Dimethyl Fumarate Disrupts Human Innate Immune Signaling by Targeting the IRAK4− Myd88 Complex

Dimethyl Fumarate Disrupts Human Innate Immune Signaling by Targeting the IRAK4− MyD88 Complex

This information is current as Balyn W. Zaro, Ekaterina V. Vinogradova, Daniel C. Lazar, of September 26, 2021. Megan M. Blewett, Radu M. Suciu, Junichiro Takaya, Sean Studer, Juan Carlos de la Torre, Jean-Laurent Casanova, Benjamin F. Cravatt and John R. Teijaro J Immunol 2019; 202:2737-2746; Prepublished online 18

March 2019; Downloaded from doi: 10.4049/jimmunol.1801627 http://www.jimmunol.org/content/202/9/2737

Supplementary http://www.jimmunol.org/content/suppl/2019/03/15/jimmunol.180162 http://www.jimmunol.org/ Material 7.DCSupplemental References This article cites 58 articles, 18 of which you can access for free at: http://www.jimmunol.org/content/202/9/2737.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 © 2019 by The American Association of Immunologists, Inc. All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. The Journal of Immunology

Dimethyl Fumarate Disrupts Human Innate Immune Signaling by Targeting the IRAK4–MyD88 Complex

Balyn W. Zaro,*,1 Ekaterina V. Vinogradova,*,1 Daniel C. Lazar,†,1 Megan M. Blewett,*,1 Radu M. Suciu,* Junichiro Takaya,* Sean Studer,† Juan Carlos de la Torre,† Jean-Laurent Casanova,‡ Benjamin F. Cravatt,* and John R. Teijaro†

Dimethyl fumarate (DMF) is a prescribed treatment for multiple sclerosis and has also been used to treat psoriasis. The electrophilicity of DMF suggests that its immunosuppressive activity is related to the covalent modiﬁcation of cysteine residues in the human proteome. Nonetheless, our understanding of the proteins modiﬁed by DMF in human immune cells and the functional consequences of these reactions remains incomplete. In this study, we report that DMF inhibits human plasmacytoid dendritic cell function through a mechanism of action that is independent of the major electrophile sensor NRF2. Using chemical proteomics, we instead identify cysteine 13 of the innate immune kinase IRAK4 as a principal cellular target of DMF. We show that DMF blocks Downloaded from IRAK4–MyD88 interactions and IRAK4-mediated cytokine production in a cysteine 13–dependent manner. Our studies thus identify a proteomic hotspot for DMF action that constitutes a druggable protein–protein interface crucial for initiating innate immune responses. The Journal of Immunology, 2019, 202: 2737–2746.

lasmacytoid dendritic cells (pDCs) are a minor DC subset also display a similar morphology as plasma cells, hence the modifier that produce large amounts of type 1 IFN in response to viral plasmacytoid (3, 4). Other hallmarks of pDCs include low MHC class http://www.jimmunol.org/ P and endogenous nucleic acids (1, 2). This cell type bridges II expression and low (mouse) or negative (human) CD11c expres- the innate and adaptive immune systems, a duality reflected in its sion (1, 5). The pDC population comprises only 0.3–0.5% of human name. pDCs share a progenitor with the more common conventional peripheral blood cells but is implicated in a variety of pathologic DCs and constitutively express the FLT3 receptor; however, pDCs conditions (1) in which pDCs tend to accumulate in affected tissues to promote local inflammation. Systemic lupus erythematosus patients display low levels of *Department of Chemistry, The Scripps Research Institute, La Jolla, CA 92037; †Department of Immunology and Infectious Disease, The Scripps Research Institute, circulating pDCs but accumulate IFN-producing pDCs in the skin ‡

La Jolla, CA 92037; and St. Giles Laboratory of Human Genetics of Infectious (6, 7). Similar results have been found in psoriasis patients, in by guest on September 26, 2021 Diseases, Rockefeller Branch, The Rockefeller University, New York, NY 10065 which pDCs infiltrate the skin and become activated, driving the 1B.W.Z., E.V.V., D.C.L., and M.M.B. contributed equally to this work. early stages of the disease (8). In a xenograft model of human ORCIDs: 0000-0002-8938-9889 (B.W.Z.); 0000-0002-1130-4590 (D.C.L.); 0000- psoriasis, blockade of IFN-a signaling or production prevented the 0001-9273-392X (S.S.); 0000-0002-8171-8115 (J.C.d.l.T.); 0000-0001-8280- 8887 (J.R.T.). development of disease (8), suggesting that inhibition of pDC function could have therapeutic value. pDCs have been identified Received for publication December 26, 2018. Accepted for publication February 26, 2019. in the cerebrospinal fluid of multiple sclerosis (MS) patients (9) This work was supported by the National Institutes of Health (Grant CA231991), a and accumulate in demyelinated lesions of inflamed MS brains Life Science Research Foundation fellowship (to E.V.V.), the American Cancer (10). It has been reported that depletion of pDCs in chronic and Society (Fellowship PF-15-142-01-CDD to B.W.Z.), the National Science Foun- dation (Fellowship DGE-1346837 to M.M.B.), and The Donald E. and Delia relapsing mouse models of MS results in the exacerbation of B. Baxter Foundation Faculty Scholar Grant (to J.R.T.). experimental autoimmune encephalomyelitis symptoms and in- + B.W.Z. contributed to the study design, conducted experiments, analyzed data, and creased CD4 T cell activation in the CNS, suggesting a negative prepared the manuscript. E.V.V. conducted experiments and analyzed data. D.C.L. regulatory role for pDCs in modulating inflammatory T cell re- contributed to the study design, conducted experiments, analyzed data, and prepared b the manuscript. M.M.B. contributed to the study design, conducted experiments, and sponses (11). Furthermore, pDCs harvested from IFN- –treated analyzed data. R.M.S., J.T., S.S., J.C.d.l.T., J.-L.C., and B.F.C. contributed to the MS patients display a lower expression of the activation markers study design, analyzed data, and prepared the manuscript. J.R.T. contributed to the CD83 and CD86 (12), and therefore IFN therapy may act at least study design, conducted experiments, analyzed data, and prepared the manuscript. All authors have read and approved the final manuscript. in part through modulating pDC function. Address correspondence and reprint requests to Dr. Benjamin F. Cravatt and Dr. John Dimethyl fumarate (DMF) is an approved oral therapy for MS and R. Teijaro, The Scripps Research Institute, 10550 North Torrey Pines Road, La Jolla, has also been used for the treatment of psoriasis for several decades CA 92037. E-mail addresses: [email protected] (B.F.C.) and [email protected] (J.R.T.) in an alternative formulation called Fumaderm, with DMF believed to be the active species (13–15). Despite its long history and use in The online version of this article contains supplemental material. multiple autoimmune conditions, the mechanism of the action of Abbreviations used in this article: ABPP, activity-based protein profiling; B-EBV, EBV immortalized B cell; BSO, buthionine sulfoximine; C13, cysteine 13; DC, DMF remains poorly understood. DMF can promote an antioxidant dendritic cell; DMF, dimethyl fumarate; DMS, dimethyl succinate; GSH, glutathione; response through activation of the NRF2–KEAP1 pathway (16–18). IA, iodoacetamide; IRAK, IL-1R–associated kinase; isoTOP, isotopic tandem orthogonal proteolysis; MMF, monomethyl fumarate; MS, multiple sclerosis; ODN, However, DMF has been found to suppress pathologic conditions in oligodeoxynucleotide; pDC, plasmacytoid dendritic cell; PEI, polyethyleneimine; Poly I:C, the experimental autoimmune encephalomyelitis model of MS in- polyinosinic-polycytidylic acid; PPI, protein–protein interaction; TEV, tobacco etch virus; dependently of NRF2 (19), indicating that the therapeutic effect of TEV tag, TEV-cleavable tag; WT, wild-type. DMF in MS and other autoimmune disorders may involve proteins Copyright Ó 2019 by The American Association of Immunologists, Inc. 0022-1767/19/$37.50 and pathways beyond the KEAP1–NRF2 system. www.jimmunol.org/cgi/doi/10.4049/jimmunol.1801627 2738 DMF INHIBITS IRAK4–MyD88 SIGNALING

DMF is an electrophilic small molecule that can react with shown in capital letters are phosphodiester, and those in lower case are nucleophilic cysteine residues in proteins. It is now appreciated that phosphorothioate (nuclease resistant). Palindrome is underlined. Influenza many immune signaling proteins harbor electrophile/oxidation- virus was propagated and titrated in Madin-Darby canine kidney cells. Human pDC were stimulated with one multiplicity of infection of influenza sensitive cysteines (20–22) and that adduction of reactive cyste- virus overnight, and IFN-a levels were measured in the supernatants by ELISA. ines in the proteome by DMF and other electrophilic compounds Isolated human pDCs were stimulated overnight with either 500 mM loxoribine may constitute a general mechanism for suppressing the activity of (tlrl-lox; InvivoGen) or 100 nM R-848 (Sigma-Aldrich). Supernatant IFN-a immune cells (20, 23, 24). We previously used a quantitative levels were measured by ELISA. CAL-1 cells were obtained from S. Kamihira, Nagasaki University. CAL-1 cells were grown in RPMI medium (Invitrogen) chemical proteomics platform to identify DMF-sensitive cysteines containing 10% FBS, 2 mM L-glutamine, 100 mg/ml streptomycin, 100 U/ml in primary human T cells, which included a CXXC motif in the C2 penicillin at 37˚ C and 5% CO2. Sendai virus, strain Cantell, was propagated at domain of PKCu that was found to contribute to CD28 binding 37˚C in 10-d-old embryonated chicken eggs. and T cell activation (20). However, the extent to which DMF Cytokine and chemokine analysis impacts the function of other immune cells and whether such effects involve reactivity with specific cysteine residues in the proteome ELISAs were performed using the VeriKine Human and Mouse IFN-a remains poorly understood. In this study, we have examined DMF ELISA Kits (R&D Systems) according to the manufacturer’s instructions. activity in pDCs, an immune cell type that plays a central role in Cellular analysis and sorting by flow cytometry psoriasis. We first show that DMF, but not structurally related nonelectrophilic analogues, inhibit IFN-a and cytokine/chemokine Cells were stained with the following anti-human Abs: PE-conjugated anti- CD123 Ab (clone 6H6; BioLegend) and allophycocyanin-conjugated anti– production from primary human pDCs. DMF-mediated suppression IFN-a Ab (clone 7N4-1; BD Pharmingen). Flow cytometry acquisition was of IFN-a production was independent of either NRF2 activation or performed with a BD FACSDiva-driven BD LSR II Flow Cytometer (Becton Downloaded from global changes in the glutathione (GSH) content of pDCs. We then Dickinson). Data were then analyzed with FlowJo software (Tree Star). used the chemical proteomic method termed isotopic tandem Generation and culture of IRAK4-deficient human EBV orthogonal proteolysis (isoTOP)–activity-based protein profiling immortalized B cells (ABPP) to map DMF-sensitive cysteines in the pDC line Cal-1. Among the .4000 total cysteines quantified by isoTOP-ABPP in Human IRAK4-deficient cells (patient identification: MB002334) were provided as a gift from the laboratory of Jean-Laurent Casanova (27). Cells Cal-1 cells, cysteine 13 (C13) of IL-1R–associated kinase IRAK4 http://www.jimmunol.org/ were cultured in RPMI 1640 medium (Life Technologies Invitrogen) sup- stood out as being among the most sensitive to DMF. Noting that plemented with heat-inactivated FBS and a penicillin–streptomycin solution C13 resides proximal to the interface involved in MyD88 binding (Life Technologies Invitrogen). Cells were maintained at a concentration of (25) and adjacent to a site of mutation in IL-1R–associated kinase 1 million cells per 1 ml of medium with fresh medium added every 2 d. (IRAK) 4 (R12C) implicated in human immunodeficiency (26), To reconstitute IRAK4 expression within the IRAK4-deficient cells, the IRAK4 open reading frame was cloned from plasmid HsCD00330298 we proceeded to show that DMF disrupts both IRAK4–MyD88 (Harvard Plasmid Information Database Repository) and inserted into the interactions and IRAK4-mediated TNF-a production in a C13- pLPC-myc-mCherry lentiviral expression system, which was provided as a dependent manner. Taken together, our data indicate that DMF gift from the Lazzerini Denchi laboratory. The C13 mutants were gener- modifies C13 of IRAK4 in human pDCs, and this reaction disrupts ated using the Q5 Site-Directed Mutagenesis Kit (New England BioLabs). MyD88 binding and IRAK4 function. These studies potentially by guest on September 26, 2021 Protein overexpression reveal a mechanistic basis for the efficacy of DMF observed in autoimmune syndromes like MS and psoriasis and point to a novel HEK293T cells were seeded 3E6 cells per dish in 10-cm dishes. The next m druggable protein–protein interaction (PPI) for controlling innate day, to an Eppendorf tube was added either 1) 2.5 g of IRAK4 construct and 2.5 mg of GFP construct or 2) 2.5 mg of IRAK4 construct and 2.5 mg immune signaling. of MyD88 construct. To each tube was added 500 ml of DMEM and 30 ml of polyethyleneimine (PEI). Twenty minutes later, the transfection solution was added dropwise to cells. Twenty hours later, DMF or DMSO vehicle Materials and Methods was added to the cells as indicated. Chemical reagents Protein labeling and “click chemistry” Assays were performed with the following reagents: DMF (242926; Sigma-Aldrich), monomethyl fumarate (MMF) (651419; Sigma-Aldrich), HEK293T cells were harvested, lysed by sonication, and diluted to a dimethyl succinate (DMS) (W239607; Sigma-Aldrich), and buthionine concentration of 2 mg protein/ml. Protein concentrations were measured sulfoximine (BSO) (14484; Cayman Chemical). The Nrf2 inhibitor ML385 with the Bio-Rad Laboratories DC protein assay reagents A and B (SML1833; Sigma Aldrich), dimethyl maleate (238198; Sigma-Aldrich), (5000113, 5000114; Bio-Rad Laboratories). The proteome sample (500 ml) diethyl fumarate (D95654; Sigma-Aldrich), diethyl maleate (D97703; was treated with 100 mM iodoacetamide (IA)-alkyne probe by adding 5 ml Sigma-Aldrich), diisobutyl fumarate (7283-69-4; TCI Chemicals), and of a 10 mM probe stock (in DMSO). The labeling reactions were incubated diisopropyl fumarate (7283-70-7; TCI Chemicals). at room temperature for 1 h, after which the samples were conjugated to isotopically labeled, tobacco etch virus (TEV)–cleavable tags (TEV tags) Isolation of human pDCs by copper-catalyzed azide-alkyne cycloaddition (click chemistry). Heavy click chemistry reaction mixture (60 ml) was added to the DMSO-treated All studies with samples from human volunteers followed protocols ap- control sample, and 60 ml of the light reaction mixture was added to the proved by The Scripps Research Institute institutional review board. Blood compound-treated sample. The click reaction mixture consisted of TEV from healthy donors (females aged 30–49) were obtained after informed tags (10 ml of a 5 mM stock; light [compound-treated] or heavy [DMSO consent. PBMCs were purified over Histopaque-1077 gradients (10771; treated]), CuSO4 (10 ml of a 50 mM stock in water), and TBTA (30 mlofa Sigma-Aldrich) according to the manufacturer’s instructions. Briefly, 1.7 mM stock in 4:1 tBuOH:DMSO). To this was added TCEP (10 mlofa blood (20 3 25 ml aliquots) was layered over Histopaque-1077 (12.5 ml), 50 mM stock). The reaction was performed for 1 h at room temperature. and the samples were then fractionated by centrifugation at 750 3 g The light- and heavy-labeled samples were then centrifuged at 16,000 3 g for 20 min at 20˚C with no brake. PBMCs were harvested from the for 5 min at 4˚C to harvest the precipitated proteins. The resulting pellets Histopaque–plasma interface and washed twice with PBS. After that were resuspended in 500 ml of cold methanol by sonication, and the heavy time, pDCs were isolated with a CD304 (BDCA-4/Neuropilin-1) cell and light samples were combined pairwise. Combined pellets were then Isolation Kit (Miltenyi Biotec) according to the manufacturer’s instructions. washed with cold methanol, after which the pellet was solubilized by Stimulation of pDCs sonication in PBS and 1.2% SDS. The samples were heated at 90˚C for 5 min and subjected to streptavidin enrichment of probe-labeled proteins, Synthesized oligodeoxynucleotides (ODNs) were purchased from InvivoGen. The sequential on-bead trypsin and TEV digestion, and liquid chromatography– sequences of the ODNs are as follows: CpG-A (ODN2216), 59-ggGGGACGA: tandem mass spectrometry, accordingtothepublishedisoTOP-ABPP TCGTCgggggg-39, and CpG-B (ODN1826), 59-tccatgacgttcctgacgtt-39. Bases protocols (28–30). The Journal of Immunology 2739

Peptide and protein identification IRAK4–MyD88 binding assay with RAW Xtractor (version 1.9.9.2; available at http://fields.scripps.edu/ DMF/MMF/DMF treatment downloads.php) was used to extract the MS2 spectra data from the raw HEK293T cells (2.5 3 106) were plated in a 10-cm plate 24 h prior to files (MS2 spectra data correspond to fragments analyzed during the sec- transfection. For transfection, DNA (5 mg, myc-tagged IRAK4 kinase-dead ond stage of mass spectrometry). MS2 data were searched against a reverse C13C, R12C, or other C13 mutants; FLAG-tagged MyD88; or FLAG- concatenated, nonredundant variant of the Human UniProt database tagged MetAP2), serum-free DMEM (500 ml; Corning), and PEI (30 ml) (release-2012_11) with the ProLuCID algorithm (publicly available at were mixed together and allowed to incubate for 30 min at room tem- http://fields.scripps.edu/downloads.php) (31). Cysteine residues were searched perature. The mixture was then added dropwise to plated cells, and cells with a static modification for carboxyamidomethylation (+57.02146) and up were rested for 48 h. Cells expressing myc-tagged proteins were treated to one differential modification for either the light or heavy TEV tags with DMSO, DMF, MMF, or DMS (10003 stock in DMSO, final con- (+464.28595 or +470.29976, respectively). Peptides were required to have centration 100 mM) and incubated for 4 h. Media from cells transfected at least one tryptic terminus and to contain the TEV modification. ProLuCID with FLAG-tagged protein was aspirated, and the cells were washed with data were filtered through DTASelect (version 2.0) to achieve a peptide false- PBS (5 ml). Cells were transferred to a 1.5 ml Eppendorf tube and washed positive rate below 1% (32). with PBS 13. Freshly prepared ice-cold lysis buffer was added to the cell pellet (400 ml; 50 mM HEPES [pH 7.4], 150 mM NaCl, 1% Triton-X 100, R value calculation and processing 1 mM DTT with protease [Mini EDTA-free; Roche] and phosphatase The quantification of heavy/light ratios (isoTOP-ABPP ratios, R values) [Phos-STOP; Roche] inhibitors). Samples were incubated for 30 min on 3 g was performed by in-house CIMAGE software (30) using default param- ice and centrifuged (10 min at 10,000 at 4˚C). Protein concentration eters (three MS1s per peak and a signal-to-noise threshold of 2.5). Site- was normalized to 2 mg/ml. FLAG-tagged protein input samples were generated (100 mg protein in 50 ml sample). FLAG beads (22.5 ml per specific engagement of electrophilic compounds was assessed by blockade 1.5 mg protein; Thermo Fisher Scientific) were prewashed with lysis buffer of IA-alkyne probe labeling. For peptides that showed a $95% reduction 3 g Downloaded from in MS1 peak area from the compound-treated proteome (light TEV tag) and pelleted by centrifugation (2 min at 1600 at 4˚C). This bead wash protocol was repeated 13. Prewashed FLAG beads were then added to when compared with the DMSO-treated proteome (heavy TEV tag), a m maximal ratio of 20 was assigned. Overlapping peptides with the same 1.5 mg lysate (750 l) and rotated (3 h at 4˚C). Meanwhile, media from labeled cysteine (for example, they had the same local sequence around the cells transfected with myc-tagged protein was aspirated, and cells were washed with PBS. Cells were then scraped and transferred to a 1.5 ml labeled cysteines but had different charge states, Multidimensional Protein Eppendorf tube and subjected to an additional wash with PBS 13. Freshly Identification Technology segment numbers, or tryptic termini) were m grouped together, and the median ratio from each group was recorded as prepared ice-cold lysis buffer was added to each cell pellet (400 l; 50 mM HEPES [pH 7.4], 150 mM NaCl, 1% Triton-X 100, 1 mM DTT with the R value of the peptide for that run. http://www.jimmunol.org/ protease [Mini EDTA-free; Roche] and phosphatase [Phos-STOP; Roche] IRAK4–MyD88 binding assay inhibitors). Cell lysates were incubated on ice for 30 min and centrifuged (10 min at 10,000 3 g at 4˚C). Protein concentration was normalized to HEK293T cells (2.5 3 106) were plated in a 10-cm plate 24 h prior to 1 mg/ml. Myc-tagged protein input samples were generated (100 mg transfection. For transfection, DNA (5 mg, myc-tagged IRAK4 kinase-dead protein in 50 ml sample). Upon completion of FLAG enrichment, beads C13C, R12C, or other C13 mutants; FLAG-tagged MyD88; or FLAG- were pelleted by centrifugation (2 min at 1600 3 g at 4˚C). Flow through tagged MetAP2), serum-free DMEM (500 ml; Corning), and PEI (30 ml) was aspirated away, and ice-cold lysis buffer was added. Tubes were were mixed together and allowed to incubate for 30 min at room tem- inverted gently to wash beads (500 ml). These washes were repeated 23. perature. The mixture was then added dropwise to plated cells and cells FLAG enrichment was resuspended in lysis buffer (100 ml) and transferred rested for 48 h. Media from cells transfected with FLAG-tagged protein to myc-tagged protein lysate (750 mg, 750 ml). The sample was then in- was aspirated, and the cells were washed with PBS (5 ml). Cells were cubated on a rotator (2 h at 4˚C). Upon completion, beads were pelleted by guest on September 26, 2021 transferred to a 1.5 ml Eppendorf tube and washed with PBS 13. Freshly by centrifugation (2 min at 1600 3 g at 4˚C), and unbound lysate was prepared ice-cold lysis buffer was added to the cell pellet (400 ml; 50 mM aspirated away. Fresh ice-cold lysis buffer was added, and the tubes were HEPES [pH 7.4], 150 mM NaCl, 1% Triton-X 100, and 1 mM DTT with inverted gently to wash beads (500 ml). This bead wash was repeated 23. protease [Mini EDTA-free; Roche] and phosphatase [Phos-STOP; Roche] Beads were resuspended in 13 loading buffer (45 ml lysis buffer and 15 ml inhibitors). Samples were incubated for 30 min on ice and centrifuged 43 loading buffer [40% glycerol, 0.4% bromophenol blue, 2.8% 2-ME (10 min at 10,000 3 g at 4˚C). Protein concentration was normalized to (pH 6.8)]), and samples were boiled to denature proteins and cleave disulfides 2 mg/ml. FLAG-tagged protein input samples were generated (100 mg (5 min, 95˚C). Samples were then loaded onto a 10% Tris-glycine gel for protein in 50 ml sample). FLAG beads (22.5 ml per 1.5 mg protein; Thermo SDS-PAGE separation (30 ml immunoprecipitation, 15 ml input). Fisher Scientific) were prewashed with lysis buffer and pelleted by centrifugation (2 min at 1600 3 g at 4˚C). This bead wash protocol was re- Western blotting 3 peated 1 . Prewashed FLAG beads were then added to 1.5 mg lysate Proteins were transferred to nitrocellulose membrane in a wet transfer cell m (750 l) and rotated (3 h at 4˚C). Meanwhile, media from cells transfected (50 V, 2.5 h). Membrane was blocked in 5% milk for 1 h followed by primary with myc-tagged protein was aspirated, and cells were washed with PBS. incubation overnight at 4˚C (1:2500 anti-myc [Cell Signaling Technology] or Cells were then scraped and transferred to a 1.5 ml Eppendorf tube and 1:2500 anti-DYKDDDDK [Cell Signaling Technology]). Membranes were 3 subjected to an additional wash with PBS 1 . Freshly prepared ice-cold washed with TBST (33, 10 min) and incubated with secondary anti-rabbit m lysis buffer was added to each cell pellet (400 l; 50 mM HEPES [pH 7.4], Ab (LI-COR Biosciences) for 1 h at room temperature. 150 mM NaCl, 1% Triton-X 100, 1 mM DTT with protease [Mini EDTA- free; Roche] and phosphatase [Phos-STOP; Roche] inhibitors). Cell lysates Kinase activity assay were incubated on ice for 30 min and centrifuged (10 min at 10,000 3 g at 4˚C). Protein concentration was normalized to 1 mg/ml. Myc-tagged IRAK4 kinase activity was evaluated by the IRAK4 Kinase Enzyme System protein input samples were generated (100 mg protein in 50 ml sample). (V9421; Promega) according to the manufacturer’s instructions. Per manufac- Upon completion of FLAG enrichment, beads were pelleted by cen- turer’s instructions, staurosporine (S6942; Sigma-Aldrich) was administered to trifugation (2 min at 1600 3 g at 4˚C). Flow through was aspirated generate a kinase inhibitor dose-response curve. away, and ice-cold lysis buffer was added. Tubes were inverted gently to wash beads (500 ml). These washes were repeated 23.FLAGen- Statistical analysis richment was resuspended in lysis buffer (100 ml) and transferred to Significance in Figs. 1–3 were determined by two-tailed unpaired t test myc-tagged protein lysate (750 mg, 750 ml). The sample was then in- from at least two independent experiments of at least four replicates per cubated on a rotator (2 h at 4˚C). Upon completion, beads were pelleted experiment using Prism 7 (GraphPad). Significance in Fig. 4 was deter- by centrifugation (2 min at 1600 3 g at 4˚C), and unbound lysate was mined by a paired t test from three independent experimental replicates aspirated away. Fresh ice-cold lysis buffer was added, and the tubes using Prism 7 (GraphPad). were inverted gently to wash beads (500 ml). This bead wash was repeated 23. Beads were resuspended in 13 loading buffer (45 mllysis buffer and 15 ml43 loading buffer [40% glycerol, 0.4% bromophenol Results blue, 2.8% 2-ME (pH 6.8)]), and samples were boiled to denature DMF inhibits IFN-a release from activated human pDCs proteins and cleave disulfides (5 min, 95˚C). Samples were then loaded onto a 10% Tris-glycine gel for SDS-PAGE separation (30 ml immu- Primary human pDCs isolated from normal human donors were noprecipitation, 15 ml input). purified, stimulated with CpG-A, and treated concomitantly with 2740 DMF INHIBITS IRAK4–MyD88 SIGNALING Downloaded from http://www.jimmunol.org/ by guest on September 26, 2021

FIGURE 1. DMF inhibits IFN-a release from human pDCs. (A) Structures of DMF, MMF, and DMS. (B) DMF, but not MMF or DMS, blocks IFN-a release from primary human pDCs. pDCs were isolated from human blood and treated with the indicated compounds (50 mM each) and concomitantly activated with CpGA. After 18 h, IFN-a was measured in the supernatant by ELISA. (C) DMF inhibits IFN-a release in a concentration-dependent manner. pDCs were isolated from human blood and treated with the indicated concentrations of DMF for 18 h with concomitant CpGA stimulation. (D) FACS dot plot showing reduced intracellular IFN-a production in DMF-treated human pDCs. pDCs harvested from human blood were stimulated with CpGA and treated concomitantly with DMF (50 mM), MMF (50 mM), or DMS (50 mM) for 6 h. Frequency of IFN-a–producing pDC (E) and mean ﬂuorescent intensity of IFN-a levels gated on IFN-a+ pDCs (F). Results are representative of at least two independent experiments. **p , 0.01, ***p , 0.005 by two-tailed unpaired t test.

DMF, the major DMF metabolite MMF, or the saturated unreactive We next evaluated additional pDC stimuli and found that, as ob- analogue DMS (50 mM each compound; Fig. 1A). DMF, but served with CpG-A, DMF also blocked IFN-a production following not MMF or DMS, produced a concentration-dependent inhibition the treatment with CpG-B, another TLR9 agonist, or influenza (TLR7 of CpG-A–induced IFN-a release, displaying an IC50 value of ∼7 mM agonist) (Fig. 2A). Interestingly, however, the impact of DMF on (Fig. 1B, 1C). The inhibitory action of DMF on IFN-a production other cytokines varied depending on the stimulus, with a broader occurred without an effect on pDC viability (Supplemental Fig. 1A) suppressive effect being observed for DMF with the TLR9 agonists and was accompanied by the suppression of multiple additional CpG-A and CpG-B but not following stimulation with influenza virus cytokines and chemokines (Supplemental Fig. 1B). We further (Supplemental Fig. 2). To further investigate the potential role of found that intracellular IFN-a was also decreased by DMF, but DMF in blocking TLR7-induced cytokine secretion independent of not MMF or DMS (Fig. 1D–F), which pointed to a mechanism a viral recognition event, we treated pDCs with the additional of action in which DMF blocks the expression, rather than TLR7/8 stimulus R848 and the TLR7-specific agonist loxoribine. secretion, of IFN-a (Fig. 1D). As we found for influenza virus, DMF treatment impaired cytokine The Journal of Immunology 2741 Downloaded from http://www.jimmunol.org/ by guest on September 26, 2021

FIGURE 2. Inhibition of IFN-a production by DMF occurs through multiple TLR signaling pathways and is mostly independent of NRF2 or GSH abundance. (A) DMF inhibits TLR7 and TLR9-induced pDC IFN-a production. pDCs were isolated from human blood and treated with 50 mM DMF for 18 h along with 1 mM CpG-A, 1 mM CpG-B, one multiplicity of infection with influenza virus, 100 nM R-848, or 500 mM loxoribine, and the levels of IFN-a were quantified by ELISA. Results are representative of at least two independent experiments. (B) Structures of DMF (Figure legend continues) 2742 DMF INHIBITS IRAK4–MyD88 SIGNALING production in response to these additional TLR7 stimuli (Fig. 2A). linker. Samples were then subject to streptavidin enrichment and These data suggest that DMF differentially impairs the activation protease cleavage, after which heavy and light samples were of innate signaling pathways acting through multiple TLRs. combined and analyzed by liquid chromatography–tandem We further explored the structure–activity relationship for the mass spectrometry to quantify IA-alkyne–labeled cysteines blockade of IFN-a production by treating pDCs with a set of using an LTQ-Orbitrap Velos instrument (Fig. 3B) (20, 21, 30). fumarate ester analogues with varied stereoelectronic properties Among the 4408 cysteines quantified by isoTOP-ABPP in Cal-1 (Fig. 2B). Although several compounds with increased steric bulk cells, ∼170 residues showed substantial sensitivity to DMF as (e.g., diethyl fumarate, diisopropyl fumarate, diisobutyl fumarate, reflected in .4-fold changes in IA-alkyne reactivity in DMF- dibenzyl fumarate) suppressed IFN-a production, none exhibited treated cells (Fig. 3C). Most of these DMF-sensitive cysteines comparable activity to DMF (50 mM of each compound; Fig. 2C). showed a greater change in IA reactivity at 4 h (Fig. 3C), likely We also tested the impact of double bond geometry and found that reflecting a time-dependent increase in modification by DMF. dimethyl maleate and diethyl maleate partially blocked IFN-a A representative example of such time-dependent change is shown production, but neither compound displayed equivalent activity for C75 of adenosine deaminase in Fig. 3D. A select subset of to DMF (Fig. 2C). To the extent that the Z-olefin arrangement cysteines, in contrast, showed a near-complete loss of IA-alkyne in dimethyl maleate and diethyl maleate would be expected to reactivity following only 1 h of DMF treatment. These DMF- increase electrophilicity, these data suggest a more complex, hypersensitive cysteines included C13 of IRAK4 (Fig. 3C, 3D), nonlinear relationship between the intrinsic reactivity and immu- a protein kinase that plays a central role in TLR-mediated innate nosuppressive effects of fumarate esters. immune cell signaling pathways that produce IFN-a (38). The

immunological functions of IRAK4 have inspired the develop- Downloaded from a DMF blockade of pDC IFN- production involves ment of several ATP-competitive inhibitors of this kinase as po- NRF2-independent mechanisms tential drugs to treat autoimmune and autoinﬂammatory disorders The pharmacological effects of DMF have been proposed to occur, (39, 40). Interestingly, however, C13 is not found in the IRAK4 at least in part, through activation of the KEAP1–NRF2 pathway active site but rather located at the interface of IRAK4 that binds (16), which is a major mediator of cellular responses to to the adaptor protein MyD88 (Fig. 3E). We next sought to un-

electrophilic/oxidative stress (33). We found, however, that DMF derstand if DMF modification of C13 in IRAK4 affects interac- http://www.jimmunol.org/ fully suppressed IFN-a production in pDCs cotreated with the tions with MyD88 and downstream signaling. NRF2 inhibitor ML385 (34) (Fig. 2D). ML385 treatment alone DMF disrupts IRAK4–MyD88 interactions produced a partial reduction in IFN-a, but this effect was much less dramatic than the complete blockade of IFN-a production by A primary function of pDCs is to produce IFN-a in response to DMF (Fig. 2D). DMF has also been shown to deplete intracellular viral or endogenous nucleic acids (1, 2). The receptors for these concentrations of GSH (14, 17, 18, 35), and we found that the nucleic acids are TLR7/8 and TLR9 (5), which in turn signal GSH synthesis inhibitor BSO modestly decreased IFN-a release through the Myddosome complex comprising MyD88, IRAK2, from pDCs (Fig. 2E). Again, this change was much lower in and IRAK4 (41). The location of the DMF-hypersensitive cysteine magnitude than the IFN-a suppression caused by DMF (Fig. 2E). in IRAK4, C13, is at the interface of the IRAK4–MyD88 PPI by guest on September 26, 2021 These data, taken together, indicate that the primary mechanism (Fig. 3E), suggesting a potential functional role for this residue. by which DMF suppresses IFN-a production is independent of This hypothesis is also supported by human genetics as a mutation NRF2 and involves biochemical pathways beyond the reduction of of the adjacent residue Arg12 (R12C) produces an immunodefi- GSH content in pDCs. ciency syndrome that manifests as an increased susceptibility to pyogenic infections in children (27, 42, 43). The R12C mutation Mapping DMF-sensitive cysteines in the pDC line model Cal-1 has been shown to disrupt binding between IRAK4 and MyD88 We next sought to identify candidate protein targets that might (44), presumably hampering pDC responses to exogenous nucleic contribute to the IFN-a suppressing activity of DMF in pDCs. We acids. We found that recombinantly expressed IRAK4 (produced previously used the chemical proteomic method isoTOP-ABPP to by transient transfection in HEK293T cells) maintained site- globally map DMF-sensitive cysteines in primary human T cells specific sensitivity to DMF at C13 (Fig. 3F), and this interaction (20). The sample requirements for such chemical proteomic ex- was disrupted by coexpression of MyD88 in HEK293T cells periments are, however, quite high (milligrams of protein) and (Fig. 3G). beyond the scale that could be prepared from primary human We next established an in vitro binding assay for measuring pDCs. We therefore turned to Cal-1 cells as a pDC model cell line the IRAK4–MyD88 interaction. We immobilized FLAG-tagged (36) for the discovery of DMF-sensitive cysteines. MyD88 on anti-FLAG beads for incubation with lysate from We first confirmed that DMF maintains IFN-a–suppressing ac- HEK293T cells expressing various R12/C13 mutants in the con- tivity in Cal-1 cells (Fig. 3A) and then applied isoTOP-ABPP to map text of a kinase-dead variant of IRAK4 (K213A/K214A; termed DMF-sensitive cysteines in these cells. In brief, Cal-1 cells were IRAK4-K2D). We chose to use the kinase-dead IRAK4 because treated with DMF (50 mM, 1 or 4 h) or DMSO control, lysed, and this form of IRAK4 has been shown to interact more stably exposed to the general cysteine-reactive probe IA-alkyne, followed with MyD88, likely indicating that IRAK4 and MyD88 engage in by click chemistry–mediated conjugation (37) to isotopically dif- a dynamic signaling complex that can be disassembled by ferentiated azide-biotin tags containing a TEV protease–cleavable autophosphorylation of IRAK4 (45–47). Consistent with expectations

analogues. DBnF, dibenzyl fumarate; DEF, diethyl fumarate; DEM, diethyl maleate; DiPrF, diisopropyl fumarate; DMenF, dimenthyl fumarate; DMM, dimethyl maleate; TMA, tetramethyl fumaramide. (C) IFN-a release from purified human pDCs stimulated with CpG-A and treated concomitantly with 50 mM of indicated compounds in (B) for 18 h. (D) IFN-a release measured by ELISA from purified human pDCs stimulated with CpG-A and treated with either the NRF2 inhibitor ML385 (5 mM), DMF (50 mM), or ML385 and DMF. (E) IFN-a release measured by ELISA from purified human pDCs stimulated with CpG-A and treated with either the GSH inhibitor BSO (2 mM), DMF (50 mM), or BSO and DMF. Results are representative of at least two independent experiments. ***p , 0.005, ****p , 0.0001 by two-tailed unpaired t test. The Journal of Immunology 2743 Downloaded from http://www.jimmunol.org/

FIGURE 3. C13 of IRAK4 is a proteomic hot spot for DMF in Cal-1 cells. (A) IFN-a release from Cal-1 cells stimulated with Sendai virus and treated concomitantly with 50 mM of indicated compounds for 18 h. **p , 0.01. (B) Schematic diagram depicting competitive isoTOP for assessing DMF-reactive cysteines. (C) Scatter plot of ratio (R) values (DMSO/DMF) for quantified cysteine residues in isoTOP-ABPP experiments from Cal-1 cells treated for 1 or 4 h with 50 mM DMF. (D) Representative parent ion (MS1) profiles for DMF-hypersensitive (C13 of IRAK4), –moderately sensitive (C75 of adenosine deaminase), and -insensitive (C152 of GAPDH) cysteines from isoTOP-ABPP experiments in Cal-1 cells. (E) Crystal structure of the Myddosome comprising six MyD88 (green), four IRAK4 (blue), and four IRAK2 (gray) molecules (Protein Data Bank accession number 3MOP). C13 of IRAK4 is in magenta. (F and G) HEK293T cells were transfected with either IRAK4 and GFP (F and G) or IRAK4 and MyD88 (G). Twenty hours later, cells were treated with DMF (50 mM) for 4 h and lysed. The isoTOP-ABPP protocol was then performed as previously described. The R values (DMSO/DMF) from the isoTOP-ABPP experiment for each quantified cysteine are shown in the bar graph. by guest on September 26, 2021 based on past work (44), we found that the R12C mutant of DMF inhibits IRAK4–MyD88 signaling and cytokine IRAK4-K2D blocked coprecipitation of this protein with FLAG- production through engagement of IRAK4 C13 tagged MyD88 (Supplemental Fig. 3A). We next treated IRAK4- One of the downstream consequences of Myddosome complex m K2D–transfected cells with DMF (100 M,4h)andfoundthat formation is the activation of the NF-kB signaling pathway. DMF this compound, but not structurally related analogues that lack has been reported to inhibit NF-kB signaling in a variety of cell IFN-a–suppressing activity (MMF, DMS), substantially blocked types (48–52). Consistent with these past findings and with the the interaction of IRAK4-K2D with MyD88 in vitro (Fig. 4A). disruption of the Myddosome complex, we found that DMF This antagonistic effect of DMF on the IRAK4-K2D–MYD88 blocks phosphorylation of the NF-kB pathway member p65 in interaction was not observed for a C13A-mutant of IRAK4-K2D pDCs stimulated with CpG-B (Supplemental Fig. 3B). (Fig. 4B), supporting that DMF reactivity with C13 is respon- We next examined the impact of DMF on IRAK4-induced cy- sible for disrupting IRAK4-K2D binding to MyD88. Interest- tokine production in human immune cells. We employed EBV ingly, we found that mutation of C13 to negatively charged immortalized B cells (B-EBV) that were generated from PBMCs of residues glutamate or aspartate, but not other amino acids, in- a patient with a large deletion in the IRAK4 gene that renders cluding glutamine, mimicked the inhibitory effect of DMF on the IRAK4-K2D–MYD88 interaction (Fig. 4C). One interpretation it nonfunctional (27). We used the B-EBV cells as a source of of these findings is that DMF, once reacted with C13 of IRAK4- IRAK4-null cells amenable to genetic complementation studies. K2D, may undergo esterolysis to present a carboxylate group IRAK4-deficient B-EBV cells were then reconstituted with either that, like the C13D and C13E mutants, impairs binding to wild-type (WT) or the C13A mutant of IRAK4 using lentiviral MyD88. transduction. Reconstitution of IRAK4-deficient B-EBV cells with a Our studies pointed to a kinase activity-independent mecha- WT-IRAK4 resulted in significantly greater TNF- production fol- nism by which covalent DMF modification of IRAK4 disrupts lowing LPS and CpG stimulation compared with IRAK4-deficient Myddosome function (i.e., disruption of IRAK4–MyD88 interac- B-EBV cells, whereas the TLR3/RIG-I/MDA5 agonist polyinosinic- tions). To more directly assess whether DMF impacts IRAK4 polycytidylic acid (Poly I:C), which signals independent of MyD88/ kinase activity, we treated purified IRAK4 (50 ng/reaction) with IRAK4, induced TNF-a production in both WT-IRAK4 and IRAK4- DMF (4 mM–2 nM) or the pan-kinase inhibitor staurosporine deficient cells (Supplemental Fig. 3C). We next discovered that DMF, (40 mM–0.02 nM) and measured residual IRAK4 activity using but not MMF or DMS, suppressed TNF-a production following a substrate assay. DMF did not alter IRAK4 kinase activity in stimulation with LPS and CpG in the B-EBV cells reconstituted with this assay, in contrast to staurosporine, which showed clear WT-IRAK4 (Fig. 4E). In contrast, DMF did not affect TNF-a pro- concentration-dependent inhibition of IRAK4 (Fig. 4D). duction following Poly I:C stimulation (Fig. 4E), suggesting that DMF 2744 DMF INHIBITS IRAK4–MyD88 SIGNALING Downloaded from http://www.jimmunol.org/ by guest on September 26, 2021

FIGURE 4. DMF disrupts IRAK4–MyD88 complexes and signaling in a C13-dependent manner. (A) DMF, but not MMF or DMS, impairs interactions between IRAK4 and MyD88. MyD88-FLAG (immunoprecipitated from HEK293T cells expressing MyD88-FLAG) bound to anti-FLAG beads was exposed to lysate from IRAK4-K2D–expressing HEK293T cells pretreated with DMF, MMF, DMS (100 mM compound, 4 h), or DMSO control. Shown Western blot is representative of three replicates used for quantitation. *p , 0.05, DMF versus MMF as determined by paired t test. (B) DMF-mediated disruption of the IRAK4–MyD88 interaction depends on C13 of IRAK4. MyD88 was immunoprecipitated from HEK293T cells expressing FLAG-tagged MyD88 and exposed to lysate from WT- or C13A–IRAK4-K2D-expressing HEK293T cells pretreated with DMF (100 mM, 4 h) or DMSO control. Shown Western blot is representative of three replicates used for quantitation. (C) Impact of mutagenesis of C13 on IRAK4 interactions with MyD88. MyD88 was immunoprecipitated from HEK293T cells expressing FLAG-tagged MyD88 and exposed to lysate from HEK293T cells expressing the indicated C13 mutants of IRAK4. Western blot is representative of three replicates used for quantitation. C13E versus C13A *p , 0.05, C13D versus C13A, *p , 0.05 as determined by paired t test. (D) Impact of DMF on IRAK4 kinase activity. A reaction of purified IRAK4 (50 ng/reaction), MBP (100 ng/reaction), and ATP (50 mM) was treated with various concentrations of either staurosporine (40 mM to 0.02 nM) or DMF (4 mM to 2 nM) to generate a dose-response curve to assess the efficacy of these compounds as inhibitors of IRAK4 kinase activity. ADP-Glo Kinase Assay measures the conversion of ATP to ADP, which correlates with overall kinase activity within the reaction. (E) IRAK4-deficient B-EBV cells were reconstituted with human WT-IRAK4 by lentiviral transduction, pretreated with DMF, MMF, DMS (50 mM, 4 h) or DMSO control, stimulated with LPS, CpG-A, or Poly I:C for 24 h, and TNF-a measured by ELISA. LPS stimulated cells, DMSO versus DMF treatment, ***p , 0.005. CpG-A treated cells, DMSO versus DMF treatment, ***p , 0.005. (F) IRAK4-deficient B-EBV cells were reconstituted with WT-IRAK4 or C13A-IRAK4 by lentiviral transduction, pretreated with DMF (50 mM, 4 h) or DMSO control, stimulated with LPS for 24 h, and TNF-a levels measured by ELISA. DMF-treated cells versus DMSO-treated cells, ****p , 0.0001. acts to suppress pDC cytokine production predominantly through larger than that of IRAK4-deficient cells (which showed a negligible IRAK4/MyD88 signaling. Finally, we confirmed that DMF does not response; see Supplemental Fig. 3C), and DMF treatment did not decrease CpG-induced TNF-a production in IRAK4-deficient cells further reduce TNF-a production in the C13A-IRAK4 cells. reconstituted with the C13A-IRAK4 mutant (Fig. 4F). We should note that B-EBV cells expressing the C13A-IRAK4 mutant showed a lower Discussion TNF-a induction than WT-IRAK4–reconstituted cells, but regardless, pDCs were first definitively characterized in 1999 and have since the TNF-a induction in C13A-IRAK4 mutant cells was still much been implicated in the initiation of psoriasis (8, 53) and the The Journal of Immunology 2745 progression of MS (9, 10, 12). Despite their low abundance in the small molecules (59) are vulnerable to enzymatic hydrolysis by blood, pDCs are the primary producers of IFN-a and can thus carboxylesterases, but it is unclear whether DMF can be hydro- drive local inflammation (1). The immunomodulatory drug DMF lyzed twice to reveal the free-acid fumarate oncometabolite in is a widely prescribed and effective treatment for MS and psori- appreciable concentrations. We have previously reported that asis, both of which are inflammatory diseases. More recently, the singly hydrolyzed metabolic product of DMF, MMF, does DMF has also been reported as a potential therapy for cutaneous not appreciably react with proteinaceous cysteines at treatment- T cell lymphoma and is at times prescribed off label to bone relevant concentrations or suppress immune cell function (20). marrow transplant patients because of its immunomodulatory Nonetheless, we cannot rule out the possibility of an increase of properties (54). The mechanism of DMF is not well understood, free fumarate or a pharmacological effect of this possible outcome but this drug has been shown to impair the activation of T cells as a result of DMF treatment. (19, 20) and conventional DCs (55). However, little is known about the effect of DMF on pDCs. Our studies indicate that pDCs Disclosures are sensitive to DMF, and that this pharmacological effect is The authors have no financial conflicts of interest. largely independent of NRF2 and, at least in part, through the NF-kB signaling pathway, with covalent adduction of C13 of IRAK4 residing at the top of the signaling cascade. Given that DMF is rel- References atively well tolerated and capable of suppressing proinflammatory 1. Reizis, B., A. Bunin, H. S. Ghosh, K. L. Lewis, and V. Sisirak. 2011. Plasmacytoid dendritic cells: recent progress and open questions. Annu. Rev. Immunol. 29: signaling in both activated T cells and now pDCs, a more detailed 163–183. understanding of its mechanisms of action may reveal potential new 2. Gilliet, M., W. Cao, and Y. J. Liu. 2008. 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