Neddylation plays an important role in the regulation of murine and human dendritic cell function

Regular Article

IMMUNOBIOLOGY

Neddylation plays an important role in the regulation of murine andhuman dendritic cell functionNathan Mathewson,1,2 Tomomi Toubai,1 Steven Kapeles,1 Yaping Sun,1 Katherine Oravecz-Wilson,1 Hiroya Tamaki,1

Ying Wang,1 Guoqing Hou,1 Yi Sun,3 and Pavan Reddy1

1Department of Internal Medicine, Division of Hematology/Oncology, University of Michigan Comprehensive Cancer Center, Ann Arbor, MI; and 2Graduate

Program in Immunology, and 3Department of Radiation Oncology, University of Michigan Medical School, Ann Arbor, MI

Key Points

• There is a role for theposttranslational modification,neddylation, in regulation ofimmune responses mediatedby dendritic cells.

• A role for neddylation inNF-kB signaling in dendriticcells was identified.

Posttranslational proteinmodifications (PTMs) are necessary for cells to function properly.

The role of PTMs in regulating immune responses, specifically thosemediated by dendritic

cells (DCs),whichare critical for both innate and adaptive immunity, is not well understood.

Utilizing multiple but complementary approaches, we determined the role of an important

but less understood typeof PTM, namely, neddylation, in regulatingDC functions. Inhibition

of neddylation suppressed the release of proinflammatory cytokines by DCs in response

to Toll-like receptor, nucleotide oligomerization domain–like receptor, and noninfectious

CD40L stimulation. These effects were more profound than those mediated by the

proteasome inhibitor bortezomib or a commonly used antiinflammatory agent, dexameth-

asone. Targeting neddylation also suppressed the ability of DCs to stimulate murine

allogeneic T cells in vitro and in vivo and human allogeneic T-cell responses in vitro.

Mechanistic studies demonstrated that inhibition of neddylation reduced both canonical

and noncanonical nuclear factor-kB (NF-kB) activity. Neddylation inhibition prevented the degradation of inhibitor-kB and thus

reduced the translocation and activation of NF-kB, but without perturbation of the mitogen-activated protein kinase/extracellular

signal-regulated kinase pathway. Thus, blocking neddylation could be a novel strategy for mitigating immune-mediated disease

processes. (Blood. 2013;122(12):2062-2073)

Introduction

Posttranslational protein modifications (PTMs) are critical for immu-nity.1 PTMs include, but are not limited to, acetylation,2 phosphory-lation, and ubiquitination.3 These PTMs are necessary for the properfunction of all cells, including dendritic cells (DCs), which are criticalfor both innate and adaptive immunity.4-6 Following an encounter withinflammatory stimuli or components of microbial or viral origin, DCsupregulate costimulatory molecules7,8 and release proinflammatorycytokines.6 These proinflammatory cytokines play an important rolein immunity in an autocrine and paracrine fashion on surroundingcells; however, the molecular processes that regulate cytokinerelease from DCs are not completely understood. Proinflammatorycytokines have profound impact on the causation or exacerbation ofmany disease states including chronic inflammatory diseases, shock,autoimmunity, and alloimmunity.9-12

Pattern recognition receptors (PRRs), such as Toll-like receptor(TLR) and nucleotide oligomerization domain (NOD)-like receptor(NLR), that are found on DCs interact with pathogen-associatedmolecular patterns (PAMPs), allowing selective recognition ofmicroorganisms.13,14 A series of signaling events leads to thephosphorylation of inhibitor-kB (IkB) kinase b, which in turnphosphorylates IkB for degradation, resulting in the activationof the transcription factor nuclear factor-kB (NF-kB).15 At basal

conditions, IkB sequesters canonical NF-kB in the cytosol. However,the critical molecular regulators of phosphorylated IkB degradationare not well understood. Activation and translocation of NF-kBresult in the transcription of the proinflammatory cytokines,tumor necrosis factor-alpha (TNF-a) and interleukin-6 (IL-6).16

Activation of NF-kB occurs through 2 distinct canonical andnoncanonical NF-kB pathways.16 NF-kB is comprised of hetero-dimers of the Rel protein family. The canonical NF-kB is a unionof p65 (RelA) and p50 and responds to numerous PRR stimuli,resulting in diverse functions. In contrast, the noncanonical pathwayconsists of RelB and p52 constituting NF-kB and responds to asubset of TNFR signals such as CD40L.17

Upon PRR stimulation, IkB is phosphorylated and subsequentlyubiquitinated, resulting in its degradation and allowing NF-kB totranslocate to the nucleus.18 Phosphorylation of IkB is followed byits ubiquitination and degradation by the E3 ligase complex. Thiscomplex consists of the proteins Skp1 (S-phase kinase associatedprotein 1), an F-box protein, Cullin1 (Cul1), and a RING box protein(Rbx1 or Rbx2, also known as SAG), which together comprisethe Cullin RING ligase-1 (CRL-1).19,20 The ability of CRL-1 toubiquitinate target proteins, including IkB, is dependent upon thecovalent PTM of the Cul1 subunit of the CRL by attachment of

Submitted February 22, 2013; accepted July 7, 2013. Prepublished online as

Blood First Edition paper, July 17, 2013; DOI 10.1182/blood-2013-02-486373.

The online version of this article contains a data supplement.

There is an Inside Blood commentary on this article in this issue.

The publication costs of this article were defrayed in part by page charge

payment. Therefore, and solely to indicate this fact, this article is hereby

marked “advertisement” in accordance with 18 USC section 1734.

© 2013 by The American Society of Hematology

2062 BLOOD, 19 SEPTEMBER 2013 x VOLUME 122, NUMBER 12

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a ubiquitin homolog called NEDD8 (neural precursor cell expressed,developmentally downregulated 8).21 PTM by the attachment ofNEDD8 is known as neddylation.22,23 Neddylation is an enzymaticprocess in which NEDD8 is activated in an ATP-dependent mannerby an E1 enzyme known as NEDD8 activating enzyme (NAE) and issubsequently transferred to the E2 enzyme, Ubc12.24 NEDD8-Ubc12transfers NEDD8 to Cullin, displacing CAND1, an inhibitor of Cullinactivity.24,25 Once activated, CRL-1 catalyzes the transfer ofubiquitin from the E2 to the substrate.24 A small molecule calledMLN4924, which has remarkable specificity and potency forinhibiting NAE, has been developed recently. MLN4924 formsa NEDD8-AMPmimetic and thereby prevents the initial activationof NEDD8.26 Currently, this small molecule is in human clinicaltrials as an anticancer agent; however, the role of neddylation inregulating immune cells, specifically DCs, is not known.

Utilizing 2 distinct but complementary approaches (chemical inhi-bition with the small molecule MLN4924 and molecular knockdownof critical CRL proteins), we examined the effect of inactivationof CRL-1 E3 ligase activity on murine and human DC function asdetermined by the release of proinflammatory cytokines and theirability to stimulate allogeneic T cells both in vitro and in vivo. Wealso compared these results with those for the known antiinflamma-tory drug dexamethasone and further determined the differentialmolecular mechanisms that underpin their antiinflammatory re-sponses.We found that inactivation of the CRL-1 E3 ligase resulted insignificantly greater suppression of proinflammatory cytokine pro-duction than dexamethasone or the proteasome inhibitor bortezomib.Also, mechanistic studies showed that in contrast to dexamethasone,inhibition of neddylation prevented the degradation of IkB as well asthe translocation and activation of NF-kB without perturbation of themitogen-activated protein kinase/extracellular signal-regulated kinase(MAPK/ERK) pathway.

Materials and methods

See supplemental Methods for details, available on the Blood website.

Reagents

RPMI1640, Dulbecco’s modified Eagle medium, alpha-minimum essentialmedium, penicillin, streptomycin, and sodium pyruvate were purchased fromGibco (Grand Island, NY); fetal calf serum from GemCell (Sacramento, CA); 2-mercaptoethanol from Sigma-Aldrich (St. Louis, MO); and murine granulocytemacrophage–colony-stimulating factor from Peprotech (Rocky Hill, NJ). Allantibodies (Abs) used for the fluorescence-activated cell sorter were purchasedfrom Biolegend (San Diego, CA). IL-6 enzyme-linked immunosorbentassay (ELISA) kits were purchased fromBDBiosciences (SanDiego, CA) andTNF-a, both mouse and human, from R&D Systems (Minneapolis, MN).Dimethylsulfoxide (DMSO) was obtained from Sigma-Aldrich, MLN4924from Active BioChem (Maplewood, NJ), and dexamethasone from APPPharmaceuticals (Schaumburg, IL); bortezomib was purchased from FisherScientific and lipopolysaccharide (LPS) from InvivoGen (San Diego, CA).

Mice

Female C57BL/6 (I-Ab; CD45.21) and BALB/c (H2d) mice were purchasedfrom the National Cancer Institute, Abb (B6.129-H2-Ab1tm1Gru N12) micewere purchased from Taconic, and bm12 (B6.C-H2-Ab1bm12/KhEg-Mc1re-J/J) mice were purchased from the The Jackson Laboratory. The age of miceused for experiments ranged from 7 to 12 weeks. All animals were cared forunder regulations reviewed and approved by the University of Michigan’sCommittee on Use and Care of Animals, based on University LaboratoryAnimal Medicine guidelines.

Isolation of bone marrow–derived DCs

DCs were obtained as described previously.27 See supplemental Methods fordetails. Studies with human cells were performed after obtaining informedconsent from the participants; informed consent was obtained in accordancewith the Declaration of Helsinki. The University of Michigan InstitutionalReview Board approved the studies (approval number: HUM00043287).

Cell culture

The DC cell line, JAWSII, was established from bone marrow (BM) cells ofa p53-knockout C57BL/6 mouse and purchased from the American TypeCulture Collection (CRL-11904; Manassas, VA). See supplemental Methodsfor details on culturing JAWSII cells.

Cytokine detection

Supernatants were collected and stored at220°C until analysis. TNF-a andIL-6 ELISA kits (mouse and human; purchased from R&D Systems) wereused per the manufacturer’s instructions and read at 450 nm using a SpectraMaxmicroplate reader (Molecular Devices, Sunnyvale, CA).

Quantitative polymerase chain reaction

See supplemental Methods for details. Briefly, BM-derived DCs (BMDCs) wereseeded on 60-mm plates with 3 3 106 cells per plate. Indicated cells were thenpretreated for 2 hours with DMSO, MLN4924, or dexamethasone at indicateddosages. Cells were then stimulated with LPS (500 ng/mL) for 4 hours. Next,RNA was extracted using an RNeasy Mini Kit (Qiagen, Hilden, Germany)according to the manufacturer’s instructions. Following validation, quantitativepolymerase chain reaction (qPCR) primers (see supplemental Methods) inconjunction with SYBR Green PCR Master Mix (Applied Biosystems,Carlsbad, CA) were used to quantify mRNA levels of cDNA (50 ng). qPCRwas performed with a Mastercycler Realplex2 (Eppendorf, Hamburg, Germany)using the DDCT method to calculate mRNA levels.

Flow cytometry

To analyze DC surface phenotype, DCs were incubated in the presence orabsence of MLN4924 or vehicle (DMSO; Sigma-Aldrich). Cells were thenharvested and stained with CD11c-conjugated allophycocyanin (APC)(clone: N418) and 1 of the following per triplicate group: Annexin V (BDBiosciences), CD80 (clone: 16-10A1), CD86 (clone: GL-1), major histo-compatibility complex (MHC) II-I-Ab (clone: AF6-120.1), PD-L1 (clone:MIH5), PD-L2 (clone: TY25). All flow cytometry Abs were purchased fromeBiosciences. Stained cells were then analyzed with an Accuri C6 flowcytometer (BD Biosciences).

Western blot and subcellular fractionation

See supplemental Methods for details. Briefly, equal amounts of protein fromwhole cell lysates were separated with sodium dodecyl sulfate–polyacrylamidegel electrophoresis and subsequently transferred to nitrocellulose membrane.See supplemental Methods for primary Abs used. Secondary Abs conjugatedto horseradish peroxidase (Jackson ImmunoResearch) were used to detectprimary Abs. Densitometric analysis was performed using ImageJ software.The cytoplasmic and nuclear fractions were isolated using the NuclearExtract Kit (Active Motif) per the manufacturer’s instructions. The extractswhere then analyzed via western blot.

Mixed lymphoctye reaction

Splenic T cells (2 3 105/well) were magnetically separated from WT-B6or WT-BALB/c mice by autoMACS using CD90.2 microbeads and sub-sequently cultured with irradiated (30 Gy) WT-B6 DC at 40:1 (53 103/well)and at 100:1 (2 3 103/well), each for 72 and 96 hours. Human mixedlymphocyte reactions (MLRs) were performed by coculture of peripheralblood mononuclear cells (PBMCs; 1 3 105/well) and monocyte-deriveddendritic cells at 1:1 (13 105) and at 10:1 (1 3 104) ratios, each for 96 and120 hours. Incorporation of 3H-thymidine (1 mCi/well) by proliferating

BLOOD, 19 SEPTEMBER 2013 x VOLUME 122, NUMBER 12 NEDDYLATION REGULATES DC FUNCTION 2063




T cells or PBMCs during the final 6 hours of culture was measured bya TopCount (PerkinElmer).

Molecular knockdown

See supplemental Methods for details. Briefly, JAWSII cells were seeded andtransfected with siRNA (3 mg) using oligofectamine reagent (Invitrogen) perthe manufacturer’s instructions for 24 hours before stimulation with LPS.RNA was extracted from cell pellets using an RNeasy Mini Kit (Qiagen) andanalyzed via qPCR.

Confocal microscopy

See supplemental Methods for details. BMDCs were seeded onto Corningglass coverslips (13 105 cells/slip; Fisher Scientific) overnight at 37°C. Coverslips were then washed, fixed with 4% paraformaldehyde for 20 minutes, andsubsequently permeabilized with 0.3% Triton X. Cells were stained withNF-kB p65 primary and Alexa Fluor 488 secondary, 4,6 diamidino-2-phenylindole (DAPI; Invitrogen), and Alexa Fluor 555 phalloidin. Coverslipswere then mounted using ProLong Gold antifade reagent (Molecular Probes),and Z-stack images were acquired at room temperature using a NikonA-1 confocal microscope (Mellville, NY) equipped with an oil immersion603 objective with a numerical aperture equal to 1.4 and imported intoNIS-Elements Software (Nikon). Excitation lasers of 405 nm, 488 nm, and561 nm were used.

Statistical analyses

All statistical analyses were performed using GraphPad Prism (GraphPadSoftware). P values were calculated using the Student’s t test.

Results

Inhibition of neddylation attenuates LPS-induced

proinflammatory cytokine production by DCs

Previous studies have shown that MLN4924 prevents the neddylationof CRLs in several cell types.28,29 Therefore, we determined whetherMLN4924 or dexamethasone could inhibit neddylation in BMDCs.BMDCs that were pretreated with MLN4924 followed by TLR4stimulation (LPS) exhibited reduced neddylation (Figure 1A). Thisinhibition of neddylation was detected using a NEDD8 Ab capableof binding NEDD8-conjugated Cullin protein at approximately90 kDa (Figure 1A, top panel).28,30 We further confirmed this withanAb specific for total Cul119 (Figure 1A,middle panel). By contrast,treatment with dexamethasone did not inhibit neddylation in BMDCs(Figure 1A).

Having established that MLN4924 inhibited neddylation, next wedetermined whether it modulated the production of proinflammatorycytokines such as TNF-a and IL-6 following stimulation of BMDCswith LPS.31 TNF-a (Figure 1B) and IL-6 (Figure 1C) cytokine levelsin the supernatants were significantly inhibited by MLN4924 in adose-dependent manner. Furthermore, MLN4924 suppressed LPS-induced inflammatory cytokines significantly more than dexameth-asone treatment (Figure 1B-C). Because LPS increases the expressionof TNF-a and IL-6 at the transcriptional level,32,33 we assessed theimpact of MLN4924 and dexamethasone on LPS-induced tran-scriptional expression of TNF-a and IL-6 mRNA. As shown inFigure 1D-E, both MLN4924 and dexamethasone inhibited LPS-induced TNF-a and IL-6 mRNA levels. The impact of MLN4924on mitigating LPS-induced increases of TNF-a and IL-6 mRNAwas dose dependent (Figure 1D-E).

Collectively, these data suggest that MLN4924 and dexameth-asone differentially affect Cul1 neddylation in DCs and that theymay not have interrelated molecular targets. More importantly, the

data show that inhibition of neddylation results in a more sig-nificant attenuation of LPS-induced TNF-a and IL-6 productionthan dexamethasone in BMDC.

Neddylation modulates DC responses to both infectious and

noninfectious stimuli

Next, in order to determine whether the effects of MLN4924 ob-served thus far were specific only to TLR4 ligation (LPS), westimulated BMDC with Pam3CSK4 (a TLR1/2 ligand), peptidogly-can (PGN; a NOD2 stimulator), and noninfectious CD40L agonist.Treatment of DCs in the presence or absence of MLN4924 ordexamethasone caused a significant reduction in the secretion ofTNF-a by both MLN4924 and dexamethasone, regardless of whetherthey were stimulated with Pam3CSK4 (Figure 2A), PGN (Figure 2B),or CD40L (Figure 2C). These data suggest that the effects on sup-pression of proinflammatory cytokines through inactivation of theCRL-1 E3 ligase are not specific to TLR4 but extend to an array ofPRR and cellular stimuli such as CD40L. Furthermore, these data alsoshow that inhibition of neddylation regulated both canonical (TLR/NLR) and noncanonical (CD40L) activated NF-kB pathways. Tofurther compare the effects of neddylation inhibition with anotherclinically used inhibitor of the proteasome and NF-kB pathway,we stimulated BMDC with LPS and concurrently treated with thediluent vehicle,MLN4924, dexamethasone, or bortezomib. As shownin Figure 2D, blocking neddylation inhibited TNF-a secretion fromthe DCs far more potently than the proteasome inhibitor bortezomib.

Inhibition of neddylation mitigates the ability of DCs to induce

proliferation of allogeneic T cells

To further characterize the effect of neddylation inhibition on thefunction of DCs, we determined the impact of treating DCs withMLN4924 on their ability to stimulate allogeneic T cells4,5 in aMLR. BALB/C T cells were cultured with C57BL/6 irradiated (30 Gy)BMDC at 40:1 and 100:1 ratios for 72 hours (Figure 3A) and 96 hours(Figure 3B). Addition of MLN4924 to the cultures upon platingcells significantly reduced T-cell proliferation (Figure 3A-B). Todetermine whether this reduction was due to a direct impact ofMLN4924 on DCs, BMDCs were preincubated with MLN4924for 6 hours, washed, and then used as stimulators in the MLR.Pretreatment of DCs with MLN4924 also reduced proliferation ofallo-T cells at both 72 hours (Figure 3A) and 96 hours (Figure 3B),suggesting that MLN4924 treatment of DCs reduced their abilityto induce proliferation of allo-T cells. To specifically determinewhether inhibition of neddylation can also suppress T cells directly,we stimulated BALB/C CD90.21 T cells with a-CD3 and a-CD28functional Abs for 48 hours following their pretreatment in thepresence or absence of MLN4924. MLN4924 significantly reducedT-cell proliferation (supplemental Figure 3A) and caused a significantdecrease in the secretion of interferon-g (supplemental Figure 3B).

Inhibition of neddylation regulates DC functions in vivo

Next we investigated the effects of neddylation inhibition on in vivoDC functions. Because host antigen–presenting cells modulategraft-vs-host disease (GVHD) and DCs are the most potent APCs, weused a clinically relevant, MHC-mismatched B6 (H-2b)→ BALB/C (H-2d) model of allogeneic bone marrow transplant (BMT) to testthe effect of neddylation inhibition on induction and severity ofGVHD. Recipient BALB/C animals were lethally irradiated with 8Gy on day21 and transplanted with 0.53 106 CD90.21 T cells and5 3 106 BM cells from either syngeneic BALB/C or allogeneic

2064 MATHEWSON et al BLOOD, 19 SEPTEMBER 2013 x VOLUME 122, NUMBER 12




MHC-mismatched C57BL/6 donors on day 0. The recipients alsoreceived either MLN4924 (20 mg/kg) or vehicle from day21 to day3 because host DCs typically cannot be recovered beyond this timefollowing allo-BMT. All syngeneic recipients survived with no signsof GVHD, demonstrating absence of nonspecific toxicity byMLN4924 (Figure 4A). The allogeneic recipients that received onlyvehicle showed signs of severe GVHD and greater mortality thansyngeneic animals (Figure 4A). By contrast, allogeneic recipientsreceiving MLN4924 exhibited significantly improved survival com-pared with vehicle control (Figure 4A), suggesting that in vivoblockade of neddylation mitigated GVHD.

Because systemic administration of MLN4924 is likely to impactnot just DCs but also several other cells, particularly donor T cellsthat are critical for GVHD,34 the reduction in GVHD mortality mightbe a reflection of its effects on these other cells in vivo. Therefore, inorder to evaluate the effects of neddylation inhibition only on the invivo function of BMDCs without the confounding effects on othertissues or expression of target antigens, we devised a model inwhich allogeneic CD41 T cells would respond only to MHC classII alloantigens on exogenously administered DCs in an acute GVHDmodel. MHC class II–deficient (Abb [H2-Ab1]) C57BL/6 mice(H2b)8 received 10 Gy total body irradiation and were injected

Figure 1. Neddylation inhibition attenuates LPS-induced TNF-a and IL-6 release and gene expression in BMDC. Protein analysis by western blot of (A, top panel)

neddylated Cullin proteins and (A, middle panel) Cul1 protein using whole cell lysate of BMDC stimulated with LPS in the presence or absence of MLN4924 or dexamethasone at the

indicated doses. Cells receiving treatment were preincubated with MLN4924 or dexamethasone for 2 hours followed by concurrent LPS stimulation (0.5 mg/mL) for 4 hours. a-tubulin

protein levels were analyzed as an equal loading control. Quantification via ELISA of cytokines TNF-a (B) and IL-6 (C) and qPCR analysis of TNF-a (D) and IL-6 (E) transcripts in

BMDC stimulated with LPS in the presence or absence of MLN4924 or dexamethasone at the indicated doses. Cells receiving treatment were preincubated with MLN4924 or

dexamethasone for 2 hours followed by concurrent LPS stimulation (0.5 mg/mL) for 4 hours. One representative experiment of 3 is shown. ***P , .0001.





with 13 107 BMDCs from syngeneic WT C57BL/6 (H2b) animalsthat were incubated overnight with either vehicle or MLN4924 in2 doses separated by 24 hours. We then injected 2 3 106 CD90.21

T cells from either syngeneic C57BL/6 or allogeneic bm12donors (see Materials and methods and supplemental Figure 2),which differ from the recipient animals by a single MHC class IIantigen. Analysis of donor T cells in the spleen at day 6 revealedfewer activated CD691 (Figure 4B) and differentiated Tbet1

(Figure 4C) CD41 T cells in animals that received DCs pretreatedwith MLN4924.

Blockade of neddylation attenuates the functions of

human DCs

To determine the clinical relevance of our observations, we deter-mined whether the effects of neddylation inhibition on DCs was alsogermane to DCs derived from healthy human PBMCs. Humanperipheral blood moDCs were harvested and cultured with andwithout MLN4924 and stimulated with LPS. The secretion of TNF-a and IL-6 by LPS from moDCs,35 were significantly reduced by

MLN4924 compared with vehicle control (Figure 4D). Similar to theeffect on murine DCs, the reduction of proinflammatory cytokineswas also significantly greater than dexamethasone (Figure 4D).

Next, we determined whether MLN4924 attenuated the ability ofhuman monocyte-derived DCs to stimulate allogeneic PBMCs.PBMCs and moDCs were obtained from 2 healthy donors andcultured in an MLR at 1:1 and 10:1 ratios in the presence or absenceof MLN4924. Following culture for 96 hours and 120 hours, asignificant decrease in proliferation of PBMCs was observed incultures incubated with MLN4924 (Figure 4E), similar to theresults observed in murine cells.

Inhibition of neddylation does not increase apoptosis or alter

the phenotype of DCs

Studies have shown that MLN4924 reduces the viability of cancercell lines.29 Therefore, we determined whether the observed effectsof neddylation inhibition through inactivation of Cul1 E3 ligase byMLN4924 was due to reduced BMDC viability. Treatment ofBMDCs with MLN4924 for 24 hours at the doses that reducedtheir function did not cause greater apoptosis and thus did not

Figure 2. Neddylation inhibition in BMDCmitigates release of non-TLR4–stimulated cytokines in vitro. ELISA quantification of TNF-a release in Pam3CSK4 (300 ng/mL)

stimulated (A), PGN (5 mg/mL) stimulated (B), and CD40L (1 mg/mL) stimulated (C) BMDC in the presence or absence of vehicle, MLN4924, or dexamethasone at the

indicated doses. (D) Comparison of TNF-a release via ELISA of cells treated with vehicle, MLN4924, dexamethasone, or bortezomib followed by concurrent stimulation with LPS

(0.5 mg/mL). Cells receiving treatment were preincubated with vehicle, MLN4924, dexamethasone, or bortezomib for 2 hours followed by concurrent stimulation for 4 hours. One

representative experiment of 3 is shown in cells treated in triplicate. *P , .05; **P , .01; ***P , .0001.





affect cell viability (supplemental Figure 1B). Next, we assessedwhether MLN4924 arrested the maturation of DCs as determinedby the expression of surface costimulatory molecules. To determinethis, we supplemented the culture media during BMDC generationwith vehicle or MLN4924 for the last 24 hours and then examined cellsurface expression of CD80, CD86, MHC II, PD-L1, and PD-L2 withflow cytometry. Addition of MLN4924 did not cause phenotypicchanges (supplemental Figure 1A). Taken together, these data suggestthat the reduction of DC function following treatment with MLN4924is not due to the result of decreased cell viability or phenotypicchange, but that these are intrinsic to inhibition of neddylation.

Inhibition of SAG reduces LPS-induced cytokine production

in DCs

The inhibition of LPS-induced cytokine production by MLN4924suggests that deneddylation is critical to this process by inactivation ofCRL-1 E3 ligase activity. To determine the specificity of the approachand to rule out any potential off-target effects of MLN4924, we usedsiRNA-mediated knockdown of RBX2/SAG, a critical and specificcomponent of the CRL-1–induced neddylation pathway. Next weperformed siRNA-mediated knockdown of RBX1 and RBX2/SAGalone and in combination in JAWSII cells. First, we confirmed theefficiency and specificity of knockdown of targeting SAG transcriptswith siRNA, as indicated by reduced levels of SAG (Figure 5A). Then

we compared TNF-a cytokine release following LPS stimulation inJAWSII cells transfected with the control-scrambled siRNA or SAG-specific siRNA. Upon LPS stimulation, JAWSII cells transfected withSAG siRNA exhibited a significant reduction in expression of TNF-acompared with the scrambled siRNA (Figure 5B). Interestingly,double knockdown of Rbx1 and SAG/Rbx2 had little or no effect oncytokine production, which could be because Rbx1 might antagonizeor cause feedback inhibition of SAG (Rbx2). Future studies willaddress this issue. Nonetheless, collectively, these data suggest thatinhibition of neddylation through inactivation of the CRL-1mediated either by siRNA knockdown of SAG or by the smallmolecule MLN4924 reduced proinflammatory cytokine releaseby DCs.

Inhibition of bTrCP but not Cul5 reduces LPS-induced cytokine

expression in DCs

Inhibition of neddylation via MLN4924 and molecular knockdown ofRbx2 results in global CRL inactivation. Therefore, to determine if theobserved results are specific to CRL-1, we silenced beta-transducinrepeat-containing protein (b-TrCP) as well as Cul5 using siRNA-mediated knockdown in JAWSII cells. First, we ensured efficientknockdown of both bTrCP (Figure 5C) and Cul5 (Figure 5D). Next,we examined the expression of TNF-a as a functional readout fromthe cells transfected with scramble, bTrCP, or Cul5 and subsequently

Figure 3. Neddylation inhibition mitigates allogeneic T-cell proliferation. BALB/C T cells were cultured with C57BL/6 irradiated (30 Gy) BMDC at 40:1 and 100:1 ratios in

the presence or absence of MLN4924 for 72 hours (A) and 96 hours (B). Where indicated, BMDC were preincubated in the presence or absence of vehicle or MLN4924 at

specified dosage for 6 hours. One representative experiment of 3 is shown of groups treated in triplicate. *P , .05; **P , .01; ***P , .0001.





stimulated with LPS. A significant decrease in TNF-a expression wasobserved in cells receiving knockdown of bTrCP, but not in cells withCul5 knockdown (Figure 5E). These data suggest that the functionof CRL-1 is critical for the underlying mechanism of immunesuppression following inhibition of neddylation.

MLN4924 inhibits translocation and activation of NF-kB

To determine if neddylation inhibition prevents transcriptional ac-tivity of LPS-induced NF-kB, we assessed p65 NF-kB translocationto the nucleus in BMDCs treated with MLN4924 or dexamethasonefollowing stimulation with LPS. As anticipated, cells cultured in

the presence of vehicle and stimulated with LPS for 30 minutes,p65 NF-kB was present in the nuclear fraction (Figure 6A, leftpanel). Cells pretreated with 500 nm dexamethasone and stimulatedwith LPS exhibited a similar level of p65 NF-kB in the nuclearfraction (Figure 6A, left, top panel). However, pretreating cellswith MLN4924 prior to LPS stimulation inhibited protein levelsof p65 NF-kB in the nuclear fraction of BMDCs (Figure 6A).Interestingly, these doses of MLN4924 and dexamethasoneinhibited both LPS-induced release and gene expression ofTNF-a and IL-6 (Figure 1B-E). These data indicate that LPS-induced nuclear accumulation of p65 NF-kB in BMDCs is blocked

Figure 4. Neddylation blockade regulates DC-mediated T-cell activation. (A) Survival of BALB/C animals lethally irradiated and transplanted with BM and CD90.21

T cells from either syngeneic BALB/C or allogeneic C57BL/6 donors. Following lethal irradiation (8 Gy) on day 1, MLN4924 (20 mg/kg) was administered in 5 daily

doses, day 1 to day 3 relative to BMT. Data are combined from 2 independent experiments (n 5 10 to 12 animals in the allogeneic groups). (B-C) Abb animals were lethally

irradiated (10 Gy) on day 1 and WT B6 BMDC (103 106) pretreated with vehicle or MLN4924 overnight and subsequently transferred in 2 doses separated by 24 hours (day 1

and day 0). CD90.21 T cells (23 106) from syngeneic WT-C57BL/6 or allogeneic bm12 animals were transferred on day 0. Following sacrifice on day 6, spleens were analyzed for

CD69 (B) and Tbet (C). (D) Quantification via ELISA of TNF-a (left) and IL-6 (right) released from human moDCs stimulated with LPS in the presence or absence of MLN4924 or

dexamethasone at the indicated doses. Cells receiving treatment were preincubated with vehicle, MLN4924, or dexamethasone for 2 hours followed by concurrent LPS stimulation

(0.5 mg/mL) for 4 hours. One representative experiment of 3 is shown of groups treated in triplicate. (E) Human PBMCs (1 3 105/well) and moDCs were obtained from 2 healthy

donors and cocultured at 1:1 and 10:1 ratios in an MLR in the presence or absence of MLN4924 for 96 and 120 hours.





by MLN4924 but not dexamethasone, suggesting that MLN4924 anddexamethasone have divergent molecular effects.

Next, in order to visualize and demonstrate the translocationof p65, we performed immunocytochemistry. BMDC culturedonly with vehicle exhibited a diffuse cytoplasmic localization ofNF-kB (Figure 6B, top row). By contrast, BMDCs stimulatedwith LPS for 1 hour and treated with vehicle showed prominentNF-kB accumulation in the nucleus (Figure 6B, middle row).However, LPS-stimulated cells treated with MLN4924 showeda dispersed cytoplasmic localization of NF-kB similar to unstimu-lated controls (Figure 6B, bottom row). These complementarymethods of examining NF-kB translocation showed prevention of

translocation of NF-kB to the nuclei in BMDCs treated withMLN4924.

Inhibition of neddylation prevents IkB degradation in BMDC

Previous studies have shown that IkB is a target of CRLs.36 Therefore,we examined the effect of MLN4924 on the degradation of IkB inBMDCs when stimulated with LPS. Similar to previous studies ofBMDCs37 and other APCs,38 stimulation with LPS resulted in thereduction of IkB protein levels within 30 minutes in vehicle-treatedBMDCs. Levels of IkB were slowly restored over a 6-hour period(Figure 7A, middle panel). The observed decrease in IkB following

Figure 5. siRNA-mediated neddylation inhibition inhibits LPS-induced TNF-a production in JAWSII cells. (A) qPCR analysis of SAG expression in JAWSII cells

transfected with indicated siRNA (3 mg). Scramble transfection performed as control. (B) qPCR analysis of TNF-a expression in JAWSII cells transfected with indicated siRNA

and stimulated with LPS (0.5 mg/mL). Scramble transfection performed as control. One representative experiment of 3 is shown. (C-E) JAWSII cells transfected with siRNA for

bTrCP, Cul5, or scramble as described in Materials and methods. Expression of bTrCP (C) and Cul5 (D) mRNA transcripts in JAWSII cells receiving indicated siRNA-

mediated knockdown. (E) Expression of TNF-a mRNA transcripts in cells transfected with indicated siRNA and cultured in the presence or absence of LPS (0.5 mg/mL)

for 4 hours. *P , .05; **P , .01; ***P , .0001.





LPS stimulation coincided with increased phosphorylation of IkB atserine-32 and serine-36 during the first 30 minutes of treatment. Theincreased p-IkB slowly returned to lower levels during the remaining5 hours of treatment (Figure 7A, top panel). Treatment of BMDCswith500 nM MLN4924 inhibited any LPS-mediated decrease in IkB(Figure 7B, middle panel), while levels of phosphorylated IkB in thesame cells were elevated within 15 minutes following LPS treatment(Figure 7B, top panel). Further, the levels of phosphorylated IkBremained elevated for the duration of the 6-hour treatment (Figure 7B).Following densitometric and statistical analyses, the decrease inIkB in vehicle-treated cells was significantly greater than inMLN4924-treated cells (Figure 7C, top panel). These data indicate

that inhibition of neddylation blocks the degradation of phosphor-ylated IkBSer32/Ser36 protein in BMDCs.

Neddylation blockade does not perturb LPS-induced activation

of MAPK/ERK pathway

Previous studies have shown that LPS stimulation of cells results inactivation of the MAPK/ERK pathway.37 Therefore, we examinedwhole cell lysates of BMDC stimulated with LPS cultured in thepresence of vehicle orMLN4924.Western blot analysis showed thatERK protein levels were similar in vehicle-treated cells (Figure 7D)andMLN4924-treated cells (Figure 7E). In addition, levels of pERK

Figure 6. Neddylation inhibition and dexamethasone differentially affect NF-kB translocation in BMDC. (A) Protein analysis by western blot (left) of p65 isoform of NF-kB

protein using nuclear (N) and cytosolic (C) fractions of BMDC in the presence or absence of 500 nm MLN4924 or 500 nm dexamethasone and stimulated concurrently with LPS

(0.5 mg/mL) for 30 minutes. Plot (right) shows densitometric and statistical analysis of p65 NF-kB presence in the nuclear fraction and cytosolic fraction. Lamin A/C and LDH proteins

were analyzed to demonstrate the presence of nuclear and cytosolic fractions, respectively. One representative experiment of 3 is shown. (B) Immunocytochemistry analysis of p65

NF-kB (column 3) localization in BMDC cultured in the presence or absence of MLN4924 and stimulated with LPS (0.5 mg/mL) for 1 hour. Cell nuclei were stained using DAPI

(column 1). Cytoplasmic actin was stained with phalloidin (column 2). Merged images of staining (column 4). One representative experiment of 3 is shown. **P , .01.





increased in a similar manner in both vehicle- andMLN4924-treatedcells (Figure 7D-E). These data suggest that MLN4924 treatmentspecifically reduced proinflammatory cytokines through regulationof the NF-kB pathway without affecting the MAPK/ERK pathway.

Discussion

PTMs modulate immunity.1 Proteins critical for an effective immuneresponse, such as NF-kB, are known to be phosphorylated andacetylated.39,40 However, the role that neddylation plays in regulating

immune cells, such as DCs, through the translocation of NF-kB isnot clear. Our results demonstrate a novel role for PTM neddylationin the regulation of DC-mediated immunity. The mechanism ofneddylation characterizes a novel molecular target for the inhibitionof secretion and transcription of certain LPS-induced proinflamma-tory cytokines in DCs. We show that following inhibition ofneddylation and stimulation via TLR and NLR, release and geneexpression of the proinflammatory cytokines TNF-a and IL-6 aregreatly mitigated (Figures 1B-E and 2).

Studies have shown the ability of MLN4924 to suppress thegrowth of tumor cell lines as well as primary human acute myeloid

Figure 7. Neddylation inhibition prevents IkB degradation in BMDC without perturbation of the MAPK/ERK pathway. Protein analysis via western blot of

phosphorylated IkBa protein (A-B, top panels) and total IkBa (A-B, middle panels) using whole cell lysate of BMDC stimulated with LPS (0.5 mg/mL) for 6 hours in the

presence of vehicle (A) or 500 nm MLN4924 (B). Densitometric and statistical analysis of total IkB (C, top) or pIkB (C, bottom) of vehicle or MLN4924-treated cells. a-tubulin

protein levels were analyzed as an equal loading control. One representative experiment of 3 is shown. Protein analysis via western blot of phosphorylated ERK protein (D-E,

top panels) and total ERK (D-E, middle panels) using whole cell lysate of BMDC stimulated with LPS for 6 hours in the presence of vehicle (D) or 500 nm MLN4924 (E).

Densitometric analysis of vehicle-treated cells (D, right) or 500 nm MLN4924-treated cells (E, right) of phosphorylated ERK and total ERK protein. a-tubulin protein levels were

analyzed as an equal loading control. One representative experiment of 3 is shown. *P , .05.





leukemia cells.19,22,30 Inhibition of the NEDD8 pathway byMLN4924 has been reported to result in apoptosis.26,41 However,our data suggest that MLN4924 did not affect BMDC viability(supplemental Figure 1B), demonstrating that the reduction in theproduction of proinflammatory cytokines was not due to loss of cellsbut was a functional consequence of neddylation inhibition. Ourdata also suggest that treatment with MLN4924 had a significantlygreater impact on suppression of the release of proinflammatorycytokines than dexamethasone or bortezomib (Figures 1 and 2) inthe nanomolar dose range. Moreover, inhibition of neddylation withMLN4924 reduced DC functions in vivo and attenuated GVHD(Figure 4A-C), suggesting that this pathway could be targeted tomitigate in vivo inflammatory diseases.

Mechanistic studies have uncovered a distinct molecular pathwayfor MLN4924-mediated suppression of DC functions when com-pared with dexamethasone. The primary molecular target of dexa-methasone is the glucocorticoid receptor.42 Binding of steroid resultsin activation of the receptor and subsequent translocation to thenucleus43 and is known to interact with transcription factors such asNF-kB and AP-1.44 These interactions repress the expression ofgenes that code a number of cytokines that play key roles in theimmune and inflammatory systems. By contrast, MLN4924 speci-fically targets NAE, a heterodimer of NAE1 and UBA3 subunits.26

By doing so, MLN4924 inactivates CRLs and impedes theubiquitination rate of the subset of proteins whose degradation isdependent on CRLs.26

Although dexamethasone significantly inhibited secretion ofTNF-a and IL-6, which are known to be regulated by NF-kB,16 itdid not directly prevent the translocation of NF-kB to the nucleusafter LPS stimulation (Figure 6A), in contrast to MLN4924. Theability of MLN4924 to directly prevent the degradation of IkB inLPS-stimulated cells (Figure 7B) suggests a specific and distinctmechanism from that of dexamethasone. The role of inhibitingneddylation, byMLN4924, in suppressing the release of TNF-a andIL-6 through the inactivation of CRL-1 causing IkBa accumulationand subsequent prevention of NF-kB activation, is further supportedby molecular inactivation of CRL-1 by the siRNA knockdownof SAG and bTrCP (Figure 5). Together, these data suggest thatMLN4924 and dexamethasone block LPS-induced cytokine releasethrough distinct mechanisms.

The results of our study are in agreement with previous reports incell lines demonstrating an effect on NF-kB activation.45 For thefirst time, we extend those observations in primary cells, DCs, andtheir functional immune responses. More importantly, we show asimilar effect on primary human DCs. Furthermore, we uncover themolecular mechanism that is dependent on IkB degradation butindependent of the MAPK/ERK pathway (Figure 7) and show that itis germane to both canonical and noncanonical NF-kB signaling-mediated release of proinflammatory cytokines. Other studies haveshown that degradation of IkB and release of proinflammatorycytokines can be altered by proteasome inhibitors.46 However, these

proteasome inhibitors also activate the transcription factor AP-1,which is known to contribute to LPS-induced cytokines.37 Ourresults show that upon treatment of LPS-stimulated BMDC withMLN4924, the MAPK/ERK pathway is unperturbed (Figure 7D-E),suggesting that AP-1 activity is unaltered by neddylation inhibition.This is likely due to the degradation of IL-1 receptor-associatedkinase 1 through its association with the E3 ligases TNF receptor–associated factor 6 and Pellino.47 Previous studies have shown thatPellino, not CRL, is a likely candidate to serve as the degradationscaffold for IL-1 receptor-associated kinase 1.48 Thus, inhibitionof the neddylation pathway may be a more specific approach ofinhibiting the degradation of IkB and thereby blocking the increasein proinflammatory cytokines as compared with the effects seen byproteasome inhibitors.

Promising preclinical results led to the advancement of MLN4924to phase 1 clinical trials for both nonhematological and hematologicalcancers.49,50 However, MLN4924’s immune-modulatory effectshave heretofore been largely unrecognized. Our results suggestthat at noncytotoxic doses, drugs such as MLN4924 that speci-fically target neddylation might provide novel therapeutic strate-gies for diseases associated with deregulated immune responsessuch as GVHD, chronic inflammatory disease, shock, autoimmu-nity, and allotransplantation.9-12,29

Acknowledgments

This work was supported by National Institutes of Health grants(National Heart, Lung and Blood Institute, HL-090775; NationalCancer Institute, CA143379; and National Institute of Allergyand Infectious Diseases, AI-075284).

Authorship

Contribution: N.M. designed and performed experiments, analyzeddata, and wrote the paper; T.T. designed and performed experimentsand analyzed data; S.K., Y.S., K.O.-W., H.T., Y.W. and G.H.performed experiments; Y.S. analyzed data, contributed reagents,and assisted with writing the paper; and P.R. designed experiments,analyzed data, and wrote the paper.

Conflict-of-interest disclosure: The authors declare no compet-ing financial interests.

Correspondence: Pavan Reddy, Department of Internal Medi-cine, Division of Hematology and Oncology, Blood and MarrowTransplantation Program, University of Michigan ComprehensiveCancer Center, 3312 CCGC, 1500 E Medical Center Dr, AnnArbor, MI, 48105-1942; e-mail: [email protected].

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online July 17, 2013 originally publisheddoi:10.1182/blood-2013-02-486373

2013 122: 2062-2073

Hiroya Tamaki, Ying Wang, Guoqing Hou, Yi Sun and Pavan ReddyNathan Mathewson, Tomomi Toubai, Steven Kapeles, Yaping Sun, Katherine Oravecz-Wilson, human dendritic cell functionNeddylation plays an important role in the regulation of murine and

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Neddylation plays an important role in the regulation of murine and human dendritic cell function