The Immunoregulatory Mechanism Of Different Phenotypic Myeloid-derived Suppressor Cells On Airway Inflammation In Asthmatic Mice

Study BackgroundAsthma has become a serious chronic disease to threaten public health. The morbidity of bronchial asthma has greatly increased over recent years although the medical level and sanitary conditions have been improved. Quite a number of asthma patients have been uncontrolled even if they have received all treatments. Researchers have been working in search of a new and effective treatment of asthma. The pathogenesis of asthma is complex. Airway inflammation is both the central pathogenic feature and the principal clinical manifestation, which is responsible for airflow obstruction and bronchial hyperresponsiveness (BHR). It is well known that Th1/Tn2imbalance plays a key role in asthmatic pathogenesis. T helper cell type2(Th2) functions strengthened and T helper cell type1(Thl) functions inhibited has been regarded as an important pathogenesis of asthma through the Th2cytokines. Interleukin-4(IL-4), secreted by Th2, induces airway inflammation by activating eosinophils as well as promoting IgE secretion. The level of IL-4was found increased in bronchoalveolar lavage fluid and airway biopsy specimen of patients with asthma. IFN-gamma, secreted by Thl, inhibits IgE secretion. Th1/Th2imbalance can not explain all the pathogenesis of asthma. there are other immunological mechanisms involved in regulating the airway inflammation of asthma. It is confirmed that regulatory T cell (Treg) plays an important role in asthmatic pathogenesis, especially CD4+CD25+Foxp3+Treg. Treg cells are essential for inducing and maintaining immunological tolerance to foreign and self-antigens (including allergens). CD4+CD25+Foxp3+Treg is a subset of CD4+T lymphocytes, which can inhibit immune response and induces immune tolerance by secretion of inhibitory cytokines (IL-10, TGF-β and IL-35) or cell-cell contact. The surface markers of Treg are CD4+and CD25+. Foxp3is a special transcription factor, which is selectively expressed by Tregs. Foxp3is a specific surface marker for Treg, which has been identified as a key factor for the development and function of Treg. Depletion of CD4+CD25+Treg cells increased airway hyperresponsiveness in mice. There are fewer number of CD4+CD25+Foxp3+Treg cells in patients with moderate-to-severe asthma compared with those of mild patients and healthy controls. CD4+CD25+Foxp3+Treg cells could prevent development of airway hypersecretion of mucus, smooth muscle hypertrophy and collagen synthesis. Treg is very important to control airway inflammation of asthma. Treg cells are apparently altered in number and function in allergic asthmatic patients.Myeloid-derived suppressor cells (MDSCs) are a heterogeneous population of cells of myeloid origin with strong immunosupression function. MDSCs are consist of myeloid progenitors and immature macrophages, immature granulocytes and immature dendritic cells. MDSCs have a remarkable ability to suppress T-cell responses by specificity and non-specificity of antigen. MDSCs are now becoming a hot area of immunotherapy because it can be used for autograft and allograft transplantation. It was confirmed that MDSCs obtained good treatment effects by adoptive transfer in many immune diseases, such as asthma, skin transplantation, inflammatory bowel disease, type1diabetes and chronic eczema in many studies.In mice, MDSCs are characterized by the co-expression of the myeloid lineage differentiation antigen Grl and CD11b. Antigen Grl is divided into antigen Ly6C and Ly6G. MDSCs in mice are divided into four phenotypes accordingly: CD11b+Ly6C+Ly6G-, CD11b+Ly6C-Ly6G+, CD11b+Ly6C+Ly6G+and CDllb+Ly6C-Ly6G-. Four phenotypic MDSCs are different in cell morphology, metabolite, proliferation effect, distribution in vivo, cytokines and immune response, which result in different effects in inflammation, tumour, infection and autoimmune diseases.MDSCs may induce proliferation of Treg cells by promoting secretion of IL-10, TGF-β, INF-γ to suppress T-cell responses. MDSCs that are triggered by IFN-γ and IL-10secrete plenty of stem cell factor, which induces proliferation of Treg cells. Proliferation of Treg can be inhibited through inhibiting stem cell factor in MDSCs. Immunosuppressive functions of MDSCs are associated with secretion of arginase1(ARG1) and inducible nitric oxide synthase (iNOS) in microenvironment. MDSCs express high levels of ARG1and iNOS. ARG1converts L-arginine into L-ornithine and urea. It leads to enhanced L-arginine catabolism, which depletes L-arginine from the microenvironment. The shortage of L-arginine inhibits T cell proliferation through decreasing CD3-ζ synthesis, inhibiting IL-2and IFN-γ synthesis, preventing the expression of T cell receptors. In addition, ARG1and iNOS induce apoptosis of T cells. iNOS can lead to an increase in the production of nitric oxide (NO). NO suppresses T-cell function through inhibiting proliferation of T cells, down-regulating the levels of IL-2, IFN-γ and IL-13, inhibiting MHC class Ⅱ expression. ARG1and iNOS plays important roles in suppresive functions of MDSCs.Many cytokines are involed in MDSC proliferation, including signal transducer and activator of transcription3(STAT3). STAT3is the main transcription factor that regulates the proliferation of MDSCs. Activation of STAT3is associated with the proliferation of MDSCs, while inhibition of STAT3expression abrogate MDSCs proliferation. STAT3promotes MDSC proliferation through inducing high expression of S100A8and S100A9proteins and inhibiting differentiation of dendritic cells. Ablating of STAT3proliferation in conditional knockout mice or selective STAT3inhibiting markedly reduced the proliferation of MDSCs. STAT3can be used as a biomarker of MDSCs proliferation. It has been widely recognized that nuclear factor-κB (NF-κB) plays a key role in MDSCs functions recently. NF-κB is an important cytokine in MDSCs activation. Suppresive activities of MDSCs is dependent on the myeloid differentiation primary-response gene88(MyD88) activated by TLRs and NF-κB activated by MyD88, which up-regulate expression of ARGland iNOS.MDSCs activation is inhibited by blocking NF-κB. NF-κB can be used as a biomarker of MDSCs activation.Many questions of MDSC research field are still unknown, such as the effect of different phenotypic MDSCs on airway inflammation of asthma, regulatory effect of different phenotypes MDSCs on Thl/Th2and CD4+CD25+Foxp3+Treg, correlation between MDSCs and ARG1, iNOS, STAT3, NF-Kβ. The Problems above mentioned remain to be elucidated.Early experiment of our research group confirmed that MDSCs stimulated by LPS gathered in BALB/c mice spleen, up-regulated CD4+CD25+Foxp3+Treg in peripheral blood, decreased the levels of IL-13in BALF and serum and reduced airway inflammation after transferring these cells into asthmatic C57BL/6mice. On this basis, we plan to separate and purify four phenotype MDSCs(Ly6C+Ly6G+, Ly6C-Ly6G+, Ly6C+Ly6G-and Ly6C-Ly6G-) which gather in BALB/c mice spleen induced by LPS in this study. Four phenotype MDSCs are adoptively transferred separately into asthmatic C57BL/6mice to observe the effect of different phenotypes MDSCs on airway inflammation of asthma, regulatory effect of different phenotypes MDSCs on Th1/Th2and CD4+CD25+Foxp3+Treg, correlation between MDSCs and ARG1, iNOS, STAT3, NF-Kβ.The study is to explore immunoregulatory mechanism of MDSCs on airway inflammation of asthma and suggest a potential MDSC-based therapeutic strategy for asthma.Part one Induction of myeloid-derived suppressor cells (MDSCs) induced by different doses lipopolysaccharidesObject:To explore the optimal dose of lipopolysaccharides(LPS) induced myeloid-derived suppressor cells(MDSCs) and optimal organ separated MDSCs in mice.Methods:A total of24BALB/c mice were randomly divided into the lng LPS group,100ng LPS group,10μg LPS group and the normal control group. Mice were intraperitoneally injected with LPS and normal saline solution respectively to induce accumulation of MDSCs in spleen and bone marrow. The number of MDSCs and proportions of four phenotypic MDSCs in spleen and bone marrow were measured by flow cytometry.Results:1. Four phenotypic MDSCs in spleen measured by flow cytometry:The percentages of Ly6C+Ly6G+, Ly6C-Ly6G+, Ly6C+Ly6G-Ly6C-Ly6G-and CD11b+MDSCs in the normal control group were60.97±3.15,3.31±0.63,15.30±3.13,22.87±1.69,3.30±0.57, respectively; The percentages of MDSCs in1ng LPS group were53.55±5.23,5.76±0.66,26.17±1.38,21.52±1.38,9.03±1.19, respectively; The percentages of MDSCs in100ng LPS group were55.39±4.87,9.81±1.03,26.95±3.04,20.64±1.22,11.96±0.67, respectively; The percentages of MDSCs in10μg LPS group were70.58±4.20,2.96±0.27,11.63±1.19,16.56±3.06,7.47±1.46, respectively. Compared with the control group, the number of CD11b+MDSCs in1ng LPS group,100ng LPS group and10μg LPS group significantly increased (P=0.000, P=0.0000, P=0.000). The number of CD11b+MDSCs in100ng LPS group significantly increased compared with that of1ng LPS group and10μg LPS group (P=0.000, P=0.000).2. Four phenotypic MDSCs in bone marrow measured by flow cytometry:The percentages of Ly6C+Ly6G+, Ly6C-Ly6G+, Ly6C+Ly6G-, Ly6C-Ly6G-and CD11b+MDSCs in the normal control group were96.43±0.55,0.26±0.04,1.52±0.04,1.45±0.22,43.84±2.73, respectively; The percentages of MDSCs in1ng LPS group were96.45±0.85,0.71±0.53,1.00±0.19,1.67±0.48,55.35±2.99, respectively; The percentages of MDSCs in100ng LPS group were97.01±0.21,0.53±0.07,1.41±0.04,1.01±0.14,58.33±2.84, respectively; The percentages of MDSCs in10μg LPS group were93.31±2.40,0.76±0.04,1.51±0.11,4.63±2.08,48.31±1.04, respectively. Compared with the control group, the number of CDllb+MDSCs in1ng LPS group,100ng LPS group and10μg LPS group significantly increased(P=0.000, P=0.000, P=0.006). The number of CD11b+MDSCs in100ng LPS group significantly increased compared with that of10μg LPS group (P=0.000).3. Most of the MDSCs in the bone marrow are Ly6C+Ly6G+MDSCs. The proportions of Ly6C+Ly6G-, Ly6C-Ly6G+and Ly6C-Ly6G-MDSCs in spleen were much more than those in bone marrow.Conclusion:1. LPS which were intraperitoneally injected could induce the accumulation of MDSCs in normal mice’s spleen and bone marrow.2. The optimal dose of LPS induced MDSCs is100ng and optimal organ separated MDSCs in mice is spleen. Part two Effects of four phenotypic myeloid-derived suppressor cells on Th1/Th2and CD4+CD25+Foxp3+regulatory T cells in vitroObject:To observe the effects of four phenotypic myeloid-derived suppressor cells on Thl/Th2and CD4+CD25+Foxp3+regulatory T cells in vitro.Methods:A total of24BALB/c mice were randomly divided into the LPS group and the in-vitro cell culture group. Mice in LPS group were intraperitoneally injected with100ng LPS to induce accumulation of MDSCs in spleen. MDSCs were separated by flow cytometry to obtain four phenotypic MDSCs:Ly6C+Ly6G+, Ly6C-Ly6G+, Ly6C+Ly6G-and Ly6C-Ly6G-MDSCs. Four phenotypic MDSCs and T cells of mice were co-cultured in vitro. The proportions of Thl/Th2and CD4+CD25+Foxp3+regulatory T cells were measured by flow cytometry.Results:1. The percentages of Thl/Th2in vitro measured by flow cytometry:Compared with the control group, Thl/Th2in Ly6C+Ly6G+group significantly increased(P=0.001); Compared with the control group, Thl/Th2differences were not statistically significant in Ly6C-Ly6G+group, Ly6C+Ly6G-group, Ly6C-Ly6G-group, CD11b+MDSCs group (P=0.975, P=1.000, P=1.000, P=0.976).2. The percentages of CD4+CD25+Foxp3+Treg in vitro measured by flow cytometry:Compared with the control group, the percentages of CD4+CD25+Foxp3+Treg in Ly6C+Ly6G+group, Ly6C-Ly6G+group, Ly6C+Ly6G-group, Ly6C-Ly6G-group, CDllb+MDSCs group significantly increased (P=0.000, P=0.000, P=0.001, P=0.000, P=0.003); Compared with Ly6C+Ly6G+group, the percentages of CD4+CD25+Foxp3+Treg in Ly6C Ly6G+group, Ly6C+Ly6G group, Ly6C-Ly6G-group, CDllb+MDSCs group significantly decreased (P=0.007, P=0.000, P=0.000, P=0.000).Conclusion:1.Ly6C+Ly6G+MDSCs up-regulate Thl/Th2, while Ly6C-Ly6G+, Ly6C+Ly6G-Ly6C-Ly6G-, CDl11b+MDSCs have no significant effect on Th1/Th2in vitro.2.Ly6C+Ly6G+, Ly6C-Ly6G+, Ly6C+Ly6G-, Ly6C-Ly6G-, CD11b+MDSCs can induce proliferation and increase number of CD4+CD25+Foxp3+Treg in vitro, especially Ly6C+Ly6G+MDSCs. Part three Effects of four phenotypic myeloid-derived suppressor cells on airway inflammation, Thl/Th2and CD4+CD25+Foxp3+regulatory T cells in asthmatic miceObject:To study the effects of different phenotypic MDSCs which were adoptive transferred into asthmatic mice on airway inflammation, Thl/Th2and CD4+CD25+Foxp3+regulatory T cells.Methods:52female BALB/c mice were selected for this study.10mice were injected intraperitoneally with LPS for preparing MDSCs and the method was described in part one.42mice were randomly divided into the normal control group, asthmatic group, Ly6C+Ly6G+MDSCs treatment group, Ly6C-Ly6G+MDSCs treatment group, Ly6C+Ly6G-MDSCs treatment group, Ly6C-Ly6G-MDSCs treatment group, CDllb+MDSCs treatment group. Mice in asthmatic and treatment group were sensitized with ovalbumin (OVA) by a combination of intraperitoneal injection and aerosol inhalation to establish the mice asthma model. Mice in treatment group were injected intravenously with different phenotypic MDSCs by adoptive transfer. Peripheral blood, bronchoalveolar lavage fluid (BALF) and lung specimens were gathered. Lung function of mice were measured with noninvasive pneumatometer. Pathology of lung specimens were observed with microscope. Inflammatory cell numbers were counted in BALF. The levels of interleukin-4in serum and BALF were measured by enzyme-linked immune-osorbent assay (ELISA). The proportions of Th1/Th2and CD4+CD25+Foxp3+regulatory T cells in peripheral blood were measured by flow cytometry.Results:1. The identification of asthmatic mice:The symptom of mice during an acute asthmatic attack, lung functions, the numbers of inflammatory cells in BALF and pathology of lung specimens indicated that the mice asthma models were successfully established.2. Lung function:when methacholine concentration was6.25mg/ml、12.5mg/ml、25mg/ml�'�50mg/ml, Penh of asthmatic group significantly increased compared with the control group (P=0.000, P=0.000, P=0.000, P=0.000); compared with the asthmatic group, Penh of Ly6C+Ly6G+group, Ly6C-Ly6G+group, CD11b+MDSCs group significantly decreased(P<0.05); Penh differences were not statistically significant in Ly6C+Ly6G-group and Ly6C-Ly6G-group compared with the asthmatic group (P>0.05).3. Cell counting in BALF:Compared with the control group, the number of total cells, and the proportions of eosinophils and neutrophils of the asthmatic group significantly increased (P=0.000,P=0.000, P=0.000); compared with the asthmatic group, the number of total cells of Ly6C+Ly6G+group, Ly6C-Ly6G+group, CD11b+MDSCs group significantly decreased(P=0.004, P=0.006, P=0.009), the proportions of eosinophils significantly decreased(P=0.000, P=0.000, P=0.000); the proportions of neutrophils significantly decreased (P=0.000, P=0.000, P=0.000); the proportions of eosinophils and neutrophils differences were not statistically significant in Ly6C+Ly6G-group and Ly6C-Ly6G-group compared with the asthmatic group(P>0.05). the lymphocytes differences were not statistically significant among seven groups(P>0.05).4. Pulmonary pathology:The lungs of the control mice displayed a clear airway structure without obvious inflammatory cells infiltration, no effusion in pulmonary alveolus. Massive inflammatory cells infiltration was present in the lungs from the asthmatic mice, Swelling of the bronchial mucosa, alveolar wall thickening, much effusion in pulmonary alveolus, goblet cell hypertrophy, mucus hypersecretion. Compared with the asthmatic group, inflammatory cells of Ly6C+Ly6G+group, Ly6C-Ly6G+group, CDllb+MDSCs group were reduced and pathologies were improved. Pulmonary pathologies of Ly6C+Ly6G-group and Ly6C-Ly6G-group are similar to those of asthmatic group.5. The levels of IL-4in serum and BALF:Compared with the control groups, the levels of IL-4in serum and BALF of asthmatic group significantly increased(P=0.000, P=0.000); compared with the asthmatic group, the levels of IL-4in serum of Ly6C+Ly6G+group, Ly6C-Ly6G+group, CDllb+MDSCs group significantly decreased(P=0.000, P=0.000, P=0.000), the levels of IL-4in BALF significantly decreased(P=0.000, P=0.000, P=0.000); the IL-4differences in serum and BALF were not statistically significant in Ly6C+Ly6G-group and Ly6C-Ly6G-group compared with the asthmatic group(P>0.05).6. Th1/Th2in peripheral blood of mice measured by flow cytometry:Compared with the control groups, Th1/Th2in asthmatic group significantly decreased(P=0.001); compared with the asthmatic group, Th1/Th2in Ly6C+Ly6G+group, Ly6C-Ly6G+group, Ly6C+Ly6G-group, Ly6C Ly6G-group, CD11b+MDSCs group significantly increased (P=0.002, P=0.004, P=0.001, P=0.001, P=0.019).7. Treg cells in peripheral blood of mice measured by flow cytometry:Compared with the control groups, the percentages of CD4+CD25+Foxp3+Treg in asthmatic group significantly decreased(P=0.000); compared with the asthmatic group, the percentages of CD4+CD25+Foxp3+Treg in Ly6C+Ly6G+group, Ly6C-Ly6G+group, Ly6C+Ly6G-group, Ly6C-Ly6G-group, CDllb+MDSCs group significantly increased (P=0.002, P=0.012, P=0.026, P=0.000, P=0.001).Conclusion:1.Ly6C+Ly6G+, Ly6C-Ly6G+and CDllb+MDSCs can suppress airway inflammation after different phenotypic MDSCs are adoptively transferred into asthmatic mice:①reduce airway responsiveness of asthmatic mice;②decrease the number of eosinophils and neutrophils in BALF;③reduce the levels of IL-4in serum and BALF;④alleviate the airway inflammation and improve pulmonary pathology.2. Ly6C+Ly6G-and Ly6C-Ly6G-MDSCs have no significant effects on airway inflammation, airway responsiveness, the number of eosinophils and neutrophils, pulmonary pathology, the levels of IL-4in serum and BALF.3. Ly6C+Ly6G+, Ly6C-Ly6G+and CD11b+MDSCs may play a role in immunosuppressive activity and asthma treatment by means of upregulating Thl/Th2and CD4+CD25+Foxp3+Treg in peripheral blood. Part four Immunoregulatory mechanism of different phenotype myeloid-derived suppressor cells on airway inflammation in asthmatic miceObject:To explore the immunoregulatory mechanism of different phenotype myeloid-derived suppressor cells on airway inflammation in asthmatic mice.Methods:The mice were laboratory mice in part three. NO in BALF were measured by ELISA. The content of ARG1and iNOS were detected by Real-time Quantitative PCR Detecting System(QPCR). The content of ARG1, iNOS, stat3and NF-κB in lungs of mice were detected by immunohistochemistry (IHC).Results:1. The results of NO in BALF:Compared with the control group, content of NO in BALF of asthmatic group significantly increased(P=0.002); Content of NO in Ly6C+Ly6G+group, Ly6C-Ly6G+group, Ly6C+Ly6G-group, Ly6C-Ly6G-group, CD11b+MDSCs group were zero.2. The contents of ARG1and iNOS detected by QPCR:Content of ARG1from high to low in turn:Ly6C-Ly6G+group, CD11b+MDSCs group, Ly6C+Ly6G+group, Ly6C+Ly6G-group, Ly6C-Ly6G-group; Content of iNOS is the highest in Ly6C+Ly6G+group and then Ly6C+Ly6G-group in the second place. Content of iNOS in Ly6C-Ly6G+group, Ly6C-Ly6G-group and CDllb+MDSCs group were zero.3. ARG1in lungs of mice detected by IHC:Much ARG1was expressed in asthmatic group while ARG1was not expressed in the control group. The expression of ARG1from high to low in turn:Ly6C-Ly6G+group, CD11b+MDSCs group, Ly6C+Ly6G+group, Ly6C+Ly6G-group. ARG1was not expressed in Ly6C-Ly6G-group.4. iNOS in lungs of mice detected by IHC:Much iNOS was expressed in asthmatic group while iNOS was not expressed in the control group. iNOS was not expressed in all other groups.5. STAT3in lungs of mice detected by IHC:STAT3was not expressed in all groups.6. NF-κB in lungs of mice detected by IHC:Much NF-kB was expressed in asthmatic group while NF-κB was not expressed in the control group. NF-κB was expressed in different phenotypic MDSCs. The expression of NF-κB from high to low in turn:Ly6C-Ly6G+group, Ly6C+Ly6G+group, CDllb+MDSCs group, Ly6C+Ly6G-group, Ly6C-Ly6G-group.Conclusion:1. Immunosuppressive functions of different phenotypic MDSCs are associated with secretion of ARG1. ARG1in Ly6C-Ly6G+group is the highest.2. Different phenotypic MDSCs by adoptive transfer into asthmatic mice are activated through NF-κB while they are not proliferated through STAT3.