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INFLAMMATION/IMMUNITY/MEDIATORS

Lipopolysaccharide activates innate immune responses in murine intestinal myofibroblasts through multiple signaling pathways

¹Department of Medicine and ²Center for Gastrointestinal Biology, University of North Carolina, Chapel Hill, North Carolina

Submitted 16 January 2008 ; accepted in final form 3 January 2009

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-B p65, I-B, Akt, p38 MAP kinase, and cyclooxygenase-2 (COX-2). LPS induced rapid phosphorylation of NF-B p65, Akt, and p38 MAPK and degradation of I-B. 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-B 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-B, 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.

intestinal mucosa; Toll-like receptor-4; innate immunity