K. Madsen, H, Jijon, H. Yeung, C. McKaigney, *C. de Simone. University of Alberta, Edmonton, Canada, *University of L’Aquila, L’Aquila, Italy.
Immune cells can distinguish prokaryotic DNA from vertebrate DNA by detecting unmethylated CpG dinucleotides. Bacterial DNA containing these CpG motifs can activate both innate and acquired immune responses, but it is not known if DNA from different bacterial species elicits differential immune responses.
In addition, intestinal epithelial cells (IECs) are key components of the mucosal immune system, but it remains to be shown if IEFs also participate in bacterial DNA recognition. Aim: The aim of this study was to examine the effect of DNA from probiotic and pathogenic bacteriaoncytokine release from epithelial cells and murine splenocytes.
Methods: DNA from VSL#3 (L. plantarum, L. delbrueckii subsp. Bulgaricus, L. casei, L. acidophilus, B. breve, B. longum, B. infantis, Streptococcus salivarius subsp. Thermophilus) or DNA from the pathogenic bacteria, Salmonella typhimurium was incubated with murine intestine for 24 hrs in the prsence and absence of LPS. IFNg levels in the supernatant were measured by ELISA. To determine if probiotic bacterial DNA could down-regulate an epithelial response to pro-inflammatory stimuli, live VSL#3 bacteria (103-107 cfu/ml) or isolated DNA (10 mg/ml) was applied to confluent HT-29 cells for 1 hr, followed by Salmonella typhimurium, TNF-a or IL-1. NF-?B-activation and IL-8 release into the supernatant were measured by EMSA and ELISA respectively. Splenocytes were isolated from IL-10 deficient mice and stimulated with fecal bacterial sonicates in the presence and absence of VSL#3 DNA or E. coli DNA. IFNg and IL-12 release were measured by ELISA. Activation of NF-kB was examined using an EMSA
. Results: In intestinal tissue, the presence of VSL#3 DNA, but not S. typhimuirum DNA, resulted in a 45% and 50% reduction in basal and LPS-stimulated IFN-g secretion respectively (n=5; p<0.05). In HT-29 monolayers, VSL#3 and VSL#3 DNA resulted in a significant (n=9;p<0.05) dose-dependent attenuation of IL-8 secretion (12-83%) and NF-kB activation in response to S. typhimurium, TNF-a, and IL-1. In splenocytes, VSL#3 DNA, but not E. coli DNA, attenuated stimulated IFN-g secretion by 98% and IL-12 by 64% (p<0.01).
Conclusions: Immune and epithelial cells can recognize and respond to DNA from probiotic bacteria with a down-regulation of pro-inflammatory cytokine secretion and, in IECs, an attenuation of the NF-kB signal pathway. This suggests a new mechanism for epithelial recognition and response to different bacterial species.
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