Acidic Residues in the Juxtamembrane Region of the Nucleotide-Sensing TLRs Are Important for UNC93B1 Binding and Signaling This information is current as of September 28, 2021. Jihee Kim, Jiwon Huh, Misun Hwang, Eun-Hye Kwon, Da-Jung Jung, Melanie M. Brinkmann, Myoung Ho Jang, Hidde L. Ploegh and You-Me Kim J Immunol 2013; 190:5287-5295; Prepublished online 12 April 2013; Downloaded from doi: 10.4049/jimmunol.1202767 http://www.jimmunol.org/content/190/10/5287 http://www.jimmunol.org/ Supplementary http://www.jimmunol.org/content/suppl/2013/04/12/jimmunol.120276 Material 7.DC1 References This article cites 51 articles, 15 of which you can access for free at: http://www.jimmunol.org/content/190/10/5287.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 © 2013 by The American Association of Immunologists, Inc. All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. The Journal of Immunology

Acidic Amino Acid Residues in the Juxtamembrane Region of the Nucleotide-Sensing TLRs Are Important for UNC93B1 Binding and Signaling

Jihee Kim,* Jiwon Huh,* Misun Hwang,* Eun-Hye Kwon,* Da-Jung Jung,* Melanie M. Brinkmann,†,‡ Myoung Ho Jang,*,x Hidde L. Ploegh,‡ and You-Me Kim*,{

TLRs are divided into two groups based on their subcellular localization patterns. TLR1, 2, 4, 5, and 6 are expressed on the cell surface, whereas the nucleotide-sensing TLRs, such as TLR3, 7, 8, and 9 stay mainly inside cells. The polytopic membrane UNC93B1 physically interacts with the nucleotide-sensing TLRs and delivers them from the endoplasmic reticulum to endolyso- somes, where the TLRs recognize their ligands and initiate signaling. In cells with nonfunctional UNC93B1, the nucleic acid–sensing

TLRs fail to exit the endoplasmic reticulum and consequently do not signal. However, the detailed molecular mechanisms that Downloaded from underlie the UNC93B1-mediated TLR trafficking remain to be clarified. All nucleotide-sensing TLRs contain acidic amino acid residues in the juxtamembrane region between the leucine-rich repeat domain and the transmembrane segment. We show that the D812 and E813 residues of TLR9 and the D699 and E704 residues of TLR3 help to determine the interaction of these TLRs with UNC93B1. Mutation of the acidic residues in TLR3 and TLR9 prevents UNC93B1 binding, as well as impairs TLR trafficking and renders the mutant receptors incapable of transmitting signals. Therefore, the acidic residues in the juxtamembrane region of the nucleotide-sensing TLRs have important functional roles. The Journal of Immunology, 2013, 190: 5287–5295. http://www.jimmunol.org/

oreign DNA and RNA derived from invading and tion of extracellular host nucleic acids that are released from bacteria are sensed by several families of innate immune damaged cells. Among TLRs, TLR3, 7, 8, 9, and 13 recognize F receptors, including TLRs, RIG-I-like receptors, and RNA, DNA, or small nucleotide analogs (10–15). Unlike the PYHIN family , all of which can activate inflammatory other TLRs that are expressed on the plasma membrane, these responses (1–3). Because endogenous nucleic acids can also ac- nucleotide-sensing TLRs are largely found inside cells (16, 17). tivate these receptors in pathogenic contexts and facilitate in- Mice engineered to artificially express TLR9 on the cell surface flammatory or autoimmune diseases, the host must have evolved succumb to fatal systemic inflammation, underscoring the physi- mechanisms to discriminate microbial nucleic acids from endog- ological importance of intracellular localization of the nucleotide- by guest on September 28, 2021 enous ones (4–7). One strategy for such discrimination is se- sensing receptors (18). questration of the nucleotide-sensing receptors inside cells (8, 9). TLR9 resides in the endoplasmic reticulum (ER) in resting cells Although capable of sensing DNA and RNA from viruses or and is recruited to endolysosomes upon stimulation with its ligands phagocytosed bacteria that enter the cytoplasm or endosomes, the (19–21). In the endolysosomes, the ectodomain of TLR9 is pro- intracellular receptors can avoid encountering a high concentra- teolytically processed by cathepsins and/or asparagine endopep- tidase, an event critical for rendering TLR9 signaling competent (22–25). This mode of regulation, involving control of traf- *Division of Integrative Biosciences and Biotechnology, Pohang University of Science ficking from the ER and subsequent proteolysis in endolysosomes, † and Technology, Pohang 790-784, Republic of Korea; Helmholtz Centre for Infection is not limited to TLR9 but applies to other nucleotide-sensing TLRs; Research, 38124 Braunschweig, Germany; ‡Whitehead Institute for Biomedical Research, Cambridge, MA 02142; xWorld Premier International Immunology Frontier it provides an additional layer of protection against unwanted { Research Center, Osaka University, 565-0871 Osaka, Japan; and Department of self DNA/RNA-triggered inflammatory responses through confine- Life Science, Pohang University of Science and Technology, Pohang 790-784, Republic of Korea ment of receptor activation to the acidic intracellular compartments (23, 24, 26, 27). Received for publication October 3, 2012. Accepted for publication March 18, 2013. The intracellular localization of each nucleotide-sensing TLR This work was supported by the Basic Science Research Program (20120001860) and the World Class University program (R31-10105) through the National Research is regulated by distinct localization motifs. A 23-aa sequence in Foundation of Korea funded by the Ministry of Education, Science and Technology the linker region (between the transmembrane and the Toll/IL-1R and by the Pohang University of Science and Technology Basic Science Research [TIR] domains) of TLR3 is important for intracellular retention, Institute Grant. whereas localization of TLR7 is determined through the trans- Address correspondence and reprint requests to Prof. You-Me Kim, Division of Integrative Biosciences and Biotechnology, Department of Life Science, Pohang membrane domain (28, 29). Localization of TLR8 to early endo- University of Science and Technology, Pohang University of Science and Technology somes requires both the transmembrane domain and the TIR Biotechnology Center, Room 270, San-31, Hyoja-dong, Nam-gu, Pohang, Kyeong- domain (30). For TLR9, the transmembrane domain and a specific buk 790-784, Republic of Korea. E-mail address: [email protected] region within the TIR domain are thought to be its localization The online version of this article contains supplemental material. determinants (31, 32). Despite the apparently different sequence Abbreviations used in this article: BMDC, –derived ; BMDM, bone marrow–derived macrophage; ER, endoplasmic reticulum; HA, hem- motifs that control the intracellular localization of the various agglutinin; ISRE, IFN-stimulated response element; LRR, leucine-rich repeat; pDC, nucleic acid–sensing TLRs, the nature of endosomal compartments plasmacytoid dendritic cell; p(I:C), polyinosinic-polycytidylic acid; POSTECH, to which the receptors are targeted seem to largely overlap (29, 31). Pohang University of Science and Technology; TIR, Toll/IL-1R. In addition, these TLRs share a requirement for UNC93B1 to exit Copyright Ó 2013 by The American Association of Immunologists, Inc. 0022-1767/13/$16.00 from the ER (33). www.jimmunol.org/cgi/doi/10.4049/jimmunol.1202767 5288 UNC93B1-BINDING RESIDUES IN THE NUCLEOTIDE-SENSING TLRs

The role of UNC93B1 in the TLR pathway was first appreciated Preparation of bone marrow–derived dendritic cells in a forward genetic screening that led to identification of the 3d Bone marrow cells were harvested by flushing the femur and tibia of wild mutant mouse (34). The 3d mutant mice have a single point type or TLR9-deficient mice using a syringe with a 23-gauge needle. After mutation (H412R) in UNC93B1 and show signaling defects of a single-cell suspension was prepared by trituration, RBCs were eliminated TLR3, 7, and 9, whereas the responses of the cell surface–local- by hypotonic lysis with ACK lysis buffer for 3 min. To induce differentiation ized TLR2 and TLR4 are unaffected. Consequently, 3d mutant of hematopoietic stem cells into dendritic cells, bone marrow cells were plated at 1–2 3 106 cells/ml in DMEM supplemented with 10% heat- mice are hypersusceptible to infection with various pathogens. inactivated FBS, penicillin/streptomycin, 50 mM 2-ME, 10 mM HEPES, Signaling defects in the nucleotide-sensing TLRs were likewise and 1% J588L/GM-CSF cell supernatant (final concentration ∼10 ng/ml observed in human pediatric patients with an autosomal deficiency GM-CSF). Half of the media was replaced with fresh media on days 2 and of UNC93B1. These patients suffer from recurrent herpes simplex 4, and bone marrow–derived dendritic cells (BMDCs) were harvested for experiments after 5–6 d of culture. encephalitis, attributable to impaired antiviral type I IFN respon- ses (35). We demonstrated that UNC93B1 binds to the trans- DNA constructs membrane domains of the nucleotide-sensing TLRs in the ER and pMSCVpuro-TLR9-GFP, pMSCVpuro-TLR9-myc, pMSCVpuro-TLR3- mediates their trafficking to endolysosomes (33, 36). The 3d myc, pMSCVpuro-TLR4-myc, pMSCVpuro-TLR9444(or TLR9N4C)- (H412R) mutation of UNC93B1 abolishes the interaction with all GFP, pMSCVneo-UNC93B1-flag-TEV-HA (referred to as UNC93B1-HA nucleotide-sensing TLRs, prevents the trafficking of TLR7 and in this article), and pMSCVneo-UNC93B-cherry constructs were de- TLR9 to endolysosomes, and consequently blocks the sensing of scribed previously (33, 36). The mutant TLR9 (D812S, E813T, D818S, or D812S/E813T) and the mutant TLR3 (D699S, E704T, or D699S/E704T) DNA and RNA by the TLRs. were generated by PCR with primers carrying the following mutations: The exact molecular nature of the interaction with UNC93B1 GAT to AGT (TLR9 D812S), GAG to ACG (TLR9 E813T), GAC to AGC Downloaded from seems to vary for different TLRs. For example, the D34A mutation (TLR9 D818S), GAC to AGC (TLR3 D699S), and GAA to ACA (TLR3 of UNC93B1 differentially affects trafficking of TLR7 versus E704T). The chimeric TLR9944-GFP construct was constructed by “PCR TLR9 to the endolysosomes and augments signaling of TLR7 sewing” of cDNA corresponding to amino acid residues 1–819 of murine TLR9 with amino acid residues 632–835 of murine TLR4. The sequences while inhibiting TLR9 (37). The UNC93B1 (D34A) mutant knock- of all constructs were verified by sequencing. in mice develop TLR7-dependent lethal systemic inflammation, Retroviral transduction demonstrating the importance of UNC93B1 in coordinating the http://www.jimmunol.org/ signaling pathways of the nucleotide-sensing TLRs (38). How- Retroviruses were generated in HEK293T cells by cotransfection of ever, the details of the molecular interactions between UNC93B1 pMSCV plasmid encoding the protein of interest along with the packaging and TLRs remain poorly understood. plasmids for VSV-G and gag/pol. At 34 h and 50 h posttransfection, me- dium containing viral particles was harvested and added to RAW cells or In this study, we found that all nucleotide-sensing TLRs have BMDCs at day 1 of BMDC culture along with 8 mg/ml polybrene. Cells acidic amino acid residues in the juxtamembrane regions and that were centrifuged for 90 min at 2200 rpm and were given fresh media the these acidic residues play an important role in the interaction with following day. UNC93B1 and receptor signaling. Point mutations of the acidic Immunoprecipitation and immunoblotting residues in TLR9 and TLR3 disrupt the interaction with UNC93B1, prevent the trafficking of the mutant TLR9 out of the ER, and Cells were lysed in 1% digitonin lysis buffer (50 mM Tris-HCl [pH 8], 150 by guest on September 28, 2021 render the receptors incapable of transmitting DNA- or RNA- mM NaCl, 5 mM EDTA, 1% digitonin) supplemented with a protease inhibitor mixture composed of leupeptin, pepstatin A, aprotinin, and PMSF. evoked signals. Collectively, our results highlight the functional The lysates were cleared of insoluble materials by centrifugation at 14,000 importance of the shared acidic amino acid residues in the juxta- rpm for 15 min. For immunoprecipitation, the lysates equalized for total membrane region of TLRs to regulate their activity. protein amounts were incubated with an appropriate Ab for 4 h and then overnight with protein A–agarose beads at 4˚C. The beads were washed three times with 0.1% digitonin lysis buffer, and the immunoprecipitated Materials and Methods proteins were eluted by mild denaturation in the SDS sample buffer for 30 Reagents min at 37˚C. Proteins were separated by SDS-PAGE, transferred to a ni- trocellulose membrane, and probed with the indicated Ab. The chemilu- CpG DNA (1826) was purchased from TIB Molbiol, and LPS (Escherichia minescence was detected with an ImageQuant LAS 4000 (GE Healthcare), coli serotype 026:B6), polyinosinic-polycytidylic acid [p(I:C)] (.500 bp), and the band intensities were quantified using National Institutes of Health and brefeldin A were purchased from Sigma. Rabbit polyclonal anti-GFP ImageJ software. (ab290) and rat monoclonal anti-hemagglutinin (HA) (clone 3F10) Abs were purchased from Abcam and Roche, respectively. Mouse monoclonal Intracellular TNF assay anti-GFP (B-2) and mouse monoclonal anti-myc (9B11) Abs were pur- BMDCs or macrophages expressing either wild type or mutant TLR9-GFP chased from Santa Cruz and Cell Signaling Technology, respectively. were stimulated with 1 mM CpG or 50 ng/ml LPS for 4 h in the presence 10 Protein A–agarose beads were from Roche. ELISA Abs and mg/ml brefeldin A. Cells were fixed with 3.7% formaldehyde for 10 min, allophycocyanin-conjugated anti-mouse TNF Ab (clone MP6-XT22) were washed, and permeabilized with 0.5% saponin in FACS buffer (PBS purchased from BD Biosciences. All common reagents were from Sigma, containing 2% BSA and 0.05% NaN3) for 10 min at room temperature. unless stated otherwise. The cells were subsequently stained with 0.5 mg/ml allophycocyanin- conjugated anti-TNF Ab (clone MP6-XT22) for 30 min at room temper- Mice and cell lines ature. Following the washing steps, the fluorescence intensity was mea- C57BL/6 wild type mice were purchased from The Jackson Laboratory (Bar sured with flow cytometers (Gallios; Beckman Coulter or Fortessa; BD Harbor, ME). TLR9 knockout mice on the C57BL/6 background were Biosciences), and the data were analyzed using FlowJo software (TreeS- purchased from Oriental Bio Service (Kyoto, Japan). All animals were tar). housed in a specific pathogen–free facility at Pohang University of Science Luciferase assay and Technology (POSTECH), and all animal experiments were approved by the Ethics Committee of POSTECH, in accordance with the institu- The pcDNA3.1–NF-kB–luciferase reporter construct was generated by tional, regional, and national guidelines. Immortalized wild type and PCR amplification of the NF-kB–luciferase cis-reporter from the TLR9-deficient bone marrow–derived macrophages (BMDMs) were ob- pNF-kB–Luc plasmid (Stratagene) and subcloning into the pcDNA3.1(+) tained from BEI Resources. Murine RAW 264.7 macrophages (ATCC TIB- vector using BglII and XhoI restriction enzyme sites. The resulting 71) and HEK293T cells (ATCC CRL-11268) were cultured in DMEM pcDNA3.1–NF-kB–luciferase plasmid no longer has the CMV promoter. containing 5% FBS. Stable cell lines were prepared by spin infection of The p–IFN-stimulated response element (ISRE)-Luc reporter construct cells with retroviruses generated with pMSCV vectors encoding the target was purchased from Stratagene. HEK293T cells were cotransfected with cDNA and selected with 100 mg/ml G418 or 5 mg/ml puromycin. either an NF-kB– or ISRE-luciferase reporter plasmid along with ex- The Journal of Immunology 5289 pression plasmids for the TLR of interest and UNC93B1 in six-well plates. The total amount of transfected DNA was kept constant by supplementing with an empty vector. After 24 h, cells were plated into a 96-well plate and stimulated the next day with p(I:C) for the indicated times. Cells were then lysed in 0.1% Triton X-100 lysis buffer, and the luciferase activity was measured on a Luminoskan Ascent DLReady Reader (Thermo Lab sys- tems) using D-luciferin (Promega) as a substrate. The level of luciferase activity was expressed as fold induction relative to the luciferase activity in unstimulated cells.

Live cell imaging BMDCs from wild type or TLR9-deficient mice were seeded in eight well– chambered coverglasses (LAB-TEK) and retrovirally transduced with various GFP- or cherry-tagged protein constructs. At day 5 or 6 of BMDC culture, the cells were imaged using a spinning disk confocal microscope equipped with an Olympus IX81 motorized inverted microscope, a Yoko- gawa CSU-X1 spinning disk confocal head, a VisiTech multilaser merge module system with four lasers (405, 488, 561, 642 nm), acousto-optic tunable filters, a Ludl MAC6000 controller for an xy stage with a Piezo z, Ludl filter wheels, and an Ando iXon EM CCD camera. During imaging, the cells were maintained in phenol red–free DMEM supplemented with

10% FBS and 25 mM HEPES (pH 7.4) at constant temperature of 37˚C Downloaded from with 5% atmospheric CO2 using a Chamlide chamber system (Live Cell Instrument). Metamorph software (Molecular Devices) was used for image acquisition, and figures were constructed using Adobe Photoshop and Il- lustrator. To quantify the degrees of colocalization of two proteins, pixel intensities of two different fluorophores were measured using a linescan tool in Metamorph, and the Pearson correlation coefficient (r) was deter- mined using Microsoft Excel. In general, $5–10 cells were subjected to image quantification. http://www.jimmunol.org/

Results FIGURE 1. Acidic amino acid residues in the juxtamembrane region of Acidic residues in the juxtamembrane region of TLR9 are TLR9 are important for interaction with UNC93B1. (A) Sequence align- important for interaction with UNC93B1 ment of the putative transmembrane and surrounding regions of mouse and human TLRs. The alignment starts with the conserved cysteine (indicated TLRs consist of an extracellular (or luminal for the intracellular in bold type) located at the end of the LRR domain; the corresponding TLRs) leucine-rich repeat (LRR) domain, a cytoplasmic TIR do- amino acid residue number of each TLR is indicated. The putative main, and a transmembrane domain that connects the LRR and membrane-inserted sequences were predicted using DG prediction server the TIR domains (39). Physical interaction between the nucleotide- V1.0 (http://dgpred.cbr.su.se/) and are represented by blue shading by guest on September 28, 2021 sensing TLRs and UNC93B1 is mediated by the transmembrane (51). Acidic amino acid residues are highlighted in red. The sequences domains of the TLRs (36). By using chimeric TLRs, we showed swapped to generate the transmembrane domain chimeras in previous that the transmembrane domains of TLR3 and TLR9, but not studies (8, 36) are underlined. (B) Wild-type (WT) or mutant TLR9-GFP TLR4, are sufficient for binding UNC93B1 (36). To identify (D812S, E813T, D818S, and D812S/E813T) was stably expressed along with wild-type UNC93B1-HA in RAW cells. TLR9 was immunoprecipi- a unique sequence feature that distinguishes the transmembrane tated with an anti-GFP Ab, and TLR9-bound UNC93B1 was probed by domains of the nucleotide-sensing TLRs from that of TLR4, we immunoblotting with an anti-HA Ab (top panel). Input lysates were probed analyzed the transmembrane domain sequences of mouse and to determine the expression levels of UNC93B1 (middle panel) and TLR9 human nucleic acid–sensing TLRs in comparison with TLR4 (Fig. (bottom panel). The relative band intensities are shown below each im- 1A). A cysteine residue at the end of the C-terminal–capping munoblot. The data shown are representative of more than three inde- module of the LRR domain (LLR-CT) is conserved among all pendent experiments. mammalian TLRs, and the membrane insertion is predicted to occur after a few intervening amino acid residues (40, 41). Be- region, we also generated the D818S mutant. In addition, we mu- cause it is not known from which residue the membrane insertion tated a few neighboring acidic amino acid residues to alanine actually starts and ends, we analyzed ∼35 amino acid residues (L811A, V814A, and S816A) for comparison. The mutant TLRs starting from the last conserved cysteine of the LRR-CT. We were expressed at a level comparable to wild type TLR9, indi- found that all of the nucleotide-sensing TLRs (TLR3, 7, 8, 9, and cating that the mutations did not grossly change the structural 13) known to bind UNC93B1 contain one or two acidic amino stability of TLR9 protein (Fig. 1B, Supplemental Fig. 1). acid residues in the short sequences between the cysteine and the To explore the interaction of TLR9 mutants with UNC93B1, expected first residue of the membrane-inserted region (the se- we stably expressed wild type or mutant TLR9-GFP along with quence referred to as the juxtamembrane region in this article). In UNC93B1-HA, immunoprecipitated TLR9-GFP with anti-GFP Ab contrast, neither human nor mouse TLR4 has an acidic amino acid from the cell lysate, and detected coimmunoprecipitated UNC93B1 residue in the same region. Because we did not find any other by immunoblotting with anti-HA Ab. Binding of UNC93B1 to salient features that are common only to the nucleotide-sensing D812S and E813T mutants was partially reduced compared with TLRs but absent in TLR4, we hypothesized that the acidic resi- wild type TLR9. Moreover, the combined mutation of D812S and dues in the juxtamembrane region may be involved in the inter- E813T (D812S/E813T or DE/ST) strongly inhibited binding of action between the nucleotide-sensing TLRs and UNC93B1. To UNC93B1 (Fig. 1B). In contrast, the D818S mutation did not alter test this hypothesis, we first generated mutant proteins of mouse UNC93B1 binding, nor did the mutation of nonacidic residues TLR9 in which the acidic residues Asp812 and Glu813 were changed affect this interaction (Fig. 1B, Supplemental Fig. 1). Thus, the to Ser and Thr, respectively (D812S and E813T). Because TLR9 acidic residues D812 and E813 in the juxtamembrane region of contains another Asp residue inside the putative membrane-inserted TLR9 play a critical role in its interaction with UNC93B1. In 5290 UNC93B1-BINDING RESIDUES IN THE NUCLEOTIDE-SENSING TLRs contrast, D818 located within the putative membrane-inserted region is not essential. Additionally, we noted that levels of the cleaved form of TLR9 differed among the mutant proteins; D812S and E813T mutant TLR9 showed lower levels of the cleaved form than did wild type TLR9. The D812S/E813T mutant shows almost no proteolytic conversion (Fig. 1B, bottom panel). The LRR do- main of TLR9 is proteolyzed by cathepsins and asparagine endo- peptidase after TLR9 reaches endolysosomes (22–27). Therefore, we suspected that the mutant proteins might differ in their ability to traffic to the endolysosomes. Acidic residues in the juxtamembrane region of TLR9 are important for trafficking to endolysosomes UNC93B1 delivers TLR9 to endolysosomes where the receptor recognizes internalized CpG DNA and initiates signaling (33). To analyze functional consequences of reduced UNC93B1 binding by the mutant TLR9 proteins, we first examined the subcellular lo- calization of the TLR9 mutants by coexpressing each mutant as a TLR9-GFP fusion protein, together with CD63-cherry, an endo- Downloaded from lysosomal marker protein, in BMDCs. We verified that CD63- cherry is localized in endolysosomes of BMDCs by comparing its localization with that of endogenous LAMP1 protein (Supplemental Fig. 2). Wild type and mutant TLR9-GFP proteins were mostly found in the ER of BMDCs at the basal state (Supplemental Fig.

3A). Upon stimulation with CpG DNA, the majority of wild type http://www.jimmunol.org/ TLR9 clearly colocalized with CD63-cherry, showing expected localization in endolysosomes (Fig. 2A). In contrast, the D812S and E813T TLR9 mutants were found in the ER, and only a small fraction colocalized with CD63-cherry in endolysosomes, indicat- ing that reduced UNC93B1 binding led to retention of the mutant receptors in the ER. No significant difference was observed in the localization of the D818S mutant, which maintains normal UNC93B1 binding. As expected from the UNC93B1-binding data, the D812S/E813T mutant TLR9 showed a more pronounced lo- by guest on September 28, 2021 calization defect compared with either D812S or E813T TLR9 mutant. Thus, the strength of UNC93B1 binding correlates with the extent to which the mutant receptors are targeted to endo- FIGURE 2. Acidic amino acid residues in the juxtamembrane of TLR9 A lysosomes. The different patterns of trafficking to endolysosomes by are important for localization in endolysosomes. ( ) Wild type or mutant TLR9-GFP and CD63-cherry were coexpressed in BMDCs, and cells were the mutant TLRs were confirmed by immunostaining of BMDCs imaged after overnight stimulation with 1 mM CpG. The results are rep- expressing the TLR9-GFP mutants with Abs against an endoge- resentative of four independent experiments. (B) Wild type or D812S/ nous ER marker protein PDI and an endolysosomal marker protein E813T TLR9-GFP was coexpressed with wild type UNC93B1-cherry in LAMP1 (Supplemental Fig. 3B). As mentioned previously, we con- TLR92/2 BMDCs, and the cells were imaged 4 h after stimulation with 1 sistently observed that the TLR9 mutants defective in UNC93B1 mg/ml LPS. The results are representative of two independent experiments. binding were also cleaved less efficiently (Fig. 1B, bottom panel). The graphs show fluorescent-intensity plots for pixels on the white dotted Because cleavage of TLR9 occurs in endolysosomes, this observation line, and the colocalization of TLR9-GFP with CD63-cherry or UNC93B1- suggests impaired trafficking of the mutant TLRs to endolysosomes, cherry was quantified by calculating the Pearson correlation coefficient of m as confirmed by our imaging data. signal intensities of two proteins. Scale bars, 10 m. Like TLR9, UNC93B1 normally traffics from the ER to endo- lysosomes. Therefore, we tested whether mislocalization of mutant TLR9 is reflected by changes in intracellular distribution of defects were attributed to the inability of mutant UNC93B1 to UNC93B1. We coexpressed UNC93B1-cherry with either wild type bind the endogenous nucleotide-sensing TLRs and deliver them to or D812S/E813T mutant TLR9-GFP in TLR9-deficient BMDCs. endolysosomes (33, 36). To test whether the mutation of TLR9, UNC93B1-cherry fully colocalized with wild type TLR9-GFP in which prevents the interaction with wild type UNC93B1, causes endolysosomes. UNC93B1 also translocated to endolysosomes in similar signaling defects, we analyzed the ability of the mutant cells that express the D812S/E813T mutant TLR9, even though the TLR9 proteins to respond to CpG DNA. First, we stably expressed mutant TLR9 remained in the ER (Fig. 2B, Supplemental Fig. 4). wild type or mutant TLR9-GFP proteins in immortalized TLR9- Thus, the disrupted interaction between TLR9 and UNC93B1 does deficient BMDMs and measured levels of CpG-induced TNF-a not affect trafficking of UNC93B1, and UNC93B1 can traffic to production by intracellular cytokine staining and ELISA (Fig. 3A, endolysosomes independently of TLR9. 3B). TLR92/2 BMDMs failed to produce TNF-a when stimulated with CpG DNA and, as expected, expression of wild type TLR9- Acidic residues in the juxtamembrane region of TLR9 are GFP in TLR9-deficient cells restored CpG-induced TNF-a pro- important for signaling duction. In contrast, cells expressing either the D812S or E813T The 3d mice bearing the H412R missense mutation in UNC93B1 TLR9 mutant, but not the D818S mutant, produced significantly have severe defects in signaling of TLR3, 7, and 9 (34). These less TNF-a than did cells expressing wild type TLR9. Thus, the The Journal of Immunology 5291 Downloaded from

FIGURE 3. Acidic residues in the juxtamembrane region of TLR9 are important for signaling. (A) TLR92/2 BMDMs were retrovirally transduced with GFP alone, wild type TLR9-GFP, or mutant TLR9-GFP. Cells were stimulated with 1 mM CpG or 50 ng/ml LPS for 4 h in the presence of 10 mg/ml a + 6 brefeldin A, and TNF- production in GFP cells was measured by intracellular staining and flow cytometry. The data (mean SEM) are representative of http://www.jimmunol.org/ at least three independent experiments. (B) TLR92/2 BMDMs stably expressing wild type or mutant TLR9-GFP were stimulated with 1 mM CpG or 50 ng/ ml LPS for 4 h, and levels of TNF-a secretion were measured by ELISA. The data (mean 6 SEM) are representative of three independent experiments. (C) TLR92/2 BMDCs were retrovirally transduced with GFP alone, wild type TLR9-GFP, or mutant TLR9-GFP. Cells were stimulated with 1 mM CpG for 4 h in the presence of 10 mg/ml brefeldin A, and TNF-a production in GFP+ cells was measured by intracellular staining and flow cytometry. The data are results of four independent experiments. The error bars represent the SE. *p , 0.05, **p , 0.01, ***p , 0.001, Student t test. acidic residues in the juxtamembrane region of TLR9 are im- well as with wild type TLR9 (Fig. 4). Thus, mutation of the acidic portant for receptor signaling. Accordingly, the D812S/E813T residues in the juxtamembrane region of TLR9 does not prevent double-mutant TLR9 was the least able to transmit the CpG- the homodimerization. We further conclude that the mutation does by guest on September 28, 2021 induced signal. Similar results were also obtained from TLR9- deficient BMDCs expressing the mutant TLR9-GFP proteins (Fig. 3C). LPS-induced TNF-a production was comparable in all cells, regardless of TLR9 expression (Fig. 3A, 3B, data not shown). The signaling defects associated with the TLR9 mutants are con- sistent with their reduced ability to bind UNC93B1 and the conse- quent retention in the ER (Fig. 2A). The acidic amino acid mutant of TLR9 retains the ability to homodimerize The x-ray structures of TLRs show that the LRR domains form ligand-dependent homo- or heterodimers (39). However, in the case of TLR9, the receptor forms constitutive homodimers, and binding of CpG DNA induces allosteric conformational changes that bring the cytosolic TIR domain from each receptor monomer in closer proximity to permit downstream signaling (42, 43). The molecular determinants that mediate the dimerization of TLR9 are not known. To exclude the possibility that the signaling defect observed with mutations of the acidic amino acid residues in the juxtamembrane region of TLR9 is attributed to disruption of re- ceptor dimerization, we tested the ability of mutant receptors to form dimers by using TLR9 fusion proteins with two different FIGURE 4. Acidic amino acid mutant of TLR9 retains the ability to epitope tags (GFP or myc). By immunoprecipitating wild type dimerize. A combination of wild type or D812S/E813T mutant form of TLR9 via the GFP tag, we recovered myc-tagged wild type TLR9, TLR9-GFP and TLR9-myc were expressed in HEK293T cells. Cells ex- pressing wild type TLR9-GFP and TLR4-myc were used as a negative but little TLR4, confirming the existence of preformed TLR9 control. TLR9-GFP was immunoprecipitated with an anti-GFP Ab, and homodimers. The observed weak binding of TLR4 with TLR9 myc-tagged TLRs were detected by immunoblotting with an anti-myc Ab might be due to overexpression of two proteins. Notably, myc- (top panel). Input lysates were probed to determine the expression levels of tagged D812S/E813T mutant protein was coprecipitated with wild myc-tagged TLRs (middle panel) and GFP-tagged TLR9 (bottom panel). type and D812S/E813T mutant TLR9-GFP, indicating that the The relative band intensities are shown below each immunoblot. The data mutant TLR9 still retains the ability to dimerize with itself, as shown are representative of two independent experiments. 5292 UNC93B1-BINDING RESIDUES IN THE NUCLEOTIDE-SENSING TLRs not cause overt structural changes, but rather specifically disrupts the juxtamembrane region, although containing the important acidic the interaction with UNC93B1. amino acid residues, is not sufficient for binding to UNC93B1; therefore, secondary sites in TLR9 must contribute to the strength The juxtamembrane region of TLR9 alone is not sufficient for of binding with UNC93B1 (Fig. 5B). We further examined locali- UNC93B1 binding zation and signaling capacity of the chimeric receptors in BMDCs. The chimeric TLR4 (composed of aa 1–625 of TLR4, aa 811–839 Similar to TLR9444, TLR9944 was located on the cell surface of of TLR9, and aa 660–835 of TLR4), in which the transmembrane BMDCs, whereas wild type TLR9 was found exclusively inside domain (including the juxtamembrane region and the putative cells (Fig. 5C). Consistent with their surface localization, we did not membrane-inserted segment) of TLR4 was replaced with that of observe efficient cleavage of the TLR9444 and TLR9944 chimeras TLR9, binds UNC93B1. Therefore, the juxtamembrane region and (Fig. 5B, bottom panel). Collectively, our data indicate that the the putative membrane-inserted region of TLR9 together (aa 811– juxtamembrane region of TLR9 alone does not determine receptor 839 of TLR9) are sufficient for interaction with UNC93B1 (36). localization. When expressed in TLR92/2 BMDCs, both chimeric Because the putative membrane-inserted segments (sequences proteins could be efficiently triggered by CpG DNA to cause pro- shaded in light blue in Fig. 1A) of both TLR9 and 4 primarily duction of TNF-a (Fig. 5D). In addition, they produced slightly consist of hydrophobic residues and do not show prominent dif- increased levels of TNF-a in the absence of exogenous CpG DNA, ferences (Fig. 1A), we next tested whether the juxtamembrane most likely due to activation of the surface-localized chimeric re- region of TLR9 (containing the acidic residues D812 and E813) is ceptors by endogenous DNA present in the cell culture supernatant sufficient for interaction with UNC93B1. The chimeric TLR9444 (8, 18). protein (aa 1–810 of TLR9 fused to aa 626–835 of TLR4), in We showed that the D812 and E813 residues in the juxta- Downloaded from which the juxtamembrane region, the putative membrane-inserted membrane region of TLR9 are important for signaling (Fig. 3). We region, and the cytosolic domain of TLR9 were replaced with believe that the primary reason for the defective signaling by the those of TLR4, did not interact with UNC93B1 and was expressed acidic amino acid mutant TLR9 is because the mutations inhibit on the cell surface (8, 33). We modified the chimera by swapping the trafficking of the mutant TLR9 to the endolysosomes. How- the juxtamembrane region of TLR4 with that of TLR9 to generate ever, we could not rule out the possibility that the D812 and E813

a chimeric receptor with the LRR domain and the juxtamembrane residues play an additional role in the initiation of signaling. For http://www.jimmunol.org/ region of TLR9 fused to the putative membrane-inserted region example, we speculated that, independently of their role in and the TIR domain of TLR4 (named TLR9944 and depicted UNC93B1 binding and receptor trafficking to endolysosomes, the schematically in Fig. 5A). We coexpressed wild type TLR9-GFP, acidic residues in the juxtamembrane region are important for TLR9444-GFP, or TLR9944-GFP together with UNC93B1-HA and CpG binding or for relaying the information of the CpG binding– examined their interaction. Wild type TLR9, but not TLR9444, induced conformational changes of the LRR domain to the TIR interacted with UNC93B1. Neither did TLR9944, indicating that domain in the other side of the membrane. We tested such pos- by guest on September 28, 2021

FIGURE 5. The luminal juxta-transmembrane region of TLR9 alone is not sufficient for binding to UNC93B1. (A) Schematic representation of wild type and chimeric TLR proteins. The portions corresponding to TLR9 and TLR4 are shown in black and gray, respectively. The D812S/E813T mutation in the juxtamembrane region of TLR9 is depicted with a white dot. (B) Wild type TLR9-GFP or chimeric TLR-GFP proteins were coexpressed with UNC93B1- HA in HEK293T cells. Cell lysates were subjected to immunoprecipitation with an anti-HA Ab, and UNC93B1-bound TLR was probed by immunoblotting with an anti-GFP Ab (top panel). Input lysates were probed to determine the expression levels of UNC93B1 (middle panel) and TLRs (bottom panel). The relative band intensities are shown below each immunoblot. The data shown are representative of three independent experiments. (C) Wild type TLR9-GFP or chimeric TLR-GFP was expressed in BMDCs, and the cells were imaged after stimulation with CpG. The results are representative of three independent experiments. Scale bars, 10 mm. (D) Wild type TLR9-GFP, D812S/E813T mutant TLR9-GFP, or chimeric TLR-GFP were expressed in TLR92/2 BMDCs. Cells were stimulated with 1 mM CpG for 4 h in the presence of 10 mg/ml brefeldin A, and TNF-a production in GFP+ cells was measured by intracellular cytokine staining and flow cytometry. The data are the results of three independent experiments. The error bars represent the SE. ***p , 0.001, Student t test. The Journal of Immunology 5293 sibilities by mutating the D812 and E813 residues in the context ciferase reporter in HEK293T cells and stimulated the cells with of TLR9944 to generate the mutant TLR9944 (D812SS/E813T) different doses of p(I:C). In cells that express wild type TLR3, p(I: protein. If the acidic residues indeed play an additional role in the C) induced luciferase activity in a dose-dependent manner. initiation of signaling, independently of their role in UNC93B1 However, in mutant TLR3-expressing cells, induction of luciferase binding and receptor trafficking to endolysosomes, we expected that was strongly impaired (Fig. 7A). the D812S/E813T double mutation would inhibit the CpG-stimulated Activation of TLR3 by p(I:C) also leads to production of type I signaling of the TLR9944 chimera. Similar to TLR9944, the mutant IFNs (10). We examined the activation of the type I IFN pathway TLR9944 (D812S/E813T) protein did not bind UNC93B1 and was by mutant TLR3 using an ISRE-luciferase reporter assay. As seen located on the cell surface. Moreover, it effectively transmitted the for activation of NF-kB–luciferase, activation of the IFN pathway CpG-derived signals to promote TNF-a production. This result shows was strongly impaired in the mutant TLR3-expressing cells (Fig. that the acidic residues are not directly involved in the ligand 7B). Thus, the acidic amino acid residues in the juxtamembrane (i.e., CpG DNA) binding or the recruitment of downstream sig- region of TLR3 are critical for receptor , as we naling molecules. It also confirms that the mutation of the acidic showed for TLR9. residues in the juxtamembrane region of TLR9 does not affect the Therefore, the acidic amino acid residues in the juxtamembrane general structural stability of the receptor. region of TLR3 and TLR9 are critical for the association of TLRs with UNC93B1, their trafficking to endolysosomes, and Acidic residues in the juxtamembrane region of TLR3 are TLR signaling. required for binding to UNC93B1

Our data collectively demonstrate that the acidic amino acid res- Discussion Downloaded from idues in the juxtamembrane region of TLR9 play an important role TLR-signaling pathways are regulated by dynamic localization of in UNC93B1 binding, trafficking to endolysosomes, and signaling. the receptors in various intracellular compartments. Upon binding Because all other nucleotide-sensing TLRs also have acidic resi- to their ligands, surface-disposed TLRs—TLR1, 2, 4, 5, and 6— dues in their luminal juxtamembrane region, we analyzed the acidic recruit signaling adaptor molecules and transmit signals for in- amino acid residues in TLR3 to determine whether they play a duction of proinflammatory primarily at the plasma similar role as their TLR9 counterparts. We generated TLR3 membrane. In contrast, signaling for production of type I IFNs by http://www.jimmunol.org/ mutants (D699S, E704T, D699S/E704T) and tested them for TLR2 and TLR4 occurs in early endosomes after internalization UNC93B1 binding. Similar to our results with TLR9 mutants, single- point mutations at D699 or E704 of TLR3 partially reduced the interaction with UNC93B1, whereas the D699S/E704T double mutation virtually abolished UNC93B1 binding (Fig. 6). Therefore, acidic residues in the juxtamembrane region of the nucleotide- sensing TLRs seem to carry a common function for mediating the interaction with UNC93B1. by guest on September 28, 2021 Acidic amino acid residues in the juxtamembrane region of TLR3 are important for signaling Next, we tested whether the mutation of the acidic residues in the juxtamembrane region of TLR3 affects its signaling. We expressed wild type or mutant TLR3 proteins together with an NF-kB–lu-

FIGURE 7. Acidic amino acid residues in the juxtamembrane region of TLR3 are important for signaling. (A) The NF-kB–luciferase reporter construct and UNC93B1-HA were transfected into HEK293T cells without FIGURE 6. Acidic residues in the juxtamembrane region of TLR3 are TLR3 (-) or together with wild type (WT) or mutant TLR3-GFP. The cells required for binding to UNC93B1. Wild type (WT) or mutant TLR3-GFP were stimulated with the indicated dose (mg/ml) of p(I:C) for 8 h or left proteins were coexpressed with wild type UNC93B1-HA in HEK293T unstimulated, and luciferase induction was measured. (B) HEK293T cells cells. The cells were lysed and subjected to immunoprecipitation with an were transfected with the ISRE-luciferase reporter construct along with anti-GFP Ab. TLR3-bound UNC93B1 was detected by immunoblotting TLR3-GFP and UNC93B1-HA expression plasmids, as in (A). ISRE ac- using an anti-HA Ab (top panel). Input lysates were probed to determine the tivation was measured 18 h after stimulation with the indicated dose (mg/ expression levels of UNC93B1-HA (middle panel) and TLR3-GFP (bottom ml) of p(I:C). The levels of luciferase activity were expressed as the fold panel). The relative band intensities are shown below each immunoblot. induction relative to the luciferase activity in unstimulated cells. The data The data shown are representative of three independent experiments. presented are representative of three independent experiments. 5294 UNC93B1-BINDING RESIDUES IN THE NUCLEOTIDE-SENSING TLRs of the receptors (44, 45). In case of the nucleotide-sensing TLRs, with secondary binding sites in the cytoplasmic surface, the identity which are normally expressed inside cells, the signaling pathways of which remains to be established. The quaternary structures of for induction of both proinflammatory cytokines and type I IFNs TLR/UNC93B1 complexes would be influenced by many factors, originate in endolysosomes. The TLR9-mediated signaling cas- such as the identity of TLR, binding of unique TLR ligands, and the cades for NF-kB–mediated proinflammatory cytokine production characteristics of intracellular organelles where the TLR/UNC93B1 versus those required for IRF3/7-mediated type I IFN induction complex is located. These higher-order structures may dictate the may be initiated in distinct endolysosomal compartments (46, 47). nature of the trafficking adaptor molecules that must be recruited It is not clear why these two signaling pathways proceed from and, thereby, the final subcellular destination of the individual separate subcellular compartments or how nucleotide-sensing TLRs TLR/UNC93B1 complexes. reach these different endolysosomes. The clathrin adaptor protein Hyperactivation of TLR7 and TLR9 contributes to the pathol- AP-3 complex is specifically required for TLR9-mediated acti- ogy of multiple autoimmune diseases, including systemic lupus vation of type I IFNs in plasmacytoid dendritic cells (pDCs), but erythematosus and psoriasis (4–6). Befitting the essential role of not for proinflammatory cytokines, suggesting that AP-3 is one UNC93B1 in the activation of TLR7 and TLR9, the 3d mutation of the critical factors that targets TLR9 to distinct signaling- in lupus-prone strains of mice suppressed autoantibody production competent subcellular compartments (47). pDCs from mice with and mortality (49). Moreover, UNC93B1 was upregulated in B mutations in the AP-3 b1 subunit (Ap3b1pearl/pearl) failed to pro- cells from patients suffering from active systemic lupus eryth- duce type I IFNs or TNF-a in response to TLR9 stimulation, but ematosus (50). Therefore, regulation of the nucleotide-sensing conventional dendritic cells from the same mice responded nor- TLR activities by means of disrupting the TLR–UNC93B1 in- mally, suggesting that AP-3 plays a cell-specific role in trafficking teraction, while maintaining the normal activities of the cell sur- Downloaded from of TLR9 (48). face–localized TLRs, might provide a new approach for TLR drug Unlike AP-3, UNC93B1 plays a universal role in signaling and discovery apart from the conventional receptor agonist/antagonist trafficking of the nucleotide-sensing TLRs. Stimulation with therapeutics. Our observation that the acidic residues in the jux- agonists for TLR3, 7, or 9 fails to induce production of TNF-a and tamembrane regions of the nucleotide-sensing TLRs determine type I IFNs in cells from 3d mutant mice. UNC93B1 was required UNC93B1 binding may facilitate the design of such therapeutic

for signaling of the nucleotide-sensing TLRs in all cells examined, reagents. http://www.jimmunol.org/ including conventional dendritic cells, pDCs, macrophages, , and epithelial cells (15, 30, 33, 34, 48). UNC93B1 Acknowledgments performs its regulatory function through a physical interaction We thank Drs. Charles Surh and Seung-Woo Lee (POSTECH) and the mem- with the nucleotide-sensing TLRs (36). Indeed, the functional 3d bers of the Hyehwa Forum for helpful discussions. mutation (H412R) of UNC93B1 disrupts TLR binding. However, the exact molecular interactions between UNC93B1 and individ- Disclosures ual TLRs have not been mapped. In this study, we identified the The authors have no financial conflicts of interest. acidic amino acid residues in the juxtamembrane region of TLR3 and TLR9 as important residues for association with UNC93B1, by guest on September 28, 2021 trafficking to endolysosomes, and receptor signaling. 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