β-glucan诱导训练免...及其在逆转免疫耐受中的应用_钟秋梅.pdf

2022 年 12 月第 30 卷 第 8 期中国实验动物学报ACTA LABORATORIUM ANIMALIS SCIENTIA SINICADecember 2022Vol.30 No.8钟秋梅,张佳,李林英,等.-glucan 诱导训练免疫及其在逆转免疫耐受中的应用 J.中国实验动物学报,2022,30(8):1013-1022.Zhong QM,Zhang J,Li LY,et al.-glucan induces trained immunity and its application in reversing immune tolerance J.Acta Lab Anim Sci Sin,2022,30(8):1013-1022.Doi:10.3969/j.issn.1005-4847.2022.08.001基金项目国家自然科学基金项目面上项目(32070904)。Funded by the National Natural Science Foundation of China(32070904).作者简介钟秋梅(1996),女,在读硕士研究生,研究方向:先天免疫与免疫耐受。Email:15882263246 通信作者郑世进(1979),男,教授,博士生导师,研究方向:先天免疫系统功能调控;先天免疫耐受;先天免疫与肿瘤治疗。Email:jamescheng -glucan 诱导训练免疫及其在逆转免疫耐受中的应用钟秋梅1,2,张佳1,2,李林英1,2,郑世进1,2(1.厦门大学生命科学学院,福建 厦门 361100;2.厦门大学细胞应激生物学国家重点实验室,福建 厦门 361100)【摘要】目的 本研究拟通过在体内和体外诱导训练免疫反应,以探讨诱导训练免疫是否可能逆转免疫耐受状态。方法以-glucan 作为训练免疫诱导剂,建立体外小鼠胫骨来源的巨噬细胞(bone marrow derived-macrophages,BMDM)训练免疫模型:40 g/mL-glucan 刺激 BMDM 24 h,随后用 PBS 洗涤后让 BMDM 在完全培养基中静息 6 d,在第 3 天的时候补充培养基,第 7 天收集细胞 100 ng/mL LPS 再刺激,4 h 后收集细胞抽提 RNA 和 24 h 后收集细胞上清;建立-glucan 体内训练免疫模型:野生型 C57BL/6J 腹腔注射 1 mg-glucan,对照组腹腔注射200 L 的 PBS,7 d 后感染金黄色葡萄球菌(每只 1 104/200 L PBS)。LPS 诱导体外免疫耐受,体外免疫耐受逆转通过在耐受的 BMDM 中加入 40 g/mL-glucan 刺激 24 h,随后让细胞静息 1 d,通过促炎因子的恢复水平来反映耐受逆转情况。盲肠结扎穿刺(cecal ligation and puncture,CLP)诱导体内免疫耐受,体内免疫耐受逆转通过向CLP 组腹腔注射 1 mg-glucan,通过再次感染非致死剂量的细菌或真菌的生存率反映耐受逆转情况。结果(1)经过-glucan 训练的 BMDM,在 LPS 再次刺激时产生更高水平的促炎因子 TNF-、IL-6 和 NO。同时,-glucan 训练的 BMDM 通过激活 m-TOR 信号通路,导致细胞代谢模式向糖酵解转变,引起细胞内乳酸增多;(2)-glucan 诱导的体内训练免疫具有保护作用;(3)-glucan 在体外能够逆转 LPS 诱导的免疫耐受,恢复 BMDM 促炎细胞因子的产生;(4)目前我们的结果尚未证实-glucan 在体内可以逆转 CLP 诱导的免疫耐受。结论 本研究成功地建立了-glucan 体外和体内训练免疫模型,并证明了在体外-glucan 能逆转 LPS 诱导的免疫耐受,但是目前结果尚未证实-glucan 在体内能够逆转 CLP 诱导的免疫耐受,提高二次感染生存率。【关键词】-葡聚糖;训练免疫;CLP;免疫耐受;耐受逆转【中图分类号】Q95-33 【文献标识码】A 【文章编号】1005-4847(2022)08-1013-10-glucan induces trained immunity and its application in reversing immune toleranceZHONG Qiumei1,2,ZHANG Jia1,2,LI Linying1,2,CHENG Shihchin1,2(1.Xiamen University School of Life Science,Xiamen 361100,China.2.Xiamen University State Key Laboratory of Cellular Stress Biology,Xiamen 361100)Corresponding author:CHENG Shihchin.E-mail:jamescheng 【Abstract】ObjectiveThe aim of this study was to examine whether immune tolerance was reversed by trained immunity in both in vivo and in vitro models.Methods Glucan is a prototypical trained immunity inducer.To establish the in vitro trained immunity model,BMDMs were stimulated with 40 g/mL-glucan for 24 h.BMDMs were washed once with warm PBS.After 24 h,they were incubated for 6 days in culture medium that was changed once at day 3.Trained BMDMs were stimulated with 100 ng/mL LPS at day 7.To establish the in vivo trained immunity model,wildtype C57BL/中国实验动物学报 2022 年 12 月第 30 卷第 8 期 Acta Lab Anim Sci Sin,December 2022,Vol.30,No.86J mice were injected intraperitoneally with 1 mg-glucan in 200 mL PBS.As a control,intraperitoneal injection of PBS alone was performed.After 7 days,mice were infected with Staphylococcus aureus intraperitoneally(1 104/200 mL PBS per mice).BMDMs were stimulated with LPS to induce immune tolerance in vitro.-glucan was added at 24 h post-LPS stimulation to reverse immune tolerance.Cytokine production capacity was determined by LPS restimulation.CLP was carried out in mice to induce immune tolerance in vivo.Intraperitoneal injection of 1 mg-glucan was performed to reverse CLP-induced tolerance.The efficacy of tolerance reversal was reflected by the survival rate post-reinfection with non-lethal doses of bacteria or fungi.Results(1)Glucan-trained BMDMs produced higher levels of proinflammatory cytokines,such as TNF-and IL-6,and more nitric oxide.The energy metabolism of trained macrophages shifted to aerobic glycolysis,leading to lactate accumulation and enhanced m-TOR activation.(2)-glucan-induced trained immunity was protective in vivo.(3)-glucan reversed LPS-induced immune tolerance in vitro.(4)Administration of-glucan did not reverse the CLP-induced immune tolerance in the current experimental setting.ConclusionThis study successfully established-glucan trained immune models in vitro and in vivo,and demonstrated that-glucan can reverse LPS-induced immune tolerance in vitro.However,the current results have not confirmed that-glucan can reverse CLP-induced immune tolerance in vivo and improve secondary infection survival.【Keywords】-glucan;trained immunity;CLP;immune tolerance;tolerance reversalConflicts of Interest:The authors declare no conflict of interest.免疫记忆指当机体初次刺激时能产生一个免疫反应,当再次遇到同种刺激时机体能产生更强大的免疫反应。1950 1960 年,免疫学家一直认为只有具有 T/B 淋巴细胞的脊椎动物才具有免疫记忆1。后天免疫反应依赖于 T/B 淋巴细胞,特异性识别病原体并建立该特定感染的免疫记忆。在第 2次遇到相同的病原体时,记忆 T/B 细胞的克隆扩增会诱导快速有效的反应。然而,在后面的研究当中发现在一些植物或非脊椎动物,甚至一些细菌和古细菌中也有免疫记忆,但显然它们的免疫记忆并不是由 T/B 淋巴细胞介导的1。研究报道,缺乏 T/B细胞的 Rag1-/-小鼠初次感染过非致死剂量的白色念珠菌(Candida albicans),当再次感染时能够抵抗感染而存活下来2。这说明先天免疫系统也能介导一种免疫记忆的产生,后来免疫学家将这种先天免疫介导的保护作用定义为训练免疫(trained immunity)3。训练免疫是指先天免疫系统在首次遇到刺激后,经过急性反应回到稳态后,当再次遇到同源或异源刺激时,机体能产生更快速的免疫应答,从而保护机体。-葡聚糖(-glucan)是训练免疫的典型诱导剂,-glucan 训练免疫模型最开始在人源单核细胞中建立。先天免疫细胞如单核细胞和巨噬细胞,当第 1 次遇到-glucan 时,细胞内糖代谢途径向有氧糖酵解转变,同时促进附近的组蛋白甲基化进行重编程,细胞内的 Akt-mTOR 信号通路也会被激活等4。当再次遇到同源