Abstract
Objectives
The purpose of this study is to characterize the association between altered epithelial barrier function, represented by changes in histology and differential expression of the mucosal water membrane permeability protein aquaporin 5 (AQP5), and the pathophysiology of chronic refractory sinusitis (CRS) in patients with and without nasal polyposis.
Study design
Prospective clinical study.
Setting
Tertiary rhinology referral center.
Participants
Sinonasal samples were obtained from seven CRS subjects with nasal polyps (CRSwNP), seven CRS without nasal polyposis (CRSsNP), and five control healthy patients.
Methods
Mucosal membrane changes were evaluated through hematoxylin and eosin staining of the membrane barrier and immunohistochemical staining of AQP5 expression, a membrane channel protein that affects trans-epithelial water permeability and tissue edema. AQP5 expression was confirmed by real-time PCR (rt-PCR) and western blot. Levels of other membrane proteins, including E-cadherin and Septin-2, were also assessed.
Results
CRSwNP patients showed substantial histologic evidence of membrane remodeling with increased edema and glandular hyperplasia. The epithelial expression of AQP5 was significantly lower in CRSwNP as compared to CRSsNP or control. There was no significant difference in the expression of E-cadherin and Septin-2.
Conclusions
Collectively, these data suggest that the mucosal epithelial barrier is compromised in the context of CRS (predominantly in CRSwNP) when compared to control and that AQP5 acts as a key tight junction protein in the maintenance of mucosal water homeostasis. We hypothesize that AQP5 plays a possible role in the pathophysiology of mucosal edema and polyp formation.
1
Introduction
The human immune response is a layered defense system against infection. It starts with a physical barrier which acts as a shield against potential environmental pathogens and is followed, first, by a non-specific innate response and, next, by a more specific adaptive response with both a celluar and humoral arm. The mucosal surface of the sinonasal tract interfaces with a considerable number of viruses and bacteria, which are usually eliminated by a mucociliary clearance mechanism. The ‘epithelial barrier hypothesis’ of chronic rhinosinusitis (CRS) posits that an intact barrier with tight epithelial junctions is necessary for a healthy nasal mucosa. Defects in this protective barrier may result in passage of pathogenic microbes across the epithelium and a subsequent dysregulated inflammatory cascade . It has been reported that the integrity of the healthy mucosal epithelium is maintained by neighboring cells, with 4 types of cell–cell contacts: tight junctions, adherens junctions, gap junctions and desmosomal junctions . The epithelial tissue in the respiratory tract also contains many important proteins, including the protease inhibitor SPINK5, the S100 family of proteins and claudins and occludins, all of which regulate the tight junction function and permeability of the barrier.
Aquaporins (AQPs) are another group of proteins found in the epithelial barrier of certain body tissues. They control tissue water transport and homeostasis and have been detected in lung tissue , but their association with the pathophysiology of CRS has not been previously studied. To date, thirteen main aquaporins have been characterized (AQP 1–13), and their expression and function correlate with tissue and cell type . Aquaporin 5 (AQP5) is found in type I pulmonary alveolar epithelial cells and submucosal gland acinar cells . Both AQP1 and AQP5 are important in water transport in the lungs and the deletion or decreased expression of these tight junction proteins is often associated with disease . AQPs 1–5 are all expressed in the human nasal mucosa, however only AQP5 is found in the apical sites of epithelial cells and acinar cells . A relationship between AQPs and polyps, the chronically inflamed sinus mucosa of CRS, has not been defined to date.
CRS is a common upper respiratory tract disease caused by a dysregulated inflammatory response to microbes that invade the mucosa of the nose and paranasal sinuses, although the full complement of microorganisms that cause CRS remains to be characterized, and the exact pathophysiology behind the inflammatory process is still a work in progress . CRS has been divided into CRS with nasal polyps (CRSwNP), which has a tendency towards T-helper type 2 (TH2) cytokine polarization, and CRS without nasal polyps (CRSsNP), which is associated with more of a TH1 type response . While the inflammatory profile is known to differ dramatically between CRSwNP and CRSsNP, little is understood about the epithelial differences between these two groups . In this work, we demonstrate that epithelial barrier function proteins are impaired in the context of an inflamed sinus mucosa when compared to control, and that the expression of AQP5 differs between CRSwNP and CRSsNP. These data provide additional insight into the pathophysiology of CRS with and without nasal polyps.
2
Materials and Methods
This is a prospective study that evaluates sinonasal tissue samples from 7 patients with CRSwNP, 7 CRSsNP patients undergoing endoscopic sinus surgery and 5 healthy controls undergoing transphenoidal or septal surgery. None of the CRS patients had previous surgery or had been taking systemic corticosteroids at the time of sample harvesting. The CRS patients had completed at least three courses of oral antibiotics and nasal corticosteroids sprays prior to surgery. Control patients were not on antibiotics or nasal corticosteroids sprays. Sinus tissue samples were taken from the ethmoids through a small Tru-cut Blakesley forceps and were about 5 mm 3 in size. Control specimens were collected, with patients’ permission, either from healthy sphenoids during trans-sphenoidal surgery for pituitary tumor or from the anterior ethmoids area during a routine septoplasty. Samples were evaluated by hematoxylin and eosin (H&E), immunohistochemistry, western blot and real-time PCR. The study was approved by the Liberty-institutional review board (IRB).
2.1
RNA isolation from human sinonasal tissue
Total RNA was isolated using the Qiagen TissueLyser LT protocol which was modified slightly for explanted sinonasal tissue. To begin RNA extraction, a 2 mL microcentrifuge tube containing 1 stainless steel bead (5 mm in diameter) was placed on dry ice for 15 min. Human sinus samples were placed into the precooled 2 mL microcentrifuge tube and cooled further for 15 min on dry ice. The tubes containing the tissue were placed into the TissueLyser adapter and incubated at RT for 2 min. After incubation, 500 μl of TriPure RNA buffer was added in addition to 500 μl sterile glass beads (0.1 mm in diameter). The TissueLyser was run at 30 Hz for 5 min. The samples were then centrifuged briefly in a cooled microcentrifuge. Two volumes of chloroform were added to the supernatant fluid, and the samples were again incubated at RT, this time for 10 min. The samples were centrifuged at 12,000 × g for 1 min at 4 °C. The top aqueous layer was removed and placed into a fresh RNase free Eppendorf tube. To this supernatant, 5 vol of 100% EtOH was added, and this mixture was pipetted into an RNA spin column (Ribopure-Bacteria, Ambion). The RNA was washed 3 × with Ribopure Wash Buffer and subsequently eluted from the column. The isolated RNA was then treated with DNase I for 30 min at 37 °C, following the manufacturer protocol (Ribopure-Bacteria Kit, Ambion). Lastly, samples were placed into sterile, RNase-free Eppendorf tubes for storage at − 80 °C until needed.
2.2
Real-time PCR for selected human sinonasal genes
Real-time PCR was conducted by SYBRgreen assay (Applied Biosystems) on a BioRad MyiQ5 Real-Time PCR detection system. PCR conditions may need to be adjusted per assay; however, the initial PCR master mix was 1 × SYBRMastermix (Applied Biosystems), 1 ng/ml DNA, and 1 μM of each primer of the appropriate primer pair. In all assays, a positive control of 100 ng purified target DNA, and a negative control consisting of water was used. If primer specificity had not been previously determined, we validated the PCR assay based on several criteria: precision of primers, accuracy, specificity, range of detection, linearity, ruggedness, and robustness of the assay. All primers were blasted against the GeneBank database in silico to ensure that their sequences are specific to each organism.
2.3
Hematoxylin and Eosin Tissue staining for histological analysis
At the time of collection, all tissues were fixed in 10% neutral buffered formalin, embedded in paraffin, sectioned at 5 μm, and stained with H&E. The severity of histopathologic changes was scored with the evaluator (RJB) blinded to the treatment groups and patient identifications. Sections were scored on a scale of 0–5 with 5 being the most severe. The following parameters for each section were evaluated: degree and type of inflammation, edema, epithelial attenuation, epithelial hyperplasia, epithelial squamous metaplasia, fibrosis, and glandular or goblet cell hyperplasia. The summed total scores (35 total possible) were compiled and compared between treatment groups.
2.4
Aquaporin 5 Immunohistochemistry
Paraffin-embedded sections of human sinonasal tissue were collected, deparaffinized, rehydrated and subjected to antigen retrieval by incubation in Target Retrieval solution, pH 6.0 (DAKO, Carpentaria, CA) for 25 min at 90 ° C, followed by a 20 min cooling period at room temperature. The sections were then treated with 0.3% hydrogen peroxide in water for 15 min to quench endogenous peroxidase activity. Following a rinse in Tris buffered saline with 1% Tween-20 (TTBS), the slides were subjected to two blocking steps: (i) 15 min incubation with 0.15 mM glycine in PBS, and (ii) 30 min incubation with 1% normal horse serum with a rinse in TTBS in between. The slides were then incubated with rabbit polyclonal antibody to human Nrf2, Guanylate Cyclase GCS (Santa Cruz Biotechnology, Santa Cruz, CA), MDA, NQO1 (Abcam, Cambridge, MA), monoclonal antibody against AQP5, mouse monoclonal antibody against glutathione (Virogen, Watertown, MA) or respective isotype IgG controls at a 1:100 dilution in blocking buffer followed by several rinses in TTBS. This was followed by 30 min incubation with biotinylated goat-anti rabbit-IgG (Vector Laboratories) and visualization of bound antibody by the Avidin-Biotin system (Vectastain; Vector Laboratories) and diaminobenzidine substrate (Dako; Carpentaria, CA). The sections were counterstained with Meyer’s hematoxylin (Scytek Laboratories; Logan, Utah), mounted with coverslips, and examined on an Olympus BX41 light microscope. Photomicrographs were acquired with an Olympus DP70 camera and the associated computer software. Microscopic lesions stained with H&E and sections stained by immunohistochemistry (IHC) were scored by the reviewer blinded to the treatment groups.
2.5
Immunohistochemical scoring
For immunohistochemical scoring purposes, each section was graded on a scale of 0–5 with 5 representing the most severe. Within the tissues sections, the overall immunohistochemical scoring was determined based on extent of staining and staining intensity. IHC for AQP5 was divided into epithelial and non-epithelial (mesencnhymal) expression, but only epithelial scores were considered in the evaluation of immunoreactivity.
2.6
Western Blot
The overall (global) expression of AQP5 in all tissue samples was determined by western blot. Specimens were homogenized with Ultra-Turrax T-25 homogenizer (Janke & Kunkel, Germany), treated with RIPA buffer (50 mM Tris HCl pH 8.0, 150 mM NaCl, 1% Triton X-100, protease and phosphatase inhibitor), and spun at 800 × g for 10 min. The supernatant was spun at 55,000 rpm with Beckman Coulter T-72Ti Centrifuge Rotor for one hour at 4 °C. After decanting the residual supernatant, the pellets were resuspended with 5 × Laemmli buffer. The protein samples were assayed using the BCA reagents (Thermo Scientific Pierce) and subjected to SDS-PAGE on 10% acrylamide gels. The gels were then transferred onto Immuno-Blot PVDF membranes (Bio-Rad) and probed for Septin-2, E-cadherin and AQP5.
2
Materials and Methods
This is a prospective study that evaluates sinonasal tissue samples from 7 patients with CRSwNP, 7 CRSsNP patients undergoing endoscopic sinus surgery and 5 healthy controls undergoing transphenoidal or septal surgery. None of the CRS patients had previous surgery or had been taking systemic corticosteroids at the time of sample harvesting. The CRS patients had completed at least three courses of oral antibiotics and nasal corticosteroids sprays prior to surgery. Control patients were not on antibiotics or nasal corticosteroids sprays. Sinus tissue samples were taken from the ethmoids through a small Tru-cut Blakesley forceps and were about 5 mm 3 in size. Control specimens were collected, with patients’ permission, either from healthy sphenoids during trans-sphenoidal surgery for pituitary tumor or from the anterior ethmoids area during a routine septoplasty. Samples were evaluated by hematoxylin and eosin (H&E), immunohistochemistry, western blot and real-time PCR. The study was approved by the Liberty-institutional review board (IRB).
2.1
RNA isolation from human sinonasal tissue
Total RNA was isolated using the Qiagen TissueLyser LT protocol which was modified slightly for explanted sinonasal tissue. To begin RNA extraction, a 2 mL microcentrifuge tube containing 1 stainless steel bead (5 mm in diameter) was placed on dry ice for 15 min. Human sinus samples were placed into the precooled 2 mL microcentrifuge tube and cooled further for 15 min on dry ice. The tubes containing the tissue were placed into the TissueLyser adapter and incubated at RT for 2 min. After incubation, 500 μl of TriPure RNA buffer was added in addition to 500 μl sterile glass beads (0.1 mm in diameter). The TissueLyser was run at 30 Hz for 5 min. The samples were then centrifuged briefly in a cooled microcentrifuge. Two volumes of chloroform were added to the supernatant fluid, and the samples were again incubated at RT, this time for 10 min. The samples were centrifuged at 12,000 × g for 1 min at 4 °C. The top aqueous layer was removed and placed into a fresh RNase free Eppendorf tube. To this supernatant, 5 vol of 100% EtOH was added, and this mixture was pipetted into an RNA spin column (Ribopure-Bacteria, Ambion). The RNA was washed 3 × with Ribopure Wash Buffer and subsequently eluted from the column. The isolated RNA was then treated with DNase I for 30 min at 37 °C, following the manufacturer protocol (Ribopure-Bacteria Kit, Ambion). Lastly, samples were placed into sterile, RNase-free Eppendorf tubes for storage at − 80 °C until needed.
2.2
Real-time PCR for selected human sinonasal genes
Real-time PCR was conducted by SYBRgreen assay (Applied Biosystems) on a BioRad MyiQ5 Real-Time PCR detection system. PCR conditions may need to be adjusted per assay; however, the initial PCR master mix was 1 × SYBRMastermix (Applied Biosystems), 1 ng/ml DNA, and 1 μM of each primer of the appropriate primer pair. In all assays, a positive control of 100 ng purified target DNA, and a negative control consisting of water was used. If primer specificity had not been previously determined, we validated the PCR assay based on several criteria: precision of primers, accuracy, specificity, range of detection, linearity, ruggedness, and robustness of the assay. All primers were blasted against the GeneBank database in silico to ensure that their sequences are specific to each organism.
2.3
Hematoxylin and Eosin Tissue staining for histological analysis
At the time of collection, all tissues were fixed in 10% neutral buffered formalin, embedded in paraffin, sectioned at 5 μm, and stained with H&E. The severity of histopathologic changes was scored with the evaluator (RJB) blinded to the treatment groups and patient identifications. Sections were scored on a scale of 0–5 with 5 being the most severe. The following parameters for each section were evaluated: degree and type of inflammation, edema, epithelial attenuation, epithelial hyperplasia, epithelial squamous metaplasia, fibrosis, and glandular or goblet cell hyperplasia. The summed total scores (35 total possible) were compiled and compared between treatment groups.
2.4
Aquaporin 5 Immunohistochemistry
Paraffin-embedded sections of human sinonasal tissue were collected, deparaffinized, rehydrated and subjected to antigen retrieval by incubation in Target Retrieval solution, pH 6.0 (DAKO, Carpentaria, CA) for 25 min at 90 ° C, followed by a 20 min cooling period at room temperature. The sections were then treated with 0.3% hydrogen peroxide in water for 15 min to quench endogenous peroxidase activity. Following a rinse in Tris buffered saline with 1% Tween-20 (TTBS), the slides were subjected to two blocking steps: (i) 15 min incubation with 0.15 mM glycine in PBS, and (ii) 30 min incubation with 1% normal horse serum with a rinse in TTBS in between. The slides were then incubated with rabbit polyclonal antibody to human Nrf2, Guanylate Cyclase GCS (Santa Cruz Biotechnology, Santa Cruz, CA), MDA, NQO1 (Abcam, Cambridge, MA), monoclonal antibody against AQP5, mouse monoclonal antibody against glutathione (Virogen, Watertown, MA) or respective isotype IgG controls at a 1:100 dilution in blocking buffer followed by several rinses in TTBS. This was followed by 30 min incubation with biotinylated goat-anti rabbit-IgG (Vector Laboratories) and visualization of bound antibody by the Avidin-Biotin system (Vectastain; Vector Laboratories) and diaminobenzidine substrate (Dako; Carpentaria, CA). The sections were counterstained with Meyer’s hematoxylin (Scytek Laboratories; Logan, Utah), mounted with coverslips, and examined on an Olympus BX41 light microscope. Photomicrographs were acquired with an Olympus DP70 camera and the associated computer software. Microscopic lesions stained with H&E and sections stained by immunohistochemistry (IHC) were scored by the reviewer blinded to the treatment groups.
2.5
Immunohistochemical scoring
For immunohistochemical scoring purposes, each section was graded on a scale of 0–5 with 5 representing the most severe. Within the tissues sections, the overall immunohistochemical scoring was determined based on extent of staining and staining intensity. IHC for AQP5 was divided into epithelial and non-epithelial (mesencnhymal) expression, but only epithelial scores were considered in the evaluation of immunoreactivity.
2.6
Western Blot
The overall (global) expression of AQP5 in all tissue samples was determined by western blot. Specimens were homogenized with Ultra-Turrax T-25 homogenizer (Janke & Kunkel, Germany), treated with RIPA buffer (50 mM Tris HCl pH 8.0, 150 mM NaCl, 1% Triton X-100, protease and phosphatase inhibitor), and spun at 800 × g for 10 min. The supernatant was spun at 55,000 rpm with Beckman Coulter T-72Ti Centrifuge Rotor for one hour at 4 °C. After decanting the residual supernatant, the pellets were resuspended with 5 × Laemmli buffer. The protein samples were assayed using the BCA reagents (Thermo Scientific Pierce) and subjected to SDS-PAGE on 10% acrylamide gels. The gels were then transferred onto Immuno-Blot PVDF membranes (Bio-Rad) and probed for Septin-2, E-cadherin and AQP5.
Stay updated, free articles. Join our Telegram channel
Full access? Get Clinical Tree
