Lipopolysaccharide activates innate immune responses in murine intestinal myofibroblasts through multiple signaling pathways.
Myofibroblasts (MF) play an important role in intestinal wound healing. A compromised epithelial barrier exposes intestinal subepithelial MF to luminal bacterial products. However, responses of murine intestinal MF to bacterial adjuvants and potential roles of intestinal MF in innate immune responses are not well defined. Our aims in this study were to determine innate immune responses and intracellular signaling pathways of intestinal MF exposed to LPS, a prototypic Toll-like receptor (TLR) ligand. Expression of TLR4 in primary murine intestinal MF cultures was confirmed by RT-PCR and Western blotting. LPS-induced secretion of prostaglandin E2 (PGE2), interleukin (IL)-6, and keratinocyte-derived chemokines (KC) was measured by ELISA. Intracellular responses to LPS were assessed by Western blotting for NF-kappaB p65, Ikappa-Balpha, Akt, p38 MAP kinase, and cyclooxygenase-2 (COX-2). LPS induced rapid phosphorylation of NF-kappaB p65, Akt, and p38 MAPK and degradation of Ikappa-Balpha. LPS induced expression of COX-2 and secretion of PGE2 (2.0+/-0.8-fold induction vs. unstimulated cells), IL-6 (6.6+/-0.4-fold induction), and KC (12.5+/-0.4-fold induction). Inhibition of phosphoinositide-3 (PI3)-kinase, p38 MAPK, or NF-kappaB pathways reduced LPS-induced PGE2, IL-6, and KC secretion. These studies show that primary murine intestinal MF respond to LPS, evidenced by activation of NF-kappaB, PI3-kinase, and MAPK signaling pathways and secretion of proinflammatory molecules. Inhibition of these pathways attenuated LPS-dependent PGE2, IL-6, and KC production, indicating that LPS activates MF by multiple signaling pathways. These data support the hypothesis that MF are a component of the innate immune system and may exert paracrine effects on adjacent epithelial and immune cells by responding to luminal bacterial adjuvants.
Walton
Kristen L W
KL
Department of Medicine, Center for Gastrointestinal Biology, University of North Carolina, Chapel Hill, NC, USA. kwalton1@missouriwestern.edu
Holt
Lisa
L
Sartor
R Balfour
RB
D000818:Animals; D002478:Cells, Cultured; D054360:Chemokine CXCL1 / Q000235:genetics / Q000378:metabolism; D004847:Epithelial Cells / Q000166:cytology / Q000276:immunology / Q000378:metabolism; D005347:Fibroblasts / Q000166:cytology / Q000276:immunology* / Q000378:metabolism; D015870:Gene Expression / Q000276:immunology; D018836:Inflammation Mediators / Q000378:metabolism; D015850:Interleukin-6 / Q000235:genetics / Q000378:metabolism; D007413:Intestinal Mucosa / Q000166:cytology / Q000276:immunology* / Q000378:metabolism; D007421:Intestine, Small / Q000166:cytology / Q000276:immunology* / Q000378:metabolism; D018950:Lipopolysaccharide Receptors / Q000235:genetics; D008070:Lipopolysaccharides / Q000494:pharmacology*; D051218:Lymphocyte Antigen 96 / Q000235:genetics; D051379:Mice; D008809:Mice, Inbred C3H; D019899:Paracrine Communication / Q000276:immunology; D015398:Signal Transduction / Q000276:immunology*; D013047:Specific Pathogen-Free Organisms; D051197:Toll-Like Receptor 4 / Q000235:genetics
D054360:Chemokine CXCL1; C475895:Cxcl1 protein, mouse; D018836:Inflammation Mediators; D015850:Interleukin-6; D018950:Lipopolysaccharide Receptors; D008070:Lipopolysaccharides; C411728:Ly96 protein, mouse; D051218:Lymphocyte Antigen 96; C493487:Tlr4 protein, mouse; D051197:Toll-Like Receptor 4