Mice

Six-to-eight-week-old wild-type female C57Bl/6J mice free of murine-specific pathogens were obtained from the Medical Experimental Animal Center of Central South University (Changsha, China). The mice were housed in the pathogen-free facility with a 12-h light/12-h dark cycle and free access to food and water. All procedures were approved by the Central South University Animal Ethics Committee.

Isolation of human peripheral blood mononuclear cells (PBMCs)

This study was approved by the ethics committee of the Third Xiangya Hospital of Central South University (No: 2022-S132). All volunteers signed informed consent forms. Blood samples were collected from 12 healthy volunteers, including six men and six women without a diagnosis of AR and who had a negative skin prick test (SPT) or specific serum IgE (sIgE) measurement. Human PBMCs were prepared from venous blood using the Ficoll–Hypaque method (TBD, China). The isolated human PBMCs were used for in vitro culture.

AR mouse model and treatment protocol

The flowchart of mouse allocation and treatments is shown in Fig. 1. Briefly, the mice were sensitized using house–dust–mite (HDM) antigen as follows: 40 µg of HDM extract (D. pteronyssinus, Greer Labs) diluted in 200 µL of sterile normal saline was administered to the mice by four intraperitoneal injections on days 1, 5, 10 and 14. Intranasal challenge was performed using 20 µg of HDM diluted in 20 µL of normal saline (NS) once a day from days 15 to 21. The allergic symptom score was calculated 15 min after the last challenge on day 21.

Fig. 1

Schematic for the establishment of AR mice and treatment schedule

In detail, 24 mice were randomly allocated into four groups: NS (n = 6), AR (n = 6), agomir (n = 6), and antagomir (n = 6). The mice in the NS group were sensitized and intranasally challenged with NS only, and those in the AR group were sensitized by intraperitoneal injections and intranasally challenged with HDM allergen. The sensitized mice in the agomir group and antagomir groups received the intranasal HDM challenge and then a nasal administration of 20 µL of 1.4 nmol miR-124-3p agomir or 20 µL of 2.8 nmol antagomir (GenePharma, Shanghai) once a day from days 15 to 21.

The mice were euthanized 24 h after the final challenge, peripheral blood was obtained by extracting the eyeball, and the nasal mucosa and spleen were isolated by precise dissection.

Measurement of HDM-specific IgE

Serum from mice was prepared by centrifugation of blood and then cryopreserved at − 80 °C. The serum level of HDM-specific IgE was measured using an enzyme-linked immunosorbent assay (ELISA) kit (Chondrex, Redmond, WA, USA) according to the manufacturers’ protocol.

Histopathology

Fresh murine nasal mucosa samples were fixed with 4% paraformaldehyde overnight, decalcified in EDTA, embedded in paraffin, dewaxed, rehydrated, and used for hematoxylin and eosin (H&E) staining, immunohistochemistry and immunofluorescence analyses. The staining was performed using standard procedures.

Immunohistochemistry (IHC)

The nasal mucosa tissue sections were blocked in blocking buffer for 1 h and then incubated with goat anti-rabbit IL-4 overnight at 4 °C. The sections were washed with PBS supplemented with 0.1% Tween-20 (PBS-T) and incubated with HRP-conjugated donkey anti-goat IgG antibody for 1 h at room temperature. After staining, all the samples were washed with PBS-T and then mounted for imaging. Images were captured using a microscope (Olympus, Japan).

Immunofluorescence (IF) staining

Freshly dissected murine nasal mucosa specimens were collected and processed as described above. Sections were deparaffinized, subjected to antigen repair, blocked with BSA blocking buffer for 30 min and then stained with rabbit anti-IL-4Rα (Thermo Fisher) overnight. Donkey–anti-rabbit FITC (Proteintech, China) was used as the secondary antibody. DAPI (Proteintech, China) was used for counterstaining. The slides were sealed with anti-fluorescence quencher. All images were captured with a confocal microscope (Leica, Germany).

Isolation of splenic lymphocytes

Splenic lymphocytes were isolated by Ficoll–Hypaque density centrifugation. Briefly, the spleens were mechanically minced into a homogenous paste with a scalpel on a dish plate and washed with PBS containing 2% FBS. After incubation in a 24-well plate for 30 min at 37 °C in a humidified incubator, cell suspensions were passed through a 70 µm Falcon nylon cell strainer, and lymphocytes were isolated from single cell suspensions of the spleen using lymphocyte separation medium (TBD, China). The isolated lymphocytes were used for further experiments, such as flow cytometry.

In vitro culture of lymphocytes and treatment protocol

Splenic lymphocytes from mice or human PBMCs were cultured in RPMI 1640 medium supplemented with 10% FBS, 1 µg/mL anti-CD3 (BioLegend, USA), 1 µg/mL anti-CD28 (BioLegend, USA) and 100 U/mL mouse IL-2 (PeproTech, USA) in a humidified incubator containing 5% CO2 at 37 °C. They were transferred to a 24-well plate at 1 × 106 cells/well in medium for 16 h and then transiently transfected with 2.5 µg of miR-124-3p mimic or mimic negative control or 5 µg of inhibitor or inhibitor negative control using Lipofectamine 3000 according to the manufacturer’s protocol for suspension cells. Each transfection was performed in triplicate in 24-well plates. Twenty-three hours after transfection, 2.5 µg of HDM antigen was added to the medium and incubated for 1 h. The cells in each well were then collected for extraction of RNA and protein.

Real-time PCR

Total RNA from murine nasal mucosa, splenic lymphocytes and human PBMCs was extracted using TRIzol reagent (Invitrogen, USA). RNA was reverse transcribed using ReverTra Ace® qPCR RT Master Mix with gDNA Remover (Toyobo, Japan). Real-time PCR was performed using KOD SYBR qPCR Mix (Toyobo, Japan), and detection was achieved using the LightCycler 480 System (Roche, Switzerland) according to the manufacturer’s instructions. All data were normalized to the β-actin levels and expressed relative to the control, and relative expression was calculated using the equation 2−ΔΔCt. The primer sequences are presented in Supplementary Table 1.

Western blot

Protein lysates from splenic lymphocytes and human PBMCs were processed in RIPA buffer (Kaiji Biotech, China) in the presence of phosphatase inhibitor and protease inhibitor cocktails. The total protein concentrations were measured by BCA assay (Kaiji Biotech, China). The samples were separated on 8–12% SDS–PAGE gels and then transferred to PVDF membranes (Millipore, USA). The membrane was incubated with primary antibody overnight at 4 °C and then with IRDye secondary antibodies (LI-COR Biosciences, USA) for 1 h at room temperature. The membrane was subsequently subjected to three 10-min washes with TBST buffer. The bands were scanned and quantified using a LI-COR Odyssey CLx scanner (LI-COR Biosciences, USA). β-Actin or Gapdh was used as an internal reference.

Flow cytometry

Flow cytometric analyses for the T cell phenotyping of splenic lymphocytes were performed as follows. Prior to cell surface staining, cells (1 × 106) were seeded into a 24-well plate and stimulated with PMA/Ionomycin mixture and BFA/monensin mixture for 4 h. For surface marker staining, cells (1 × 106) were stained with anti-CD4 conjugated with APC-Cyanine7 (eBioscience, USA) for 30 min on ice and then washed in PBS containing 5% FBS. For intracellular cytokine staining, cells (1 × 106) were fixed with the FoxP3/Transcription Factor Staining Buffer Kit (eBioscience, USA) and then incubated with anti-IL4 conjugated with PE-Cyanine7 and anti-IFN-γ conjugated with PerCP-Cyanine5.5 (eBioscience, USA) for 30 min. Cytokine level in CD4+ lymphocytes was analyzed using FlowJoV10 (Tree Star, Ashland, OR).

Dual-luciferase reporter assays

IL-4Rα was predicted to be the underlying target of miR-124p by an online bioinformatics analysis (PicTar, TargetScan and miRBase). An interaction diagram of mmu-miR-124 and wild-type IL-4Rα-UTR is shown in Fig. 2a. Dual-luciferase assays were implemented using the luciferase reporter assay system in accordance with the manufacturer’s instructions (Promega). The mutated (Mut) or wild-type (WT) IL-4Rα-3′-UTR sequence including the mmu-miR-124 targeting site was inserted into the XhoI/BamHI sites of the pLUC vector to construct pLUC-luc-IL-4Rα. All constructs were verified by sequence analysis (Supplementary Fig. 1). HEK-293T cells were prepared, seeded in 96-well plates and then transfected with pLUC-luc-IL-4Rα (0.2 µg), mmu-miR-124 negative or mimic control (0.45 µg) or Renilla luciferase (0.15 µg) by adopting FuGENE® HD (Roche, Switzerland). The transfections were performed in duplicate, and each experiment was repeated in triplicate. Luciferase activity was detected after 48 h using the Luciferase Reporter Assay System (Promega). IL-4Rα-3′-UTR activity is expressed as a ratio of firefly luciferase activity to Renilla luciferase activity.

Statistical analyses

All quantification results are shown as the means (± SEMs) of at least three independent experiments. Statistical comparisons between two groups were conducted by unpaired two-tailed Student’s t tests. One-way analysis of variance (ANOVA) was used for comparisons of multiple groups. The statistical analyses were performed using GraphPad Prism 8 software (GraphPad Software Inc.). A p value < 0.05 was considered to indicate statistical significance.