Isolation And Identification Of Monocyte And Granulocyte Derived Myeloid-derived Suppressor Cells From Bone Marrow Of Sepsis Mice

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, Arginaseâ… (Argâ… )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 Argâ… and 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 / + mice3. 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.