Effects Of TLR4-ligands Lipopolysaccharide And Paclitaxel On CD11b~+Gr-1~+MDSCs Derived From The Spleen Of Lewis Lung Carcinoma Bearing Mice

Background and purposesImmunotherapy in cancer treatment was gradually rising, but its clinical effect was unsatisfactory partly because of tumor-induced immune tolerance. Researches showed that the generation of immune tolerance was associated with accumulation of MDSCs. MDSCs were a subset of immunosuppressive cells, which could inhibit immuno-functions through several manners, including the expression of immunosuppressive enzymes. Once dysdifferentiation and accumulation of MDSCs happened in tumor microenvironment, it would be detrimental to anti-tumor immunity. Currently, studies on the regulation of MDSCs generation, differentiation and function were becoming the focus of tumor immunology filed.Toll like receptor 4 (TLR4) was an important immune molecule, which could recognize Lipopolysaccharide (LPS), a component of the outer membrane of bacteria. TLR4 mainly located in the surface of myeloid cells, which could regulate immuno-functions of myeloid cells. Recently, TLR4 was aslo found on the surface of MDSCs from 4T1 mammary adenocarcinoma bearing mice, and which was possibly involved in the regulation of MDSCs generation, differentiation and function. Paclitaxel was a newly identified potential TLR4 ligand, which might affect anti-tumor immunity by regulating TLR4-expressing immunocytes and this process had been confirmed by studies on dendritic cell and macrophages. Then paclitaxel whether could affect anti-tumor immunity via regulating TLR4-expressing MDSCs? Moreover, the same as the TLR4 ligand, whether there were different effects between paclitaxel and LPS on the MDSCs? To understand these questions, we identified and sorted MDSCs from Lewis lung carcinoma bearing mice, detected the expression of TLR4 on such MDSCs, then investigated the changes in MDSCs apoptosis, differentiation and immune function after treatment with LPS or paclitaxel. The findings of our research would be conducive to clarity the relationship between TLR4 and MDSCs, and offered a new perspective for anti-tumor immunity function of paclitaxel.Methods1. Fluorescence conjugated monoclonal antibodies and flow cytometry were used to detect the expression of surface markers CD11b and Gr-1, differentiation antigens CD11c and F4/80, major histocompatibility complex-Ⅱ, costimulatory molecules CD86 on the splenic mononuclear cells of Lewis lung carcinoma bearing mice or normal mouse. (Identify the phenotype of MDSCs).2. Sorted the MDSCs from splenic mononuclear cells of Lewis lung carcinoma bearing mice by utilizing immunomagnetic beads.3. Set up mixed lymphocyte reaction (MLR) to assay the immunosuppression function of the sorted MDSCs in vitro.4. RT-PCR detected the TLR4 gene expression in MDSCs; FCM detected the TLR4 protein expression on the MDSCs surface.5. Annexin V and PI staining to detect the apoptosis of MDSCs after treatment with LPS or paclitaxel; FCM detected the alteration of the percentage of MDSCs in splenic mononuclear cells of Lewis lung carcinoma bearing mice after intraperitoneal injection of paclitaxel.6. FCM detected the expression of MHC-Ⅱand CD86 on the surface of MDSCs after treatment with LPS or paclitaxel.7. MLR detected immunosuppression function of MDSCs after treatment with LPS or paclitaxel.8. Western Blot detected the expression of immunosuppressive arginasel (ARG1) in MDSCs.Results1. CDllb/Gr-1 double positive cells increased in the Spleen of Lewis lung carcinoma bearing mice, which accounting for (42.53±6.26)% of splenic mononuclear cells. The expression levels of CD11c, F4/80, MHC-Ⅱand CD86 on such double positive cells were lower in Lewis lung carcinoma bearing mice compare with nomal mice ((11.67±3.01)% vs. (24.67±4.02)%, p=0.01; (14.33±3.33)% vs. (27.00±4.21)%, p=0.01; (8.66±2.33)% vs. (30.00±3.51)%, p=0.001; (14.67±3.76)% vs. (32.00±4.73)%,p=0.007, respectively) 2. Used CD11b immunomagnetic beads to sort CD11b/Gr-1 double positive cells from splenic mononuclear cells of Lewis lung carcinoma bearing mice. The percentage of double positive cells in the sorted positive cells reached over 90%, and the percentage of double positive cells in the remained negative cells was less than 1%.3. Lymphocyte proliferation in experimental group contained CD11b/Gr-1 double positive cells was lower than control group ((14.67±3.76)% vs. (45.30±7.26)%, p=0.02).4. TLR4 was expressed on the surface of CD11b+Gr-1+MDSCs from the spleen of Lewis lung carcinoma bearing mice.5. The apoptosis of CD11b+Gr-1+MDSCs after treatment with 0.1μg/ml LPS or 1μg/ml LPS for 48 hours was lower than control group ((9.83±2.36)% vs. (23.93±4.67)%,p0.1μg/ml=0.007; (8.00±2.64)% vs. (23.93±4.67)%,p1μg/ml=0.004). There was no statistically significant difference between 0.1μg/ml LPS treatment group and 1μg/ml LPS treatment group. There was no statistically significant difference among 0.1μg/ml LPS treatment group, 1μg/ml LPS treatment group and control group after treatment with LPS for 24 hours.6. The expression of MHC-Ⅱand CD86 on CD11b+Gr-1+MDSCs after treatment with 1μg/ml LPS was lower than control group ((15.33±3.78)%vs. (34.66±3.78)%, pMHC-Ⅱ=0.002; (18.48±3.51)% vs. (31.23±3.60)%,pCD86=0.01). There was no statistically significant difference between 0.1μg/ml LPS treatment group and control group.7. The inhibition function of CD11b+Gr-1+MDSCs on lymphocytes proliferation was improved after treatment with 1μg/ml LPS compared with the control group, the inhibition ratios were (44.33±9.71)% vs. (19.33±5.52)%, p1:1=0.01; (34.66±7.76)% vs. (17.66±4.52)%,p1:2=0.03, respectively (1:1 and 1:2 represent the dilution of CD11b+Gr-1+MDSCs in MLR system). There is no statistically significant difference between 0.1μg/ml LPS treatment group and control group.8. 0.1μg/ml LPS or 1μg/ml LPS exerted an effect on stimulating ARG1 expression in CD11b+Gr-1+MDSCs.9. The percentage of CD11b+Gr-1+MDSCs in splenic mononuclear cells of Lewis lung carcinoma bearing mice after intraperitoneal injection of 20mg/kg paclitaxel was significantly lower than untreatment group ((28.64±5.45)% vs. (42.58±5.06)%, p=0.03). 10.30ng/ml paclitaxel or 300ng/ml paclitaxel increased the apoptotic rate of CDllb+Gr-1+MDSCs compare with control group, The apoptotic rate were (21.46±6.76)%vs. (4.53±0.90)%, p24小时/30ng/ml=0.01; (38.96±5.47)%vs. (4.53±0.90)%,p24小时/300ng/ml=0.001; (38.70±4.21)% vs. (23.93±4.67)%; p48小时/30ng/ml=0.04; (55.03±6.65)% vs. (23.93±4.67)%, p48小时/300ng/ml=0.009, respectively. There were significant differences between 30ng/ml paclitaxel group and 300ng/ml paclitaxel group (P24小时=0.01,p48小时=0.02).11. The inhibition function of CDllb+Gr-1+MDSCs on lymphocytes proliferation was reduced after 30ng/ml paclitaxel or 300ng/ml paclitaxel treatment compared with the control group, inhibition ratios were (12.0±4.87)% vs. (25.56±6.56)%, p30ng/ml=0.04; (10.22±4.52)%vs. (25.56±6.56)%p300ng/ml=0.02, respectively.12.30ng/ml paclitaxel or 300ng/ml paclitaxel treatment did not exert an effect on ARG1 expression in CD11b+Gr-1+MDSCs.Conclusions1. CDllb/Gr-1 double positive cells accumulated in the spleen of Lewis lung carcinoma bearing mice instead of naive mouse. These double positive cells derived from Lewis lung carcinoma bearing mice expressed the similar characteristics as MDSCs, such as dysdifferentiation, dysmaturity, and immunosuppression function.2. TLR4 was expressed on the surface of CD11b+Gr-1+MDSCs from the spleen of Lewis lung carcinoma bearing mice. the TLR4 ligand LPS had protective effect on CD11b+Gr-1+MDSCs apoptosis, inhibited the maturation of CD11b+ Gr-1+MDSCs, stimulated the expression of ARG1 in CD11b+Gr-1+MDSCs, improved the immunosuppression activity of CD11b+Gr-1+MDSCs.3. Effects of paclitaxel on CD11b+Gr-1+MDSCs were difference with LPS. paclitaxel could kill CD11b+Gr-1+MDSCs in vivo and vitro, reduced the immunosuppression activity of CD11b+Gr-1+MDSCs, but never affect the maturation of CD11b+Gr-1+MDSCs and ARG1 expression.