Reticulum Via Association with UNC93B1 Sensing Tlrs from The

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

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The Journal of Immunology is published twice each month by The American Association of Immunologists, Inc., 1451 Rockville Pike, Suite 650, Rockville, MD 20852 Copyright © 2015 by The American Association of Immunologists, Inc. All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. 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 domain 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 endosome 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 from Invivogen; G3PDH (forward: 5 -GAGTCAACGGATTTGGTCGT-3 , reverse: 5 -TT- 9 9 and Polybead polystyrene 3.0-mm microspheres were from Polysciences. GATTTTGGAGGGATCTCG-3 ); TICAM-1 (forward: 5 -AGCGCCTT- CGACATTCTAGGT-39, reverse: 59- AGGAGAACCATGGCATGCA-39); Plasmids LRRC59 (forward: 59-GAATGAGGTCCCGGTGAAGG-39,reverse:59-TG- AGGCCACACAGAAATCCGAC-39); IFN-b (forward: 59-CAACTTGCTT- cDNAs for human TLR3 and TLR8 were cloned in our laboratory by RT- GGATTCCTACAAAG-39, reverse: 59-TATTCAAGCCTCCCATTCAA- PCR from total RNA of monocyte-derived immature dendritic cells and TTG-39); IL-6 (forward: 59-CTCCAGGAGCCCAGCTATGA-39, reverse: were ligated into the cloning site of the expression vector, pEF-BOS, 59-CCCAGGGAGAAGGCAACTG-39). which was provided by Dr. S. Nagata (Kyoto University). The FLAG-tag Downloaded from or HA-tag was inserted into the C terminus of pEF-BOS expression vectors Confocal microscopy for hTLR3 or hTLR8. The truncated TLR3 mutant DCYT (1–866 aa) was HeLa cells (5.0 3 104 cells/well) were plated onto microcover glasses generated by PCR with Pfu Turbo DNA polymerase (STRATAGENE) (Matsunami, Tokyo, Japan) in a 12-well plate. HEK293 cells (2.0 3 104 using specific primers as described previously (23). A plasmid encoding cells/well) were plated onto poly-L-lysine–coated coverslips (BD Biosci- cDNA of human UNC93B1 (pMD2/UNC93B1) and the expression plas- ences) in a 24-well plate. The following day, cells were transfected with mids for human TLR4 (pEF-BOS/TLR4) and MD-2 (pEF-BOS/MD-2) siRNAs. In some cases, expression vector for TLR3 DCYT truncated were provided by Dr. K. Miyake (University of Tokyo). The pEF-BOS/

mutant were transfected 24 h after siRNA transfection. Forty-eight hours http://www.jimmunol.org/ TLR9-Myc expression plasmid was made from a pBluescript II/human after siRNA transfection, cells were fixed with 4% paraformaldehyde for TLR9 vector that was provided by Dr. S. Akira (Osaka University). The 15 min and permeabilized using PBS containing 0.02% Triton and 1% HA-tag was inserted into the C terminus of the pEF-BOS expression vector BSA for 15 min. Fixed cells were labeled with indicated primary Abs for human UNC93B1. The human UNC93B1 mutant, hUNC93B1 (H412R), (1/100–1/200) overnight at 4˚C. Then Alexa Fluor 488– or 594-conjugated in which the arginine residue at position 412 was substituted for a histidine secondary Abs (1/400) were used to visualize the primary Abs. Prolong residue, was made by site-directed mutagenesis (15). cDNA for human Gold was used for staining the nuclei. Cells were visualized at 363 LRRC59 was cloned in our laboratory by RT-PCR from total RNA of magnification using an LSM510 META microscope (Zeiss, Jena, Ger- HEK293 cells and was ligated into the cloning site of the expression vector, many). Colocalization coefficient between TLR3 DCYT and CT-B was pEF-BOS. Flag-tag was inserted into the C terminus of pEF-BOS expression calculated as in a previous report (24). vector for LRRC59. by guest on October 1, 2021 Luciferase reporter assay Proximity ligation assay 3 4 HEK293 cells (2 3 105 cells/well) cultured in 24-well plates were trans- HeLa cells (5.0 10 cells/well) were plated onto microcover glasses in a 12-well plate. HEK293 cells (2.0 3 104 cells/well) were plated onto poly- fected with indicated expression vectors together with the reporter plasmid L-lysine–coated coverslips in a 24-well plate. The following day, cells were (100 ng/well) and an internal control vector, phRLTK (5 ng/well; Promega, Madison, WI) using Lipofectamine 2000 (Invitrogen). The p-125 lucif- transfected with the expression vector for HA-tagged UNC93B1. To detect erase reporter containing the human IFN-b promoter was provided by the interaction between LRRC59 and UNC93B1 upon poly(I:C) stimula- Dr. T. Taniguchi (University of Tokyo). A luciferase-linked NF-kBre- tion, we used a Duolink In Situ Proximity Ligation Assay (PLA) Kit porter gene was from Stratagene. The reporter plasmid containing the according to the manufacturer’s protocol (Olink Bioscience). After fixation ELAM-1 promoter was constructed in our laboratory. The total amount and permeabilization, cells were stained with anti-LRRC59 pAb and anti- HA mAb. In situ PLA probe Anti-Mouse PLUS and Anti-Rabbit MINUS of DNA (500 ng/well) was kept constant by adding empty vector. After were directed against primary Abs. After ligation, PL+ signals were am- 24 h, cells were reseeded in 96-well plates and stimulated or left un- + treated. Thereafter, cells were lysed in lysis buffer (Promega), and firefly plified and visualized by Duolink In Situ Detection Reagents Red. PL and Renilla luciferase activities were determined using a Dual-Luciferase signals were detected using microscopy and quantified by Duolink Image reporter assay kit (Promega). The firefly luciferase activity was normalized Tool. to the Renilla activity and expressed as the fold stimulation relative to the Statistical analysis activity of unstimulated cells. All assays were performed in triplicate. Statistical analyses were made with the Student t test. The p value of RNA interference significant differences was noted. Small interfering RNA (siRNA) duplexes [TICAM-1, catalog no. s45114; LRRC59, catalog no. s30849 (siLRC59-1) s30851 (siLRC59-2); negative Results control, catalog no. AM4635] were obtained from Applied Biosystems (Ambion). HEK293 cells or HeLa cells cultured in 24-well plates were Knockdown of LRRC59 inhibits TLR3-mediated signaling transfected with siRNA (5 pmol) using Lipofectamine iMAX (Invitrogen). To investigate the role of LRRC59 in TLR3-mediated signaling, Forty-eight hours after transfection, total RNA was extracted by TRIzol we examined whether LRRC59 knockdown affects TLR3-mediated (Invitrogen), or cells were lysed with lysis buffer including 1% Nonidet b P-40 for quantitative PCR (qPCR) or immunoblotting, respectively. IFN- promoter activation in response to poly(I:C) in HEK293 cells. First, we checked knockdown efficiency of two types of Immunoprecipitation and immunoblotting siRNA for LRRC59, siLRC59-1, and siLRC59-2, and found HEK293 cells (5 3 105 cells/well) cultured in six-well plates were siLRC59-2 to be more effective. Then we used siLRC59-2 for transfected as described earlier with the indicated plasmids. The total the following experiments. In cells transfected with siRNA for amount of DNA (2.0 mg/well) was kept constant with an empty vector. LRRC59, poly(I:C)-induced IFN-b promoter activation was reduced After 24 h, cells were stimulated or left untreated and lysed in lysis buffer [20 mM Tris-HCl, pH 7.5, containing 150 mM NaCl, 1% digitonin, 10 mM by half compared with control siRNA–transfected cells, whereas EDTA, 2 mM PMSF, 5 mM Na3VO4, and a protease inhibitor mixture knockdown of TICAM-1 completely abolished poly(I:C)-induced (Roche Diagnostics)]. Lysates were precleared with protein G-Sepharose signaling (Fig. 1A). LRRC59 localizes to the ER and nuclear The Journal of Immunology 4935 Downloaded from http://www.jimmunol.org/ by guest on October 1, 2021

FIGURE 1. LRRC59 is involved in TLR3-mediated signaling upstream of TICAM-1 activation. (A) HEK293 cells were transfected with the indicated siRNAs. Twenty-four hours after siRNA transfection, cells were transfected with an empty vector or expression plasmid for TLR3, together with the IFN-b promoter reporter and phRL-TK. Twenty-four hours after plasmid transfection, culture medium was removed and 2, 10, or 50 mg/ml poly(I:C) was added with fresh medium. Luciferase activitywasmeasured6hafterstimulationandshownasmeanfoldindexinductioncomparedwithnonstimulatedcells(left panel). Knockdown efﬁciency was measured48haftersiRNAtransfectionbyqPCRatthemRNAlevel.Expression of each gene was normalized to G3PDH mRNA expression (middle panels). Knockdown efﬁciency of LRRC59 at the protein level was assessed by immunoblotting with anti-LRRC59 Ab and anti-(b-actin) Ab (right panel). (B) Twenty-four hours after siRNA transfection, cells were transfected with an empty vector or expression plasmid for TICAM1 (0.25, 2.5, or 25 ng) together with the IFN-b promoter reporter and phRL-TK. Luciferase activity was measured 24 h after plasmid transfectionandshownasmeanfoldindexinductioncomparedwithemptyvector transected cells. (C) Twenty-four hours after siRNA transfection, cells were transfected with the IFN-b promoter reporter and phRL-TK. After 24 h, Lipofectamine- conjugated poly(I:C) (0.01, 0.1, 1.0, or 10.0 mg) was transfected to cells. Luciferase activity was measured 24 h after stimulation and (Figure legend continues) 4936 LRRC59 REGULATES ER EXIT OF TLR3 Downloaded from http://www.jimmunol.org/ by guest on October 1, 2021

FIGURE 2. LRRC59 is involved in NA-sensing TLRs, but not in TLR4-mediated signaling. HEK293 cells were transfected with scramble siRNA or siRNA for LRC59. Twenty-four hours after siRNA transfection, cells were transfected with an empty vector or expression plasmid for TLR4 and MD2 (A and B), TLR8 (C), TLR9 (D), or TLR3 (E), with or without increasing amounts of UNC93B1 plasmid (D and E), together with the IFN-b promoter reporter (A and E), NF-kB reporter (B), or ELAM promoter reporter (C and D) and phRL-TK. Twenty-four hours after plasmid transfection, culture medium was removed and 0.1 or 1.0 mg/ml LPS (A and B), 2.0 or 10.0 mg/ml CL075 (C), 5.0 mM ODN2006 (D), or 10 mg/ml poly(I:C) was added with fresh medium. Luciferase activity was measured 12 (A–C), 24 (D), or 6 h (E) after stimulation and shown as mean fold index induction compared with vector-transfected cells (A and B) or unstimulated cells (C–E). Representative data from three independent experiments are shown (mean 6 SD). *p , 0.05. membrane; therefore, it was assumed that LRRC59 would play a positively regulates TLR3 signaling before TLR3 interacts with role in TLR3 trafﬁcking from the ER to Golgi, or downstream signal TICAM-1 to cause TICAM-1 dimerization (Fig. 1B). The subcel- transduction, including nuclear import of transcription factors. We lular localization of LRRC59 suggested that LRRC59 was involved then measured TICAM-1–induced IFN-b promoter activation in in modulation of signal transduction from cytoplasmic RIG-I-like TICAM-1–overexpressed HEK293 cells to determine whether receptors. We experimentally conﬁrmed that LRRC59 was inde- LRRC59 functions upstream or downstream of TICAM-1 in TLR3- pendent of RIG-I–like receptor–mediated signaling in poly(I:C)- mediated signaling. Knockdown of LRRC59 did not affect TICAM- transfected HEK293 cells (Fig. 1C). To test the biological impor- 1–mediated IFN-b promoter activation, indicating that LRRC59 tance of LRRC59, we performed knockdown of LRRC59 in human

shown as mean fold index induction compared with nonstimulated cells. Representative data from three independent experiments are shown (mean 6 SD). (D) Monocyte-derived macrophages were transfected with control siRNA or siRNA for LRRC59 at days 4 and 5 of differentiation from monocytes. Twenty-four hours after siRNA transfection, cells were stimulated with poly(I:C) for 1 or 3 h and total RNAs were extracted. The mRNA levels of IFN-b, IL-6, LRRC59, and G3PDH were measured by qPCR. Expression of each gene was normalized to G3PDH mRNA expression. *p , 0.05. The Journal of Immunology 4937 Downloaded from http://www.jimmunol.org/ by guest on October 1, 2021

FIGURE 3. LRRC59 associates with UNC93B1 in a poly(I:C) stimulation-dependent manner. (A and C) HEK293 cells were transfected with FLAG- tagged LRRC59 and HA-tagged UNC93B1 wild type (WT) (A and C) or H412R mutant (C). After 24 h, cells were stimulated with poly(I:C) (25 mg/ml) for 30 min. Then cells were lysed and UNC93B1 was immunoprecipitated (IP) using anti-HA pAb. The immunoprecipitants were resolved on SDS-PAGE (12.5 and 7.5% gel) under reducing conditions followed by immunoblotting (IB) with anti-FLAG mAb or anti-HA mAb. Whole cell lysate (WCL) was subjected to immunoblotting with anti-FLAG mAb to detect protein expression of LRRC59. (B) HEK293 cells were stimulated with 25 mg/ml poly(I:C). Thirty minutes after stimulation, cells were lysed and endogenous LRRC59 was immunoprecipitated using an anti-LRRC59 pAb. The immunoprecipitants were resolved on SDS-PAGE (12.5 and 7.5% gel) under reducing conditions followed by immunoblotting with anti-UNC93B1 pAb or anti-LRRC59 pAb. WCLs were subjected to immunoblotting with anti-UNC93B1 pAb and anti-LRRC59 pAb. (D) HEK293 cells were transfected with HA-tagged UNC93B1. Twenty-four hours after transfection, cells were stimulated with poly(I:C) 50 mg/ml for 30 min and then ﬁxed and permeabilized. After staining with anti- LRRC59 pAb and anti-HA mAb, cells were incubated with an Alexa Fluor 488– and 594–conjugated secondary Ab and analyzed using confocal microscopy. Red, LRRC59; green, HA-tagged UNC93B1; light blue, nuclei with DAPI; yellow, merged LRRC59 with HA-tagged (Figure legend continues) 4938 LRRC59 REGULATES ER EXIT OF TLR3

FIGURE 4. Endocytic event triggers the association of LRRC59 and UNC93B1. (A–D) HEK293 cells were transfected with FLAG- Downloaded from tagged LRRC59 and HA-tagged UNC93B1. After 24 h, cells were stimulated with poly(I:C) (25 mg/ml) (A, B,andD), CL075 (5 mg/ml)

(A), M362 (10 mg/ml) (A), Pam2CSK4 (100 nM) (A), LPS (100 ng/ml) (A and C), polystyrene bead (5 ml/ml) (D), or OVA m D B C (10 g/ml) ( ) for 30 min. ( and )Cells http://www.jimmunol.org/ were incubated with DMSO or cytochalasin D (0.4 mg/ml) for 1 h before ligand stimulation. Then cells were lysed and UNC93B1 was immunoprecipitated (IP) using anti-HA pAb. The immunoprecipitants were resolved on SDS-PAGE (12.5 and 7.5% gel) under reducing conditions followed by immunoblotting (IB) with anti- FLAG mAb or anti-HA mAb. Whole cell lysate (WCL) was subjected to immuno- by guest on October 1, 2021 blotting with anti-FLAG mAb to detect protein expression of LRRC59.

monocyte-derived macrophages and analyzed TLR3-mediated sig- IL-6 mRNA expression were signiﬁcantly reduced in LRRC59- naling. LRRC59 was efﬁciently knocked down in macrophages at the knockdown macrophages compared with control macrophages mRNA level (Fig. 1D, right panel). Poly(I:C)-induced IFN-b and (Fig. 1D, left and middle panels). These results indicate that LRRC59

UNC93B1. Scale bar, 10 mm. (E) HEK293 or HeLa cells cultured on cover glasses were transfected with HA-tagged UNC93B1. Twenty-four hours after transfection, cells were stimulated with 50 mg/ml poly(I:C) for 30 min and then ﬁxed and permeabilized. After staining with anti-LRRC59 pAb and anti-HA mAb, interaction between LRRC59 and HA-tagged UNC93B1 was measured by PLA. Red, PL+ signals; light blue, nuclei with DAPI. Using Duolink Image Tool, the number of PL+ signal was counted in ∼200 HeLa cells from 15 areas or 700 HEK293 cells from 10 areas. The average number of PL+ signal per single cell in unstimulated and stimulated cells is shown. Scale bar, 10 mm. The Journal of Immunology 4939 Downloaded from http://www.jimmunol.org/ by guest on October 1, 2021

FIGURE 5. LRRC59 helps TLR3 trafficking from the ER to endosomes. (A) HeLa cells were transfected with control siRNA or siRNA for LRRC59. Forty-two hours after transfection, cells were pretreated with 400 IU/ml IFN-b for 6 h and then fixed and permeabilized. After staining with anti-TLR3 mAb and anti-EEA1 pAb (left panels) or anti-LRRC59 (right upper panels), cells were incubated with an Alexa Fluor 488– and 594–conjugated secondary Ab and analyzed using confocal microscopy. Red, EEA1 or LRRC59; green, TLR3; light blue, nuclei with DAPI; yellow, merged TLR3 with EEA1. Scale bar, 10 mm. Colocalization between EEA1 and TLR3 was analyzed by counting the number of cells bearing merged TLR3 with EEA1 in 50 cells. The data are shown as percentage of cells with colocalization between TLR3 and EEA1 (right lower panel). (B) HEK293 cells were transfected with control siRNA or siRNA for LRRC59. After 24 h, expression vector for the TLR3 DCYT truncated mutant was transfected. Twenty-four hours after plasmid transfection, cells were fixed and permeabilized. After staining with anti-TLR3 mAb and Alexa 488–conjugated CT-B (left panels) or anti-LRRC59 (right panels), cells were incubated with an Alexa Fluor 594–conjugated secondary Ab and then analyzed using confocal microscopy. Representative images are shown. Red, TLR3 DCYT or LRRC59; green, CT-B; light blue, nuclei with DAPI; yellow, merged TLR3 DCYT with CT-B. Colocalization (Figure legend continues) 4940 LRRC59 REGULATES ER EXIT OF TLR3

regulates TLR3-mediated signaling in primary immune cells in- including CL075 (TLR8 ligand), M362 (TLR9 ligand), Pam2CSK4 cluding macrophages. (TLR2/6 ligand), and LPS (TLR4 ligand), clearly enhanced this association independent of TLRs, indicating that NAs and their Knockdown of LRRC59 inhibits NA-sensing, TLR-mediated derivatives, as well as LPS/lipopeptides, can promote LRRC59 to signaling, but not TLR4-mediated signaling bind UNC93B1 (Fig. 4A). These results correspond to other pre- We examined whether LRRC59 had a role in other TLR-mediated vious reports that trafficking of TLR7/9 from the ER to endosomes/ signaling in HEK293 cells using a reporter assay. Knockdown of lysosomes is not ligand specific (14); for example, LPS-induced LRRC59 did not affect the IFN-b promoter and NF-kB activation endosomal expression of TLR7 has been reported in TLR42 cells upon LPS stimulation in HEK293 cells overexpressing TLR4 (14). To examine whether LRRC59 association with UNC93B1 and MD2 (Fig. 2A, 2B). TLR8- or TLR9-mediated ELAM pro- depends on the internalization of ligands, we treated cells with moter activation was decreased when LRRC59 was knocked down cytochalasin D, a phagocytosis inhibitor. Pretreatment of cells with (Fig. 2C, 2D). Because trafficking of TLR4 from the ER to the cell cytochalasin D abolished the enhancement of interaction between surface is independent of UNC93B1, unlike NA-sensing TLRs LRRC59 and UNC93B1 by poly(I:C) or LPS (Fig. 4B, 4C), sug- (12), LRRC59 appears to participate in the UNC93B1-dependent, gesting that endocytosis elicits interaction of LRRC59 with TLR-mediated signaling. TLR9-mediated signaling is low in the UNC93B1. We further examined whether any endocytic event triggers HEK293 cells used in this study; thus, UNC93B1 was forcibly the association. We used OVA and Polybead polystyrene microspheres expressed in HEK293 cells, and the effect of LRRC59 knockdown as stimulators, which are known to be endocytosed and phagocytosed was determined. TLR9-mediated ELAM promoter activation was into cells, respectively. Both stimuli induced interaction between decreased by half in LRRC59-knockdown cells compared with LRRC59 and UNC93B1 (Fig. 4D). Thus, endocytic/phagocytic events control cells, even in the presence of UNC93B1 (Fig. 2D). In trigger the association of LRRC59 and UNC93B1 by some unknown Downloaded from the case of TLR3 signaling, UNC93B1-dependent upregulation mechanisms. of TLR3 signaling was efficiently impaired by knockdown of LRRC59 (Fig. 2E). These results suggest that LRRC59 positively LRRC59 is required for TLR3 translocation from the ER to regulates NA-sensing, TLR-mediated signaling in a UNC93B1- endosomes dependent manner. Considering that LRRC59 resides on the ER To determine the ability of LRRC59 to facilitate TLR3 trafficking membranes and NA-sensing TLRs traffic from the ER to the from the ER, we performed confocal microscopic analysis to http://www.jimmunol.org/ endosomes dependent on UNC93B1 either under the steady-state observe the endogenous TLR3 localization in LRRC59 knock- or ligand stimulation, LRRC59 might facilitate ER egress of NA- down HeLa cells. Cells were preincubated with IFN-b to upreg- sensing TLRs. ulate TLR3 expression. Compared with cells treated with control LRRC59 interacts with UNC93B1 by poly(I:C) stimulation siRNA, cells silencing LRRC59 reduced the colocalization of TLR3 and early endosome marker EEA1 (Fig. 5A). These re- Stimulation with TLR ligands triggers translocation of NA- sults were confirmed in HEK293 cells transiently expressed sensing TLRs to endosomes/lysosomes (25, 26). We investi- with TLR3 (Supplemental Fig. 1). Because endosomal locali- gated the interaction between LRRC59 and UNC93B1 by zation of TLR3 was sparse even in control HeLa cells treated coimmunoprecipitation assay. LRRC59 barely associated with by guest on October 1, 2021 with IFN-b, we used TLR3 DCYT truncated mutant that pre- UNC93B1 when they were overexpressed in HEK293 cells and dominantly localizes on the cell surface after exit from the ER their interaction was greatly increased by stimulation with (23) to verify the role of LRRC59 in TLR3 trafficking. As ex- poly(I:C) (Fig. 3A). The poly(I:C)-induced association of en- pected, overexpressed TLR3 DCYT colocalized with CT-B, a dogenous LRRC59 with UNC93B1 was also detected in clear plasma membrane marker, in control HEK293 cells (Fig. 5B). HEK293 cells (Fig. 3B). However, their interaction appears On the other hand, TLR3 DCYT mutant rarely localized to the to be independent of TLR3-mediated signaling, because HEK293 plasma membrane when LRRC59 was knocked down (Fig. 5B). cells are TLR32. TLR-independent association of LRRC59 with Quantitative analyses showed that colocalization coefficient between UNC93B1 was further examined using UNC93B1 H412R mutant TLR3 DCYT and CT-B was decreased in LRRC59 knockdown cells that is unable to bind NA-sensing TLRs. LRRC59 could bind (Fig.5B).Notably,forciblyexpressedTLR3DCYT colocalized to H412R mutant similar to wild-type UNC93B1 in response with UNC93B1 at the cell surface but not with LRRC59 that to poly(I:C) stimulation (Fig. 3C), which never affects the remained on the ER (Fig. 5C), suggesting a different role in trafficking and signaling of the TLRs. Immunofluorescence TLR sorting between UNC93B1 and LRRC59. These results analysis showed that LRRC59 colocalized with UNC93B1 in a indicate that LRRC59 is involved in ER exit of TLR3 cooper- stimulation-dependent manner (Fig. 3D). Poly(I:C)-induced colo- atively with UNC93B1, but its regulatory mechanism is differ- calization of LRRC59 and UNC93B1 was quantitatively analyzed ent from that of UNC93B1. in HEK293 and HeLa cells using PLA. Average number of PL+ signal per single cell was increased in poly(I:C)-stimulated cells compared with unstimulated cells (Fig. 3E). These results indicate Discussion that poly(I:C) triggers interaction of LRRC59 and UNC93B1. Endosomal localization of NA-sensing TLRs is an excellent strategy to avoid recognition of host-derived NAs (27–30). The LRRC59 interacts with UNC93B1 in a TLR-independent optimum level of functional TLRs in endosomes/lysosomes is manner important for induction of prompt antimicrobial immune re- The association between LRRC59 and UNC93B1 was investigated sponses, as well as prevention of undesired autoactivation. Mac- by similar coimmunoprecipitation assay in HEK293 cells stimu- rophages and dendritic cells express high levels of UNC93B1 to lated with other TLR agonists. Notably, all tested TLR ligands, provide functional cleaved/associated forms of TLR3, 7, 8, and 9 coefficient of TLR3 DCYT with CT-B was calculated using LSM510 ZEISS LSM Image examiner software in 10 images (4–5 cells/image) of control or LRRC59 knockdown cells. **p , 0.01. (C) HEK293 cells were transfected with an expression plasmid for TLR3 DCYT. Twenty-four hours after plasmid transfection, fixed/permeabilized cells were stained with anti-TLR3 mAb, anti-UNC93B1 Ab, and anti-LRRC59 Ab. Scale bar, 10 mm. The Journal of Immunology 4941 in the endosomes/lysosomes, whereas nonimmune cells such as with UNC93B1 via the cytoplasmic region and assists the loading MEFs and epithelial cells have low levels of UNC93B1, resulting of NA-sensing TLRs into COPII vesicles. Upon poly(I:C) stimu- in the predominance of the uncleaved form of these TLRs in the lation, an endogenous LRRC59 colocalized with Sar1, which is a ER (5, 11, 31, 32). In the steady-state, UNC93B1 controls traf- key player in assembly of the COPII coat on the ER membrane, in ficking of NA-sensing TLRs from the ER through binding to their both HeLa and HEK293 cells (Supplemental Fig. 2). However, it transmembrane domains and the juxtamembrane region at the ER remains undetermined how stimulation with TLR ligands promotes (12–14, 33), and coexists with translocated TLRs. In particular, LRRC59 binding to UNC93B1 and influences the TLR sorting from ER egress of TLR7/9 is tightly regulated via the N-terminal region the ER. ER stress followed by internalization of nonself molecules of UNC93B1 to balance their expression, which prevents the in- including TLR ligands may cause the alteration of protein transport duction of autoimmunity (34, 35). Although several reports have and localization. Ligand-induced, LRRC59-dependent trafficking of shown that NA-sensing TLRs are translocated from the ER NA-sensing TLRs might be beneficial for augmentation of antimi- to lysosomes upon ligand stimulation (25, 26), the molecular crobial immune responses, as well as restraint on the TLR-mediated mechanism of ligand-dependent trafficking of the TLRs is response in sterile/inflammatory states. largely unknown. In this study, we demonstrate that an ER-resident LRRC59 Acknowledgments protein regulates NA-sensing, TLR-mediated signaling. In human We are grateful to laboratory members for valuable discussions. We also macrophages, TLR3-mediated IFN-b and IL-6 mRNA expression thank Drs. K. Miyake and T. Taniguchi (University of Tokyo), Dr. S. Akira is reduced by knockdown of LRRC59 (Fig. 1D). LRRC59 helps (Osaka University), and Dr. S. Nagata (Kyoto University) for providing the UNC93B1-mediated TLR trafficking through ligand-induced plasmids. binding to UNC93B1. Furthermore, ligand-endocytosis pro- Downloaded from cess, but not TLR signal itself, affects this LRRC59–UNC93B1 Disclosures interaction and TLR trafficking. Because endosomal localiza- The authors have no financial conflicts of interest. tion of endogenous TLR3 or cell-surface localization of the TLR3 DCYT mutant is reduced by LRRC59 knockdown (Fig. 5A, 5B, Supplemental Fig. 1), LRRC59 appears to participate in TLR3 References

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stimulation. J. Immunol. 173: 1179–1183. 38. D’Arcangelo, J. G., K. R. Stahmer, and E. A. Miller. 2013. Vesicle-mediated http://www.jimmunol.org/ 27. Marshak-Rothstein, A. 2006. Toll-like receptors in systemic autoimmune dis- export from the ER: COPII coat function and regulation. Biochim. Biophys. Acta ease. Nat. Rev. Immunol. 6: 823–835. 1833: 2464–2472. by guest on October 1, 2021 HEK293 +DIC/DAPI EEA1/Rab7 TLR3-flag Merge

si control

si LRC59

Supplementary Figure 1. LRRC59 is required for endosomal localization of TLR3. HEK293 cells were transfected with control siRNA or siRNA for LRRC59. Twenty-four hours after siRNA transfection, cells were transfected with an expression plasmid for TLR3-Flag. After 24 h, fixed/permeabilized cells were stained with anti-TLR3 mAb, anti-EEA1 Ab, and anti-Rab7 Ab. Red, EEA1 and Rab7; green, TLR3; light blue, nuclei with 4’ ,6-diamidino-2-phenylindole; yellow, merged TLR3 with EEA1/Rab7. Scale bar, 10 µm. LRRC59 Sar1 +DIC/DAPI endogenous endogenous Merge A HEK293

unstimulation

poly(I:C) 30 min

B HeLa LRRC59 Sar1 +DIC/DAPI endogenous endogenous Merge

unstimulation

poly(I:C) 30 min

Supplementary Figure 2. LRRC59 co-localizes with Sar1 in a poly(I:C) stimulation-dependent manner in HEK293 cells and HeLa cells. A,B, HEK293 cells (A) or HeLa cells (B) were stimulated with 50 µg/ml poly(I:C) for 30 min and then fixed and permeabilized. After staining with anti-LRRC59 pAb and anti-Sar1 mAb, cells were incubated with an Alexa Fluor-488- and -594-conjugated secondary Ab and analyzed using confocal microscopy. Red, LRRC59; green, Sar1; light blue, nuclei with 4’ ,6-diamidino-2-phenylindole; yellow, merged LRRC59 with Sar1. Scale bar, 10 µm.