基质金属蛋白酶对MRSA脓毒症患者NK细胞的调控作用

Matrix metalloproteinase regulates natural killer cells function in methicilin-resistant staphylococcus aureus sepsis

目的观察耐甲氧西林金黄色葡萄球菌(MRSA)脓毒症患者外周血自然杀伤(NK)细胞亚群和功能,探讨基质金属蛋白酶(MMP)对MRSA脓毒症患者NK细胞功能的影响。方法选择2017年1月至2018年6月本科收治的MRSA脓毒症患者21例和健康对照者(NC)11例,分离外周血单个核细胞(PBMC),流式细胞术检测NK细胞亚群比例。通过检测PBMC与靶细胞共培养体系中CD107a、CD69、CD16的表达评估NK细胞功能。分选NK细胞,实时定量PCR法检测MMPmRNA,使用MMP抑制剂刺激NK细胞,检测共培养体系中CD107a变化。组间比较采用成组t检验或配对t检验。结果总NK细胞比例在NC和MRSA脓毒症患者之间的差异无统计学意义(P>0.05),MRSA脓毒症患者CD56brightCD16-NK细胞[(5.36±1.02)%vs(4.30±0.89)%]和CD56-CD16+NK细胞[(24.04±2.92)%vs(9.70±1.54)%]升高(P<0.05),CD56dimCD16+NK细胞(71.22±13.03)%vs(87.64±7.05%)]降低(P<0.01).NK细胞通过不同受体介导相应靶细胞杀伤后,MRSA脓毒症患者NK细胞中CD107a[(33.55±3.84)%w(25.34±6.20%)]和CD69[(14.96±1.47%)vs(18.80±1.49)%]比例低于NC(P<0.01),CD16 MFI[(247.1±50.31)vs(189.4±57.54)]高于NC(P<0.01)。MMP-1/2/3/9 mRNA在MRSA脓毒症患者NK细胞中的相对表达量升高(P<0.01)。抑制MRSA脓毒症患者NK细胞MMP使CD107a比例升高[(33.67±8.03%)vs(25.87±6.23)%,P=0.018]。结论MRSA脓毒症患者存在NK细胞亚群失衡和功能衰竭,这可能与MMP诱导的抗体依赖细胞介导的毒性作用降低有关。

Objective To investigate the characteristics of natural killer(NK)cell subsets and function in methicillin-resistant staphylococcus aureus(MRSA)sepsis,and to assess the influence of matrix metalloproteinase(MMP)to NK cell function in MRSA sepsis patients.Methods Twenty-one MRSA sepsis patients who were hospitalized in our department between January 2017 and June 2018 were enrolled.Eleven healthy individuals were served as healthy controls.Peripheral blood mononuclear cells(PBMCs)were isolated.NK cell subsets were investigated by flow cytometry.NK cell function was assessed by measuring CD107a,CD69,and CD16 expression in co-culture system between PBMCs and different target cells.MMP mRNA was semi-quantified by real-time PCR in purified NK cells.The influence of NK cell fiinction was assessed by measuring CD 107a expression in co-culture system between NK cells with MMP inhibitor stimulation and target cells.Results There was no significant difference of total NK cell percentage between healthy controls and MRSA sepsis patients(P>0.05).CD56brightCD16^+NK[(5.36±1.02)%(4.30±0.89)%]and CD56^-CD16^+NK.[(24.04±2.92)%vs(9.70±1.54)%]percentage was elevated(P<0.05),while CD56dimCD16^NK:percentage[(71.22±13.03)%w(87.64±7.05)%,P<0.01]was reduced in MRSA sepsis.NK cells recognized and killed target cells via different receptors upon activation.CD107a[(33.55±3.84)%vs(25.34±6.20)%]and CD69 percentage[(14.96±1.47)% vs(18.80±1.49)%]was decreased(P<0.0001),while CD16 MFI was increased[(247.1±50.31)vs(189.4±57.54),P<0.01]in MRSA sepsis patients in comparison with healthy controls.MMP-1/2/3/9 mRNA relative levels were elevated in purified NK cells from MRSA sepsis patients(P<0.01).Inhibition of MMP in NK cells from MRSA sepsis patients promoted CD107a percentage[(33.67±8.03)%vs(25.87±6.23)%,P=0.018].Conclusions NK cell subsets imbalance and exhaustion is existed in MRSA sepsis,which might be due to the MMP-induced down-regulation of antibody-dependent cell-mediated cytotoxicity.