Absence of Toll-like receptor 4 explains endotoxin hyporesponsiveness in human intestinal epithelium.

Abstract

The Toll protein in Drosophila regulates dorsal ventral patterning during embryogenesis, and participates in antibacterial and antifungal host defense. Mammalian homologues are termed Toll-like receptors and, to date, nine have been cloned (TLRI-9) in humans. They are characterized by extracellular leucine-rich repeats and a cytoplasmic domain similar to the interleukin 1 receptor. Both TLR2 and TLR4 recognize various bacterial cell wall components including lipopolysaccharide (LPS). This results in the activation of the NFkappaB pathway. Peripheral blood mononuclear cells (PBMCs) express both TLR2 and TLR4. The authors hypothesized that the expression of TLR 2 and TLR4 in human intestinal epithelial cells differs from PBMCs because of the abundance of LPS in the intestinal lumen. Epithelial cells were isolated from Caco-2 cells, fetal gut explants, and small bowel resection specimens using Hanks/ethylenediamine tetraacetic acid solution. PBMCs were used as positive controls. Ribonucleic acid (RNA) was isolated using the TRIzol method. Standard reverse transcription-polymerase chain reaction examined TLR2 and TLR4 messenger RNA (mRNA) expression. NFkappaB expression was determined using a luciferase reporter assay. TLR2 mRNA was highly expressed in PBMCs and was present in all human intestinal epithelial cells. TLR4 mRNA was detected only in PBMCs. TLR4 is not present in epithelium from children with inflammatory bowel disease. In Caco-2 cells, significant NFkappaB activation in response to LPS occurred only in the presence of TLR4 introduced by complementary deoxyribonucleic acid transfection. Absence of TLR4 is associated with endotoxin hyporesponsiveness of intestinal epithelial cells. TLR4 is not directly involved in inflammation of the intestinal epithelium. Although TLR2 is normally present in the epithelial cell, it plays a limited role in inflammation. It may be activated during conditions in which bacterial cell wall concentrations within the intestine are pathologically high.