Long Non Coding RNA FOXD3‑AS1 Alleviates Allergic Rhinitis by Elevating the Th1/Th2 Ratio via the Regulation of Dendritic Cells

ABSTRACT This article aimed to explore whether the regulation of Th1/Th2 immune responses by FOXD3-AS1 is associated with dendritic cells (DCs) in allergic rhinitis (AR). HE staining was performed to assess the pathological changes in the nasal mucosa; ELISA was performed to measure the levels of Th1/Th2-related cytokines; flow cytometry was performed to analyze Th1/Th2 cells and MHC-II-, CD80-, and CD86-positive DCs; and qRT‒PCR and western blotting were performed to measure mRNA and protein expression levels, respectively. Our data revealed that LV-FOXD3-AS1 improved AR and increased the Th1/Th2 cell ratio in AR model mice. LV-FOXD3-AS1 further inhibited DC maturation both in vivo and in vitro. Moreover, the coculture system of DCs and CD4+ T cells demonstrated that LV-FOXD3-AS1 increased the Th1/Th2 cell ratio by inhibiting the maturation of DCs. In addition, LV-FOXD3-AS1 reduced the level of phosphorylated STAT6 in DCs derived from healthy mice, and STAT6 overexpression eliminated the inhibitory effect of LV-FOXD3-AS1 on the maturation of DCs. In summary, LV-FOXD3-AS1 ameliorated AR by increasing the Th1/Th2 cell ratio by inhibiting DC maturation via the inhibition of STAT6 phosphorylation. Our data confirmed the protective effect of FOXD3-AS1 in AR and provided a novel idea for the treatment of this disease.


Introduction
Allergic rhinitis (AR) is a noninfectious disease of the nasal mucosa that is mainly mediated by IgE after exposure to allergens, and it is also a refractory disease with a high prevalence in rhinology (Eguiluz-Gracia et al. 2019). AR causes many diseases, reduces the size of the workforce, poses substantial socioeconomic burdens, and has been a global health problem. Thus, elucidating the pathogenesis of AR and exploring effective prevention strategies are necessary.
AR is a chronic inflammatory disease, and an abnormal immune system is closely associated with the development of AR (Leaker et al. 2015). It has been proven that T cells play a key role in AR, and the core pathogenesis is the imbalance of T helper (Th)1/Th2 cells (Xu et al. 2018;Yanyun et al. 2014). Our previous study also confirmed an abnormal increase Th2 cells and related cytokines, such as interleukin (IL)-4 and IL-13, in the serum of AR patients ). However, dendritic cells (DCs) also play a crucial role in AR. DCs not only present antigens to T cells to stimulate the immune response but also guide the differentiation of Th cells, and DCs act as the only antigen-presenting cells that activate T cells (Re and Strominger 2004). DCs go through two different stages during differentiation and development: immature DCs (imDCs) and mature DCs (mDCs). ImDCs express low levels of costimulatory molecules and adhesion molecules, and they have a poor ability to stimulate the proliferation of naive T cells, but they differentiate into mDCs after ingesting antigens or being stimulated. mDCs exhibit high expression of MHC-II, CD80, and CD86 and secrete a variety of cytokines associated with immune regulation; thus, mDCs can present antigens and stimulate immune responses (Banchereau et al. 2000). In a previous study, Alex Kleinjan et al. (2006) revealed that, compared with healthy subjects, AR patients exhibited a higher number of DCs in the epithelium and lamina propria of the nasal mucosa, and the number of mDCs was notably higher than imDCs in AR patients. It was further demonstrated that the inhibition of DC maturation significantly attenuated symptoms and allergic inflammation by regulating Th1/Th2 cells in AR Niu et al. 2020), suggesting that mDCs contribute to the development of AR.
Long noncoding RNAs (lncRNAs) are a type of noncoding RNA that are more than 200 nucleotides in length, and they are involved in the regulation of gene transcription and posttranscription processes (Marchese et al. 2014). The abnormal expression of lncRNAs is closely associated with various diseases, and lncRNAs have become a focus of research on immune diseases such as AR and asthma (Bocchetti et al. 2021;Yang et al. 2020;Zheng et al. 2020). Our previous study confirmed that lncRNA FOXD3-AS1 was notably decreased in the nasal mucosa of AR patients and further revealed that FOXD3-AS1 suppressed Th2-type immunoreactions by inhibiting IL-25 expression ; however, whether the regulation of the Th1/Th2 immune response by FOXD3-AS1 in AR is also associated with DCs is unknown. This will be the focus of this study.

Construction of a lentiviral vector overexpressing FOXD3-AS1
cDNA encoding FOXD3-AS1 was amplified and subcloned into the vector pcDNA3.1, generating the vector pcDNA-FOXD3-AS1 (RIBOBIO, Guangzhou, China). The empty pcDNA3.1 vector was used as the control. E. coli were then transformed with these plasmids, which were then isolated and verified through DNA sequencing. The gene encoding FOXD3-AS1 was amplified using PCR with the template pcDNA-FOXD3-AS1. This PCR product was cloned into a lentivirus vector, which was then transfected into 293T cells at an MOI of 100:1. Forty-eight hours after transfection, the constructed viral vector (named LV-FOXD3-AS1) was harvested by subjected the cells to multiple freeze-thaw cycles and purified by polyethylene glycol. The control was named LV-NC.

The establishment of the AR model and experimental group
Five-week-old BALB/c mice, each weighing approximately 20-30 g, were used as experimental animals. The mice were maintained in a specific pathogen-free animal room at a temperature of 22-24 C with a 12-h light/12-h dark cycle. Animal protocols were approved by the Committee of Animal Use and Care of the Second Affiliated Hospital of Nanchang University. The method for establishing the AR model is shown in Supplementary 1. Briefly, mice were first intraperitoneally injected with 100 μg ovalbumin (OVA, Invitrogen, Carlsbad, CA, USA) plus 2 mg aluminum hydroxide (Invitrogen) in 100 μL normal saline once a week 3 times. Then, the mice were treated daily with 20 μL OVA solution (40 mg/mL) via the nostrils for 7 days. Mice treated with normal saline without OVA served as the controls. To examine the effect of FOXD3-AS1 on AR, LV-FOXD3-AS1 and LV-NC were injected into the mice via tail vein before the establishment of the AR model. All mice were divided into four groups (n = 6): control, AR, AR+LV-NC, and AR+LV-FOXD3-AS1. Twenty-four hours after the last nasal stimulation, the number of times each mouse scratched its nose or sneezed was recorded every 5 minutes.

Hematoxylin-eosin (HE) staining
HE staining was performed to observe the pathological changes in the nasal mucosa and the infiltration of inflammatory cells using an HE staining kit (Abcam, Cambridge, MA, USA).

Eosinophil cell count
Peripheral blood was obtained and allowed to clot for 30 min at 37°C. Total counts were performed, and blood smears were used to estimate the differential counts (Liu stain kit, Solarbio, Beijing, China), where a minimum of 100 cells were counted and classified as eosinophil cells based on standard morphological criteria.

Enzyme-linked immunosorbent assay (ELISA)
IgE, IL-4, IL-5, IL-13, IL-6, and IFN-γ levels in the peripheral blood of mice were measured using ELISA according to the manufacturer's instructions. All ELISA kits were purchased from MSKBIO (Wuhan, China).

Isolation of CD4+ T cells and DCs
Peripheral blood was obtained from mice and then centrifuged at 1500 × g for 5 min to obtain the cells. The cells were subsequently resuspended in PBS and stained with FITClabeled anti-CD4 (BD Biosciences, New Jersey, USA) or APC-labeled anti-CD11c (BD Biosciences) antibodies in the dark for 30 min at 4°C. Subsequently, a BD FACSCanto II flow cytometer (BD Biosciences) was utilized to separate CD4+ T cells and DCs. CD4+ T cells were cultured in RPMI-1640 medium (Sangon Biotech, Shanghai, China) supplemented with 10% fetal bovine serum (Sangon Biotech), 100 U/mL penicillin and 100 μg/mL streptomycin sulfate. DCs were cultured in RPMI-1640 medium supplemented with 10% fetal bovine serum, 100 U/mL penicillin, 100 μg/mL streptomycin sulfate, 10 ng/ml IL-4, and 20 ng/ml recombinant mouse GM-CSF (R&D Systems, Abingdon, UK). All cells were grown at 37°C in a humidified atmosphere with 5% CO 2 .

Flow cytometric analysis of Th1/2 cells and MHC-II-, CD80-, CD86-positive DCs
Flow cytometry was performed to analyze the proportion of Th1 and Th2 cells and the expression of MHC-II, CD80, and CD86 on the surface of DCs. To detect Th1 and Th2 cells, CD4+ T cells isolated from the peripheral blood of mice were stained with APC-labeled anti-IFN-γ (BD Biosciences) and PE-labeled anti-IL-4 (BD Biosciences). To analyze the maturation of DCs, DCs were stained with PE-labeled anti-MHC-II (BD Biosciences), PElabeled anti-CD80 (BD Biosciences), and APC-labeled anti-CD86 (BD Biosciences). During the process, the cells were incubated with the above antibodies in the dark for 30 min at 4°C. Finally, the percentages of Th1 and Th2 cells and the percentages of MHC-II-, CD80-, and CD86-positive cells were analyzed utilizing a BD FACSCanto II flow cytometer (BD Biosciences) with Flow Jo v7.6 software (TreeStar Inc., San Carlos, CA, USA).

Cell transfection
DCs isolated from normal and AR model mice were transfected with LV-FOXD3-AS1 or LV-NC using Lipofectamine 2000 (Invitrogen) following the manufacturer's protocol. The transfected cells were harvested after 48 to 72 h.

Coculture of CD4+ T cells and DCs
The coculture of CD4+ T cells and DCs was conducted using a Transwell system (Corning, Acton, MA, USA). CD4+ T cells were plated into the upper chamber of the Transwell chamber, and DCs were seeded into the bottom chamber for 24 h. Subsequently, the ratio of Th1/Th2 cells and the expression of related molecules by CD4+ T cells were measured.

Quantitative realtime PCR (qRT-PCR)
qRT-PCR was performed to measure the mRNA expression of different genes. Total RNA was extracted from CD4+ T cells using TRIzol reagent (Invitrogen). The RNA was reverse transcribed to complementary DNA using the PrimeScript™ RT Reagent Kit (Takara, Tokyo, Japan). qRT-PCR was carried out using the SYBR Green PCR Mix Kit (Takara) according to the manufacturer's instructions. The results were analyzed using the 2 −∆∆CT (cycle threshold) method for quantification. The primer sequences of the genes were as follows:

Western blotting
Total protein was separated from DCs using RIPA lysis buffer (Invitrogen). Then, the concentration of protein was determined using a BCA Protein Assay Kit (Thermo Fisher Scientific, Waltham, MA, USA). After that, 25 μg of protein was separated in a 12% SDS-PAGE gel and transferred to PVDF membranes. The membranes were then incubated with 5% nonfat milk for 1 h at room temperature followed by incubation with anti-STAT6 (Abcam) and anti-p-STAT6 antibodies (Abcam) overnight at 4°C. The next day, the membranes were incubated with secondary goat anti-rabbit (Abcam) for 1 h at room temperature. Finally, an enhanced chemiluminescence kit (ECLplus, Amersham, Little Chalfont, UK) was utilized to visualize the protein bands, and the optical densities of the western blot bands were analyzed using Image-Pro Plus 6.0 software (Media Cybernetics, Bethesda, MD, USA). The relative expression of STAT6 and p-STAT6 was normalized to that of β-actin.

Statistical analysis
All the experiments were independently repeated at least three times. All the values are presented as the mean ± standard deviation and were analyzed by SPSS 22.0 statistical software (SPSS Inc., Chicago, IL, USA). Unpaired Student's t test and one-way ANOVA were used to compare the differences between groups. P < .05 was considered statistically significant.

LV-FOXD3-AS1 significantly increased the Th1/Th2 ratio in the peripheral blood of AR model mice
Our previous study demonstrated that FOXD3-AS1 inhibited Th2-type immunoreactions in AR, but that study was performed in vitro. Here, we used AR model mice to further evaluate the effect of FOXD3-AS1 on Th1/Th2 cells. Actually, FOXD3-AS1 was also named linc1623 in mouse owing to the similar genomic structure of mouse and human FOXD3-AS1 (linc1623) ). In the control group, the nasal mucosa was intact, and the cilia were arranged neatly. We observed obvious tissue edema and inflammatory cell (eosinophil) infiltration in the nasal mucosa of AR model mice, and these effects were markedly ameliorated in the AR+LV-FOXD3-AS1 group, suggesting that LV-FOXD3-AS1 effectively inhibited AR development ( Figure 1a); these results were further confirmed by eosinophil counts (Figure 1b) and the number of times each mouse scratched its nose or sneezed every 5 minutes (Figure 1c). We also found that LV-FOXD3-AS1 reduced the IgE levels in the peripheral blood of AR model mice (Figure 1d). In addition, the serum levels of IL-4, IL-5, IL-13, IL-6, and IFN-γ, which are important cytokines for Th1/2 cells, were significantly upregulated in AR model mice, but LV-FOXD3-AS1 effectively reversed this upregulation (Figure 1e-i). Furthermore, LV-FOXD3-AS1 significantly reduced Th2 cells in the peripheral blood of AR model mice but had no significant effects on Th1 cells, resulting in an increase in the Th1/Th2 ratio (Figure 1j,k). The above data indicated that LV-FOXD3-AS1 significantly increased the ratio of Th1/Th2 cells, owing to the reduced Th2 cells, and improved AR.

LV-FOXD3-AS1 significantly inhibited the maturation of DCs in vivo and in vitro
Furthermore, we measured the expression of surface markers of mDCs in the blood of mice, including CD86, MHC-II, and CD80. The results showed that the percentages of CD80-positive DCs, CD86-positive DCs and MHC-II-positive DCs were all increased in AR model mice but this was significantly reversed by LV-FOXD3-AS1 (Figure 2). In addition, in vitro experiments were further performed to assess the effect of FOXD3-AS1 on the maturation of DCs in AR. LPS and TSLP were used to stimulate the maturation of DCs. The results revealed that LV-FOXD3-AS1 significantly reduced the percentages of CD80-positive DCs, CD86-positive DCs and Figure 1. LV-FOXD3-AS1 improved AR and increased the Th1/Th2 ratio in the peripheral blood of AR model mice. Twenty-four hours after the last nasal application, the nasal mucosa and the peripheral blood of mice in different groups (n = 6) were collected. (a) HE staining was performed to assess the pathological changes in the nasal mucosa, and the red arrows indicate the infiltration of eosinophils. (b) Liu staining was performed to examine eosinophil counts in the peripheral blood of mice. (c) the number of times each mouse scratched its nose or sneezed was recorded every 5 minutes. ELISA was performed to measure the levels of IgE (d), IL-4 (e), IL-5 (f), IL-13 (g), IL-6 (h), and IFN-γ (i) in the peripheral blood of mice. Flow cytometry was carried out to measure the percentage of Th1/Th2 cells (j) and the Th1/Th2 cell ratio (k) in the peripheral blood of mice. *P < .05, compared with Control; #P < .05, compared with AR +LV-NC. All the experiments were independently repeated at least three times.
MHC-II-positive DCs, regardless of LPS or TSLP treatment (Figure 3a-f), and these results were similar to the in vivo results shown in Figure 2. Overall, these data indicated that LV-FOXD3-AS1 inhibited the maturation of DCs. Twenty-four hours after the last nasal application, the peripheral blood of mice in different groups (n = 6) was collected. Flow cytometry was performed to measure the percentage of CD80-, CD86-, and MHC-IIpositive DCs in the peripheral blood of mice. *P < .05, compared with Control; #P < .05, compared with AR +LV-NC. All the experiments were independently repeated at least three times.

LV-FOXD3-AS1 increased Th1/Th2 ratio via inhibition of DC maturation
Based on the above findings and the regulation of Th1/Th2 cells by DCs, we wondered whether LV-FOXD3-AS1 increased the Th1/Th2 ratio by inhibiting DC maturation in AR. Next, we constructed a coculture system of DCs with CD4+ T cells. CD4+ T cells were isolated from healthy mice and cocultured with normal DCs (isolated from healthy mice), AR-DCs (isolated from AR model mice), AR-DCs transfected with LV-NC or LV-FOXD3-AS1, and LPS-or TSLP-treated AR-DCs transfected with LV-FOXD3-AS1. The results showed that normal DCs significantly increased the Th1/Th2 ratio, but AR-DCs induced the opposite results, indicating the regulation of Th1/Th2 cell differentiation by DCs in AR (Figure 4a,b). Furthermore, the AR-DC-mediated reduced Th1/Th2 ratio was obviously reversed by LV-FOXD3-AS1-transfected AR-DCs, which was further enhanced by LPS but was weakened by TSLP (Figure 4a,b). The above results were further manifested by IL-4, IL-5, IL-13, and IFN-γ levels (Figure 4c-f) and T-bet and GATA3 mRNA expression (Figure 4g,h) in CD4+ T cells. Thus, in consideration of the results revealed by Figures 1-4, we concluded that LV-FOXD3-AS1 increased the Th1/Th2 cell ratio to alleviate AR by inhibiting the maturation of DCs.

LV-FOXD3-AS1 inhibited DC maturation by suppressing STAT6 phosphorylation
Finally, we explored the related mechanisms by which LV-FOXD3-AS1 inhibited the maturation of DCs. Our results indicated that, compared with normal DCs transfected with LV-NC, the level of phosphorylated STAT6 was notably downregulated in normal DCs transfected with LV-FOXD3-AS1 (Figure 5a,b). Then, normal DCs were pretreated with LPS, followed by cotransfection with LV-FOXD3-AS1 and STAT6. The results showed that STAT6 overexpression effectively reversed the decrease in the percentages of CD80-, CD86-, and MHC-II-positive DCs induced by LV-FOXD3-AS1 (Figure 5c,d). These data revealed that LV-FOXD3-AS1 inhibited the maturation of DCs by inhibiting the phosphorylation of STAT6.

Discussion
Th1/Th2 cell imbalance is the pathophysiological basis of a series of inflammatory reactions (Moss et al. 2005). After Th1 cells differentiate, the transcription factor T-bet is highly expressed, and the cytokine IFN-γ is secreted, forming a positive feedback loop; however, after Th2 cells differentiate, the transcription factor GATA-3 is highly expressed, and the cytokine IL-4 is secreted, also forming a positive feedback loop. A large number of inflammatory cells and cytokines further trigger IgE synthesis and induce allergic reactions such as basophilic granulocyte and mast cell degranulation (Lin et al. 2016). Our previous study revealed that lncRNA FOXD3-AS1 markedly inhibited Th2-type immunoreactions in CD4+ T cells cultured with LPS-treated nasal epithelial cells (NECs) .
Here, our work further demonstrated that LV-FOXD3-AS1 significantly reduced Th2 cells in the peripheral blood of AR model mice but had no significant effects on Th1 cells, resulting in an increase in the Th1/Th2 ratio. In addition, LV-FOXD3-AS1 also reversed the significant alterations in the levels of the Th1/Th2 cell-related cytokines IL-4, IL-5, IL-13, IL-6, and IFN-γ in the peripheral blood of AR model mice. However, the specific mechanisms by which LV-FOXD3-AS1 affected the Th1/Th2 ratio in AR model mice were not clear. DCs are the most potent antigen-presenting cells in vivo, are the initiator of the immune response and play an important role in the differentiation of Th cells (Liu et al. 2016;Srivastava et al. 2019). LPS can induce the maturation of DCs, and mDCs not only present antigens to T cells but also secrete cytokines such as IFN-γ, contributing to acute inflammatory responses and the migration of activated T cells to Th1 cells (Wan et al. 2006). TSLP can also activate DCs to promote antigen presentation, can induce the differentiation of activated T cells into Th2 cells, and is considered the master switch and regulator inducing Th2 hypersensitivity (Melum 2014). CD80, CD86, and MHC-II are known mature markers of DCs. Accumulating evidence has revealed that DCs play a vital role in allergic airway diseases, such as asthma and AR (Chen et al. 2011;Lee et al. 2015;Murakami et al. 2016). HLA-DR and CD86 expressed by DCs were remarkably upregulated in AR model mice (Xie et al. 2005). Similarly, in this study, we found that CD80, CD86, and MHC-II were obviously upregulated in AR, but these phenomena were all effectively reversed by LV-FOXD3-AS1, both in vivo and in vitro. Several studies have revealed that the maturation of dendritic cells usually enhances Th1 cells in various diseases (Chen et al. 2009;Jiang et al. 2017;Zhou et al. 2017). However, the relationship between the maturation of dendritic cells and Th2 cells was also reported in allergic airway diseases. Pilette et al. (2013) reported that DC numbers increased in airway and skin tissues exposed to allergens and produced lower levels of IL-10, IL-12, and IFN-γ both locally and in the blood of AR patients. Sun et al. (2019) revealed that the silencing of CD86 in DCs significantly reduced the production of the cytokines IL-4 and IL-5 in T cells from AR patients. Similarly, in this study, in addition to the significant alterations in the levels of IL-4, IL-5, IL-13, and IFN-γ, the AR-DC-mediated decrease in the Th1/Th2 ratio was obviously reversed by LV-FOXD3-AS1-transfected AR-DCs, which was mainly due to the reduced Th2 cell numbers. LPS further enhanced the increase in the Th1/ Th2 ratio induced by LV-FOXD3-AS1-transfected AR-DCs owing to the increased Th1 cell number, which was markedly weakened by TSLP due to the increased Th2 cell numbers. The TSLP-DC pathway plays an important role in the occurrence and development of Th2 inflammation in asthma. It has been reported that TSLP is a key factor in the initiation of asthma (Zhou et al. 2005); it activates dendritic cells and upregulates the expression of costimulatory molecules in dendritic cells to promote the maturation of dendritic cells, thus promoting the differentiation of naive CD4+ T cells into Th2 cells and producing inflammatory factors such as IL4, IL-5 and IL-13, aggravating airway inflammation (Melum 2014). Thus, based on the above results, we concluded that LV-FOXD3-AS1 inhibited the maturation of DCs to increase the Th1/ Th2 cell ratio, mainly by reducing Th2 cell numbers, thus improving AR. Furthermore, the mechanisms of the TSLP-DC-Th2 pathway in AR are shown in Supplementary 2.
STATs are closely associated with T-cell differentiation. IL-4 and IL-13 can activate STAT6, and activated STAT6 induces the secretion of Th2 cytokines, thus resulting in the occurrence of Th2 inflammation (Thomas et al. 2016). Liang et al. (2019) reported that Morin ameliorated ovalbumin-induced AR via inhibition of the STAT6 and GATA3/T-bet signaling pathways in BALB/c mice. In addition, cisplatin and oxaliplatin inhibited the phosphorylation of STAT6 during the differentiation and maturation of DCs (de Haas et al. 2019). Thus, we speculated that LV-FOXD3-AS1 may affect DC maturation by regulating the phosphorylation of STAT6 in AR. As expected, our data revealed that LV-FOXD3-AS1 markedly inhibited the phosphorylation of STAT6 in DCs, and overexpressing STAT6 effectively reversed the decrease in the percentages of CD80-, CD86-, and MHC-II-positive DCs induced by LV-FOXD3-AS1. Regrettably, we only examined the change in STAT6. It will be better to clarify whether STAT6 is the major transcription factor involved in the regulation of DC maturation in AR by assessing other STATs.
In conclusion, our data revealed that LV-FOXD3-AS1 markedly improved AR by elevating the Th1/Th2 cell ratio via inhibition of DC maturation. Mechanistically, LV-FOXD3-AS1 inhibited DC maturation by suppressing the phosphorylation of STAT6. Our research may provide a novel therapeutic strategy for AR.

Disclosure statement
No potential conflict of interest was reported by the author(s).