Recombinant Lactococcus lactis delivering hCAP18 cDNA alleviates DNBS-induced colitis in C57BL/6 mice by promoting IL17A and IL10 cytokine expression

In this study, we attempted to alleviate chemically induced colitis in mice using a recombinant probiotic strain that delivered hCAP18 cDNA under the control of the cytomegalovirus promoter (LL-Probi-H1:hCAP18) to the host cells. We investigated whether the alleviation of symptoms could be explained through modification of the host gut microbiota by hCAP18. Feces from mice were collected at day 7, before DNBS colitis induction, for 16S DNA sequencing. Total bacterial DNA was extracted from mouse fecal samples according to the protocol described by Godon et al. (Godon et al., Appl Environ Microbiol., 63(7):2802-13, 1997). The V3-V4 hyper-variable region of the 16S rRNA gene was amplified by using the following primers: forward primer CTTTCCCTACACGACGCTCTTCCGATCTACGGRAGGCWGCAG and reverse primer GGAGTTCAGACGTGTGCTCTTCCGATCTTACCAGGGTATCTAATCCT. The PCR reactions were carried out using 10 ng of fecal DNA, 0.5 μM primers, 200 µM dNTP, and 0.5 U of the DNA-free Taq-polymerase, MolTaq 16S DNA Polymerase (Molzym). Amplifications started at 94°C for 60 sec, followed by 30 cycles at 94°C for 60 s, 65°C for 60 s, 72°C for 60 s, and finishing with a step at 72°C for 10 min. The resulting PCR products were purified, quantified and sent to the @BRIDGe platform (INRAE, Jouy-en-Josas) for sequencing using Illumina MiSeq technology (Illumina, CA, USA).

本研究旨在借助一株命名为LL-Probi-H1:hCAP18的重组益生菌菌株，该菌株可在巨细胞病毒启动子（cytomegalovirus promoter）的调控下，将人阳离子抗菌肽18（hCAP18）的互补脱氧核糖核酸（cDNA）递送至宿主细胞，以缓解化学诱导型小鼠结肠炎。本研究同时探究hCAP18是否可通过修饰宿主肠道菌群，实现结肠炎症状的缓解。我们于二硝基苯磺酸（DNBS）结肠炎造模前第7天收集小鼠粪便，用于16S脱氧核糖核酸（16S DNA）测序。我们参照Godon等人发表的实验方案（Godon et al., Appl Environ Microbiol., 63(7):2802-13, 1997），从小鼠粪便样本中提取总细菌DNA。我们采用下述引物扩增16S rRNA基因的V3-V4高变区：上游引物为CTTTCCCTACACGACGCTCTTCCGATCTACGGRAGGCWGCAG，下游引物为GGAGTTCAGACGTGTGCTCTTCCGATCTTACCAGGGTATCTAATCCT。PCR反应体系包含10 ng粪便DNA、0.5 μM引物、200 μM脱氧核糖核苷三磷酸（dNTP），以及0.5 U无外源DNA的Taq DNA聚合酶MolTaq 16S DNA聚合酶（Molzym公司）。扩增反应程序设置为：94℃预变性60秒，随后进行30个循环：94℃变性60秒、65℃退火60秒、72℃延伸60秒，最终于72℃终延伸10分钟。扩增得到的PCR产物经纯化、定量后，送至@BRIDGe平台（法国国家农业食品与环境研究院INRAE，茹伊昂若萨），采用Illumina MiSeq测序技术（美国加利福尼亚州Illumina公司）完成测序。