Human Nod1 Confers Responsiveness to Bacterial Lipopolysaccharides

Abstract

The immune response to microbial pathogens is initiated by recognition of specific pathogen components by host cells both at the cell surface and in the cytosol. While the response triggered by pathogen products at the surface of immune cells is well characterized, that initiated in the cytosol is poorly understood. Nod1 is a member of a growing family of intracellular proteins with structural homology to apoptosis regulators Apaf-1/Ced-4 and a class of plant disease-resistant gene products. Here we show that bacterial lipopolysaccharides, but not other pathogen components tested, induced TLR4-and MyD88-independent NF-B activation in human embryonic kidney 293T cells expressing trace amounts of Nod1. Nod2, another Nod family member, also conferred responsiveness to bacterial components but with a response pattern different from that observed with Nod1. As it was reported for plant diseaseresistant R proteins, the leucine-rich repeats of Nod1 and Nod2 were required for lipopolysaccharide-induced NF-B activation. A lipopolysaccharide binding activity could be specifically coimmunopurified with Nod1 from cytosolic extracts. These observations suggest that Nod1 and Nod2 are mammalian counterparts of plant diseaseresistant gene products that may function as cytosolic receptors for pathogen components derived from invading bacteria.The innate immune system regulates the immediate response to microbial pathogens in multiple organisms including humans. The innate immune response is initiated by recognition of specific pathogen components by host immune cells. Mammalian cells have cell surface receptors and intracellular mechanisms that initiate the defense response against microbial pathogens (1, 2). Toll-like receptors (TLRs) 1 comprise a family of cell surface receptors that are related to the Drosophila Toll protein, a molecule involved in defense against fungal infection in the fly (1). Ten mammalian TLRs have been identified (1). Two members of the family, TLR2 and TLR4, have been better characterized and shown to mediate the response to multiple bacterial cell wall components including lipopolysaccharide (LPS), lipopeptides, peptidoglycans (PGN), and lipoteichoic acid (LTA) (3-7). Mammalian TLRs have multiple leucine-rich repeats in the ectodomain and an intracellular Toll-IL1 receptor domain that mediates a signaling cascade to the nucleus (1). Stimulation of TLR2 and TLR4 leads to the recruitment of the adaptor molecule MyD88 and the serine kinase IL1R-associated kinase, two signaling components that, together with TRAF6, mediate activation of NF-B (1).Plants have several classes of genes that regulate the defense against invading pathogens. An important class of these molecules is termed disease-resistant (R) proteins, and members include both membrane-bound and cytosolic proteins. These are essential for the defense against multiple pathogens including bacteria, fungi, and viruses (8). The cytosolic type of R proteins, which include the tobacco N gene product and up to 200 gene products in Arabinopsis thaliana...

RICK/RIP2 Mediates Innate Immune Responses Induced through Nod1 and Nod2 but Not TLRs

“…N-myristoyl (C 14 ) ␥-D-glutamyl-meso-diaminopimelic acid (iE-DAP; in a modified form denoted KF1B), a Nod1 activator, has been described (17). The amount of Nod1 and Nod2 stimulatory activity in LPS preparations was determined as described (15,18) and given as units per microgram of weight. One unit of the Nod1 and Nod2 stimulatory activity is equivalent to that of 1 ng of synthetic iE-DAP and MDP, respectively.…”

“…These studies suggested that RICK is involved in TLR signaling. However, many preparations of bacterial components contain Nod1-and Nod2-stimulating molecules (4,5,7,15,16) and the presence of such contaminants could also explain reduced TLR signaling in RICK-deficient macrophages. In the present report, we have studied the role of RICK in TLR signaling in macrophages and in the animal.…”

Bacterial c-di-GMP Is an Immunostimulatory Molecule

“…The total RNA was converted to cDNA and QPCR was performed to determine the number of copies of IL-8. For Nod studies, experiments examining the synergistic activation of NF-B by c-di-GMP in cells overexpressing Nod1 or Nod2 were conducted as previously described (32). Briefly, HEK 293 T cells were transfected overnight with 30 ng of Nod1 or Nod2 plus 75 ng of Ig luciferase reporter plasmid.…”

NLRP2, an Inhibitor of the NF-κB Pathway, Is Transcriptionally Activated by NF-κB and Exhibits a Nonfunctional Allelic Variant

“…When indicated, cells were also cotransfected with pcDNA3-p53, pcDNA3-Gfi (both provided by G. Nunez, University of Michigan, Ann Arbor, MI), pRc/CMVp65 (provided by I. Udalova, University of Oxford, U.K.), pcDNA3-Stat3C (17), pcDNA3-E2F1 (18), or pcDNA3 empty vector. In another set of experiments, HEK293T cells were cotransfected with NLRP2 cDNA cloned into pHA-EAK vector (15), 0.25 g of the reporter plasmid pBVIxLuc, containing six NF-B recognition sites within the promoter sequence linked to the luciferase gene (19), and 0.2 g of pRSV-␤-galactosidase. Polymorphic variants of NLRP2 were generated by site-directed mutagenesis of the pHA-EAK-NLRP2 vector (15) with 18-bp primers containing the nucleotide change in the middle of the sequence.…”