Abstract
Purpose
Nasal polyposis (NP) is a chronic inflammatory disease that is characterized by increased populations of Th17 cells and impairment of Treg cells function in Chinese patients. Recent studies have shown that signal transducer and activator of transcription 3 (STAT3) and STAT5 are indispensable in the development and maintenance of Th17 and Treg cells. We investigated the roles of STAT3 and STAT5 in the imbalance of Th17 and Treg cells in NP.
Materials and methods
The levels of IL-6, IL-2, pSTAT3, pSTAT5, SOCS3, RORc, Foxp3, IL-17A, and TGF- β 1 were measured in patients with atopic NP, patients with nonatopic NP, and controls. We also evaluated the local distribution of Th17 and Treg cells by double immunofluorescence staining and the correlations between activated STAT3/STAT5 and Th17/Treg cell development were assessed.
Results
Increased levels of IL-6, pSTAT3, SCOS3, RORc, IL-17A, and CD4 + RORc + cells, and decreased levels of IL-2, pSTAT5, Foxp3, TGF- β 1, and CD4 + Foxp3 + cells were detected in both NP groups compared to controls ( P < .05). The differences in all expression levels (except for IL-6) were significant between atopic and nonatopic patients ( P < .05). There was a positive correlation between pSTAT3/pSTAT5 levels and Th17/Treg development and a negative correlation between SOCS3 and pSTAT3 in NP ( P < 0.01).
Conclusions
The results suggest that STAT3 and STAT5 may function through the IL-6 and IL-2 pathways to play a role in the imbalance of Th17/Treg in NP. An even more exaggerated imbalance of Th17/Treg caused by atopy may be correlated to the improper ratio of activated STAT3/STAT5.
1
Introduction
Nasal polyposis (NP) is characterized by the appearance of hyperplastic polyps in the nasal cavity and paranasal sinuses that can be seen by endoscopy and computed tomography (CT) scanning . The pathophysiology of this disease has been a research focus in the field of otorhinolaryngology owing to its high prevalence and lack of effective treatments . Although NP has been widely studied for many years, its pathogenesis is still poorly understood. Previous studies found that Chinese patients showed a predominantly Th17 response, unlike white patients with the same illness, and that both Chinese and white patients had impaired regulatory T-cell (Treg) function . These findings suggested that misregulation of T-effector cells and Treg cells may play a crucial role in the pathogenesis of NP. More recently, Shen et al showed that there is an imbalance of Th17/Treg cells in Chinese patients with NP and that the imbalance was more severe in the atopic group. Although this study provided a deeper understanding of the pathophysiology of NP, the exact mechanism of disease progression remains unknown.
Signal transducers and activators of transcription (STATs) are a family of cytokine- and growth factor–inducible transcription factors that are known to play critical roles in Th cell differentiation . These proteins are phosphorylated at specific tyrosine residues in response to the binding of cytokines to their appropriate receptors. This results in their dimerization and translocation to the nucleus, where they modulate gene expression . Among the STAT family of proteins, STAT3 has been implicated in Th17 differentiation via its activation in response to cytokines such as interleukin 6 (IL-6). The IL-6/STAT3 pathway has been illustrated to play a pivotal role in chronic inflammation and autoimmune disorders by promoting Th17 and inhibiting Treg cells . Conversely, STAT5, another member of STAT family, is activated by interleukin 2 (IL-2) and is essential for Treg cell development and has a negative effect on Th17 cell differentiation . Recent studies have focused on the reciprocal effect of STAT3 and STAT5 on T cells, and it appears that STAT3 and STAT5 activities regulate the Th17/Treg balance . As an imbalance of Th17/Treg exists in NP, we speculate that the reciprocal effects of STAT3 and STAT5 may contribute to imbalance of Th17/Treg in NP. The aggravated imbalance of Th17/Treg caused by atopy may be correlated to more severe improper ratio of activated STAT3/STAT5.
To test this hypothesis, we measured the expressions of IL-6, IL-2, pSTAT3, pSTAT5, and suppressor of cytokine signaling 3 (SOCS3), a key inhibitor of the STAT3 pathway, in an atopic NP group, a nonatopic NP group, and controls, to determine the level of activation of the STAT pathways. We then evaluated the expression levels of the transcription factors; retinoid acid–related orphan receptor C (RORc) and forehead box P3 (Foxp3); levels of CD4 + RORc + cell, CD4 + Foxp3 + cell, IL-17A, and TGF- β 1 as indicators of Th17; and Treg cell development. Finally, we analyzed the correlations between STAT3/STAT5 activities and development of Th17/Treg. To our knowledge, this is the first study on the relationship between STAT pathways and the imbalance of Th17/Treg in NP.
2
Materials and methods
2.1
Patients
This study was approved by the ethical committee of Chongqing Medical University and informed consent was obtained from all patients and controls. Forty patients with NP were recruited for this study, and the enrollment standard and the diagnosis of atopic status were according to established criteria from previous studies . Clinical data about patients included sex, age, duration of disease, history of asthma, and recurrence. Symptom scores were assessed according to a visual analog scale . The preoperative CT scans were graded according to the classification by Lund and Kennedy . The preoperative nasal endoscopy scores were graded according to the classification by Lanza and Kennedy . Patients who had antrochoanal polyps, cystic fibrosis, primary ciliary dyskinesia, fungal sinusitis, or gastroesophageal reflux disease were excluded. Fifteen patients with a deviated septum were recruited as controls. These patients had no history of respiratory disease or atopy, and their skin prick test results were negative. Oral and topical applications of corticosteroids or antihistamines were withheld for at least 3 months, and all patients received 3 days of antibiotics before surgery . Patients received surgery only when medical treatment had failed. Nasal polyposis samples were obtained from ethmoidal polyp tissue, and the inferior turbinates from patients undergoing septoplasty or rhinoseptoplasty were used as controls.
2.2
Histologic analysis
The paraffin sections of tissue samples (4–5 μm thick) were stained with hematoxylin-eosin. The overall histopathologic features were observed under low-power field magnification (× 100) and then high-power field (HPF) magnification (× 400). We used an image analyzer to measure the maximal basement membrane (BM) thickness. This included the subepithelial basal part and the thickened fibrosis beneath the basal part in the most severely thickened regions. The absolute number of eosinophils was counted in 10 randomly selected HPFs of 1 section, and these 10 counts were averaged to obtain the mean number of eosinophils per HPF. All analyses were conducted by 2 independent observers who were blinded to the diagnosis or clinical data of the patients from whom the tissue samples had been obtained.
2.3
Immunohistochemistry
Paraffin sections were dewaxed and dehydrated in alcohol, and nonspecific binding was blocked with 2% bovine serum albumin before immunohistochemical staining. The monoclonal antibodies to pSTAT3 (1:100 dilution), pSTAT5 (1:50 dilution), and SOCS3 (1:100 dilution) (Abcam, UK) were used for staining via the streptavidin-biotin complex method. Nonimmune serum IgG and phosphate-buffered saline were used as the negative controls. We analyzed the number of stained cells at a magnification of × 400 as an indicator of protein expression levels. The counting method was the same as that for determining the number of eosinophils.
2.4
Western blotting
We extracted proteins from sample tissue with the Nuclear and Cytoplasmic Protein Extraction Kit (Beyotime, China) using the manufacturer’s instructions, and supernatants were separated and stored at − 80º C for analysis. Samples containing 25 μg of protein were boiled, separated by acrylamide gel electrophoresis, and transferred electrophoretically to polyvinylidene fluoride membranes (Beyotime). The membranes were blocked with Western blocking buffer (Beyotime) for 1 hour at room temperature and then incubated with rabbit anti-human pSTAT3, pSTAT5, SOCS3, and β -actin polyclonal antibodies (Abcam) at dilutions of 1:1000. After washing, the membranes were incubated with a secondary antibody linked to horseradish peroxidase (mouse anti-rabbit immunoglobulin G, 1:2000 dilution). Proteins were detected with the BeyoECL Plus (Beyotime) kit according to the manufacturer’s instructions. β -Actin was used to normalize the results in each lane.
2.5
Quantitative real-time PCR
We analyzed the mRNA levels of Foxp3 and RORc by means of real-time polymerase chain reaction (PCR). Total RNA was isolated via TRIzol extraction (Invitrogen, USA), and all assays were performed in accordance with the manufacturer’s instructions. Total RNA (1000 ng) was reverse transcribed to cDNA using random hexamer primers (Invitrogen), and SYBR Premix Taq (TaKaRa, China) was used to perform the real-time PCR. The following primer sequences were used for RORc: 5′-GCT GTG ATC TTG CCC AGA ACC-3′ (forward) and 5′-CTG CCC ATC ATT GCT GTT AAT CC-3′ (reverse); for Foxp3: 5′-GAG AAG CTG AGT GCC ATG CA-3′ (forward) and 5′-AGG AGC CCT TGT CGG ATG AT-3′ (reverse). The PCR protocol consisted of 2 cycles at 95 °C for 30 seconds followed by 40 cycles at 95 °C for 5 seconds and 63 °C for 20 seconds. All PCR reactions were performed in duplicate. The comparative CT method was used to calculate the relative gene expression levels . Glyceraldehyde-3-phosphate dehydrogenase was used as the housekeeping gene for normalization, and a no-template sample was used as the negative control.
2.6
Enzyme-linked immunosorbent assay
Samples were weighed and homogenized in 1 mL 0.9% sodium chloride solution on ice per 100 mg of tissue. Then, they were centrifuged at 4 °C and 3000 rpm for 10 minutes. After that, the supernatants were collected and stored at − 80 °C. We used cytokine-specific enzyme-linked immunosorbent assay kits (R&D Systems, USA) to assay the levels of IL-2, IL-6, IL-1 7A, and TGF- β 1 in the samples according to the manufacturer’s instructions. We performed the assay of each sample in duplicate, and all data are expressed in picograms per milliliter.
2.7
Double immunofluorescence staining
We detected CD4 and RORc or CD4 and Foxp3 by double immunofluorescence staining to investigate the distribution of Th17 and Treg cells in local tissue. Deparaffinized sections were heated at 95 °C for 10 minutes in the presence of 10 mmol/L citric acid sodium for antigen retrieval and then incubated in blocking solution (10% normal goat serum in phosphate-buffered saline) for 10 minutes. The sections were incubated overnight with the primary antibodies anti-CD4 (1:100 dilution) (Santa Cruz, USA), anti-Foxp3 (1:200 dilution), and anti-RORc (1:200 dilution) at 4 °C. After washing, a fluorescein isothiocyanate–labeled antibody against CD4 (1:100 dilution) (Santa Cruz) and then a CY3-labeled antibody against Foxp3 or RORc (1:200) were incubated with the sections away from light. This was followed by nuclear staining with 4′,6-diamidino-2-phenylindole (1:1500 dilution) (Santa Cruz) for 1 hour. Negative control sections were obtained by omission of the primary antibody. The method of cell counting was the same as described above.
2.8
Statistical analysis
We used IBM SPSS version 20.0 (IBM SPSS, Chicago, IL) with the data represented by medians and interquartile ranges for statistical analysis. We used 1-way analysis of variance to analyze the statistical significance of the differences between groups. A Kruskal-Wallis H test was used when the equal variance test failed, the Mann–Whitney U test was used for comparisons between groups, and the Spearman test was used to determine correlations. The significance level was set at P < .05.
2
Materials and methods
2.1
Patients
This study was approved by the ethical committee of Chongqing Medical University and informed consent was obtained from all patients and controls. Forty patients with NP were recruited for this study, and the enrollment standard and the diagnosis of atopic status were according to established criteria from previous studies . Clinical data about patients included sex, age, duration of disease, history of asthma, and recurrence. Symptom scores were assessed according to a visual analog scale . The preoperative CT scans were graded according to the classification by Lund and Kennedy . The preoperative nasal endoscopy scores were graded according to the classification by Lanza and Kennedy . Patients who had antrochoanal polyps, cystic fibrosis, primary ciliary dyskinesia, fungal sinusitis, or gastroesophageal reflux disease were excluded. Fifteen patients with a deviated septum were recruited as controls. These patients had no history of respiratory disease or atopy, and their skin prick test results were negative. Oral and topical applications of corticosteroids or antihistamines were withheld for at least 3 months, and all patients received 3 days of antibiotics before surgery . Patients received surgery only when medical treatment had failed. Nasal polyposis samples were obtained from ethmoidal polyp tissue, and the inferior turbinates from patients undergoing septoplasty or rhinoseptoplasty were used as controls.
2.2
Histologic analysis
The paraffin sections of tissue samples (4–5 μm thick) were stained with hematoxylin-eosin. The overall histopathologic features were observed under low-power field magnification (× 100) and then high-power field (HPF) magnification (× 400). We used an image analyzer to measure the maximal basement membrane (BM) thickness. This included the subepithelial basal part and the thickened fibrosis beneath the basal part in the most severely thickened regions. The absolute number of eosinophils was counted in 10 randomly selected HPFs of 1 section, and these 10 counts were averaged to obtain the mean number of eosinophils per HPF. All analyses were conducted by 2 independent observers who were blinded to the diagnosis or clinical data of the patients from whom the tissue samples had been obtained.
2.3
Immunohistochemistry
Paraffin sections were dewaxed and dehydrated in alcohol, and nonspecific binding was blocked with 2% bovine serum albumin before immunohistochemical staining. The monoclonal antibodies to pSTAT3 (1:100 dilution), pSTAT5 (1:50 dilution), and SOCS3 (1:100 dilution) (Abcam, UK) were used for staining via the streptavidin-biotin complex method. Nonimmune serum IgG and phosphate-buffered saline were used as the negative controls. We analyzed the number of stained cells at a magnification of × 400 as an indicator of protein expression levels. The counting method was the same as that for determining the number of eosinophils.
2.4
Western blotting
We extracted proteins from sample tissue with the Nuclear and Cytoplasmic Protein Extraction Kit (Beyotime, China) using the manufacturer’s instructions, and supernatants were separated and stored at − 80º C for analysis. Samples containing 25 μg of protein were boiled, separated by acrylamide gel electrophoresis, and transferred electrophoretically to polyvinylidene fluoride membranes (Beyotime). The membranes were blocked with Western blocking buffer (Beyotime) for 1 hour at room temperature and then incubated with rabbit anti-human pSTAT3, pSTAT5, SOCS3, and β -actin polyclonal antibodies (Abcam) at dilutions of 1:1000. After washing, the membranes were incubated with a secondary antibody linked to horseradish peroxidase (mouse anti-rabbit immunoglobulin G, 1:2000 dilution). Proteins were detected with the BeyoECL Plus (Beyotime) kit according to the manufacturer’s instructions. β -Actin was used to normalize the results in each lane.
2.5
Quantitative real-time PCR
We analyzed the mRNA levels of Foxp3 and RORc by means of real-time polymerase chain reaction (PCR). Total RNA was isolated via TRIzol extraction (Invitrogen, USA), and all assays were performed in accordance with the manufacturer’s instructions. Total RNA (1000 ng) was reverse transcribed to cDNA using random hexamer primers (Invitrogen), and SYBR Premix Taq (TaKaRa, China) was used to perform the real-time PCR. The following primer sequences were used for RORc: 5′-GCT GTG ATC TTG CCC AGA ACC-3′ (forward) and 5′-CTG CCC ATC ATT GCT GTT AAT CC-3′ (reverse); for Foxp3: 5′-GAG AAG CTG AGT GCC ATG CA-3′ (forward) and 5′-AGG AGC CCT TGT CGG ATG AT-3′ (reverse). The PCR protocol consisted of 2 cycles at 95 °C for 30 seconds followed by 40 cycles at 95 °C for 5 seconds and 63 °C for 20 seconds. All PCR reactions were performed in duplicate. The comparative CT method was used to calculate the relative gene expression levels . Glyceraldehyde-3-phosphate dehydrogenase was used as the housekeeping gene for normalization, and a no-template sample was used as the negative control.
2.6
Enzyme-linked immunosorbent assay
Samples were weighed and homogenized in 1 mL 0.9% sodium chloride solution on ice per 100 mg of tissue. Then, they were centrifuged at 4 °C and 3000 rpm for 10 minutes. After that, the supernatants were collected and stored at − 80 °C. We used cytokine-specific enzyme-linked immunosorbent assay kits (R&D Systems, USA) to assay the levels of IL-2, IL-6, IL-1 7A, and TGF- β 1 in the samples according to the manufacturer’s instructions. We performed the assay of each sample in duplicate, and all data are expressed in picograms per milliliter.
2.7
Double immunofluorescence staining
We detected CD4 and RORc or CD4 and Foxp3 by double immunofluorescence staining to investigate the distribution of Th17 and Treg cells in local tissue. Deparaffinized sections were heated at 95 °C for 10 minutes in the presence of 10 mmol/L citric acid sodium for antigen retrieval and then incubated in blocking solution (10% normal goat serum in phosphate-buffered saline) for 10 minutes. The sections were incubated overnight with the primary antibodies anti-CD4 (1:100 dilution) (Santa Cruz, USA), anti-Foxp3 (1:200 dilution), and anti-RORc (1:200 dilution) at 4 °C. After washing, a fluorescein isothiocyanate–labeled antibody against CD4 (1:100 dilution) (Santa Cruz) and then a CY3-labeled antibody against Foxp3 or RORc (1:200) were incubated with the sections away from light. This was followed by nuclear staining with 4′,6-diamidino-2-phenylindole (1:1500 dilution) (Santa Cruz) for 1 hour. Negative control sections were obtained by omission of the primary antibody. The method of cell counting was the same as described above.
2.8
Statistical analysis
We used IBM SPSS version 20.0 (IBM SPSS, Chicago, IL) with the data represented by medians and interquartile ranges for statistical analysis. We used 1-way analysis of variance to analyze the statistical significance of the differences between groups. A Kruskal-Wallis H test was used when the equal variance test failed, the Mann–Whitney U test was used for comparisons between groups, and the Spearman test was used to determine correlations. The significance level was set at P < .05.
3
Results
3.1
Clinical data and histologic characteristics of patients
The clinical characteristics of the study subjects are shown in Table 1 . Compared with the nonatopic NP and control groups, the CT score and endoscopy score were significantly higher in the atopic NP group ( P < .05). On the contrary, there were no statistically significant differences in symptom score, age, sex, duration of disease, or recurrence among the 3 groups ( P > .05). Subjects with asthma were only found in the atopic NP group. The eosinophil counts and BM thicknesses increased significantly in both NP groups compared to controls, and statistically significant differences were seen between the atopic NP and nonatopic NP groups ( P < .05).
| Control | Atopic NP | Nonatopic NP | P value | |
|---|---|---|---|---|
| Age (year), median (range) | 28 (21–50) | 36 (22–64) | 37 (23–67) | NS |
| Gender | 9M/6F | 12M/8F | 10M/10F | NS |
| Asthma | 0/15 | 2/20 | 0/20 | NS ★ |
| Duration (year) | 0 | 4 (2–7) | 3 (1–5) | NS |
| Recurrence | 0 | 5/20 | 3/20 | NS ★ |
| Symptom score | 0 | 10 (9–14) | 9 (8–13) | NS |
| CT score | 0 | 12.5 (11.25–14.75) | 10 (9–12) | < .05 |
| Endoscopy score | 0 | 7.5 (5.25–9.75) | 4 (3–6) | < .05 |
| Eosinophils (/HPF) | 2.9 (2.0–4.3) | 39.7 (33.5–48.9) | 14.8 (12.1–19.8) | < .05 ⁎ † ‡ |
| BM thickness (μm) | 1.33 (0.90–1.43) | 21.13 (16.22–27.04) | 16.33 (11.87–22.93) | < .05 ⁎ † ‡ |
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