The Effect Of Mesenchymal Stem Cells On CD4~+CD25~+ Regulatory T Cells And Airway Inflammation In Asthmatic Mice

It's demonstrated that asthma is characterized of chronic atopic airway inflammation in which the imbalance of Th1/Th2 plays a role. However, the classical theory of the Th1/Th2 imbalance can't completely explain the immunological pathogenesis of asthma. Some other mechanisms may exist. Recent studies have suggested that Regulatory T cells(Treg),which are a subset of CD4+ T lymphocytes, play an important role in the regulation of the immune system through suppressing the function of other immunocytes and the immunoreaction. Asthma is a chronic atopic inflammatory disease of the airways, and a disease which is insufficient in peripheral immunotolerance. More and more evidences suggested that Treg are closely associated with the atopic inflammation of asthma and the maintenance of peripheral immunotolerance. CD4+CD25+Treg are first described by Sakaguchi, also known as naturally occurring Treg or thymus derived Treg. As a major composition of Treg, they play an important role in the immunological pathogenesis of asthma.Mesenchymal stem cells (MSCs) are another class of bone marrow stem cells which are different from hematopoietic stem cells. Mesenchymal stem cells can differentiate along multiple lineages such as osteoblasts, chondrocytes, myocytes, tendon cells, neurocytes, adipocytes, etc.In addition, resent studies indicated that MSCs exert an immunoregulatory capacity both in vitro and in vivo. MSCs can upregulate the proportion of CD4+CD25+Treg, thus suppress the proliferative response of the T lymphocytes in the mixed lymphocyte reaction or stimulated by the mitogen PHA. Mesenchymal stem cells don't express major histocompatibility complexⅡ, Fas ligand and costimulatory molecules of T cells such as B7-1,B7-2, CD40,CD40L, so that they are not easily recognized by the immunocytes of the host and able to avoid rejection by the host immune system. Heterogenic MSCs can be implanted into the immune organs of the host and survive in a long term. In spite of the advantage in the cytotherapy, it is still unkown whether MSCs can upregulate CD4+CD25+Treg of asthmatic mice and improve its airway inflammation, and furthermore be used in asthmatic therapy. So we design this experiment to observe the influence of MSCs to the peripheral blood CD4+CD25+Treg and the airway inflammation in BALB/c asthmatic mice.The study includes three parts:1.Isolation,culture and identification of mouse mesenchymal stem cells from bone marrow2. Mesenchymal stem cells up-regulating CD4+CD25+ regulatory T cells of peripheral blood and alleviating airway inflammation in asthmatic BALB/c mice3.The effect of mesenchymal stem cells on airway inflammation in asthmatic BALB/c mice deleted of CD4+CD25+ regulatory T cells一,Isolation,culture and identification of mouse mesenchymal stem cells from bone marrow0bjective:To establish an efficient method for isolation and culture of mouse mesenchymal stem cells (mMSCs) from bone marrow, then proliferate and identify the cells in vitro.Methods:The mononuclear cells were isolated from BALB/c mouse bone marrow and mMSCs were enriched and expanded by using bone marrow adherent culture.Osteogenic and adipogenic induction was performed on the mMSCs. The phenotype was analyzed by Flow Cytometry (FCM) and morphology of the mMSCs was observed under a microscope.Results : The mMSCs were the adherent cells of similar fibroblastoid morphology. The expressions of CD45,CD11b,CD44 and CD29 were 3.34%,2.41%,98.46%and 99.36%. The mMSCs of the fourth generation could be induced to differentiate into osteoblasts or adipocytes.Conclusion:The mMSCs can be isolated and expanded by using bone marrow adherent culture which is efficient and stable. The cells had been identified by morphology,biological characteristics and differentiation potentia1 in vitro. We isolated and cultured mouse MSCs from bone marrow in vitro successfully, making it possible for further study.二,Mesenchymal stem cells up-regulating CD4+CD25+ regulatory T cells of peripheral blood and alleviating airway inflammation in asthmatic BALB/c mice.0bjective:To observe the effect of MSCs on the CD4+CD25+ regulatory T Cells (Treg) of peripheral blood and the airway inflammation in asthmatic BALB/c mice. Methods:Thirty BALB/c mice were randomly divided into three groups (10 mice in each group): the normal control group (group A), the asthmatic group (group B) and the MSCs group (group C). Group B and group C were sensitized by intraperitoneal injection of 0.2 ml 500μg/ml ovalbumin (OVA) and challenged by inhalation of nebulized 5% OVA solution to establish asthma models. In group A, normal saline of the equal volume was given instead of OVA. Group C was intravenously administered MSCs on the 10th day after sensitization. The proportion of CD4+CD25+ Treg/lymphocytes in peripheral blood was detected by Flow Cytometry. The number of total cells, eosinophils, lymphocytes and neutrophils in BALF was counted to analyze the degree of airway inflammation together with pathological sections.Results:The proportion of CD4+CD25+ Treg/lymphocytes in peripheral blood was (5.81±0.76)% in group A,(3.21±0.74)% in group B, and (5.12±0.34)% in group C. The difference was significant when group A was compared with group B(P<0.01), and when group B with group C(P<0.01). The number of total cells and eosinophils in BALF was (5.93±1.86)×104 /ml and(0.06±0.04)×104 /ml in group A(,51.15±7.42)×104 /ml and(14.24±2.84)×104/ml in group B(,24.87±3.15)×104/ml and (4.34±0.44)×104 /ml in group C. The numbers were significantly different when group A was compared with group B(P<0.01), and group B with group C(P<0.01). Lung inflammation was examined in HE stained sections. There was no obvious infiltration of inflammatory cells in the airways of group A. However, in group B, remarkable infiltration of inflammatory cells, proliferation and damage of airway epithelia and more mucus in the lumen could be observed in the sections, and the inflammation was attenuated remarkably in group C.Conclusions:MSCs can up-regulate CD4+CD25+ Treg of peripheral blood and relieve the airway inflammation in asthmatic BALB/c mice. The mechanism that MSCs relieve the airway inflammation in asthmatic BALB/c mice may be up-regulation of CD4+CD25+ Treg.三,The Effect of Mesenchymal Stem Cells on Airway Inflammation in Asthmatic BALB/c Mice Depleted of CD4+CD25+ Regulatory T CellsObjective:To study the effect of Mesenchymal Stem Cells on the airway inflammation in asthmatic BALB/c mice depleted of CD4+CD25+ Regulatory T Cells.Methods:Forty BALB/c mice were randomly divided into five groups(8 mice in each group): the normal control group (group A),the asthmatic group (group B), the asthmatic group depleted of CD4+CD25+Treg (group C),the MSCs group (group D),and the asthmatic group depleted of CD4+CD25+Treg and administrated with MSCs (group E). Except group A, other groups were sensitized by intraperitoneal injection of 0.2 ml 500μg/ml ovalbumin (OVA) and challenged by inhalation of nebulized 5% OVA solution to establish asthma models. In group A, normal saline of the equal volume was given instead of OVA. In group C and group E, each mouse was treated with two doses of 0.25 mg anti-CD25+ mAb– one dose was intravenously injected on the previous day before the OVA challenge, and another was intraperitoneally injected on the third day after the OVA challenge. Group D and group E were intravenously administered with MSCs on the tenth day after sensitization, while other groups were treated with PBS instead. The proportion of CD4+CD25+Treg in peripheral blood was detected by Flow Cytometry. The number of total cells, eosinophils, lymphocytes and neutrophils in BALF was counted to analyze the degree of airway inflammation together with pathological sections.Results:The proportion of CD4+CD25+Tregs in peripheral blood was (5.86±0.48)% in group A, (3.60±0.41)% in group B, (0.32±0.11)% in group C, (4.72± 0.23)% in group D and(0.33±0.11)% in group E. The proportion was significantly lower in group B than that in group A(P<0.01), as well as in group C than that in group B (P<0.01). The number of total cells and eosinophils in BALF was(4.93±1.68)×104 /ml and(0.03±0.01)×104 /ml in group A(,62.84±9.67)×104 /ml and(18.93±3.40)×104 /ml in group B(,80.59±11.34)×104 /ml and (29.26±5.43)×104 /ml in group C,(27.68±4.92)×104 /ml and (4.87±0.55)×104 /ml in group D, and (29.59±6.67)×104 /ml and (5.51±1.75)×104 /ml in group E. The proportion was significantly different when group A was compared with other groups(P<0.01), and the difference was still significant among group B,C,D and E(P<0.01). The pulmonary pathological score of the group A was 0(0～1), group B was 3(2～4), group C was 4 (3～4),group D was 2(1～3) and group E was 2 (1～3). There was significant difference among the scores of the five groups(H=32.527,P<0.01). The score was significantly lower when group A was compared with other groups(P<0.01),when group D and group E with group B and group C, and when group B with group C (P<0.05). There was no significant difference between the scores of group D and group E(P>0.05).Conclusions : The asthmatic BALB/c mice have more severe airway inflammation when CD4+CD25+Treg are depleted from peripheral blood. MSCs can up-regulate the CD4+CD25+Treg and thus inhibit the airway inflammation in asthmatic BALB/c mice. This effect is weakened in the mice depleted of CD4+CD25+Treg, compared with those not depleted, even though the difference is not significant. These findings suggest that low level of CD4+CD25+Treg may be an important mechanism of the airway inflammation in asthmatic mice. Increasing the level of CD4+CD25+Treg is just one of the mechanisms of MSCs in alleviating the airway inflammation. Further studies are needed to understand other mechanisms.