基于果蝇口腔感染的肠道训练免疫模型研究

An Intestinal Trained Immunity Model Based on Drosophila melanogaster Oral Infection

目的利用果蝇作为遗传工具从个体和分子层面研究果蝇的训练免疫效应,并为后续深入研究其分子机制提供依据。方法首先构建无菌果蝇模型,在此基础上构建果蝇成虫及跨发育阶段训练免疫模型,用两种革兰氏阴性菌——胡萝卜软腐欧文氏菌(Erwinia carotovora carotovora 15)及铜绿假单胞菌(Pseudomonas aeruginosa)分别经口腔感染果蝇。在第一次感染完全消退后进行再次感染,然后通过比较果蝇在两个感染阶段的存活率和细菌量来衡量训练免疫的潜在效果。通过实时荧光定量PCR检测相应先天免疫相关基因的表达水平,研究革兰氏阴性菌对免疫缺陷(IMD)通路的诱导作用。结果果蝇成虫及幼虫初次感染均可提高二次感染后的生存率、细菌清除效率及死亡时能承受的最高细菌负荷;二次感染的果蝇中,IMD通路中免疫反应基因的基础表达比未感染的高,这提供了获得感染抗性的分子基础;果蝇的免疫反应主要发生在中肠,二次免疫比初次免疫的效应更迅速且剧烈;二次免疫的果蝇中,肠道干细胞的数量显著多于初次感染。结论果蝇肠道中强大的训练免疫可由同源或异源革兰氏阴性菌口腔感染引发,且免疫记忆可在整个发育阶段持续存在;其可能作用于染色质,通过染色质修饰将免疫记忆储存在相关基因位点。免疫记忆跨发育阶段传递的一种潜在方式是通过肠道干细胞的JNK/STAT通路激活,这些干细胞可能在从幼虫到成虫的发育阶段携带免疫印记。

Objective To study the trained immunity effect of Drosophila melanogaster at the individual and molecular level,and provide a basis for the follow-up in-depth study of the molecular mechanism of trained immunity using genetic tools available to Drosophila.Methods Firstly,a germ-free Drosophila culture model was constructed.Subsequently,the Drosophila adult and crossdevelopmental trained immunity models were constructed.Two Gram-negative bacteria,Erwinia carotovora carotovora 15 and Pseudomonas aeruginosa,were respectively used to infect Drosophila orally.With a repeated infection elicited after the first infection completely subsided,the effects of potential trained immunity is demonstrated by comparing the survival rate and bacterial load of Drosophila melanogaster during the two infection phases.The induction of immune deficiency(IMD)pathway by Gramnegative bacteria as the expression level of corresponding innate immunity-related genes was detected by real-time quantitative PCR.Results The primary infection of during either adults or larva developmental stage can significantly improve the survival rate of secondary challenge.A higher bacterial clearance efficiency and maximum bacterial load of death is consistently observed after a second infection.The basal expression of immune response genes in IMD signaling pathway is boosted prior to secondary infection than naive animal,explaining the molecular basis of gained infection resistance.Midgut is examined to be primary anatomic site of immune response,and the effects of secondary immunization were faster and more intense than those of primary infection.The numbers of intestinal stem cells in the midgut were significantly higher during the second infection compared with the first one.Conclusion A robust trained immunity in Drosophila melanogaster intestine can be triggered by oral infection of either homologous or heterologous Gram-negative bacteria,and the immunological memory can persist across developmental stages.It may act on chromatin and store immunologic memory at relevant gene loci through chromatin modifications.A potential way for the passage of immunologic memory across developmental stages is through JNK/STAT activation of intestinal stem cells,which may carry on the immune imprint from larval to adult developmental stages in the gut.