| Summary: | 碩士 === 臺灣大學 === 生理學研究所 === 98 === Background: Gram (-) bacterial lipopolysaccharide (LPS) induces innate immune responses via recognition by LPS receptor complex (CD14/TLR4/MD2) on cells of monocytic lineage. LPS binds to cell surface CD14 causing activation of a lipid secondary messenger ceramide and PKCζ to recruit adjacent TLR4 into lipid raft domains to form a receptor complex which initiates downstream signaling pathways including MyD88, IκB-α and MAPK families (e.g. JNK, ERK, and p38). In contrast to monocytes, distinct expression patterns of LPS receptors were identified in human enterocytes. The gut lumen normally harbors a large amount of commensal bacteria, for which barrier and suppressive mechanism at the epithelial level is important to downregulate unnecessary inflammatory reactions and to maintain gut homeostasis. Our previous studies indicated that luminal LPS triggered epithelial apoptosis via a ceramide/PKCζ-dependent pathway in human intestinal Caco-2 cells that express only CD14 but not TLR4 proteins. The aim of the current study is to investigate whether luminal LPS challenge and commensal bacteria overgrowth stimulate colonic enterocytic apoptosis in gene-deficient mouse models. Materials and methods: Wild type mice (WT; BALB/c and C57C57BL/6), mice with spontaneous mutation in TLR4 gene (TLR4-m), and mice with targeted mutation (knock out) of CD14 gene (CD14-m) were used. Mouse colonic tissues were mounted on Ussing chambers for luminal challenge with PBS or LPS (obtained from nonpathogenic E.coli; 5 and 50 μg/mL) for 2 hrs. The level of epithelial apoptosis was analyzed by TUNEL assay and cell death ELISA. Phosphorylation levels of PKCζ, IκB-α, JNK and ERK in the colonic mucosa were assessed by immunofluoresenct staining and western blotting. In the next experiment, colons of WT BALB/c and TLR4-m mice were either sham-operated or obstructed by thread ligation for 24 hrs to induce enteric bacterial overgrowth, and the apoptotic levels of colonic enterocytes were determined. Total and G (-) bacterial colony forming units (CFU) in the intestine, liver, and spleen was calculated on fresh blood and MacConkey agar plates. Caco-2 cells were apically exposed to live or dead nonpathogenic E.coli, and the levels of apoptosis and necrosis were examined. Results: Luminal LPS challenge significantly increased the levels of colonic epithelial apoptosis in TLR4-m mice, whereas no effect was seen in WT BALB/c, WT C57BL/6 and CD14-m mice. A slight decrease of LPS-induced mucosal apoptosis levels was seen in TLR4-m colonic tissues pretreated with neutralizing anti-CD14 compared with isotype antibody controls. LPS induced epithelial PKCζ phosphorylation in colonic tissues of WT BALB/c, C57BL/6 and TLR4-m mice. Enhanced mucosal phosphorylation of JNK and IκB-α was evident after luminal LPS challenge in WT C57BL/6 and WT BALB/c mice, but absent in CD14-m and TLR4-m mice. Moreover, colonic obstruction resulted in increase of total and G (-) bacterial counts in the intestines in both WT BALB/c and TLR4-m mice, suggesting obstruction-induced commensal bacterial overgrowth irrespective of mouse strain. Increased proinflammatory cytokines (TNF-α and IFN-γ) in intestinal mucosa was caused by colonic obstruction in BALB/c mice, but not in TLR4-m mice. No difference of mucosal cell apoptotic levels was seen between sham-operation and obstruction groups in WT BALB/c mice. However, a three-fold increase in mucosal cell apoptosis was evident in obstructed guts compared to sham operation in TLR4-m mice. Increased epithelial PKCζ phosphorylation was seen in the obstructed guts of WT BALB/c and TLR4-m mice. Enteric bacterial translocation was evidenced by increased total bacterial CFU in the liver and spleen after colonic obstruction in both WT BALB/c and TLR4-m mice, indicating gut barrier dysfunction. The total bacterial counts in the liver and spleen in obstructed TLR4-m mice were 40 times higher than those of obstructed WT BALB/c mice. However, G (-) bacterial counts in the liver and spleen after sham operation and colonic obstruction were 0 CFU/g in both mouse strains. Luminal challenge with live or dead E.coli induced Caco-2 cell apoptosis in a dose-dependent manner. Conclusions: Luminal LPS challenge and commensal bacterial overgrowth induced colonic epithelial cell apoptosis in mice deficient of TLR4 signaling. LPS-induced epithelial apoptosis may be mediated via CD14/PKCζ-dependent pathways.
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