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) beta1. Nuclear factor kappaB (NFkappaB) pathway activation was assessed by inhibitory kappaBalpha (IkappaBalpha) degradation and NFkappaB promoter activity. Fibroblast contractility was measured using a fibroblast-populated collagen lattice. Smad-7, a negative regulator of TGF-beta1 signalling, and connective tissue growth factor (CTGF) expression were assessed using reverse transcriptase-polymerase chain reaction and western blot. The NFkappaB pathway was inhibited by IkappaBalpha transfection.


RESULTS
TLR-4 was present on the surface of intestinal fibroblasts. LPS treatment of fibroblasts induced IkappaBalpha degradation, enhanced NFkappaB promoter activity and increased collagen contraction. Pretreatment with LPS (before TGF-beta1) significantly increased CTGF production relative to treatment with TGF-beta1 alone. LPS reduced whereas TGF-beta1 increased smad-7 expression. Transfection with an IkappaBalpha plasmid enhanced basal smad-7 expression.


CONCLUSIONS
Intestinal fibroblasts express TLR-4 and respond to LPS by activating NFkappaB and inducing collagen contraction. LPS acts in concert with TGF-beta1 to induce CTGF. LPS reduces the expression of the TGF-beta1 inhibitor, smad-7.
Burke
J P
JP
Department of Surgery, St Vincent's University Hospital, Dublin, Ireland.
Cunningham
M F
MF
Watson
R W G
RW
Docherty
N G
NG
Coffey
J C
JC
O'Connell
P R
PR
D000368:Aged; D000369:Aged, 80 and over; D002478:Cells, Cultured; D003110:Colonic Neoplasms / Q000378:metabolism; D055513:Connective Tissue Growth Factor / Q000096:biosynthesis; D005347:Fibroblasts / Q000187:drug effects* / Q000378:metabolism; D006801:Humans; D051550:I-kappa B Kinase / Q000378:metabolism; D008070:Lipopolysaccharides / Q000494:pharmacology*; D008875:Middle Aged; D016328:NF-kappa B / Q000378:metabolism; D051906:Smad7 Protein / Q000378:metabolism; D051197:Toll-Like Receptor 4 / Q000378:metabolism*; D053773:Transforming Growth Factor beta1 / Q000494:pharmacology*; D014945:Wound Healing / Q000502:physiology*
D008070:Lipopolysaccharides; D016328:NF-kappa B; D051906:Smad7 Protein; D051197:Toll-Like Receptor 4; D053773:Transforming Growth Factor beta1; D055513:Connective Tissue Growth Factor; D051550:I-kappa B Kinase