Bacterial lipopolysaccharide promotes profibrotic activation of intestinal fibroblasts

Background: Fibroblasts play a critical role in intestinal wound healing. Lipopolysaccharide (LPS) is a cell wall component of commensal gut bacteria. The effects of LPS on intestinal fibroblast activation were characterized. Methods: Expression of the LPS receptor, toll-like receptor (TLR) 4, was assessed in cultured primary human intestinal fibroblasts using flow cytometry and confocal microscopy. Fibroblasts were treated with LPS and/or transforming growth factor (TGF) beta 1. Nuclear factor kappa B (NE kappa B) pathway activation was assessed by inhibitory kappa B alpha (I kappa B alpha) degradation and NF kappa B promoter activity. Fibroblast contractility was measured using a fibroblast-populated collagen lattice. Smad-7, a negative regulator of TGF-beta 1 signalling, and connective tissue growth factor (CTGF) expression were assessed using reverse transcriptase-polymerase chain reaction and western blot. The NF kappa B pathway was inhibited by I kappa B alpha transfection. Results: TLR-4 was present on the surface of intestinal fibroblasts. LPS treatment of fibroblasts induced I kappa B alpha degradation, enhanced NF kappa B promoter activity and increased collagen contraction. Pretreatment with LPS (before TGF-beta 1) significantly increased CTGF production relative to treatment with TGF-beta 1 alone. LPS reduced whereas TGF-beta 1 increased smad-7 expression. Transfection with an I kappa B alpha plasmid enhanced basal smad-7 expression. Conclusion: Intestinal fibroblasts express TLR-4 and respond to LPS by activating NF kappa B and inducing collagen contraction. LPS acts in concert with TGF-beta 1 to induce CTGF. LPS reduces the expression of the TGF-beta 1 inhibitor, smad-7.