that the inflammatory milieu might be the critical driver of the defective ciliogenesis and abnormality of ultrastructural markers in NP.Building on our previous research,we sought to systematically investigate the expression patterns of four ciliary ultrastructural markers(RSPH1,RSPH4A,RSPH9 and DNAH5)and two ciliogenesis markers(CP110 and FOXJ1)for their manifestation of ciliary impairment in NPs.Our findings might help elucidate the roles of ciliogenesis and ciliary ultrastructural markers in NP pathogenesis,and whether chronic airway inflammation is responsible for the abnormal expression of ciliary ultrastructural markers.MethodsNP biopsy samples were obtained from 97 NP patients and inferior turbinate from 32 healthy controls.Immunofluorescence staining,quantitative polymerase chain reaction and single-cell cytospin staining were performed.We classified the patterns of radial spoke head protein(RSPH)1,4A(RSPH4A),9(RSPH9)and dynein axonemal heavy chain 5(DNAH5)localization.Immunofluorescence imaging of RSPH 1,RSPH4A,RSPH9,and DNAH5 shared three patterns:(i)Pattern A,markers located throughout the entire axoneme;(ii)Pattern B,markers partly missing at distal axoneme.(iii)Pattern C,markers completely missing throughout the axoneme.To determine the magnitude of abnormality of ciliary ultrastructural markers,we developed a semi-quantitative scoring system for which 0 denoted pattern A>70%,1 denoted patterns A+B>70%,and 2 denoted pattem C≥30%to assess co-localization.The semi-quantitative scoring system was used to assess their expression patterns and associations with ciliogenesis markers[centrosomal protein 110(CP110)and forkhead box J1(FOXJ1)].Results(1)Based on our semi-quantitative scoring system,the median(the 1st and 3rd quartile)scores of RSPH1,RSPH4A,RSPH9 and DNAH5 were 0.2(0.2,0.5),0.2(0,0.6),0.2(0,0.8)and 0.4(0.2,0.7)in IT for paraffin sections,respectively.Conversely,the median(the 1st and 3rd quartile)scores of RSPH1,RSPH4A,RSPH9 and DNAH5 were significantly higher[1.0(0.6,1.5),1.2(0.8,1.6),1.3(0.8,1.8)and 1.2(0.8,1.6)]in patients with NPs(all P<0.001),particularly in eosinophilic NPs.Expression pattern scores of RSPH1,RSPH4A,RSPH9 and DNAH5 correlated positively with each other in both groups.(2)In single-cell cytospin slides,the percentage of pattern A-C was 50.0%,22.0%and 28.0%for RSPH1,58.0%,12.0%and 30.0%for RSPH4A,48.0%,18.0%and 34.0%for RSPH9,and 51.5%,13.5%and 35.0%for DNAH5 in patients with NP,respectively.However,the percentage of patterns A-C among control subjects was 72.0%,18.0%and 10.0%for RSPH1,88.0%,6.0%and 6.0%for RSPH4A,72.0%,12.0%and 16.0%for RSPH9,and 74.0%,14.0%and 12.0%for DNAH5,respectively(all P<0.05).(3)CP110 staining showed a localized and thin pattern in control subjects,while a diffuse and thick pattern was seen in NP.The median(the 1st and 3rd quartile)of TFI for CP110 was significantly higher in NP than in IT[1.3(1.1,1.6)vs.0.7(0.5,0.9)×106 arbitrary units,P<0.01].FOXJ1 was stained within the nucleus of ciliated and non-ciliated epithelial cells.Compared with control subjects,the TFI for FOXJ1 staining was markedly greater in NP than in IT[2.7(2.1,3.7)vs.1.7(1.3,2.1)×106 arbitrary units,P<0.01].The total fluorescence intensity of CP110 and FOXJ1 correlated positively with expression pattern scores of RSPH1,RSPH4A,RSPH9 and DNAH5.A trend towards lengthened cilia was observed in NP.ConclusionIn conclusion,a greater prevalence of absence of RSPH1,RSPH4A,RSPH9,and DNAH5 expressions is observed in NP,which is associated with the up-regulation of ciliogenesis markers(CP110 and FOXJ1)and greater cilia length in the chronic inflammatory milieu.Our integrated findings may help elucidate the roles and events leading to the manifestation of impaired ciliogenesis that may lead to cilia ultrastructural abnormalities in driving NP formation.Given the chronic inflammation and significant tendency of recurrence even after surgery,ciliary functions should be comprehensively appraised and managed as an important therapeutic strategy for patients with NP.BackgroundAsthma is a debilitating,heterogeneous inflammatory disease that often co-exists with upper airway diseases.The healthcare burden of frequent exacerbations and hospitalizations is substantial in patients with severe asthma(SA)who frequently remain symptomatic despite standardized treatment.The airway epithelium is made up of ciliated cells--the barrier against pathogens,xenobiotics and aeroallergens.When this barrier is breached,alarmins(i.e.,thymic stromal lymphopoietin,interleukin-33 and interleukin-25)will induce T1 or T2 pathways leading to neutrophilic or eosinophilic inflammation,which are key features of asthma.Apart from inflammation and airway remodeling,impaired mucociliary clearance may have an important role in modulating asthma pathogenesis.Impaired mucociliary clearance,where the cilia beat asynchronously,arises from defective expression of ultrastructural markers that stemmed from genetic defects leading to ciliary dyskinesia,and/or secondary causes related to chronic airway inflammation.Impaired mucociliary clearance is tightly linked to the persistence and severity of asthma because it predisposes patients to further infections and allergies.Thus,research in impaired mucociliary clearance can lead to better understanding of the cross-talk between upper and lower airway in driving asthma persistence and severity.In view of the concep of "the same airway,the same disease",the assessment of motile ciliary disorder(MCD)may provide a more systematic appraisal of asthma on the basis of asthma severity,upper airway diseases and airway inflammatory endotype assessment.Whether motile ciliary disorders of nasal epithelium correlate with asthma is unclear.MethodsNasal brushing cytospins were obtained from 41 asthmatic patients(33 with severe and 8 with mild-moderate asthma)and 10 healthy controls.We collected the demographics,smoking history,medication usage,details on atopy and upper and lower airway diseases[e.g.asthma,Allergic rhinitis(AR)and/or Chronic Rhinosinusitis(CRS)with or without nasal polyps].Upper airway:Hematoxylin-eosin and immunohistochemistry staining was employed for enumeration of eosinophils and neutrophils,and we analyzed immunofluorescence-staining patterns of DNAH5 and DNAI1 for nasal brushing cytospin slides.Lower airway:Asthmatic subjects underwent sputum induction and performed Hematoxylin-eosin and immunohistochemistry staining to assess the inflammatory endotype,and pulmonary function test was performed to evaluate the pulmonary function of sasthmatic patients.By comprehensively analyzing the results above,we assessed the factors associated with MCD in asthma,and systematically evaluated asthma by incorporating the information of MCD.Results(1)AR and CRS accounted for 61.0%and 78.0%of asthmatic patients.Of 33 asthmatic patients with CRS,24 had nasal polyps.Only three asthmatic patients(7.3%)had neither AR nor CRS.(2)Sputum and nasal inflammatory endotypes were heterogeneous,with sputum eosinophilia and nasal neutrophilia being more prominent in severe asthma.(3)Abnormal immunofluorescence staining patterns of DNAH5 and DNAI1 were significantly more common in asthmatic patients than in healthy subjects(both p<0.05),but not in severe compared with non-severe asthma(both p>0.05).(4)34.8%of asthmatic patients(including those without upper airway disease)compared with no healthy control had abnormal staining patterns of dual markers.(5)The prevalence of abnormal staining patterns of ciliary markers was greater in severe asthma with upper airway diseases than those without.Abnormal staining patterns were more common in mild-to-moderate asthma with upper airway diseases than those without.ConclusionWe have identified:(1)the high prevalence of upper airway diseases(AR,CRS and nasal polyps)in asthma;(2)the significant heterogeneity of upper and lower airway inflammatory endotypes which cannot solely interpret the clinical characteristics of asthma;(3)the higher prevalence of MCD in asthmatic patients without upper airway diseases compared with healthy controls;(4)a trend towards more prominent MCD in patients with upper airway disease who had SA and MMA.So endotyping of asthma by incorporating ciliary assessment of upper airways may provide important complementary insights into asthma pathogenesis. |
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