Bone Marrow Mesenchymal Stem Cells Regulate IL4 and INF-γ Expression in AR Mouse Model

Bone marrow mesenchymal stem cells can promote the recovery of immune balance and regulate the balance of Th1/2 cells. Allergic rhinitis is a disease with Th1/2 imbalance mediated by IgE. It’s unclear whether BMSCs could regulate AR disease. In this study, the possible role of BMSCs was explored. AR mouse model was established by ovalbumin (OVA). 18 models were randomly divided into three groups: AR-sensitized, Stem-cell-returned, Medium-returned; six unsensitized mouses named normal-control. IgE, IL-4 and INF-γ levels were measured by Elisa. Observing migration of BMSCs by immunouorescence. Flow cytometry used to detect changes of Th1/2. STAT 4/6 protein level was detected by Western-blot. while Th2 decreased.


Background
Allergic rhinitis (AR), is an common chronic in ammatory disorder of the nasal airways with Th1/Th2 imbalance 1  By reading the literature, we found that BMSCs can promote the recovery of immune balance. It inhibits T cell, B cell, nature killer(NKC) and dendritic cell(DC) activities, and regulate Th1/2 cell balance, then achieve immune regulation function through a variety of mechanisms 6 . Smail Ogulur reported that intranasal application of embryonic stem cells (ESCs) and induced pluripotent stem cells (iPSCs) can inhibit airway in ammation in a mouse model of acute asthma 7 . Stem cells regulate the immune environment by inhibiting the over activation of Th 2 cells and the production of Tregs, alleviated the symptoms of immune disease and reduced the secretion of Th 2 cells secreting cytokines (including IL-4, IL-5 and IL-13), as well as decreased IgE levels and mucus production8. Its regulation mechanism has a highly relevance with the pathogensis of AR, but there are few studies related to AR.
We hypothesized that BMSCs can alleviate AR symptoms by regulating the STAT 4/6 signaling pathway, and designed this experimental program: AR mouse model, was established, BMSCs were isolated and cultured, then BMSCs or BMSCs medium was returned through mouse tail vein, to study its action in AR. At the same time, we veri ed that whether the mechanism of BMSCs is through differentiation or paracrine.

Material and animal models
Mature healthy male mouse purchased from National Rodent Laboratory Animal Resources (Shanghai, China). All animal care and experimental procedures were ethical and approved by the Tongji University Institution Animal Care and Use Committee. The AR mouse model was prepared as follows. A total of 25 mature healthy male mice were used to establish AR mouse model. Each mouse was rst sensitized intraperitoneally with 0.3 mg of OVA and 30 mg of AL(OH)3 every other day for a total of seven times. From day 15, the mouses were treated with 0.5% OVA aerosol to stimulate AR symptoms for ve times. The symptoms were observed and the frequencies of scratching and sneezing were assessed using the procedure previously described by Al Suleimani M but with modi cations. Finally, 22 AR mouse model were obtained. We randomly excluded 4 of them and divided the remaining into 3 groups (6 in each group), named Stem cell returned group (SCRg), Medium returned group (MRg), AR-sensitized group (ARg). Meanwhile, the other 6 mature healthy male mouse were named Normal control group (NCg).

BMSCs preparation and intervention therapy
Four weeks old mice were selected, sacri ced by cervical dislocation, then soaked in 75% ethanol for 15 min. Quickly cut the skin and muscles of the hind limbs under aseptic conditions, and the bone marrow is taken. The obtained BMSCs were cultured in a low-sugar DMEM culture medium (containing 10% FBS, 100 u/ml penicillin, and 100 mg/ml streptomycin), and changed every 3 days until the cells were up to about 90% , and the cells were digested and passaged. After digesting with 0.25% trypsin, it was inoculated into a new medium, and the inoculation density was 0.5-1.0*10^4 pieces/cm2. A total of 2 passages were taken, p2 cells were taken and counted, and the supernatant was taken. The collected cells were resuspended again with PBS and controlled to contain 1.0*10^6 cells per 0.2 ml.
On the 21st day, the SCRg group was transfused with BMSCs via the tail vein, which were labeled with CM-Dil and a total of 1.0×10^6 of p2 generation, once daily for 5 days; MRg group was transfused with the same dose of medium supernatant which were taken during BMSCs culture. AR g and NC groups returned the same dose of PBS.

Determination of serum IgE and cytokines IL-4 and INF-γ
The mouses were anesthetized by intraperitoneal administration of pentobarbital (40 mg/kg). These animals were sacri ced by rapid decapitation, and then blood and nasal mucosa were collected. Part of the nasal mucosa was taken from the front of the inferior turbinate and the anterior segment of the nasal septum, and immediately placed in liquid nitrogen.The levels of IgE, IL-4 and INF-γ in guinea pigs were determined by ELISA (RB Inc., Maryland, USA). The latter part of the mouse head was retained and examined for decalci cation staining.
Western blot analyses of STAT 4/6 The nasal mucosa, which was frozen in liquid nitrogen, was homogenized in 1 mL of protein lysis buffer (PBS containing 0.1% Triton X-100) and centrifuged at 14,000 g for 10 min at 4 °C. Nasal mucosa lysates from each group were analyzed by Western blot.The blot was blocked with PBS-T containing 1% skim milk and then incubated with a 1:1000 diluted STAT 4 antibody (AbCAM, ab68153) or a 1:1000 diluted STAT 6 antibody (AbCAM, ab32520) at room temperature. After three additional washes, the blots were incubated with anti-mouse secondary antibody (1:5000) then conjugated to horseradish peroxidase for 1h at room temperature. The bands were visualized using EZ-ECL detection reagents.The second batch of images was quanti ed using Quantity One software. Β-actin was employed as an endogenous control for protein normalization. The experiments were performed in duplicate.

Results
Immuno uorescence of BMSCs in nasal mucosa and Nasal sinus decalci cation staining Immuno uorescence of BMSCs in nasal mucosa Tissue from the specimens were xed in 10% buffered formalin. Immunohistochemical stain were performed on formalin-xed and para n-embedded 4um sections. Fluorescence microscope was applied to observe and photograph the migration of CM-Dillabeled BMSCs in nasal mucosa. It can be seen that there was red uorescence of BMSCs in the nasal mucosa, and the nasal mucosa cells were stained with DAPI and displayed in blue Fig. 1). At the same time, we decalci ed the cross section of the nasal sinus of the animal model. It can be observed that the AR mouse model group is thicker than the nasal mucosa basement membrane and the submucosal lamina propria in ammatory cell in ltration is signi cantly increased. After intervention with BMSCs or the medium of BMSCs, the in amematory cell in ltration of the lamina propria was not as severe as in the AR group, and there was no statistical difference between the SCRg and MRg Fig.2 .

Reduction of IgE levels in serum
The IgE level in the serum of the AR-sensitized group was higher than those in the control group (p<0.05). Compared with the AR group, IgE levels were signi cantly lower after treatment with BMSCs or the medium of BMSCs (p < 0.01) (Fig. 3), but there was no statistical difference between the SCRg and MRg groups.

Reduction of IL-4 and INF-γ levels in serum
The level of IL-4 in the AR-sensitized group was higher than that in the control group (p<0.05). Compared with the ARg, IL-4 levels were signi cantly decreased after after treatment with BMSCs or the medium of BMSCs (p < 0.01) (Fig. 4), but there was no statistical difference between the SCRg and MRg groups. After sensitization with OVA, the level of INF-γ is lowered, after treatment with BMSCs or the medium of BMSCs, INF-γ levels were elevated in SCRg and MRg. Also there was no statistical difference between the SCRg and MRg. (Fig. 5) Flow cytometry to detect changes in Th1 / 2 cell content In the NCg, the content of Th1 cells was signi cantly higher than that of Th2 cells. After OVA sensitization, the content of Th2 cells increased (including the group of ARg, SCRg, MRg). And after treatment with BMSCs or the medium of BMSCs, the content of Th1 cells were signi cantly more than that in ARg, and the content of Th2 cells were decreased. But there was no statistical difference between the two groups. (Fig.6) Western blot results of STAT 4/6 expression pathway protein As shown in Fig 7 Western blot analysis showed that the expression level of STAT 6 in the AR-sensitized group were higher than that in the control groups (p<0.05), and the expression level of STAT 6 in the SCRg and MRg group were lower, but there was no statistical difference between the SCRg and MRg. The expression level of STAT 4 in the AR-sensitized group were lower than that in the control groups (p<0.05), while the expression of STAT 4 in the SCRg and MRg was higher than those in ARg and NC group, also there was no statistical difference between the SCRg and MRg . (Fig. 7)

Discussion
The pathophysiology of AR is chronic airway in ammation, with in ltration of eosinophils, mast cells, and CD4+ T lymphocytes, expressing many cytokines, such as IgE, IL-4, and INF-γ. Lymphocytes develop into Th1 or Th2 cells. They can be classi ed based on their cytokine production in the immune system.
Th2 cells produce IL-4, IL-10, and other cytokines that are primarily involved in IgE-mediated delayed type-1 hypersensitivity and contribute to the recruitment of eosinophils 9,10 .
BMSCs are adult stem cells derived from mesoderm. Which have the potential for self-renewal and multidirectional differentiation. BMSCs can differentiate into various cells, such as osteoblasts, chondrocytes, nerve cells and hepatocytes, etc 11,12 . Due to its easy separation and culture, low risk of conversion to cancer and low immunogenicity 7,13-15 , BMSCs have become a hotspots of tissue engineering research and clinical application experimental research. At present, it has been extensively reported that BMSCs have been used for cell replacement therapy, inhibiting in ammation and promoting tissue repair. In addition, they can also replicate the target gene to the host as a cell carrier, which provides much of evidence for diseases treatment 16,17 . Currently, BMSCs have been shown to have weak immunogenicity and unique immuno-negative properties, which can inhibit the activity of T cells, B cells, natural killer cells (NKC) and dendritic cells (DC) at different levels 18,19 . In vivo, BMSCs have a complex regulatory effects on the imbalance of Th1/Th2 immune response. Stem cells can modulate the immune environment by inhibiting excessive activation of Th2 cells and production of Tregs. Reversal of disease symptoms and reduction of Th2 secreting cytokines (including IL-4, IL-5 and IL-13) and reduction of IgE levels and mucus production can be observed. Additionally, it has been reported that BMSCs can exert immunosuppressive effects by reducing the activation and differentiation of B and T cells 20,21 .
It was found that BMSCs can regulate the immune response by reducing IL-4, IL-6, and then increase the levels of IL-10 and INF-r, thereby regulating the differentiation of various T cell subtypes. This is consistent with our ndings. After stem cell re-infusion and culture medium re-infusion22, IL-4 levels in the blood of AR mice decreased, while INF-r levels increased, indicating that the immune response was down-regulated. However, there was no difference of IL-4 and INF-r between the SCRg and MRg. This suggests that both approaches can perform the similar effect on immune system. Furthermore, it was found that the content of IgE was decreased; and the symptoms of AR mouse was alleviated, evaluated by behavior. This con rmed the result again. However, there was still no statistically signi cant difference between the two groups and not clear which aspect played a role.
After stem cell re-infusion and medium-return intervention, we used proteomics to detect the stat protein levels in the nasal mucosa. We found that the stat4/6 protein content was signi cantly higher in ARsensitized group, indicating that the stat pathway was activated, a, and participated in the AR immune response. After stem cell reinfusion and medium-return intervention, the stat4/6 protein activation was reduced. It suggested that those interventions can regulate STAT 4 pathway in AR immune response.
However, there was no statistically signi cant difference between the two groups and the mechanism was not clear.
We investigated the effect of BMSCs on AR immune response and its possible mechanism, using Elisa, ow cytometry, and proteomics analysis to detect expression changes of related cytokines and possible signaling pathways in AR mouse model after stem cell reinfusion and medium-return intervention. We found that these interventions can reduce the IgE level and IL-4 cytokines in AR mouse and increase INF-r level to balance Th1/Th2, and inhibit the AR immune response. In addition, stem cell re-infusion and medium re-infusion interventions activate the STAT4/6 signaling pathway. It was also found that the two interventions achieved similar results with no statistical difference between them. therefore, it can be concluded that BMSCs may perform effects on AR immune response through a large amount of mediators produced by direct secretion or the paracrine pathway, which regulated the cytokines and further regulated the immune response. However, the mechanism is complex and further in-depth experimental research is still needed.

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Treatment of BMSCs and the medium of BMSCs can reduce the expression of IL-4, promote the expression of INF-γ and regulate the expression of Th cell in AR mouse model, and its mechanism is closely related to the STAT 4/6 signaling pathway. Moreover, the mechanism of action of BMSCs in regulating immune balance may be achieved by paracrine function rather than multi-directional differentiation potential.

Authors' contributions
All the authors conceived and designed the study. Chuanliang Zhao and Wentao Zou participated and carried out the experiment. Chuanliang Zhao wrote the rst draft of the manuscript, interpreted the data and Jiaxiong Zhang modi ed and con rmed the nal version. Jingwen Sun and Xiaojing Cai also participated and carried out the experiment. The migration of BMSCs was observed under a uorescence microscope. BMSCs were visualized in the nasal mucosa (red light), and the nasal mucosa cells were stained with DAPI and displayed in blue.

Figure 3
After OVA sensitization, IgE level in the serum increased signi cantly(including the group of ARg, SCRg, MRg ) compared with NCg. After treatment with BMSCs or the medium of BMSCs, the IgE level in the SCRg and MRg groups decreased, but there was no statistical difference between the two groups.

Figure 5
The level of INF-γ in the AR-sensitized group (including the group of ARg, SCRg, MRg ) is lower than that in NCg, but the levels of INF-γ in SCRg and MRg are higher than in ARg.

Figure 7
As shown in Fig, WB showed that the level of STAT 6 in the AR-sensitized group (including the group of ARg, SCRg, MRg ) were higher than that in NCg, and the expression level of STAT 6 in the SCRg and MRg were lower than that in ARg, but there was no statistical difference between the SCRg and MRg. the level of STAT 4 in the AR-sensitized group (including the group of ARg, SCRg, MRg ) were lower than that in NCg.