骨髓单核系、粒系来源的髓系抑制细胞的分离及鉴定
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摘要
目的:由于缺乏有效的分离方法,目前对于髓系来源的免疫抑制细胞(Myeloid derived suppressor cells, MDSCs)的研究均停留在混合细胞水平。本实验旨在利用Gfi1:GFP基因敲入小鼠感染性休克模型,采用流式细胞分选技术分离骨髓单核系和粒系来源的MDSCs,并对其表型和体外免疫抑制功能加以鉴定。
方法:1)感染性休克小鼠模型的构建:利用LPS连续腹腔注射方法构建Gfi1:GFP基因敲入小鼠感染性休克模型,分别于注射后第4天、第7天检测模型小鼠血浆细胞因子分泌情况,以鉴定造模是否成功;2)骨髓单核系、粒系来源的MDSCs的分离:采用流式细胞分选技术,分离感染性休克小鼠模型骨髓中单核来源的MDSCs (CD11b~+Gr1~(med) GFP~-)和粒系来源的MDSCs (CD11b~+Gr1~(med) GFP~+)两群细胞,并进行形态学鉴定;3)表型和功能鉴定:动态观察单核来源的MDSCs (CD11b~+Gr1~(med) GFP~-)和粒系来源的MDSCs (CD11b~+Gr1~(med) GFP~+)两群细胞在模型小鼠骨髓细胞中的比例变化和表型差异,并利用体外共培养技术,将分离纯化的单核系和粒系来源的MDSCs亚群分别与CFSE标记的活化CD4+T细胞共培养,体外检测二者对CD4+T细胞增殖的抑制功能,同时采用real-time PCR方法检测二者促炎因子IFN-γ和抑炎因子IL-4、IL-10、IL-13、TGF-β等细胞因子的表达情况,以及发挥免疫抑制作用所需的精氨酸酶(ArginaseⅠ,ArgⅠ)和一氧化氮合酶2(nitric oxide synthetase2, NOS2)的表达水平。
结果:1)与正常对照小鼠相比,感染急性期(LPS腹腔注射24小时)模型小鼠外周血各种促炎、抑炎以及趋化因子均明显增高;与急性感染模型小鼠相比,感染性休克小鼠模型(LPS连续腹腔注射7天)外周血血浆中各种促炎因子TNF-α、IL-6、IFN-γ、趋化因子MCP-1水平明显降低,而抑炎因子IL-10的水平略有下降,但仍明显高于正常对照小鼠;2)利用Gfi1:GFP转基因小鼠,根据Gfi1基因在单核系、粒系来源的MDSCs中的差异表达,可分选出纯度大于99%的骨髓单核系来源的MDSCs(其表型为CD11b~+Gr1~(med) GFP~-)和粒系来源的MDSCs(其表型为CD11b~+Gr1~(med) GFP~+),经Wright-Giemsa染色形态学鉴定, CD11b~+Gr1~(med) GFP+细胞主要为以中晚幼粒细胞为主的幼稚粒细胞;而CD11b~+Gr1~(med) GFP-细胞主要是幼稚单核细胞;3)感染性休克模型小鼠骨髓中CD11b~+的髓系细胞较正常小鼠明显增多,在这些增多的髓系细胞中,尤以粒系来源的MDSCs为主。与正常小鼠相比, LPS连续腹腔注射4天模型小鼠骨髓粒系来源的MDSCs细胞膜表面CD124(IL-4受体)、CD210(IL-10受体)、以及Toll样受体TLR-2和TLR-4的表达有所增加,CD62L表达有所下降,而CD80和CD86的表达则无明显差别;单核系来源的MDSCs细胞表面TLR-2表达减弱, CD124、CD210以及TLR-4表达增强,其他分子表达无显著差异; LPS连续腹腔注射7天模型小鼠骨髓粒系来源的MDSCs细胞膜表面CD124、CD210和TLR-2的表达有所增加,而CD80和CD86的表达无显著差异;单核系来源的MDSCs细胞表面TLR-2表达减弱,其他表型无明显差别;CD11b~+Gr1~(med) GFP~+与CD11b~+Gr1~(med) GFPˉ细胞均能不同程度地影响经CD3/28刺激活化的CD4+T淋巴细胞增殖,且该抑制作用随共培养体系中MDSCs比例的增加而呈现递增现象。粒系和单核系来源的骨髓MDSCs细胞均能降低CD4~+T细胞的增殖活性,且尤以粒系来源的MDSCs的增殖抑制作用显著;粒系来源的MDSCs(CD11b~+Gr1~(med) GFP~+)和单核系来源的MDSCs(CD11b~+Gr1~(med) GFP-)细胞亚群的IL-4、IL-10、IL-13、IFN-γ等细胞因子以及ArgⅠ、NOS2等酶的RNA水平表达均增强,说明两个细胞亚群均可通过分泌各种抑制性细胞因子以及这两种酶参与感染性休克免疫抑制。
结论:
1.利用LPS腹腔连续给药的方法,成功建立了感染性休克小鼠模型;
2.利用Gfi1:GFP基因敲入小鼠,采用流式细胞分选技术,可以获得高纯度单核系和粒系来源的骨髓MDSCs;
3.感染性休克小鼠模型中,骨髓MDSCs显著增高,且以粒系来源的MDSCs为主;
4.感染性休克模型小鼠骨髓粒系来源的MDSCs(CD11b~+Gr1~(med) GFP~+)和单核系来源的MDSCs(CD11b~+Gr1~(med) GFP-)两个细胞亚群均能抑制CD4~+T细胞增殖;且两群细胞均能上调抑炎因子IL-4、IL-10、IL-13和发挥免疫抑制作用的关键酶ArgI、NOS2的表达。
Objectives:
For lacking of the effective separation methods, most of the studys on MDSCs still keep in a mixed cellular levels. Our experiments were designed to isolate monocyte and granulocyte derived MDSCs (Myeloid-derived suppressor cells, MDSCs) from bone marrow of septic shock Gfi1: GFP knock-in mice, and to further identify their phenotypes and immune suppressive functions in vitro.
Methods:
1) We seted up Gfi1: GFP knock-in mice septic shock models through continually intraperitoneal injection of LPS (10mg/kg) and detect the concentration of plasma cytokines. 2) We isolate the monocyte(CD11b~+Gr1~(med) GFP-)and granulocyte (CD11b~+Gr1~(med) GFP~+) derived MDSCs from septic shock mice bone marrow by the technology of flow cytometry sorting system, and identified their morphology with Wright-Giemsa staining. 3) We observed the proportions and phenotypes of monocyte and granulocyte derived MDSCs in septic shock mice with FACS and identify their immunosuppressive functions with co-culturing of CFSE-labeled CD4+ T cells in different ratios. The expression of the pro-inflammatory, anti-inflammatory factors, ArginaseⅠ(ArgⅠ)and nitric oxide synthetase 2 ( NOS2) within the above MDSCs subsets were also determined with Realtime PCR.
Results:
1) Comparing with the normal mice, the concentration of the pro-inflammatory and anti-inflammation cytokines increased within the plasma of acute-phase infection (LPS intraperitoneal injection for 24 hours); the pro-inflammatory factors decreased significantly until LPS intraperitoneal injection for 7 days. Accordingly, the level of anti-inflammatory factor IL-10 reduced slightly but still much higher than that in normal controls. 2) Monocyte and granulocyte derived MDSCs can be separated with BD FACS Aria accoding to the different expression of transcriptional factor Gfi1. We have successfully isolated these two MDSC subsets from septic shock mice, with the phenotype of CD11b~+Gr1~(med) GFP~- for monocyte derived MDSC and CD11b~+Gr1~(med) GFP+ for granulocyte derived MDSCs respectively. The morphology analysis identified their lineage features. 3) The CD11b~+Gr1~(med) myeloid cells in septic shock bone marrows were increased obviously. The most important of all, the main part of the increased cells was granulocyte derived MDSCs. 4) Comparing with the normal control, the expression of CD124, CD210, TLR-2 and TLR-4 were up-regulated and CD62L was down-regulated in granulocyte derived MDSCs from septic shock models , no obvious changes of CD80 and CD86 were observed; the expression patterns in monocyte derived MDSCs were similar to the above, except for the slight down-regulation of TLR2. 5) The CD4+T cells proliferation suppression experiments indicated that both granulocyte and monocyte derived MDSCs possessed immunosuppressive functions in vivo. 6)The expression of anti-inflammatory factors IL-4、IL-10、IL-13 and key enzymes ArgⅠand NOS2 were elevated significantly in both MDSCs subsets. However, TGF-β1 expression was down-regulated slightly.
Conclusions:
1. We had successfully constructed a septic shock model with Gfi1: GFP knock-in mice (Gfi1GFP / +) and LPS intraperitoneal injection successively.
2. We have obtained highly purified monocyte and granulocyte derived MDSCs from bone marrow of septic shock Gfi1GFP / + mice
3. The proportions of MDSCs in septic shock mice elevated significantly, and most of which were granulocyte derived MDSCs.
4. Both monocyte and granulocyte derived MDSCs could suppress the proliferation of activated CD4+T cells and up-regulate the expression of anti-inflammatory factor IL-4, IL-10, IL-13 and enzymes ArgI、NOS2.
方法:1)感染性休克小鼠模型的构建:利用LPS连续腹腔注射方法构建Gfi1:GFP基因敲入小鼠感染性休克模型,分别于注射后第4天、第7天检测模型小鼠血浆细胞因子分泌情况,以鉴定造模是否成功;2)骨髓单核系、粒系来源的MDSCs的分离:采用流式细胞分选技术,分离感染性休克小鼠模型骨髓中单核来源的MDSCs (CD11b~+Gr1~(med) GFP~-)和粒系来源的MDSCs (CD11b~+Gr1~(med) GFP~+)两群细胞,并进行形态学鉴定;3)表型和功能鉴定:动态观察单核来源的MDSCs (CD11b~+Gr1~(med) GFP~-)和粒系来源的MDSCs (CD11b~+Gr1~(med) GFP~+)两群细胞在模型小鼠骨髓细胞中的比例变化和表型差异,并利用体外共培养技术,将分离纯化的单核系和粒系来源的MDSCs亚群分别与CFSE标记的活化CD4+T细胞共培养,体外检测二者对CD4+T细胞增殖的抑制功能,同时采用real-time PCR方法检测二者促炎因子IFN-γ和抑炎因子IL-4、IL-10、IL-13、TGF-β等细胞因子的表达情况,以及发挥免疫抑制作用所需的精氨酸酶(ArginaseⅠ,ArgⅠ)和一氧化氮合酶2(nitric oxide synthetase2, NOS2)的表达水平。
结果:1)与正常对照小鼠相比,感染急性期(LPS腹腔注射24小时)模型小鼠外周血各种促炎、抑炎以及趋化因子均明显增高;与急性感染模型小鼠相比,感染性休克小鼠模型(LPS连续腹腔注射7天)外周血血浆中各种促炎因子TNF-α、IL-6、IFN-γ、趋化因子MCP-1水平明显降低,而抑炎因子IL-10的水平略有下降,但仍明显高于正常对照小鼠;2)利用Gfi1:GFP转基因小鼠,根据Gfi1基因在单核系、粒系来源的MDSCs中的差异表达,可分选出纯度大于99%的骨髓单核系来源的MDSCs(其表型为CD11b~+Gr1~(med) GFP~-)和粒系来源的MDSCs(其表型为CD11b~+Gr1~(med) GFP~+),经Wright-Giemsa染色形态学鉴定, CD11b~+Gr1~(med) GFP+细胞主要为以中晚幼粒细胞为主的幼稚粒细胞;而CD11b~+Gr1~(med) GFP-细胞主要是幼稚单核细胞;3)感染性休克模型小鼠骨髓中CD11b~+的髓系细胞较正常小鼠明显增多,在这些增多的髓系细胞中,尤以粒系来源的MDSCs为主。与正常小鼠相比, LPS连续腹腔注射4天模型小鼠骨髓粒系来源的MDSCs细胞膜表面CD124(IL-4受体)、CD210(IL-10受体)、以及Toll样受体TLR-2和TLR-4的表达有所增加,CD62L表达有所下降,而CD80和CD86的表达则无明显差别;单核系来源的MDSCs细胞表面TLR-2表达减弱, CD124、CD210以及TLR-4表达增强,其他分子表达无显著差异; LPS连续腹腔注射7天模型小鼠骨髓粒系来源的MDSCs细胞膜表面CD124、CD210和TLR-2的表达有所增加,而CD80和CD86的表达无显著差异;单核系来源的MDSCs细胞表面TLR-2表达减弱,其他表型无明显差别;CD11b~+Gr1~(med) GFP~+与CD11b~+Gr1~(med) GFPˉ细胞均能不同程度地影响经CD3/28刺激活化的CD4+T淋巴细胞增殖,且该抑制作用随共培养体系中MDSCs比例的增加而呈现递增现象。粒系和单核系来源的骨髓MDSCs细胞均能降低CD4~+T细胞的增殖活性,且尤以粒系来源的MDSCs的增殖抑制作用显著;粒系来源的MDSCs(CD11b~+Gr1~(med) GFP~+)和单核系来源的MDSCs(CD11b~+Gr1~(med) GFP-)细胞亚群的IL-4、IL-10、IL-13、IFN-γ等细胞因子以及ArgⅠ、NOS2等酶的RNA水平表达均增强,说明两个细胞亚群均可通过分泌各种抑制性细胞因子以及这两种酶参与感染性休克免疫抑制。
结论:
1.利用LPS腹腔连续给药的方法,成功建立了感染性休克小鼠模型;
2.利用Gfi1:GFP基因敲入小鼠,采用流式细胞分选技术,可以获得高纯度单核系和粒系来源的骨髓MDSCs;
3.感染性休克小鼠模型中,骨髓MDSCs显著增高,且以粒系来源的MDSCs为主;
4.感染性休克模型小鼠骨髓粒系来源的MDSCs(CD11b~+Gr1~(med) GFP~+)和单核系来源的MDSCs(CD11b~+Gr1~(med) GFP-)两个细胞亚群均能抑制CD4~+T细胞增殖;且两群细胞均能上调抑炎因子IL-4、IL-10、IL-13和发挥免疫抑制作用的关键酶ArgI、NOS2的表达。
Objectives:
For lacking of the effective separation methods, most of the studys on MDSCs still keep in a mixed cellular levels. Our experiments were designed to isolate monocyte and granulocyte derived MDSCs (Myeloid-derived suppressor cells, MDSCs) from bone marrow of septic shock Gfi1: GFP knock-in mice, and to further identify their phenotypes and immune suppressive functions in vitro.
Methods:
1) We seted up Gfi1: GFP knock-in mice septic shock models through continually intraperitoneal injection of LPS (10mg/kg) and detect the concentration of plasma cytokines. 2) We isolate the monocyte(CD11b~+Gr1~(med) GFP-)and granulocyte (CD11b~+Gr1~(med) GFP~+) derived MDSCs from septic shock mice bone marrow by the technology of flow cytometry sorting system, and identified their morphology with Wright-Giemsa staining. 3) We observed the proportions and phenotypes of monocyte and granulocyte derived MDSCs in septic shock mice with FACS and identify their immunosuppressive functions with co-culturing of CFSE-labeled CD4+ T cells in different ratios. The expression of the pro-inflammatory, anti-inflammatory factors, ArginaseⅠ(ArgⅠ)and nitric oxide synthetase 2 ( NOS2) within the above MDSCs subsets were also determined with Realtime PCR.
Results:
1) Comparing with the normal mice, the concentration of the pro-inflammatory and anti-inflammation cytokines increased within the plasma of acute-phase infection (LPS intraperitoneal injection for 24 hours); the pro-inflammatory factors decreased significantly until LPS intraperitoneal injection for 7 days. Accordingly, the level of anti-inflammatory factor IL-10 reduced slightly but still much higher than that in normal controls. 2) Monocyte and granulocyte derived MDSCs can be separated with BD FACS Aria accoding to the different expression of transcriptional factor Gfi1. We have successfully isolated these two MDSC subsets from septic shock mice, with the phenotype of CD11b~+Gr1~(med) GFP~- for monocyte derived MDSC and CD11b~+Gr1~(med) GFP+ for granulocyte derived MDSCs respectively. The morphology analysis identified their lineage features. 3) The CD11b~+Gr1~(med) myeloid cells in septic shock bone marrows were increased obviously. The most important of all, the main part of the increased cells was granulocyte derived MDSCs. 4) Comparing with the normal control, the expression of CD124, CD210, TLR-2 and TLR-4 were up-regulated and CD62L was down-regulated in granulocyte derived MDSCs from septic shock models , no obvious changes of CD80 and CD86 were observed; the expression patterns in monocyte derived MDSCs were similar to the above, except for the slight down-regulation of TLR2. 5) The CD4+T cells proliferation suppression experiments indicated that both granulocyte and monocyte derived MDSCs possessed immunosuppressive functions in vivo. 6)The expression of anti-inflammatory factors IL-4、IL-10、IL-13 and key enzymes ArgⅠand NOS2 were elevated significantly in both MDSCs subsets. However, TGF-β1 expression was down-regulated slightly.
Conclusions:
1. We had successfully constructed a septic shock model with Gfi1: GFP knock-in mice (Gfi1GFP / +) and LPS intraperitoneal injection successively.
2. We have obtained highly purified monocyte and granulocyte derived MDSCs from bone marrow of septic shock Gfi1GFP / + mice
3. The proportions of MDSCs in septic shock mice elevated significantly, and most of which were granulocyte derived MDSCs.
4. Both monocyte and granulocyte derived MDSCs could suppress the proliferation of activated CD4+T cells and up-regulate the expression of anti-inflammatory factor IL-4, IL-10, IL-13 and enzymes ArgI、NOS2.
引文
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[38] Movahedi K,Guilliams M,Van den Bossche J,et a1.Identification of discrete tumor-induced myeloid-derived suppressor cell subpopulations with distinct T-cell suppressive activity[J].Blood,2008,111(8):4233-4244.
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[1] Ward E. Bullock, Elaine M. Carlson, et al. The evolution of immunosuppressive cell populations in experimental mycobacterial infection[J]. J.Immunol, 1978,120:1709-1716.
[2] Lisa P. Seung, Donald A. Rowley, et al. Synergy between T-cell immunity and inhibition of paracrine stimulation causes tumor rejection[J]. Proc.Natl.Acad.Sci.U.S.A, 1995,92:6254-6258.
[3] Matthew J.Delano, et al. MyD88-dependent expansion of an immature GR-1+CD11b+population induces T cell suppression and Th2 polarization in sepsis[J]. J. Exp. Med, 2007,204:1463-147.
[4] Dmitry I. Gabrilovich, et al. The Terminology Issue for Myeloid-Derive Suppressor Cells[J]. American Association for Cancer Research, 2007,67:425-426.
[5] Srinivas Nagaraj, Dmitry I. Gabrilovich, et al. Myeloid-derived suppressor cells as regulators of the immune system[J]. Nature, 2009,9:162-174.
[6] Augusto C. Ochoa, Arnold H.Zea, et al. Arginase, prostaglandins, and myeloid-derived suppressor cells in renal cell carcinoma[J]. Clin.CancerRes, 2007,13:721-726.
[7] Ilaria Marigo, Luigi Dolcetti, et al. Tumor-induced tolerance and immune suppression by myeloid derived suppressor cells[J]. Immunological Reviews,2008,222:162-179.
[8] Craig Murdoch, Munitta Muthana, et al. The role of myeloid cells in the promotion of tumour angiogenesis[J]. Nat.cancer, 2008,8:618-631.
[9] Paulo C. Rodriguez, et al. Arginase I–Producing Myeloid-Derived Suppressor Cells in Renal Cell Carcinoma Are a Subpopulation of Activated Granulocytes[J]. Cancer Res, 2009,69:1553-1560.
[10] Maciej M Markiewski, Robert A DeAngelis, et al. Modulation of the antitumor immune response by complement[J]. Nat. Immunol, 2008,9:1225-1235.
[11] Vincenzo Bronte, Paola Zanovello, et al. Regulation of immune responses.by L-arginine metabolism[J]. Nat. Imm, 2005,5:641-654.
[12] Je-In Youn, et al. Subsets of myeloid-derived suppressor cells in tumor-bearing mice[J]. J. Immunol, 2008,181:5791–5802.
[13] Antonio Sica, Vincenzo Bront , et al. Altered macrophage differentiation and immune dysfunction in tumor development[J]. J.Clin.Invest, 2007,117:1155-1166.
[14] Sarah Glennie, et al. Bone marrow mesenchymal stem cells induce division arrest anergy of activated T cells[J]. Blood, 2005,105:2821-2827.
[15] Hequan Li, et al. Cancer-expanded myeloid derived suppressor cells induce anergy of NK cells through membrane-bound TGF-β1[J]. J. Immunol, 2009,182:240-249.
[16] Cunren Liu, et al. Expansion of spleen myeloid suppressor cells represses NK cell cytotoxicity in tumor-bearing host[J]. Blood, 2007,109:4336-4342.
[17] Norman Nausch, et al. Monocyte myeloid-derived“suppressor”cells express RAE-1 and activate natural killer cells[J]. Blood, 2008,112:4080-4089.
[18] Xiao-Xia Jiang, et al. Human mesenchymal stem cells inhibit differentiation and function of monocyte-derived dendritic cells[J]. Blood, 2005,105:4120-4126.
[19] Suzanne Ostrand-Rosenberg, et al. Immune Surveillance: A Balance Between Pro- and Anti-tumor Immunity[J]. Curr Opin Genet Dev, 2008,18:11-18.
[20] Suzanne Ostrand-Rosenberg, Pratima Sinha, et al. Myeloid-Derived SuppressorCells: Linking Inflammation and Cancer[J]. The Journal of Immunology, 2009,182:4499-4506.
[21] Steffen Massberg, et al. Immunosurveillance by hematopoietic progenitor cells trafficking through blood, lymph, and peripheral tissues[J]. Cell, 2007,131:994-1008.
[22] Analía V. Ezernitchi, et al. TCR Down-Regulation under Chronic Inflammation Is Mediated by Myeloid Suppressor Cells Differentially Distributed between Various Lymphatic Organs[J]. Journal of immunology. 2006,177:4763-4772.
[23] Valeriya P. Makarenkova, et al. CD11b+/Gr-1+ myeloid suppressor cells cause T cell dysfunction after traumatic stress[J]. J. Immunol, 2006,176:2085-2094.
[24] Oscar Go?i, et al. Immunosuppression during acute Trypanosoma cruzi infection: involvement of Ly6G (Gr1+)CD11b+ immature myeloid suppressor cells[J]. Int. Immunol, 2002,14:1125-1134.
[25] Mathieu-Beno?t Voisin, et al. Both expansion of regulatory GR1+CD11b+ myeloid cells and anergy of T lymphocytes participate in hyporesponsiveness of the lungassociated immune system during acute toxoplasmosis[J]. Infect. Immun, 2004,72:5487-5492.
[26] Cord Sunderk?tter, et al. Subpopulations of mouse blood monocytes differ in maturation stage and inflammatory response[J]. J. Immunol, 2004,172:4410-4417.
[27] Lea Brys, et al. Reactive oxygen species and 12/15-lipoxygenase contribute to the antiproliferative capacity of alternatively activated myeloid cells elicited during helminth infection[J]. J. Immunol, 2005,174:6095-6104.