Extracellular Nucleic Acids Secreted by Lactobacillus Regulate TLR9 Expression

Abstract

Objective: The study was designed to determine the immune mechanism of nucleic acid sequences present in the medium filtrate of lactobacillus DM8909 at exponential growth phase. The effect of extracellular nucleic acids on TLR9 on the expression of IL-6 was also investigated.Methods: Three groups were formed as nucleic acid group, CpG-ODN group and control group. Extracellular nucleic acids were examined by TA cloning and sequencing technology. RT-PCR and ELISA were used to detect the expressions of TLR9 and NF-κBp65 in HT-29 cells and the presence of IL-6 in the medium.Results: The nucleic acids detected in the medium filtrate of lactobacillus DM8909 at exponential growth phase were found to be RNAs. A total of 39 RNA sequences were identified as lactobacillus delbrueckii subsp. Bulgaricus with a size of 49 bp, 23S ribosomal RNA and another RNA with a size of 52 bp identified as lactobacillus rhamnosus, ATCC 53103, tRNA-Leu sequence. TLR9 expressions in CpG-ODN and nuclei acid groups were higher compared to the control group (TLR-9 values were 3.2386, 0.4984 and 0.1780 respectively). The same results were obtained for NF-κBp65 where the expressions in CpG-ODN and nucleic acid groups were higher than the control group (relative values were 1.6671, 0.6501 and 0.3094 respectively). Furthermore, IL-6 expressions in CpG-ODN and nucleic acid groups were significantly higher than the control group (P <0.05, (relative values were 23.6735 ± 1.4515, 20.2041 ± 1.6820 and 15.7143 ± 0.7047 respectively).Conclusion: The extracellular nucleic acids in the medium of lactobacillus DM8909 are RNAs and they could elevate the level of IL-6 through the activation of NF-κB and TLR9 in vitro. These molecules have similar immune activity with CpG-ODN.