Dependence of pathogen molecule-induced toll-like receptor activation and cell function on Neu1 sialidase.
The signaling pathways of mammalian Toll-like receptors (TLR) are well characterized, but the initial molecular mechanisms activated following ligand interactions with the receptors remain poorly defined. Here, we show a membrane controlling mechanism that is initiated by ligand binding to TLR-2, -3 and-4 to induce Neu1 sialidase activity within minutes in live primary bone marrow (BM) macrophage cells and macrophage and dendritic cell lines. Central to this process is that Neu1 and not Neu2,-3 and-4 forms a complex with TLR-2,-3 and-4 on the cell surface of naïve macrophage cells. Neuraminidase inhibitors BCX1827, 2-deoxy-2,3-dehydro-N-acetylneuraminic acid (DANA), zanamivir and oseltamivir carboxylate have a limited significant inhibition of the LPS-induced sialidase activity in live BMC-2 macrophage cells but Tamiflu (oseltamivir phosphate) completely blocks this activity. Tamiflu inhibits LPS-induced sialidase activity in live BMC-2 cells with an IC(50) of 1.2 microM compared to an IC(50) of 1015 microM for its hydrolytic metabolite oseltamivir carboxylate. Tamiflu blockage of LPS-induced Neu1 sialidase activity is not affected in BMC-2 cells pretreated with anticarboxylesterase agent clopidogrel. Endotoxin LPS binding to TLR4 induces Neu1 with subsequent activation of NFkappaB and the production of nitric oxide and pro-inflammatory IL-6 and TNFalpha cytokines in primary and macrophage cell lines. Hypomorphic cathepsin A mice with a secondary Neu1 deficiency respond poorly to LPS-induced pro-inflammatory cytokines compared to the wild-type or hypomorphic cathepsin A with normal Neu1 mice. Our findings establish an unprecedented mechanism for pathogen molecule-induced TLR activation and cell function, which is critically dependent on Neu1 sialidase activity associated with TLR ligand treated live primary macrophage cells and macrophage and dendritic cell lines.
Department of Microbiology & Immunology, Queen's University, Kingston, ON, K7L3N6, Canada.
Schammim Ray
SR
Amith
Preethi
P
Jayanth
Susan
S
Franchuk
Sarah
S
Siddiqui
Volkan
V
Seyrantepe
Katrina
K
Gee
Sameh
S
Basta
Rudi
R
Beyaert
Alexey V
AV
Pshezhetsky
Myron R
MR
Szewczuk
D000818:Animals; D001854:Bone Marrow Cells / Q000166:cytology; D002462:Cell Membrane / Q000187:drug effects / Q000201:enzymology; D002470:Cell Survival / Q000187:drug effects; D002478:Cells, Cultured; D016207:Cytokines / Q000097:blood; D003713:Dendritic Cells / Q000166:cytology / Q000187:drug effects / Q000201:enzymology*; D004791:Enzyme Inhibitors / Q000494:pharmacology; D006801:Humans; D047468:Immunoprecipitation; D018836:Inflammation Mediators / Q000378:metabolism; D008024:Ligands; D008070:Lipopolysaccharides / Q000494:pharmacology; D008264:Macrophages / Q000166:cytology / Q000187:drug effects / Q000201:enzymology*; D051379:Mice; D009439:Neuraminidase / Q000037:antagonists & inhibitors / Q000172:deficiency / Q000378:metabolism*; D009569:Nitric Oxide / Q000096:biosynthesis; D053139:Oseltamivir / Q000494:pharmacology; D051192:Receptors, Pattern Recognition / Q000378:metabolism*; D051193:Toll-Like Receptors / Q000276:immunology*
D016207:Cytokines; D004791:Enzyme Inhibitors; D018836:Inflammation Mediators; D008024:Ligands; D008070:Lipopolysaccharides; D051192:Receptors, Pattern Recognition; D051193:Toll-Like Receptors; D053139:Oseltamivir; D009569:Nitric Oxide; C110128:Neu1 protein, mouse; D009439:Neuraminidase