> Pathogénie microbienne moléculaire - INSERM U389
• Summary
• Saga Shigella
• Objectives
• Genetics
• Cell Biology
• Immunology
• Vaccines
3 – Rupture, invasion and inflammatory destruction of the epithelial barrier by Shigella: the Yin and Yang of innate immunity.
Permanent scientists : Philippe Sansonetti (Professeur à l’Institut Pasteur), Régis Tournebize. (Chargé de Recherche INSERM), Laurence Arbibe (Chargé de Recherche INSERM).
Post-doctoral scientist : Stephen Girardin.
Engineer: Thierry Pédron.
A unique and detailed analysis of how a pathogen may subvert the complex, integrated structure of an epithelial barrier has been provided, thanks to the studies carried out with Shigella. For this in vitro and in vivo assays have been developed to model the epithelial barrier. A paradigm has been unravelled in which Shigella uses inflammation to destabilise the epithelial barrier and to facilitate its invasion of the intestinal tissue. The cost for the host is an even increased inflammatory response, which causes severe tissue destruction, but eventually eradicates the microorganisms ( , ). Two discoveries have dominated this area: the demonstration that Shigella cause apoptosis of macrophages ( ) which represents a pro-inflammatory process, and the demonstration of an intracellular system of microbial sensing in epithelial cells leading to their re-programming to produce pro-inflammatory mediators ( ).
M-cells of the follicle-associated epithelium (FAE) act as a « Troyan Horse » permitting Shigella to cross the epithelium. Central role of macrophage apoptosis in bacterial survival and initiation of inflammation.
Identifying the initial entry site of a pathogen is essential to understand the entire chain of the infectious process, but also to better define strategies for vaccine development. Crossing of the FAE ( ) through M cells, first suggested from data obtained in monkeys, was confirmed in a rabbit model of intestinal invasion ( , ). The demonstration that Shigella enters its host through the inductive sites of mucosal immunity is a strong incentive to use this organism in a genetically attenuated form, as a vaccine.
Phagocytosis of the incoming bacteria by macrophages is the immediate consequence of FAE being crossed. Following demonstration of the rapid killing of macrophages infected in vitro by Shigella ( ), it was showed that this killing process corresponded to apoptosis of the macrophages ( ) and occurred massively in vivo ( ). This was the first demonstration of a bacterial pathogen causing apoptosis of a phagocyte. Apoptosis of macrophages appears to be caused by the Shigella protein IpaB ( ) and, unexpectedly, results in the initiation of a pro-inflammatory cascade leading to the massive release of IL-1b ( ). This led to discover that IpaB was activating caspase-1 (a homologue to Ced3 of Caenorhabditis elegans), with two consequences: (i) maturation and release of IL-1b and IL-18, (ii) and apoptosis of the infected cell ( ). IL-1b and IL-18 play antagonistic roles. IL-1b induces the rupture of tissue barriers and facilitates bacterial diffusion, while IL-18 programs the eradication of Shigella through IFNb induction ( , ).
Diffusion of inflammation and bacterial invasion, the central role of epithelial cells.
In an ongoing approach at understanding how Shigella invade and cause the inflammatory destruction of the intestinal epithelial barrier, the essential role plaid by the infected epithelial cell itself has been confirmed, as well as the key functions of IL-8, IL-1bband TNFb. The strategy combines in vitro and in vivo approaches. Under the effect of pro-inflammatory cytokines, and in the presence of apical LPS that they absorb ( , ), but even more, once invaded by Shigella, ( ), epithelial cells produce massive amounts of pro-inflammatory mediators, particularly IL-8. This chemokine attracts polymorphonuclear cells that disrupt the permeability of the epithelial barrier and allow access of apical bacteria to the baso-lateral pole of the epithelium that is permissive for invasion ( , ). Intracellular Shigella cause the activation of NF-bB and the stress-associated kinase JNK. Nod1, a member of a growing family of modular cytoplasmic proteins with C-terminal leucine repeats and N-terminal caspase-activating (CARD) domains, is able to sense bacterial patterns and to cause activation of NF-bB in a RICK-dependent manner ( ). This is the first demonstration that eukaryotic cells can recognize bacteria intracellularly. Nod proteins, are mammalian homologues of plant resistance proteins. It appears that this newly recognised signalling system is a cytosolic equivalent of Toll-Like Receptors (TLR).
Global transcriptional analysis of epithelial cells infected by Shigella has been carried out with the AFFYMETRIX system and defined a transcriptional profile that confirms the essentially pro-inflammatory orientation taken by invaded epithelial cells. In an in vivo model, the level of endotoxicity of the lipid A plays a major role in the rupture of the epithelial barrier. This was demonstrated following mutagenesis of the two msbB genes whose products carry out additional acylation of the lipid A. Complete connection between in vitro and in vivo observations now needs to be established ( ).
Beyond the specific understanding of its pathogenesis, Shigella has proven to be a great tool for studying basic principles of intestinal inflammation and further understanding of Shigella-host interactions may provide some insights into the pathogenesis of inflammatory bowel diseases (i.e. Crohn’s Disease and Ulcerative Colitis), and, possibly, provide potential therapies for these diseases.
In order to better analyze the dynamics of infectious processes in vivo, we are developing methods for real time analysis of infectious processes, based on magnetic resonance imaging (MRI) and intravital microscopy. In collaboration withe the MRI group of Jean-Claude Beloeil (CNRS, Gif), the proper conditions for imaging the kinetics of development of a pneumonia have been established.