Effects Of Lactobacillus Rhamnosus On β-defensin9Expression And Its Signal Transduction Pathway In Chicken Small Intestinal Epithelial Cells

Endogenous antimicrobial peptides with broad-spectrum antibacterial action exists widely in respiratory, digestive, and reproductive tract of the animal body, plays an important role in the animal’s innate and acquired immunities. Avian B-defensin9(AvBD9), an important antimicrobial peptide expressed in chicken tissues, especially in the digestive tract and immune tissues, plays an important role in chicken anti-infection immunity. Lactobacilli as the dominant microflora in the small intestine, peptidoglycan, lipoteichoic acid etc of its surface can be specifically identified by Toll-like receptors (TLRs) of intestinal epithelial cells (IEC), and then activating the downstream signaling pathways regulating the immune response.In this study, five cellular components:the whole cell wall peptidoglycan (WPG), lipoteichoic acid (LTA), whole cell wall composition (WCW), the cell wall protein (CWP), Lactobacilli culture supernatant (S) were extracted from Lactobacillus rhamnosus (L. rhamnosus) MLGA. AvBD9gene expression in primary cultured chicken IEC stimulated by five cellular components from L. rhamnosus MLGA were measured by quantitative PCR. The results showed that compared with the control group, the cellular components from L. rhamnosus MLGA significantly promoted AvBD9expression with different potential between five cellular components. WPG had the most strong potential in inducing AvBD9expression in chicken IEC among the five cellular components, and WPG promoted AvBD9expression in a dose-and time-dependent manner.The possible signal transduction pathway of AvBD9gene expression induced by L. rhamnosus MLGA and its cellular component-WPG was futher explored using anti-TLR2specific antibody blocking TLR2, electrophoretic mobility shift assay (EMSA), specific inhibitors to block signaling pathways and other methods. L. rhamnosus MLGA, heat-inactivated L. rhamnosus MLGA and its cell wall component WPG improved TLR2mRNA expression in different extent. AvBD9mRNA expression levels were decreased to different extents in each group with the biggest decline degree in WPG group when TLR2was blocked by specific anti-TLR2antibody. Result of EMSA showed that the activities of nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) and activator protein1(AP-1) were increased by stimulation with L. rhamnosus MLGA, heat-inactivated L. rhamnosus MLGA and its cell wall component WPG with peptidoglycan group being strongly positive. AvBD9induction by L. rhamnosus MLGA, heat-inactivated L. rhamnosus MLGA and its cell wall componentWPG was blocked or reduced by treatment of the chicken IEC with SP600125, a selective inhibitor of c-Jun N-terminal kinase (JNK) which is one of mitogen-activated protein kinase (MAPK) signal pathway molecules, and PDTC, a selective inhibitor of NF-κB. Furthermore the inhibition extent of AvBD9expression by PDTC was bigger than by SP600125. In conclusion, L. rhamnosus MLGA, heat-inactivated L. rhamnosus MLGA and its cell wall componenWPG-induced AvBD9gene expression in chicken IEC was mediated by TLR2-NF-κB/AP-1signaling pathway with the TLR2-NF-κB being the main signal transduction pathway.