IL-37a facilitates allergen immunotherapy in a house dust mite-induced mouse model of allergic asthma

Abstract

In recent years, the incidence of allergic asthma has increased dramatically. Allergen immunotherapy (AIT) is an effective approach to achieve long-term remission of allergic asthma and has gained widespread attention. However, current AIT suffers from low efficacy and long treatment duration. In this study, we found that IL-37a transgenic mice with allergic asthma exhibited reduced pulmonary inflammation, decreased Th2 cytokines, and lower allergen-specific IgE following AIT. Mechanistic studies revealed increased IL-37⁺ Bregs, suggesting that IL-37a may promote AIT through Bregs. Our study demonstrates the promoting effect of IL-37 in allergen immunotherapy and suggests that Bregs may underlie this mechanism, providing insights for overcoming the limitations of AIT in the future.

Supplementary Information

The online version contains supplementary material available at https://doi.org/10.1186/s12865-026-00869-x.

Introduction

Allergic diseases are the most common chronic disorders worldwide, afflicting up to 22% of the global population. Annual spending on anti-allergic medications alone exceeds US $8 billion, and the World Health Organization has ranked allergy among the three priority diseases requiring intensive prevention and control in the twenty-first century [1]. House dust mite (HDM) is the most prevalent allergen among allergic asthma and rhinitic patients in China [2], and the level of sensitization to Dermatophagoides pteronyssinus (D. pteronyssinus) strongly correlates with increasing asthma severity [3]. T helper (Th) 2 cells, eosinophils, innate lymphoid cells type 2 (ILC2), M2 macrophages, inflammatory dendritic cells (DC) and natural killer T (NKT) cells accumulate in the tissue, creating the chronic features of allergic asthma. Repeated cycles of injury and repair enhance connective-tissue deposition and structural airway changes—basement-membrane thickening and smooth-muscle hypertrophy—culminating in airway remodeling [4].

Allergen immunotherapy (AIT) involves the repeated administration of gradually increasing doses of allergen extracts to allergic patients, thereby enhancing their tolerance to the allergen. It is the only etiology-targeted treatment for allergic diseases that can alter the natural course of the disease [5]. Currently, AIT requires a long course, typically lasting three to five years. Treatment with standardized allergen vaccines markedly reduces sensitivity to HDM in asthmatic patients, alleviates allergic symptoms, diminishes airway hyper-responsiveness, lowers the need for anti-allergic and anti-asthmatic medication, and can even prevent the development of new sensitizations [6].

Although AIT has been used for more than a century, its mechanisms of action remain incompletely understood. Current evidence indicates that desensitization may (1) reverse the dominant Th2 response by driving a compensatory Th1 reaction and (2) induce allergen-specific immune tolerance through progressively increasing antigen doses, thereby preventing recurrence and progression of antigen-driven asthma. Mechanistically, AIT shifts the immune balance from a Th2-skewed profile toward Th1/Th2 equilibrium and expands regulatory populations including regulatory T (Treg), B (Breg) and dendritic (DCreg) cells [7], serum allergen-specific IgE decreases, whereas “blocking” IgG4 and IgA increase, and eosinophil, basophil and mast-cell numbers and activity decrease [8], tolerogenic cytokines IL-10, transforming growth factor (TGF)-β and IL-35 increase while Th2 cytokines (IL-4, IL-5, IL-9, IL-13) decrease [9]. Bregs contribute centrally to this process by releasing IL-10 and TGF-β1, which directly suppress T-cell responses, inhibit dendritic-cell maturation, promote Tregs expansion and drive IgG4 production [9]. Thus, Bregs are pivotal mediators of AIT-induced tolerance.

IL-37 is a recently discovered immunoregulatory factor that is widely expressed in epithelial cells and various immune cell [10]. As a member of the IL-1 family, IL-37 exerts dual anti-inflammatory effects both extracellularly and intracellularly. The IL-37 precursor can be secreted extracellularly, where it binds to the membrane-bound heterodimeric receptor complex IL-1R8/IL-18Rα, blocking myeloid differentiation primary response gene 88 (MyD88) recruitment and activation of downstream inflammatory pathways, thereby exerting broad-spectrum anti-inflammatory effect [11]. Additionally, IL-37 can bind to phosphorylated SMAD family member 3 (pSmad3) and translocate to the nucleus, where it inhibits the expression of pro-inflammatory factors such as TGF-β to achieve anti-inflammatory effects [12]. Interestingly, unlike humans, mice have lost the IL-37 gene during evolution but still retain the IL-37 receptor, and can bind human IL-37 to mediate immunosuppression, providing a valuable in vivo model for studying human IL-37 function [13]. Studies have shown that IL-37 is induced by inflammatory factors and can effectively inhibit multiple inflammatory signaling pathways, including metabolic pathways, Toll-like receptor (TLR), nuclear factor kappa- B (NF-κB), mitogen-activated protein kinase (MAPK), etc., and may play an important protective role in respiratory diseases such as asthma, pulmonary fibrosis, and acute pneumonia [14–17].

The IL-37 gene contains six exons and can express five isoforms (a-e) through alternative RNA splicing. The IL-37a isoform consists of exons 3–6 (192 amino acids) and exhibits substantial structural differences from other isoforms. Previous work has found that, like IL-37b, IL-37a can also regulate immunity by binding to the IL-1R8/IL-18Ra receptor. The difference is that IL-37a carries a unique nuclear localization sequence (exon 3) and may translocate to the nucleus through different mechanisms to inhibit inflammatory gene activation [18]. Compared with IL-37b, IL-37a exhibits a more pronounced inhibitory effect on inflammatory cytokine production [19]. These results suggest that IL-37a may play an important and unique regulatory role in asthmatic inflammatory diseases.

In our study, we used IL-37a transgenic mice (IL-37atg) to model allergic asthma and AIT, combined with recombinant human IL-37a protein (rhIL-37a) as adjunctive therapy. We found that IL-37atg asthmatic mice showed attenuated pulmonary inflammation, reduced Th2 cytokine levels, and decreased HDM-specific IgE. Moreover, AIT was more effective in IL-37atg asthmatic mice, possibly via increased IL-37⁺ Bregs. Our study preliminarily validated the role of IL-37a in allergic asthma and its treatment, advancing AIT research.

Materials and methods

Generation and identification of IL-37atg mice

All animal procedures received prior approval from the Wuhan Fourth Hospital Institutional Animal Care and Use Committee. The IL-37a transgenic (IL-37atg) mice used in this study were originally generated by Prof. Damo Xu’s laboratory and kindly provided to us by Prof. Xu. The generation and characterization of these mice have been previously described in detail [18]. Briefly, the human IL-37a coding sequence (exons 3–6, encoding 192 amino acids) was cloned into a transgenic expression vector under the control of a ubiquitous promoter. The linearized transgene was then microinjected into fertilized C57BL/6J mouse oocytes via standard pronuclear microinjection. Founder mice were identified by PCR genotyping and subsequently backcrossed onto the C57BL/6J background for at least six generations. Heterozygous IL-37atg mice were used for all experiments, with wild-type C57BL/6J littermates serving as controls.

For routine genotyping, approximately 0.5 cm of ear tissue was collected and put into a PCR tube, add 200 µL of 0.05 M NaOH, and incubate at 95°C for 1 h in a PCR machine (Thermo Fisher Scientific, Waltham, MA, USA). After heating, add 17 µL of pH 6.8 Tris-HCl, mix gently. Conventional PCR was performed using 2.5 µL of the extracted genomic DNA as template. The PCR reaction mixture (20 µL total volume) contained: 10 µL of Premix Taq buffer (Takara, Catalog Number: RR901), 0.5 µL of each primer (10 µM), and 6.5 µL of nuclease-free water. The following primers were used to amplify the human IL-37a transgene: Forward 5’-ATGTCAGGCTGTGATAGGAGGGAAACAG-3’ and Reverse 5’-TGCTATGAGATTCCCAGAGTCCAGGACC-3’, yielding a 150-bp amplicon. The thermal cycling conditions were: initial denaturation at 95 °C for 3 min, 35 cycles of 94 °C for 30 s, 62 °C for 30 s, and 72 °C for 30 s; followed by a final extension at 72 °C for 5 min. After amplification, PCR products were separated by electrophoresis on a 2% agarose gel containing ethidium bromide. Electrophoresis was performed using a Bio-Rad electrophoresis system (PowerPac Basic Power Supply and Sub-Cell GT Horizontal Electrophoresis Cell, Bio-Rad Laboratories, Hercules, CA, USA) at 100 V for 40 min. Gels were visualized under UV light using a Thermo Fisher Scientific gel documentation system (Thermo Fisher Scientific, Waltham, MA, USA). Wild-type C57BL/6J mice served as negative controls.

Murine model of allergic asthma and allergen immunotherapy

Four-week-old female C57BL/6J (wild type control) and IL-37atg mice were randomly divided into four groups: Ctrl (A), Asthma (B), AIT (C) and rhIL-37a plus AIT (D). Except for group A, mice in the other groups received intraperitoneal injections of 150 µL sensitization solution (50 µg HDM crude extract and 2 mg Al (OH)₃,) on days 0, 7, and 14. Group A received equivalent volumes of PBS. The HDM crude extract (Dermatophagoides pteronyssinus) was purchased from Stallergenes Greer (Catalog No. XPB91D3A25). From days 36 to 40 (5 consecutive days), mice in Group C (AIT) received subcutaneous HDM-AIT once daily, consisting of 50 µg HDM crude extract in 100 µL PBS. Mice in Group D (AIT+rhIL-37a) received intranasal rhIL-37a (1 µg per mouse in 50 µL PBS) once daily, followed 30 min later by subcutaneous HDM-AIT (same dose as Group C) [20]. The rhIL-37a protein was a generous gift from Prof. Damo Xu, and its generation and characterization have been described previously [18]. Groups A and B received equivalent volumes of PBS via the same routes at corresponding time points. Starting on day 55, all groups were intranasally challenged with HDM every other day for a total of three challenges (days 55, 57, and 59). Each challenge consisted of 25 µg HDM crude extract in 50 µL PBS. Mice were lightly anesthetized by isoflurane inhalation (3–5% for induction, 1.5–2% for maintenance) in an induction chamber, after which the required volume was slowly instilled into the nostrils. The experiment ended on day 61. Mice were euthanized for tissue collection. All animals were first deeply anesthetized with 5% isoflurane inhalation in an induction chamber, maintained until the breathing rate slowed significantly and the pedal reflex was completely absent. Mice were then administered an intraperitoneal injection of sodium pentobarbital (100 mg/kg body weight) to ensure deep anesthesia. Following confirmation of complete loss of consciousness and absence of response to toe pinch, blood was collected via cardiac puncture, after which the mice were exsanguinated and cervical dislocation was performed as a secondary physical method to ensure death. The lungs and other tissues were immediately harvested for subsequent analysis. All procedures were carried out in accordance with institutional guidelines for animal care and euthanasia.

Airway resistance

Anesthetized mice are tracheostomized and mechanically ventilated via a flexiVent system (SCIREQ, Montreal, Canada), where airway resistance (Rrs) is calculated in real-time from simultaneously recorded peak transpulmonary pressure (ΔP, cmH₂O) and peak flow (mL·s⁻¹) using the equation Rrs = ΔP/Flow. Following a 5-minute stabilization period (tidal volume 10 mL/kg, 150 breaths/min, PEEP 3 cmH₂O), baseline measurements are acquired, then dose-dependent bronchoconstriction is induced by aerosolizing methacholine (3.125–50 mg/mL) through an in-line nebulizer, with Rrs computed from the pressure-flow relationship at each concentration to generate dose-response curves and quantify airway hyperresponsiveness.

ELISA for cytokines

Cytokine levels in BALF were measured using commercial ELISA kits according to the manufacturers’ instructions. The following kits were used: IL-4 (Invitrogen, Catalog No. 88-7044-88), IL-5 (Invitrogen, 88-7054-88), IL-13 (Invitrogen, 88-7137-86), IL-10 (Invitrogen, 88-7105-88), IL-33 (Invitrogen, 88-7333-88), TGF-β1 (Invitrogen, 88-8350-86), and human IL-37 (R&D Systems, DuoSet ELISA, Catalog No. DY1975). All Invitrogen Uncoated ELISA Kits contain pre-titrated capture and detection antibodies. According to the manufacturer’s instructions, capture antibodies and detection antibodies were used at a 1:250 dilution. For the R&D Systems DuoSet ELISA, the capture antibody was used at 2 µg/mL and the detection antibody at 200 ng/mL. The assay procedure was as follows: 96-well plates were coated with capture antibody overnight at 4 °C, blocked with 1% BSA in PBS for 1 h at room temperature, and then incubated with standards or samples (100 µL per well) for 2 h at room temperature. After washing, detection antibody was added for 2 h, followed by streptavidin-HRP for 20 min. Substrate solution (TMB) was added for 15 min, and the reaction was stopped with 2 N H₂SO₄. Absorbance was read at 450 nm using a microplate reader (Thermo Fisher Scientific, Waltham, MA, USA). Standard curves were generated using a four-parameter logistic (4PL) curve fit, performed with SoftMax Pro software (Molecular Devices, San Jose, CA, USA). Sample concentrations were calculated by interpolating from the standard curve. All samples were measured in duplicate, and the mean values were used for analysis.

ELISA for immunoglobulins

Dissolved HDM at 2 µg/mL in carbonate-bicarbonate buffer (CBS) and coat a 96-well plate with 100 µL per well; incubate overnight at 4 °C. Blocked with 1% BSA in 0.05% PBST (100 µL per well) for 1 h at 37 °C. Added serum samples (100 µL per well) and incubate for 2 h at 37 °C. Washed five times with PBST, then add HRP-conjugated anti-mouse secondary antibody diluted 1:1000 in 1% BSA/0.05% PBST (100 µL per well); incubate for 1 h at 37 °C. Washed five times with PBST, added 100 µL per well of TMB substrate, and incubated at room temperature for 5 min; stopped the reaction with stop solution. Read absorbance at 450 nm (Thermo Fisher Scientific, Waltham, MA, USA).

Hematoxylin and eosin staining

After the mice were euthanized under anesthesia, the lung tissues were collected and fixed in 4% paraformaldehyde at room temperature for 24 h. Following dehydration, clearing, paraffin infiltration, and embedding, the tissues were sectioned at 4 μm thickness. H&E staining was performed using a commercial kit (Solarbio, Beijing, China; Catalog No. G1120) according to the manufacturer’s instructions. Briefly, sections were deparaffinized in xylene, rehydrated through a graded ethanol series, stained with hematoxylin for 5 min and eosin for 2 min, then dehydrated and mounted. Inflammatory cell infiltration was examined under a light microscope. Lung inflammation was scored as follows: 0, normal tissue; 1, few inflammatory cells; 2, inflammatory cell cuff 1 cell layer thick; 3, inflammatory cell cuff 2–4 cell layers thick; 4, inflammatory cell cuff > 4 cell layers thick [21]. For each mouse, three non-adjacent sections (spaced at least 100 μm apart) were analyzed, and five randomly selected fields of view were examined per section. All sections were scored by two independent observers blinded to the experimental groups. All images were captured using an Olympus IX71 microscope (Olympus Corporation, Tokyo, Japan).

Periodic acid–schiff staining

PAS staining was performed using a commercial kit (Solarbio, Beijing, China; Catalog No. G1281) according to the manufacturer’s instructions. Briefly, sections were oxidized in 0.5% periodic acid for 5 min, rinsed, stained with Schiff’s reagent for 15 min, counterstained with hematoxylin for 1 min, then dehydrated and mounted. Goblet-cell metaplasia was examined under a light microscope. PAS-stained images were analyzed with ImageJ software (National Institutes of Health, Bethesda, MD, USA, https://imagej.net/contribute/citing): a segment of bronchial epithelium of defined length was outlined, the total area of this epithelial segment was measured, and the area occupied by PAS-positive (red) goblet cells within that segment was then calculated. Goblet-cell hyperplasia in airway epithelium was graded on a 5-point scale: 0 = normal; 1 = < 25%; 2 = 25–50%; 3 = 50–75%; 4 = > 75% epithelial involvement [22]. The same number of sections and fields of view were analyzed as for H&E staining, and scoring was performed by the same two blinded observers. All images were captured using an Olympus IX71 microscope (Olympus Corporation, Tokyo, Japan).

Giemsa staining

After the mice were euthanized under anesthesia, the trachea was exposed via a midline incision and cannulated with a 22-gauge catheter. Bronchoalveolar lavage (BAL) was performed by instilling a total of 1.0 mL of sterile PBS into the lungs in two aliquots of 0.5 mL each. After each instillation, the PBS was gently aspirated, and the two recovered fractions were pooled. The typical recovery volume was 0.8–0.9 mL (80–90% recovery). The pooled BAL fluid was centrifuged at 500 × g for 10 min at 4 °C. The supernatant was collected and stored at -80 °C for subsequent cytokine analysis. The cell pellet was resuspended in 100 µL PBS for cytospin preparation. The resuspended cell suspension was loaded into a cytocentrifuge (Cytospin 4, Thermo Fisher Scientific, Waltham, MA, USA) and spun at 500 rpm for 5 min onto glass slides. The slides were then air-dried at room temperature. Giemsa staining was performed using a commercial kit (Solarbio, Beijing, China; Catalog No. G1015) according to the manufacturer’s instructions. Briefly, air-dried slides were fixed in methanol for 5 min, immersed in diluted Giemsa staining solution (1:10 dilution in PBS) for 20 min, rinsed gently with distilled water, and air-dried again. Stained slides were examined under a light microscope (Olympus IX71, Tokyo, Japan). Slides were counted by two independent blinded observers. Eosinophils were identified by their characteristic bilobed nucleus and red cytoplasmic granules. The proportion of eosinophils among total cells was calculated as (eosinophil count / total cells counted) × 100%. All counts were performed by two independent observers blinded to the experimental groups.

Flow cytometry

Mouse spleens were harvested and placed in RPMI 1640 medium on ice. Each spleen was gently mashed through a 70 μm cell strainer (Falcon, Corning, NY, USA) using the plunger of a 1 mL syringe. The strainer was rinsed with cold PBS, and the resulting single-cell suspension was collected. Red blood cells were lysed using ACK Lysis Buffer (Solarbio, Beijing, China; Catalog No. R1013) according to the manufacturer’s instructions. Briefly, 2 mL of ACK Lysis Buffer was added to the cell pellet, gently mixed, and incubated for 3 min at room temperature. The lysis reaction was stopped by adding 10 mL of cold PBS, and the cells were centrifuged at 300 × g for 5 min at 4 °C. The supernatant was discarded, and the cell pellet was washed once more with 10 mL of cold PBS. Cells were then resuspended in cold PBS, counted using a hemocytometer, and adjusted to approximately 1 × 10⁶ cells per tube. Cells were blocked with purified Rat Anti-Mouse CD16/CD32 (Mouse BD Fc Block™, BD Biosciences, Catalog No. 553142, clone 2.4G2; 1:100 dilution) for 15 min on ice in the dark to prevent non-specific binding of antibodies to Fc receptors. For surface staining, cells were incubated with LIVE/DEAD™ Fixable Aqua Dead Cell Stain Kit (Invitrogen, Catalog No. L34966, 1:1000 dilution) for 30 min on ice in the dark to exclude dead cells. After washing with cold PBS, cells were stained with the following antibodies (all from BioLegend, San Diego, CA, USA): FITC anti-mouse CD19 (clone 1D3/CD19, Catalog No. 152404, 1:200 dilution), Brilliant Violet 421™ anti-mouse CD5 (clone 53 − 7.3, Catalog No. 100618, 1:200 dilution), and APC-Fire750™ anti-mouse CD1d (clone 1B1, Catalog No. 123525, 1:200 dilution). The staining reaction was carried out on ice for 30 min in the dark. After surface staining, cells were washed twice with cold PBS and then fixed and permeabilized using the Cytofix/Cytoperm™ Fixation/Permeabilization Kit (BD Pharmingen, Catalog No. 554714) according to the manufacturer’s instructions. Briefly, cells were resuspended in 250 µL of Cytofix/Cytoperm buffer and incubated for 20 min at 4 °C. Cells were then washed twice with 1× Perm/Wash buffer. For intracellular staining, cells were incubated with Brilliant Violet 605™ anti-mouse IL-10 (clone JES5-16E3, BioLegend, Catalog No. 505031, 1:100 dilution) and PE mouse anti-human IL-37 (clone 37D12, Invitrogen, Catalog No. 12-7379-42, 1:50 dilution) in 1× Perm/Wash buffer for 30 min at room temperature in the dark. After a final wash with Perm/Wash buffer, cells were filtered through a 40 μm mesh and acquired on a BD LSRFortessa flow cytometer (BD Biosciences, San Jose, CA, USA). Data were analyzed using FlowJo software (TreeStar, Ashland, OR, USA). The gating strategy was presented in Supplementary Figure S1.

Statistics and analysis

To confirm reproducibility, every assay was run in a minimum of three biologically independent replicates. Summary statistics (mean ± SD) were computed with SPSS. Pairwise group differences were evaluated by two-tailed Student’s t-test; experiments with more than two conditions were examined by one-way ANOVA followed by Tukey post-hoc correction. Differences were judged significant when p < 0.05. Final plots were assembled in GraphPad Prism.

Results

IL-37a enhanced allergen immunotherapy in allergic asthma

To evaluate the role of IL-37 in allergen immunotherapy of allergic asthma, we generated and expanded IL-37atg mice. RT-PCR screening revealed a 150-bp transgene-specific band in IL-37atg mouse spleen cells that was absent in wild-type mice (Fig. 1A). Four-week-old female C57BL/6J and IL-37atg mice were randomly assigned to four groups (n = 4–7 per group): Ctrl, Asthma, Allergen Immunotherapy (AIT), or AIT plus rhIL-37a (AIT+rhIL-37a) (Fig. 1B). The rhIL-37a used were described previously [18]. Airway resistance measured 24 h after the final allergen challenge was lower in IL-37atg asthmatic mice than in wild-type controls. AIT further reduced resistance in both genotypes, whereas supplemental rhIL-37a did not further improve lung function beyond AIT alone (Fig. 1C). Bronchoalveolar-lavage fluid (BALF) collected at sacrifice was cytospun and stained with Giemsa. IL-37atg allergic mice showed a reduced percentage of eosinophils, which fell further after AIT, again, rhIL-37a co-administration did not augment this effect (Fig. 1D). Histological examination of the right middle lobe (H&E and PAS) revealed diminished peribronchial inflammation, fewer goblet-cell metaplasia and less subepithelial fibrosis in IL-37atg asthmatic animals. These improvements were even more pronounced when AIT was given (Fig. 1E). Collectively, the data indicate that IL-37a not only attenuates allergic airway disease but also enhances the efficacy of AIT.

Fig. 1.

Experimental design and lung status of mice with allergic asthma and AIT. (A). Genomic IL-37a transgene detected by PCR. A 150-bp band was present in IL-37atg mice but absent in wild-type C57BL/6J controls. All mice used in this study were genotyped, and a representative gel was shown. (B). Schematic timeline of the experimental design. Female wild-type C57BL/6J and IL-37atg mice were randomly assigned to four groups (n = 4–7 per group): Ctrl (PBS sensitization + HDM challenge), Asthma (HDM sensitization + HDM challenge), AIT (HDM sensitization + HDM-AIT + HDM challenge), and AIT+rhIL-37a (HDM sensitization + HDM-AIT + rhIL-37a + HDM challenge). HDM sensitization was performed intraperitoneally on days 0, 7, and 14 using 50 µg HDM crude extract plus 2 mg Al (OH)₃ in 150 µL PBS. From days 36 to 40 (5 consecutive days), the AIT group received subcutaneous HDM-AIT (50 µg HDM in 100 µL PBS) once daily, the AIT+rhIL-37a group received intranasal rhIL-37a (1 µg in 50 µL PBS) followed 30 min later by subcutaneous HDM-AIT, the Asthma and Ctrl groups received equivalent volumes of PBS via the same routes. Intranasal HDM challenge (25 µg HDM crude extract in 50 µL PBS) was administered on days 55, 57, and 59 (every other day, three challenges total). All mice were euthanized on day 61 for sample collection. (C). Airway resistance in each group. (D). Eosinophil infiltration in the lung assessed by Giemsa staining. (E). Pulmonary inflammation level evaluated by H&E staining and goblet-cell metaplasia detected by PAS staining. Experiments were repeated three times independently. Data are presented as mean ± SD (n = 4–7 mice per group). Each data point represents an individual mouse. Statistical significance: ns, p ≥ 0.05 (not significant), *p < 0.05, **p < 0.01, ***p < 0.001

IL-37a attenuated Th2-type cytokines production

BALF was collected from animals at the end of the protocol. ELISA showed that Th2 cytokines were markedly lower in both IL-37atg asthmatic and AIT groups relative to WT controls, whereas IL-10 and TGF-β levels remained comparable across genotypes (Fig. 2A–F). IL-37 itself was significantly reduced in IL-37atg asthmatic mice but rebounded after AIT (Fig. 2G). Administration of exogenous rhIL-37a failed to provide any additional benefit to AIT (Fig. 2).

Fig. 2.

Cytokine levels in BALF measured by ELISA. Concentrations of (A) IL-4, (B) IL-5, (C) IL-13, (D) IL-33, (E) IL-10, (F) TGF-β, and (G) IL-37 in BALF from each group. All samples were measured in duplicate (technical replicates). Experiments were repeated three times independently. Data are presented as mean ± SD (n = 4–7 mice per group). Each data point represents an individual mouse. Statistical significance: ns, p ≥ 0.05 (not significant), *p < 0.05, **p < 0.01, ***p < 0.001

IL-37a reduced HDM-specific IgE in serum

Serum were collected and analyzed for HDM-specific IgE (sIgE), sIgG1, and sIgG2a. In both the asthma and AIT groups, sIgE levels were significantly lower than in the respective controls (Fig. 3A), whereas sIgG1 and sIgG2a titers remained unchanged (Fig. 3B, C).

Fig. 3.

Serum allergen-specific immunoglobulin levels measured by ELISA. Serum levels of (A) HDM-specific IgE (sIgE), (B) HDM-specific IgG2a (sIgG2a), and (C) HDM-specific IgG1 (sIgG1) in each group. All samples were measured in duplicate (technical replicates). Experiments were repeated three times independently. Data are presented as mean ± SD (n = 4–7 mice per group). Each data point represents an individual mouse. Statistical significance: *p < 0.05

IL-37⁺ Bregs increased in AIT mice

Although IL-37 was up-regulated during AIT, its expression within Bregs remained undefined. We therefore used flow cytometry to quantify IL-10⁺ and IL-37⁺ fractions among splenic Bregs. In WT mice, the IL-10⁺ Breg subset declined in Asthma mouse and was restored by AIT (Fig. 4A). IL-37atg mice displayed the same pattern for IL-37⁺ and IL-10⁺IL-37⁺ double-positive Bregs, whereas IL-10⁺ Bregs showed no significant change (Fig. 4B).

Fig. 4.

Identification of IL-10⁺ and IL-37⁺ Bregs in splenocytes by flow cytometry. (A). Frequency of CD19⁺CD5⁺CD1d^hiIL-10⁺ cells in wild-type mice splenocytes. (B). Frequencies of CD19⁺CD5⁺CD1d^hiIL-10⁺IL-37⁻, CD19⁺CD5⁺CD1d^hiIL-10⁻IL-37⁺, and CD19⁺CD5⁺CD1d^hiIL-10⁺IL-37⁺ subsets in IL-37atg mice splenocytes. Experiments were repeated three times independently. At least 10,000 events were acquired per sample.Data are presented as mean ± SD (n = 4–7 mice per group). Each data point represents an individual mouse. Statistical significance: *p < 0.05, **p < 0.01

Discussion

Most studies to date have focused on IL-37b, whereas the functions of the other IL-37 isoforms remain unknown. IL-37a differs from IL-37b in structure, nuclear translocation, protein maturation and functional activity. Like IL-37b, IL-37a can trigger comparable immunoregulatory responses through the IL-37 receptor; however, IL-37a additionally harbours a unique nuclear-localization sequence that enables it to enter the nucleus. There it activates histone phosphatases, thereby promoting expression of immunoregulatory factors such as peroxisome-proliferator-activated receptor-γ (PPARγ) and consequently suppressing inflammatory-gene activation [23]. Thus, IL-37a is a cytokine that operates both intracellularly and extracellularly, exerting immunomodulatory effects via receptor-dependent and receptor-independent pathways. IL-37b has been reported to protect against respiratory diseases, attenuating asthma, pulmonary fibrosis and acute pneumonia by directly inhibiting the activation and function of asthma-related pathological cells—mast cells, eosinophils and macrophages—through its receptor [24, 25], and by inducing tolerogenic dendritic cells and Foxp3⁺ regulatory T cells [26, 27]. Whether IL-37a plays a role in allergic asthma or AIT is still unknown. We established HDM-induced allergic asthma and AIT models in wild-type and IL-37atg mice and monitored changes in lung histopathology, serum allergen-specific Ig levels, and cytokine profiles. IL-37atg asthmatics exhibited markedly attenuated pulmonary inflammation, with fewer infiltrating eosinophils and reduced goblet-cell metaplasia. After AIT, these protective effects were even more pronounced, leaving IL-37atg mice with significantly milder lung pathology than their wild-type counterparts. Our study provides the demonstration that IL-37a not only markedly attenuates allergic asthma but also markedly potentiates AIT in asthmatic mice.

The phenomenon of reduced IL-37 in allergic asthma has been confirmed by multiple independent studies. The levels of IL-37 are decreased in the sputum and nasal secretions of human asthma patients [28]. IL-37 levels in peripheral blood mononuclear cells (PBMCs) of asthma patients are lower than those in healthy controls and are negatively correlated with disease severity indicators such as FeNO and ACT scores [29]. Furthermore, several serum-based studies have consistently shown that serum IL-37 levels are significantly lower in patients with asthma and allergic rhinitis compared to healthy controls [30, 31]. Our study found reduced IL-37 levels in animal experiments. Collectively, these findings support a protective role for IL-37 in allergic asthma, and its reduced level may be an important factor in the development and progression of the disease.

A key finding of this study is that IL-37 levels in BALF were significantly elevated in IL-37atg asthmatic mice following AIT. It should be noted that currently available commercial ELISA kits cannot specifically distinguish IL-37a from other isoforms, and the detected signal represents total IL-37 levels. Since IL-37atg mice only harbor the transgenic human IL-37a gene, the expressed human IL-37 protein is theoretically exclusively the IL-37a isoform; therefore, the detected IL-37 signal can reflect the expression/accumulation level of human IL-37a. This result forms an interesting contrast with a recent clinical study in patients with allergic rhinitis. Jakwerth et al. [32] longitudinally measured various cytokines in nasal secretions from grass pollen-allergic patients undergoing three years of AIT and found that IL-37 levels continuously decreased during treatment rather than increased. However, several differences exist between the two studies. First, Jakwerth et al. detected IL-37 in nasal secretions (local upper airway compartment), whereas the present study measured IL-37 in BALF (local lower airway compartment). Although both represent local airway compartments, the upper and lower airways may respond differently to AIT. Second, Jakwerth et al. detected endogenous human IL-37 (predominantly the IL-37b isoform), while the present study detected a human IL-37a transgenic product. IL-37a and IL-37b exhibit substantial differences in structure, nuclear translocation mechanisms, and functional activities [18, 19], and may be subject to distinct post-transcriptional regulatory controls, which could represent the underlying molecular basis for the differential dynamics observed following AIT. Third, the Jakwerth et al. study was an observational cohort in which patients were concurrently exposed to natural pollen season allergen stimulation during AIT. In contrast, AIT mice in the present study received immediate allergen challenge and sampling following treatment completion, potentially capturing an acute upregulation response of IL-37a, whereas Jakwerth et al. measured steady-state levels during chronic treatment. Collectively, the regulation of IL-37 family members by AIT may exhibit isoform-specific, time-dependent, and anatomical site-heterogeneous characteristics. The differential dynamics of IL-37a and IL-37b during AIT reveal the functional heterogeneity of this family. Future studies should develop detection tools capable of specifically distinguishing IL-37 isoforms and dynamically monitor the expression changes of IL-37 isoforms in different anatomical compartments (BALF, nasal secretions, serum) and at different treatment stages to comprehensively understand the mechanisms of IL-37 in AIT.

In BALFs of allergic asthmatic mice, IL-37 was markedly reduced, they rose again after AIT, yet no significant difference was observed between mice receiving AIT alone and those co-treated with rhIL-37a protein. Regardless of genetic background, AIT substantially suppressed Th2-type cytokine expression in both wild-type and IL-37atg mice. After AIT, wild-type animals displayed significant increases in IL-10 and TGF-β, whereas IL-10 showed no significant increase. in IL-37atg transgenics, suggesting that IL-37a may inhibit IL-10 production.

Our previous work identified IL-37⁺ Bregs in humans that can be induced by rIL-37 or Cytosine-phosphate-Guanine (CpG) through distinct mechanisms without eliciting IL-10. The frequency of these IL-37⁺ Bregs is markedly reduced in patients with psoriasis, underscoring their potential role in inflammatory diseases [33]. In this study, we have demonstrated in vivo that IL-37⁺ Bregs were associated with a beneficial effect during AIT. However, the optimal inductive regimen and their precise mechanisms of action remain to be elucidated, and causal evidence requires further functional studies.

In summary, we showed that asthmatic IL-37atg mice receiving AIT revealed markedly attenuated airway inflammation, suppressed Th2 cytokine production and reduced allergen-specific IgE. This enhanced therapeutic response was associated with a selective expansion of IL-37⁺ Bregs, raising the hypothesis these cells may mediate the link between IL-37a and improved AIT efficacy. Our findings provide the evidence that IL-37a accelerates AIT and point to IL-37⁺ Bregs as a cell type warranting further investigation. Future studies employing adoptive transfer or Breg-specific depletion will be required to determine whether these cells are necessary and sufficient for the observed effect.

Abbreviations

AIT

Allergen immunotherapy

BALF

Bronchoalveolar lavage fluid

Breg

Regulatory B cell

CBS

Carbonate-bicarbonate buffer

CpG

Cytosine-phosphate-Guanine

DC

Dendritic cell

DCreg

Regulatory dendritic cell

HDM

House dust mite

IL-37atg

IL-37a transgenic mice

ILC2

Innate lymphoid cells type 2

MAPK

Mitogen-activated protein kinase

MyD88

Myeloid differentiation primary response gene 88

NF-κB

Nuclear factor kappa- B

NKT

Natural killer T

PPARγ

Peroxisome-proliferator-activated receptor-γ

pSmad3

Phosphorylated SMAD family member 3

rhIL-37a

Recombinant human IL-37a protein

Rrs

Airway resistance

sIgE

HDM-specific IgE

TGF

Transforming growth factor

TLR

Toll-like receptor

Treg

Regulatory T cell

Authors’ contributions

Conceptualization: Zheng Jin; Zhenhua Zhu; methodology: Zheng Jin; Xiaopeng Jing; software: Zheng Jin; Xiaopeng Jing; validation: Zhenhua Zhu; Ji Zeng; formal analysis: Zheng Jin; Xiaopeng Jing; investigation: Ji Zeng; Xiaopeng Jing; resources: Zheng Jin; Zhenhua Zhu; data curation: Zheng Jin; Zhenhua Zhu; writing— original draft preparation: Zheng Jin; writing—review and editing: Zhenhua Zhu; Ji Zeng; visualization: Zhenhua Zhu; Ji Zeng; supervision: Zhenhua Zhu; Ji Zeng; funding acquisition: Zheng Jin; Zhenhua Zhu. All authors have read and agreed to the published version of the manuscript.

Funding

This work was supported by Wuhan Municipal Health Commission Chenxing program, Wuhan 2023 Medical Research Project (Health Development No. WX23Q17), Wuhan Natural Science Foundation Exploration Plan (Chenguang Project, No. 2024040801020374, 2025020701020268), Hubei Natural Science Foundation (No. 2024AFB482, 2024AFB479) and Wuhan 2024 Talent Program (Outstanding Young Talents).

Data availability

All data generated or analyzed during this study are included in this article. Further enquiries can be directed to the corresponding author.

Declarations

Ethics approval and consent to participate

All experiments and methods were performed in accordance with the relevant guidelines and regulations, and all experimental protocols, including animal experimental designs and procedures, were reviewed and approved by the Institutional Ethics Committee and the Animal Welfare Committee of Wuhan Fourth Hospital.

Consent for publication

Not applicable.

Competing interests

The authors declare no competing interests.