5-HT7 receptorsare over-expressed in patients with nasal polyps

Abstract

Nasal polyposis (NP) is an inflammatory disease of the paranasal sinuses and nasal cavity. The primary purpose of our study is to determine the expression of 5-HT₇ receptors both in nasal polyps and in healthy tissue in the nasal cavity. The subsequent aim is to compare the expression of 5-HT₇ receptors in patients with NP and in inferior turbinate tissue (control).The study included 60 participants (40 with NP and 20 controls) aged 35 to 62 years. Nasal polyp samples were collected from all patients and relative 5-HT₇ receptor expression analyses were performed. RT-PCR analysis of nasal polyps and control tissue identified 5-HT₇ receptor expression in the nasal cavities of controls. This expression was approximately 67 times higher in nasal polyp tissue than in healthy tissue. Our study identifies the expression of 5-HT₇ receptors in the nasal cavity for the first time. It is also the first demonstration of increased 5-HT₇ receptor expression in tissue from nasal polyps, which occur in the paranasal sinuses and nasal cavity.

Introduction

Nasal polyposis (NP) is a chronic inflammatory disease of the mucosa lining the nasal cavities and paranasal sinuses. ^1,2 Its prevalence is 1% to4% in the general population, and it leads to complications such as nasal obstruction and rhinorrhea. ^3,4 Although it is a well-known and easily diagnosed pathologic condition, its etiopathogenesis still requires discussion. Previously, NP was suggested to occur because of allergic diseases of the nasal mucosa; however, it is now thought that many factors contribute to its development, including chronic infection, genetic polymorphism, asthma, cystic fibrosis, epithelium cell defects, and mucosa epithelium breakdown. ⁵

Histologically, NP is recognized as an inflammatory disease that is characterized by the migration of inflammatory cells, such as eosinophils and neutrophils. ^6,7,8 Additionally, studies have shown that the immune system and oxidative stress play important roles in the formation and development of NP. ^1,9,10 Many studies have been conducted in a search for treatments for NP, and many antioxidant and anti-inflammatory agents have been investigated as potential treatments for this disease.

Currently, NP is treated with systemic or local steroids or the surgical removal of polyp tissue. However, the symptoms can continue even after treatment with steroids and the disease can quickly reappear even after surgical treatment. ¹¹ Therefore, experimental and clinical studies investigating the mechanisms leading to NP are continually being conducted.

Serotonin, which is a vasoactive amine, is an important neurotransmitter and is involved in many behavioral and psychological phenomena, such as pain, appetite, mood, and sleep. ¹² Additionally, serotonin has immunomodulatory activities that occur via serotonergic receptoractivation. ^13,14 Serotonergic receptors have been characterized in lymphocytes, monocytes, macrophages, and dendritic cells ¹⁵ these receptors are divided into seven classes based on their structures, and have been found to cause biologic effects via different signal-transduction pathways. ¹⁶ One of these receptor types is the recently discovered 5-HT₇ receptor, which is commonly found in the brain and has peripheral effects that are not completely known. ^{17 -22}

The 5-HT₇ receptor is expressed in several vascular beds (the pulmonary and coronary arteries and the aorta), has been identified in antigen-presenting dendritic cells, and regulates the activation and function of T lymphocytes. ^{23 -28} 5-HT₇ receptors have also been shown to modulate the expression of some inflammatory cytokines. ²⁹ In previous studies, we showed that 5-HT₇ receptors have important roles in systemic inflammatory events, such as sepsis, and in the acute inflammation that develops in rats with paw edema. ^30,31 We have also shown that inflammatory cytokines and oxidant parameters decreased as a result of the stimulation of 5-HT₇ receptors with an agonist. Our studies demonstrate that serotonin has anti-inflammatory and antioxidant effectsvia5-HT₇receptors.The important effects that 5-HT₇ receptors have on the immune system and inflammation suggest that 5-HT₇ receptors could be involved in nasal polyp development. Additionally, serotonin expression has been evaluated in nasal polyp tissue, but the presence and amount of 5-HT₇ receptors in the nasal cavity and nasal polyp tissue were not assessed. ³² Therefore, identifying 5-HT₇receptor expression in the nasal cavity will be an important contribution to the field.

In light of this information, the primary purpose of our study is to demonstrate the presence of 5-HT₇ receptors in nasal polyps and in inferior turbinate tissue. The subsequent aim is to compare the expression of 5-HT₇receptorsin tissue from patients with nasal polyps with control tissue.

Materials and Methods

Study population. The study included 60 participants 35 to 62 years old. Forty were patients with a diagnosis of chronic rhinosinusitis with nasal polyp (CRSwNP) according to the standard criteria issued in the European Position Paper on Rhinosinusitis and Nasal Polyps 2012. ³³ The patients with a diagnosis of NP were included in the study after histopathologic confirmation of disease. Twenty control subjects without any sinonasal diseases who were undergoing septoplasty and inferior turbinoplasty because of anatomic variations were recruited. None of the participants had received medications (i.e., antihistamines, antileukotriene agents, antibiotics, or topical or oral steroids) for at least 1 month before surgery. Patients and controls who had fungal sinusitis, antrochoanal polyps, acute rhinosinusitis, and underlying chronic disorders such as Wegener granulomatosis, sarcoidosis, or cystic fibrosis were excluded.

Polyp tissues from patients with CRSwNP and inferior turbinate mucosa from control subjects were obtained through endoscopic examination during surgery.

All patients and controls provided written informed consent before participating in the study. Those who approved and opted for surgical therapy underwent operations.

Ethical considerations. The study was approved in a meeting of the Ataturk University Faculty of Medicine Noninvasive Clinic Researches Ethical Committee.

Sample collection. Polyp tissues from patients with CRSwNP and inferior turbinate mucosa from controls were obtained during endoscopic examination during surgery. Immediately after surgery, nasal polyp tissue from all patients was collected and stored in ice for transfer to the laboratory. After being transferred to the laboratory, the samples were kept at −80°C in a deep freezer until molecular analyses were conducted.

Molecular investigations. Total RNA extraction and cDNA synthesis were performed according to our previously publishedmethods. ³¹ Briefly, the tissue (20 mg) was stabilized in RNA stabilization reagent (RNAlater, Qiagen, Hilden, Germany) and the cells were lysed using the TissueLyser II (2 x 2.5 minutes for nasal polyps). Total RNA was purified using an RNeasy Mini Kit (Qiagen) according to the manufacturer’s instructions in a QIAcube (Qiagen). The RNA samples were reverse-transcribed into complementary DNA with a High Capacity cDNA Reverse Transcription Kit (Applied Biosystems, Foster City, Calif.). Then, 10 µl of total RNA were treated with 2 µl of 10 X RT Buffer, 0.8 µl of 25 X dNTPs mix, 2 µl of 10X RT Random Primers, 1 µl of MultiScribe Reverse Transcriptase, and 4.2 µl of DEPC-H₂O. Reverse transcription was carried out at 25°C for 10 minutes, followed by 37°C for 120 minutes and finally, 85°C for 5 minutes, using a Veriti 96-well thermal cycler (Applied Biosystems). The cDNA concentration and quality were assessed and quantified using an Epoch spectrophotometer system and a Take3 plate (Biotek, Highland Park, Vt.).

Relative 5-HT₇ receptor expression analyses were performed with StepOnePlus Real Time Polymerase Chain Reaction (PCR) System technology (Applied Biosystems) on cDNA synthesized from nasal polyp RNA. The qPCR was run using the TaqMan Probe Mix and TaqMan probe-based technology (Applied Biosystems). Real-time PCR was performed using primers generated for human 5-HT₇ receptors forward, 5′-TTC TCT CCG TCT GGC TTC TC-3′; and reverse, 5′-GCA CAC CTT ATC ATC ATT TAC ATT CT-3′; and for human β-actin forward, 5′-GCA AGC AGG AGT ATG ACG AGT-3′; and reverse, 5′- CAA GAA AGG GTG TAA CGC AAC TAA-3′. The results are expressed as relative to the inferior turbinate tissue from healthy patients. β-actin was used as the reference gene against which the data were normalized. Primers and probes for 5-HT₇ receptors and β-actin were designed by Primerdesign (Southampton, UK). For each tissue sample, triplicate determinations were performed in a 96-well optical plate for both targets using 9 µl of cDNA (100 ng), 1 µl of Primer Perfect Probe mix, and 10 µl of QuantiTect Probe PCR Master Mix (Qiagen, Hilden, Germany) in each 20-µl reaction. The plates were heated for 2 minutes at 50°C and 10 minutes at 95°C, after which 40 cycles of 15 seconds at 94°C and 60 seconds at 60°C were applied. All data are expressed as the fold-change in expression compared to that in the healthy group using the 2^-ΔΔCt method. ³⁴

Statistical analysis. The results obtained from the statistical analyses were expressed as the mean ± standard deviation (SD), and p values > 0.05 were not considered statistically significant. Statistical significance of the difference between the groups was tested using a paired comparison test (Student t test). A p value <0.05 was considered to be significant.

Results

5-HT₇receptor expression. Real time PCR analyses were performed on nasal polyps and control tissues. As shown in the Table 1, the 5-HT₇ receptor was expressed in the inferior turbinate control tissues (1.09-fold, SD: 0.21). This expression was increased by 66.2-fold (SD: 4.9) in nasal polyp tissue compared with healthy tissue(figures 1 and 2). This increase was statistically significant (p < 0.001).

Table 1.

Threshold cycle(CT) values of nasal tissues obtained from healthy subjects and patients with nasal polyposis.

Groups	CT means	ΔCT means	2’ΔΔCT means	SD
Control	31,48849606	4,6208432303533	1,0903484066669	0,21511392883088
Nasal	30,49128317091	1,5477222866484	66,227365866518	4,98140996299814

polyps

Key: CT = threshold cycle, SD = standard deviation.

Figure 1.

This graph shows 5-HT₇ receptor expression in inferior turbinate mucosa (control) and nasal polyps (Unit: fold change). *p < 0.05.

Figure 2.

This graph depicts 5-HT₇ receptor expression in inferior turbinate mucosa and nasal polyps. (Unit: fold change).

Discussion

In our study, we examined 5-HT₇ receptor expression in NP, a disease that leads to complications such as nasal obstruction, rhinorrhea, and anosmia and whose pathogenesis has not been completely described.

As a multifactorial chronic inflammatory disease of the paranasal sinuses and nasal cavity mucosa, the etiology and pathogenesis of NP still require examination. Several theories have been proposed to explain its development. ³⁵ All the theories proposed for the pathogenesis of NP acknowledge that the fundamental pathology that leads to polyp formation is mucosal edema, and these theories attempt to elucidate the cause underlying this edema. ^36,37 The immune system, inflammatory mediators, cytokines, and adhesion molecules all play a role in the development of mucosal edema. Despite the many possible treatments for nasal polyps today, problems can still occur after treatment, such as continuing symptoms or disease recurrence. Therefore, new research is continuously being conducted to determine the possible mechanisms underlying the pathophysiology of nasal polyps.

Serotonin has been shown to be released in peripheral tissues, mast cells, basophils, and enterochromaffin cells. The extracellular serotonin levels increase under inflammatory conditions. ³⁸ Therefore, serotonin can be considered to be an immunomodulator. Activation of 5-HT₁ and/or 5-HT₇ receptors in serotonin monocytes prevents monocytic apoptosis by triggering increases in cyclic adenosine monophosphate levels and cytokine production. ^39,40 A recent study detailed 5-HT expression in spleen T cells and showed that naïve T cells selectively express5-HT₇receptors. ²⁸ Additionally, the reduction of T cell proliferation caused by the suppression of serotonin synthesis was reversed via treatment with AS-19, a 5-HT₇ receptor agonist. This finding suggests that serotonin plays a role in autocrine signaling through activation of 5-HT₇ receptors in naïve T cells. Additionally, when we examined the immunologic pattern in more detail, 5-HT₇ receptors were found in both humans ⁴¹ and rats ^42,25 and in immune-response cells such as monocytes, ³⁹ T cells, ²⁸ thymus, peripheral lymphocyte, spleen, and mitogen-activated spleen cells. ⁴² Consequently, the roles of 5-HT₇ receptors in peripheral diseases are currently being investigated. However, little information is available on this subject.

In a study related to ours, serotonin levels were evaluated in patients with nasal polyps but the presence and expression of 5-HT₇ receptors were not presented. Our study is the first to show5-HT₇ receptor expression in control tissue, and it showed that 5-HT₇ receptor expression was increased nearly 67-fold in nasal polyp tissue compared with its expression in healthy tissue. In our previous studies, we showed that stimulation of 5-HT₇ receptors with an agonist in the experimental sepsis model created with cecal ligation and puncture caused a recovery in the levels of inflammatory cytokines and oxidative stress parameters, such as TNF-α, IL-1β, and IL-6. ^30,31 Likewise, in a previous study, we showed that 5-HT₇ receptor expression was increased in acute inflammation that was induced via carrageenan and that the inflammation decreased following the application of a5-HT₇receptor agonist. All of these studies support the theory that serotonin plays an important role in the immune system via5-HT₇receptors and that it has anti-inflammatory and antioxidant effects. This result suggests that 5-HT₇ receptors might play a role in the pathophysiology of nasal polyps.

Our study is the first to demonstrate the expression of 5-HT₇ receptors in the nasal cavity. It is also the first to show that 5-HT₇ receptors are highly expressed in NP. It is crucial to conduct experimental and clinical studies to examine the role of 5-HT₇receptors in the formation and development of nasal polyps. In the future, 5-HT₇ agonists and antagonists could be examined as potential novel therapeutic agents for the treatment of nasal polyps.