The Inhibition of Group II Innate Lymphoid Cell Response by IL-27 in Allergic Rhinitis

Objectives Interleukin-27 (IL-27) has been reported to inhibit type 2 T helper cell (Th2) response in allergic rhinitis (AR). However, its effects on group II innate lymphoid cells (ILC2) in AR are not fully understood. Methods Nineteen patients with AR and nineteen controls were enrolled in this study. The effects of IL-27 on ILC2 differentiation and function as well as the regulation of the IL-27 receptor (IL-27R) were analyzed by tritiated thymidine incorporation, enzyme-linked immunosorbent assay (ELISA), and real-time polymerase chain reaction (PCR), respectively. AR mice were used to confirm the role of IL-27 in vivo. Results The serum IL-27 protein expression in AR patients was significantly lower compared with controls. IL-27 decreased the ILC2 proliferation and type II cytokine secretion through the interaction with IL-27R. IL-27 also inhibited systemic and nasal ILC2 response of AR mice. Conclusion IL-27 inhibited the proliferation and function of ILC2 in AR, implying that IL-27 may be used as new treatment target in AR.


Introduction
Allergic rhinitis (AR), one of the most common diseases in otorhinolaryngology, affects 10%-20% of the whole population [1]. Clinical symptoms of AR included nasal obstruction, itchy nose, sneezing, and runny nose, which affect the quality of life of patients and consume huge social and medical costs.
Group II innate lymphoid cells (ILC2), distributed in various organs, play key roles in the early stage of immune response [12]. Studies have found that the frequency of ILC2 in the peripheral blood of AR patients sensitized to pollen increased in pollen season [13]. Moreover, Fan et al.'s study found that ILC2 may exert different roles in AR subtypes mediated by dust mites and mugwort [14]. Moro et al. and Mchedlidze et al.'s studies had proven that IL-27 treatment inhibited ILC2 proliferation and cytokine production and significantly reduced their accumulation in mouse models [15,16].
In this study, we aimed to explore the role of IL-27 on ILC2 differentiation and type II cytokine production using cell and animal models.

Methods
2.1. Subjects. All 19 AR patients were confirmed by a positive skin prick test (SPT) or specific immunoglobulin to Dermatophagoides pteronyssinus and/or Dermatophagoides farinae as well as typical symptoms. The 19 healthy controls had no history of allergic symptoms, and the allergen test was negative. Subjects with usage of systemic corticosteroids in the previous one month, smoking history, immune diseases, pregnancy, or lactation were excluded. Our study was approved by local ethical committee boards, and written informed consent was obtained (Approval number 20190134).

Clinical Severity.
A total nasal symptom score (TNSS) was obtained by scoring the symptoms such as nasal obstruction, itchy nose, sneezing, and runny nose from a 0 to 3 scale (0, none; 1, mild; 2, moderate; and 3, severe). The scores were averaged in an 8-week observation period as described in a previous study [17].
Various concentrations of IL-27 (10-100 ng/mL), anti-IL-27 (100 ng/mL), and IL-10 (100 ng/mL) were provided in some experiments described in a previous study [15]. All stimulators and inhibitors were purchased from R&D Systems. The ILC2 proliferation was measured by using tritiated thymidine incorporation. The expression of gp130 and WSX-1 by ILC2 was detected by staining of the antibody to gp130 and WSX-1 (BD Bioscience, NJ) using flow cytometry.

Quantitative Real-Time Polymerase Chain Reaction
(qRT-PCR). The total RNA from ILC2s was obtained using a TRIzol reagent (Life Technologies, California). The RNA (1 μg) was reversely transcribed using a cDNA kit (QIA-GEN). The cDNA was synthesized using an Oligo(dT) [12][13][14][15][16][17][18] Primer and SuperScript III Reverse Transcriptase (Life Tech-nologies). PCR was carried out using an ABI PRISM 7300 Detection System. The expression target genes were calculated using the change-in-cycling-threshold (Ct) method and normalized to the housekeeping gene as described in a previous study [19].
2.6. Enzyme-Linked Immunosorbent Assay (ELISA). The concentrations of cytokines from the supernatant were detected using ELISA kits (R&D Systems, USA) as described by instructions. The detection sensitivity for cytokines was listed as follows: IL-5, 3.9 pg/mL; IL-13, 125 pg/mL; and IL-27, 156 pg/mL.

Animal Model.
Six-to eight-week-old BALB/cJ mice (30 mice) were raised in a specific pathogen-free animal facility provided with food and water. On day 0 and day 7, the mice were sensitized with 100 μg of Der p1 and 2 mg of aluminium hydroxide intraperitoneally. Intranasal challenge of Der p1 (100 μg) was done on days 15, 16, and 19. The control mice received intranasal PBS or IL-27 (0.5 μg, R&D Systems) 1 hour before rechallenge with Der p1. Nasal tissue was collected and prepared for further experiments. All animal care and experimental protocols were approved by local ethics committee boards.
2.9. Statistical Analysis. Statistical analysis was performed by SPSS 11.0. The Kruskal-Wallis H test or the nonparametric Mann-Whitney U test was done. Correlations were analyzed by the Spearman rank correlation analysis. A P value of less than 0.05 was considered as statistically significant.

The IL-27 Serum Levels and Their Correlation with ILC2
and Clinical Severity in AR. The characteristics of participants are summarized in Table 1. To explore the correlation between IL-27 and the proportion of ILC2 as well as clinical  Journal of Immunology Research severity, we measured the mRNA and protein levels of serum IL-27 and found that they were downregulated in AR patients compared with controls (Figures 1(a) and 1(b)), whereas the proportion of ILC2 in PBMCs was significantly elevated in AR patients compared with controls (Figures 1(c) and 1(d)). The IL-27 protein levels were negatively correlated with the  (Figures 2(a) and 2(b)), whereas the proportion of ILC2 was positively correlated with TNSS score (r = 0:57, P < 0:01) (Figure 2(c)).   Journal of Immunology Research (gp130 and WSX-1) by ILC2 were significantly upregulated after IL-27 stimulation (Figures 3(a)-3(c)). However, IL-27 receptor expression by healthy control and patient samples under steady state was undetectable. Next, we investigated the proliferation and function of ILC2 regulated by IL-27. We found that IL-27 inhibited the proliferation of ILC2 as well as GATA binding protein-3 (GATA-3) and Retinoic Acid-Related Orphan Receptor α (RORα) production by ILC2 directly in a dose-dependent manner (Figures 4(a)-4(c)). Moreover, the production of IL-5 and IL-13 by ILC2 were also inhibited after IL-27 stimulation in a dose-dependent manner (Figures 4(d) and 4(e)).

IL-27 Inhibits Allergic Responses in Mouse Model.
To prove the in vivo effect of IL-27, we treated allergic mice with IL-27. The IL-27 reduced the Der p1-specific IgE level ( Figure 5(a)) and decreased the proportion of nasal ILC2 in total cells of inferior turbinate tissue (Figures 5(b) and 5(c)) and type II cytokine production ( Figures 5(d)-5(f)) by nasal ILC2s compared with control mice.

Discussion
In this study, our data demonstrated that IL-27 inhibited the proliferation and function of ILC2s, providing a new mechanism for the pathogenesis of AR.
Recent studies suggest that IL-27 has broad effects on Th1, Th2, and Th17 subsets of T cells and regulatory T cells [20,21]. For example, IL-27 can inhibit Th2 responses in AR patients. In animal models, intranasal administration of IL-27 decreased nasal allergic responses and symptoms even after the establishment of allergic rhinitis [22,23]. These 5 Journal of Immunology Research results suggested a natural protective role of IL-27, of which signaling deficiency induces a Th17-type hyperresponse that further aggravates the Th2-dominant allergic response [24].
For ILC2s, IL-27 has been reported to suppress ILC2 cells in a manner dependent on the transcription factor signal transducer and activator of transcription 1 (STAT1) during lung inflammation induced by Alternaria alternata [15]. Moreover, IL-27 inhibited ILC2 proliferation and cytokine production and significantly inhibited their accumulation in papain-induced airway inflammation [16]. However, no study explored the effect and mechanism of IL-27 on ILC2 in AR. Our in vitro results suggested that IL-27 can inhibit the proliferation and function of ILC2 directly by regulation of IL-27R on ILC2. We first reported a dose-dependent manner of IL-27R expression regulated by IL-27, proving the direct effect of IL-27 on ILC2. Consistently, Mchedlidze et al.'s study also suggested that IL-27 receptor signaling directly regulates ILC2 responses. Our study provided a novel mechanism for the negative regulation of ILC2 immune responses in AR [16].
GATA-3 is needed for the maintenance of mature ILC2 cells as well as their precursors in bone marrow. Moreover, GATA-3 has been shown to be necessary for the production of IL-5 and IL-13 by human ILC2 cells. RORα is also essential  Journal of Immunology Research for ILC2 development, as RORα-deficient mice lack ILC2 cells [25]. Our data also confirmed that IL-27 inhibited both expressions of GATA-3 and RORα, which contributed to the decreased proliferation and cytokine production by ILC2. Similarly, the allergic responses in the mouse model were significantly abrogated when IL-27 were added and presented with reduced Der p1-specific IgE level and decreased proportion of nasal ILC2 and type II cytokines. Consistently, Mchedlidze et al.'s study demonstrated that IL-27 deficiency is correlated with increased mucosal infiltration of ILC2 in an inflammatory lung disease model. Moreover, IL-27 treatment inhibited ILC2 proliferation and cytokine production and significantly inhibited their accumulation in vivo [16].
In this study, we used human ILC2 to investigate the effect of IL-27 on ILC2 in vitro. However, it needed further studies to prove if IL-27 suppresses AR in vivo in humans. In summary, our study proved the effect of IL-27 in the regulation of ILC2s through in vitro and in vivo studies. Our results implied that IL-27 may be used as a promising treatment target in AR.

Data Availability
The datasets used and/or analyzed during the current study are available from the corresponding author on reasonable request.

Ethical Approval
The study protocols were approved by local ethics committee boards.

Consent
Written informed consent was obtained from all participants.