Effect Of LPS-induced Myeloid-derived Suppressor Cells On CD4⁺ Treg Cells And Airway Inflammation In Asthmatic Mice

Study BackgroundThe incidence of atopy and asthma has greatly increased over recent years,concomitant with an improved hygienic status in the industrialized world.The characteristics of bronchial asthma are reversible airflow obstruction and chronic airway inflammation.The etiology of asthma is complex,but it is well known that Th1/Th2 unbalanced plays a key role in asthmatic pathogenesis.The lymphocytes helper 2（Th2）functions strengthened and lymphocytes helper 1（Th1）functions inhibited has been regarded as an important pathogenesis of asthma.The level of IL-13 was found increased in bronchoalveolar lavage fluid of patients with asthma.The IL-13 can participate in the maintenance of the inflammation of bronchial asthma and induce airway hyperresponsiveness by activating eosinophils as well as promoting IgE secretion.But the important reasons of the Th1/Th2 imbalance is the damaged immunological tolerance to allergens in patients with asthma.Nearly,evidences that regulatory T cells（Treg）can induce immune reactivity against self-antigens and non-self-antigens by various mechanisms,which may play an important role in the pathogenesis and treatment of asthma.The expression of Foxp3-mRNA（Foxp3/beta-actin）and the levels of TGF-β in asthmatic children was significantly lower than that in the control children in both PBMC and induced sputum.There may be a serious skin rashes,asthma and food allergies in patients with X-associated autoimmune syndrome of allergic disorders with a lack of Treg cells.The recent study confirmed that BCG vaccinations inhibited de novo allergic inflammatory responsiveness in a mouse model of asthma,associated with the increase of CD4+CD25+Treg cells and Foxp3 expression,accompanied by an increased CTLA-4 expression and cytokine IL-10 and TGF-β levels.All of the evidences above showed that an increase of Treg cells may be an important way of prevention and treatment of asthma.CD4+CD25+Foxp3+regulatory T cells are a subset of CD4+T lymphocytes.The forkhead/winged helix transcription factor（Foxp3）is a specific surface marker for Treg cells,which has been identified as a key regulatory gene for the development and function of Treg.The other cells,such as CD4+CD25T cells,B cells and CD8+T cells,are expressing little or no Foxp3.Unlike the other cell surface markers used to identify Treg（e.g.,CD25,CD45RB,CTLA4,and GITR）,Foxp3 is not up-regulated on T-cell activation and thus discriminates Treg cells from activated effector T cells.Myeloid-derived suppressor cells（MDSCs）,a group of myeloid cells in the early differentiation stage,have a strong immune regulatory activities.The study showed that MDSCs exerted an immunosupperssive action by influencing the normal differentiation of dendritic cells,inhibiting NK cell innate immune response and the proliferation and killing ability of T cells directly.In recent years,myeloid-derived suppressor cells in the study of immunotherapy has been reported widely.Studies confirmed that MDSCs can be implanted into same models of animals,such as alopecia areata,inflammatory bowel disease,allograft,autoimmune encephalomyelitis,type 1 diabetes,in order to prevent the body from excessive immune response generated by the pathological damage.In Arora’s research revealed that the LPS-induced myeloid cells MDSCs can suppress the lever of IL-13 resulting in tempering the airway inflammation and airway hyperresponsiveness.Then in Huang’s research discovered that MDSCs are able to exerte the immunosupperssive action in tumor-bearing mice via inducing the development of Foxp3+T regulatory cells（Treg）in vivo by secreting IL-10,TGF-B,and arginase（TGF-β independ）.These all revealed that MDSCs have a special advantage in the cytotherapy.Although accumulating evidence suggests that Tregs and MDSCs are associated with tumor-mediated suppression,it has not been established whether MDSCs can upregulate CD4+CD25+Foxp3+Treg cells of asthmatic mice and improve its airway inflammation,and furthermore be used in asthmatic therapy.The phenotypes of MDSCs in mice is usually both CD11b and Gr-1,but they can also only expresse CD11b or Gr-1,which depended on the mouse strain or disease states.Studies confirmed the populations are play an important role in tumor-immune escape,but it is rarely reported that whether the CD11b+Gr-1+cells which induced by other ways also belong to the MDSCs subsets with immunosuppressive ability.Then in Arora’s research revealed that continual exposure to LPS induced the generation of a suppressive myeloid cell type that expressed CD11b,Gr-1 at intermediate levels,and F4/80,the combination of which distinguished it from neutrophils,macrophages,and dendritic cells（DCs）.The cells resembled previously described hematopoietic stem and progenitor cell-derived myeloid cells in specific tissues including the lung whose numbers were shown to expand in the presence of Toll-like receptor（TLR）agonists such as LPS.We design this experiment to confirm LPS-induced CD11b+Gr-1+myeloid cells are MDSCs described in study reports with immunosuppressive ability by analysising of phenotypes,morphology,and the ability of inhibiting the proliferation of T lymphocytes.We hypothesize that LPS induce the accumulation of MDSCs that not only can inhibit clonal expansion of activated effector T cells but also induce specific Treg to further establish and maintain T-cell tolerance in the asthmatic mice.We design this experiment to observe the influence of MDSCs which separated and purified in vitro to the peripheral blood CD4+CD25+Foxp3+Treg and the lever of IL-13 and the effect on airway inflammation in asthmatic mice,and furthermore be used in asthmatic therapy.Part one.Induction,isolation and identification of myeloid-derived suppressor cells（MDSCs）Object:To isolate the LPS-induced MDSCs with immunomagnetic beads and identity the cells in vitro.Method:BALB/c mice were randomly divided into the LPS group and the normal control group,and were injected intraperitoneally with LPS and normal saline solution respectively to induce accumulation of MDSCs in spleen.MDSCs were separated with CD11b immunomagnetic beads from the spleen extract of mice.The characteristic molecules on the cell surface were identified by flow cytometry and the morphological characterization of MDCSs was observed via Wright-Giemsa staining.Effect of MDSCs on the proliferation of T cells in vitro was determined by MTT method.Result:1.Purily of MDSCs in spleen tissues detected by flow cytometry:The proportion of MDSCs in the spleen of the LPS group was about（28.35±8.57）%,much more than that of the normal control group（6.25±2.73）%;t=5.489,P=0.003).The CD11b+Gr-1+MDSCs could be separated from the spleen of the mice injected with LPS by means of CD11b immunomagnetic beads at the high purity of 88.82%.2.The morphology of MDSCs:MDSCs were showing the presence of ring-shaped or lobular-shaped nuclei after Wright-Giemsa staining.3.MDSCs can inhibit the proliferation of T lymphocytes in spleen:MTT method showed that the proliferation of T cells were suppressed after co-cultivation with CD11b+MDSCs.Compared with the control group,the numerous A of the MDSCs groups decreased significantly and it had a positively correlation with the number of MDSCs（F=52.211,P=0.000）.All the results above showed that LPS-induced MDSCs had a dose-dependent inhibitory effect on the proliferation of the spleen T lymphocytes.Conclusion:1.LPS could cause the accumulation of CD11b+Gr-1+MDSCs in mice’s spleen in the short term.2.The high purity of LPS-induced myeloid-derived suppressor cells could be separated with CDllb immunomagnetic beads.LPS-induced CD11b+Gr1+myeloid cells are MDSCs described in study reports with immunosuppressive ability.Prat two Effect of LPS-induced myeloid-derived suppressor cells on CD4+regulatory T cells and airway inflammation in asthmatic miceObject:To study the effect of LPS-induced CD11b+Gr-1+MDSCs transplantation on the airway inflammation in asthmatic mice and to explore the role of MDSCs on improving the airway inflammation in asthmatic mice by upregulatation of CD4+CD25+Foxp3+Treg cells and inhibition of the Th2 response.Methods:Thirty-four female BALB/c mice were selected for this study.Of them,four mice were injected intraperitoneally with LPS for preparing MDSCs and the method was described in part one.Thirty mice were randomly divided into the normal control group,the asthmatic group and the cells treated group.Mice in asthmatic and the cells treated group were sensitized with ovalbumin（OVA）by a combination of intraperitoneal injection and repeated OVA solution challenges to establish an mouse asthma model.In the normal control group,normal saline of the equal volume was given instead of OVA.On the 14th and 21th day after sensitization,mice in the cells treated group were injected intravenously with LPS-induced MDSCs which separated with immunomagneticbeads.Twenty-four hours after the last allergen challenge,inflammatory cell numbers and morphological identification of leucocytes in BALF were measured to analyze the degree of inflammation of the airway together with pathological section.The levels of T-helper 2（Th2）cytokine interleukin-13 in mice bronchoalve-olar lavage fluid（BALF）and serum were measured by enzymelinked immune-osorbent assay（ELISA）.The number of CD4+CD25+Foxp3+regulatory T cells in peripheral blood was detected by flow cytometry.Results:1.The behavior changes of mice after sensitizationg and rechallenge:Mice in asthmatic group displayed various types of allergic responses,such as scratching head and face,dyspnea,oral lip cyanosis,active decreased as well as gatism,while the control group showed no such symptoms.2.The identification of asthmatic mice:The manifestation of mice during an acute asthmatic attack,the change of BALF tatal cell numbers and differentials,and the pulmonary pathology all indicated that the mice model was successfully established.3.Cell population of BALF:Compared with the control group,the number of total cells,and the percentage of neutrophils and eosinophils of BALF in the asthmatic groups significantly increased（P=0.000,P=0.000,P=0.020）;The number of total cells,the percentage of neutrophils and eosinophils of BALF in MDSCs treated group decreased compared with the asthmatic groups（P=0.002,P=0.000,P=0.033）.4.Pulmonary pathology:The lungs of the control mice displayed a clear airway structure without any significant inflammatory cell infiltration.Massive inflammatory infiltration was present in the lungs from the asthmatic mice,alveolar wall thickening,epithelial damage,structural disorder,goblet cell hypertrophy,mucus hypersecretio.Compared with the asthmatic group,there were significantly tempered inflammation in the cells treated group.5.The results of IL-13 in serum and BALF:Compared with the control groups,the lever of IL-13 of serum and BALF in asthmatic groups significantly higher（P=0.000,P=0.000）which significantly lower in the MDSCs treated groups（P=0.000,P=0.000）.6.The number of Treg cells in peripheral blood was detected by flow cytometry:The proportion of CD4+CD25+Foxp3+Tregs accounte for CD4+T lymphocytes of peripheral blood in asthmatic mice significantly lower compared with the control groups.In the cells treated groups,the proportion was significantly higher than that in the asthmatic groups.There was significant difference between the groups（F=17.682,P=0.000）.Conclusions:Adoptively transferred LPS-induced CD11b+Gr-1+myeloid-derived suppressor cells may improve airway inflammation by means of upregulating CD4+CD25+Foxp3+Treg of peripheral blood and reducing the level of IL-13 in BALF and serum in asthmatic mice.That just one of the mechanisms of MDSCs in alleviating the airway inflammation,further studies should be started to do to understand other mechanisms.Therefore,to explore appropriate strategies to induce the accumulation and immunosuppressive function enhanced of MDSCs is expected to become a new strategy on prevention and treatment of clinical allergic asthma.