title: Cell-specific TLR9 trafficking in primary APCs of transgenic TLR9-GFP mice.
abstract: Recognition of nucleic acids by TLR9 requires its trafficking from the endoplasmic reticulum to endolysosomal compartments and its subsequent proteolytic processing. Both processes depend on interactions of TLR9 with the polytopic endoplasmic reticulum-resident protein UNC93B1. To examine the intracellular behavior of TLR9 in primary APCs, we generated transgenic mice expressing a TLR9-GFP fusion. The TLR9-GFP transgene is functional and is proteolytically processed in resting bone marrow-derived macrophages (BMDMs), dendritic cells, and B cells. Inhibition of cleavage impairs TLR9-dependent responses in all primary APCs analyzed. The kinetics of TLR9-GFP processing in BMDMs and B cells differs: in B cells, proteolysis occurs at a faster rate, consistent with an almost exclusive localization to endolysosomes at the resting state. In contrast to the joint requirement for cathepsins L and S for TLR9 cleavage in macrophages, TLR9-GFP cleavage depends on cathepsin L activity in B cells. As expected, in BMDMs and B cells from UNC93B1 (3d) mutant mice, cleavage of TLR9-GFP is essentially blocked, and the expression level of UNC93B1 appears tightly correlated with TLR9-GFP cleavage. We conclude that proteolysis is a universal requirement for TLR9 activation in the primary cell types tested, however the cathepsin requirement, rate of cleavage, and intracellular behavior of TLR9 varies. The observed differences in trafficking indicate the possibility of distinct modes of endosomal content sampling to facilitate initiation of TLR-driven responses in APCs.
authors.affiliation: Whitehead Institute for Biomedical Research, Cambridge, MA 02142, USA.
authors.forename: Ana M
authors.initials: AM
authors.lastname: Avalos
authors.forename: Oktay
authors.initials: O
authors.lastname: Kirak
authors.forename: J Margit
authors.initials: JM
authors.lastname: Oelkers
authors.forename: Marina C
authors.initials: MC
authors.lastname: Pils
authors.forename: You-Me
authors.initials: YM
authors.lastname: Kim
authors.forename: Matthias
authors.initials: M
authors.lastname: Ottinger
authors.forename: Rudolf
authors.initials: R
authors.lastname: Jaenisch
authors.forename: Hidde L
authors.initials: HL
authors.lastname: Ploegh
authors.forename: Melanie M
authors.initials: MM
authors.lastname: Brinkmann
mesh_terms: D000818:Animals; D000938:Antigen-Presenting Cells / Q000378:metabolism*; D001402:B-Lymphocytes / Q000378:metabolism; D001854:Bone Marrow Cells / Q000378:metabolism; D002460:Cell Line; D004721:Endoplasmic Reticulum / Q000378:metabolism; D049452:Green Fluorescent Proteins / Q000235:genetics / Q000378:metabolism; D006801:Humans; D008247:Lysosomes / Q000378:metabolism; D008264:Macrophages / Q000378:metabolism; D026901:Membrane Transport Proteins / Q000235:genetics / Q000378:metabolism; D051379:Mice; D008822:Mice, Transgenic; D055550:Protein Stability; D021381:Protein Transport; D059748:Proteolysis; D015398:Signal Transduction; D051217:Toll-Like Receptor 9 / Q000235:genetics* / Q000378:metabolism*; D019076:Transgenes
chemical_list: D026901:Membrane Transport Proteins; D051217:Toll-Like Receptor 9; C512254:UNC93B1 protein, mouse; D049452:Green Fluorescent Proteins