Microbial products activate monocytic cells through detergent-resistant membrane microdomains.
Patients with cystic fibrosis suffer recurrent pulmonary infections that are characterized by an overactive yet ineffective and destructive inflammatory response that is associated with respiratory infections by Pseudomonas aeruginosa, a pathogen that produces a number of phlogistic molecules. To better understand this process, we used exoenzyme S (ExoS), one of the key P. aeruginosa-secreted exoproducts, which is known to stimulate cells via the Toll-like receptor (TLR) pathway. We found that ExoS induced proinflammatory cytokine production via the NF-kappaB, Erk1/2, and Src kinase pathways. Because Src kinases are concentrated within cholesterol-containing, detergent-resistant membrane microdomains (DRM) (also called lipid rafts) and DRM act as signaling platforms and amplifiers on the surface of cells, we addressed the role of DRM in ExoS signaling. ExoS bound directly to a subset of DRM and induced the phosphorylation of multiple proteins within DRM, including Src kinases. Disruption of DRM by cholesterol extraction prevented NF-kappaB and Erk 1/2 activation and TNF-alpha production in response to ExoS. Activation of monocytic cells by other TLR and Nod-like receptor agonists, such as lipoteichoic acid, lipopolysaccharide, and peptidoglycan, were also dependent on DRM, and disruption prevented TNF-alpha production. Disruption of DRM did not prevent ExoS binding but did release the Src kinase, Lyn, from the DRM fraction into the detergent-soluble fraction, a site in which Src kinases are not active. These studies show that ExoS, a TLR agonist, requires direct binding to DRM for optimal signaling, which suggests that DRM are possible therapeutic targets in cystic fibrosis.
Epelman
Slava
S
Department of Microbiology and Infectious Diseases, University of Calgary, Alberta, Canada.
Berenger
Byron
B
Stack
Danuta
D
Neely
Graham G
GG
Ma
Ling Ling
LL
Mody
Christopher H
CH
D036002:ADP Ribose Transferases / Q000494:pharmacology*; D001427:Bacterial Toxins / Q000494:pharmacology*; D002460:Cell Line; D003902:Detergents / Q000494:pharmacology*; D006801:Humans; D020672:I-kappa B Proteins / Q000378:metabolism; D018950:Lipopolysaccharide Receptors / Q000378:metabolism; D008070:Lipopolysaccharides / Q000494:pharmacology; D021962:Membrane Microdomains / Q000187:drug effects* / Q000201:enzymology; D019950:Mitogen-Activated Protein Kinase 1 / Q000378:metabolism; D048052:Mitogen-Activated Protein Kinase 3 / Q000378:metabolism; D009000:Monocytes / Q000166:cytology* / Q000187:drug effects* / Q000201:enzymology; D016328:NF-kappa B / Q000378:metabolism; D010766:Phosphorylation / Q000187:drug effects; D011485:Protein Binding / Q000187:drug effects; D015398:Signal Transduction / Q000187:drug effects; D013682:Teichoic Acids / Q000494:pharmacology; D051193:Toll-Like Receptors / Q000819:agonists; D014409:Tumor Necrosis Factor-alpha / Q000096:biosynthesis; D047392:beta-Cyclodextrins / Q000494:pharmacology; D019061:src-Family Kinases / Q000378:metabolism
D001427:Bacterial Toxins; D003902:Detergents; D020672:I-kappa B Proteins; D018950:Lipopolysaccharide Receptors; D008070:Lipopolysaccharides; D016328:NF-kappa B; D013682:Teichoic Acids; D051193:Toll-Like Receptors; D014409:Tumor Necrosis Factor-alpha; D047392:beta-Cyclodextrins; C009900:lipoteichoic acid; D036002:ADP Ribose Transferases; C444820:exoenzyme S; C060902:lyn protein-tyrosine kinase; D019061:src-Family Kinases; D019950:Mitogen-Activated Protein Kinase 1; D048052:Mitogen-Activated Protein Kinase 3; C031215:betadex