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Dependence of pathogen molecule-induced Toll-like receptor activation and cell function on Neu1 sialidase

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Abstract

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 IC50 of 1.2 μM compared to an IC50 of 1015 μM 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 NFκB and the production of nitric oxide and pro-inflammatory IL-6 and TNFα 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.

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Abbreviations

(TLR):

Toll-like receptors

(PAMPS):

pathogen-associated molecular patterns

(oseltamivir phosphate):

Tamiflu

(DANA):

2-deoxy-2,3-dehydro-N-acetylneuraminic acid

(IL-6):

interleukin-6

(TNFα):

tumor necrosis factor

(LPS):

lipopolysaccride

(IC50):

50% inhibition concentration

(ECD):

ectodomain

(poly:IC):

polyinosinic-polycytidylic acid

(TS):

T. cruzi trans-sialidase

(BM):

bone marrow

(M-CSF):

monocyte colony-stimulating factor

(4-MUNANA (4-MU)):

[2′-(4-methylumbelliferyl)-α-N-acetylneuraminic acid]

(PVDF):

polyvinylidene fluoride

(NO):

nitric oxide

(Neu1-CathA KD):

hypomorphic cathepsin A mice with the secondary ~90% reduction of the Neu1 activity

(CathA KI):

normal Neu1 sialidase bound to inactive cathepsin A Ser190Ala mutant

(Neu4 KO):

Neu4 knockout

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Acknowledgements

These studies are partially supported by grants to MRS from Natural Sciences and Engineering Research Council of Canada (NSERC), the Harry Botterell Foundation for Neuroscience Research, ARC, and the Garfield Kelly Cardiovascular Research and Development Fund. S.R.A. is a recipient of the Queen’s University Research Award, the Robert J. Wilson Fellowship and the Ontario Graduate Scholarship. P.J. is a recipient of the Queen’s Graduate Award and the Robert J. Wilson Fellowship. SF is a recipient of the Ontario Graduate Scholarship in Science and Technology (OGSST). Work on KD mice is supported by grant to A.V.P. from Canadian Institutes of Health (CIHR). Research work on the TLR transfected cell lines is supported by grants to R.B. from the ‘Interuniversitaire Attractiepolen’ (IAP6/18), the ‘Fonds voor Wetenschappelijk Onderzoek-Vlaanderen’ (FWO; grant 3G010505), and the ‘Geconcerteerde Onderzoeksacties’ of the Ghent University (GOA; grant 01G06B6).

Author Contributions M.R.S. and S.R.A. wrote the paper, designed and performed experiments; P.J. performed the WB and EMSA blots; S.F. did the neuraminidase inhibitor studies; S.S. performed the NO experiments; V.S. generated Neu1-deficient, Neu1-CathA, Neu4 KO mice; K.G., S.B., R.B. and A.V.P. helped with experiments and writing the paper; M.R.S. supervised the research design and the writing of the paper. All authors read and commented on the manuscript, and declare no competing financial interests.

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Authors and Affiliations

  1. Department of Microbiology & Immunology, Queen’s University, Kingston, ON, K7L3N6, Canada

    Schammim Ray Amith, Preethi Jayanth, Susan Franchuk, Sarah Siddiqui, Katrina Gee, Sameh Basta & Myron R. Szewczuk

  2. Departments of Pediatrics and Biochemistry, Montreal University, Service de Genetique, Ste-Justine Hospital, 3175 Cote-Ste-Catherine, H3T1C5, Montreal, QC, Canada

    Volkan Seyrantepe & Alexey V. Pshezhetsky

  3. Department for Molecular Biomedical Research, VIB, Unit for Molecular Signal Transduction in Inflammation, Technologiepark 927, B-9052, Zwijnaarde, Belgium

    Rudi Beyaert

  4. Department of Biomedical Molecular Biology, Ghent University, Ghent, Belgium

    Rudi Beyaert

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  1. Schammim Ray Amith
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  2. Preethi Jayanth
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Correspondence to Myron R. Szewczuk.

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Amith, S., Jayanth, P., Franchuk, S. et al. Dependence of pathogen molecule-induced Toll-like receptor activation and cell function on Neu1 sialidase. Glycoconj J 26, 1197–1212 (2009). https://doi.org/10.1007/s10719-009-9239-8

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