Pseudomonas aeruginosa LPS or Flagellin Are Sufficient to Activate TLR-Dependent Signaling in Murine Alveolar Macrophages and Airway Epithelial Cells

Abstract

BackgroundThe human lung is exposed to a large number of airborne pathogens as a result of the daily inhalation of 10,000 liters of air. Innate immunity is thus essential to defend the lungs against these pathogens. This defense is mediated in part through the recognition of specific microbial ligands by Toll-like receptors (TLR) of which there are at least 10 in humans. Pseudomonas aeruginosa is the main pathogen that infects the lungs of cystic fibrosis patients. Based on whole animal experiments, using TLR knockout mice, the control of this bacterium is believed to occur by the recognition of LPS and flagellin by TLRs 2,4 and 5, respectively.Methodology/Principal FindingsIn the present study, we investigated in vitro the role of these same TLR and ligands, in alveolar macrophage (AM) and epithelial cell (EC) activation. Cellular responses to P. aeruginosa was evaluated by measuring KC, TNF-α, IL-6 and G-CSF secretion, four different markers of the innate immune response. AM and EC from WT and TLR2, 4, 5 and MyD88 knockout mice for were stimulated with the wild-type P. aeruginosa or with a mutant devoid of flagellin production.Conclusions/SignificanceThe results clearly demonstrate that only two ligand/receptor pairs are necessary for the induction of KC, TNF-α, and IL-6 synthesis by P. aeruginosa-activated cells, i.e. TLR2,4/LPS and TLR5/flagellin. Either ligand/receptor pair is sufficient to sense the bacterium and to trigger cell activation, and when both are missing lung EC and AM are unable to produce such a response as were cells from MyD88−/− mice.

Disparities in TLR5 Expression and Responsiveness to Flagellin in Equine Neutrophils and Mononuclear Phagocytes

“…In the relatively few studies published to date, flagellin activates mononuclear cells and neutrophils isolated from other species and does so by interacting with TLR5 (21). For example, human peripheral blood monocytes express moderate amounts of TLR5 (46), and flagellin induces marked proinflammatory cytokine gene expression in these cells (47).…”

“…1). This finding was unexpected, as human monocytes and murine alveolar macrophages are sensitive to flagellin stimulation, producing cytokines upon flagellin treatment in vitro (21,39). As a lack of response to flagellin could be due to degradation of the flagellin, we resolved the flagellin by SDS-PAGE and demonstrated a single band of 55 kDa indicative of an intact protein (data not shown).…”

“…For example, both flagellin and Abs recognizing flagellin have been detected in human patients and a murine model of sepsis (16,17). Furthermore, concentrations of Abs directed against flagellin are increased in human patients with Crohn's disease (18), the results of in vitro studies indicate that flagellin induces the production of NF-kB-dependent proinflammatory mediators by human monocytes, murine alveolar macrophages, and epithelial cells (19)(20)(21), and in vivo administration of flagellin to mice leads to the development of hypotension, acute pulmonary inflammation, and vascular hypocontractility that resemble findings in septic shock (16). Finally, flagellin induces colitis in TLR4 2/2 mice (22).…”

Inhibition of TLR2 signaling by small molecule inhibitors targeting a pocket within the TLR2 TIR domain

“…TLR2 dimerization with TLR1 or TLR6 allows for recognition of both gram-positive and certain gram-negative bacteria (e.g., S. aureus, Streptococcus pneumoniae, Pseudomonas aeruginosa, Francisella tularensis) (4,6,7,9). C29 significantly inhibited heat-killed P. aeruginosa-induced and heat-killed S. aureus (HKSA)-induced IL-8 mRNA in HEK-TLR2 cells (Fig.…”

“…TLR2 heterodimerizes with TLR6 or TLR1 to recognize diacyl lipopeptides or triacyl lipopeptides, respectively (2,3), present in gram-positive and gram-negative bacteria (4)(5)(6)(7)(8)(9).…”