title: Contribution of MINCLE-SYK Signaling to Activation of Primary Human APCs by Mycobacterial Cord Factor and the Novel Adjuvant TDB.
abstract: Trehalose-6,6-dimycolate (TDM), the mycobacterial cord factor, is an abundant cell wall glycolipid and major virulence factor of Mycobacterium tuberculosis. Its synthetic analog trehalose-6,6-dibehenate (TDB) is a new adjuvant currently in phase I clinical trials. In rodents, the C-type lectin receptors Mincle and Mcl bind TDB/TDM and activate macrophages and dendritic cells (DC) through the Syk-Card9 pathway. However, it is unknown whether these glycolipids activate human innate immune cells through the same mechanism. We performed in vitro analysis of TDB/TDM-stimulated primary human monocytes, macrophages, and DC; determined C-type lectin receptor expression; and tested the contribution of SYK, MINCLE, and MCL by small interfering RNA knockdown and genetic complementation. We observed a robust chemokine and cytokine release in response to TDB or TDM. MCSF-driven macrophages secreted higher levels of IL-8, IL-6, CCL3, CCL4, and CCL2 after stimulation with TDM, whereas DC responded more strongly to TDB and GM-CSF-driven macrophages were equally responsive to TDB and TDM. SYK kinase and the adaptor protein CARD9 were essential for glycolipid-induced IL-8 production. mRNA expression of MINCLE and MCL was high in monocytes and macrophages, with MINCLE and MCL proteins localized intracellularly under resting conditions. Small interfering RNA-mediated MINCLE or MCL knockdown caused on average reduced TDB- or TDM-induced IL-8 production. Conversely, retroviral expression in murine Mincle-deficient DC revealed that human MINCLE, but not MCL, was sufficient to confer responsiveness to TDB/TDM. Our study demonstrates that SYK-CARD9 signaling plays a key role in TDB/TDM-induced activation of innate immune cells in man as in mouse, likely by engagement of MINCLE.
authors.affiliation: Mikrobiologisches Institut, Klinische Mikrobiologie, Immunologie und Hygiene, Universitätsklinikum Erlangen, Friedrich-Alexander-Universität Erlangen-Nürnberg, 91054 Erlangen, Germany;
authors.forename: Jenny
authors.initials: J
authors.lastname: Ostrop
authors.affiliation: Mikrobiologisches Institut, Klinische Mikrobiologie, Immunologie und Hygiene, Universitätsklinikum Erlangen, Friedrich-Alexander-Universität Erlangen-Nürnberg, 91054 Erlangen, Germany;
authors.forename: Katrin
authors.initials: K
authors.lastname: Jozefowski
authors.affiliation: Department of Biomolecular Systems, Max Planck Institute of Colloids and Interfaces, 14476 Potsdam, Germany; Free University of Berlin, Institute of Chemistry and Biochemistry, 14195 Berlin, Germany; and.
authors.forename: Stephanie
authors.initials: S
authors.lastname: Zimmermann
authors.affiliation: Mikrobiologisches Institut, Klinische Mikrobiologie, Immunologie und Hygiene, Universitätsklinikum Erlangen, Friedrich-Alexander-Universität Erlangen-Nürnberg, 91054 Erlangen, Germany;
authors.forename: Katharina
authors.initials: K
authors.lastname: Hofmann
authors.affiliation: Transfusionsmedizinische und Hämostaseologische Abteilung, Universitätsklinikum Erlangen, Friedrich-Alexander-Universität Erlangen-Nürnberg, 91054 Erlangen, Germany.
authors.forename: Erwin
authors.initials: E
authors.lastname: Strasser
authors.affiliation: Department of Biomolecular Systems, Max Planck Institute of Colloids and Interfaces, 14476 Potsdam, Germany; Free University of Berlin, Institute of Chemistry and Biochemistry, 14195 Berlin, Germany; and.
authors.forename: Bernd
authors.initials: B
authors.lastname: Lepenies
authors.affiliation: Mikrobiologisches Institut, Klinische Mikrobiologie, Immunologie und Hygiene, Universitätsklinikum Erlangen, Friedrich-Alexander-Universität Erlangen-Nürnberg, 91054 Erlangen, Germany; roland.lang@uk-erlangen.de.
authors.forename: Roland
authors.initials: R
authors.lastname: Lang
mesh_terms: D000276:Adjuvants, Immunologic / Q000737:chemistry / Q000378:metabolism; D000818:Animals; D015153:Blotting, Western; D053468:CARD Signaling Adaptor Proteins / Q000235:genetics / Q000378:metabolism; D002478:Cells, Cultured; D018925:Chemokines / Q000276:immunology / Q000378:metabolism; D003311:Cord Factors / Q000737:chemistry / Q000276:immunology* / Q000378:metabolism; D016207:Cytokines / Q000276:immunology / Q000378:metabolism; D003713:Dendritic Cells / Q000276:immunology / Q000378:metabolism; D005434:Flow Cytometry; D015870:Gene Expression / Q000276:immunology; D057809:HEK293 Cells; D006801:Humans; D047908:Intracellular Signaling Peptides and Proteins / Q000235:genetics / Q000276:immunology* / Q000378:metabolism; D037181:Lectins, C-Type / Q000235:genetics / Q000276:immunology* / Q000378:metabolism; D008264:Macrophages / Q000276:immunology / Q000378:metabolism; D018345:Mice, Knockout; D009000:Monocytes / Q000276:immunology / Q000378:metabolism; D009169:Mycobacterium tuberculosis / Q000737:chemistry / Q000276:immunology; D011485:Protein Binding / Q000276:immunology; D011505:Protein-Tyrosine Kinases / Q000235:genetics / Q000276:immunology* / Q000378:metabolism; D034622:RNA Interference; D011971:Receptors, Immunologic / Q000235:genetics / Q000276:immunology* / Q000378:metabolism; D020133:Reverse Transcriptase Polymerase Chain Reaction; D015398:Signal Transduction / Q000276:immunology; D000072377:Syk Kinase
chemical_list: D000276:Adjuvants, Immunologic; D053468:CARD Signaling Adaptor Proteins; C505855:CARD9 protein, human; C482593:CLEC4D protein, human; D018925:Chemokines; D003311:Cord Factors; D016207:Cytokines; D047908:Intracellular Signaling Peptides and Proteins; D037181:Lectins, C-Type; D011971:Receptors, Immunologic; D011505:Protein-Tyrosine Kinases; C000606896:SYK protein, human; D000072377:Syk Kinase; C000606897:Syk protein, mouse