The Study On The Mediating Role Of Myeloid-derived Suppressor Cell (MDSC) In Susceptibility To Lung Cancer In Chronic Intermittent Hypoxia (CIH) And Its Mechanism

Objective:To examine the mediating role of myeloid-derived suppressor cell(MDSC)in susceptibility to lung cancer in chronic intermittent hypoxia(CIH)and its mechanism.Methods:(1)The peripheral blood specimens were obtained from the patients with obstructive sleep apnea syndrome(OSAS)and the health people(control)of the corresponding age group.A C57BL/6J mouse chronic intermittent hypoxia(CIH)model was established.Then the tail vein blood and spleen specimens derived from the CIH mice and the normal mice(control)were obtained.The number of CD14+HLA-DR-/low MDSC in human peripheral blood(OSAS vs health)was analyzed by flow cytometry.The number of CD11b+Gr1+ MDSC in mouse peripheral blood and spleen(CIH vs normal)was also analyzed by flow cytometry.(2)The CD14+HLA-DR-/low MDSC and T cells in the peripheral blood of OSAS patients and health people was isolated by flow cytometric sorting and a human T cell negative isolation kit,respectively.The CD11b+Gr1+ MDSC in the spleen of CIH and normal mice were isolated by a MDSC positive isolation kit.The T cells in the spleen of CIH and normal mice were purified by a nylon wool fiber column method.The CD4+T and CD8+T cells were further negatively isolated by Dynabeads.(3)For T-cell proliferation assays in vitro,the carboxyfluorescein diacetate succinimidyl ester(CFSE)-labebled T cells were incubated with autologous CD14+HLA-DR-/low MDSC derived from the peripheral blood of OSAS patients or health people.The CFSE-labebled T cells and CD4+T or CD8+T cells were incubated with autologous CDllb+Grl+MDSC derived from the spleen of CIH or normal mice.The CFSE-labebled T cells and CD4+T or CD8+T cells derived from normal mice were incubated with CD11b+Gr1+MDSC derived from CIH or normal mice.(4)The mature bone marrow(BM)-derived dendritic cells(DC)(BM-DC)of C57BL/6J mice were generated and then pulsed with chicken ovalbumin(OVA)protein to prepare an OVA-loaded DC(DCOVA)vaccine.The CIH and normal(control)mice were immunized with DCOVA.The normal mice were immunized with DCOVA plus CIH mice spleen-derived MDSC(MDSClH)or normal mice spleen-derived MDSC(MDSCnOrmal)(control).In addition,the CIH mice were pretreated with anti-Grl blocking antibody(MDSC-)or isotype control antibody(MDSC+)(control)and then immunized with DCOVA.Six days post immunization,the OVA-specific CD8+CTL in peripheral blood were analyzed using Tetramer by flow cytometry.(5)The splenocytes of C57BL/6J mice were labeled with either high or low concentration of CFSE.These high CFSE-labeled(CFSEhigh)cells were pulsed with H-2Kb-restricted OVAI peptide and served as OVA-specific CFSEhigh target cells.These low CFSE-labeled(CFSElow)cells were pulsed with irrelevant H-2Kb-restricted Mutl peptide and served as negative control target cells.These CFSEhigh and CFSElow target cells were intravenously co-injected into the immunized mice(DCOVA-immunized CIH mice vs DCOVA-immunized normal mice;DCovA+MDSCcIH-immunized normal mice vs DCOVA+MDSCnormal-immunized normal mice)six days after immunization.The cytotoxic effect of OVA-specific CD8+CTL was then analyzed by flow cytometry.(6)The pcDNA3.1+/OVA eukaryotic expressing plasmid expressing chicken OVA was constructed.The pcDNA3.1+/OVA plasmid was then transfected into a mouse lewis lung cancer(LLC)cell line and selected with a drug neomycin(Neo).The Neo-resistant pcDNA3.1+/OVA-derived transfectant was further moncloned and the transgene expression of OVA was identified by qRT-PCR,flow cytometric and Western blot analysis,leading to generation of LLC-OVA transgenic mouse lung cancer cells expressing high level of OVA(used as a OVA-specific tumor target cell line).Six days after immunization,the immunized mice(DCOVA-immunized CIH mice vs DCOVA-immunized normal mice;DCOVA+MDSCCIH-immunized normal mice vs DCOVA+MDSCnormal-immunized normal mice)were subcutaneously injected with LLC-OVA mouse lung cancer cells expressing OVA.The tumor growth was then monitored.(7)The expression of cytokines including interleukin-6(IL-6),tumor necrosis factor-a(TNFa),IL-10 and transforming growth factor-?1(TGF?1)in CD14+HLA-DR-/low MDSC from human peripheral blood(OSAS vs health)or CD11lb+Gr1+MDSC from mouse spleen(CIH vs normal)was evaluated by enzyme-linked immunosorbent assay(ELISA).The expression of PD-L1 on CD14+HLA-DR-/low MDSC from human peripheral blood(OSAS vs health)or CD11b+Gr1+MDSC from mouse spleen(CIH vs normal)was analyzed by flow cytometry.The expression of arginase 1(Argl),induced nitric oxide synthase(iNOS)and hypoxia-inducible factor-la(HIF1a)in CD14+HLA-DR-/low MDSC from human peripheral blood(OSAS vs health)or CDllb+Grl+MDSC from mouse spleen(CIH vs normal)was determined by qRT-PCR and Western blot analysis.(8)The CIH and normal mice were pretreated with anti-Grl blocking antibody(MDSC-)or isotype control antibody(MDSC+)(control)and then subcutaneously injected with LLC mouse lung cancer cells.The tumor growth was monitored and the promoting effect of CIH(OSAS)-conditioned MDSC on tumorgenesis and tumor growth was finally analyzed.Results:Section ? The effect of OSAS(CIH)on quantity of MDSCThe quantity of MDSC in OSAS patients and CIH mice is increased.Compared with health people,the number of CD14+HLA-DR-/low MDSC in peripheral blood of OSAS patients was obviously increased(p<0.05).The number of CD14+HLA-DR-/low MDSC in both peripheral blood and spleen of CIH mice was also significantly higher than that in normal mice(p<0.05).Section ? The effect of OSAS(CIH)-conditioned MDSC on T-cell immune function(1)OSAS patient-or CIH mice-derived MDSC exhibits a stronger suppressive effect in T-cell proliferation in vitro.OSAS patient-derived CD14+HLA-DR-/low MDSC more efficiently inhibited autologous T-cell proliferation in vitro than health people-derived CD14+HLA-DR-/low MDSC did(p<0.05).CIH-derived CDllb+Grl+MDSC also more efficiently suppressed autologous or allogous(normal mice-derived T cells)T-cell,CD4+T-cell and CD8+T-cell proliferation in vitro than normal mice-derived CD11b+Gr1+MDSC did(p<0.05).(2)CIH mice-derived MDSC exhibits a stronger suppressive effect in DCOVA-stimulated OVA-specific CD8+CTL responses in vivo.Compared with that in DCOVA-immunized normal mice,the percentage of FITC-CD8+PE-Tetramer+double positive cells in total CD8+T-cell population in DCOVA-immunized CIH mice was significantly reduced.DCOVA-immunized CIH mice exhibited much poorer OVA-specific CD8+CTL responses(p<0.05).Compared with that in DCOVA+MDSCnormal-immunized normal mice,the OVA-specific CD8+CTL responses in DCOVA+MDSCciH-immunized normal mice were also attenuated(p<0.05).(3)Depletion of MDSC,to a great extent,rescues the ability of DCOVA-stimulated OVA-specific CD8+CTL responses.Pre-depletion of MDSC was capable of,to a great extent,rescuing the capability of DCOVA-stimulated OVA-specific CD8+CTL responses in CIH mice(p<0.05).CIH-conditioned MDSC was crucial for suppression of DCOVA-stimulated OVA-specific CD8+ CTL responses.(4)CIH mice-derived MDSC exhibits a stronger suppressive effect in DCOVA-stimulated OVA-specific CD8+CTL-mediated cytotoxic activity.Compared with that in DCOVA-immunized normal mice,DCOVA-stimulated OVA-specific CD8+CTL in CIH mice exhibited much poorer cytotoxicity to OVA-specific target cells(p<0.05).Compared with that in DCOVA+MDSCnormal-immunized normal mice,DCOVA+MDSCCIH-stimulated OVA-specific CD8+CTL-mediated OVA-specific cytotoxic activity in normal mice was also impaired(p<0.05).CIH mice-derived MDSC more efficiently inhibited generation of OVA-specific effector CTL.(5)CIH mice-derived MDSC exhibits a stronger suppressive effect in DCOVA-stimulated OVA-specific CD8+CTL-induced preventive antitumor immunity.Compared with that in DCOVA-immunized normal mice,DCOVA-stimulated OVA-specific CD8+CTLs in CIH mice exhibited much poorer preventive antitumor immunity(p<0.05).Compared with that in DCOVA+MDSCnormal-iMMunized normal mice,DCOVA+MDSCCIH-stimulated OVA-specific CD8+CTL-induced preventive antitumor immunity in normal mice was also weakened(p<0.05).Section ? The mechanistic study on OSAS(CIH)-conditioned MDSC-elicited an enhanced immunosuppressive function(1)OSAS patient-or CIH mice-derived MDSC secretes much higher levels of IL-6,TNF?,IL-10 and TGF?1.The secretory level of cytokines such as IL-6,TNFa,IL-10 and TGF?1 in OSAS patient-derived CD14+HLA-DR-/low MDSC and CIH mice-derived CD11b+Gr1+MDSC was much higher than that in health people-and normal mice-derived ones(p<0.05).(2)OSAS patient-or CIH mice-derived MDSC expresses much higher levels of PD-L1.The expression level of immune checkpoint molecule PD-L1 in OSAS patient-derived CD14+HLA-DR-/low MDSC and CIH mice-derived CD11b+Grl+MDSC was much higher than that in health people-and normal mice-derived ones(p<0.05).(3)OSAS patient-or CIH mice-derived MDSC expresses much higher levels of Argl,iNOS and HIFla.The expression level of metabolism enzymes including Argl and iNOS,and critical signaling molecule HIF1a in OSAS patient-derived CD14+HLA-DR-/low MDSC and CIH mice-derived CD11b+Gr1+MDSC was much higher than that in health people-and normal mice-derived ones(p<0.05).Section ? The study on the mediating role of OSAS(CIH)-conditioned MDSC in susceptibility to lung cancerThe susceptibility of CIH mice to lung cancer is increased and OSAS(CIH)-conditioned MDSC plays an important role in the process.Compared with normal mice,LLC mouse lung cancer cells subcutaneously implanted in CIH mice were more easily tumorigenic(p<0.05).Moreover,the growth of forming tumor was accelerated in CIH mice(p<0.05).Importantly,depletion of MDSC impaired the tumorigenic potential in CIH mice(p<0.05).Conclusions:OSAS(CIH)is capable of inducing increase and generation of MDSC as well as upregulating expression of immunosuppressive molecules possibly via activation of HIF1a signaling in MDSC,leading to enhanced immunoinhibitory ability.MDSC is a critical mediator for CIH-induced susceptibility to lung cancer.