LRRC59 Regulates Trafficking of Nucleic Acid−Sensing TLRs from the Endoplasmic Reticulum via Association with UNC93B1 This information is current as Megumi Tatematsu, Kenji Funami, Noriko Ishii, Tsukasa of October 1, 2021. Seya, Chikashi Obuse and Misako Matsumoto J Immunol 2015; 195:4933-4942; Prepublished online 14 October 2015; doi: 10.4049/jimmunol.1501305 http://www.jimmunol.org/content/195/10/4933 Downloaded from Supplementary http://www.jimmunol.org/content/suppl/2015/10/14/jimmunol.150130 Material 5.DCSupplemental http://www.jimmunol.org/ References This article cites 37 articles, 15 of which you can access for free at: http://www.jimmunol.org/content/195/10/4933.full#ref-list-1 Why The JI? Submit online. • Rapid Reviews! 30 days* from submission to initial decision • No Triage! 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The Journal of Immunology LRRC59 Regulates Trafficking of Nucleic Acid–Sensing TLRs from the Endoplasmic Reticulum via Association with UNC93B1 Megumi Tatematsu,* Kenji Funami,* Noriko Ishii,* Tsukasa Seya,* Chikashi Obuse,† and Misako Matsumoto* Compartmentalization of nucleic acid (NA)–sensing TLR3, 7, 8, and 9 is strictly regulated to direct optimal response against microbial infection and evade recognition of host-derived NAs. Uncoordinated 93 homolog B1 (UNC93B1) is indispensable for trafficking of NA-sensing TLRs from the endoplasmic reticulum (ER) to endosomes/lysosomes. UNC93B1 controls loading of the TLRs into COPII vesicles to exit from the ER and traffics with the TLRs in the steady state. Ligand-induced translocation also happens on NA-sensing TLRs. However, the molecular mechanism for ligand-dependent trafficking of TLRs from the ER to endosomes/lysosomes remains unclear. In this study, we demonstrated that leucine-rich repeat containing protein (LRRC) 59, an Downloaded from ER membrane protein, participated in trafficking of NA-sensing TLRs from the ER. Knockdown of LRRC59 reduced TLR3-, 8-, and 9-mediated, but not TLR4-mediated, signaling. Upon ligand stimulation, LRRC59 associated with UNC93B1 in a TLR- independent manner, which required signals induced by ligand internalization. Endosomal localization of endogenous TLR3 was decreased by silencing of LRRC59, suggesting that LRRC59 promotes UNC93B1-mediated translocation of NA-sensing TLRs from the ER upon infection. These findings help us understand how NA-sensing TLRs control their proper distribution in the infection/inflammatory state. The Journal of Immunology, 2015, 195: 4933–4942. http://www.jimmunol.org/ oll-like receptors recognize pathogen-associated molec- NA-sensing TLRs from the ER to the Golgi/endosomes (12–15). ular patterns derived from microorganisms and induce UNC93B1 interacts with TLR3, 7, 8, and 9 and appears to regulate T production of type I IFN and inflammatory cytokines their packaging into COPII-coated vesicles on ER membranes (16). through the adapter molecule MyD88 or Toll/IL-1R domain- Besides UNC93B1, several chaperones such as gp96 and protein containing adapter molecule-1 (TICAM-1) (also called TRIF) associated with TLR4 A (PRAT4A) are required for proper folding (1, 2). All TLRs except for TLR3 activate MyD88, and TLR3 and and maturation of TLR7 and TLR9 to allow them to exit the ER TLR4 can activate TICAM-1. TLR3, 7, 8, and 9 sense endocy- (17, 18). These chaperones are also involved in the cell-surface– by guest on October 1, 2021 tosed nucleic acids (NAs) in endosomal compartments (3). Endo- expressing TLRs, gp96 plays a role in trafficking of TLR1, 2, 4, 5, somal localization and intracellular trafficking of these TLRs are 7, and 9, and PRAT4A is required for TLR1, 2, 4, 7, and 9 traf- strictly regulated to evade recognition of self-RNA/DNA (4), which ficking. So far, chaperones necessary for translocation of TLR3 is also necessary for generation of functional cleaved/associated from the ER to the Golgi have not been discovered. Also, the de- form of receptors (5–11). After synthesis, NA-sensing TLRs are tailed mechanism of UNC93B1-mediated trafficking of NA-sensing retained in the endoplasmic reticulum (ER) and translocate to the TLRs remains unclear. endosomal compartment. ER membrane protein uncoordinated 93 The leucine-rich repeat (LRR) containing protein (LRRC) 59/ homolog B1 (UNC93B1) plays an important role in trafficking of p34 is a type II transmembrane protein with a short C-terminal domain facing the ER lumen and four LRRs and coiled-coil do- main facing the cytosol. LRRC59 resides in the ER and nuclear *Department of Microbiology and Immunology, Hokkaido University Graduate membrane, and is reported to have the function of nuclear import of School of Medicine, Sapporo 060-8638, Japan; and †Division of Molecular Life Science, Graduate School of Life Science, Hokkaido University, Sapporo 001- fibroblast growth factor (19, 20) and CIP2A (21) at the nuclear 0021, Japan membrane. However, the role of LRRC59 in the ER has rarely Received for publication June 8, 2015. Accepted for publication September 11, 2015. been discussed. Because Geo Profile data show that stimulation This work was supported by grants-in-aid from the Ministry of Education, Science, of mouse macrophages and bone marrow–derived dendritic cells and Culture, the Ministry of Health, Labor, and Welfare of Japan, and the Akiyama with polyinosinic:polycytidylic acid [poly(I:C)] or CpG oligo- Life Science Foundation. deoxynucleotide induces LRRC59 mRNA expression (ID: 6823478, Address correspondence and reprint requests to Dr. Misako Matsumoto, Department 12161365, and 12161366), we investigated the function of LRRC59 of Microbiology and Immunology, Hokkaido University Graduate School of Medi- cine, Kita 15, Nishi 7, Kita-ku, Sapporo 060-8638, Japan. E-mail address: matumoto@ in TLR-mediated signaling. We demonstrated that LRRC59 is a pop.med.hokudai.ac.jp novel member of ER-resident proteins that control endosomal lo- The online version of this article contains supplemental material. calization of NA-sensing TLRs. Abbreviations used in this article: CT-B, cholera toxin subunit B; EEA1, early endo- some Ag 1; ER, endoplasmic reticulum; LRR, leucine-rich repeat; LRRC, leucine- rich repeat containing protein; NA, nucleic acid; pAb, polyclonal Ab; PL, proximity Materials and Methods ligation; PLA, proximity ligation assay; poly(I:C), polyinosinic:polycytidylic acid; Cell culture and reagents PRAT4A, protein associated with TLR4 A; qPCR, quantitative PCR; siRNA, small interfering RNA; TICAM-1, Toll/IL-1R domain-containing adapter molecule-1; HEK293 cells were maintained in DMEM low glucose (Invitrogen) sup- UNC93B1, uncoordinated 93 homolog B1. plemented with 10% heat-inactivated FCS (Thermo Scientific) and anti- biotics. HeLa cells were maintained in MEM (Nissui, Tokyo, Japan) Copyright Ó 2015 by The American Association of Immunologists, Inc. 0022-1767/15/$25.00 supplemented with 1% L-glutamine and 5% heat-inactivated FCS. Human www.jimmunol.org/cgi/doi/10.4049/jimmunol.1501305 4934 LRRC59 REGULATES ER EXIT OF TLR3 monocytes were isolated from PBMCs obtained from healthy individuals (GE Healthcare) and incubated with 0.5 mg anti-tag Abs, control rabbit with a magnetic cell sorting system using anti-CD14–coated microbeads IgG (1:200), or anti-LRRC59 pAb (1:200). Immunocomplexes were re- (Miltenyi Biotec). Monocyte-derived macrophages were differentiated covered by incubation with protein G-Sepharose, washed four times with from CD14+ monocytes by culturing with 20 ng/ml recombinant human wash buffer (20 mM Tris-HCl, pH 7.5, containing 100 mM NaCl, 0.5% GM-CSF (Peprotech) for 6 d. The anti-human TLR3 mAb (clone TLR3.7) Nonidet P-40, 10 mM EDTA, 10% glycerol), and resuspended in dena- was generated in our laboratory (22). Anti-FLAG M2 mAb, anti-HA turing buffer. Samples were analyzed by SDS-PAGE (7.5 and 12.5% gel) polyclonal Ab (pAb), anti–b-actin mAb (A2228), and OVA were pur- under reducing conditions followed by immunoblotting with indicated chased from Sigma; anti-HA mAb was from Covance (Emeryville, CA); Abs. anti-LRRC59 pAb was from GeneTex; anti-UNC93B1 pAb was from ProSci; anti-Sar1 mAb was from Abcam; anti-Rab7 mAb (D95F2) and Quantitative PCR anti–early endosome Ag 1 (EEA1) mAb (C45B10) were from Cell Sig- naling; Alexa Fluor 488–conjugated cholera toxin subunit B (CT-B) and Total RNA was extracted using TRIzol reagent and reverse transcribed using Alexa Fluor 488– and 594–conjugated secondary Abs were from Invi- the High Capacity cDNA Reverse Transcription kit (Applied Biosystems) and random primers. qPCR was performed using the following primers and trogen. Poly(I:C) was purchased from GE Bioscience. Pam2CSK4 was synthesized by Biologica Co. Ltd (Nagoya, Japan). LPS and cytochalasin the Step One Real-Time PCR system (Applied Biosystems). Primers for 9 9 9 D were purchased from Sigma; CL075 and M362 were