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

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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).