Effect of Vibrio cholerae on chemokine gene expression in HT29 cells and its modulation by Lactobacillus GG.
Epithelial cells participate in the innate immune response to pathogenic bacteria by elaborating chemokines. This study examined the effect of Vibrio cholerae and Lactobacillus rhamnosus GG on inflammatory chemokine gene expression in the HT29 human intestinal epithelial cell line. HT29 cells were exposed to V. cholerae 0139, Lactobacillus or both for 2 h and cultured further thereafter for 4 h. RNA was extracted from the cells and expression of genes for chemokines and related molecules was quantitated by real time PCR using a pathway-focused PCR array. TLR4 was silenced using shRNA and output of interleukin-8 (IL-8) into the media quantitated with and without V. cholerae exposure. NFkappaB and p38 MAP kinase activation were determined by immunoblotting for IkappaBalpha and phosphorylated p38. Vibrio cholerae significantly upregulated gene expression for the neutrophil chemoattractant CXCL chemokines, IL-8, CXCL and CXCL in HT29 cells, while downregulating the expression of macrophage-attracting C-C chemokines. TLR4 silencing did not reduce IL-8 output from HT29 cells in response to V. cholerae. IkappaBalpha degradation was noted in the HT29 cells soon after exposure to V. cholerae and this recovered over time after removal of bacteria. p38 MAP kinase activation was not noted. Vibrio cholerae upregulated the expression of neutrophil attractant chemokines, most prominently IL-8, in HT29 cells, but downregulated macrophage-attracting chemokines. Probiotic lactobacilli modulated the IL-8, but not the other chemokine gene changes, in response to V. cholerae.
Nandakumar
N S
NS
Department of Gastrointestinal Sciences, Christian Medical College, Vellore, India.
Pugazhendhi
S
S
Madhu Mohan
K
K
Jayakanthan
K
K
Ramakrishna
B S
BS
D015153:Blotting, Western; D018925:Chemokines / Q000096:biosynthesis* / Q000235:genetics* / Q000276:immunology; D002771:Cholera / Q000235:genetics / Q000276:immunology* / Q000382:microbiology; D015536:Down-Regulation; D004789:Enzyme Activation; D015870:Gene Expression; D019073:HT29 Cells; D006801:Humans; D052201:Lacticaseibacillus rhamnosus / Q000276:immunology*; D016328:NF-kappa B / Q000378:metabolism; D012333:RNA, Messenger / Q000096:biosynthesis / Q000235:genetics; D020133:Reverse Transcriptase Polymerase Chain Reaction; D051197:Toll-Like Receptor 4 / Q000378:metabolism; D015854:Up-Regulation; D014734:Vibrio cholerae / Q000276:immunology*; D048051:p38 Mitogen-Activated Protein Kinases / Q000378:metabolism
D018925:Chemokines; D016328:NF-kappa B; D012333:RNA, Messenger; C495345:TLR4 protein, human; D051197:Toll-Like Receptor 4; D048051:p38 Mitogen-Activated Protein Kinases