title: Thymoquinone-induced Neu4 sialidase activates NFκB in macrophage cells and pro-inflammatory cytokines in vivo.
abstract: Thymoquinone (TQ) derived from the nutraceutical black cumin oil has been reported to be a novel agonist of Neu4 sialidase activity in live cells (Glycoconj J DOI 10.1007/s10719-010-9281-6). The activation of Neu4 sialidase on the cell surface by TQ was found to involve GPCR-signaling via membrane targeting of Gαi subunit proteins and matrix metalloproteinase-9 activation. Contrary to other reports, TQ had no anti-inflammatory effects in vitro. Here, we show that MyD88/TLR4 complex formation and subsequent NFκB activation are induced by the Neu4 activity associated with TQ-stimulated live primary bone marrow (BM) macrophage cells from WT and Neu1-deficient mice, HEK-TLR4/MD2 cells and BMC-2 macrophage cell line but not with primary macrophage cells from Neu4-knockout mice. Tamiflu (oseltamivir phosphate), pertussis toxin (PTX), a specific inhibitor of Gαi proteins of G-protein coupled receptor (GPCR) and the broad range inhibitor of matrix metalloproteinase (MMP) galardin applied to live primary BM macrophage cells completely block TQ-induced MyD88/TLR4 complex formation. Using immunocytochemistry and western blot analyses, Tamiflu, galardin and PTX inhibit NFκB activation induced by Neu4 activity associated with TQ-stimulated BMC-2 cells, HEK-TLR4/MD2 cells and primary BM macrophages from WT mice. EMSA analyses on HEK-TLR4/MD2 nuclear cell extracts confirm the nuclear localization and DNA binding of TQ-induced NFκB activation in a biphasic manner within 30 min. Co-immunoprecipitation experiments reveal for the first time that MMP-9 may be an important intermediate link in the TQ-induced Neu4 activity circuitously targeting TLR4 receptors. Central to this process is that Neu4 forms a complex with MMP-9, which is already bound to TLR4 receptors. Fluorescence spectrophotometer analyses of live CD14-THP1 cells treated with TQ show Neu4 sialidase activity over 5 min. Using flow cytometry analyses, CD14-THP1 cells treated with TQ express stable protein levels of Neu4, TLR4 and MMP9 on the cell surface over 30 min except for a marked diminution of MMP9 at 15 min. Using cytokine array profiling analyses of serum, Neu4-knockout mice respond poorly to TQ in producing pro-inflammatory cytokines and chemokines after 5-h treatment compared to the wild-type or hypomorphic cathepsin A mice with a secondary 90% Neu1 deficient mice. Our findings establish an unprecedented signaling paradigm for TQ-induced Neu4 sialidase activity. It signifies that MMP-9 forms an important molecular signaling platform in complex with TLR4 receptors at the ectodomain and acts as the intermediate link for TQ-induced Neu4 sialidase in generating a functional receptor with subsequent NFκB activation and pro-inflammatory cytokine production in vivo.
authors.affiliation: Department of Microbiology & Immunology, Queen's University, Kingston, ON K7L3N6, Canada. tmfinlay@ucalgary.ca
authors.forename: Trisha M
authors.initials: TM
authors.lastname: Finlay
authors.forename: Samar
authors.initials: S
authors.lastname: Abdulkhalek
authors.forename: Alanna
authors.initials: A
authors.lastname: Gilmour
authors.forename: Christina
authors.initials: C
authors.lastname: Guzzo
authors.forename: Preethi
authors.initials: P
authors.lastname: Jayanth
authors.forename: Schammim Ray
authors.initials: SR
authors.lastname: Amith
authors.forename: Katrina
authors.initials: K
authors.lastname: Gee
authors.forename: Rudi
authors.initials: R
authors.lastname: Beyaert
authors.forename: Myron R
authors.initials: MR
authors.lastname: Szewczuk
mesh_terms: D000818:Animals; D016227:Benzoquinones / Q000494:pharmacology*; D001854:Bone Marrow Cells / Q000166:cytology; D002478:Cells, Cultured; D016207:Cytokines / Q000097:blood; D004151:Dipeptides / Q000494:pharmacology; D004789:Enzyme Activation / Q000187:drug effects; D004790:Enzyme Induction / Q000187:drug effects; D006801:Humans; D018836:Inflammation Mediators / Q000378:metabolism*; D051218:Lymphocyte Antigen 96 / Q000378:metabolism; D008264:Macrophages / Q000187:drug effects* / Q000201:enzymology*; D020780:Matrix Metalloproteinase 9 / Q000378:metabolism; D051379:Mice; D053594:Myeloid Differentiation Factor 88 / Q000378:metabolism; D016328:NF-kappa B / Q000378:metabolism*; D009439:Neuraminidase / Q000037:antagonists & inhibitors / Q000096:biosynthesis*; D053139:Oseltamivir / Q000494:pharmacology; D037342:Pertussis Toxin / Q000494:pharmacology; D010766:Phosphorylation / Q000187:drug effects; D011485:Protein Binding / Q000187:drug effects; D021381:Protein Transport / Q000187:drug effects; D043562:Receptors, G-Protein-Coupled / Q000378:metabolism; D013997:Time Factors; D051197:Toll-Like Receptor 4 / Q000378:metabolism
chemical_list: D016227:Benzoquinones; D016207:Cytokines; D004151:Dipeptides; D018836:Inflammation Mediators; C495377:LY96 protein, human; D051218:Lymphocyte Antigen 96; D053594:Myeloid Differentiation Factor 88; C078131:N-(2(R)-2-(hydroxamidocarbonylmethyl)-4-methylpentanoyl)-L-tryptophan methylamide; D016328:NF-kappa B; D043562:Receptors, G-Protein-Coupled; D051197:Toll-Like Receptor 4; D053139:Oseltamivir; D037342:Pertussis Toxin; C481539:Neu4 protein, mouse; D009439:Neuraminidase; D020780:Matrix Metalloproteinase 9; C003466:thymoquinone