Mesenchymal stromal cells ameliorate acute allergic rhinitis in rats

Mesenchymal stromal cells (MSCs) have been extensively investigated as a potential antiinflammatory treatment in many inflammatory‐related diseases; however, it remains unclear whether MSCs could be used to treat acute allergic rhinitis. A rat model of allergic rhinitis was treated with MSCs. The effect of MSCs on the inflammation of allergic rhinitis was evaluated by sneezing, nose rubbing, the pathology of the nasal mucosa, and the expression of interleukin 4, tumour necrosis factor alpha, and immunoglobulin E in the serum of rats. Also, the population of MSCs isolated from umbilical cords of humans was evaluated to determine if they could inhibit the symptoms and inflammation of acute allergic rhinitis in a rat model. We observed that this population of cells inhibited sneezing, nose rubbing, and changes in the pathology of the nasal mucosa. Intriguingly, we observed that MSCs reduced the expression of interleukin 4, tumour necrosis factor alpha, and immunoglobulin E in the serum. Furthermore, MSCs reduced the expression of histamine and the recruitment of macrophages in the nasal mucosa of allergic rhinitis rats. We reasoned that the effect of MSCs on allergic rhinitis might be through its regulation of the secretion of related cytokines from macrophages during the process of acute allergic rhinitis. This work suggested that MSCs from the umbilical cords of humans could be used as a positive clinical therapy for the human disease.

Allergic rhinitis is an immune disorder, and it is caused by hypersensitivity towards all kinds of allergens. 1,7 Recent studies have suggested that T helper type 2 (Th2) cells and mast cells play crucial roles in the pathogenesis and process of allergic reactions during AR. 8 T helper type 2 cells mediate the activation and maintenance of the allergens and secrete a population of Th2 cytokines, such as interleukin 4 (IL-4). Then, more T helper cells are recruited into the allergic response and promote the synthesis and secretion of immunoglobulin E (IgE) from B lymphocytes. 8,9 Once IgE binds to the high-affinity receptor (FcεRI) on the surface of mast cells, special IgE responses to antigens are switched on. 10 Subsequently, the binding of IgE-FcεRI activates mast cells, which leads to a degranulation response and the secretion of allergic mediators, including inflammatory cytokines and histamine, and results in the infiltration of inflammatory cells, followed by acute or chronic inflammation of the nasal mucosa. Moreover, IL-4 suppresses Th1 and Th17 cell responses through the upregulation of transcriptional repressors of interferon gamma (IFN-γ) and IL17 that play important roles in the process of AR. 11,12 Thus, the regulation of the disordered inflammatory cytokine secretion could be a potential strategy to cure AR.
Human mesenchymal stromal cells (MSCs) are multilineage stem cells that can be isolated from bone marrow, umbilical cord, amniotic membrane, fat aspirate, and dental pulp. 13 In recent years, this population of cells has been reported to inhibit inflammation as a promising therapeutic strategy to many inflammatory diseases and cancers [14][15][16] ; however, no study has been conducted to reveal the efficacy and possible mechanisms of human MSCs in the treatment of AR.
In the present study, we isolated MSCs from human umbilical cord and aimed to evaluate their effect on allergic inflammatory responses in an ovalbumin (OVA)-induced AR model in rats. Intriguingly, we observed that MSCs from human umbilical cord appear to have significant therapeutic efficacy in inhibiting the symptoms of sneezing and nose rubbing in acute AR rats. We further analysed that the anti-AR effect of MSCs might be associated with the regulation of cytokine secretion from macrophages during the process of AR. Our study provides hope for the therapy of AR using MSCs from human umbilical cord.

| Cells
Mesenchymal stromal cells were isolated with high efficiency from umbilical cord under the agreement of informed consent in the hospital and expanded in vitro with Eagle's minimal essential medium (Gibco/ BRL, USA), supplemented with 10% foetal bovine serum and 1 × L-glutamine, according to the method reported by Izaskun Ferrin et al. 13 The Institution Review Board of Tsinghua University approved the entirety of the project and found it concurrent with all related protocols (no: 20170013).

| Ovalbumin-induced allergic rhinitis rat models
The rats were sensitized by OVA by using a standard protocol. Briefly, OVA and aluminium hydroxide gel diluted in normal saline (1 mg/ mL + 30 mg/mL + 1 mL) were used to sensitize the rats 3 times via injection in 5 sites (double back toes, bilateral inguinal subcutaneous region, and abdominal cavity, with 0.2 mL for each site) on days 1, 5, and 10. The same amount of normal saline was injected into the 5 sites and the peritoneum in the rats of the normal group. From days 15 to 21, the sensitized rats were intranasally challenged by daily droppings with OVA diluted with sterile physiological saline (50 μL/nostril, 100 mg/mL, 10% OVA). The normal group rats were intranasally challenged by daily droppings with normal saline.
After the OVA-induced AR model was successfully established, the rats were randomly divided into 5 groups (n = 12 per group). In addition to the normal group and model group, the other 3 groups of rats received different frequencies of MSC treatment by intraperitoneal injection: group A (rats treated with MSCs once 7 days prior to AR rat model construction), group B (rats treated with MSCs once a day after AR rat model construction), and group C (rats treated with MSCs weekly for 4 consecutive weeks after AR rat model construction). Approximately 5 × 10 6 MCSs were suspended in 0.5 mL of saline and intraperitoneally injected into each rat.

| Evaluation of nasal symptoms
On the 30th day from the first injection of MSC cells, the rats were stimulated by the dropping of OVA (20 μL/nostril, 50 mg/mL) dissolved in a physiological saline solution into the bilateral nasal cavities, and maintained in the observation cage. Instances of sneezing and nose scratching were counted for 10 minutes after the OVA intranasal stimulation to evaluate the degree of allergic responses. The control rats were processed with saline.

| Histological analysis of nasal mucosa
7After the experiment, the rats were sacrificed by drawing out all the blood in the hearts after isoflurane anaesthesia, and the nasal mucosa was separated and fixed in 4% paraformaldehyde at room temperature. After fixation, the tissues were routinely paraffin embedded, sectioned at 4 μm thickness, and stained with haematoxylin and eosin to examine eosinophils, lesions, and swelling of the nasal mucosa.

| Serum and plasma
Abdominal aorta blood samples (at least 10 mL) were collected in vacutainer tubes under sterile conditions from the normal, model, and treatment groups of rats. Serum and plasma were obtained following rapid centrifugation and stored at −80°C.

| Statistical analysis
Statistical analyses for the experimental studies were carried out by using Student t tests. The values are shown as the mean ± standard error from at least 3 experiments. Statistical significance was set at a P value of less than .05.

| Mesenchymal stromal cells reduce the expression of interleukin 4, tumour necrosis factor alpha, and immunoglobulin E in the serum of allergic rhinitis rats
To elucidate the mechanism underlying the therapeutic effect of MSCs on AR, we examined the production of cytokines that mediate the symptoms of the disease. We measured the expression level of IL-4, IL-17, TNF-α, and IFN-γ, as those have been demonstrated to play a key role in AR. 7,11,12 The results showed that the level of IL-4 was dramatically decreased by the multiple dosage treatment ( Figure 3A, group C), and the other 2 strategies were also effective ( Figure 3A, groups A and B). Furthermore, the level of TNF-α was decreased significantly after the different treatments with MSCs ( Figure 3B). It appeared that all of the treatments had similar significant effects on the inhibition of the production of TNF-α. On the other hand, the levels of IL-17 and IFN-γ were not significantly changed after the MCS treatments, although IL-17 tended to decrease ( Figure 3C) and INF-γ tended to increase slightly ( Figure 3D). Together, these results indicate that MSCs play a role in the regulation of the production of inflammatory cytokines, in particular, IL-4 and TNF-α.

| Mesenchymal stromal cells reduce histamine in the plasma of allergic rhinitis rats
Antigen-induced IgE and histamine release from mast cell degranulation are the features of AR. 8,10 To further examine the therapeutic effect of MSCs on AR, we measured the expression level of IgE in the serum and histamine in the plasma in each group of rats via ELISA.
Our results displayed that the multiple dosage treatment significantly decreased the level of IgE ( Figure 4A) and inhibited histamine release caused by OVA stimulation in AR rats ( Figure 4B). These results suggest that MSCs function in inhibiting mast cells, preventing them from secreting histamine. These results are consistent with the symptom amelioration of acute AR in rats.

| DISCUSSIONS
Allergic rhinitis is a common inflammatory disease worldwide. 1,4,5 Patients have a well-defined phenotype, including increased airway resistance, oral breathing difficulty, sleep disturbance, and problems with social activities. 2,6 Current chemical drugs towards AR are limited to antihistamines, antiallergens, antileukotrienes, and intranasal corticosteroids that only alleviate allergic symptoms but fail to regulate the allergic reaction. Occasionally, these drugs have side effects, such as headache, throat irritation, and nasal dryness. [17][18][19] Thus, the development of more safe and effective therapeutic agents is needed.
Mesenchymal stromal cells have been proven to possess immunomodulatory and antiinflammatory properties. 20 Currently, human MSCs hold great promise in regenerative medicine for the therapy of human diseases. [14][15][16] Human MCSs can be acquired from many tissues, such as umbilical cord, bone marrow, and chorionic membrane. 13  The MSCs used in this study were isolated from human umbilical cord. The successful study of the pathogenesis and pathophysiology is dependent upon the establishment of a successful animal model of AR. Animal models induced by intraperitoneal injections of allergens vary greatly from the natural processes of AR. It has been reported that MSCs mediate their immunomodulatory effects by interacting with adaptive immunity systems; an altered Th1/Th2 balance in allergic diathesis has recently been termed a "procrustean paradigm". 24 T helper type 2 are activated by proinflammatory cytokines such as  have no statistical significance between the OVA-induced acute allergic rhinitis rats and the MCS treatment groups, the results correspond with the prospective tendency. *P < .05, **P < .01, and ***P < .001 IL-4 that are produced in the process of AR. 25 The ability of MSCs to significantly decrease the secretion of IL-4 is the main reason why the injection of MSCs is effective in AR. Furthermore, macrophages are another key player in AR, and we found that MSCs achieve their antiinflammatory effects by reducing the secretion of TNF-α.
Here, we investigated the role of umbilical cord-derived human MSCs in AR. Based on our studies, MSCs present a promising immediate curative effect to the inflammatory reaction in AR rats. Both AR symptoms and AR-related inflammatory cytokines have been inhibited by the intraperitoneal injection of MSCs. Although the continuous weekly therapy of MSCs for 1 month presents the strongest effects of AR in rats, the specification of therapeutic frequency, the dosage of MSCs, and the potential mechanism still need to be investigated further. Additionally, MSC therapy in AR needs to undergo innovations and improvements to be a more effective and safer treatment option for AR patients in the future.

CONFLICT OF INTERESTS
The authors declare no conflicts of interest.