Frequency of CD4⁺ and CD8⁺ T cells in Iranian chronic rhinosinusitis patients

Patients suffering from CRS were recruited from the ENT and Head and Neck clinic at Hazrat Rasoul Akram Hospital, Iran University of Medical Sciences. Twenty-three patients with CRSwNP and 12 patients with CRSsNP enrolled in our study. Fifteen inferior turbinate samples were collected as controls from subjects undergoing septoplasty due only to nasal septum deviation without a history of CRS or asthma. The diagnosis of CRS was made according to the current European Position Paper on Rhinosinusitis and nasal polyps [17], fulfilling two or more of the following criteria: blockage/congestion/obstruction, nasal discharge, facial pain/pressure, decrease or loss of smell for at least 4 weeks. In addition, CT scan and endoscopic scores were recorded to confirm nasal polyp(s). Demographic characteristics of population study are summarized in Table 1. Lund-Kennedy nasal endoscopy scores [18], preoperative Lund-Mackay CT scores [19], as well as preoperative and postoperative 22-item Sinonasal Outcome Test (SNOT-22) [20], were recorded to calculate clinical scores of each CRS patient. This study was approved by the ethics committee of Iran University of Medical Sciences (IR.IUMS.REC 1395.95-03-30-27364) and all patients filled written informed consent for tissue sample collection. We performed ethical clearance according to the ethical standards of the relevant national and institutional guidelines on human experimentation and with the Helsinki Declaration of 1975, as revised in 2008. Exclusion criteria are as follow: CRS patients (1) with immunodeficiencies, cystic fibrosis, bronchiectasis, chronic obstructive pulmonary disease, diabetes mellitus, neoplasia, or fungal rhinosinusitis; (2) during pregnancy or lactation, and (3) with upper airway infections within 1 month ago. All the patients enrolled for surgery had previously failed to respond to adequate medical treatments. None of the subjects had used systemic or nasal corticosteroids, antibiotics, antihistamines, decongestants, and anti-leukotrienes 4 weeks before biopsy/surgery.

Tissue samples were divided into three sections; the first section was stored at − 80 °C for subsequent RNA isolation and the second section was used for protein isolation. The last section was fixed overnight for immunohistochemistry in a freshly prepared fixative containing 4% paraformaldehyde in PBS (pH 7.4) and was embedded in paraffin wax.

We used Hematoxylin & Eosin (H&E) staining to evaluate the pathologic characteristic of the tissues. To this aim, paraffin sections (5 mm) were stained with H&E. Air-dried sections were stained with H&E for 60 min at room temperature (RT). Then, the stained sections were analyzed by a pathologist who was blind to the clinical data. The number of eosinophils, neutrophils, mononuclear cells, total inflammatory cells (eosinophils, neutrophils, T cells, macrophages and mononuclear cells), goblet cells, and mucosal glands in the lamina propria was counted at high power field (HPF) 400×, using an Olympus CX-40 microscope (Olympus, Tokyo, Japan) and 5 random HPFs were selected and observed. Results were presented as cells or glands per HPF [8]. CRSwNP patients were categorized as eosinophilic when eosinophils consisted of more than 10% of total inflammatory cells, and as non-eosinophilic when eosinophils consisted of less than 10% of the total inflammatory cells [8].

CD4 (a marker representing Th cells), CD8 (a marker representing CD8⁺ T cells), and CD68 (a marker representing macrophages) were evaluated by immu-nohistochemistry. Sinonasal tissues were dehydrated and embedded in the paraffin. Tissue samples were sectioned at 3 µm. Then, they were rehydrated through a xylene and ethanol series and were immersed in Target Retrieval solution (low pH, Dako, Glostrup, Denmark) and autoclaved at 121 °C for 20 min for the retrieval of antigens. Endogenous peroxidase activity was blocked with 3% H2O2/methanol. After washing, sections were incubated for 30 min in a blocking solution (PBS, pH 7.4, containing 2% bovine serum albumin [Sigma-Aldrich, Darmstadt, Germany], 0.1% Triton X-100, and 0.1% sodium azide) at RT to decrease nonspecific bindings [21], then incubated with: Monoclonal Mouse Anti-Human CD4 (1:100, Clone 4B12, Dako), Monoclonal Mouse Anti-Human CD8 (1:200, Clone C8/144B, Dako), Monoclonal Mouse Anti-Human CD68 (1:100, Clone KP1, Dako) for 1 h at RT. After the incubation process, all slides were washed with Tris-buffered saline (TBS) for 10 min and incubated for another 45 min at 30 °C with EnVision™ (Dako), using an Autostainer (Dako). The samples were counterstained with Mayer’s hematoxylin stain and mounted in Faramount Mounting Medium (Dako), prior to analysis by light micros-copy. The number of positive cells in tissue sections was counted by a light microscope at a magnification of 400×, using an Olympus CX-40 microscope (Olympus, Tokyo, Japan) [22].

Total RNA was isolated from sinonasal tissues with Trizol (Invitrogen, USA) according to the manufacturer’s instructions. RNA integrity and the success of the reverse transcription reaction were monitored by PCR amplification of glyceraldehyde-3-phosphate dehydrogenase transcripts and denaturing agarose gel 2%. Genomic DNA was removed from total RNA, using RNase-free DNase Set (Qiagen, Chatsworth, CA, USA). Then, 500 ng of total RNA from each sample was utilized to prepare cDNA, using PrimeScript™ RT reagent Kit (TaKaRa, Korea). Reverse transcription reaction was done in total 20 µL of a reaction mixture containing 2.5 U of MML-V (RT; GIBCO BRL, Grand Island, NY) and 50 pmol of random hexanucleotides at 42 °C for 60 min, followed by 85 °C for 5 s to inactivate reverse transcriptase enzyme. Each real-time PCR reaction was carried out in a final volume of 20 µL, including 10 µL of 2 × SYBR Green Real-time PCR Master Mix (TaKaRa, Korea), 1 µL of cDNA, and 1 µL of the forward and reverse 200 nM primers. Instead of cDNA, nuclease-free water was added to each negative control microtube. The mRNA expression levels of specific transcription factors for Th1 (T-bet), Th2 (GATA3), Th17 (Ror-γt), and Treg (FoxP3) cells were subsequently determined using extracted RNA from tissue biopsies using quantitative real-time PCR. The primer sequences are listed in Table 2. Real-time PCR was performed using Rotor-Gene Q (Qiagen, Hilden, Germany). Conditions for 40 cycles of PCR were 95 °C denaturation for 5 s, 60 °C annealing-extension for 30 Sec. Experiments were assayed in duplicate for each sample. The mean threshold cycle values were normalized to beta actin (β-actin) expression levels, and the relative mRNA levels of target genes were calculated by 2^−ΔΔCt method [23, 24].

In this study, 23 patients (6 females and 17 males) with CRSwNP, 12 patients with CRSsNP (4 females and 8 males), and 15 healthy controls (5 females and 10 males) were evaluated. The median (min–max) age of the population study was as follows: 32 (14–67) for CRSwNP, 28 (10–43) for CRSsNP, and 27 (17–44) for controls. We also evaluated and compared CT score, endoscopic score, and the VAS score in CRS patients, demonstrating sinus involvement in comparison to age- and sex-matched controls. There was a significant difference between CRSwNP and CRSsNP patients in CT score (< 0.0001), endoscopic score (< 0.001), and the VAS score (< 0.05). Five patients in CRSwNP group had asthma who were diagnosed according to the history. Approximately all CRSwNP patients with concomitant asthma represented high eosinophil infiltration. The demographic and clinical characteristics of CRS patients and controls are shown in Table 1.

In this study, H&E findings showed that CRSwNP group was eosinophil dominant (Fig. 1, Table 3) whereas CRSsNP group was neutrophil dominant (Fig. 1, Table 3). The number of eosinophils was higher in CRSwNP patients than CRSsNP patients and controls (CRSwNP vs. CRSsNP vs. controls: Pv < 0.0001 for CRSwNP vs. controls, Pv = 0.003 for CRSsNP vs. controls, and Pv = 0.013 for CRSsNP vs. CRSwNP) (Fig. 2, Table 3). It was only observed that the number of eosinophils positively correlated with the number of total inflammatory cells (r = 0.82, Pv < 0.0001), but no relationship was found between eosinophils and other cells (Fig. 3). The number of total inflammatory cells in CRSwNP, CRSsNP, and control groups was 91.5 (68.5–129), 42.25 (34–54.5), and 12.5 (10–20), respectively (Fig. 1, Table 3). According to the proportion of eosinophils to the total inflammatory cells, we divided CRSwNP patients into 16 eosinophilic CRSwNP (ECRS) patients and 7 non-eosinophilic CRSwNP (N-ECRS) patients (Table 4).

The frequency of infiltrating neutrophils was significantly elevated in CRSwNP and CRSsNP groups in comparison to controls (Pv = 0.001 and Pv < 0.0001, respectively). However, the number of neutrophils was higher in CRSsNP group than CRSwNP group (Pv = 0.025) (Fig. 2, Table 3). ECRS patients had fewer neutrophils, while the N-ECRS patients presented significant neutrophil infiltration (Fig. 4, Table 5).

CRSwNP group showed enhanced infiltration of CD4⁺ T cells into the sinonasal mucosa in comparison to CRSsNP group and controls (Pv < 0.0001 for CRSwNP vs. controls, Pv = 0.016 for CRSsNP vs. controls, and Pv = 0.006 for CRSsNP vs. CRSwNP) (Figs. 1 and 2, Table 3). In comparison to controls, both CRSwNP and CRSsNP groups indicated increased infiltration of CD8⁺ T cells into the sinonasal mucosa (both groups Pv < 0.0001). Meanwhile, there was no significant difference between CRSwNP and CRSsNP groups (Figs. 1, and 2, Table 3). Furthermore, the number of macrophages was significantly increased in CRS patients in comparison to controls (Pv < 0.0001 for CRSwNP vs. controls, Pv = 0.003 for CRSsNP vs. controls, and Pv = 0.013 for CRSsNP vs. CRSwNP) (Figs. 1 and 2, Table 3). We also found a significant difference in the number of macrophages between ECRS and CRSsNP patients (Pv = 0.004) (Fig. 4, Table 5).

It was shown that mRNA expression of GATA3 was increased in CRSwNP patients compared with CRSsNP and control groups (Pv < 0.0001 for CRSwNP vs. controls, Pv = 0.001 for CRSsNP vs. controls) (Fig. 5). It represents Th2 dominance in CRSwNP patients. There was no significant difference in T-bet mRNA expression among three groups (Fig. 5). It was found that mRNA expression of Ror-γt was increased in CRSsNP patients compared with CRSwNP and control groups (Pv = 0.001 for CRSsNP vs. controls and also for CRSsNP vs. CRSwNP). No significant difference was found between CRSwNP and control groups (Fig. 5). It demonstrates Th17 dominance in CRSsNP patients. In comparison to healthy controls, both CRSwNP and CRSsNP groups showed decreased FoxP3 mRNA expression (Pv = 0.001 and Pv < 0.0001, respectively). There was no significant difference between CRSsNP and CRSwNP groups (Fig. 5).