DISCUSSION
We have verified that the AR mouse model was successfully established. We first showed that intranasal delivery of MSCs-Exo could not only relieve the main AR symptoms, sneezing and runny nose, but also decrease various inflammation indicators, including the spleen index, tissue staining, and the expression of inflammation-related cytokines. Moreover, we found that the optimal concentration of MSCs-Exo was 4×108/mL. Besides, we demonstrated that the MAPK and NF-κB pathways play an important role in inflammation, and that MSCs-Exo ameliorated AR by inhibiting the expression of MAPK pathway proteins, which modulated the immune response in the AR mouse model.
MSCs exert immunoregulatory effects by inhibiting T cell proliferation, inactivating allogeneic T cells, influencing T cell apoptosis22, and inhibiting B cell proliferation to decrease antibody secretion23. Furthermore, MSCs inhibit the differentiation and maturation of dendritic cells, resulting in a decline in the ability to activate T cells24. MSCs exert the above immunomodulatory effects mainly through the paracrine secretion of exosomes25. The presence of microvilli and columnar cells in the nasal cavity can intensify drug absorption26, and intranasal administration is not only safe but also convenient to operate. These advantages increase its feasibility for clinical application. Therefore, we chose to study the therapeutic effect of MSCs-Exo on AR by intranasal delivery; our results verify that intranasal administration of MSCs-Exo works not only locally but also systemically.
After exposure to the specific allergen, IgE mediates mast cell degranulation, causing itching and sneezing and further promoting hemangiectasis and increasing secretion of submucosal glands, thus resulting in obstruction and rhinorrhea. The immunohistological characteristics of AR include the infiltration of eosinophils and the predominant expression of cytokines secreted by Th2 cells, with a reduced activity level of Th1 cells, which contributes to the late-phase response such as nasal obstruction and hyperreactivity27, 28. Our results correspond with the mechanism underlying AR described above. In the AR model, IgE, OVA-IgE, IgG1, and histamine levels in BALF and serum and Th2 cytokine levels increased, corresponding to severe local and systematic symptoms. After intranasal administration of MSCs-Exo, the decrease in IgE, OVA-IgE, and histamine levels relieved the classic symptoms; the decrease in CCL-11 levels is linked to lower eosinophil infiltration; the change in ICAM-1 and Th2 cytokine levels is linked to reduced inflammation; and increased IFN-γ levels can stimulate the expression of IgG2a and inhibit the production of IgG3, IgG1, IgG2b, and IgE29, thus further exerting a therapeutic effect on AR. Many studies have also demonstrated that after intravenous treatment with different kinds of exosomes in an asthma mouse model, inflammation of lung tissues decreased, and Th2 cytokine and inflammatory cytokine levels were lower in serum and BALF30-32. All these results imply that intranasal delivery of MSCs-Exo has an effect similar to that of intravenous delivery to suppress allergic reactions.
In order to pave the way for clinical application in the near future, we examined the effects of different doses, and found that the optimal concentration is 4×108/mL. When the concentration of MSCs-Exo is lower than 4×108/mL, the efficacy decreases significantly. Once the concentration is higher than 4×108/mL, the curative effects do not further increase significantly. The data show that MSCs-Exo works in a dose-dependent manner, which supports future clinical research.
The activation of NF-κB plays an important role in allergic diseases, inducing the accumulation of inflammatory cells33. When activated by inflammatory signals, the released NF-κB induces the transcription of many inflammatory cytokines, which are related to the pathogenesis of asthma34. MAPKs are involved in many different cellular events, including allergic diseases35. Mammalian MAPKs are mainly divided into three categories: extracellular signal-regulated kinase (ERK), c-Jun N-terminal kinase (JNK), and p3836. The activation of MAPK is critical for the production of inflammatory cytokines37, and functional differentiation into Th1 or Th2 subsets38. Notably, inhibition of ERK promotes the transition of Th2 lymphocytes to Th139. Our results showed that the expression of genes related to the NF-κB and MAPK pathways increased significantly in AR mice, but after MSCs-Exo treatment, the expression of genes related to the NF-κB pathway did not decrease significantly, while MSCs-Exo treatment significantly inhibits ERK, JNK, JUN, and FOS expression. The results confirm that the NF-κB and MAPK pathways play important roles in AR and indicate that MSCs-Exo do not reduce the activation of NF-κB to alleviate the symptoms of AR. MSCs-Exo inhibit inflammation by restricting OVA from affecting the MAPK pathway in AR mice.
While our results have demonstrated that intranasal delivery of MSCs-Exo is an effective therapy for AR, the specific mechanism has not been adequately revealed. It has not been researched how the MAPK pathway regulates the expression of cytokines to play a therapeutic role in AR. The specific functional components of MSCs-Exo have not been identified, and it is unknown how long they remain active. Previously, the effects of intranasal and intravenous delivery of MSCs-Exo on the brain were compared, and intranasal delivery was more effective40. For AR, no direct comparisons between intravenous and intranasal delivery have been made. Note also that we only studied intranasal administration of MSCs-Exo isolated from human umbilical cord; and while this extends the current treatment methods, it will be interesting to test exosomes secreted by other cells to examine the different effects.
Intranasal administration of MSCs-Exo has been researched in various diseases, including complete spinal cord injury41, microglia-mediated neuroinflammation42, and autism spectrum disorders43. It is noteworthy that intranasal delivery of MSC-Exo could substantially expand pulmonary IL-10-producing interstitial macrophages to protect against allergic asthma in mice44. However, research on intranasal delivery of MSC-Exo in AR is still scarce. Our results offer a theoretical and experimental basis for the future clinical local application of MSCs-Exo in the nasal cavity for the treatment of AR, which can effectively alleviate pain in AR patients and has significant clinical and social value.