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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.

The signaling pathways of mammalian Toll-like receptors (TLRs) are well characterized, but the precise mechanism(s) by which TLRs are activated upon ligand binding remains poorly defined. Recently, we reported a novel membrane sialidase-controlling mechanism that depends on ligand binding to its TLR to induce mammalian neuraminidase-1 (Neu1) activity, to influence receptor desialylation, and subsequently to induce TLR receptor activation and the production of nitric oxide and proinflammatory cytokines in dendritic and macrophage cells. The ␣-2,3-sialyl residue of TLR was identified as the specific target for hydrolysis by Neu1. Here, we report a membrane signaling paradigm initiated by endotoxin lipopolysaccharide (LPS) binding to TLR4 to potentiate G protein-coupled receptor (GPCR) signaling via membrane G␣ i subunit proteins and matrix metalloproteinase-9 (MMP9) activation to induce Neu1. Central to this process is that a Neu1-MMP9 complex is bound to TLR4 on the cell surface of naive macrophage cells. Specific inhibition of MMP9 and GPCR G␣ i -signaling proteins blocks LPS-induced Neu1 activity and NFB activation. Silencing MMP9 mRNA using lentivirus MMP9 shRNA transduction or siRNA transfection of macrophage cells and MMP9 knock-out primary macrophage cells significantly reduced Neu1 activity and NFB activation associated with LPS-treated cells. These findings uncover a molecular organizational signaling platform of a novel Neu1 and MMP9 cross-talk in alliance with TLR4 on the cell surface that is essential for ligand activation of TLRs and subsequent cellular signaling.The mammalian Toll-like receptors (TLRs) 11 are one of the families of sensor receptors that recognize pathogen-associated molecular patterns. Not only are TLRs crucial sensors of microbial infections for innate immune cells; they play important roles in the pathophysiology of infectious, inflammatory, and autoimmune diseases. Thus, the intensity and duration of TLR responses with these diseases must be tightly controlled. It follows that the structural integrity of TLR receptors, their ligand interactions, and their signaling components are important for our understanding of subsequent immunological responses.Although the signaling pathways of TLR sensors are well characterized, the parameters controlling interactions between TLRs and their ligands have remained poorly defined until now. We have recently identified a novel paradigm of TLR activation by its natural ligand, which has not been observed previously (1). This paradigm suggests that ligand-induced TLR activation is tightly controlled by Neu1 activation. The data indicate that Neu1 is already in complex with either TLR2, -3, or -4 receptors and is induced upon ligand binding to their respective receptors. In addition, activated Neu1 specifically hydrolyzes ␣-2,3-sialyl residues linked to ␤-galactosides, which are distant from ligand binding. This desialylation process is proposed to remove steric hindrance to TLR4 dimerization, MyD88-TLR4 complex recruitment, NFB activation, and p...

Mammalian Toll-like receptors (TLRs) are a family of receptors that recognize pathogen-associated molecular patterns. Not only are TLRs crucial sensors of microbial (e.g., viruses, bacteria and parasite) infections, they also play an important role in the pathophysiology of infectious diseases, inflammatory diseases, and possibly in autoimmune diseases. Thus, the intensity and duration of TLR responses against infectious diseases must be tightly controlled. It follows that understanding the structural integrity of sensor receptors, their ligand interactions and signaling components is essential for subsequent immunological protection. It would also provide important opportunities for disease modification through sensor manipulation. Although the signaling pathways of TLR sensors are well characterized, the parameters controlling interactions between the sensors and their ligands still remain poorly defined. We have recently identified a novel mechanism of TLR activation by its natural ligand, which has not been previously observed 1,2 . It suggests that ligand-induced TLR activation is tightly controlled by Neu1 sialidase activation. We have also reported that Neu1 tightly regulates neurotrophin receptors like TrkA and TrkB 3 , which involve Neu1 and matrix metalloproteinase-9 (MMP-9) cross-talk in complex with the receptors 4 . The sialidase assay has been initially use to find a novel ligand, thymoquinone, in the activation of Neu4 sialidase on the cell surface of macrophages, dendritic cells and fibroblast cells via GPCR Gαi proteins and MMP-9 5 . For TLR receptors, our data indicate that Neu1 sialidase is already in complex with TLR-2, -3 and -4 receptors, and is induced upon ligand binding to either receptor. Activated Neu1 sialidase hydrolyzes sialyl α-2,3-linked β-galactosyl residues distant from ligand binding to remove steric hinderance to TLR-4 dimerization, MyD88/TLR4 complex recruitment, NFkB activation and pro-inflammatory cell responses. In a collaborative report, Neu1 sialidase has been shown to regulate phagocytosis in macrophage cells 6 . Taken together, the sialidase assay has provided us with powerful insights to the molecular mechanisms of ligand-induced receptor activation. Although the precise relationship between Neu1 sialidase and the activation of TLR, Trk receptors has yet to be fully elucidated, it would represent a new or pioneering approach to cell regulation pathways. Before resurrecting frozen cells from the -80°C freezer, one needs to prepare culture medium using sterile filtered Dulbecco's Modified Eagle's Medium (DMEM) supplemented with 10% fetal bovine serum (FBS) and 5 μg/mL of plasmocin. Plasmocin is an antibiotic solution used in our research to eliminate and prevent mycoplasma contamination of cell cultures. 2. For one vial of frozen cells, one adds 4 mL, 1 mL of the cultured medium to a 25 cm 2 cell culture flask in a sterile biohazard containment hood. 3. When the vial of frozen cells are taken from the -80°C freezer, they are quickly thawed by hand warming in the...

The Opioid Crisis in Canada-The Need for More Robust Psychosocial InterventionsThe opioid crisis continues unabated in Canada. In 2019, there were 3,923 reported deaths from opioid overdose, and 94% of these deaths were unintentional (Government of Canada, 2019; https://health-infobase.canada.ca/substancerelated-harms/opioids). Moreover, it appears that opioid overdose death rates have increased since the beginning of the COVID-19 pandemic. 1 Clearly, there are multiple factors contributing to this crisis, and the limited accessibility to opioid agonist treatments (OATs), combined with a lack of utilization of psychosocial interventions (PSIs), further complicates this issue. Needless to say, the opioid crisis is a complex issue that will need nuanced solutions. We argue that while we have excellent evidence-based medication treatments for opioid use disorder (OUD), they are probably less effective when they are not appropriately combined with PSIs. As the death toll from Canada's opioid crisis continues to mount, it is thus imperative that empirically based solutions, which combine medications with PSIs, are widely implemented and are accessible to all patients with OUD.