viral cell death inhibitor mc159 enhances innate immunity against

JOURNAL OF VIROLOGY, Oct. 2010, p. 10467–10476 Vol. 84, No. 200022-538X/10/$12.00 doi:10.1128/JVI.00983-10Copyright © 2010, American Society for Microbiology. All Rights Reserved.

Viral Cell Death Inhibitor MC159 Enhances Innate Immunityagainst Vaccinia Virus Infection�

Sreerupa Challa,1,2 Melissa Woelfel,1 Melissa Guildford,1David Moquin,1,2 and Francis Ka-Ming Chan1,2,3*

Department of Pathology,1 Immunology and Virology Program,2 and Diabetes and Endocrinology Research Center,3 University ofMassachusetts Medical School, 55 Lake Avenue North, Worcester, Massachusetts 01655

Received 5 May 2010/Accepted 27 July 2010

Viral inhibitors of host programmed cell death (PCD) are widely believed to promote viral replication bypreventing or delaying host cell death. Viral FLIPs (Fas-linked ICE-like protease [FLICE; caspase-8]-likeinhibitor proteins) are potent inhibitors of death receptor-induced apoptosis and programmed necrosis.Surprisingly, transgenic expression of the viral FLIP MC159 from molluscum contagiosum virus (MCV) inmice enhanced rather than inhibited the innate immune control of vaccinia virus (VV) replication. This effectof MC159 was specifically manifested in peripheral tissues such as the visceral fat pad, but not in the spleen.VV-infected MC159 transgenic mice mounted an enhanced innate inflammatory reaction characterized byincreased expression of the chemokine CCL-2/MCP-1 and infiltration of �� T cells into peripheral tissues.Radiation chimeras revealed that MC159 expression in the parenchyma, but not in the hematopoietic com-partment, is responsible for the enhanced innate inflammatory responses. The increased inflammation inperipheral tissues was not due to resistance of lymphocytes to cell death. Rather, we found that MC159facilitated Toll-like receptor 4 (TLR4)- and tumor necrosis factor (TNF)-induced NF-�B activation. Theincreased NF-�B responses were mediated in part through increased binding of RIP1 to TNFRSF1A-associatedvia death domain (TRADD), two crucial signal adaptors for NF-�B activation. These results show that MC159is a dual-function immune modulator that regulates host cell death as well as NF-�B responses by innateimmune signaling receptors.

Successful immunity against pathogenic challenges is centralto the survival of all organisms. Metazoans employ a wide arrayof innate and adaptive immune responses to control variouspathogens. In response, pathogens have developed variousstrategies to evade detection and elimination by the immunesystem. Programmed cell death (PCD) plays an important rolein host defense against pathogens by directly eliminating in-fected cells to limit the viral factory. A role for host cell deathin antiviral responses is highlighted by the identification of viralinhibitors of apoptosis (3). In addition to apoptosis, nonapop-totic PCD pathways, such as necrosis and autophagy haverecently been shown to participate in host defense againstpathogens (31, 44). For instance, we recently showed that ge-netic ablation of an essential necrosis mediator, RIP3, resultedin severely impaired innate immune responses against vacciniavirus (VV) infection characterized by the lack of virus-inducedtissue necrosis and inflammation (11). In addition, certainvFLIPs (viral Fas-linked ICE-like protease [FLICE; caspase-8]-like inhibitor proteins) are potent inhibitors of programmednecrosis (6, 8). These results indicate that host PCD machin-eries play important roles in controlling the viral factory anddissemination of the virus within the infected host.

Despite the widely accepted view that inhibition of host celldeath is an important viral immune evasion strategy, relativelyfew in vivo studies have been performed to directly test this

hypothesis. This is due partly to the lack of suitable animalmodels in which specific components of host apoptotic machin-ery are inhibited. For instance, germ line inactivation of manyof the components of the PCD machinery, such as Fas-associ-ated via death domain (FADD) and caspase-8, resulted inembryonic lethality (50, 55, 57), thus preventing in vivo virusinfection studies from using these animal models. Anotherapproach that was widely used was transgenic expression ofviral apoptosis inhibitors, such as poxvirus CrmA, baculovirusp35, and vFLIPs. However, since expression of these inhibitorswas restricted mostly to the lymphoid compartment (26, 28, 34,46, 51, 54, 58), they do not permit evaluation of the role of hostcell death in the parenchyma in antiviral responses. Cell deathin the stromal compartment could impact the innate inflam-matory reaction, cross-priming of antigens, and viral dissemi-nation to other tissues. Because cells in the parenchyma are theprimary targets for many virus infections, it is important todetermine the contribution of cell death in the parenchyma inantiviral responses.

The vFLIPs were first identified as inhibitors of caspase-dependent apoptosis. They share homology with caspase-8 andcaspase-10 in the tandem death effector domains (DEDs) atthe amino termini. However, vFLIPs lack the caspase enzymedomain at the carboxyl termini. Thus, binding of vFLIPs toFADD and caspase-8/-10 via DED-mediated homotypic inter-action led to inhibition of FasL-, tumor necrosis factor (TNF)-,and TNF-related apoptosis-inducing ligand (TRAIL)-inducedapoptosis (18, 19). Importantly, certain vFLIPs, includingMC159 and E8, are also potent inhibitors of programmednecrosis induced by TNF-like death cytokines (8). These re-

* Corresponding author. Mailing address: Room S2-125, Depart-ment of Pathology, University of Massachusetts Medical School,Worcester, MA 01655. Phone: (508) 856-1664. Fax: (508) 856-1665.E-mail: [email protected].

� Published ahead of print on 11 August 2010.

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sults suggest that viral inhibitors could inhibit multiple hostPCD pathways to avoid elimination by the host immune sys-tem.

In order to determine the effect of vFLIPs on host responsesagainst viral infections, we generated transgenic mice express-ing vFLIP MC159 under the control of the ubiquitous H2-Kb

promoter (53). We previously showed that transgenic expres-sion of MC159 did not alter lymphocyte functions and devel-opment, but rather caused a mild form of lymphoproliferationthat resembled that seen with the lpr mice with Fas/CD95/Apo-1 mutations (53). Here, we show that MC159 transgenicmice exhibited enhanced innate immune responses to VV in-fections, which led to enhanced viral clearance in peripheraltissues. Surprisingly, the enhanced control of VV productionwas not due to enhanced lymphocyte survival. Rather, VV-induced expression of the chemokine CCL-2/MCP-1 washighly elevated in MC159 transgenic mice and was accompa-nied by enhanced recruitment of �� T cells to peripheral tis-sues. MC159 promotes the binding between TRADD andRIP1, two crucial signal adaptors for NF-�B activation. Con-sequently, MC159 transgenic fibroblasts exhibited enhancedNF-�B activation to TNF and Toll-like receptor 4 (TLR4)stimulation. These results reveal a previously unappreciatedeffect of MC159 on NF-�B activation and indicate that viralcell death inhibitors could impact innate immune responsesthrough mechanisms beyond cell death regulation.

MATERIALS AND METHODS

Mice and virus. The MC159 transgenic mice have been described (53). For allexperiments, unless otherwise stated, mice between 12 to 14 weeks of age wereused. All experiments were performed according to protocols approved by theUniversity of Massachusetts Medical School animal care and use committee. ForVV infections, one million PFU of the Western Reserve (WR) strain were usedto infect mice via the intraperitoneal route. Viral titers were determined byplaque assays using Vero cells and crystal violet staining as described before (8).Briefly, the whole organ/tissue was ground up in 1 ml of RPMI 1640 medium.Viral titers were determined by serial dilutions of the tissue supernatants. Theplaque counts obtained were used to determine the viral load for the whole organby using the formula N � Y/(vx), where N is the final titer per ml, Y is the numberof plaques at the dilution used, v is the volume plated in ml, and x is the dilutionfactor. For lipopolysaccharide (LPS)-induced septic shock, mice were injectedwith 50 �g/kg of LPS (Sigma) and 1,000 mg/kg of D-galactosamine (Sigma) viathe intraperitoneal route. Sera and liver tissues were collected 5 h after LPSadministration for alanine aminotransferase (ALT) and caspase assays. Thenumber of apoptotic nuclei was scored in a double-blind manner by averaging thenumber of condensed nuclei in six different fields. For bone marrow chimeras, 4-to 5-week-old mice were irradiated (950 rads) and reconstituted with 5 � 106

bone marrow cells depleted of B and T cells. Four months later, the chimeraswere challenged with VV as described above and analyzed for lymphocyte infil-tration 24 h postinfection and viral titers 4 days postinfection.

Caspase assay. One hundred micrograms of liver cell extracts were diluted in200 �l of iTFB buffer (10% sucrose, 30 mM HEPES [pH 7.4], 10 mM CaCl2 and5 mM dithiothreitol [DTT]) containing 25 �M N-acetyl-Asp-Glu-Val-Asp-7-amino-4-methylcoumarin (Ac-DEVD-AMC) (Biomol). The release of fluores-cent AMC was measured with a Fluostar multiwell plate reader at 340 nm forexcitation and 450 nm for emission over 3 h. The rate of conversion was calcu-lated by including 25 �M free AMC as the control.

Bone marrow-derived dendritic cell (BMDC), macrophage, and T-cell isola-tion. For fat pad lymphocyte isolation, visceral fat pads were incubated in theenzyme digestion buffer (0.5 mg/ml collagenase type II [Sigma], 100 units/ml typeI DNase [Sigma], and 10% fetal calf serum in Hank’s buffered saline solution[Invitrogen]) for 45 min at 37°C. For isolation of splenic dendritic cells (DCs),spleens were incubated at 37°C for 30 min in 2 mg/ml collagenase D (Roche)dissolved in 10 mM HEPES (pH 7.4), 150 mM NaCl, 5 mM KCl, 1 mM MgCl2,and 1.8 mM CaCl2. For terminal deoxynucleotidyltransferase-mediated dUTP-biotin nick end labeling (TUNEL) staining, lymphocytes were incubated in a

37°C CO2 incubator for 1 h prior to staining. For annexin V staining, lymphocyteswere incubated in a 37°C CO2 incubator for 5 h prior to staining. For annexin Vstaining of splenic DCs, cells were incubated at 37°C for 60 min before staining.

Intracellular cytokine staining. Lymphocytes were stimulated with 10 �g ofthe VV-specific peptide B8R, K3L, or A47L (32) in a 37°C CO2 incubator for 5 h.For intracellular cytokine staining, cells were fixed using a BD cytofix/cytopermkit (BD Pharmingen) per the manufacturer’s instructions. Staining antibodieswere obtained from BD Pharmingen or eBioscience. Peptides were synthesizedby EZBiolab.

MEFs, BMDCs, and macrophages. Mouse embryonic fibroblasts (MEFs) weregenerated from day 12 to 14 embryos. BMDCs were obtained by culturing bonemarrow cells in 10 ng/ml granulocyte-macrophage colony-stimulating factor(GM-CSF) and 5 ng/ml interleukin-4 (IL-4) for 7 days (43). Bone marrow-derived macrophages (BMMs) were obtained by culturing bone marrow cells inL929-conditioned medium for 7 days (15). Nonadherent cells were carefullytransferred to a new culture dish, rested for 24 h, and stimulated with 0.1 �g/mlLPS (for BMDCs and BMMs). Total-cell extracts were prepared by lysis inNP-40 lysis buffer (150 mM NaCl, 10 mM Tris [pH 7.5], 1% Nonidet P-40 and 1�Complete protease inhibitor cocktail [Roche]). Cell extracts were resolved with4 to 12% NuPAGE gels (Invitrogen), blotted onto nitrocellulose membranes,and probed with antibodies to phosphorylated I�B� (p-I�B�), I�B� (Cell Sig-naling), or �-actin (BD Pharmingen). The antibody against E3L was a kind giftfrom J. W. Yewdell (56). For electrophoretic mobility shift assays (EMSA),nuclear extracts binding to 32P-labeled oligonucleotides were performed as de-scribed previously (7). Oligonucleotide sequences used were 5-AGTTGAGGGGACTTTCCCAGGC-3 and 5-GCCTGGGAAAGTCCCCTCAACT-3 (forNF-�B) and 5-GAGGTGGGTGGAGTTTCGCG-3 and 5-CGCGAAACTCCACCCACCTC-3 (for the GT box).

Transfections and reporter assays. Transfections with 293T cells were per-formed using Fugene 6 transfection reagent (Roche) per the manufacturer’sinstructions. For measuring NF-�B activation, cells were transfected with theplasmids pNF-�B-luc (Stratagene) and pTK-�gal (Promega). Luciferase activitywas normalized to �-galactosidase activity by using kits from Promega. Forfluorescence-activated cell sorter (FACS) analysis, a cyan fluorescent protein(CFP) reporter was amplified by PCR to replace the luciferase gene in thepNF-�B-luc vector. For immunoprecipitations, cells were lysed in 150 mM NaCl,20 mM Tris (pH 7.5), and 1% NP-40 supplemented with protease inhibitorcocktails (Roche). Antibodies against TRADD and hemagglutinin (HA) werepurchased from Millipore and Covance, respectively.

Quantitative PCR analyses. Total RNA was isolated from visceral fat pads byusing an RNeasy kit from Qiagen. cDNA was generated using an Omniscriptreverse transcription (RT) kit (Stratagene). Real-time quantitative PCR wasperformed using SYBR green (Bio-Rad). Signals were normalized to 18S RNA.Primer sequences used were as follows: CCL-2, 5-TGCTACTCATTCACCAGCAA-3 and 5-GTCTGGACCCATTCCTTCTT-3; IL-6, 5-CGGAGAGGAGACTTCACAGA-3 and 5-CCAGTTTGGTAGCATCCATC-3; and 18S RNA,5-TGGTGGAGGGATTTGTCTGG-3 and 5-TCAATCTCGGGTGGCTGAAC-3.

Statistical analyses. Statistically analyses were performed using unpaired Stu-dent’s t tests. One-way analysis of variance was used for Fig. 6B to D.

RESULTS

MC159 expression conferred protection against LPS-in-duced cell injury. We have previously shown that transgenicexpression of the viral cell death inhibitor MC159 under theubiquitously expressed H2-Kb promoter results in protectionof lymphocytes from cell death induced by TNF-like cytokines(53). Since MC159 was also expressed in nonhematopoietictissues (53), we asked whether parenchymal tissues in theMC159 transgenic mice were also protected from cytokine-induced PCD. To this end, we used an LPS-induced sepsismodel, because TNF plays a central role in liver cell injury inthis model (16, 37, 41). Transgenic expression of MC159 pro-tected hepatocytes from LPS-induced liver injury as deter-mined by a reduced serum ALT level (Fig. 1A) and reducedcaspase-3 activity in liver cell extracts (Fig. 1B) compared withresults for wild-type littermates. Moreover, apoptosis markedby nuclear condensation was completely absent in the MC159

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transgenic hepatocytes (Fig. 1C, compare panels c and d, andFig. 1D). However, LPS-induced mortality in MC159 trans-genic mice was comparable to that of their wild-type counter-parts (Fig. 1E). This is consistent with the requirement forother cytokines, including IL-1�, in LPS-induced systemic or-gan failure (52). These results show that transgenic expressionof MC159 inhibited death cytokine-induced PCD in the liverbut did not inhibit signaling by other cytokines.

MC159 transgenic mice exhibited enhanced control of VVinfection. Having established that MC159 expression protectedcells in the parenchyma against death cytokine-induced PCD,we examined whether antiviral responses are affected in theMC159 transgenic mice. We recently showed that TNF-medi-ated programmed necrosis plays a crucial role in the innateimmune protection against VV infections (11). Since MC159 isa potent inhibitor of death receptor-induced apoptosis and

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FIG. 1. MC159 protects against LPS-induced liver injury. (A) Serum ALT level or (B) caspase-3 activity was determined 5 h after LPS plusD-galactosamine treatment. (C) Hematoxylin and eosin staining of liver sections of wild-type and MC159 transgenic mice treated with phosphate-buffered saline (PBS) or LPS for 5 h. Note that the extensive apoptotic nuclei in the wild-type hepatocytes (indicated by the arrows) were absentin the LPS-treated transgenic liver. Pictures shown are representative of images taken from two experiments with three different mice each. 200�magnification. (D) Decreased apoptosis in LPS-treated transgenic liver. The number of condensed apoptotic nuclei was counted in a double-blindmanner. Each circle represents the average number of apoptotic nuclei from six different fields from one mouse. The results shown arerepresentative of those of two experiments with at least three mice in each group. (E) Kaplan-Meier survival curve of LPS-treated wild-type andtransgenic mice. WT, wild type; Tg, transgenic.

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programmed necrosis (8), we expected that MC159 expressionwould result in defective control of VV production. Surpris-ingly, viral titers of the visceral fat pads (Fig. 2A) and livers(Fig. 2B) of transgenic mice were significantly reduced com-pared with those of wild-type mice. The reduction in virusproduction in the transgenic mice was observed 4 days postin-fection, prior to the onset of acute CD8 T-cell responses.Importantly, virus production in the spleen was unaffected inthe transgenic mice (Fig. 2C), indicating that the enhancedcontrol of viral production by the MC159 transgene was re-stricted to nonlymphoid tissues. The reduction in viral titerswas not due to lack of productive infection, since the VVantigen E3L was readily detected in the fat pads (Fig. 2D, lanes1 to 6), livers (Fig. 2D, lanes 7 to 10), and spleens (Fig. 2D,lanes 11 to 14) of wild-type and transgenic mice 24 h postin-fection. Furthermore, reduction of viral titers in the transgenicfat pad was also observed 5 days postinfection (Fig. 2E). Thus,MC159 transgenic mice exhibited enhanced control of VVinfection.

Increased �� T-cell infiltration in MC159 transgenic miceduring VV infection. The reduction in viral replication by 4days postinfection suggests that innate immune responsesagainst VV were enhanced in the MC159 transgenic mice.Indeed, within 24 h postinfection, the infected livers of trans-genic mice exhibited a conspicuous increase in the number ofinflammatory foci compared with that of wild-type littermatecontrols (Fig. 3A, compare panels b and d with panels c and e).A similar increase in inflammation was detected in the visceralfat pads of the transgenic mice (Fig. 3B, compare panels b andd with panels c and e). The chemokine CCL-2/MCP-1 andinflammatory cytokine IL-6 were highly upregulated in the

visceral fat pads of VV-infected mice (Fig. 3C and D). Strik-ingly, the expression of the chemokine CCL-2/MCP-1 in thetransgenic mice further increased about 3-fold compared withthat in wild-type mice (Fig. 3C). In contrast, IL-6 productionwas comparable in wild-type and MC159 transgenic mouse fatpads (Fig. 3D). CCL-2/MCP-1 is crucial for the recruitment ofinnate immune effectors, including �� T cells (13, 36). A flowcytometric analysis shows that a higher percentage of CD3 Tcells was detected in the visceral fat pads of MC159 transgenicmice (Fig. 3E, compare panel a [4.23% in wild-type mice] withpanel b [9.9% in transgenic mice]). Importantly, 65% of thetransgenic CD3 T cells recovered from the visceral fat padsexpressed the �� T-cell receptor (TCR) (Fig. 3F, panel b). Thisis in contrast to CD3 lymphocytes isolated from the fat padsof wild-type mice, which only contained 27.8% of �� T cells(Fig. 3F, panel a). These results indicate that �� T-cell infil-tration accounts for the enhanced inflammation detected inthe peripheral tissues of the transgenic mice. The increased ��T-cell infiltration correlated with increased killing of virus-infected cells, since cell death in the transgenic liver as mea-sured by the serum ALT level (Fig. 3G) and by caspase-3activity (Fig. 3H) was elevated compared with that in wild-typecontrols. These results are consistent with the crucial role of ��T cells in the innate immune control of VV replication (45).

Since MC159 protects cells from the cytotoxic effects ofTNF-like death cytokines, we next examined whether the in-creased �� T-cell infiltration in peripheral tissues was attrib-uted to their enhanced survival. Surprisingly, �� T cells isolatedfrom the fat pads of wild-type and transgenic mice exhibitedsimilar levels of TUNEL staining when they were analyzedafter 1 h of incubation ex vivo (Fig. 3I, compare panels a and

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FIG. 2. Enhanced clearance of VV in the peripheral tissues of MC159 transgenic mice. Wild-type and MC159 transgenic mice were intra-peritoneally infected with 1 � 106 PFU of WR VV. Viral titers per the whole tissue or organ were determined 4 days postinfection for the visceralfat pad (A), liver (B), and spleen (C) using a Vero cell plaque assay as described previously (8). The titers shown for different tissues weredetermined from the same group of mice. (D) Expression of the VV antigen E3L in infected tissues. Fat pads (lanes 1 to 6), livers (lanes 7 to 10),and spleens (lanes 11 to 14) were harvested from wild-type or transgenic mice 24 h postinfection. Tissue lysates were analyzed by Western blottingfor expression of E3L, green fluorescent protein (GFP; for MC159-GFP), and �-actin as indicated. Uninfected tissues were included as controlsas indicated. (E) Viral titers for wild-type transgenic mice infected with VV were determined 5 days after infection as in panels A to C.



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b). The low level of TUNEL staining is likely due to theefficient clearance of dying cells by professional phagocytes (1).To further evaluate if there were any differences in cell deathsin the transgenic lymphocytes, we incubated lymphocytes iso-

lated from the fat pads of infected mice at 37°C for 5 h to allowthe expression of the apoptotic marker annexin V in cells thatwere committed to the death program (1). Under these con-ditions, lymphocyte cell death levels were still indistinguishable

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FIG. 3. Enhanced �� T-cell infiltration in VV-infected MC159 transgenic mice. Hematoxylin and eosin staining of livers (A) and visceral fatpads (B) of uninfected wild-type control mice (a), VV-infected wild-type mice (b and d), or VV-infected transgenic mice (c and e). Tissues wereharvested 24 h after infection. Panels a to c, 40� magnification; panels d and e, 200� magnification of the boxed area in panels b and c. The blackarrows indicate the inflammatory cells. Images taken were representative of results of two experiments, each with four animals in each group.(C) Elevated expression of the chemokine CCL2 in the transgenic fat pad. The induction of CCL2 in VV-infected wild-type and transgenic fat padswas determined by quantitative PCR using 18S RNA as the internal control. Fold induction was determined by comparing the expression ininfected mice with that in uninfected mice. (D) Transgenic and wild-type mice exhibited similar induction of IL-6 upon VV infection. IL-6expression in visceral fat pads was determined as in panel C. (E and F) Enhanced �� T-cell infiltration in the visceral fat pads of MC159 transgenicmice upon VV infection. Fat pad lymphocytes were isolated from wild-type (a) or transgenic (b) littermates 24 h after VV infection. (E) VV-infected transgenic fat pad lymphocytes were enriched in CD3 T cells. Fat pad lymphocytes were harvested by enzyme digestion and stained withCD3 and Ly6G/C. The numbers represent the percentages of cells in the respective quadrants. (F) Increased �� T cells in VV-infected transgenicfat pad lymphocytes. CD3 cells were gated and analyzed for the expression of �� TCR. The numbers represent the percentages of �� TCR Tcells. (G and H) Increased cell death in the livers of VV-infected MC159 transgenic mice. (G) The serum ALT level was determined 3 dayspostinfection. (H) Increased caspase-3 activation in VV-infected MC159 transgenic mice. Liver cell extracts were harvested 24 h postinfection andanalyzed for active caspase-3 using a fluorogenic substrate assay as described in Materials and Methods. (I and J) Fat pad lymphocytes from MC159transgenic mice exhibited normal cell death markers during VV infection. (I) Fat pad lymphocytes were harvested 24 h after VV infection,incubated for 1 h at 37°C, and stained with CD3, �� TCR, and TUNEL. The percentages of TUNEL-positive CD3 �� TCR cells are indicated.(J) Fat pad lymphocytes were harvested as for panel I and incubated at 37°C for 5 h prior to staining with CD3 and annexin V. The percentagesof cells that were annexin V positive are indicated. FACS plots and histology were representative of those of at three experiments with threeanimals in each experiment.



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between wild-type and transgenic mice (Fig. 3J). Since MC159inhibits only extrinsic PCD pathways, the lack of a differencebetween TUNEL and annexin V staining suggests that theintrinsic PCD pathway might contribute to lymphocyte celldeath during VV infection. Taken together, these results sug-gest that the enhanced inflammation and control of viral rep-lication in the MC159 transgenic mice were caused by in-creased �� T-cell infiltration into the peripheral tissues ratherthan enhanced lymphocyte survival.

MC159 expression enhanced TNF- and TLR4-inducedNF-�B activation. NF-�B is a proinflammatory transcriptionfactor that plays crucial roles in innate inflammatory responses.Since inhibition of cell death does not appear to contribute tothe enhanced inflammation observed for the MC159 trans-genic mice, we explored whether MC159 might promote innateinflammatory responses through NF-�B. TNF induced a bi-phasic NF-�B activation with an early wave of signal withinminutes followed by a more sustained, second wave of activa-tion hours after TNF stimulation (10). This biphasic pattern ofNF-�B activation was revealed by I�B� phosphorylation andI�B� degradation in wild-type and transgenic primary mouseembryonic fibroblasts (MEFs) (Fig. 4A). Although earlyNF-�B activation in wild-type MEFs was similar to that intransgenic MEFs, transgenic MEFs exhibited a more sustainedI�B� phosphorylation and a higher level of I�B� expressionlate during stimulation (Fig. 4A, compare lanes 7 to 9 andlanes 16 to 18). Since I�B� is a transcriptional target of NF-�B(27), the higher I�B� expression level and sustained I�B�phosphorylation indicate that transgenic MEFs exhibitedstronger NF-�B activation.

Enhanced NF-�B activation in the form of sustained I�B�phosphorylation was also observed when transgenic MEFs

were stimulated with the TLR4 agonist LPS (Fig. 4B, comparelanes 2 to 5 with lanes 7 and 8). However, unlike TNF, TLR4stimulation causes production of cytokines, including TNF,which causes further I�B� degradation and contributed to thereduction in I�B� expression in the LPS-treated transgenicMEFs (Fig. 4B, lanes 6 to 10). A similar reduction in I�B�expression was observed with LPS-treated bone marrow-de-rived dendritic cells (BMDCs) (Fig. 4C, compare lanes 7 and 8with lanes 15 and 16). Enhanced NF-�B activation in responseto LPS stimulation in transgenic bone marrow-derived macro-phages (BMMs) was further revealed by EMSA. As has beenreported previously (15), untreated BMMs often exhibitedstrong basal NF-�B DNA binding activity (Fig. 4D). Whilewild-type BMMs exhibited little to no NF-�B DNA bindingactivity 8 h after LPS treatment, strong nuclear NF-�B DNAbinding activity was detected with transgenic BMMs (Fig. 4D,compare lanes 4 and 9). These results show that in addition toinhibiting host cell death, MC159 could modulate host immuneresponses by promoting NF-�B activation.

MC159 regulates NF-�B induction via RIP1 in a concentra-tion-dependent manner. We next sought to understand themechanism by which MC159 promotes NF-�B activation. Tothis end, we first validated the NF-�B-promoting effect ofMC159 by transient expression of MC159 in Jurkat cells usingan NF-�B-driven CFP reporter. Using this system, we foundthat MC159 expression alone was sufficient to induce a modestamount of NF-�B activity (Fig. 5A, compare panels a and c).Consistent with the observations for transgenic MEFs,BMDCs, and BMMs, MC159 significantly increased the TNF-induced NF-�B activity in Jurkat cells (Fig. 5A, compare pan-els b and d). The adaptor proteins TRADD (9, 17, 38) andRIP1 (14, 23, 30) are crucial mediators for NF-�B activation

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FIG. 4. MC159 promotes NF-�B activation in parenchymal and hematopoietic cells. (A) Wild-type and transgenic MEFs were stimulated withrecombinant mouse TNF for the indicated times. I�B� phosphorylation and degradation were analyzed by Western blotting. �-Actin was used asthe internal control. (B) MEFs were stimulated with 100 pg/ml of LPS. I�B� phosphorylation and degradation were analyzed as in panel A.(C) BMDCs were stimulated with 100 ng/ml LPS for the indicated times. I�B� degradation was monitored by Western blotting. �-Actin is shownas the control. (D) BMMs were stimulated with LPS for the indicated times. An EMSA was performed with nuclear extracts isolated from wild-typeor transgenic BMMs. Results are representative of those of two experiments. NS, nonspecific band.



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for multiple receptors, including TNF receptor 1 (TNFR-1)and TLR4. We therefore tested whether MC159 might en-hance NF-�B activation through TRADD and RIP1. Strik-ingly, we found that MC159 significantly increased the bindingof RIP1 to TRADD (Fig. 5B, compare lanes 6 and 7). Incontrast, the binding of TRADD to another TNFR-1 signaladaptor, TNFR-associated factor 2 (TRAF2), was not affectedby the presence of MC159 (Fig. 5B). These results are consis-tent with a model in which MC159 facilitates NF-�B activationby enhancing RIP1 recruitment to TRADD and the TNFR-1complex.

Our results differ from those in an earlier report that showsthat MC159 could inhibit TNF-induced NF-�B activation (33).The discrepant results could be attributed to the differentexpression levels of MC159 achieved in this early report. Totest whether the expression level of MC159 might influence itseffect on NF-�B activation, we coexpressed RIP1 with increas-ing doses of MC159 in HEK 293T cells. Similar to the resultsfor Jurkat cells (Fig. 5A), expression of MC159 alone wassufficient to induce a low level of NF-�B-driven luciferaseactivity (Fig. 5C, compare lanes 1 and 2). We found that lowdoses of MC159 synergized with RIP1 to promote NF-�Bactivation (Fig. 5C, compare lanes 3 and 4). However, at higherdoses of expression, MC159 suppressed NF-�B induction (Fig.5C, lanes 5 to 8). Thus, we conclude that the expression levelof MC159 determines whether it enhances or inhibits NF-�Bactivation.

The enhanced antiviral responses in MC159 transgenicmice were due to expression of MC159 in the parenchyma.Since cells of both hematopoietic and nonhematopoietic ori-gins exhibited increased NF-�B activation, we created radia-tion chimeras to determine the contribution of these compart-ments to the enhanced anti-VV responses observed withMC159 transgenic mice. Successful reconstitution of the he-matopoietic compartment of irradiated wild-type mice withtransgenic bone marrow and the reciprocal chimeras was con-firmed by flow cytometry (Fig. 6A and data not shown). Inter-estingly, transgenic hosts, but not wild-type hosts, reconstitutedwith transgenic bone marrow exhibited enhanced control ofvirus production in the visceral fat pads (Fig. 6B). Moreover,transgenic hosts reconstituted with wild-type bone marrow ex-hibited control of virus production that was similar in effec-tiveness to that of hosts that received transgenic bone marrow(Fig. 6B), suggesting that expression of MC159 in the stromalparenchyma, but not in the hematopoietic compartment, is

HA-TRADDHA-RIP1

HA-TRAF2HA-MC159

----

+---

-+--

--+-

---+

+++-

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TRAF2

TRADD

MC159

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TRAF2

TRADD

MC159

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B

C

1.67% 9.59%

36%8.6%

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orM

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9Untreated TNF

CFP (NF-kB)

a b

c d

MC159

RIP1MC159

--

-+

+-

+ + ++ +

10

20

30

RLU

0

MC159

RIP1

WB

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FIG. 5. MC159 promotes NF-�B induction in a dose-dependentmanner by facilitating the binding between RIP1 and TRADD.(A) MC159 enhances NF-�B activation in Jurkat cells. Jurkat cellswere transfected with an NF-�B responsive CFP reporter and either anempty GFP vector (a and b) or MC159-GFP (c and d). Sixteen hourslater, cells were stimulated with 10 ng/ml human recombinant TNF for8 h. CFP expression as an indicator of NF-�B activity was analyzed inthe transfected GFP population by flow cytometry. The numbersrepresent the percentages of CFP cells in the GFP populations.(B) MC159 enhances RIP1 binding to the TNFR-1 adaptor TRADD.HEK 293T cells were transfected with the indicated HA-tagged plas-mids. TRADD immune complexes were isolated (top panel) withTRADD-specific antibody (immunoprecipitation [IP]). The presenceof RIP1, TRAF2, and MC159 was examined by Western blotting (WB)

with anti-HA antibody. The bottom panel shows the expression of theproteins in whole-cell extracts (WCE). (C) MC159 enhances RIP1-dependent NF-�B activation in a dose-dependent manner. HEK 293Tcells were transfected with NF-�B-driven luciferase reporter, a consti-tutive active �-galactosidase reporter, a RIP1 expression plasmid, andincreasing amounts of MC159 expression plasmid (lanes 4 to 8, 50 ng,100 ng, 200 ng, 500 ng, and 800 ng, respectively). Luciferase activitywas normalized to �-galactosidase activity and is shown as relative lightunits (RLU). The averages and standard errors of the means (SEM)from experiments performed in triplicate are shown. The bottom pan-els show the Western blot results for expression of RIP1 and MC159in the corresponding samples. Results are representative of those ofthree experiments.



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crucial for the enhanced innate immune control of VV infec-tions. Furthermore, the enhanced �� T-cell recruitment to thevisceral fat pad also tracks with expression of MC159 in thenonhematopoietic parenchyma (Fig. 6C and D). Taken to-gether, these results show that although MC159 enhancedTNF- and TLR4-induced NF-�B activation in hematopoieticand stromal cells, its expression in the parenchyma was neces-sary and sufficient to drive the enhanced innate inflammatoryresponses against VV infections.

DISCUSSION

In this report, we show that transgenic mice expressing theviral PCD inhibitor MC159 exhibited enhanced control of VVproduction in peripheral tissues such as the visceral fat pad andliver, but not in the spleen. The enhanced clearance of VVcorrelated with increased cell death in the liver and wasmarked by the increased expression of the chemokine CCL-2/MCP-1. This is accompanied by an early infiltration of �� Tcells, which is consistent with the role of �� T cells in the innateimmune control of VV replication (5, 45, 47). The differentialprotection by MC159 in peripheral tissues might reflect therequirement to recruit innate immune effector cells to periph-eral sites. In contrast, the abundance of immune effectorsmight mitigate the requirement for chemokine-driven recruit-ment of �� T cells to the infected spleen. Although MC159 isa potent inhibitor of death cytokine-induced apoptosis andprogrammed necrosis (4, 8, 24, 48), perforin/granzyme-medi-ated cell death could bypass inhibition by MC159 to eliminate

virus-infected cells in the transgenic mice. This notion is sup-ported by the normal responses of MC159 transgenic mice tolymphocytic choriomeningitis virus (LCMV) (53), whose clear-ance requires the coordinated function of perforin/granzymeand Fas (40). Perforin/granzyme-mediated target cell killingmight also explain why MC159 transgenic hepatocytes wereprotected from LPS-induced liver injury mediated by TNF butremained sensitive to cell-mediated cytotoxicity during VVinfections.

MC159 is known predominantly as an inhibitor of deathcytokine-induced apoptosis and programmed necrosis (8).However, MC159 was unable to inhibit apoptosis induced bystaurosporine (49) and by overexpression of caspase-8/FLICE(24), which bypass the death receptors. Similarly, MC159transgenic lymphocytes exhibited normal cell death markers,such as annexin V and TUNEL, during VV infection. Theseresults are reminiscent of the lack of inhibition of CD8 T-celldeath by pan-caspase inhibitors during VV infection (35). Al-though we cannot rule out the possibility that the transgeniclymphocytes could still undergo programmed necrosis, our re-sults do indicate that the enhanced �� T-cell infiltration andinnate immune control in MC159 transgenic mice during VVinfection could not be attributed to inhibition of apoptosis.Since our previous work indicates that lymphocyte activationto various stimuli, including that during LCMV infection, wasnormal in MC159 transgenic mice (53), lymphocyte-intrinsiceffects are unlikely to account for the enhanced clearance ofVV in the MC159 transgenic mice.

How might MC159 expression in the parenchyma facilitate

FIG. 6. Expression of MC159 in the parenchyma, but not in the hematopoietic compartment, contributes to the enhanced �� T-cell infiltrationand control of viral replication. (A) Flow cytometric analysis of splenocytes isolated from radiation chimeras. Representative results from aTg3wild-type chimera shows that �84% of lymphocytes were of the donor origin after reconstitution. (B) Radiation chimeras were generated asdescribed in Materials and Methods. Four months after reconstitution with the indicated bone marrow cells, the mice were challenged with VV.Viral titers for the whole visceral fat pads were determined 3.5 days postinfection. (C) Radiation chimeras were created by transferring transgenicor wild-type bone marrow into wild-type recipient mice. Four months after bone marrow reconstitution, the chimeras and age-matched transgenicmice were challenged with VV. Fat pad lymphocytes were harvested 24 h postinfection and analyzed for the percentage of �� TCR cells withinthe CD3 population by flow cytometry. (D) Transgenic mice reconstituted with wild-type bone marrow cells and age-matched transgenic micewere analyzed for �� T-cell infiltration to the visceral fat pads by flow cytometry 24 h post-VV infection. Results are representative of those of twoexperiments.



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innate immune responses? Our results indicate that transgenicfibroblasts exhibited heightened NF-�B activation in responseto TNF and TLR4 stimulation. MC159 appears to specificallyaffect late-phase NF-�B activation while having little effect onearly NF-�B induction. Interestingly, CCL-2 is a transcrip-tional target of NF-�B (21), and TNF and TLR4 signaling havebeen shown to play crucial roles in the innate immune defenseagainst VV (8, 11, 25). Thus, increased NF-�B signaling toTNF or TLR4 stimulation could explain the increased CCL-2expression and inflammatory leukocyte infiltration in the trans-genic fat pad in response to VV infection. Although enhancedNF-�B activation was also observed for cells of hematopoieticorigin (e.g., BMDCs and BMMs), radiation chimeras indicatethat enhanced NF-�B activation in hematopoietic cells had aminimal contribution to the enhanced innate immunity againstVV infections. Collectively, these results demonstrate an im-portant role for signaling by cells in the parenchyma in innateimmune responses to pathogens.

It is noteworthy that another vFLIP, K13 from human her-pesvirus 8 (HHV-8), has also been reported to enhance NF-�Bactivation through an unknown mechanism (12, 29). Interest-ingly, we found that MC159 enhanced the binding betweenRIP1 and TRADD, two essential adaptors for NF-�B activa-tion, by multiple TLRs and TNF receptors (9, 17, 38). Wepropose that enhanced interaction between RIP1 and TRADDmight underlie the mechanism by which MC159 promotesNF-�B signaling by innate immune receptors. However, thepositive effect of MC159 on NF-�B activation was observedonly when it is expressed at a low level. When expressed athigher levels, MC159 inhibited NF-�B activation. The inhibi-tory effect of MC159 at higher expression levels was reminis-cent of several previous reports in which MC159 impairedTNF- and PKR-induced NF-�B activation (20, 33), double-stranded RNA (dsRNA)-mediated interferon regulatory factor7 (IRF7) signaling (2), and T-cell activation (54). The molec-ular mechanism by which MC159 inhibits NF-�B activation isunclear at the moment. Based on our results, we propose thatat low levels of expression, MC159 promotes RIP1 binding tothe TRADD. However, as its expression level increases,MC159 might bind to and inhibit other cellular targets thatlead to inhibition of NF-�B and other immune functions.

TNF-TNF receptor signaling is particularly important forthe innate immune control of poxvirus infections, since TNFR-1�/� and TNFR-2�/� mice were highly susceptible to VV andectromelia virus infections (8, 39, 42). In contrast, molluscumcontagiosum virus (MCV) is readily controlled in immunocom-petent individuals but causes persistent lesions in immunocom-promised individuals, such as those infected with HIV (22).Due to the ability of MC159 to promote or inhibit immunefunctions, it is tempting to speculate that MC159 might coop-erate with other MCV-encoded immunoregulatory moleculesto differentially modulate host responses in healthy and immu-nocompromised individuals.

ACKNOWLEDGMENTS

We thank R. Welsh and L. Selin for critical readings of the manu-script and T. McQuade for help with the scoring of histology sections.

F.K.C. is a member of the UMass DERC (DK32520). D.M. issupported by a NIH predoctoral training grant (T32 AI07349). Thiswork was supported by NIH grants AI083497, AI042845, AI017672,and AI042845. Core resources supported by the Diabetes Endocrinol-

ogy Research Center Grant DK32520 were also used. F.K.C. was arecipient of investigator awards from the Smith Family Foundationand the Cancer Research Institute.

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