miR-224-5p Attenuates Allergic Responses in Mice with Allergic Rhinitis by Modulating the Th1/Th2 Response

Background Allergic rhinitis (AR) is a common chronic respiratory disease that has become a global health problem. miRNAs play an important role in multiple immune and inflammatory diseases, including AR. In this work, the mechanism by which miR-224-5p regulates AR in vivo and in vitro was examined. Methods Human nasal epithelial cells (HNEpCs) were used to establish an AR cell model induced by Der P1, and C57BL/6 mice were used to establish an AR animal model induced by OVA (ovalbumin). RT-qPCR was used to determine the level of miR-224-5p; western blot analysis was used to determine GATA3; ELISA was used to determine the levels of OVA-specific IgE, IFN-γ, IL-4, IL-5, and IL-13; flow cytometry was used to determine the differentiation of Th1 and Th2 cells; and HE and PAS staining was used to observe the histopathological alterations in the mouse nasal mucosa and spleen. Results miR-224-5p was downregulated in nasal mucosa from mice with AR and an AR cell model. Overexpressed miR-224-5p can improve AR development and attenuate AR symptoms by regulating GATA3-mediated Th1/Th2 responses. Conclusion miR-224-5p attenuates allergic reactions in mice with AR by regulating the Th1/Th2 response.


Introduction
Allergic rhinitis (AR) is a common chronic respiratory disease mediated by immunoglobulin E (IgE), which is characterized by nasal clogging, nasal discharge, sneezing, nasal friction, and nasal pruritus [1].AR is a worldwide health problem affecting approximately 40% of the total population worldwide [2].Current AR therapies incorporate allergen avoidance, medication, and immunotherapy [3].Nevertheless, treatment failure or unsatisfactory results may still occur with these regimens [4].Therefore, it is important to explore AR etiopathogenesis, find novel remedial targets, and develop new therapeutic drugs.
The imbalance of T helper type 1 (Th1) cells and T helper type 2 (Th2) cells was proposed as a major inducer of IgEmediated allergic inflammation [5][6][7].A previous study reported that the fundamental cause of allergic diseases may be the mutual effect of genetic and ambient factors [8].When the body is exposed to a certain concentration of allergen in the environment for a long time, antigen-presenting cells submit the allergen to CD4 T lymphocytes, and CD4 T cells liberate cytokines to provoke B cells to differentiate into plasma cells, which ultimately promotes the generation of IgE and restrains the Th1 reaction [9].It causes the release of inflammatory cytokines that induce a Th2 response when IgE antibodies combine with receptors on mast cells and eosinophils [10].AR manifests as increased Th2 cells and decreased Th1 cells [11].The imbalance in CD4 T-cell subsets and the accumulation of eosinophils and mast cells in the nasal mucosa are important features of AR.Therefore, normalizing the Th1/Th2 ratio and inhibiting the accumulation of eosinophils and mast cells are key for AR treatment.
MicroRNAs (miRNAs) are one of the fundamental regulatory mechanisms of gene expression and bind complementary sequences of the 3′UTR on target mRNA transcripts to control posttranscriptional gene expression [12].Studies have shown that miRNAs play an important role in many immune and inflammatory diseases, including AR [13,14].miRNAs accelerate the evolution of allergic diseases by influencing the Th1/Th2 balance, facilitating inflammatory responses in the epithelium and tissues, and activating innate immune cells.miR-125a, miR-21, and miR-140 have been demonstrated to be related to symptom severity and Th1/Th2 imbalance in AR [15,16].Recent studies have shown that miR-224-5p plays an important role in AR progression [17].Nevertheless, the latent mechanism of miR-224-5p in AR evolution remains unclear.
GATA3, a key regulator of Th2 cells, has been reported to be intimately linked to Th2 proliferation and differentiation [18].GATA3 is a target gene of miRNA and is involved in regulating immune cell proliferation and differentiation and in regulating congenital and accommodative immune responses [18][19][20].For example, miR-466a-3p attenuates AR in mice by targeting GATA3 [21].miR-135a regulates eosinophils and mast cells and allergic inflammation in AR by targeting GATA3 [22].Notably, it was predicted that miR-224-5p could directly target GATA3 through the starBase website (https://starbase.sysu.edu.cn/),so we conjectured that miR-224-5p might control the generation and progression of AR by GATA3.
The aim of this study was to identify the mechanism by which miR-224-5p attenuates the allergic response in mice with AR by targeting GATA3 to regulate the Th1/Th2 response and provide a reference for the development of effective and novel remedies for AR.

Materials and Methods
2.1.Cell Culture.Human nasal epithelial cells (HNEpCs) were purchased from Huatuo Biology (Shenzhen, China).HNEpCs were cultured in human airway epithelial cell growth medium (Procell, Wuhan, China).CD4 T cells were purchased from IPHASE Biotechnology Co., Ltd.(isolated from normal human peripheral blood; the purity of the cells was ≥85%) and cultured in RPMI-1640 complete substrate (Gibco, USA).All cell culture media contained 1% penicillin/streptomycin (Gibco, USA).RPMI-1640 complete substrate contained 10% fetal serum.The incubator conditions were 37°C and 5% CO 2 .Microscopic observation of cell morphology, routine medium change, and passaging were performed.

Establishment of the AR Cell
Model.An AR cell model was established by treating HNEpC cells with 1 μg/mL Der P1 (Sigma, USA) for 24 hr [23].The treated HNEpCs were cocultured with CD4 T cells, flow cytometry was performed to observe the differentiation of Th1 and Th2 cells, and the levels of Th1/Th2-related cytokines were analyzed by ELISA.The AR mouse model was generated by ovalbumin (OVA, Sigma, USA) induction, and 100 μL of saline containing 50 μg of OVA and 2 mg of Al(OH) 3 was injected intraperitoneally on days 0, 7, and 14.Then, 20 μL of saline containing 400 μg OVA was directly injected intranasally every 4 days from days 21 to 28.After 28 days, nasal mucosa and spleen tissues were collected for analysis after sacrifice [24,25].
The miR-224-5p mimic group mice were intraperitoneally sensitized and intranasally challenged with OVA, similar to that of the AR group.On days 18, 21, 24, and 27, the mimic group was treated intranasally with 20 μL saline containing 50 μg miR-224-5p mimic to intervene with the incidence of AR. miR-224-5p mimic was provided by Sangon Biotech (Shanghai, China).

Assessment of Allergy Symptoms.
The number of mice sneezing and with nasal rubbing was counted, and the frequency within 15 min was calculated after the last OVA challenge.The average of the observations was used in the final results to assess the AR symptoms of mice.
2.5.RT-qPCR.Total RNA was extracted from the AR mouse nasal mucosa and spleen and the AR cell model by TRIzol Reagent (Beyotime, China).Then, the RNA quality and concentration were measured.Transcribed RNA was reverse transcribed into cDNA.RT-qPCR was performed with U6 as an internal control.The detailed primer sequences are shown in Table 1, and differential gene expression was analyzed by the 2 −ΔΔCt method.
2.6.Western Blot.Proteins were isolated from cells or nasal mucosa with RIPA buffer containing 1% protease inhibitors.Protein concentrations were determined according to the specifications of the bicinchoninic acid assay (BCA, Solarbio, China) kit.50 µg of protein was loaded per lane, and total proteins were separated by SDS-PAGE (5% concentrated gel, 10% separating gel), transferred to PVDF membranes (Millipore, USA) and then blocked with 5% nonfat milk powder for 1.5 hr at room temperature.The following diluted primary antibodies were added and incubated overnight at 4°C: anti-β-actin (1 : 1,000, ab8226 Abcam, UK) and anti-GATA3 (ab106625, 1 : 1,000, Abcam, UK).Next, the membranes were incubated with secondary antibodies (1 : 4,000, ab97051) for 1 hr at room temperature and developed with an ECL kit.Finally, the bands were semiquantitatively analyzed by ImageJ software.
2.7.Flow Cytometry.Cells were harvested and suspended in medium with 10% FBS.The designated cells were collected and suspended after culturing for 72 hr.The cell suspensions were treated with PMA (25 ng/mL), ionomycin (1 μg/mL), and Golgistop (5 ng/mL) for 4 hr at 37°C.Th1 and Th2 cell 2.8.ELISA.The corresponding ELISA kits were used according to the protocol (Solarbio, China) to determine the levels of IFN-γ, IL-4, IL-5, and IL-13 in the supernatants of treated HNEpCs cocultured with CD4 T cells and the levels of OVA-specific IgE, IFN-γ, IL-4, IL-5, and IL-13 in serum from mice.
2.10.Histological Analysis.Nasal mucosa and spleen tissues were isolated from mice and immobilized with 4% paraformaldehyde for 24 hr at room temperature.The dehydrated tissue specimens were paraffin-embedded and cut into 4-5 μm sections.Pathological changes in the nasal mucosa and spleen were observed by HE and PAS staining.

Statistical Analysis.
The analytical results are expressed as the mean AE SD.All statistical analyses were performed with GraphPad Prism 8.0.All cell experiments were performed with three parallel groups and repeated three times.
All animal experiments were performed with five parallel groups and repeated three times.One-way ANOVA and T test were used for the analysis by Tukey's test post hoc tests.The data were statistically significant if P <0:05.
To assess the influence of miR-224-5p on AR development and progression, miR-224-5p was overexpressed in the CD4 T cells and cocultured with AR cell model, and its effect on CD4 T differentiation was observed.First, RT-qPCR showed successful transfection of miR-224-5p (Figure 2(a)).Compared to the NC group, IFN-γ was observably decreased, and IL-4, IL-5, and IL-13 were observably elevated in the supernatant of the AR cells cocultured with CD4 T cells.Compared to the AR group, IFN-γ was prominent, while the expression of IL-4, IL-5, and IL-13 was observably decreased after the addition of the miR-224-5p mimic (Figure 2(b)).Flow cytometry showed that compared to the NC group, the amount of Th1 cells was observably decreased, and the amount of Th2 cells was observably increased in the AR group.The Th1/Th2 balance was reversed after overexpression of miR-224-5p (Figures 2(c) and 2(d)).Next, the influence of miR-224-5p overexpression was assessed in an AR mouse model.RT-qPCR showed that miR-224-5p expression was observably upregulated in mice after overexpression of miR-224-5p (Figure 2(e)).The results showed that compared to the normal group, the frequency of sneezing and nasal friction in the mice with AR was observably elevated, while the frequency of snoring and nasal friction in the mice with AR was decreased after miR-224-5p mimic transfection (Figures 2(f) and 2(g)).ELISA showed that compared to the normal group, the levels of OVA-specific IgE, IL-4, IL-5, and IL-13 were observably elevated, while the content of IFN-γ was observably decreased, and the opposite results were observed after the addition of the miR-224-5p mimic (Figure 2     .HE staining showed that edema and lymphocyte proliferation were observably alleviated after overexpressing miR-224-5p, while edema and lymphocyte proliferation were prominently aggravated after overexpressing miR-224-5p and GATA3 in the spleen (Figure 5(j)).
The consequences indicated that miR-224-5p regulates the Th1/Th2 response and inhibits AR inflammation, allergic response, and other symptoms to alleviate AR progression through GATA3.

Discussion
AR has become a global health problem, although it does not directly threaten the lives of patients [26].The latest studies have shown that the AR pathological process is extremely intricate, incorporating multifarious immune cells, inflammatory mediators, and cytokines [25].Further elucidation of the mechanism of AR occurrence and progression and the development of latent targets for AR treatment are needed.It was confirmed that miR-224-5p attenuates allergic responses in mice with AR by regulating Th1/Th2 responses and that GATA3 is critical for miR-224-5p-mediated regulation of AR development.miRNAs have been shown to be joint regulators of AR development [27].For example, miR-29 suppresses allergic reactions and symptoms in mice with AR [28].miRNA-345-5p attenuates AR progression through TLR4/NF-κB pathwaymediated inflammation [29].miR-133b improves allergic inflammation in an AR model [30].It has been shown that miR-224-5p is involved in the regulation of multifarious inflammatory diseases.For example, miR-224-5p lessens microglial inflammation by regulating NLRP3 to influence the progression of obstructive sleep apnea and type 2 diabetes [31].In recent studies, miR-224-5p was confirmed to alleviate AR in mice [17].In this study, miR-224-5p was decreased in OVA-induced AR mouse nasal mucosa and a Der P1-induced AR cell model, and it has an anti-inflammatory effect in AR development by restraining the expression of IgE, IL-4, IL-5, and IL-13 and facilitating IFN-γ expression.Moreover, overexpressed miR-224-5p moderated allergic reactions and lessened sneezing and nasal friction events in mice with AR.
Th1/Th2 imbalance has been proven to be a pivotal factor in AR pathogenesis [32].Abnormal cytokines generated by IgE-mediated immune responses, inflammatory cell diseases, and Th1/Th2 imbalance have been shown to be involved in AR development [33].Pathogenic memory Th2 cells play a pivotal role in AR pathogenesis.In general, the levels of IgE, IL-4, IL-5, and IL-13 facilitate Th2 responses to reduce the integrity of nasal epithelial cells in AR [34].Th1 mainly secretes TNF-α and IFN-γ, and mediates cellular immune response; Th2 mainly secretes IL-4, IL-5, and IL-10, and mediates humoral immune response.Studies have shown that the two responses are antagonistic.Th1 cytokines (e.g., IFN-γ) inhibit the Th2 response, and Th2 cytokines restrain  Analytical Cellular Pathology the Th1 response [35].Hence, it is important to identify the key molecules regulating the Th1/Th2 balance in AR therapy.The GATA3 transcription factor has been shown to be a prime regulator of Th2 differentiation.GATA3 mediates the expression and activation of IL-4, IL-5, IL-13, and other cytokines to condition Th2 cell differentiation [36].It has been reported that T-bet, a transcription factor mediating Th1 cell differentiation, can combine with GATA3 to suppress Th2 cell differentiation [37].In recent years, numerous studies have shown that GATA3 levels can be regulated by miRNAs.For instance, Chen et al. [21] certified that miR-466a-3p targets GATA3 to alleviate allergic nasal inflammation; miR-24 and miR-27 cooperate to restrain Th2 reactions by targeting IL-4 and GATA3 [38].MiR-224-5p immediately targets GATA3, as predicted by starBase.Moreover, it was found that overexpressed miR-224-5p restrained GATA3 levels, which facilitated CD4 T-cell differentiation into Th1 cells and restrained CD4 T-cell differentiation into Th2 cells in the AR cell model.miR-224-5p regulates the Th1/Th2 response by negatively regulating GATA3, which alleviates eosinophil infiltration and goblet cell proliferation in the nasal mucosa and improves edema and lymphocyte proliferation in the spleen in mice with AR. through GATA3.Overexpressed miR-224-5p can attenuate allergic reactions and symptoms in mice with AR, and miR-224-5p can serve as a latent remedial target for AR progression.Moreover, our findings provide an academic reference for elucidating the pathogenesis of AR.

2. 3 .
Establishment of the AR Mouse Model.Female C57BL/6 (18-20 g, 6-8 weeks old) mice were purchased from the Animal Experimental Center of Kunming Medical University.

FIGURE 1 :FIGURE 2 :
FIGURE 1: Downregulation of miR-224-5p in mice with AR and a cell model: (a) RT-qPCR for testing the miR-224-5p level in the nasal mucosa of normal control mice and mice with AR and (b) RT-qPCR for testing the miR-224-5p level in normal control and AR cell models.** * P <0:001.