Nod1 is an essential signal transducer in intestinal epithelial cells infected with bacteria that avoid recognition by toll-like receptors

Abstract

The transcription factor NF-kappaB in human intestinal epithelial cells plays a central role in regulating genes that govern the onset of mucosal inflammatory responses following intestinal microbial infection. Nod1 is a cytosolic pattern recognition receptor in mammalian cells that senses components of microbial peptidoglycans and signals the activation of NF-kappaB. The aim of these studies was to assess the functional importance of Nod1 in activating NF-kappaB and NF-kappaB proinflammatory target genes in human intestinal epithelium. Human colon epithelial cells that constitutively express Nod1 were used as a model intestinal epithelium. These cells do not signal through Toll-like receptor 4 (TLR4) or respond to bacterial lipopolysaccharide, but they express functional TLR5 and interleukin 1 (IL-1) receptors that signal the activation of NF-kappaB in response to bacterial flagellin or IL-1 stimulation. Stable expression of dominant negative (DN) Nod1 in colon epithelial cells prevented IkappaB kinase and NF-kappaB activation in response to infection with enteroinvasive Escherichia coli. In contrast, DN Nod1 did not eliminate IL-1 or flagellin-stimulated NF-kappaB activation. Inhibition of NF-kappaB was accompanied by inhibition of NF-kappaB target genes that provide signals for the mucosal influx of neutrophils during intestinal infection. We conclude that signaling through Nod1 is required for activating NF-kappaB in human intestinal epithelial cells infected with gram-negative enteric bacteria that can bypass TLR activation. Signaling through Nod1 provides the intestinal epithelium with a backup mechanism for rapidly activating innate immunity during infection with a group of highly invasive pathogenic gram-negative bacteria.

FIG. 1.

Human colon epithelial cells constitutively express Nod1 mRNA. (A) RT-PCR analysis of Nod1 and β-actin mRNA transcripts in freshly isolated human colon epithelial cells (IEC), seven human intestinal epithelial cell lines, human embryonic kidney cells (HEK293T), HeLa cells, and a human macrophage cell line (U937). (B) Ratio of Nod1 to β actin transcripts constitutively expressed by the same cells, as assayed by real-time RT-PCR. (C) Nod1 mRNA expression by Caco-2 and T84 cells that were not stimulated or were stimulated for 3 h with proinflammatory cytokines, as indicated above the lanes. (D) Nod1 and Nod2 mRNA expression in Caco-2 cells that were not infected or were infected for up to 24 h with enteroinvasive E. coli 029:NM.

FIG. 2.

IKK activation in response to enteroinvasive E. coli infection in wild-type Caco-2 cells and Caco-2 cells expressing DN Nod1. Control Caco-2, Caco-2 cells that express an empty vector (CEV1), and Caco-2 cells that stably express DN Nod1 (CDN10) cells were infected with enteroinvasive E. coli O29:NM, after which IKK activity was determined by an in vitro kinase assay at different times after infection by using a glutathione S-transferase-IκBα fusion protein as the substrate. IKKα levels assessed by immunoblotting revealed equal loading (data not shown).

FIG. 3.

Inhibition of E. coli-induced NF-κB activation in cells that express DN Nod1. (Top panel) Caco-2 cell lines that stably express DN Nod1 (CDN1 and CDN10) or control vector (CEV1) were transiently transfected with a 3XNF-κB-luc reporter construct and either infected with enteroinvasive E. coli O29:NM (solid bars) or not infected (open bars). The data indicate the fold increases in luciferase activity compared to the luciferase activity of uninfected cells. The data are from three independent experiments, and the error bars indicate standard errors of the means. An asterisk indicates that the P value is <0.05 for a comparison with CEV1-infected cells. (Bottom panel) Myc expression was assessed by immunoblotting as an indicator of DN Nod1 expression in cell lysates from uninfected CEV1, CDN1, and CDN10 cells.

FIG. 4.

RelA/p65 immunostaining of control and E. coli-infected cells. Control Caco-2 cells (top row), Caco-2 cells stably transfected with an empty vector (CEV1) (middle row), and Caco-2 cells stably transfected with DN Nod1 (CDN10) (bottom row) were not infected (left column) or were infected with enteroinvasive E. coli O29:NM (middle column) for 45 min and then immunostained with anti RelA/p65 antibody. Staining with an isotype control antibody is shown in the right column.

FIG. 5.

DN Nod1 does not eliminate NF-κB activation in response to stimulation with IL-1 or bacterial flagellin. (A) Control Caco-2 and CDN10 cells were infected with enteroinvasive E. coli O29:NM as described in Materials and Methods or stimulated with IL-1α (20 ng/ml) or were not infected or stimulated (control lanes). NF-κB DNA binding was assessed by an EMSA 45 min after infection or stimulation. (B) Caco-2 and CDN10 cells were stimulated with H7 flagellin (100 ng/ml) for different times or were not stimulated for 60 min, after which NF-κB DNA binding was determined by an EMSA.

FIG. 6.

NF-κB DNA binding in E. coli-infected Caco-2 cells. Caco-2 cells were not infected or were infected with E. coli O29:NM. The specificity of NF-κB DNA binding was assessed by using an excess of a specific oligonucleotide competitor (100× NF-κB) or a nonspecific oligonucleotide competitor (100× AP-1). Supershifts obtained with antibodies to RelB, cRel, p52, p50, and RelA/p65 are indicated on the right.

FIG. 7.

DN Nod1 inhibits NF-κB target gene expression in E coli-infected Caco-2 cells. (A) CEV1, CDN1, and CDN10 cells were infected with enteroinvasive E. coli O29:NM. CXCL8 (open bars) and CXCL5 (solid bars) mRNA levels were assessed by real-time PCR 4 h after infection. The results are from three independent experiments, and the error bars indicate standard errors of the means. An asterisk indicates that the P value is <0.05 for a comparison with CEV1-infected cells. (B) CEV1 cells (open bars) and CDN10 cells (closed bars) were infected with E. coli O29:NM for 1 h, and CXCL8 secretion in culture supernatants was assessed at different times after infection. The results are from three or more independent experiments, and the error bars indicate standard errors of the means. An asterisk indicates that the P value is <0.05 for a comparison with CEV1-infected cells.