USP15 Deubiquitinates CARD9 to Downregulate C-Type Lectin Receptor−Mediated Signaling

Wenting Xu, Jason S. Rush, Daniel B. Graham, Zhifang Cao and Ramnik J. Xavier Downloaded from

ImmunoHorizons 2020, 4 (10) 670-678 doi: https://doi.org/10.4049/immunohorizons.2000036 http://www.immunohorizons.org/content/4/10/670

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Innate Immunity

USP15 Deubiquitinates CARD9 to Downregulate C-Type Lectin Receptor–Mediated Signaling

Wenting Xu,*,†,‡,1 Jason S. Rush,§ Daniel B. Graham,*,†,§ Zhifang Cao,*,†,§ and Ramnik J. Xavier*,†,§ *Gastrointestinal Unit and Center for the Study of Inflammatory Bowel Disease, Massachusetts General Hospital and Harvard Medical School, Boston, MA 02114; †Department of Molecular Biology, Massachusetts General Hospital and Harvard Medical School, Boston, MA 02114; Downloaded from ‡Department of Gastroenterology, The First Affiliated Hospital of Nanchang University, Nanchang, Jiangxi 330006, China; and §Broad Institute of MIT and Harvard, Cambridge, MA 02142 http://www.immunohorizons.org/ ABSTRACT Posttranslational modifications are efficient means to rapidly regulate function in response to a stimulus. Although ubiquitination events and the E3 ubiquitin ligases involved are increasingly characterized in many signaling pathways, their regulation by deubiquitinating enzymes remains less understood. The C-type lectin receptor (CLR) signaling adaptor CARD9 was previously reported to be activated via TRIM62-mediated ubiquitination. In this study, we identify the deubiquitinase USP15 as a novel regulator of CARD9, demonstrating that USP15 constitutively associates with CARD9 and removes TRIM62-deposited ubiquitin marks. Furthermore, USP15 knockdown and knockout specifically enhance CARD9-dependent CLR signaling in both mouse and human immune cells. Altogether, our study identifies a novel regulator of innate immune signaling and provides a blueprint for the identification of additional deubiquitinases that are likely to control these processes. ImmunoHorizons, 2020, 4: 670–678. by guest on September 26, 2021 INTRODUCTION (10–14), although it remains unclear whether CARD9 is important in defense against microbes other than fungi and microalgae in Human genetic studies have uncovered that CARD9 is important humans. for immune-microbial interactions at mucosal surfaces, as CARD9 In IBD, CARD9 variants have been associated with both risk polymorphisms are associated with disorders such as immunode- and protection. A rare splicing mutation IVS11+1G.C, resulting in ficiency, inflammatory bowel diseases (IBD), and increased a C-terminally truncated CARD9, plays a protective role against susceptibility to fungal infection (1–3). To date, more than two IBD and ankylosing spondylitis (15, 16). A common coding variant, dozen CARD9-coding mutations have been reported in clinical CARD9 S12N (single-nucleotide polymorphism rs4077515), re- studies to predispose individuals to diseases associated with fungal sults in higher CARD9 mRNA expression (17) and is associated or microalgal infection (4–9). CARD9 has also been reported to be with increased risk of IBD (15, 17), ankylosing spondylitis (18), Ig critical during bacterial and viral infections in mouse models A nephropathy (19), and primary sclerosing cholangitis (20). This

Received for publication May 13, 2020. Accepted for publication October 7, 2020. Address correspondence and reprint requests to: Dr. Zhifang Cao and Dr. Ramnik J. Xavier, Massachusetts General Hospital, 185 Cambridge Street, Simches 7, Boston, MA 02114. E-mail addresses: [email protected] (Z.C.) and [email protected] (R.J.X.) ORCIDs: 0000-0003-3019-2763 (Z.C.); 0000-0002-5630-5167 (R.J.X.). 1Current address: Department of Gastroenterology, The First Affiliated Hospital, College of Medicine, Zhejiang University, Hangzhou, China. This work was supported by funding from the Helmsley Charitable Trust and the National Institutes of Health (Grant AI137325 to R.J.X.). W.X. was supported by the China Scholarship Council. R.J.X. and Z.C. conceived the project. W.X., J.S.R., and Z.C. performed experiments. R.J.X., Z.C., and D.B.G. supervised the project. R.J.X. and Z.C. wrote the manuscript. All authors discussed the results and commented on the manuscript. Abbreviations used in this article: BMDC, bone marrow–derived dendritic cell; CBM, CARD9–BCL10–MALT1; CLR, C-type lectin receptor; HA, hemagglutinin; IBD, inflammatory bowel disease; KO, knockout; sgRNA, single-guide RNA; shRNA, short hairpin RNA; TDM, trehalose 6,69-dimycolate; USP15, ubiquitin-specific peptidase 15; WGP, whole glucan particle. The online version of this article contains supplemental material. This article is distributed under the terms of the CC BY 4.0 Unported license. Copyright © 2020 The Authors

670 https://doi.org/10.4049/immunohorizons.2000036

ImmunoHorizons is published by The American Association of Immunologists, Inc. ImmunoHorizons USP15 DEUBIQUITINATES CARD9 TO DOWNREGULATE CLR SIGNALING 671 variant, however, also appears to have protective effects in some downstream of CARD9, suggesting that it is an important contexts, such as primary immune thrombocytopenia (21). regulator of this innate immune signaling pathway. Mechanistically, when expressed at equivalent levels, the S12N allele enhances CARD9-mediated cytokine production in murine bone marrow–derived dendritic cells (BMDCs) stimulated with MATERIALS AND METHODS fungal products relative to the wild-type allele (22), indicating that S12N is a gain-of-function mutant. CARD9 S12N reportedly Reagents and plasmids enhances IL-5 production in macrophages; accordingly, PBMCs Whole glucan particles (WGP) were purchased from InvivoGen from three S12N homozygous patients with allergic bronchopul- (catalog no. tlrl-wgp). TDM was obtained from Enzo Life Sci- monary aspergillosis produced higher levels of IL-5 than those ences (catalog no. AL-581-210-M001). TDM was dissolved in from an allergic bronchopulmonary aspergillosis patient carrying chloroform/methanol/water(90:10:1, 1 mg/ml) and further diluted wild-type CARD9 (23). with isopropanol. LPS was purchased from InvivoGen (catalog no. CARD9 is an essential component of innate immune signaling tlrl-peklps). through multiple C-type lectin receptors (CLRs), such as Dectin-1, Lentivirus-based short hairpin RNA (shRNA) constructs were Dectin-2, Dectin-3, and Mincle (2). These receptors are generally generated in a pLKO.1 vector by the Genetic Perturbation Platform Downloaded from expressed on myeloid cells and recognize a broad range of fungal at the Broad Institute of MIT and Harvard. A lentiviral vector cell wall components (24). Mincle is additionally activated by pCDH-CMV from Addgene (no. 72265) was used to generate lenti- nuclear protein SAP130 released from damaged cells (25) and based USP15 constructs with a puromycin–T2A–3xHA tag in the trehalose 6,69-dimycolate (TDM; also known as cord factor) N terminus. All other constructs were either cloned into a pCMV present in the cell membranes of mycobacteria (26), suggesting a vector (Clontech Laboratories) or pcDNA4/TO–FLAG–StrepII as http://www.immunohorizons.org/ broad role for CLRs in innate immune recognition of fungi, described previously (22). USP15 variants were originally ampli- damaged cells, and certain bacteria. Upon engagement, CLRs fied from a Clontech Laboratories immune cDNA panel and activate the tyrosine kinase Syk to facilitate the formation of the subcloned into indicated tagged vectors by PCR. CARD9–BCL10–MALT1 (CBM) complex (27), which is essential for downstream NF-kB signaling and related immune responses. Immunoprecipitation and Western blotting Notably, CBM architecture in signaling cascades is not limited to The experimental methods were previously described in detail CARD9 in myeloid cells but also exists with CARD10 in (22). Briefly, cells were lysed in standard lysis buffer (50 mM Tris- nonhematopoietic cells, CARD11 in lymphoid cells, and CARD14 HCl [pH 7.5], 150 mM NaCl, 0.5 mM EDTA, 1% Nonidet P-40, in keratinocytes. Although these CBM complexes are highly Halt phosphatase inhibitor single-use mixture [Pierce Biotech], specific to cell types and stimuli, their activity likely involves and protease inhibitor tablets [Roche Diagnostics]). For immuno- by guest on September 26, 2021 similar regulatory events. precipitation of Flag–StrepII-tagged protein, cell lysates were Posttranslational modifications are known to directly regulate incubated with StrepII Sepharose beads (catalog no. 2-1201-010; the assembly of CBM complexes. In the case of CARD9, IBA) for 1 h at 4°C. For immunoprecipitation of endogenous phosphorylation directly regulates the formation of the CBM CARD9 in BMDCs, cell lysates were incubated with anti-CARD9 complex: protein kinase C d phosphorylates CARD9 on T231, Ab or control IgG overnight, followed by pulldown with protein facilitating the interaction between CARD9 and BCL10 (28). In A/G magnetic beads (catalog no. LSKMAGAG02; MilliporeSigma) contrast, protein phosphatase 1 interacts with CARD9 via incubation for 30 min. For ubiquitination blots, cells were lysed DOK3 to dephosphorylate CARD9 threonine phosphorylation, in standard lysis buffer with 10 mM N-ethylmaleimide (catalog which negatively regulates CARD9-dependent signaling in no. E3876-5G; Sigma-Aldrich). neutrophils (29). Furthermore, casein kinase 2 negatively The following Abs were used: anti-FLAG M2 Ab (catalog no. regulates CARD9 via phosphorylation of the CARD9 C F1804; Sigma-Aldrich), anti–b-actin Ab (catalog no. 8226; Abcam), terminus at T531 and T533 (30). We previously uncovered that anti–hemagglutinin (HA) Ab (catalog no. 901502; BioLegend), the E3 ubiquitin ligase TRIM62 is a binding partner of CARD9 anti-Myc Ab (catalog no. 626802; BioLegend), anti-V5 Ab (catalog that might associate with the CBM complex (22). TRIM62- no. 903801; BioLegend), anti-CARD9 (A-8) Ab (catalog no. mediated, K27-linked CARD9 ubiquitination at K125 is critical sc0374569; Santa Cruz Biotechnology), anti-CARD9 Ab (mouse for CARD9-mediated NF-kB signaling and cytokine production preferred, catalog no. 12283; Cell Signaling Technology), anti- in myeloid cells as well as antifungal immunity in vivo. These USP15 (D1K6S) Ab (catalog no. 66310; Cell Signaling Technology), findings revealed a previously unknown ubiquitin-dependent anti-USP15 (2D5) Ab (catalog no. sc-100629; Santa Cruz Bio- regulatory mechanism for CARD9 function. However, the technology), and normal mouse IgG2b isotype control (catalog no. regulation of this process and the involvement of potential sc-3879; Santa Cruz Biotechnology). HRP-conjugated anti-mouse deubiquitinases for CARD9 remained unclear. In this study, we and anti-rabbit Abs (Dako) were used as secondary Abs. identified ubiquitin-specific peptidase 15 (USP15) as a novel CARD9 interactor by proteomics. We show that USP15 Cell culture and lentiviral production removes TRIM62-mediated CARD9 ubiquitination. Addition- HEK293T cells were maintained at 37°C and 5% CO2 in DMEM ally, we directly show the impact of USP15 on signaling supplemented with 10% FCS and 15 mg/ml gentamicin sulfate. To

https://doi.org/10.4049/immunohorizons.2000036 672 USP15 DEUBIQUITINATES CARD9 TO DOWNREGULATE CLR SIGNALING ImmunoHorizons prepare lentivirus for infection, protocols from the Broad CRISPR-generated USP152/2 THP-1 cell lines and NF-kB 2 2 Institute’s RNAi Consortium shRNA Library were used as luciferase assay. The USP15 / THP-1 cell lines were generated described previously (22). USP15 shRNA constructs in pLKO.1- by a CRISPR-based approach using USP15 single-guide RNA puro vector were obtained from the Broad Institute. Control (sgRNA) A1 (59-CCAAGTTACTTAGGCCACAG-39)andsgRNAB2 shRNA construct in pLKO.1-puro vector was from Sigma-Aldrich (59-AAGGTGTTCCTTAAGTGACT-39). Briefly, THP-1 cells were (catalog no. SHC002). USP15 shRNA constructs are as follows: infected with lentivirus carrying CARD9 sgRNA. Trans-activating mouse USP15 shRNA 1 target sequence, 59-TGTCTATGGAGAT CRISPR RNAs for USP15 sgRNAs A and B were purchased from GAAGTTAT-39; mouse USP15 shRNA 2 target sequence, 59- Integrated DNA Technologies. Integrated DNA Technologies Alt-R TGAGAGGTGAAATAGCTAAAT-39;humanUSP15shRNAA CRISPR–Cas9 system was applied for preparation of the ribonu- target sequence, 59-CCTTGGAAGTTTACTTAGTTA-39;andhu- cleoprotein complex containing trans-activating CRISPR RNA, man USP15 shRNA B target sequence, 59-GCTGACACAATAGA CRISPR RNA (Integrated DNA Technologies), and Cas9 protein TACAATT-39. (PNA Bio). The ribonucleoprotein complex and 0.5 mgpmaxGFP were transfected with 1 M THP-1 cells by nucleofection according to BMDC culture and functional assay the manufacturer’s protocol (Lonza). Two days postnucleofection, Preparation and infection of BMDCs were described previously GFP-positive THP-1 cells were sorted by flow cytometry in the flow Downloaded from (22). Functional assays were performed on day 9 in 96-well plates. cytometry facility at the Broad Institute. Seven days postsorting, cells BMDCs were stimulated with WGP, LPS, precoated TDM, or were diluted and plated into wells at a ratio of one cell per well in 2 2 corresponding solvent. Twenty-four hours after stimulation, 96-well plates. USP15 / cell lines were validated by Western blot. cytokine production in the supernatant was detected using For the NF-kB luciferase assay, three independent pools of the appropriate ELISA kits (BD Biosciences) according to the same THP-1 cells were transduced with Dectin-1 (NM_022570) http://www.immunohorizons.org/ manufacturer’sprotocol. and a lentivirus-based NF-kB luciferase reporter (31). Three

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FIGURE 1. USP15 binds the N terminus of CARD9. (A) Schematics of CARD9 variants used. D385–423, deletion of aa 385–423; CC, coiled-coil domain; WT, wild-type. (B and C) HEK293T cells were transfected with HA-tagged USP15 and Flag–StrepII-tagged CARD9 WT, CARD9 2–420 (N terminus), or either CARD9 416–536 (C terminus) (B) or CARD9 D385–423 (C). Samples were immunoprecipitated with anti-StrepII and probed for HA (USP15). Total lysates were probed for HA and FLAG (CARD9). (D) WT, primary murine BMDCs were unstimulated or stimulated with 100 mg/ml of the Dectin-1 agonist WGP for 25 and 50 min. The unstimulated sample was divided and immunoprecipitated with normal IgG2b isotype and anti-CARD9 (A-8). Stimulated samples were immuno- precipitated with anti-CARD9 (A-8). All samples were probed for endogenous USP15 and CARD9.

https://doi.org/10.4049/immunohorizons.2000036 ImmunoHorizons USP15 DEUBIQUITINATES CARD9 TO DOWNREGULATE CLR SIGNALING 673

2 2 independent pools of the same USP15 / cells overexpressing HA–ubiquitin (Boston Biochem), 25 nM CARD9, 12.5 nM UBE1 Dectin-1 and the NF-kB luciferase reporter were reconstituted (LifeSensors), and 200 nM UBE2D2 (LifeSensors). After in- with indicated USP15 constructs in parallel. Cells with cubation at room temperature for 30 min, the reactions were re-expressed USP15 were expanded for at least four passages quenched by the addition of 1 ml 0.5 M EDTA, followed by the prior to luciferase assay. Cells were treated with WGP for 24 h addition of 1 mlof2mMHis6-USP15 (E-594, lot 30271815A; Boston prior to luciferase assay. Reporter luciferase activity was exam- Biochem). Incubation was continued for 1 h, after which the ined by according to the manufacturer’s protocol (Steadylite reaction was quenched by the addition of 23 SDS loading buffer. Plus; PerkinElmer). The luciferase activity of each sample was normalized by calculating the fold change to the luciferase activity CARD9 proteomics of the corresponding untreated samples. JAWSII dendritic cells overexpressed with Flag–StrepII-tagged human CARD9 were harvested and washed twice with PBS. In vitro deubiquitination assay Cellular extracts were prepared by cell disruption through 15 Invitroubiquitinationreactionsweresetupona50-mlscaleas strokes in a tight-fitting Dounce homogenizer, followed by two previously described (22) and contained 2.5 nM TRIM62, 5 mM freeze–thaw cycles in standard lysis buffer. The cell extract was Downloaded from A http://www.immunohorizons.org/

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FIGURE 2. USP15 regulates TRIM62-mediated CARD9 ubiquitination. (A) Schematics of USP15 variants used. The catalytic C298 residue and catalytically inactivating C298A mutation are indicated. USP15 variant 2 was identified from a Clontech Laboratories immune cDNA library harboring the indicated frameshift insertion. DUSP, domain present in ubiquitin– specific protease; UBL, ubiquitin-like; WT, wild-type. (B) HEK293T cells were transfected with Flag–StrepII-tagged CARD9, V5-tagged TRIM62, Myc- tagged ubiquitin (Ub), and HA-tagged USP15 WT, USP15 C298A, or USP15 variant 2. Samples were immunoprecipitated with anti-StrepII and probed for Myc. Total lysates were probed for Flag (CARD9), V5 (TRIM62), and HA (USP15). (C) Ubiquitination of CARD9 was initiated by incubating purified Flag–StrepII-tagged CARD9 with V5-tagged TRIM62 and required ubiquitination machinery UBE1, UBE2D2, and Ub. Ubiquitinated CARD9 was then incubated with and without USP15, and USP15-mediated CARD9 deubiquitination was assessed by Western blot.

https://doi.org/10.4049/immunohorizons.2000036 674 USP15 DEUBIQUITINATES CARD9 TO DOWNREGULATE CLR SIGNALING ImmunoHorizons

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FIGURE 3. USP15 suppresses CARD9-mediated signaling. (A) Wild-type (WT), primary murine BMDCs were transduced with lentivirus expressing the indicated control or USP15 shRNAs. BMDCs were stimulated with the Dectin-1 agonist WGP, precoated Mincle agonist TDM, or TLR4 agonist LPS for 24 h before TNF-a levels were assessed by ELISA. Knockdown of protein expression was confirmed by Western blot. Data were obtained from three independent experiments (n = 3 mice). Error bars represent mean 6 SD. Comparisons are relative to stimulated cells transduced with control shRNA. *p , 0.05, ***p , 0.005. (Continued)

https://doi.org/10.4049/immunohorizons.2000036 ImmunoHorizons USP15 DEUBIQUITINATES CARD9 TO DOWNREGULATE CLR SIGNALING 675 incubated with 25 mlofMagStrep“type3” XTbeads(IBA)for1hat BMDCs, this interaction appeared constitutive and largely un- 4°C. Beads and bound were washed three times with lysis affected by stimulation with the Dectin-1 agonist WGP (Fig. 1D). buffer and eluted with SDS-PAGE loading buffer. Proteins of interest were separated by SDS-PAGE and stained using Colloidal USP15 removes ubiquitin marks deposited by TRIM62 Coomassie GelCode Blue (Pierce Biotech). Gel sections were cut Given the well-characterized role of USP15 as a deubiquitinase from the gel and sent for mass spectrometry analysis at the Taplin (32), we examined whether USP15 regulates TRIM62-mediated Biological Mass Spectrometry Facility of Harvard Medical School. CARD9 ubiquitination. To this end, we coexpressed CARD9, TRIM62, and ubiquitin with either the wild-type USP15, Statistical analysis catalytically inactive USP15 C298A mutant, or USP15 variant 2 in Unpaired two-tailed Student t tests were used for comparisons. All HEK293T cells (Fig. 2A). USP15 variant 2 is a C-terminal statistical comparisons were made between multiple independent truncation that lacks a zinc-binding motif Cys-x-x-Cys (aa experiments performed in parallel. 809–812) required for the activity of ubiquitin-specificproteases (32). We confirmed ubiquitination of CARD9 by TRIM62 at K125 and further showed that wild-type USP15 drastically reduces

RESULTS Downloaded from CARD9 ubiquitination (Fig. 2B, Supplemental Fig. 2). Coexpres- sion with neither the USP15 C298A mutant nor USP15 variant 2 USP15 is a CARD9-binding partner affected CARD9 ubiquitination, indicating that the removal of fi Previously, we identi ed TRIM62 as an E3 ubiquitin ligase ubiquitin is linked to the catalytic activity of USP15. responsible for optimal CARD9 activation through K27-linked We next investigated the deubiquitinase activity of USP15 on ubiquitination at the CARD9 K125 residue (22). We speculated that CARD9 in a reconstituted in vitro system. CARD9 was incubated http://www.immunohorizons.org/ there must be a deubiquitinase to reverse TRIM62-mediated with TRIM62 and ubiquitination machinery UBE1, UBE2D2, and ubiquitination and activation of CARD9. To identify potential ubiquitin. Ubiquitinated CARD9 was then incubated with and – deubiquitinases, we overexpressed Flag StrepII-tagged CARD9 in without USP15. We observed that incubation with USP15 led to the murine BMDC line JAWSII and employed a proteomics marked cleavage of the polyubiquitin chains present on CARD9 approach (Materials and Methods, Supplemental Fig. 1). In (Fig.2C),confirming that USP15 is sufficient for the removal of addition to the known binding partner TRIM62, immunoprecip- ubiquitin from CARD9. itation of overexpressed CARD9 and subsequent mass spectrom- etry analysis identified the deubiquitinase USP15 as a top hit based USP15 suppresses CARD9-mediated signaling on high peptide counts and percentage of protein coverage To determine whether USP15 can directly modulate CARD9- (Supplemental Fig. 1). mediated signaling, we used shRNA to knockdown endogenous To validate the interaction between USP15 and CARD9, we USP15 in wild-type BMDCs (Fig. 3A). USP15 knockdown by guest on September 26, 2021 overexpressed Flag–StrepII-tagged, full-length CARD9 as well as enhanced TNF-a production upon stimulation with WGP and N-terminal and C-terminal fragments together with USP15 in TDM, but not LPS, demonstrating that USP15 has a specific HEK293T cells (Fig. 1A). Full-length CARD9 and the N-terminal inhibitory effect on the CARD9-dependent, WGP- and TDM- fragment, but not the C-terminal fragment, coimmunoprecipitated induced pathways but is not involved in the regulation of MyD88- with USP15 (Fig. 1B). This result supports that CARD9 interacts and TRIF-dependent LPS recognition in primary murine cells. with USP15 and suggests that the N terminus of CARD9 harbors We next tested whether USP15 is also able to inhibit CARD9- the USP15 interaction domain. Furthermore, we found that dependent signaling in human cells. We first knocked down USP15 CARD9 D385–423 (22) (Fig. 1A) largely lost binding affinity with by shRNA in THP-1 cells overexpressing Dectin-1 and an NF-kB USP15 (Fig. 1C), indicating that this very short end region of the luciferase reporter. As in murine BMDCs, we observed that CARD9 coiled-coil domain is critical for interaction with USP15. knockdown of USP15 increased WGP-induced signaling measured We next tested whether USP15 binds to CARD9 endogenously in through NF-kB luciferase activity (Fig. 3B). We further explored primary murine BMDCs. We immunoprecipitated endogenous the effect of USP15 on CARD9 function in USP15 knockout (KO) USP15 with endogenous CARD9, demonstrating that the USP15– THP-1 cells generated by a CRISPR-based approach (Materials CARD9 interaction occurs under physiological conditions. In and Methods). USP15 KO also resulted in a higher fold induction of

(B and C) Endogenous USP15 in human THP-1 cells was knocked down by shRNA (B) or knocked out with a CRISPR-based approach (C). Indicated THP-1 cells were stimulated with WGP for 24 h, and NF-kB reporter activity was assessed by a luciferase assay. Western blots show protein levels of USP15 (B and C) and CARD9 (C) expressed in the cells used. Three independent pools of the same cells [THP-1 in (B) and USP152/2 in (C)] were transduced with Dectin-1 and NF-kB luciferase reporter and used in parallel for the subsequent treatments and assays (n = 3). Error bars represent mean 6 SD. **p , 0.01. (D) USP15 KO THP-1 cells were reconstituted with either empty vector, USP15 WT, or USP15 C298A. Cells were stimulated with WGP for 24 h, and NF-kB reporter activity was assessed by a luciferase assay. The Western blot shows USP15 protein levels expressed in the reconstituted cells. Three independent cell pools were transduced with Dectin-1 and NF-kB luciferase reporter and used in parallel for the subsequent treatments and assays (n = 3). Error bars represent mean 6 SD. **p , 0.01.

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NF-kB luciferase upon WGP treatment (Fig. 3C). To further Importantly, we observed that USP15 binds to CARD9 confirm that the observation in USP15 KO THP-1 cells is specific, in BMDCs in the absence of stimulation and that this we reconstituted the cells with either empty vector, wild-type interaction is largely unaffected by Dectin-1 ligand binding, USP15, or USP15 C298A. As expected, reconstitution with wild- indicating that USP15 constitutively interacts with CARD9 type USP15 dramatically reduced WGP-induced NF-kBluciferase and may act to prevent aberrant CARD9 signaling at steady- activity, whereas the catalytically inactive USP15 C298A mutant state. The negative regulation demonstrated in this study had no effect (Fig. 3D). These results suggest that USP15 catalytic mediated by the USP15–CARD9 association could occur at activity counters that of TRIM62, inhibiting CARD9 via the multiple levels—deactivation of CARD9 after ubiquitination removal of TRIM62-mediated ubiquitination. and modulation of CARD9-mediated signaling intensity, duration, or both—with the dynamic balance between TRIM62 and USP15 enzymatic activities likely critical in controlling DISCUSSION the overall output of this pathway. Interestingly, mutations in NEMO that affect the binding of A20 can lead to immune Posttranslational modifications play a critical role in virtually all phenotypes (42), raising the interesting possibility that some signaling pathways, and ubiquitination in particular is crucial for CARD9 mutations alter USP15 binding. More broadly, com- Downloaded from the regulation of innate immune signaling (33). Previously, we pounds that modulate the strength of CARD9–USP15 interac- identified the E3 ligase TRIM62 as a key activator of CARD9 tions may also represent an avenue for the specific regulation of through the deposition of K27-linked ubiquitin. This finding raised this signaling pathway in fungal infections and inflammatory or the question of the existence and identity of the deubiquitinase autoimmune syndromes. involved in the regulation of CARD9 activation. In this study, http://www.immunohorizons.org/ through a mass spectrometry approach using CARD9 as bait, we DISCLOSURES demonstrate that USP15 is the deubiquitinase responsible for this regulation. Our in vitro and cell-based assays confirm that USP15 removes TRIM62-deposited ubiquitin and limits the extent of R.J.X. cofounded Jnana Therapeutics and Celsius Therapeutics. CARD9-dependent NF-kB activation and cytokine production. These organizations had no role in this work. The other authors fi fl Although we only assessed the effects of USP15 on CLR-mediated have no nancial con icts of interest. signaling, it is important to note that the architecture of the CBM complex is conserved across multiple cell types and signaling ACKNOWLEDGMENTS pathways, suggesting that these or other E3 ligase/deubiquitinase pairs may regulate their signaling. We thank Nonghua Lu (The First Affiliated Hospital of Nanchang by guest on September 26, 2021 Several deubiquitinases have been directly associated with University, China) for supervising W.X. throughout the project, Jacques immune processes through the identification of mutations in Deguine for guidance with manuscript preparation, and Theresa Reimels human patients, for example A20/TNFAIP3, USP18, and OTULIN for manuscript preparation. (34), but overall, our knowledge of the deubiquitinases involved in specific immune processes remains more limited. 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