scholarly journals Comparison of Subtyping Approaches and the Underlying Drivers of Microbial Signatures for Chronic Rhinosinusitis

mSphere ◽  
2019 ◽  
Vol 4 (1) ◽  
Author(s):  
Kristi Biswas ◽  
Raewyn Cavubati ◽  
Shan Gunaratna ◽  
Michael Hoggard ◽  
Sharon Waldvogel-Thurlow ◽  
...  
Keyword(s):  
Tight Junction ◽  
Chronic Rhinosinusitis ◽  
Inflammatory Markers ◽  
Nasal Polyps ◽  
Epithelial Barrier ◽  
Clear Understanding ◽  
Microbial Composition ◽  
Data Set ◽  
Heterogeneous Condition ◽  
Gene And Protein Expression

ABSTRACT Chronic rhinosinusitis (CRS) is a heterogeneous condition characterized by persistent sinus inflammation and microbial dysbiosis. This study aimed to identify clinically relevant subgroups of CRS patients based on distinct microbial signatures, with a comparison to the commonly used phenotypic subgrouping approach. The underlying drivers of these distinct microbial clusters were also investigated, together with associations with epithelial barrier integrity. Sinus biopsy specimens were collected from CRS patients (n = 23) and disease controls (n = 8). The expression of 42 tight junction genes was evaluated using quantitative PCR together with microbiota analysis and immunohistochemistry for measuring mucosal integrity and inflammation. CRS patients clustered into two distinct microbial subgroups using probabilistic modelling Dirichlet (DC) multinomial mixtures. DC1 exhibited significantly reduced bacterial diversity and increased dispersion and was dominated by Pseudomonas, Haemophilus, and Achromobacter. DC2 had significantly elevated B cells and incidences of nasal polyps and higher numbers of Anaerococcus, Megasphaera, Prevotella, Atopobium, and Propionibacterium. In addition, each DC exhibited distinct tight junction gene and protein expression profiles compared with those of controls. Stratifying CRS patients based on clinical phenotypic subtypes (absence or presence of nasal polyps [CRSsNP or CRSwNP, respectively] or with cystic fibrosis [CRSwCF]) accounted for a larger proportion of the variation in the microbial data set than with DC groupings. However, no significant differences between CRSsNP and CRSwNP cohorts were observed for inflammatory markers, beta-dispersion, and alpha-diversity measures. In conclusion, both approaches used for stratifying CRS patients had benefits and pitfalls, but DC clustering provided greater resolution when studying tight junction impairment. Future studies in CRS should give careful consideration to the patient subtyping approach used. IMPORTANCE Chronic rhinosinusitis (CRS) is a major human health problem that significantly reduces quality of life. While various microbes have been implicated, there is no clear understanding of the role they play in CRS pathogenesis. Another equally important observation made for CRS patients is that the epithelial barrier in the sinonasal cavity is defective. Finding a robust approach to subtype CRS patients would be the first step toward unravelling the pathogenesis of this heterogeneous condition. Previous work has explored stratification based on the clinical presentation of the disease (with or without polyps), inflammatory markers, pathology, or microbial composition. Comparisons between the different stratification approaches used in these studies have not been possible due to the different cohorts, analytical methods, or sample sites used. In this study, two approaches for subtyping CRS patients were compared, and the underlying drivers of the heterogeneity in CRS were also explored.

scholarly journals Comparison of subtyping approaches and the underlying drivers of microbial signatures for chronic rhinosinusitis

2018 ◽  
Author(s):  
Kristi Biswas ◽  
Raewyn Cavubati ◽  
Shan Gunaratna ◽  
Michael Hoggard ◽  
Sharon Waldvogel-Thurlow ◽  
...  
Keyword(s):  
Tight Junction ◽  
Chronic Rhinosinusitis ◽  
Nasal Polyps ◽  
Expression Profiles ◽  
Alpha Diversity ◽  
Careful Consideration ◽  
Future Studies ◽  
Microbial Dysbiosis ◽  
Gene And Protein Expression ◽  
Protein Expression Profiles

Chronic rhinosinusitis (CRS) is a heterogeneous condition characterised by persistent sinus inflammation and microbial dysbiosis. This study aimed to identify clinically relevant sub-groups of CRS patients based on distinct microbial signatures, with a comparison to the commonly used phenotypic subgrouping approach. The underlying drivers of these distinct microbial clusters were also investigated, together with associations with epithelial barrier integrity. Sinus biopsies were collected from CRS patients (n=23), and disease controls (n=8). Expression of 42 tight junction genes was evaluated using quantitative PCR, together with microbiota analysis and immunohistochemistry for measuring mucosal integrity and inflammation. CRS patients clustered into two distinct microbial sub-groups using probabilistic modelling Dirichlet (DC) multinomial mixtures. DC1 exhibited significantly reduced bacterial diversity, increased dispersion, and was dominated by Pseudomonas, Haemophilus, and Achromobacter. DC2 had significantly elevated B-cells, incidence of nasal polyps, and higher numbers of Anaerococcus, Megasphaera, Prevotella, Atopobium, and Propionibacterium. In addition, each DC exhibited distinct tight junction gene and protein expression profiles compared with controls. Stratifying CRS patients based on clinical phenotypic subtypes (absence or presence of nasal polyps (CRSsNP or CRSwNP respectively) or with cystic fibrosis (CRSwCF)) did account for a larger proportion of the variation in the microbial dataset compared with DC groupings. However, no significant differences between CRSsNP and CRSwNP cohorts were observed for inflammatory markers, beta-dispersion and alpha diversity measures. In conclusion, both stratification approaches used had benefits and pitfalls, but DC clustering did provide greater resolution when studying tight junction impairment. Future studies in CRS should give careful consideration into the patient subtyping approach used.

scholarly journals Microbiome profiling of uncinate tissue and nasal polyps in patients with chronic rhinosinusitis using swab and tissue biopsy

PLoS ONE ◽  
2021 ◽  
Vol 16 (4) ◽  
pp. e0249688
Author(s):  
Sung-Woo Cho ◽  
Dong-Young Kim ◽  
Sungmi Choi ◽  
Sungho Won ◽  
Hye-Ryun Kang ◽  
...  
Keyword(s):  
Chronic Rhinosinusitis ◽  
Nasal Polyps ◽  
Sampling Methods ◽  
Clinical Severity ◽  
Rrna Gene ◽  
Microbial Composition ◽  
Tissue Samples ◽  
Tissue Biopsy ◽  
Two Samples ◽  
Nasal Swabs

Chronic rhinosinusitis (CRS) is characterized according to the presence or absence of nasal polyps (NPs) and displays nasal microbiota dysbiosis. However, optimal sampling methods of the nasal microbiome in CRS have not been identified. We aimed to assess the microbial composition in patients with CRS, comparing different sampling methods (swab and tissue biopsy), tissue types (uncinate tissue and NP), and disease subtypes. Samples were obtained by swabbing the middle meatus and taking a biopsy of uncinate tissue (UT) in patients with CRS with (CRSwNP, N = 8) or without NP (CRSsNP, N = 6) and controls (N = 8). NPs were also harvested in CRSwNP. DNAs were extracted from fifty-two samples and analyzed by 16S rRNA gene amplicon sequencing. As a result, a great interpersonal variance was observed in nasal swabs, while UT samples presented distinct microbiome with low inter-personal differences. Moreover, the UT microbiomes were further differentiated into three clusters which are associated with disease status (control, CRSsNP, and CRSwNP). Compared to UT, NP revealed a unique microbiome profile with significantly less bacterial diversity. Prevotella was the genus whose abundance was negatively correlated with disease severity in NP. In conclusion, tissue samples are better specimens than nasal swabs for assessing the microbiomes of CRS patients. Several bacteria in UT and NP tissues revealed an association with clinical severity of CRSwNP.

Faecal Proteases from Pouchitis Patients Activate Protease Activating Receptor-2 to Disrupt the Epithelial Barrier

2019 ◽  
Vol 13 (12) ◽  
pp. 1558-1568 ◽  
Author(s):  
Sarit Hoffman ◽  
Nathaniel Aviv Cohen ◽  
Ian M Carroll ◽  
Hagit Tulchinsky ◽  
Ilya Borovok ◽  
...  
Keyword(s):  
Tight Junction ◽  
Proteolytic Activity ◽  
Epithelial Barrier ◽  
Dependent Manner ◽  
Epithelial Permeability ◽  
Tight Junction Proteins ◽  
Microbial Composition ◽  
Junction Protein ◽  
Activating Receptor ◽  
Junction Proteins

Abstract Background and Aims The pathogenesis of pouch inflammation may involve epithelial barrier disruption. We investigated whether faecal proteolytic activity is increased during pouchitis and results in epithelial barrier dysfunction through protease activating receptor [PAR] activation, and assessed whether the intestinal microbiome may be the source of the proteases. Methods Faecal samples were measured for protease activity using a fluorescein isothiocyanate [FITC]-casein florescence assay. Caco-2 cell monolayers were exposed to faecal supernatants to assess permeability to FITC-dextran. Tight junction protein integrity and PAR activation were assessed by immunoblot and immunofluorescence. A truncated PAR2 protein in Caco-2 cells was achieved by stable transfection using CRISPR/Cas9 plasmid. PAR2 activation in pouch biopsies was examined using antibodies directed to the N-terminus of the protein. Microbial composition was analysed based on 16S rRNA gene sequence analysis. Results Ten pouchitis patients, six normal pouch [NP] patients and nine healthy controls [HC] were recruited. The pouchitis patients exhibited a 5.19- and 5.35-fold higher faecal protease [FP] activity [p ≤ 0.05] compared to the NP and HC participants, respectively. The genus Haemophilus was positively associated with FP activity [R = 0.718, false discovery rate < 0.1]. Faecal supernatants from pouchitis patients activated PAR2 on Caco-2 monolayers, disrupted tight junction proteins and increased epithelial permeability. PAR2 truncation in Caco-2 abrogated faecal protease-mediated permeability. Pouch biopsies obtained from pouchitis patients, but not from NP patients, displayed PAR2 activation. Conclusions Protease-producing bacteria may increase faecal proteolytic activity that results in pouch inflammation through disruption of tight junction proteins and increased epithelial permeability in a PAR2-dependent manner. This mechanism may initiate or propagate pouch inflammation.

Tissue and Exosomal Serine Protease Inhibitors Are Significantly Overexpressed in Chronic Rhinosinusitis With Nasal Polyps

2019 ◽  
Vol 33 (4) ◽  
pp. 359-368 ◽  
Author(s):  
S. K. Mueller ◽  
A. L. Nocera ◽  
S. T. Dillon ◽  
T. A. Libermann ◽  
O. Wendler ◽  
...  
Keyword(s):  
Western Blot ◽  
Protease Inhibitors ◽  
Chronic Rhinosinusitis ◽  
Liquid Biopsy ◽  
Nasal Polyps ◽  
Data Set ◽  
Western Blots ◽  
Institutional Review ◽  
Proteolytic Cascade ◽  
Whole Transcriptome Sequencing

Background The fibrinolysis pathway has been previously implicated in the etiopathogenesis of chronic rhinosinusitis with nasal polyps (CRSwNP). Objective The purpose of this study was (1) to explore protein derangements of selected protease inhibitors of the serpin superfamily in CRSwNP and (2) to correlate the protease inhibitor derangements of the fibrinolysis pathway in tissue with exosomal samples to evaluate the potential of an exosomal noninvasive “liquid biopsy” for CRSwNP. Methods Institutional review board approved study in which matched tissue and mucus exosomal proteins (SerpinB2, SerpinF2, SerpinG1, and SerpinE1) were compared between control and CRSwNP patients using Western Blot analysis (n = 6/group) and immunohistochemistry (IHC). Transcriptome analysis (n = 10/group) on the same proteins was performed using whole transcriptome sequencing. Semiquantitative analysis of the Western Blots was performed using the Whitney–Mann U test. Results The transcriptomic data set showed multiple differentially expressed genes including SerpinB2 (fold changes [FC] 7.38), SerpinE1 (FC 1.42), SerpinF2 (FC 2.03), and SerpinG1 (FC 0.72). Western Blot and IHC analysis showed an overexpression of the Serpin protease inhibitors in tissue (SerpinB2, P < .01; SerpinE1, P < .01; SerpinF2, P < .01; and SerpinG1, P < .01) indicating a downregulation of the fibrinolysis cascade. The mucus exosomal serpin proteins exhibited similar findings. Conclusion Our analysis supported that protease inhibitors of the fibrinolysis pathway, especially SerpinB2, SerpinF2, and SerpinG1, are highly deranged in patients with CRSwNP. These findings suggest a downregulation of the fibrinolysis pathway via proteolytic cascade imbalance leading to excessive polyp fibrin deposition. Our data further supported our hypothesis that exosomal proteomic analyses may be used as noninvasive “liquid biopsy” for CRSwNP and a novel method to study chronic sinonasal inflammation.

scholarly journals Mucosal expression of aquaporin 5 and epithelial barrier proteins in chronic rhinosinusitis with and without nasal polyps

2014 ◽  
Vol 35 (3) ◽  
pp. 377-383 ◽  
Author(s):  
Alan H. Shikani ◽  
Venkataramana K. Sidhaye ◽  
Randall J. Basaraba ◽  
Henry J. Shikani ◽  
Mohanned A. Alqudah ◽  
...  
Keyword(s):  
Chronic Rhinosinusitis ◽  
Nasal Polyps ◽  
Epithelial Barrier ◽  
Aquaporin 5

scholarly journals Microbiome profiling reveals distinct microbial compositions of nasal polyps in chronic rhinosinusitis

2020 ◽  
Author(s):  
Sung-Woo Cho ◽  
Dong-Young Kim ◽  
Sungmi Choi ◽  
Hye-Ryun Kang ◽  
Hana Yi
Keyword(s):  
Chronic Rhinosinusitis ◽  
Nasal Polyps ◽  
Sampling Methods ◽  
Clinical Severity ◽  
Rrna Gene ◽  
Microbial Composition ◽  
Principal Coordinates Analysis ◽  
Principal Coordinates ◽  
Nasal Swabs ◽  
Disease Subtypes

Abstract Background: Chronic rhinosinusitis (CRS) is characterized according to the presence or absence of nasal polyps (NPs) and displays nasal microbiota dysbiosis. However, optimal sampling methods of the nasal microbiome in CRS have not been identified. We therefore aimed to assess the microbial composition in patients with CRS, comparing different sampling methods (swab and tissue biopsy), tissue types (uncinate tissue and NP), and disease subtypes.Methods: Samples were obtained by swabbing the middle meatus and taking a biopsy of uncinate tissue (UT) in 8 patients with CRS with NP (CRSwNP), 6 patients with CRS without NP (CRSsNP), and 8 controls. NPs were also harvested in CRSwNP. Extracted DNA samples were analyzed by 16S rRNA gene amplicon sequencing.Results: The microbiome diversity did not significantly differ between disease subtypes in nasal swabs or UT samples. However, a principal coordinates analysis based on weighted UniFrac distances revealed a greater interpersonal variance for nasal swabs than for UT. UT samples presented with a distinct pattern and composition in CRSwNP compared to CRSsNP or control. Compared to UT, NP revealed a unique microbiome profile with significantly less bacterial diversity. Prevotella was the most abundant genus in the UT regardless of the disease subtype and its prevalence was significantly reduced in NP. Prevotella abundance was negatively correlated with disease severity as measured by Lund-Mackay scoring.Conclusion: Tissue samples are better specimens than nasal swabs for assessing the microbiomes of CRS patients. Microbiomes of NP tissues revealed an association with clinical severity of CRSwNP.

scholarly journals Inflammatory markers in the nasal mucosa and polyp-modified tissues in chronic rhinosinusitis with nasal polyps

2021 ◽  
Vol 10 (3) ◽  
pp. 29-38
Author(s):  
D.G. Pavlush ◽  
◽  
I.V. Dyuizen ◽  
Keyword(s):  
Chronic Rhinosinusitis ◽  
Nasal Mucosa ◽  
Inflammatory Markers ◽  
Nasal Polyps ◽  
Chemical Structure ◽  
Comparison Group ◽  
Pathological Process ◽  
Molecular Rearrangement ◽  
Clinical Prognosis ◽  
Neurokinin Receptors

Introduction. To date, chronic rhinosinusitis with nasal polyps (CRSwNP) has not yet been extensively studied: the molecular factors and mechanisms involved in the initiation of polypous transformations in nasal mucosa (NM) and sustaining their recurrence probability are still to be determined. Simultaneously, it is necessary to understand the molecular rearrangement in NM tissues to make clinical prognosis and choose an adequate therapeutic or surgical strategy for CRSwNP treatment. The aim of the study was to identify the features of how inflammatory markers localize and are distributed in the NM and polyps in various morphological CRSwNP types. Materials and methods. We studied morphological and chemical structure of nasal polyps and mucosa of the inferior turbinates. The material was obtained during surgical management of patients diagnosed with CRSwNP. The comparison group involved the patients with a deviated septum who underwent septorhi-noplasty and had neither polyposis nor concomitant inflammatory/allergic pathology. The NM removed in surgeries was used to compare morphological and chemical changes. Immunohistochemistry was applied to determine the localization and distribution of SP, NK1, nNOS, iNOS, and IL1b in the tissues. Results. The formation of nasal polyps was found to be accompanied by morphological and chemical altera-tions in the mucous membrane of the inferior turbinates. In polyps of different morphological types, the changes in the activity of inflammatory markers were specific. Conclusion. The data obtained indicate that changes in the NM of the inferior turbinates, which accompany polyposis development, give certain pathological causes that induce and maintain the pathological process. We have revealed the features of the specific signaling microenvironment in the nasal cavity, which provide special conditions for the formation of polyps of various types. The specificity of the activity and distribu-tion of inflammatory markers in the polyps of different morphological types may serve as a prerequisite for the development of personalized therapy for the disease. Keywords: chronic rhinosinusitis with nasal polyps, inflammation, neurokinin receptors, substance P, nitric oxide

scholarly journals Upregulation of Basonuclin1 Is Associated with p63-Involved Epithelial Barrier Impairment and Type-2 Helper T-cell Inflammation in Chronic Rhinosinusitis with Nasal Polyps

2021 ◽  
pp. 1-12
Author(s):  
Yunbo Gao ◽  
Jingyun Li ◽  
Jian Jiao ◽  
Ying Li ◽  
Chengshuo Wang ◽  
...  
Keyword(s):  
T Cell ◽  
Chronic Rhinosinusitis ◽  
Nasal Polyps ◽  
Mrna Level ◽  
Mucosal Inflammation ◽  
Epithelial Barrier ◽  
Helper T Cell ◽  
Downstream Target ◽  
Nasal Tissue

<b><i>Background:</i></b> Tumor protein p63 has been shown to be important for epithelial dysfunction, including epithelial barrier defects and mucosal inflammation, in the development of chronic rhinosinusitis with nasal polyps (CRSwNP). Basonuclin1 (BNC1), an epithelial-specific transcriptional factor, is a direct downstream target of p63 and thus might be involved in the pathogenesis of CRSwNP. <b><i>Objective:</i></b> We sought to investigate whether BNC1 was associated with p63-mediated epithelial barrier defects and nasal mucosal inflammation in CRSwNP. <b><i>Methods:</i></b> Nasal tissue biopsies were obtained from 91 patients to CRSwNP, 49 chronic rhinosinusitis without nasal polyps (CRSsNP) patients, and 28 control subjects. Immunohistochemistry and immunofluorescence staining were used to determine the distribution of BNC1 in tissues and localization in cells, respectively. Quantitative PCR was performed to detect the expression levels of <i>BNC1</i>, <i>TP63</i>, epithelial barrier proteins, and type-2 helper T-cell inflammation-related genes. <b><i>Results:</i></b> <i>BNC1</i> mRNA expression was significantly elevated in the tissues in CRSwNP patients compared with CRSsNP (1.96-fold, <i>p</i> = 0.0003) and control groups (2.40-fold, <i>p</i> &#x3c; 0.0001). <i>BNC1</i> staining was strongly positive in the nasal epithelium and co-localized with p63-positive epithelial cells. The expression of <i>BNC1</i> mRNA was strongly correlated with <i>TP63</i> mRNA level both in tissue biopsies (<i>r</i> = 0.78, <i>p</i> &#x3c; 0.0001) and epithelial scrapings (<i>r</i> = 0.97, <i>p</i> &#x3c; 0.0001). <i>BNC1</i> expression was also positively correlated with epithelial barrier protein genes (<i>CDH1</i>, <i>CLDN1</i>, <i>CLDN4</i>, <i>TJP1,</i> and <i>TJP2</i>) and epithelial genes involved in T<sub>H</sub>2 inflammation (<i>IL33</i>, <i>CCL26</i>, <i>CLC</i>, and <i>ALOX15</i>). <b><i>Conclusions:</i></b> Overexpression of <i>BNC1</i> may be associated with increased expression of <i>TP63</i>, and possibly contribute to the epithelial barrier defects and T<sub>H</sub>2 inflammation in CRSwNP.

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