Exosomes from bone marrow mesenchymal stem cells regulate the balance of Foxp3+ Treg/Th17 in asthmatic mice

doi:10.3969/j.issn.2095-4344.0871

Abstract

Abstract:

BACKGROUND: Imbalance of Th1/Th2 immune response is an crucial pathophysiological manifestation of asthma, but recent studies have proved that asthma also has a close correlation with the imbalance of Foxp3⁺ Treg/Th17. Accumulating evidence indicate that the immunoregulatory capacity of mesenchymal stem cells are mainly related to exosomes secreted by the cells.
OBJECTIVE: To observe the effect of bone marrow mesenchymal stem cell exosomes on Foxp3⁺ Treg cells, Th17 T cells and airway inflammation of asthmatic mice as well as cytokines in the bronchoalveolar lavage fluid.
METHODS: Twenty-seven BALB/c mice of SPF grade were randomly divided into three groups: normal control group, asthmatic model group, and exosomes group. Except the normal control group, each mouse in the other groups was sensitized by ovalbumin to establish asthma models. In the exosomes group, each mouse was intravenously administrated with exosomes at 21 days of sensitization. At 24 hours after the final administration of ovalbumin, the proportion of Foxp3⁺ Treg and Th17 in the sleep of asthmatic mice as well as the expression of CTLA-4 and PD-1 in Foxp3⁺ Treg cells were detected by flow cytometry. The total number of inflammatory cells, and the number of eosinophils, lymphocytes, monocytes and neutrophils in the bronchoalveolar lavage fluid were counted to analyze the degree of airway inflammation in the combination with pathological observation. We also detected the expression of interleukin-4, 5, 13, 10, 17 and interferon-γ in the bronchoalveolar lavage fluid as well as p27^{kip1 in CD4+ T cells.

RESULTS AND CONCLUSION: (1) The proportion of Foxp3⁺ Treg in splenic lymphocytes and the CTLA-4 and PD-1 expression in Foxp3⁺Treg were significantly higher in the exosomes group than the asthmatic model group (P < 0.01). (2) The proportion of Th17 in splenic lymphocytes was ranked as follows: asthmatic model group > exosomes group > normal control group (P < 0.01). (3) The total number of inflammatory cells and the number of eosinophils, lymphocytes, neutrophils and monocytes in the bronchoalveolar lavage fluid were ranked as follows: asthmatic model group > exosomes group > normal control group (P < 0.01). (4) Pathological observation of the lung showed that the asthmatic mice appeared to have severest airway inflammation. However, mesenchymal stem cell exosomes could significantly alleviate the airway inflammation. (5) The detection of cytokines in the bronchoalveolar lavage fluid showed that levels of interleukin 13 and interleukin 17 were significantly reduced (P < 0.05, P < 0.01), while the level of interleukin 10 increased (P < 0.01). (6) The p27^kip1 expression in the CD4⁺ T cells was obviously higher in the exosomes group than the asthmatic model group. In conclusion, exosomes from bone marrow mesenchymal stem cells can reverse the imbalance of Foxp3⁺ Treg/Th17 and significantly inhibit the airway inflammation in asthmatic mice.

中国组织工程研究杂志出版内容重点：干细胞；骨髓干细胞；造血干细胞；脂肪干细胞；肿瘤干细胞；胚胎干细胞；脐带脐血干细胞；干细胞诱导；干细胞分化；组织工程}

Key words: Asthma, Mesenchymal Stem Cells, Exosomes, Tissue Engineering

CLC Number:

R394.2

Lin Ying, Hu Jin-zhang, Zhuansun Yong-xun, Ran Pi-xin, Chen Rui, Du Yu-mo, Lin Lin, Li Jian-guo. Exosomes from bone marrow mesenchymal stem cells regulate the balance of Foxp3⁺ Treg/Th17 in asthmatic mice[J]. Chinese Journal of Tissue Engineering Research, 2018, 22(17): 2637-2643.

Figures/Tables 3

2.1 外泌体鉴定结果图1A为电镜下的外泌体，呈中间凹陷的杯口样形态，直径50-150 nm，背景清晰，少见杂质。图1B为Western blot鉴定外泌体表面标记分子CD9，CD63，CD81的表达结果阳性，符合外泌体的生物学特征。 2.2 各组小鼠支气管肺泡灌洗液炎症细胞计数结果的比较哮喘模型组、外泌体处理组炎症细胞总数，嗜酸性粒细胞计数，单核巨噬细胞计数均显著高于正常对照组(P < 0.01)，外泌体处理组炎症细胞总数，嗜酸性粒细胞计数较哮喘模型组有所下降(P < 0.01)，但高于正常对照组(P < 0.01)，见表1。 2.3 肺病理组织学变化哮喘模型组支气管、细支气管上皮下和小血管周围可见明显炎症细胞浸润，气道上皮部分脱落，管腔内渗出增加，肺泡间质增厚，亦可见炎症细胞浸润；正常对照组细小气管上皮黏膜完整，气道纤毛上皮排列整齐，气管、血管周围无炎性细胞浸润、无充血水肿；外泌体处理组支气管、细支气管和伴行小血管周围炎症细胞浸润程度减轻，充血水肿状况明显改善，见图2。 2.4 肺泡灌洗液白细胞介素4，5，13，10，17及干扰素γ水平变化哮喘模型组白细胞介素4，5水平较正常对照组明显升高(P < 0.01)，白细胞介素13水平稍许升高(P < 0.01)。外泌体处理组较哮喘模型组白细胞介素4，5，13水平下降(P < 0.01)。白细胞介素10水平哮喘模型组低于正常对照组(P < 0.01)，经外泌体处理后白细胞介素10水平有所提高(P < 0.01)。白细胞介素17哮喘模型组明显高于正常对照组(P < 0.01)，经外泌体处理后显著下降(P < 0.01)。干扰素γ水平正常对照组明显高于哮喘模型组(P < 0.01)，外泌体处理组干扰素γ水平略高于哮喘模型组(P < 0.05)，见图3。 2.5 各组小鼠Foxp3+ Treg比例及其CTLA-4，PD-1表达正常对照组，哮喘模型组，外泌体处理组脾中CD4+T细胞中Foxp3+ Treg细胞比例分别为(17.54±2.10)%，(4.95± 0.51)%及(17.10±2.20)%，可以看出哮喘模型组Foxp3+ Treg比例较正常对照组明显下降(P < 0.01)，经外泌处理后Foxp3+ Treg比例回升(P < 0.01)。正常对照组，哮喘模型组，外泌体处理组Foxp3+ Treg表达CTLA-4分别为(8.41±1.01)%，(5.39±0.54)%及(6.07±0.71)%，PD-1分别为(45.67±3.91)%，(41.12±4.41)%及(65.00±6.74)%。哮喘模型组Foxp3+ Treg表达CTLA-4，PD-1较正常对照组有所下降(P < 0.01)，经外泌体处理后表达提高(P < 0.01)，见图4。 2.6 各组小鼠Th17比例正常对照组、哮喘模型组、外泌体处理组Th17细胞分别为(1.04±0.12)%，(3.21± 0.41)% 及(2.21±0.31)%，从图5可以看出哮喘模型组较正常对照组Th17比例显著提高(P < 0.01)，经外泌体处理后比例有所减低(P < 0.01)。 2.7 CD4+T细胞分选及p27kip1的表达分选后的CD4+T细胞纯度高达(98.01±1.44)%, 从图中可以看出哮喘小鼠CD4+T细胞中p27kip1表达较正常小鼠明显减低，经外泌体处理后p27kip1表达提高，见图6，7。"

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