Philpott, Dana

Introduction

Epithelial cells comprising mucosal surfaces constitute the first line of defense against microbial pathogens. These cells are in the unique position of being in constant contact with bacteria and bacterial products yet these factors that are normally pro-inflammatory for other cell types do not induce epithelial cells to initiate a defensive response. However, epithelial cells are capable of pro-inflammatory responses since infection with pathogenic bacteria can induce NF-κB and secretion of cytokines. Our findings suggest that a discriminatory system has evolved in these cells based on the inside versus outside presentation of bacterial products known collectively as "pathogen-associated molecular patterns" or "PAMPs". Epithelial cells are refractory to extracellular bacteria and PAMPs yet when presented inside the cell, an inflammatory response is initiated. We have shown that a cytosolic protein called Nod1 (also known as CARD4) plays a role in this intracellular detection system in epithelial cells leading to the activation of pro-inflammatory pathways involving NF-κB.

Nod1 is a member of a new family of mammalian proteins ( photo1 ). Strikingly, this protein is similar to the plant disease resistance proteins "N" from tobacco and "RPS2" of Arabidopsis. Similar to these proteins, Nod1 has an C-terminal leucine-rich repeat (LRR) and a central nucleotide binding site (NBS). Nod1 also possesses an N-terminal CARD domain (for "caspase activating and recruitment domain") found also in the pro-apoptotic proteins, CED4 from C. elegans and APAF-1 in mammals. One homologue of Nod1, Nod2, was recently identified to be the first susceptibility gene involved in the chronic inflammatory bowel disease, Crohn's disease. Surprisingly, mutations in Nod2 are also responsible for another human genetic disease called Blau syndrome in which sufferers also present symptoms of inflammation. This information in conjunction with our findings implicates this family of proteins in the induction of inflammation. Furthermore, our studies suggest the possibility that this family of proteins represents human homologues of the plant disease-resistance proteins, and like their plant counterparts, are also involved in pathogen detection.

Bacterial ligands and potential co-receptors for Nod1 and Nod2. Student: Jérôme Viala. Post-doctoral fellow: Rafika Athman. In collaboration with Stephen Girardin and Philippe Sansonetti, Pathogénie Microbienne Moléculaire.

Our initial studies showed that presentation of bacterial products to the cytosolic compartment of epithelial cells was sufficient to activate Nod1 and Nod2 and initiate pro-inflammatory signal transduction through the induction of NF-κB. Our goal is to now identify the bacterial motifs recognised by Nod1 and Nod2 and to identify possible co-receptors for these bacterial products. Our findings with Nod2 have shown that this molecule is a general sensor of peptidoglycan through the recognition of muramyldipeptide (MDP), the minimal bioactive peptidoglycan motif common to all bacteria. Nod2 is mainly expressed in macrophages, which is consistent with our findings that these cells, but not epithelial cells, can detect microinjected MDP. Moreover, the 3020insC frameshift mutation, the most frequent Nod2 variant associated with Crohn's disease patients, fully abrogates Nod2-dependent detection of peptidoglycan and MDP. Together, these results impact on the understanding of Crohn's disease development. Additionally, the characterization of Nod2 as the first pathogen-recognition molecule that detects MDP will help to unravel the well-known biological activities of this immunomodulatory compound.

Characterisation of the signalling pathway activated down-stream of Nod proteins. Technician: Muguette Jéhanno.

We first implicated a role for the kinase RICK in sensing of invasive Shigella flexneri in the Nod1 pathway ( photo2 ). RICK is a kinase that, like IRAK1 in the TLR/IL-1 pathway, does not require the kinase activity for its ability to interact with IKKγ and activate NF-κB. We showed that RICK associates transiently with Nod1 following entry of S. flexneri and also with the IKK complex. Our goal now is to understand the regulation of the RICK/Nod1 complex and the RICK/IKK complex that is activated following bacterial infection by examining potential signal-induced modifications of RICK.

Role of Nod proteins during bacterial infection in vitro and in vivo. Students: Jérôme Viala and Leticia de Albuquerque Carneiro. In collaboration with Richard Ferrero, Unité Pathogénie Bactérienne des Muqueuses.

In order to test the role of Nod1 in bacterial infection we obtained Nod1-deficient mice from the group headed by Dr. John Bertin at Millennium Pharmaceuticals. Our aims are to 1) test different infection paradigms, including Salmonella typhimurium and Helicobacter pylori and 2) characterise primary intestinal epithelial cells isolated from these mice and infect these cultures in vitro with different bacterial pathogens.

Photo 1. NBS/LRR family of mammalian proteins showing the 8 mammalian proteins characterised to date and the domain structure of these proteins.

Photo 2 Schematic diagram of the Nod1 signaling pathway leading to the activation of NF-κB.

Keywords: Nod proteins; innate immunity; bacterial infection; NF-kappaB; Crohn's disease