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 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 IL-19 Induced by IL-13/IL-17A in the Nasal Epithelium of Patients with Chronic Rhinosinusitis Upregulates MMP-9 Expression via ERK/NF-kB Signaling Pathway

2020 ◽  
Author(s):  
Xia Li ◽  
Jiancong Huang ◽  
Xiaohong Chen ◽  
Xiaoping Lai ◽  
Zizhen Huang ◽  
...  
Keyword(s):  
Western Blot ◽  
Chronic Rhinosinusitis ◽  
Nasal Polyps ◽  
Tissue Remodeling ◽  
Tissue Inhibitors Of Metalloproteinases ◽  
Tissue Samples ◽  
Nasal Tissue ◽  
Human Nasal Epithelial Cells ◽  
Pathway Activation

Abstract Background: Tissue remodeling is a crucial characteristic of chronic rhinosinusitis (CRS). Imbalance between matrix metalloproteinases (MMPs) and tissue inhibitors of metalloproteinases (TIMPs) is crucial for the pathologic tissue remodeling in CRS. Elevation of interleukin (IL)-19 or MMP-9 levels in patients with CRS had been proven in previous studies. Here, we aimed to investigate the role of IL-19 in mediating MMP-9 expression in CRS. Methods: Nasal tissue samples were collected from 45 individuals having chronic rhinosinusitis with nasal polyps (CRSwNP), 24 CRS without nasal polyps (CRSsNP), and 17 controls. Expression of IL-19, its receptors (IL-20R1/IL-20R2), and MMP-9 were investigated using RT-qPCR and Immunofluorescence. Human nasal epithelial cells (HNECs) were stimulated by IL-19; ERK phosphorylation, NF-kB pathway activation, and MMP-9 level were detected by RT-qPCR, ELISA, western blot and Immunofluorescence. We also explored the effect of type1/2/3 cytokines on IL-19 production by RT-qPCR, and western blot. Results: Expression levels of IL-19, its receptors (IL-20R1/IL-20R2), and MMP-9 were increased in nasal tissues from individuals with CRSwNP compared to those with CRSsNP as well as the controls. IL-19 significantly elevated the production of MMP-9 in HNECs. Furthermore, IL-19 could activate the ERK and NF-kB pathways, accompanied by increased MMP-9 production in HNECs. Conversely, both ERK and NF-kB inhibitors significantly attenuated the role of IL-19 in MMP-9 production. siRNA knockdown of IL-20R1 suppressed ERK and NF-kB pathway activation, thereby decreasing MMP-9 expression. IL-13 and IL-17A were found to stimulate IL-19 production in HNECs.Conclusion: IL-19, promoted by IL-13 and IL-17A, contributes to the upregulation of secretion of the tissue remodeling factor MMP-9 in patients with CRS.

The Role of Relaxin-2 in Tissue Remodeling of Chronic Rhinosinusitis With Nasal Polyps

2019 ◽  
Vol 33 (5) ◽  
pp. 490-499 ◽  
Author(s):  
Yimin Li ◽  
Guojing Tan ◽  
Jie Liu ◽  
Xia Ke ◽  
Yang Shen ◽  
...  
Keyword(s):  
Western Blot ◽  
Chronic Rhinosinusitis ◽  
Nasal Polyps ◽  
Ex Vivo ◽  
Tissue Remodeling ◽  
Collagen I ◽  
Enzyme Linked Immunosorbent Assay ◽  
Type I ◽  
Relaxin 2 ◽  
Tgf Β1

Background Relaxin is a small peptide hormone that regulates extracellular matrix remodeling and reduces fibrosis in a number of organs. Little is known about its impact on chronic rhinosinusitis with nasal polyps (CRSwNP); thus, we aimed to determine the expression of human H2 relaxin (relaxin-2) and its role in tissue remodeling in CRSwNP. Methods Patients were enrolled and divided into the following groups: CRS with NP (CRSwNP; n = 20), CRS without NP (CRSsNP; n = 20), and controls (n = 15). Tissue samples were analyzed by Masson trichrome staining for collagen, while the location and expression of relaxin-2, transforming growth factor beta 1 (TGF-β1), and phosphorylated (p) Smad2/Smad3 were analyzed by immunohistochemistry and Western blot. The expression of relaxin-2, Smad2, Smad3, and TGF-β1 mRNA was tested by quantitative polymerase chain reaction (qPCR). Ex vivo NP were treated with relaxin-2 (n = 15) or TGF-β1 (n = 15). Collagen type I (collagen I), relaxin-2, and TGF-β1 levels in the culture supernatants were examined by enzyme-linked immunosorbent assay, while pSmad2/Smad3 in culture pellets was analyzed by Western blot, and the expression of Smad2 and Smad3 mRNA was tested by qPCR. Results The collagen, relaxin-2, TGF-β1, and pSmad2/Smad3 protein expression levels were significantly decreased in the CRSwNP group compared with the CRSsNP group ( P < .05). The expression of relaxin-2, Smad2, Smad3, and TGF-β1 mRNA in the CRSsNP group was significantly higher than in the CRSwNP and control groups ( P < .05). Compared with the ex vivo controls, in CRSwNP, the levels of TGF-β1, collagen I, pSmad2/Smad3, Smad2, and Smad3 were markedly decreased after relaxin-2 treatment. However, relaxin-2, collagen I, pSmad2/Smad3, Smad2, and Smad3 were remarkably increased after TGF-β1 treatment. Conclusions The antifibrotic effects of relaxin-2 may play a role in tissue remodeling in CRSwNP, but the detailed mechanism deserves further study.

scholarly journals IL-19 Induced by IL-13/IL-17A in the Nasal Epithelium of Patients with Chronic Rhinosinusitis Upregulates MMP-9 Expression via ERK/NF-kB Signaling Pathway

2020 ◽  
Author(s):  
Xia Li ◽  
Jiancong Huang ◽  
Xiaohong Chen ◽  
Xiaoping Lai ◽  
Zizhen Huang ◽  
...  
Keyword(s):  
Western Blot ◽  
Chronic Rhinosinusitis ◽  
Nasal Polyps ◽  
Tissue Remodeling ◽  
Tissue Inhibitors Of Metalloproteinases ◽  
Tissue Samples ◽  
Nasal Tissue ◽  
Human Nasal Epithelial Cells ◽  
Pathway Activation

Abstract Background: Tissue remodeling is a crucial characteristic of chronic rhinosinusitis (CRS). Imbalance between matrix metalloproteinases (MMPs) and tissue inhibitors of metalloproteinases (TIMPs) is crucial for the pathologic tissue remodeling in CRS. Elevation of interleukin (IL)-19 or MMP-9 levels in patients with CRS had been proven in previous studies. Here, we aimed to investigate the role of IL-19 in mediating MMP-9 expression in CRS. Methods: Nasal tissue samples were collected from 45 individuals having chronic rhinosinusitis with nasal polyps (CRSwNP), 24CRS without nasal polyps (CRSsNP), and 17 controls. Expression of IL-19, its receptors (IL-20R1/IL-20R2), and MMP-9 were investigated using RT-qPCR and Immunofluorescence. Human nasal epithelial cells (HNECs) were stimulated by IL-19; ERK phosphorylation, NF-kB pathway activation, and MMP-9 level were detected by RT-qPCR, ELISA, western blot and Immunofluorescence. We also explored the effect of type1/2/3 cytokines on IL-19 production by RT-qPCR, and western blot. Results: Expression levels of IL-19, its receptors (IL-20R1/IL-20R2), and MMP-9 were increased in nasal tissues from individuals with CRSwNP compared to those with CRSsNP as well as the controls. IL-19 significantly elevated the production of MMP-9 in HNECs. Furthermore, IL-19 could activate the ERK and NF-kB pathways, accompanied by increased MMP-9 production in HNECs. Conversely, both ERK and NF-kB inhibitors significantly attenuated the role of IL-19 in MMP-9 production. siRNA knockdown of IL-20R1 suppressed ERK and NF-kB pathway activation, thereby decreasing MMP-9 expression. IL-13 and IL-17A were found to stimulate IL-19 production in HNECs.Conclusion: IL-19, promoted by IL-13 and IL-17A, contributes to the upregulation of secretion of the tissue remodeling factor MMP-9 in patients with CRS.

Protein Microarray Analysis of Nasal Polyps from Aspirin-Sensitive and Aspirin-Tolerant Patients with Chronic Rhinosinusitis

2009 ◽  
Vol 23 (3) ◽  
pp. 268-272 ◽  
Author(s):  
Kelly A. Zander ◽  
Milene T. Saavedra ◽  
James West ◽  
Victor Scapa ◽  
Linda Sanders ◽  
...  
Keyword(s):  
Protein Expression ◽  
Western Blot ◽  
Chronic Rhinosinusitis ◽  
Nasal Polyp ◽  
Blot Analysis ◽  
Western Blot Analysis ◽  
Nasal Polyps ◽  
Protein Microarray ◽  
Control Group ◽  
Microarray Technology

Background The purpose of this study was to apply protein microarray technology to the study of sinonasal tissue and to identify differential protein expression in nasal polyps from aspirin-sensitive (AS) versus aspirin-tolerant (AT) patients with chronic rhinosinusitis (CRS) and CRS with nasal polyps (CRSwNPs). Methods Nasal polyp specimens were prospectively obtained from two groups of patients with CRSwNP. The test group (AS) consisted of five patients that were diagnosed with CRSwNP and intolerance to aspirin based on medical history and physical exam. The control group (AT) consisted of four AT patients with CRSwNP. Protein was extracted and labeled from harvested polyps and the Sigma Panorama Antibody Microarray–Cell Signaling Kit was used to identify differences in protein expression between the two polyp groups. Western blot analysis was used to validate the results of the protein microarray. Results The protein microarray showed a greater than twofold change in expression of both beta-adaptin and heat shock protein 70 (HSP70). Western blot analysis confirmed up-regulation of beta-adaptin and HSP70 in nasal polyp tissue from AS patients. Conclusion Pooled samples of AS and AT nasal polyps evaluated by protein microarray show distinct protein expression profiles in the stress response and receptor-mediated endocytosis pathways. This study establishes the successful application of protein microarray technology to study nasal polyposis, which in turn can be validated by Western blot analysis.

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