Interleukin-37b Suppressed ILC2s in Children with Allergic Rhinitis

Background Interleukin-37b is a fundamental inhibitor of innate and acquired immunity. Type 2 innate lymphoid cells (ILC2s) can secret type 2 cytokines and regulate allergic rhinitis (AR). However, the role of IL-37b in ILC2s in children with AR was not clear. Methods We recruited 15 AR children and controls. The serum IL-37b levels and its relation with the frequency and functional phenotype of ILC2s. The regulation of IL-37b on ILC2s proliferation and function was confirmed using flow cytometry and enzyme-linked immunosorbent assay (ELISA). The mRNA expression of IL-1R8, IL-18Rα, and ICOSL was examined using RCR. The change of IL-37b protein level in serum during subcutaneous allergen immunotherapy (SCIT) was determined by ELISA. Results We have demonstrated that both of the frequencies of blood ILC2s, IL-5+ILC2s, and IL-13+ILC2s in AR children were elevated compared with controls. The serum protein level of IL-37b was downregulated in AR, and it was negatively related to the frequency of ILC2s, IL-5+ILC2s, and IL-13+ILC2s. IL-37b increased the mRNA levels of IL-1R8, IL-18Rα, and ICOSL expressed by ILC2s. IL-37b suppressed the proliferation of ILC2s and the secretion of IL-5 and IL-13 from ILC2s. Finally, we found that IL-37b was increased in AR children after 3 years' SLIT, especially in the good response group. Conclusion Our findings highlight the role of IL-37b in the suppression of ILC2s and establish a new therapeutic target in AR.


Introduction
Allergic rhinitis (AR) is a common disease that can be found in 15-25% of the population worldwide. AR often occurs in childhood, and the occurrence increases with increasing age [1]. The prevalence rate of children and adolescents was reported to be 44.23% [2]. The high incidence of AR has a significant financial impact on the management of the disease both direct and indirect. Allergen immunotherapy (AIT), mechanism-oriented therapy, is increasingly recognized as a most valuable therapy for AR patients, especially in the early phases [3,4]. Type 2 innate lymphoid cells (ILC2) are innate immune cells that can secrete type II cytokines dependent on epithelium-derived cytokines IL-33, IL-25, and thymic stromal lymphopoietin (TSLP) [5]. The frequency of blood ILC2s was increased in AR patients and correlated with disease severity [6]. ILC2s can also express inducible T cell costimulator ligand (ICOSL), which mediated the interaction between ILC2s CD4+ T helper cell subsets [7][8][9].
We previously found that IL-37b had an antiinflammatory effect in children with AR through downregulating with Th2 cytokines expressed by peripheral blood mononuclear cells [18]. Studies have also found IL-37b alleviates allergic symptoms by inhibiting Th2 and Th17 inflammation and suppressing the levels of IgE [13]. However, immunological roles and underlying mechanisms of IL-37b to ILC2s have remained elusive in allergic settings, especially in children with AR. Herein, it is worthy to explore the suppression role of IL-37b in ILC2s in AR children.

Methods
2.1. Subjects. All 15 AR children were recruited and confirmed according to disease history, physical examination, and positive skin prick test ( Table 1). The 15 healthy controls matched age and gender. Subjects with severe systematic diseases and drug use history (oral or nasal corticosteroids, antiepileptics, and immunosuppressants) one month before the study were not included. This study received approval from the local Ethics Committee and the children's guardian.

Samples Collection and Cell
Culture. The serum samples were achieved through 15 minutes centrifugation of 10 ml venous blood at 1000 × g. Ficoll density gradient was used for the purification of peripheral blood mononuclear cells (PBMC) from the blood of AR and controls.

The mRNA Level
Detected by qRT-PCR. The total RNA from ILC2s was isolated using RNeasy Mini Kit. The reverse transcription of RNA (1 μg) was done by cDNA kit (Qiagen). The cDNA synthesis was performed using an Oligo Primer and SuperScript III Reverse Transcriptase (Invitrogen). The target gene expression was calculated by the formula 2 −ΔΔCt and normalized to the endogenous gene [19].
2.5. Allergen Immunotherapy. Standardized allergen extracts (50% of Dermatophagoides pteronyssinus and 50% of Dermatophagoides farinae) (Allergopharma) were administered as described by the manufacturer's instruction. Briefly, at the initial buildup stage, the subjects received injections weekly from doses of 0.2 in no. 1 vials to 1.0 mL in the no. 3 vial. At the maintenance stage, 1.0 mL in the no. 3 vial was administered at 4-6 weeks interval. The efficacy of SCIT was determined using symptom medication score (SMS), which is the sum of total nasal symptom score (TNSS) and rescue medication score (RMS). The TNSS was scored from 0 to 3 (no, slight, moderate, and severe symptoms) according to severity. The RMS was scored according to medication use (1 for an oral or intranasal antihistamine and 2 for an intranasal corticosteroid) [20]. The evaluation and serum collection were performed at baseline and 3 years after SCIT. Children who obtained 2.6. Statistical Analysis. The data were provided as the mean ± standard deviation or medium and interquartile interval and analyzed by nonparametric Mann-Whitney U test. Intergroup comparisons were performed followed by the post hoc Tukey's test. The Spearman correlation analysis was performed t. Statistical significant difference was set as 0.05.

Comparisons of the Frequency and Functional
Phenotype of ILC2 between AR and Control Children. Despite heterogeneity across donors, the frequency of ILC2s was higher in AR than in controls ( Figure 1). As type 2 cytokines are associated with the pathophysiology of AR, we studied subsets of ILC2s and confirmed that the percentages of IL-5 + ILC2s and IL-13 + ILC2s were increased in the AR group than controls (Figure 1).

IL-37b
Levels and Its Correlation with ILC2s and Type 2 Cytokines in AR. We next assessed the levels of IL-37b in   Mediators of Inflammation serum from participants. Reduction of IL-37b in individuals with AR was found when compared with normal controls (Figure 2). We observed that the serum level of IL-37b was strongly negatively related to the frequency of ILC2s, IL-5 + ILC2s, and IL-13 + ILC2s (Figure 2), suggesting a potential suppression effect of IL-37b in type 2 immune response.

IL-37b Suppressed the Proliferation and Function of
ILC2s. As expected, the proliferation of ILC2s treated with IL-37b was suppressed and the production of IL-5 and IL-13 was clearly reduced (Figure 3). We also detected enhanced expression of IL-1R8, IL-18Rα, and ICOSL in ILC2s ( Figure 3) in a dose-dependent manner.

IL-37b
Increased in AR Children after 3 Years' SCIT. The TNSS, RMS, and SMS after 3 years' SCIT decreased compared with the baseline (Table 2) in agreement with previous reports [20]. As to the serum level of IL-37b, we found an increase following the 3-year SCIT treatment, especially in the good response group (Figure 4). Moreover, the IL-37b expression and SMS score were negatively correlated after 3-year SCIT treatment ( Figure 4).

Discussion
ILC2s play specific roles in promoting the development of AR. We found that the proliferation of ILC2s and their functional phenotype were increased in AR children. IL-37 functions as a suppressor in both innate and adaptive immune responses. Here, we confirmed that IL-37b was decreased in AR, and it can function as a critical inhibitor of ILC2s cells and the secretion of IL-5 and IL-13. Moreover, the study revealed that IL-37b participated in the pathogenesis of SCIT in children with AR via suppressing of ILC2s.
Allergens such as house dust mites can activate pattern recognition receptors in nasal epithelium to start innate immune responses by releasing alarmins (IL-33, TSPL, or IL-25) which could activate ILC2s to rapidly produce type 2 cytokine. These indicated that ILC2s have an important role in maintaining type 2 adaptive immune response [21].

Mediators of Inflammation
Thus, we first confirmed the enhancement of ILC2s and functional phenotype in children with AR. IL-37 can be secreted from diverse cells such as PBMCs and dendritic cells [11]. We inspected that the levels of IL-37b in children with AR were downregulated which is consistent with the previous reports [17,18]. Our in vitro study found that IL-37b can regulate the proliferation and function of ILC2s negatively and directly.
Consistently, rIL-37b treatment could reduce type 2 cytokines expression in the BALF of asthmatic mice [22].
IL-37 plays its role by binding to IL-18Rα and IL-1R8 extracellularly [13]. We found that IL-37b could increase the levels of not only IL-18Rα and IL-1R8 but also ICOSL in ILC2s, implying that IL-37b may regulate the interaction between ILC2 and Th2 cells.
For the clinical scenario, our data detected that the protein level of IL-37b was significantly upregulated in HDMsensitive AR children after 3-year treatment with SCIT, which suggests that IL-37b might be involved in allergenspecific immunotherapy mediated by ILC2s. Our data also suggested that IL-37b levels were negatively correlated with disease severity, implying that IL-37b may be used as a biomarker during allergen-specific immunotherapy.