Introduction: Tolerance to intestinal microbiota is one of the key aspects of gut homeostasis. In fact, intestinal disorders such as necrotizing enterocolitis and inflammatory bowel diseases may result from the failure to establish or maintain tolerance to luminal bacteria. Thus, understanding the pathogenesis of these diseases critically depends on our ability to define the mechanisms that govern mucosal tolerance. In this study, we sought to elucidate the role of MKP-1, the mitogen-activated kinase (MAPK) phosphatase that negatively regulates inflammatory signaling via MAPK, in tolerance to toll-like receptor (TLR) ligands in enterocytes.

Methods: Enterocyte cell lines were treated with various TLR ligands for 0-4 h. Expression of MKP-1, phosphorylation of p38, and degradation of inhibitory subunits (IkB) of nuclear factor kappaB (NF-kB) were examined by Western blotting. MKP-1 was localized intracellularly using immunofluorescence microscopy. Binding of NF-kB and activation of MKP-1 promoter were examined using chromatin immunoprecipitation and transcriptional reporter.

Results: Ligands of TLR3 (dsRNA), TLR4 (lipopolysaccharide, LPS), TLR5 (flagellin, Fla), and TLR9 (CpG DNA), but not TLR2 (peptydoglycan, Pgn), transiently induce MKP1, coincident with dephosphorylation of p38 following peak TLR ligand-induced phosphorylation, and preceded by transisent degradation of IkB (Figure). Inhibitors of NF-kB, but not MAPK, block LPS-induced expression of MKP-1, whereas siRNA knockdown of IkB? prolongs expression of MKP-1. Rat MKP-1 promoter contains two NF-kB-binding sites. Mutational inactivation of these sites abrogates LPS-induced transcription from the MKP-1 promoter. In the small intestine, MKP-1 is expressed in the crypts, the epithelial compartment that also displays bacteria-dependent activating phosphorylation of p38.

Conclusion: TLR ligand induced, NF-kB-mediated expression MKP-1 may promote rapid establishment of tolerance to TLR ligands in enterocytes via deactivation of p38.