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     Innate Host Defense and Inflammation - Inserm E336

  Director : CHIGNARD MICHEL (chignard@pasteur.fr)



Our studies concern the innate defense and inflammation of the lung and are based on different in vitro approaches as well as animal models. In the context of infection, we investigate the role of epithelial cells, alveolar macrophages, polymorphonuclear neutrophils and monocytes. A specific endeavor is focused on different molecules such as the Toll-like receptors (TLRs), the urokinase receptor (CD87) and also the phospholipases A2 (PLA2) (see figure).



The lung is the site of various diseases for which mechanisms of innate defense and inflammation play a major role. Acute respiratory distress syndrome (ARDS), chronic obstructive pulmonary disease (COPD), cystic fibrosis or infectious pneumonias from bacterial, fungal or viral origins, are typical lung pathologies. The induction of innate defense is a beneficial process but its exacerbation may lead to a pathologic inflammatory status. Therefore, the major aim of our research is to contribute to the qualitative and quantitative understanding of the mechanisms involved in these diseases, which would allow to target the events enhancing innate immunity without exacerbating the inflammatory process.

Cellular interactions in pulmonary airspaces (Michel Chignard, Dominique Pidard and Mustapha Si-Tahar)

Among the microbial stimuli that trigger an innate immune response, we focused on LPS, a cell wall component of Gram-negative bacteria and on double-stranded RNA (dsRNA), a replicative intermediate of viruses. LPS is a potent activator of TLR4 (and/or TLR2 for certain species) while dsRNA is a strong agonist of TLR3. The in vivo role of those TLRs in defense against microorganisms such as the opportunistic Gram- bacteria Pseudomonas aeruginosa and the influenza A virus (IAV) were not well-studied. In that context, we first assessed the role of LPS as it is exposed by intact P. aeruginosa by evaluating the susceptibility of mice lacking TLR4 or both TLR2 and TLR4 in a model of acute Pseudomonas pneumonia. These mutant mice were not hypersusceptible to the Pseudomonas challenge and mounted an effective innate response that cleared the organism despite low levels of TNF-alpha and KC in the airways. Mice deficient in MyD88, an adaptor molecule associated to the signaling of all TLRs but TLR3 were, however, hypersusceptible, with 100% of mice dying within 48 h with a lower dose of P. aeruginosa. Thus, the susceptibility of mice to P. aeruginosa acute lung infection does not go through TLR2 or TLR4, implying that Pseudomonas LPS is not the most important virulence factor in acute pneumonia caused by this organism (collaboration with the Unité de Recherche et d'Expertise Histotechnologie et Pathologie).

Next, we focused on IAV, the etiological agent of highly contagious acute respiratory disease that causes epidemics and considerable mortality annually. Previously, we demonstrated, using an in vitro approach, that TLR3 plays a key role in the immune response of lung epithelial cells to IAV. In view of these data and the fact that the functional role of TLR3 in vivo is still debated, we designed an investigation to better understand the role of TLR3 in the mechanisms of IAV pathogenesis and host immune response, using an experimental murine model. First, we found that the pulmonary expression of TLR3 is constitutive and markedly up-regulated following IAV infection in control mice. Notably, when compared to wild-type mice, infected TLR3-/- animals displayed significantly reduced inflammatory mediators, including RANTES, IL-6 and IL-12p40/p70 as well as a lower number of CD8+ T lymphocytes in the bronchoalveolar airspace. More importantly, in spite of a persistent viral production in the lungs, mice deficient in TLR3 had an unexpected survival advantage. Hence, our findings suggest that TLR3-IAV interaction critically contributes to the debilitating effects of a detrimental host inflammatory response.

An experimental model of the innate immune response of the lung to infection induced by Aspergillus fumigatus, a fungus responsible for invasive pulmonary aspergillosis (IPA) in immunosuppressed patients, was also set up in mice. Our approach was centered on the evaluation of different parameters of the innate response during the progression of IPA as a function of the expression of different TLRs. We showed that TLR2 plays an important role in the immune response of the host to A. fumigatus. Thus, during in vivo infection, the respiratory distress and the pathogen burden were higher in the TLR2-deficient mice and their survival was shorter (collaboration with the Unité des Aspergillus and the Unité de Recherche et d'Expertise Histotechnologie et Pathologie). We also showed that alveolar macrophages from TLR2-deficient mice produced less cytokines and chemokines than those from wild-type animals in response to the fungus. Along with macrophages, we have started to study more specifically the response of respiratory epithelial cells (human cell lines) to A. fumigatus infections.

Archetypal molecules of the innate defense against pathogens are the antimicrobial peptides (AMPs). We have started a research project looking at the production of AMPs by respiratory epithelial cells challenged by different TLR agonists. Different cationic peptides potentially bearing antimicrobial activities, have been identified by a proteomic approach (collaboration with platform of Protein Microsequencing and Analysis).

During the process of infection and inflammation in the lungs, proteinases released by either or both the host cells (alveolar macrophages, polymorphonuclear neutrophils, epithelial cells) and bacterial pathogens (such as P. aeruginosa in cystic fibrosis patients) play a major role as effectors of immunity, but also as deleterious factors in various pathologies, such as ARDS and cystic fibrosis. A membrane receptor ubiquitously expressed in the lung, the urokinase receptor (uPAR/CD87), has an important place in the recruitment of inflammatory cells and in tissue repair. Its activity is known to be regulated by proteinases, either positively (expression of a chemotactic motif) or negatively (loss of functional domains for cell adherence). We have (i) analyzed the capacity of proteinases secreted by leucocytes, epithelial cells or bacteria to cleave CD87, (ii) identified for each of them the cleavage sites (collaboration with the Platform of Proteomics), and (iii) started to investigate the impact of the cleavage on some of the receptor functions (cell adherence and chemotaxis). We have also established that a soluble form of CD87 is present in the bronchoalveolar lavage fluid (BAL) of patients with ARDS, which concentration is related to the presence of certain leucocyte proteinases.

Mechanisms of regulation and roles of phospholipases A2 in lung inflammatory diseases (Lhousseine Touqui)

PLA2s are a wide family of enzymes that catalyze the hydrolysis of phospholipids at the sn-2 position, generating lysophospholipids and free fatty acids. Besides these fatty acids, arachidonic acid (AA) is the precursor of prostaglandins which are produced by cyclooxygenase (COX) existing in two different forms, a constitutive one (COX-1) and an inducible one (COX-2). Prostaglandins play a major role in the modulation of lung inflammation. Clinical studies showed higher concentrations of AA metabolites in BAL of patients with cystic fibrosis and ARDS as compared to normal individuals.

Mammalian PLA2s comprise cytosolic PLA2 (cPLA2) and secreted PLA2 (sPLA2). The latter class is composed by various types including among others the sPLA2-IIA. sPLA2-IIA is involved in the pathogenesis of various inflammatory diseases and in innate defense against pathogenic bacteria. We showed that sPLA2-IIA is able to kill germinated spores of Bacillus anthracis, the etiological agent of anthrax. An anthracidal activity linked to sPLA2-IIA was found in BAL of ARDS patients and medium of alveolar macrophages, the major source of this enzyme in lung tissues. Interestingly, the synthesis of sPLA2-IIA by these cells is significantly inhibited by the B. anthracis lethal toxin at a transcriptional level by an unknown mechanism. Finally, we showed that transgenic mice overexpressing sPLA2-IIA or wild type mice treated with this enzyme survive to infection by B. anthracis infection, in contrast to untreated wild type mice that died within 3 days after infection. These results suggest that sPLA2-IIA plays a role in host defense against B. anthracis infection and that the lethal toxin may help the bacteria to escape from the bactericidal activity of sPLA2-IIA by inhibiting the production of this enzyme.

In parallel, we examined the potential role of AA and its metabolites in lung inflammation in cystic fibrosis. We showed that the human respiratory epithelial cell line CFT-2 which bears the ÆF508 mutation on CFTR, released more AA than the control cell line, NT-1. This release was accompanied by an enhanced PGE2 release and sPLA2-IIA expression by CFT-2 as compared to NT-1 cells during stimulation with P. aeruginosa LPS. We also observed that intranasal instillation of this LPS to mice induced an increase of PGE2 concentrations in BAL that was higher in CFTR-/- mice than in littermate controls. We established a link between the ÆF508 mutation of CFTR and the overproduction of PGE2 by epithelial cells and showed that this overproduction was not due to the mutation per se but to the resulting absence of CFTR in plasma membrane of cells bearing this mutation. Indeed, pharmacological approaches that induce CFTR membrane translocation reduced PGE2 production to values similar to those of control NT-1 cells. Our results showed that a relationship exists between CFTR mutation and overproduction of PGE2 and suggested that this process may play a role in the exacerbation of lung inflammation in patients with cystic fibrosis. Studies are in progress to investigate the role of different PLA2 and prostaglandins in mucus hypersecretion by lung of CFTR -/- mice. Indeed, mucus abundance associated to absence of mucociliary clearance are key processes in the pathogenicity of cystic fibrosis.

Keywords: innate immunity/inflammation, infection, epithelial cells, leukocytes, Toll-like receptors, proteinases, phospholipases A2, CD87, pneumopathy, cystic fibrosis


puce Publications 2005 of the unit on Pasteur's references database


  Office staff Researchers Scientific trainees Other personnel
  VILLENEUVE Josiane (jvillene@pasteur.fr) CHIGNARD Michel, INSERM, (DR1, chignard@pasteur.fr)

PIDARD Dominique, CNRS, (CR1, dpidard@pasteur.fr)

SI-TAHAR Mustapha, INSERM, (CR1, sitahar@pasteur.fr)

TOUQUI Lhousseine, IP, (Chef de Laboratoire, touqui@pasteur.fr)

BOUTILLON Florence, Bachelor degree

BEAUFORT Nathalie, PhD student

CHIOU Shean-Jaw, Visiting Professor

DIF Fariel, Post-doctoral fellow

LABEGUERIE Marylène, Master 2

LE GOFFIC Ronan, Post-doctoral fellow

MEDJANE Samir, PhD student

MOUBARECK Carole, Post-doctoral fellow

RAYMOND Benoît, PhD student

BALLOY Viviane, (Assistant-Ingeneer, INSERM, vballoy@pasteur.fr)

LEDUC Dominique, (Technician, IP, dleduc@pasteur.fr)

Activity Reports 2005 - Institut Pasteur

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