Study participants
Patients admitted to the Otorhinolaryngology Department, Renji Hospital, Shanghai Jiaotong University School of Medicine, were selected. This study was approved by the ethics committee of Renji Hospital. All participants signed the informed consent form before enrolment. A total of 41 patients (Table 1) who underwent nasal mucosal inflammation surgery were included. The diagnosis was based on endoscopic and computed tomography scores and in accordance with the guidelines and consensus for the diagnosis and treatment (Chinese guidelines for diagnosis and treatment of chronic rhinosinusitis. 2018) of nasal mucosal inflammation. These patients were divided into nasal mucosal inflammation with NP (N = 28) and nasal mucosal inflammation without NP (N = 13) groups, exhibiting nonoverlapping grouping. A total of 15 patients (with NP:without NP = 10:5) underwent immunofluorescence staining and 26 patients (with NP:without NP = 18:8) underwent flow cytometry. The nasal mucosa was harvested from the nasal mucosa adjacent to NPs and the uniform mucosa of patients without NP during the surgery of functional endoscopic sinus. All patients with asthma and aspirin intolerance triad were excluded according to their medical histories and were not treated with antibiotics or glucocorticoids 4 months before the surgery.
Study animals
For this study, 8-week-old male C57 BL/6 mice from the same litter, weighing 18–25 g, with a genotype of Senp3 fl/fl and Senp3 cKO were selected. The SENP3 knockout mice were obtained by mating Senp3 fl/fl mice with Senp3 cKO mice. The control group consisted of mice that were littermates of Senp3 fl/fl mice. The gene knockout methods and sources were the same as those described in published studies17. All experimental mice were raised in the specific-pathogen free (SPF) animal room of Shanghai Jiaotong University School of Medicine, with free access to food and water. All animal experiments were performed in strict accordance with the guidelines for the care and use of experimental animals issued by the Ministry of Science and Technology of the People’s Republic of China (Guidelines on the Humane Treatment of Laboratory Animals.2006). The program was approved by the Animal Protection and Use System Committee of Shanghai Jiaotong University School of Medicine (license number: A-2019-041). After continuous administration of nasal drops for 12 weeks, the mice were sacrificed 30 min after the last nasal cavity stimulation. Then, the hearts were rinsed with 20 mL of PBS and fixed with 20 mL of 4% paraformaldehyde. After the fixation, the nasal specimens were harvested according to the procedures described by Dunston et al.23. The scalp was peeled, the zygoma and mandible were cut, and the nose was dissected from the posterior part of the eyelid. The primary BMDMs were extracted and induced in the same manner as described by Weischenfeldt et al. 24, with some changes in terms of the incubation method. Also, after the mice were sacrificed, the femur and tibia were separated, the muscles were removed, and the bilateral metaphyses were opened with a sterile ophthalmoscope. Then, a 1-mL syringe was punched out from one metaphysis end to the other end with α-minimum Eagle’s medium (α-MEM; Gibco, 12571-063, USA), followed by filtering with a 70-µm MACS SmartStrainer (Miltenyi, 130-098-462, Germany) and incubation.
Culture of BMDMs
The culture method was as described by Weischenfeldt et al24. The cells were incubated with α-MEM, 10% fetal bovine serum (Hyclone, sv30087.03, USA), and 50 ng/mL M-CSF (Sino Biological, 511112-MNAH, China), and the medium was replaced once every 2 days. After culture for 7 days, the cells were seeded in a six-well plate and co-stimulated with 20 ng/mL IL-4 (Sino Biological, 51084-MNAE) and 10 ng/mL IL-13 (Sino Biological, 50225-MNAH) on day 7 for 48 h.
Allergic rhinitis modeling
The model was constructed as described by Kim et al25 and Wang et al26. A model of allergic rhinitis with Th2 immune response was used. The whole process was carried out in the SPF animal room, with no bacteria or fungi colonized in the nasal cavity, to eliminate the uncertain effects of bacteria or fungi on macrophages and NPs. The model was constructed using four Senp3 fl/fl mice and five Senp3 cKO mice.
Sensitization phase: The animals were intraperitoneally injected with 100 µL of chicken OVA (Sigma, A5503-1g, USA) dissolved in PBS (75 µg/mouse) and Imject Alum Adjuvant (Thermo Scientific, 77161, USA; 50 µL/mouse), once every alternate day for seven times.
Stimulating phase: The mice were dripped with 25 µg/µL OVA, once a day, for 12 weeks, 10 µL per nostril, and inverted for 5 min after administering nasal drops to prevent OVA from entering the airway. The symptoms of the mice were observed 10 min after each stimulation. Nine mice showed behaviors of scratching the nose and ears and sneezing. The symptom scores met the criteria for the allergic rhinitis model25,26. Luna staining revealed eosinophil infiltration.
Immunofluorescence staining
Macrophages and SENP3 protein obtained from the nasal mucosa of patients were subjected to immunofluorescence co-localization staining, while the macrophages in the nasal specimens of mice with allergic rhinitis were subjected to immunofluorescence staining. The specimens were fixed with 4% paraformaldehyde, embedded in paraffin, and then separately incubated with CD68 antibody 1:2000 (Service, GB13067-M-2, China), CD206 antibody 1:200 (Service, GB11062), and SENP3 antibody 1:5000 (5591S, CST, USA) overnight at 4°C in the dark. Subsequently, the specimens were incubated with horseradish peroxidase–labeled goat anti-mouse or anti-rabbit antibody 1:500 (Service, GB2330) at room temperature for 50 min and then incubated with Future Innovation Technology Creativity (FITC), CY3, and 1:200 594 goat anti-rabbit fluorescence secondary antibody (Service) at room temperature in the dark for 10 min, followed by counterstaining with 4',6-diamidino-2-phenylindole (DAPI; Service, G1012) and observation under a microscope. The wavelength of DAPI ultraviolet light was 330–380 nm (blue light), FITC was 465–495 nm (green light), CY3 was 510–560 nm (red light), and 594 was 594 nm (rose red). The immunofluorescence staining of nasal specimens obtained from mice followed a slightly different procedure. After fixation with 4% paraformaldehyde, the specimens were decalcified with 10% ethylenediaminetetraacetic acid for 2 weeks, followed by paraffin embedding. The immunofluorescence staining was performed using CD206 (Service, GB11062). An upright fluorescence microscope (Nikon Eclipse C1, Japan) and imaging system (Nikon DS-U3) were used for observation.
Luna staining
The Luna staining procedure referred to the method described by Matthijs et al27. The cells were stained for 5 min using the mixture of Weiger's iron hematoxylin (0.005% hematoxylin and 0.6% ferric chloride incubated in 2% hydrochloric acid) and Biebrich scarlet solution (Biebrich scarlet and 0.1% acid fuchsin incubated in 1% acetic acid for 5 min) in a ratio of 9:1. They were then differentiated with 1% hydrochloric ethanol and soaked in 0.5% lithium carbonate solution for 1 min, followed by washing, dehydration, mounting, and observation under a light microscope. The cells specifically stained scarlet were eosinophils.
Flow cytology
1. Digestion of the nasal mucosa of patients before testing
The digestive fluid was formulated using 250 U DNaseI (Sigma, D5025-15KU), 5-mg type 2 collagenase (Sigma, C6885-100MG), and 2.2 mL of Dulbecco's Modified Eagle Medium (DMEM) (Gibco, C11960500BT). After filtration and washing, the suspension was incubated with 1:1000 Fixable Viability Stain 510 (BD Pharmingen, 564406, USA) at room temperature in the dark for 15 min, mixed with 1:50 Fc Block (BD Pharmingen, 564219), and sealed at 4°C for 5 min. Further, it was mixed with CD11b antibody 1:100 (BD Pharmingen, 557396A) and CD80 antibody 1:100 (BD Pharmingen, 557227) and incubated at room temperature in the dark for 30 min, followed by membrane rupture using a membrane rupture kit (BD Pharmingen, 554714) at 4°C for 20 min. Then, it was incubated with CD68 antibody 1:100 (BD Pharmingen, 564943) and CD206 antibody 1:100 (BD Pharmingen, 550889) at room temperature in the presence of light for 30 min, washed, and tested with Beckman CytoFlex flow cytometry.
2. Quantitative analysis of BMDMs by flow cytometry
After the cultured BMDM controls were seeded in a six-well plate, the cells were carefully scraped off using a cell scraper. After washing, the suspension was sealed with 1:50 Fc Block (BD Pharmingen, 553141) at 4°C for 5 min and incubated with F4/80 antibody 1:100 (eBioscience, 48-4801-82, USA) at room temperature in the dark for 30 min, followed by membrane rupture using a membrane rupture kit (BD Pharmingen, 554714) at 4°C for 20 min. Further, it was incubated with CD206 (BD Pharmingen, 141710) at room temperature in the light for 30 min, washed, and tested using a Beckman CytoFlex flow cytometer.
Western blot analysis
The cultured BMDM controls were seeded in a six-well plate. The protein was extracted with 150 µL of 4× Sodium Dodecyl Sulfate (SDS) per well and quantified using a NanoDrop2000 spectrophotometer. The gel was prepared using a 10% Polyacrylamide Gel Electrophoresis (PAGE) gel kit (PG112, EpiZyme). Each well was loaded with 5–10 µL of the gel, and 5 µL of prestained protein ladder (Thermo Scientific, 00594048) was added on both sides, followed by electrophoresis at a constant voltage of 90 V for 90 min. The suspension was sealed with 5% skimmed milk powder and then incubated with SENP3 antibody 1:1000 (CST, 5591S) and β-actin antibody 1:1000 (Sigma, MAB8929) overnight at 4°C. Subsequently, 5% skimmed milk powder was separately added to dissolve SENP3 antibody 1:5000 (Jackson ImmunoResearch, 111-035-003, USA) and anti-β-actin antibody 1:5000 (Jackson ImmunoResearch, 115-035-003). The β-actin was developed using Electrochemiluminescence (ECL) colored solution (Pierce, 34580, USA), and SENP3 was developed using an ultra-sensitive colored solution (Merck, wbkls0500, Germany).
Statistical analysis
Data were analyzed using SPSS 23 software. Data from flow cytometry were acquired and analyzed using CytExpert. Statistical analyses were performed using t tests.