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. We investigate in a pathophysiological context, the role of epithelial cells, alveolar macrophages, neutrophils and some of their receptors (Toll-like receptors (TLR), proteinase-activated receptors (PAR) and CD87. Another aspect of our research deals with the synthesis and effects particularly the interactions with the surfactant, of phospholipases A2 (PLA2) secreted by alveolar macrophages (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 such as invasive pulmonary aspergillosis, 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. 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 concerning the expression of TLR in pulmonary epithelial cells per se, their distribution within the cell, their function as well as the signaling pathways involved. We demonstrated by RT-PCR and immunoblot that TLR4 is constitutively expressed in distinct human alveolar and bronchial epithelial cells. We further characterized by flow cytometry, biotinylation/precipitation and confocal microscopy the intracellular localization of TLR4 in these cells. Despite this intracellular compartmentalization of TLR4, pulmonary epithelial cells were responsive to the TLR4 activator lipopolysaccharide (LPS), a potent Gram-negative bacteria-associated molecular pattern. Using respiratory epithelial cells isolated from TLR4 knockout- and wild type mice, we demonstrated that TLR4 is the actual activating receptor for LPS in these cells. Furthermore, we showed that this cell response to LPS involves a signaling complex including the kinases IRAK, p38, Jnk and Erk1/2. Moreover, using vectors expressing dominant-negative forms of MyD88 and TRAF6, we established that LPS-induced activation of respiratory epithelial cells is largely dependent on TLR4 signaling intermediates. Altogether, these data demonstrate that TLR4 is a key element in the response of pulmonary epithelial cells to molecules derived from Gram-negative bacteria.
Macrophages and polymorphonuclear neutrophils also participate actively to inflammatory and infectious pulmonary diseases. Concerning neutrophils, three secretable serine proteinases particularly account for their effects, namely elastase, cathepsin G and proteinase 3. We focused our research on the analysis of the effects of these proteinases on different membrane receptors involved in the innate defense and inflammatory processes. Thus, our studies showed an effect of elastase and cathepsin G on leukocytes themselves, through their capacity to proteolytically lower the membrane expression of CD87/uPAR (urokinase-type plasminogen activator receptor), a glycoprotein also expressed by epithelial cells, and involved in mechanisms of cell adhesion and migration, as well as in tissue repair. Experiments revealed that these proteinases also cleave and disarm PAR-2, a receptor expressed at the surface of pulmonary epithelial cells. Such a receptor, for which the (patho)physiological agonist(s) are not yet characterized, is most likely involved in the mechanisms of innate defense and inflammation. For both receptors, proteomic approaches allowed to delineate the precise cleavage sites (collaboration with platforms of Protein Microsequencing and Analysis, and of Proteomics). Our current studies consider also the potential activity of bacterial proteinases on these receptors, particularly that of the elastase secreted by Pseudomonas aeruginosa, one of the major opportunistic bacteria responsible for severe pulmonary infections in patients with cystic fibrosis.
An experimental model of study of the innate immune response of the lung to infection was set up in mice challenged by Aspergillus fumigatus. This fungus is 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 Aspergillus Unit and the Histopathology Unit).
Mechanisms of regulation and roles of phospholipases A2 in lung inflammatory diseases (Carine Mounier, Lhousseine Touqui)
PLA2 catalyse the hydrolysis of phospholipids at the sn-2 position, leading to the generation of cytotoxic lysophospholipids and free fatty acids such as arachidonic acid. The latter is the precursor of leukotrienes and prostaglandins endowed with various biological activities and involved in a number of inflammatory diseases. Moreover, hydrolysis of cell membrane phospholipids by PLA2 induces the reorganisation of the structure of these membranes and changes of their physico-chemical properties. Recent studies showed the existence of several types of PLA2 whose genes have been cloned and classified in several families including intracellular and secretory PLA2 (sPLA2). The type IIA sPLA2 (sPLA2-IIA) is suggested to play a significant role in different human inflammatory diseases, such as allergic rhinitis, rhumatoïd arthritis, septic shock or ARDS. We developed an animal model which reproduces the anatomopathological criteria of human ARDS. This model is based on the intratracheal administration of LPS to guinea pigs. We showed that LPS causes an acute pulmonary inflammation accompanied by an increased expression of sPLA2-IIA and its release in the alveolar spaces. Alveolar macrophages are the main source of this enzyme whose expression is induced by an autocrine/paracrine process mediated by TNF-α. Stimulation of sPLA2-IIA synthesis by LPS is due to induction of the expression of this enzyme at the transcriptional level via a process involving the activation of the transcriptional factor NF-κB. Finally, we showed that sPLA2-IIA is involved in the hydrolysis of surfactant phospholipids and suggested that this process may play a role in the deterioration of the pulmonary surfactant. Similar results were obtained using another model of ARDS induced by intratracheal instillation of P. aeruginosa to rats. This deterioration is a typical characteristic of ARDS, leading to the permanent affixing of the alveolar walls, thus blocking the diffusion of oxygen. We also showed that hydrolysis of surfactant phospholipids is controlled by the surfactant protein A (SP-A), which inhibits the catalytic activity of sPLA2-IIA via a specific and calcium-dependent interaction. Thus, hydrolysis of surfactant phospholipids was enhanced both in vitro and in vivo in SP-A-/- mice as compared to wild-type mice. SP-A was also able to inhibit the enzymatic activity of sPLA2-V and -X, but not that of sPLA2-IB, -IID and -IIE. These properties of SP-A may represent a mechanism by which the lung protects surfactant against the deleterious effect of sPLA2. More recently, we showed that sPLA2-IIA released from guinea pig alveolar macrophages or present in the airspaces of patients with ARDS, exhibits bactericidal activities toward Gram-positive bacteria. Altogether, these studies suggest that sPLA2-IIA plays not only a pro-inflammatory role but is also involved in host defense of the lung toward invading bacteria. On the other hand, we investigated the consequences of CFTR gene deletion on the pulmonary expression of sPLA2-IIA and its pathophysiological significance in cystic fibrosis. The results showed an increased secretion of prostaglandin E2 (PGE2) in the airspaces of CFTR-/- mice treated with LPS. Such a difference was also observed in vitro in LPS-stimulated human bronchial epithelial cells bearing the ?F508 mutation of CFTR. This was accompanied by an elevated expression of sPLA2-IIA and an abnormal activation of NF-κB. These effects were also observed in PGE2-stimulated epithelial cells. These studies suggest that the ?F508 mutation of CFTR results in enhanced expression of sPLA2-IIA via a PGE2-dependent mechanism and that NF-κB may play a role in the process.
Keywords: innate immunity/inflammation, epithelial cells, neutrophils, macrophages, Toll-like receptors, proteinases phospholipases A2, surfactant, pneumopathy