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Human foetal intestinal fibroblasts are hyper-responsive to lipopolysaccharide stimulation

Okogbule-Wonodi, AC;Li, G;Anand, B;Luzina, IG;Atamas, SP;Blanchard, T;

Intestinal myofibroblasts contribute to immune regulation in adults with inflammatory bowel disease but have not been characterised in neonatal intestinal inflammatory diseases. To compare lipopolysaccharide (LPS)-stimulated interleukin-8 (IL-8) production between human foetal and mature intestinal myofibroblasts in vitro. Foetal, neonatal and adult cells were stimulated with increasing concentrations of E. coli LPS. In LPS stimulated foetal myofibroblasts, Toll-like receptor 4 mRNA expression was assessed by real-time PCR whilst Toll-like receptor 4 receptor activity was determined using anti-Toll-like receptor 4 antibody. Mitogen activated protein kinase pathway activity was assessed using chemical inhibitors and Western blotting. IL-8 production was measured by quantitative ELISA. IL-8 production by LPS stimulated foetal myofibroblasts occurred in a dose dependent manner. Toll-like receptor 4 expression was constitutive and Toll-like receptor 4 receptor blockade reduced IL-8 production by 42% (P=0.0262). C-Jun N-terminal kinase, p38 and NF-B inhibitors significantly attenuated LPS stimulated IL-8 production by 42%, 33% and 2%, respectively. Mitogen activated protein kinase activity was confirmed by the presence of phosphorylated proteins on Western blots. These data demonstrate increased IL-8 production by foetal myofibroblasts that is partially mediated by Toll-like receptor 4, mitogen activated protein kinase and NF-B cell signalling pathways. Intestinal myofibroblasts cells may contribute to the dysregulated inflammatory response in the immature intestine and may form targets that lead to new therapies to prevent neonatal intestinal inflammatory bowel diseases.