Effect of mesenchymal stem cells on airway inflammation in asthmatic mice depleted of CD4+CD25+ regulatory T cells

doi:10.3969/j.issn.2095-4344.2017.05.015

Abstract

Abstract:

BACKGROUND: Mesenchymal stem cells have an immunoregulatory capacity to suppress the airway inflammation of asthmatic mice, but the mechanism remains unclear. Our preliminary studies indicated that mesenchymal stem cells can upregulate the CD4⁺CD25⁺ regulatory T cells (Treg) of the asthmatic mice.
OBJECTIVE: To study the effect of mesenchymal stem cells on the airway inflammation in asthmatic BALB/c mice depleted of CD4⁺CD25⁺ Treg.
METHODS: Forty BALB/c mice were randomly divided into five groups: normal control group (group A), asthmatic group (group B), asthmatic group depleted of CD4⁺CD25⁺Treg (group C), mesenchymal stem cells group (group D), and asthmatic group depleted of CD4⁺CD25⁺Treg and administrated with mesenchymal stem cells (group E). Except group A, mice in the other groups were sensitized and challenged by ovalbumin to establish asthma models. In group C and group E, each mouse was treated with anti-CD25⁺ monoclonal antibody to deplete CD4⁺CD25⁺Treg. In group D and group E, mice were intravenously administered with 0.2 mL mesenchymal stem cells 1×10⁹/L) at 10 days after sensitization. At 24 hours after the final activation, the number of CD4⁺CD25⁺Treg in the peripheral blood was detected by flow cytometry. The total cell number of inflammatory cells in the bronchoalveolar lavage fluid, and the number of eosinophils, lymphocytes and neutrophils were counted to analyze the degree of inflammation of the airway together with pathological observation.
RESULTS AND CONCLUSION: (1) The proportion of CD4⁺CD25⁺Treg in peripheral lymphocytes was ranged as follows: group B < group D < group A (P < 0.01). (2) Number of inflammatory cells in the bronchoalveolar lavage fluid: Regarding the total cell number and the number of eosinophils in the bronchoalveolar lavage fluid, ranking was as follows: groups B, C, D, E were significantly higher than group A (P < 0.01), group C was significantly higher than group B ( P < 0.01), group D was significantly lower than group B (P < 0.01), group E was significantly lower than group C (P < 0.01). (3) Pathological observation of the lung: Mesenchymal stem cells could significantly inhibit the airway inflammation of asthmatic mice. The asthmatic mice appeared to have severer airway inflammation when CD4⁺CD25⁺Treg were depleted from the peripheral blood. However, mesenchymal stem cells could also significantly inhibit the airway inflammation of asthmatic mice depleted of CD4⁺CD25⁺Treg. Taken together, our data indicate that mesenchymal stem cells can significantly inhibit the airway inflammation of asthmatic mice depleted of CD4⁺CD25⁺Treg.

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: Stem Cells, Mesenchymal Stem Cells, Asthma, Tissue Engineering

CLC Number:

R394.2

Zhuansun Yong-xun, Zhang Wei, Du Yu-mo, Ran Pi-xin, Chen Rui, Lin Lin, Li Jian-guo. Effect of mesenchymal stem cells on airway inflammation in asthmatic mice depleted of CD4+CD25+ regulatory T cells[J]. Chinese Journal of Tissue Engineering Research, 2017, 21(5): 742-747.

Figures/Tables 1

2.1 MSCs细胞形态观察及鉴定结果 MSCs的原代培养：骨髓细胞接种于塑料培养瓶后，细胞在培养瓶散在分布，呈圆形；24 h后就见有细胞贴壁；72 h后，首次全量换液去除未贴壁细胞，此时贴壁的细胞为单个，形态大部分为圆形；第1次换液后5-7 d，可见由几十个到数百个细胞组成的集落，细胞形态呈梭形、三角形或星形，其中夹杂一些圆形细胞。原代培养10 d左右，细胞快速增殖，形成细胞小集落，每个小集落中的细胞不断增殖，呈“漩涡状”；培养2周左右，每个小集落形成约数百致上千个细胞的大集落，集落交界处出现重叠、融合，细胞形态呈梭形、三角形或星形，仍有圆形细胞夹杂其中。 MSCs的传代培养：经胰酶消化后的MSCs呈圆形，胞体较大，传代24 h内完全贴壁伸展，呈成纤维细胞样形态，即呈梭形、三角形、星形，传代后的MSCs增殖能力不断增加，第1，2代细胞约经7 d左右融合，第3代以后细胞经4 d左右融合。第1代细胞仍可见很多圆形细胞夹杂其中；第2代细胞圆形细胞明显减少；第3代以后细胞均呈梭形、三角形、星形，未见圆形细胞。细胞融合时，MSCs细胞呈成纤维细胞样细胞形成的集落(图1)。 MSCs鉴定：①细胞黏附于塑料培养瓶的特性，集落样生长，呈成纤维细胞样集落，细胞形态为成纤维细胞样形态，具有快速增殖能力；②MSCs表面标志CD44及CD29抗原表达为阳性，造血干细胞表面标志CD45、CD11b抗原表达为阴性，表达阳性率如下：CD44 (98.46%)、CD29 (99.36%)、CD45(3.34%)、CD11b (2.41%)，见图2；③诱导分化：MSCs可诱导分化为成骨细胞，茜素红S染色见大量钙化的细胞外基质沉淀，见图3A；MSCs也可诱导分化为脂肪细胞，油红O染色见大量脂肪空泡，见图3B。 2.2 实验动物数量分析造模过程中无动物死亡，40只BALB/c小鼠均进入结果分析。 2.3 外周血CD4+CD25+Treg比例流式细胞仪检测示，A、B、C、D、E组外周血CD4+CD25+Treg占淋巴细胞比例分别为(5.86±0.48)%、(3.60±0.41)%、(0.32±0.11)%、(4.72±0.22)%及(0.33±0.11)%；B组外周血CD4+CD25+ Treg占淋巴细胞比例较A组显著减少(P < 0.01)；D组外周血CD4+CD25+Treg占淋巴细胞比例较B组显著提高(P < 0.01)，较A组仍显著减少(P < 0.01)。 2.4 支气管肺泡灌洗液炎症细胞计数 B组﹑C组﹑D组﹑E组支气管肺泡灌洗液炎症细胞总数﹑嗜酸性粒细胞计数均显著高于A组(P < 0.01)，C组支气管肺泡灌洗液炎症细胞总数﹑嗜酸性粒细胞计数显著高于B组(P < 0.01)，D组支气管肺泡灌洗液炎症细胞总数﹑嗜酸性粒细胞计数显著低于B组(P < 0.01)，E组支气管肺泡灌洗液炎症细胞总数﹑嗜酸性粒细胞计数显著低于C组(P < 0.01)，D组和E组炎症细胞总数﹑嗜酸性粒细胞计数比较差异无显著性意义(P > 0.05)，见表1。 2.5 肺组织病理变化镜下观察，B组﹑C组﹑D组﹑E组支气管、细支气管上皮下和小血管周围可见明显炎性细胞浸润，气道上皮细胞部分脱落，管腔内渗出增加，上皮下充血水肿明显，肺泡间质增厚，亦可见炎性细胞浸润；与其他4组比较，A组支气管、小支气管、伴行小动脉及肺泡结构清晰，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；D组﹑E组气道炎症改变较B组和C组有所减轻，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，充血水肿状况改善；C组较B组炎症改变有所加重；D组和去E组炎症改变无显著差异，见图4。"

References

[1] Martinez FD,Vercelli D.Asthma.Lancet.2013; 382(9901): 1360-1372.

[2] Bianco P. "Mesenchymal" stem cells.Annu Rev Cell Dev Biol. 2014;30:677-704.

[3] Sala E,Genua M,Petti L,et al. Mesenchymal Stem Cells Reduce Colitis in Mice via Release of TSG6,Independently of Their Localization to the Intestine. Gastroenterology. 2015; 149(1):163-176.

[4] Meier RP,Mahou R,Morel P,et al.Microencapsulated human mesenchymal stem cells decrease liver fibrosis in mice.J Hepatol.2015;62(3):634-641.

[5] 李建国,颛孙永勋,冉丕鑫,等.骨髓间充质干细胞对哮喘小鼠CD4+CD25+调节性T细胞及气道炎症的影响[J].中国组织工程研究与临床康复,2008,12(47):9302-9305.

[6] 李建国,颛孙永勋,冉丕鑫,等.小鼠骨髓间充质干细胞的分离、培养及鉴定[J].中国比较医学杂志,2008,18(12):28-31.

[7] Meirelles LS,Nardi NB.Murine marrow-derived mesenchymal stem cell: isolation, in vitro expansion, and characterization.Br J Haematol.2003;123(4):702-711.

[8] Long TT,Nakazawa S,Onizuka S,et al.Influence of CD4+CD25+ T cells on Plasmodium berghei NK65 infection in BALB/c mice.Int J Parasitol.2003;33(2):175-183.

[9] Smigiel KS,Srivastava S,Stolley JM,et al.Regulatory T-cell homeostasis: steady-state maintenance and modulation during inflammation.Immunol Rev.2014;259(1):40-59.

[10] Bin Dhuban K,Kornete M,S Mason E,et al.Functional dynamics of Foxp3(+) regulatory T cells in mice and humans[J]. Immunol Rev,2014,259(1):140-158.

[11] Plitas G,Rudensky AY.Regulatory T Cells: Differentiation and Function.Cancer Immunol Res.2016;4(9):721-725.

[12] Kanamori M,Nakatsukasa H,Okada M,et al.Induced Regulatory T Cells: Their Development, Stability, and Applications.Trends Immunol.2016. [Epub ahead of print]

[13] Stelmaszczyk-Emmel A.Regulatory T cells in children with allergy and asthma: it is time to act.Respir Physiol Neurobiol. 2015;209:59-63.

[14] Ismail AM,Aly SS,Fayed HM,et al.Serum 25-hydroxyvitamin D and CD4+CD25(+high) FoxP3+ Regulatory T cell as Predictors of Severity of Bronchial Asthma in Children. Egypt J Immunol.2015;22(1):9-18.

[15] Chauhan A,Singh M,Agarwal A,et al.Correlation of TSLP, IL-33, and CD4 + CD25 + FOXP3 + T regulatory (Treg) in pediatric asthma.J Asthma.2015;52(9):868-872.

[16] Donma M,Karasu E,Ozdilek B,et al.CD4(+), CD25(+), FOXP3 (+) T Regulatory Cell Levels in Obese, Asthmatic, Asthmatic Obese, and Healthy Children. Inflammation.2015;38(4): 1473-1478.

[17] Noval RM,Chatila TA.Regulatory T cells in allergic diseases.J Allergy Clin Immunol.2016;138(3):639-652.

[18] Kinoshita T,Baatjes A,Smith SG,et al.Natural regulatory T cells in isolated early responders compared with dual responders with allergic asthma.J Allergy Clin Immunol.2014;133(3): 696-703.

[19] Vega-Letter AM,Kurte M,Fernandez-O'Ryan C,et al. Differential TLR activation of murine mesenchymal stem cells generates distinct immunomodulatory effects in EAE.Stem Cell Res Ther,2016;7(1):150.

[20] Holan V,Hermankova B,Bohacova P,et al.Distinct Immunoregulatory Mechanisms in Mesenchymal Stem Cells: Role of the Cytokine Environment.Stem Cell Rev.2016. [Epub ahead of print]

[21] Karimi MH,Barzkar Z,Babaee M,et al. Evaluating Effect of Mesenchymal Stem Cells on Expression of TLR2 and TLR4 in Mouse DCs.Adv Pharm Bull.2016;6(2):179-186.

[22] Li X,Lu X,Sun D,et al. Adipose-Derived Mesenchymal Stem Cells Reduce Lymphocytic Infiltration in a Rabbit Model of Induced Autoimmune Dacryoadenitis.Invest Ophthalmol Vis Sci.2016;57(13):5161-5170.

[23] Fierabracci A,Del F A,Muraca M.The Immunoregulatory Activity of Mesenchymal Stem Cells: 'State of Art' and 'Future Avenues'.Curr Med Chem. 2016. [Epub ahead of print]

[24] Gazdic M,Volarevic V,Arsenijevic N,et al.Mesenchymal stem cells: a friend or foe in immune-mediated diseases.Stem Cell Rev.2015;11(2):280-287.

[25] Wang Y, Chen X, Cao W,et al.Plasticity of mesenchymal stem cells in immunomodulation: pathological and therapeutic implications.Nat Immunol.2014;15(11):1009-1016.

[26] Liu X,Ren S,Qu X,et al.Mesenchymal stem cells inhibit Th17 cells differentiation via IFN-gamma-mediated SOCS3 activation.Immunol Res.2015;61(3):219-229.

[27] Farini A,Sitzia C,Erratico S,et al.Clinical applications of mesenchymal stem cells in chronic diseases.Stem Cells Int.2014;2014:306573.

[28] Zhang Y,Cai W,Huang Q,et al.Mesenchymal stem cells alleviate bacteria-induced liver injury in mice by inducing regulatory dendritic cells. Hepatology.2014;59(2):671-682.

[29] Yan Z,Zhuansun Y,Chen R,et al.Immunomodulation of mesenchymal stromal cells on regulatory T cells and its possible mechanism.Exp Cell Res.2014;324(1):65-74.

[30] 温冰,颛孙永勋,颜富德,等.血红素加氧酶1在间充质干细胞上调哮喘患者外周血调节性T细胞中的作用[J].中国组织工程研究与临床康复,2011,15(27):5053-5057.