Regulatory effects of the Nocardia rubra cell wall skeleton on the biological function of human neutrophils
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摘要:
目的 探讨红色诺卡菌细胞壁骨架（Nr-CWS）对人中性粒细胞生物学功能的调节作用及其机制。 方法 采用实验研究方法。2022年5―10月招募于苏州市体检中心体检的成年健康志愿者15名（男7名、女8名，年龄24~45岁），采集外周静脉血，采用免疫磁珠分选法提取中性粒细胞。将细胞分为不进行任何处理的正常对照组、仅用终质量浓度为60 ng/mL Nr-CWS处理的单纯Nr-CWS组、仅用终质量浓度1 μg/mL内毒素/脂多糖（LPS）刺激的单纯LPS组、用同前LPS刺激后再用Nr-CWS处理的LPS+Nr-CWS组。培养1 h，采用改良后的琼脂糖趋化模型检测中性粒细胞的趋化距离、趋化细胞百分比、趋化指数、最大趋化速度、趋化功能评分；采用流式细胞术检测发生吞噬的细胞占比与荧光强度，活性氧水平，颗粒蛋白CD35、CD66b、CD63的蛋白表达水平，以及细胞培养上清液中炎症因子白细胞介素2（IL-2）、IL-4、IL-6、IL-10、IL-17A、肿瘤坏死因子α（TNF-α）、γ干扰素浓度。以上实验各组样本数均为15。对数据行析因设计方差分析与独立样本 t检验。 结果 培养1 h，正常对照组、单纯Nr-CWS组、单纯LPS组、LPS+Nr-CWS组细胞趋化功能评分分别为15.0、（14.5±0.5）、（1.5±0.5）、（12.0±1.5）分；与正常对照组比较，单纯LPS组与LPS+Nr-CWS组细胞趋化距离、趋化细胞百分比、趋化指数、最大趋化速度与趋化功能评分均显著降低（ t值分别为18.36、18.88、54.28、18.36、46.77，10.58、14.74、6.84、10.58、4.24， P<0.05）；与单纯LPS组比较，LPS+Nr-CWS组细胞上述5项趋化功能指标均显著升高（ t值分别为11.47、14.65、11.62、11.47、13.75， P<0.05）。培养1 h，与正常对照组比较，单纯Nr-CWS组发生吞噬的细胞占比与荧光强度均显著升高（ t值分别为6.86、6.73， P<0.05），单纯LPS组（ t值分别为7.35、22.72， P<0.05）与LPS+Nr-CWS组（ t值分别为21.37、13.10， P<0.05）发生吞噬的细胞占比与荧光强度均明显降低。培养1 h，与正常对照组比较，单纯LPS组细胞活性氧水平显著升高（ t=6.64， P<0.05）；与单纯LPS组比较，LPS+Nr-CWS组细胞活性氧水平明显降低（ t=5.46， P<0.05）。培养1 h，与正常对照组比较，单纯LPS组与LPS+Nr-CWS组细胞CD35、CD66b、CD63蛋白表达均显著升高（ t值分别为16.75、17.45、10.82，5.70、19.35、15.37， P<0.05）；与单纯LPS组比较，LPS+Nr-CWS组细胞CD35、CD66b、CD63蛋白表达均明显下降（ t值分别为4.92、5.72、3.18， P<0.05）。培养1 h，与正常对照组比较，单纯LPS组细胞培养上清液中IL-2、IL-4、IL-6、IL-10、IL-17A、TNF-α、γ干扰素浓度均显著升高（ t值分别为22.10、9.50、7.21、10.22、24.88、8.43、47.48， P<0.05），LPS+Nr-CWS组细胞培养上清液中IL-6、IL-10、IL-17A、TNF-α、γ干扰素浓度均显著上升（ t值分别为4.68、5.12、8.02、5.58、7.13， P<0.05）；与单纯LPS组比较，LPS+Nr-CWS组细胞培养上清液中IL-2、IL-4、IL-6、IL-10、IL-17A、TNF-α、γ干扰素浓度均显著降低（ t值分别为5.39、2.83、5.79、2.90、5.87、4.88、39.64， P<0.05）。 结论 Nr-CWS能够提高正常状态下人中性粒细胞的吞噬能力，改善感染状态下人中性粒细胞的趋化功能及活性氧、脱颗粒蛋白、炎症因子水平，在先天免疫层面通过调控人中性粒细胞生物学行为，提高抗感染能力。 Abstract:Objective To investigate the regulatory effects and mechanism of Nocardia rubra cell wall skeleton (Nr-CWS) on the biological function of human neutrophils. Methods The experimental research method was used. Fifteen healthy adult volunteers (7 males and 8 females, aged 24 to 45 years) were recruited from Suzhou Physical Examination Center for physical examination from May to October 2022, the peripheral venous blood was collected, and neutrophils were extracted by immunomagnetic bead sorting. The cells were divided into normal control group without any treatment, Nr-CWS alone group treated with Nr-CWS of final mass concentration 60 ng/mL alone, endotoxin/lipopolysaccharide (LPS) alone group stimulated with LPS of final mass concentration 1 μg/mL alone, and LPS+Nr-CWS group stimulated with LPS first and then treated with Nr-CWS as before. After 1 h of culture, the chemotaxis distance, chemotactic cell percentage, chemotactic index, maximum chemotactic speed, and chemotactic function score of neutrophils were detected using the modified agarose chemotactic model; the proportion and fluorescence intensity of phagocytosis cells, the level of reactive oxygen species (ROS), the protein expression levels of granular protein CD35, CD66b, and CD63, and the concentrations of inflammatory cytokines of interleukin 2 (IL-2), IL-4, IL-6, IL-10, IL-17A, tumor necrosis factor alpha (TNF-α), and interferon-γ in cell culture supernatant were detected by flow cytometry. The number of samples in each group in the above experiments was 15. Data were statistically analyzed with analysis of variance for factorial design and independent sample t test. Results After 1 h of culture, the chemotactic function score of cells in normal control group, Nr-CWS alone group, LPS alone group, and LPS+Nr-CWS group were 15.0, 14.5±0.5, 1.5±0.5, 12.0±1.5, respectively. Compared with those in normal control group, the chemotaxis distance, chemotactic cell percentage, chemotactic index, maximum chemotactic speed, and chemotactic function score of cells were significantly decreased in LPS alone group and LPS+Nr-CWS group (with t values of 18.36, 18.88, 54.28, 18.36, 46.77, 10.58, 14.74, 6.84, 10.58, and 4.24, respectively, P<0.05); compared with those in LPS alone group, the five chemotactic function indexes as above in LPS+Nr-CWS group were significantly increased (with t values of 11.47, 14.65, 11.62, 11.47, and 13.75, respectively, P<0.05). After 1 h of culture, compared with those in normal control group, the proportion and fluorescence intensity of phagocytosis cells were significantly increased in Nr-CWS alone group (with t values of 6.86 and 6.73, respectively, P<0.05), and the above two indexes were significantly decreased in LPS alone group (with t values of 7.35 and 22.72, respectively, P<0.05) and LPS+Nr-CWS group (with t values of 21.37 and 13.10, respectively, P<0.05). After 1 h of culture, compared with that in normal control group, the level of ROS of cells in LPS alone group was significantly increased ( t=6.64, P<0.05); compared with that in LPS alone group, the level of ROS of cells in LPS+Nr-CWS group was significantly decreased ( t=5.46, P<0.05). After 1 h of culture, compared with those in normal control group, the protein expressions of CD35, CD66b, and CD63 of cells were significantly increased in LPS alone group and LPS+Nr-CWS group (with t values of 16.75, 17.45, 10.82, 5.70, 19.35, and 15.37, respectively, P<0.05); compared with those in LPS alone group, the protein expressions of CD35, CD66b, and CD63 of cells were significantly decreased in LPS+Nr-CWS group (with t values of 4.92, 5.72, and 3.18, respectively, P<0.05). After 1 h of culture, compared with those in normal control group, the concentrations of IL-2, IL-4, IL-6, IL-10, IL-17A, TNF-α, and interferon-γ in cell culture supernatant were significantly increased in LPS alone group (with t values of 22.10, 9.50, 7.21, 10.22, 24.88, 8.43, and 47.48, respectively, P<0.05), and the concentrations of IL-6, IL-10, IL-17A, TNF-α, and interferon-γ in cell culture supernatant were significantly increased in LPS+Nr-CWS group (with t values of 4.68, 5.12, 8.02, 5.58, and 7.13, respectively, P<0.05); compared with those in LPS alone group, the concentrations of IL-2, IL-4, IL-6, IL-10, IL-17A, TNF-α, and interferon-γ in cell culture supernatant were significantly decreased in LPS+Nr-CWS group (with t values of 5.39, 2.83, 5.79, 2.90, 5.87, 4.88, and 39.64, respectively, P<0.05). Conclusions Nr-CWS can enhance the phagocytosis ability of neutrophils in normal condition and improve the chemotactic function, ROS level, degranulation protein level, and inflammatory factor level of human neutrophils in infectious condition. Nr-CWS can enhance the anti-infection ability of human neutrophils by regulating its biological behavior in innate immunity. -
Key words:
- Infection /
- Phagocytosis /
- Chemotaxis /
- Neutrophils /
- Biological function /
- Nocardia rubra cell wall skeleton
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1 4组人中性粒细胞培养1 h趋化功能。1A、1B、1C、1D.分别为正常对照组、单纯Nr-CWS组、单纯LPS组、LPS+Nr-CWS组趋化功能展示，图1A细胞趋化距离、趋化细胞数较图1C、1D明显增加，图1D细胞趋化距离、趋化细胞数较图1C明显增加；1E、1F、1G、1H、1I.分别为趋化距离、趋化细胞百分比、趋化指数、最大趋化速度、趋化功能评分比较（样本数为15，
注：图1E、1F、1G、1H、1I中横坐标1、2、3、4分别表示正常对照组、单纯红色诺卡菌细胞壁骨架（Nr-CWS）组、单纯内毒素/脂多糖（LPS）组、LPS+Nr-CWS组；趋化距离、趋化细胞百分比、趋化指数、最大趋化速度、趋化功能评分Nr-CWS处理因素主效应，F值分别为94.95、30.76、76.19、94.95、189.00，P值均<0.001；LPS处理因素主效应，F值分别为428.40、690.50、427.40、428.40、466.70，P值均<0.001；两者交互作用，F值分别为104.60、38.36、99.62、104.60、189.00，P值均<0.001；与正常对照组比较，aP<0.05；与单纯LPS组比较，bP<0.05
2 4组人中性粒细胞培养1 h吞噬功能比较（样本数为15，
注：图2A、2B中横坐标1、2、3、4分别表示正常对照组、单纯红色诺卡菌细胞壁骨架（Nr-CWS）组、单纯内毒素/脂多糖（LPS）组、LPS+Nr-CWS组；发生吞噬的细胞占比和荧光强度Nr-CWS处理因素主效应，F值分别为26.59、49.05，P值均<0.001；LPS处理因素主效应，F值分别为192.30、350.20，P值均<0.001；两者交互作用，F值分别为45.66、29.52，P值均<0.001；与正常对照组比较，aP<0.05
3 4组人中性粒细胞培养1 h活性氧水平比较（样本数为15，
注：Nr-CWS为红色诺卡菌细胞壁骨架，LPS为内毒素/脂多糖；图3B中横坐标1、2、3、4分别表示正常对照组、单纯Nr-CWS组、单纯LPS组、LPS+Nr-CWS组；活性氧水平Nr-CWS处理因素主效应，F=23.08，P=0.001；LPS处理因素主效应，F=64.94，P<0.001；两者交互作用，F=26.31，P<0.001；与正常对照组比较，aP<0.05；与单纯LPS组比较，bP<0.05
4 4组人中性粒细胞培养1 h颗粒蛋白表达。4A、4C、4E.分别为CD35、CD66b、CD63蛋白表达流式直方图；4B、4D、4F.分别为CD35、CD66b、CD63蛋白表达条图（样本数为15，
注：Nr-CWS为红色诺卡菌细胞壁骨架，LPS为内毒素/脂多糖；图4B、4D、4F中横坐标的1、2、3、4分别表示正常对照组、单纯Nr-CWS组、单纯LPS组、LPS+Nr-CWS组；CD35、CD66b、CD63蛋白表达水平Nr-CWS处理因素主效应，F值分别为24.23、24.81、15.51，P值分别为0.001、0.001、0.004；LPS处理因素主效应，F值分别为24.23、641.60、374.70，P值均<0.001；两者交互作用，F值分别为23.47、33.69、7.70，P值分别为0.001、<0.001、0.024；与正常对照组比较，aP<0.05；与单纯LPS组比较，bP<0.05
5 4组中性粒细胞培养1 h上清液中7种炎症因子浓度比较（样本数为15，
注：Nr-CWS为红色诺卡菌细胞壁骨架，LPS为内毒素/脂多糖，IL为白细胞介素，TNF-α为肿瘤坏死因子α；IL-2、IL-4、IL-6、IL-10、IL-17A、TNF-α、γ干扰素浓度Nr-CWS处理因素主效应，F值分别为23.80、5.47、18.81、8.80、16.32、17.65、324.20，P值分别为0.001、0.048、0.003、0.018、0.004、0.003、<0.001；LPS处理因素主效应，F值分别为4.98、33.43、114.10、59.91、126.40、17.65、613.90，P值分别为0.039、<0.001、<0.001、<0.001、<0.001、<0.001、<0.001；两者交互作用，F值分别为27.41、0.55、0.10、0.60、2.22、139.60、191.50，P值分别为<0.001、0.481、0.767、0.804、0.175、0.712、<0.001；与正常对照组比较，aP<0.05；与单纯LPS组比较，bP<0.05
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