Unit: Innate Host Defense and Inflammation - Inserm E336
Director: Chignard Michel
Our studies concern the innate defense and the inflammation of the lung and use 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 Toll-like receptors (TLR), the urokinase receptor (CD87) and also secreted phospholipases A2 (PLA2) (see figure).
The lung is the site of various diseases for which the mechanisms of innate defense and lung 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)
Pulmonary cells are continuously exposed to microbial challenges as a result of breathing. For instance, influenza A virus causes pulmonary inflammation and exacerbates chronic lung diseases. Bronchial epithelial cells play an important role in the pathogenesis of this viral infection. It is recognized that immune myeloid cells express TLR, which play a major role in detecting microbes and initiating innate immune responses. In contrast, little is known about the expression of TLR in pulmonary epithelial cells per se, their distribution within the cell, their function as well as the signaling pathways involved. Moreover, while many of the molecular events in influenza A virus replication have been described, the underlying mechanisms by which virus-epithelium interaction triggers the inflammation process have yet to be fully characterized. The discovery of TLR3 as a key receptor for viral double-stranded RNA (dsRNA) led us to investigate the contribution of this receptor to the activation of pulmonary epithelial cells by dsRNA and influenza A virus. We showed: (i) that TLR3 is constitutively expressed in respiratory epithelial cells in an intracellular compartment; (ii) that TLR3 expression is upregulated either by influenza A virus or by purified dsRNA, but not by other major inflammatory mediators such as TNFα and IL-1α (iii) that TLR3 plays a central role in the immune response of bronchial epithelial cells triggered by these stimuli; and (iv) that influenza A virus and dsRNA induce epithelial cell activation through MAPK, PI3-K/Akt signaling and TRIF- but not MyD88-dependent activation of the transcription components NF-κB and IRF/ISRE. Ultimately, this signal transduction elicits an epithelial response that includes the secretion of the cytokines IL-8, IL-6, RANTES and interferon-α and the up-regulation of the major adhesion molecule ICAM-1.
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 Pseudomonas 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 lungs, 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) analysed the capacity of proteinases secreted by leucocytes, epithelial cells or bacterias to cleave CD87, (ii) identified for each of them the cleavage sites (collaboration with the Plate-forme de Protéomique), and (iii) started to investigate the impact of the cleavage on some of the receptor functions (cell adherence and chemotaxis). We 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, and which could bear biological activities, which remain to be evaluated.
Two experimental models of the innate immune response of the lung to infection were set up in mice. First of all, an infection induced by Aspergillus fumigatus, a fungus responsible for invasive pulmonary aspergillosis (IPA) in immunosuppressed patients. Our approach was centered on the evaluation of different parameters of the innate response during the progression of IPA as a function of two different immunosuppressive treatments, namely by a corticosteroid, and by a chemotherapeutic agent. At different time points after infection, host responses were compared in terms of survival, pulmonary production of pro- and anti-inflammatory cytokines, airway leukocyte trafficking, lung injury, respiratory distress and fungal development. It was found that according to the type of immunosuppression, the pathogenesis of IPA involves a predominant role of either the development of the fungus or the adverse response of the host. We also showed that TLR2 plays an important role in the immune response of the host to A. fumigatus. Thus, alveolar macrophages from TLR2-deficient mice produced less cytokines and chemokines than those from wild-type animals in response to the fungus. We also showed that 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). With the second model, we studied the host response to Pseudomonas aeruginosa infection. The same parameters were evaluated and the results evidenced that LPS expressed by these bacteria and recognized as a virulence factor, does not play a role in the death of the animals. Thus, TLR4-deficient mice, as well as TLR2-deficient mice, showed a survival time similar to that of wild-type animals. Nonetheless, the synthesis of different cytokines was suppressed, while that of others was maintained. It is suggested that some patterns expressed by P. aeruginosa are compulsory for its recognition by host cells and trigger the synthesis of specific cytokines that are beneficial for the fight of the infection (collaboration with the Unité de Recherche et d'Expertise Histotechnologie et Pathologie).
Mechanisms of regulation and roles of phospholipases A2 in lung inflammatory diseases (Lhousseine Touqui)
PLA2s are a family of enzymes that catalyze the hydrolysis of phospholipids at the sn-2 position, generating lysophospholipids and free fatty acids, especially arachidonic acid (AA). Mammalian PLA2s can be divided into two major classes according to their molecular mass and location: cytosolic PLA2 (cPLA2) and secreted PLA2 (sPLA2). This class is composed by various types such as sPLA2-I, sPLA2-II, sPLA2-III, sPLA2-V and sPLA2-X. sPLA2-IIA, the best-known enzyme of this group, is involved in the pathogenesis of various inflammatory diseases. We examined the ability of sPLA2-IIA to kill Bacillus anthracis, the etiological agent of anthrax. Our results showed that both germinated B. anthracis spores and encapsulated bacilli were sensitive to the bactericidal activity of recombinant sPLA2-IIA in vitro. In contrast, non-germinated spores were resistant. This bactericidal effect was correlated to the ability of sPLA2-IIA to hydrolyze bacterial membrane phospholipids. Alveolar macrophages, the major pulmonary source of sPLA2-IIA released enough sPLA2-IIA to kill extracellular B. anthracis. The production of sPLA2-IIA in these cells was significantly inhibited by the B. anthracis lethal toxin. Human BALF from ARDS patients are known to contain sPLA2-IIA; Bactericidal activity against B. anthracis was detected in a high percentage of these samples. This anthracidal activity was correlated to the levels of sPLA2-IIA and was abolished by a sPLA2-IIA inhibitor. These results suggest that sPLA2-IIA may play a role in the innate host defense against B. anthracis infection and that the lethal toxin may help the bacteria to escape from the bactericidal action of sPLA2-IIA by inhibiting the production of this enzyme. The mechanisms involved in this inhibition are under investigation.
AA is the precursor of prostaglandins produced by cyclooxygenase (COX), an ubiquitous enzyme which exists in cells in two different forms, a constitutive one named COX-1 and an inducible one named COX-2. Previous studies showed high levels of AA metabolites in the BAL of patients with cystic fibrosis as compared to nomal subjects. Cystic fibrosis is characterized by an exacerbated inflammatory pulmonary response with excessive production of inflammatory mediators. We investigated the impact of cystic fibrosis transmembrane conductance regulator (CFTR) dysfunction on prostaglandin E2 (PGE2) production and sPLA2-IIA expression. We showed that the human respiratory epithelial cell line CFT-2, which bears the Δ F508 mutation on CFTR, released more PGE2 than control cell line NT-1. This release was attenuated after experimentally-induced re-trafficking of the Δ F508-CFTR at the plasma membrane. In parallel, we observed that intranasal instillation of LPS to mice induced an increase of PGE2 concentrations in BAL which was higher in CFTR-/- mice than in littermate controls. Next, we showed that sPLA2-IIA expression occurred at higher levels in CFT-2 than in NT-1 cells and that this expression was enhanced by LPS and PGE2. Higher activation of NF-κ B was observed in CFT-2 cells compared to NT-1 cells. We suggest that the lack of the Δ F508-CFTR in the plasma membrane results in a PGE2 overproduction and an enhanced sPLA2-IIA expression. This expression is up-regulated by NF-κ B and amplified by PGE2 via an as yet unidentified signalling pathway.
Keywords: innate immunity/inflammation, infection, epithelial cells, leukocytes, Toll-like receptors, proteinases phospholipases A2, CD87, pneumopathy, cystic fibrosis