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Molecule-Dependent and -Independent through MyD88 AdaptorMacrophages by Japanese Encephalitis Virus Functional Modulation of Dendritic Cells and

Koanhoi Kim and Seong Kug EoRahman, Seon Ju Kim, Sang Bae Han, Byung Sam Kim, Abi G. Aleyas, Junu A. George, Young Woo Han, M. M.

http://www.jimmunol.org/content/183/4/2462doi: 10.4049/jimmunol.0801952July 2009;

2009; 183:2462-2474; Prepublished online 27J Immunol 

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Print ISSN: 0022-1767 Online ISSN: 1550-6606. Immunologists, Inc. All rights reserved.Copyright © 2009 by The American Association of1451 Rockville Pike, Suite 650, Rockville, MD 20852The American Association of Immunologists, Inc.,

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Functional Modulation of Dendritic Cells and Macrophages byJapanese Encephalitis Virus through MyD88 AdaptorMolecule-Dependent and -Independent Pathways1

Abi G. Aleyas,* Junu A. George,2* Young Woo Han,2* M. M. Rahman,* Seon Ju Kim,*Sang Bae Han,† Byung Sam Kim,‡ Koanhoi Kim,§ and Seong Kug Eo3*

Dendritic cells (DCs) are potent initiators of T cell-mediated immunity that undergo maturation during viral infections. However,few reports describing the interactions of DCs with Japanese encephalitis virus (JEV), which remains the most frequent cause ofacute and epidemic viral encephalitis, are available. In this study, we investigated the interaction of JEV with DCs and macro-phages. JEV replicated its viral RNA in both cells with different efficiency, and JEV infection of macrophages followed the classicalactivation pathway of up-regulation of tested costimulatory molecules and proinflammatory cytokine production (IL-6, TNF-�,and IL-12). On the contrary, JEV-infected DCs failed to up-regulate costimulatory molecules such as CD40 and MHC class II. Ofmore interest, along with production of proinflammatory cytokines, DCs infected by JEV released antiinflammatory cytokineIL-10, which was not detected in macrophages. Moreover, signaling through MyD88 molecule, a pan-adaptor molecule of TLRs,and p38 MAPK in JEV-infected DCs was found to play a role in the production of cytokines and subversion of primary CD4�

and CD8� T cell responses. We also found that IL-10 released from JEV-infected DCs led to a reduction in the priming of CD8�

T cells, but not CD4� T cells. Taken together, our data suggest that JEV induces functional impairment of DCs through MyD88-dependent and -independent pathways, which subsequently leads to poor CD4� and CD8� T cell responses, resulting in boostingviral survival and dissemination in the body. The Journal of Immunology, 2009, 183: 2462–2474.

M acrophages and dendritic cells (DCs)4 are major play-ers in early immune responses to many viruses (1–4).Both cell types produce several cytokines, including

TNF-� and IL-6, in response to viral infection (5). Additionally,both cell types serve as APCs, and DCs, in particular, function invivo as potent APCs and play crucial roles in the enhancement andregulation of cell-mediated immune reactions (1–4). Since DCsexpress various costimulatory and adhesion molecules, they canefficiently activate naive T cells in primary responses. Upon en-counters with pathogens, immature DCs undergo maturation pro-cesses that are characterized by the production of proinflammatory

cytokines (TNF-�, IL-12, and IL-6), up-regulation of costimula-tory molecules (CD40, CD80, and CD86), alteration of chemokinereceptors (CCR2, CCR5, and CCR7), and enhanced Ag presenta-tion (6, 7). Because these events are crucial in the development ofoptimal antiviral responses, many viruses target these events toprevent the development of antiviral immunity and boost viralsurvival.

The recognition mechanisms that initiate and control innate re-actions to viruses remain poorly understood. Accumulating evi-dence suggests that membrane-bound cell-surface or intracellularTLRs form part of the surveillance system (8). Indeed, several of13 recognized mammalian TLRs are known to be involved in therecognition of viral components (8). Additionally, cytosolic non-TLR dsRNA sensors, including protein kinase R, melanoma dif-ferentiation-associated gene 5 (MDA5), and retinoic acid-induc-ible gene I (RIG-I), have been found to play roles in DC activation(9–12). All TLRs, with the exception of TLR3, use MyD88 as themain adaptor molecule to activate the downstream signaling path-way. Activation of this pathway, in turn, leads to the activation ofNF-�B and MAPKs, such as stress-activated protein kinase/JNKand p38, which subsequently leads to the production of inflamma-tory cytokines (13, 14). Furthermore, the MyD88-independent or-dependent pathway results in production of IFN-�/� in responseto stimulation of TLR3, TLR4, TLR7, TLR8, and TLR9 (15, 16).TLR3 and TLR4 can signal via Toll/IL-1R domain-containingadaptor-inducing IFN-� (TRIF), which induces IFN-� gene tran-scription partly through IFN regulatory factor 3 (IRF3) activation(17). TLR7, TLR8, and TLR9 activate some IFN-� genes throughformation of the MyD88-TNFR-associated factor 6 (TRAF6)-IFNregulatory factor 7 (IRF7) complex (18, 19). However, NF-�B andMAPK are also activated by the MyD88-independent pathway.The point of intersection between the MyD88-dependent and -in-dependent pathways is thought to be TRAF6 (20, 21).

*Laboratory of Microbiology, College of Veterinary Medicine and Bio-Safety Re-search Institute, Chonbuk National University, Jeonju, Republic of Korea; †College ofPharmacy, Chungbuk National University, Cheonju, Republic of Korea; ‡Immuno-modulation Research Center, University of Ulsan, Ulsan, Republic of Korea; and§Department of Pharmacology, School of Medicine, Pusan National University,Busan, Republic of Korea

Received for publication June 16, 2008. Accepted for publication June 14, 2009.

The costs of publication of this article were defrayed in part by the payment of pagecharges. This article must therefore be hereby marked advertisement in accordancewith 18 U.S.C. Section 1734 solely to indicate this fact.1 This work was supported by Grant RTI05-03-02 from the Regional TechnologyInnovation Program of the Ministry of Commerce, Industry, and Energy (MOCIE), aresearch grant from the Bio-Safety Research Institute, Chonbuk National University,and by the Brain Korea 21 Project in 2008, Republic of Korea.2 J.A.G. and Y.W.H. contributed equally to this work.3 Address correspondence and reprint requests to Dr. Seong Kug Eo, Laboratory ofMicrobiology, College of Veterinary Medicine and Bio-Safety Research Institute,Chonbuk National University, Jeonju City 561-756, Republic of Korea. E-mail ad-dress: vetvirus@chonbuk.ac.kr4 Abbreviations used in this paper: DC, dendritic cell; bmDC, bone marrow-derivedDC; bmM�, bone marrow-derived macrophage; gB, glycoprotein B; JEV, Japaneseencephalitis virus; MFI, mean fluorescence intensity; NS1, nonstructural protein 1;p.i., postinfection; TCID50, 50% tissue culture-infective dose.

Copyright © 2009 by The American Association of Immunologists, Inc. 0022-1767/09/$2.00

The Journal of Immunology

www.jimmunol.org/cgi/doi/10.4049/jimmunol.0801952

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Japanese encephalitis virus (JEV), which is a mosquito-bornemember of the genus Flavivirus, such as West Nile and dengueviruses, is responsible for most acute and epidemic cases of viralencephalitis (22, 23). Approximately 60% of the world populationinhabits JEV endemic areas, and the virus is continuing to spreadto previously unaffected regions due to global warming (24–26). Itis estimated that 30,000–50,000 cases of JEV occur each year,resulting in 10,000–15,000 deaths, although this number may beunderestimated (22, 23). Additionally, 30–60% of surviving pa-tients suffer from serious long-term neuropsychiatric sequelae (27,28). However, the pathogenesis of JEV-associated disease in hu-mans and in mice has not been completely elucidated. In manycases, the virus is not directly involved in the destruction of braintissue, but instead causes indirect damage through cell-mediatedimmune responses (29). Activated inflammatory cells secrete cy-tokines such as IL-1 and TNF-�, which can cause apoptosis ofneuronal cells. Similarly, JEV infection leads to the production ofhigh levels of cytokines such as macrophage-derived chemokinefactor, TNF-�, and IL-8 in the serum and cerebrospinal fluid (30–33). Increased levels of such cytokines may play protective rolesagainst infection or initiate irreversible immune responses. JEVmultiplies in macrophages and DCs of the periphery, which causesinitial viremia before entry into the CNS (34). Therefore, moststudies that have been conducted to evaluate the pathogenesis ofJEV infection have examined the interaction of the virus with mac-rophages and CNS cells (30–33). These CNS cells include micro-glia and astrocytes, which are major contributors to the productionof inflammatory cytokines and CNS degeneration.

However, there is presently little information describing the in-teraction between JEV and DCs, which play a major role in im-mune responses. Therefore, in this study we have compared theinteraction of JEV with macrophages and DCs. Our data demon-strate that JEV infection induces differing modulations of cytokineproduction and phenotypes in both DCs and macrophages. We alsodefined certain roles of the MyD88 adaptor molecule in the cyto-kine production by DCs and macrophages and in the initiation ofprimary CD4� and CD8� T cell responses. Additionally, wegained insight into the contribution of IL-10 to primary immuneresponses following JEV infection. We suggest that imbalancedactivation and modulation of macrophages and DCs in JEV infec-tion are critical events that determine the immunopathological out-comes in the CNS and inadequate immune responses.

Materials and MethodsAnimals

C57BL/6 mice (H-2b), 5 to 6 wk old, were purchased from Koatech. OT-Iand OT-II mice, which are transgenic for the V�2/V�5 TCR that recognizesthe H-2Kb-restricted peptide (OVA257–264, SIINFEKL) and the I-Ab-re-stricted peptide (OVA323–339, ISQAVHAAHAEINEAGR) of chickenOVA, were obtained from The Jackson Laboratory. MyD88-deficient mice(H-2b) were a gift from the Immunoregulatory Research Center (IRC),Ulsan, Korea. The investigators adhered to the guidelines set by the Com-mittee on the Care of Laboratory Animal Resources, Chonbuk NationalUniversity. The animal facility of the Chonbuk National University is fullyaccredited by the National Association of Laboratory Animal Care.

Cells and viruses

JEV Beijing-1 strain was obtained from the Green Cross Research Institute(Suwon, Korea) and propagated by suckling mice brain passage. The titerof the virus in clarified brain lysates (HBSS-10% BSA) was determined bya cytopathic assay using Vero cells (CCL81; American Type Culture Col-lection). Similarly, brain lysates from healthy mice were prepared and usedto inoculate a control group of mock-infected mice. HSV-1 strain 17 wasgrown in Vero cells using DMEM supplemented with 2% FBS, penicillin(100 U/ml), and streptomycin (100 U/ml). Virus stocks were concentratedby centrifugation at 50,000 � g, titrated by a plaque assay, and then storedin aliquots at �80°C until needed.

Abs and peptides

The following mAbs were obtained from eBioscience or BD Biosciencesfor flow cytometric analysis and other experiments: FITC-anti-CD40 (3/23), CD80 (16-10A1), CD86 (GL1), MHC class II (25-9-17), MHC classI (28-14.8), PE-anti-CD4 (GK1.5), CD8� (53-6.7), CD11b (M1/70), andCD11c (N418). Anti-mouse IL-10 Ab (JES5-2A5) was used to neutralizethe IL-10 activity. The mAb against the nonstructural protein 1 (NS1) ofJEV was obtained from Abcam. The defined peptides of chicken OVA,OVA257–264 (SIINFEKL) and OVA323–339 (ISQAVHAAHAEINEAGR),and immunodominant peptide (glycoprotein B (gB)498–505, SSIEFARL) ofHSV-1 gB were chemically synthesized at Peptron. JEV-specific primersfor the detection of viral RNA (JEV10,564–10,583 forward, 5�-CCC TCAGAA CCG TCT CGG AA-3� and JEV10,862–10,886 reverse, 5�-CTA TTCCCA GGT GTC AAT ATG CTG T-3�) (35) were synthesized at Bioneer.

Preparation of bone marrow-derived DCs and macrophages

DCs derived from bone marrow cells (bmDCs) were prepared as previ-ously described (36) with some modifications. Briefly, bone marrow cellsfrom femurs and tibiae were cultured in RPMI 1640 supplemented with 2ng/ml GM-CSF and 10 ng/ml IL-4. On days 5 and 8 the culture was re-plenished with 5 ml of fresh media containing cytokines. Cells were har-vested on day 10 for use and then characterized by flow cytometrc analysis,which revealed that the culture generally consisted of �75% CD11c� cells(25% CD11c�CD11b� and 65% CD11c�CD8��). Bone marrow-derivedmacrophages (bmM�) were prepared by culturing bone marrow cells inDMEM containing 30% conditioned culture media of L929 cells (37).bmM� was harvested by using trypsin digestion following a 7-day incu-bation. The prepared bmM� was composed of �85% F4/80� cells thatconsisted of 99.2% F4/80�CD11b� and �1% F4/80�CD11c� cells.

Immunohistochemistry

bmDCs and bmM� were fixed with 3% ice-cold formaldehyde 48 h afterJEV infection and then blocked with PBS containing 10% healthy mouseserum for 1 h at 4°C. Following quenching by endogenous peroxidase with0.2% H2O2 in methanol, infected cells were stained by overnight incuba-tion at 4°C with a HRP-conjugated mAb against JEV E protein (38). Afterrinsing with PBS the color was developed with 0.3% H2O2 and 3,3�-dia-minobenzidine tetrahydrochloride. JEV E protein expression in infectedcells was then checked and captured using a light microscope equippedwith digital imaging equipment.

Quantitative SYBR Green-based real-time PCR for viralreplication

Relative levels of viral RNA in JEV-infected cells or the spleens of micewere determined by conducting quantitative real-time PCR analysis on aMini Opticon system (Bio-Rad Laboratories) using a DyNAmo SYBRGreen qPCR kit (Finnzymes) following reverse transcription of total RNAisolated from infected samples. The reaction mixture contained 2 �l oftemplate cDNA, 10 �l of 2� Master Mix, 1.5 mM MgCl2, and 100 nMprimers at a final volume of 20 �l. The reactions were denatured at 95°Cfor 10 min and then subjected to 50 cycles of 95°C for 30 s, 58°C for 30 s,and 72°C for 30 s. After the reaction cycle was completed the temperaturewas increased from 65°C to 95°C at a rate of 1°C/min, and the fluorescencewas measured every 15 s to construct a melting curve. A control samplethat contained no template DNA was run with each assay, and all deter-minations were performed at least in duplicate to ensure reproducibility.The authenticity of the amplified product was determined by melting curveanalysis. The relative ratio of viral RNA in the infected samples to unin-fected samples was determined. All data were analyzed using the OpticonMonitor version 3.1 analysis software (MJ Research).

Cytokine ELISA/ELISPOT

Sandwich ELISA was used to determine the levels of cytokines in theculture supernatants. The ELISA plates were coated with IL-2 (JES6-1A12), IL-4 (11B11), IL-6 (MP5-20F3), IL-10 (JES5-16E3), IL-12p70(C18.2), IFN-� (R4-6A2), and TNF-� (1F3F3D4) anti-mouse Abs pur-chased from eBioscience and BD Bioscience, and then incubated overnightat 4°C. The plates were washed three times with PBS containing 0.05%Tween 20, after which they were blocked with 3% nonfat-dried milk for 2 hat 37°C. The culture supernatant and standards for recombinant cytokineproteins (PeproTech) were added to the plates and incubated for 2 h at37°C. The plates were then washed again and the biotinylated IL-2 (JES6-5H4), IL-4 (BVD6-24G2), IL-6 (MP5-32C11), IL-10 (JES5-2A5), IL-12(C17.8), IFN-� (XMG1.2), and TNF-� (polyclonal Ab) Abs were added.Next, the mixtures were incubated overnight at 4°C followed by washing

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and subsequent incubation with peroxidase-conjugated streptavidin(eBioscience) for 1 h. Color development was then performed by the ad-dition of a substrate (ABTS) solution. Cytokine concentrations were de-termined with an automated ELISA reader and SoftMax Pro4.3, accordingto comparisons with two concentrations of standard cytokine proteins.

The IFN-�-producing CD4� T cells in response to HSV-1 Ag was enu-merated by ELISPOT (39). Briefly, CD4� T cells purified from HSV-immunized mice were stimulated with HSV Ag-pulsed APCs on IFN-�capture Ab-coated 96-well ELISPOT plates (Millipore). After 72 h of in-cubation, the plates were washed and biotinylated IFN-� Ab was added andincubated for 2 h. After washing, streptavidin-alkaline phosphatase wasadded and the mixture was incubated for an additional 30 min. Spotswere visualized by adding a 5-bromo-4-chloro-3-indolyl phosphate/tet-ra-NBT substrate solution (Promega) and counted 24 h later using astereomicroscope.

Intracellular cytokine staining and flow cytometric analysis

Conventional surface staining was used for flow cytometric analysis.Briefly, cells were suspended with PBS containing 1% BSA and 0.05%NaN3 at a concentration of 2 � 106 cells, followed by incubation at 4°C for30 min with properly diluted mAbs. After staining, the cells were washedtwice by spinning at 1200 rpm, 4°C for 5 min. To detect Ag-specific CD8�

T cells, intracellular cytokine staining was performed following an 8-hstimulation of synthetic peptide in the presence of 2 �M monensin (Sigma-Aldrich). For the intracellular IFN-� staining, cells were stained for surfacemarker, washed, permeabilized, and stained with PE-conjugated anti-IFN-�. Following fixation, cells were resuspended in PBS and then ana-lyzed using FACSCalibur equipped with the CellQuest program (BD Bio-sciences) and WinMDI 2.8 software.

Proliferation of Ag-specific CD4� and CD8� T cells

The proliferation of CD4� and CD8� T cells was assessed by measuringthe viable cell ATP bioluminescence (40). Briefly, Ag-specific CD4� andCD8� T cells were purified from OT-II and OT-I mice, respectively, usinga MACS LS column (Miltenyi Biotec) according to the manufacturer’sinstructions. The purified CD4� and CD8� T cells (5 � 105 cells/ml) werethen cultured together with stimulator cells at different ratios. JEV-infectedDCs were used as stimulator cells following pulsing with cognate antigenicpeptides (OVA323–339 peptide for CD4� T cells, and OVA257–264 for CD8�

T cells). Anti-mouse IL-10 mAb (10 �g/ml) was incorporated in someexperiments to neutralize the IL-10 bioactivity. The culture was incubatedfor 3 days at 37°C in a humidified 5% CO2 incubator. Replicate cultureswere transferred to V-bottom 96-well culture trays that were subsequentlycentrifuged to collect the cells. The proliferated cells were then evaluatedusing a Vialight cell proliferation assay kit (Cambrex Bio Science) accord-ing to the manufacturer’s instructions.

In vivo CTL killing assay

An in vivo CTL assay was conducted as reported elsewhere (41). Briefly,syngeneic splenocytes of naive mice were pulsed with gB498–505 peptide(SSIEFARL, 1 �M) of HSV-1 and then labeled with CFSE (2.5 �M). Tocontrol for Ag specificity, peptide-unpulsed syngeneic splenocytes werelabeled with a lower concentration of CFSE (0.25 �M). A 1:1 mixture ofeach target cell population was then injected i.v. into mice for evaluation.Splenocytes were then collected from recipient mice 24 h after adoptivetransfer of target cells and analyzed by flow cytometry. Each populationwas distinguished by its respective fluorescence intensity. The percentagekilling of target cells in immunized animals was calculated using the fol-lowing equation: ratio � (percentage CFSElow/percentage CFSEhigh). Per-centage specific lysis was determined as: [1 � (ratio of naive/ratio ofimmunized)]/100.

Zosteriform infection of herpes simplex virus

A zosteriform challenge experiment was performed as described by Gi-erynska et al. (42). Briefly, the left flank area was depilated before chal-lenge using a combination of hair clipping and a depilatory chemical. Theanimals were then anesthetized with Avertin (2,2,2-tribromoethanol) and2-methyl-2-butanol (Sigma-Aldrich), and a total of five scarifications weremade on an �4-cm2 area of the left flank region. A total dose of 10 �l ofHSV-1 strain 17 (containing 1 � 106 PFU) were then applied to the scar-ifications, after which the area was gently massaged. The animals wereinspected daily for the development of zosteriform ipsilateral lesions, gen-eral behavioral changes, encephalitis, and mortality.

Statistical analysis

Where specified, the data were analyzed for statistical significance using aStudent’s t test. A p value of �0.05 was considered significant. Kaplan-Meier curves were also generated for mice that survived the zosteriformchallenge with HSV-1. The p values were then computed using the �2

method. The survival rates of the two groups were considered to be sig-nificantly different if the two-sided p value was �0.05.

ResultsDCs and macrophages are permissible for viral replicationof JEV

JEV can multiply in murine macrophages without causing cyto-pathic changes (43). However, few reports regarding interaction ofDCs with JEV are available so far. To examine the interaction ofDCs and macrophages with JEV, bmDCs and bmM� were mor-phologically observed following JEV infection. As shown in Fig.1A, bmDCs infected with JEV showed morphological changes,including rounding and detachment from the culture surface at48 h postinfection (p.i.). Conversely, there was no apparent changein JEV-infected bmM�. When immunohistochemical staining ofthe infected cells was conducted to identify the biosynthetic ex-pression of viral proteins, both bmDCs and bmM� were found tobe permissible for the expression of viral E protein (Fig. 1B). Tofurther identify the interaction of both bmDCs and bmM� withJEV, the titers of infectious progeny viruses in the culture super-natants of both cell types were determined daily by cytopathicassays using Vero cells. bmM� induced the productive release ofinfectious progeny viruses with levels that peaked at 4–5 days p.i.,while the productive release of infectious virus in bmDCs was notdetected in cytopathic assays (Fig. 1C). However, quantitative re-al-time PCR indicated that JEV viral RNA was capable of repli-cating in both cells, even though the viral RNA levels of bmDCswere markedly lower than those of bmM� (Fig. 1D). To evaluatethe ratio of infected bmDCs and bmM�, we determined the per-centage of JEV-infected bmDCs and bmM� using mAb againstJEV NS1 protein, which is largely retained within infected cellsand involved in RNA replication (44). As shown in Fig. 1E, �50%of bmM� were infected with JEV, but fewer bmDCs were foundto be infected. Furthermore, to examine in vivo replication of JEV,we determined viral RNA load in splenocytes of JEV-infectedmice. The viral RNA load was found to peak at 7 days p.i., afterwhich it declined (Fig. 1F). Additionally, splenic DCs (CD11chigh

cells) and macrophages (F4/80high cells) were observed to be in-fected with JEV, as confirmed by intra- and extracellular stainingwith NS1 Ab (Fig. 1G). The expression of NS1 protein peaked at3 days p.i., and splenic F4/80high macrophages showed more ef-fective expression of NS1 protein than did splenic CD11chigh DCs.However, the difference of JEV NS1 expression in CD11chigh DCsubsets (CD11chighCD8�� and CD11chighCD8��) was not ob-served (data not shown). Taken together, theses results indicatethat both DCs and macrophages can be infected with JEV, but thatthere are differences in permissiveness of viral replication.

The profiles of cytokine production in DCs and macrophagesinfected with JEV

DCs and macrophages play an important role in primary defensesby generating and regulating adaptive immunity (1–4). Virusesmust evolve ways to modulate DC and macrophage function thatenable them to evade detection and elimination by hosts. We ex-amined the pattern of pro- and antiinflammatory cytokines pro-duced by JEV-infected bmDCs and bmM� to investigate JEVmodulation on DC and macrophage function. As shown in Fig. 2A,bmDCs produced high levels of IL-6, IL-10, IL-12, and TNF-� inresponse to JEV infection. The production of IL-6, IL-10, and

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TNF-� were detected as early as 6 h p.i. and peaked at 48 h p.i.However, IL-12 production showed later kinetics, being detectableonly after 24 h p.i. The peak levels of IL-10 were observed at 24 hp.i., after which they declined to a level that was higher than thatof mock-infected bmDCs. With regard to JEV-infected bmM�,cytokine production showed a similar pattern to that observed inbmDCs. Importantly, however, IL-10 production was not detectedat any of the time points (Fig. 2A). Additionally, JEV-infectedbmM� produced greater amounts of TNF-� than did bmDCs,whereas higher amounts of IL-6 and IL-12 were produced in JEV-infected bmDCs than bmM�. Moreover, the amount of cytokines

produced after infection of both bmDCs and bmM� with differentdoses of JEV varied depending on the infection doses (Fig. 2B). Todetermine whether viral replication is required for the productionof cytokines by bmDCs and bmM�, we infected bmDCs andbmM� with equivalent doses of live and UV-irradiated/heat-inac-tivated JEV. As shown in Fig. 2C, JEV inactivated by UV irradi-ation and heat failed to induce the production of any cytokines,which indicates that viral replication was necessary for cytokineinduction. Therefore, these results suggest that the interaction ofJEV with DCs and macrophages may differ, leading to the pro-duction of divergent cytokines.

FIGURE 1. Permissiveness of DCs and macrophages for JEV replication. A, Morphological changes in DCs and macrophages infected with JEV. DCs(bmDCs) and macrophages (bmM�) derived from bone marrow cells were infected with JEV Beijing-1 strain (5 � 105 50% tissue culture-infective dose(TCID50)/ml) and then observed for morphological changes at 48 h p.i. B, Immunohistochemistry for the expression of JEV E protein. The expression ofJEV E protein in infected bmDCs and bmM� was checked by immunohistochemistry at 48 h p.i. using mAb against JEV E protein. C, The levels ofinfectious progeny virus in the supernatants of infected bmDCs and bmM�. The supernatants of JEV-infected bmDCs and bmM� were harvested on theindicated days p.i., after which infectious viral titers were quantified by cytopathic assays using Vero cells (n � 3). D, Viral RNA levels of JEV-infectedbmDCs and bmM�. The total RNA extracted from JEV-infected bmDCs and bmM� harvested on the indicated days p.i. was used to quantify JEV RNAby real-time PCR. The levels of viral RNA were expressed as relative levels to uninfected cells (n � 3). E, Percentage of bmDCs and bmM� infected withJEV. The percentage of infected bmDCs and bmM� was determined by flow cytometric analysis at the indicated time using mAb against NS1 protein ofJEV (n � 3). F, In vivo kinetics of viral RNA load in the spleen of mice infected with JEV. The viral RNA load in splenocytes collected from JEV-infectedmice (n � 3) on the indicated days p.i. was ascertained by quantitative real-time PCR. The bars in the graph represent the average levels SD of viralRNA relative to those in naive mice. G, The expression of NS1 in splenic DCs and macrophages of mice infected with JEV. Splenocytes of JEV-infectedmice (n � 4) were stained with CD11c or F4/80 Ab at the indicated days p.i., after which the expression of NS1 in CD11chigh and F4/80high cells wasdetermined by flow cytometric analysis following intra- and extracellular staining with NS1 mAb. The relative mean fluorescence intensity (MFI) levels SD of NS1 in cells gated on CD11chigh (left) or F4/80high (right) are shown.

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FIGURE 2. The profiles of cytokines produced by DCs and macrophages infected with JEV. A, The pattern of cytokine production in JEV-infected DCs andmacrophages. DCs (bmDCs) and macrophages (bmM�) derived from bone marrow cells were infected with JEV Beijing-1 strain (5 � 105 TCID50/ml), after whichthe cytokine levels in culture supernatants harvested at the indicated times p.i. were determined by ELISA. B, Dependence of cytokine production on infectiondoses. The levels of cytokines in the culture supernatants of bmDCs and bmM� infected with the indicated doses of JEV were determined at 24 h p.i. C, Viralreplication is required for the production of cytokines by bmDCs and bmM�. Bone marrow-derived cells were infected with live JEV (5 � 105 TCID50/ml) orequivalent amounts of viruses that were inactivated by UV irradiation and heat (heating at 95°C for 10 min), and the cytokine levels in culture supernatant werethen quantified by ELISA at 24 h p.i. Data represent the means SD from wells evaluated in quadruplicate. n.d., Not detected.

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Differential phenotypic modulation of DCs and macrophagesinfected with JEV

Costimulatory molecules expressed on APCs are also critical tothe optimal development of adaptive immune responses. Co-stimulatory molecules are up-regulated after activation of APCsupon pathogen exposure, which enables APCs to provide ade-quate immune responses. Therefore, alteration of costimulatorymolecules expressed on APCs may be a major target of viralimmune evasion. To determine whether JEV induced alterationof costimulatory activation markers in both DCs and macro-phages, we examined the expression levels of CD40, CD80,CD86, MHC class I, and MHC class II molecules in JEV-in-fected DCs and macrophages by flow cytometric analysis. Asshown in Fig. 3A, JEV infection resulted in differential modu-lation of costimulatory molecules in both bmDCs and bmM�.Specifically, CD40 and MHC class II expression in bmDCs wasprofoundly down-regulated by JEV infection, while CD80 andCD86 showed enhanced expression (Fig. 3A). The enhancedexpression of MHC class I molecule was also observed in JEV-infected bmDCs, as seen in other flaviviral infection (45). Incontrast, after JEV infection bmM� showed clearly enhancedexpression of all CD40, CD80, CD86, and MHC classes I and

II molecules that were evaluated in this study (Fig. 3A). More-over, when we examined the number of splenic DC subsets andmacrophages in mice infected with JEV, the significantly re-duced number of splenic CD11chighCD8�� DC subset was ob-served at 7 days p.i., the time when viral RNA load was foundto peak. However, there was no change in the number of plas-macytoid DC (CD11chighB220�) and macrophages (F4/80highCD11b�) (supplemental Table I).5 Also, JEV infectioninduced similar in vivo modulation of costimulatory moleculesin splenic CD11chigh DCs and F4/80high macrophages. With theexception of CD80, CD86, and MHC class I molecules, theexpression of CD40 and MHC class II molecules was consis-tently reduced in splenic CD11chigh DCs, which followed thepattern observed with in vitro experiments (Fig. 3B). In con-trast, splenic F4/80high macrophages showed enhanced expres-sion of all tested activation markers following JEV infection(Fig. 3B). These results suggest that JEV induces differentialalteration of the expression of costimulatory activation markersin DCs and macrophages.

5 The online version of this article contains supplemental material.

FIGURE 3. Phenotypic changes in DCs and macrophages infected with JEV. A, Phenotypic changes of bmDCs and bmM�. DCs (bmDCs) andmacrophages (bmM�) derived from bone marrow cells were infected with JEV Beijing-1 strain (5 � 105 TCID50/ml) and used to stain activation phenotypicmarkers (CD40, CD80, CD86, MHC classes I and II) at 24 h p.i. The values in histograms denote the relative MFI levels of the indicated phenotypicmarkers. B, The in vivo activation of splenic DCs and macrophages in JEV-infected mice. Splenocytes of C57BL/6 mice infected i.p. with JEV (103

TCID50) were prepared by digestion with collagenase 7 days p.i. and used to stain surface activation markers. The histogram of the expression of theindicated molecule in cells gated on CD11chigh and F4/80high cells is representative of four mice, and the values in histograms are the average of relativeMFI levels obtained from each mouse.

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The cytokine production and phenotypic alteration ofJEV-infected DCs is partially dependent on MyD88and p38 MAPK molecules

APCs have a range of innate receptors, including TLRs andnon-TLRs, that recognize pathogens (8). To examine the rolethat TLRs play in the induction of cytokines by DCs and mac-rophages following JEV infection, we used DCs and macro-phages derived from mice that lacked the MyD88 molecule,which acts as an adaptor molecule in signal transduction fromall TLRs except TLR3. As shown in Fig. 4A, bmDCs preparedfrom MyD88-deficient mice showed reduced production ofIL-6, IL-10, IL-12, and TNF-� following JEV infection, whencompared with bmDCs from wild-type mice. Similarly, JEV-infected bmM� showed reduced production of inflammatorycytokines in the absence of the MyD88 adaptor molecule. Thesefindings suggest that cytokine production from JEV-infectedDCs and macrophages is partially dependent on signal trans-duction by MyD88 adaptor molecule. In particular, IL-6, IL-10,and IL-12 production was markedly reduced in the absence ofMyD88 adaptor molecule, whereas TNF-� production byMyD88-deficient bmDCs was not significantly reduced (Fig.4A). Furthermore, bmDCs and bmM� derived from MyD88-deficient mice also showed altered phenotypes of costimulatorymolecules following JEV infection (Fig. 4B). However, MyD88-deficient bmDCs and bmM� failed to show more apparent changes

in any of the tested comstimulatory molecules in response to JEVinfection when compared with wild-type DCs. Consistent withthese results, the production of cytokines by splenic CD11chigh

DCs and F4/80high macrophages was found to depend on MyD88adaptor molecule when splenic DCs and macrophages purifiedfrom wild-type and MyD88-deficient mice were used for JEV in-fection (Fig. 4C). These findings indicate that MyD88 adaptormolecule and possibly TLRs play a role in shaping innate andadaptive immune responses against JEV. Moreover, these resultssuggest that other MyD88-independent pathways contribute tofunctional modulation of DCs and macrophages following JEVinfection, since the complete disappearance of cytokine productionand the expression of costimulatory molecules in MyD88-deficientcells was not observed. To further characterize the signal trans-duction involved in the production of cytokines by JEV-infectedDCs, we used several inhibitors of MAPK, including p38, ERK,JNK, and MEK-1. Of these inhibitors, treatment with p38 MAPKinhibitor (SB203580) resulted in a marked reduction of IL-6, IL-10, IL-12, and TNF-� production by wild-type bmDCs followingJEV infection (Fig. 4D), which suggests that the p38 MAPK path-way plays a pivotal role in cytokine production by JEV infection.Taken together, these results demonstrate that the MyD88-depen-dent signal pathway may be involved in cytokine production byJEV-infected DCs along with other MyD88-independent cellularsignal pathways.

FIGURE 4. Involvement of MyD88 adaptor molecule and p38 MAPK in the production of cytokine by DCs and macrophages infected with JEV. A, Theproduction of cytokines from JEV-infected DCs and macrophages is partially dependent on the MyD88 adaptor molecule. bmDCs and bmM� prepared fromwild-type (C57BL/6) and MyD88-deficient (MyD88 KO) mice were infected with JEV Beijing-1 strain (5 � 105 TCID50/ml). The cytokine levels in culturesupernatants of the infected bmDCs were quantified by ELISA at 24 h p.i. B, The phenotypic changes of MyD88-deficient DCs and macrophages followingJEV infection. The expression levels of activation phenotypic markers (CD40, CD80, CD86, MHC classes I and II) were determined by flow cytometricstaining using the appropriated Abs at 24 h p.i. The bars in graph show the average SD of the relative MFI obtained from the treated group (n � 4).C, The cytokine production of splenic DCs and macrophages purified from wild-type and MyD88-deficient mice following JEV infection. The levels ofcytokines produced by splenic CD11chigh DCs and F4/80high macrophages of wild-type and MyD88-deficient mice were determined by ELISA 24 hfollowing JEV infection. D, Dependence of cytokine production by JEV-infected DCs on p38 MAPK. DCs derived from bone marrow cells of C57BL/6mice were infected with JEV in the presence or absence of p38 inhibitor (SB203580; 500, 50, and 5 �M). The cytokine levels in the culture supernatantof infected DCs were determined by ELISA at 24 h p.i. �, p � 0.05; ��, p � 0.01; ���, p � 0.001 compared with the levels of wild-type DCs infectedwith JEV; n.d., not detected.

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JEV-infected DCs show defects in the priming of Ag-specificCD4� and CD8� T cells

DCs are key players in the generation of adaptive T cell responsesthrough presentation of cognate Ags to T cells using MHC mol-ecules. The quality and quantity of T cell responses is determinednot only by the level of Ag presented but also by the costimulatorysignals stimulated by the interaction of costimulatory molecules onAPCs with their ligands and the cytokine milieu. In a view of thealtered phenotype and cytokine production that occur in responseto infection, JEV-infected DCs may have important effects on Tcell priming and proliferation and the subsequent generation ofeffector functions. To explore the ability of JEV-infected DCs toprime Ag-specific CD4� and CD8� T cells, we used a TCR-trans-genic model system of OT-II and OT-I mice from which we iso-lated a homogeneous population of naive Ag-specific CD4� andCD8� T cells. As shown in Fig. 5, A and B, the primary prolifer-ation and cytokine production of CD4� and CD8� T cells wereassessed after JEV-infected bmDCs that had been pulsed with cog-

nate peptide were cocultured with CD4� and CD8� T cells puri-fied from OT-II and OT-I mice, respectively. CD4� and CD8� Tcells primed with JEV-infected bmDCs showed significantly lessproliferation than did those that were primed with mock-infectedbmDCs. The differences were more apparent when the ratio ofDCs to T cells was low, due to increased competition for DCs.Similarly, CD4� and CD8� T cells primed with JEV-infected bm-DCs were found to produce reduced amounts of the cytokinesIL-2, IL-4, and IFN-� (Fig. 5, A and B). To further confirm thedefective ability of JEV-infected DCs to prime CD4� and CD8�

T cells, the ability of splenic CD11chigh DCs from JEV-infectedmice to support CD4� and CD8� T cell proliferation was com-pared with that of splenic CD11chigh DCs from uninfected mice(Fig. 5C). Splenic CD11chigh DCs from JEV-infected mice werefound to have a decreased ability to induce the proliferation ofAg-specific CD4� and CD8� T cells. Therefore, these results in-dicate that JEV induces defective T cell responses through mod-ulation of the function of DCs.

FIGURE 5. The ability of JEV-infected DCs to prime Ag-specific CD4�/CD8� T cells. A and B, Priming of Ag-specific CD4� (A) and CD8� (B) Tcells with JEV-infected bmDCs. DCs derived from bone marrow cells were infected with JEV Beijing-1 strain (5 � 105 TCID50/ml) and then used to primeCD4�/CD8� T cells 24 h later. CD4� and CD8� T cells, which were purified from corresponding OT-II and OT-I mice, were incubated with JEV- andmock-infected bmDCs in the presence of OVA323–339 peptide (500 nM) for CD4� T cells and OVA257–264 peptide (100 nM) for CD8� T cells at differentratios. The levels of cytokine IFN-�, IL-4, and IL-2 in culture supernatant were determined by ELISA at the indicated time. C, Splenic CD11chigh DCsobtained from JEV-infected mice have a reduced ability to stimulate CD4� and CD8� T cells. CD4� and CD8� T cells were purified from correspondingOT-II and OT-I mice and stimulated with splenic CD11chigh DCs purified from mice that were previously infected with JEV (103 TCID50). The proliferationof CD4� and CD8� T cells was assessed by a bioluminescence assay following 72 h of incubation. The means SD of RLUs from wells evaluated inquadruplicate are shown. �, p � 0.05; ��, p � 0.01; ���, p � 0.001 compared with mock-infected group.

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CD8, but not CD4, T cells primed by JEV-infected DCs showreduced proliferation in an IL-10-dependent manner

As with costimulatory molecules on the surface of DCs and sol-uble mediators in the microenvironment, several factors determinethe optimal priming of CD4� and CD8� T cells by DCs. Con-versely, the presence of soluble factors such as IL-10 may exertnegative influences on the priming of CD4� and CD8� T cells.Furthermore, since DCs may function as an IL-10 source in JEVinfection through MyD88-dependent and -independent pathways,we employed the neutralization of IL-10 biological activity with anIL-10-neutralizing Ab to investigate the role that IL-10 plays inimpaired priming of CD4� and CD8� T cells by JEV-infectedDCs. The incorporation of neutralizing IL-10 Ab in the coculturemedia of purified CD4� T cells and JEV-infected bmDCs inducedno significant changes in the suppression of the proliferation ofCD4� T cells (Fig. 6A). However, suppression of the proliferationof CD8� T cells by JEV-infected bmDCs was reversed by theincorporation of neutralizing IL-10 Ab (Fig. 6B). Therefore, thissuggests that IL-10 from JEV-infected DCs mediates reducedpriming of CD8� T cells, but not CD4� T cells. IL-10 acts onAPCs and causes down-regulation of their activation status. To testif the incorporated IL-10 Ab affected the altered phenotype ofJEV-infected bmDCs, we examined the phenotypic modulation ofJEV-infected bmDCs in the presence of neutralizing IL-10 Ab. Asshown in Fig. 6C, the neutralization of IL-10 activity led to partialrecovery of the expression of CD40 and MHC class II moleculeson infected bmDCs. Taken together, these results suggest thatIL-10 produced by JEV-infected DCs can mediate impaired re-sponses of CD8� T cells. However, the reduced priming of CD4�

T cells by JEV-infected DCs may occur through unknownpathways.

JEV infection induces the reduced CD4� and CD8� T cellresponses depending on MyD88 molecules

To test the involvement of MyD88 molecule in defective prim-ing of CD4� and CD8� T cells by JEV-infected DCs, when weexamined the capacity of MyD88-deficient DCs to prime CD4�

and CD8� T cells after JEV infection, MyD88-deficient DCsshowed higher proliferation of CD4� and CD8� T cells thandid wild-type DCs (data not shown), which indicates thatMyD88 adaptor molecule may mediate impairment of JEV-in-fected DCs to prime CD4� and CD8� T cells. Therefore, todetermine the involvement of MyD88 adaptor molecule in invivo defective CD4� and CD8� T cell responses in JEV-in-fected mice, we investigated the generation of CD4� and CD8�

T cell responses using an HSV-1 challenge model. As shown inFig. 7, wild-type and MyD88-deficient mice were immunizedwith HSV-1 seven days after JEV infection because viral RNAload and reduced numbers of DC subsets were found to peak atthis time. The responses of CD4� and CD8� T cells were thenevaluated. Mice infected with JEV had significantly decreasedHSV-specific proliferation of purified CD4� T cells and 2-foldfewer IFN-�-producing CD4� T cells in response to HSV-1antigenic stimulation than did the mock-infected group (Fig.7A). Such suppressed responses of CD4� T cells by JEV in-fection were observed to be more obvious in wild-type micethan those in MyD88-deficient mice, which indicated that theMyD88 adaptor molecule plays a role in generation of Ag-spe-cific responses following JEV infection. It is also interesting tonote that MyD88-deficient mice showed reduced responses ofCD4� T cells when compared with wild-type mice. Similarly,mice that received JEV infection showed markedly suppressedCD8� T cell responses when HSV-specific CD8� T cells were

observed by in vivo CTL killing activity (Fig. 7B). JEV infec-tion resulted in an average of 27% lysis of specific targets inspleen, whereas 97% lysis of the targets was observed in mock-infected mice. In the absence of MyD88 adaptor molecule, suchdifferences of CD8� T cell responses between JEV- and

FIGURE 6. Neutralization of IL-10 rescues the suppressed proliferationof CD8� T cells mediated by JEV-infected bmDCs, but not CD4 T cells.A and B, The proliferation of Ag-specific CD4� (A) and CD8� (B) T cellswith JEV-infected bmDCs in the presence of IL-10 neutralizing Ab. DCsderived from bone marrow cells of wild-type C57BL/6 mice were infectedwith JEV Beijing-1 strain (5 � 105 TCID50/ml) and then pulsed with cog-nate antigenic peptide of CD4� and CD8� T cells obtained from OT-II(CD4) and OT-I (CD8) mice 24 h later. The proliferation of CD4� andCD8� T cells was assessed by using a bioluminescence assay following a96-h incubation with treated bmDCs in the presence of IL-10 neutralizingAb. The means SD of RLUs from wells evaluated in quadruplicate areshown. C, Phenotypic change in bmDCs following JEV infection in thepresence of IL-10-neutralizing Ab. DCs were infected with JEV in thepresence or absence of IL-10-neutralizing Ab and used for flow cytometricstaining of the surface activation markers (CD40, CD80, CD86, MHCclasses I and II) 24 h later. The bars in graph show the average SD ofrelative MFI obtained from each treated group (n � 4). ��, p � 0.01; ���,p � 0.001 compared with JEV plus isotype-treated group.

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mock-infected mice were significantly reduced, and MyD88-deficient mice mounted comparable HSV-specific CD8� T cellresponses to wild-type mice (Fig. 7B). Consistent with theseresults, JEV-infected mice were found to have a decreased num-ber of IFN-�-producing CD8� T cells following stimulationwith immunodominant epitope of HSV-1 (gB498 –505, SSIEF-ARL), which resulted in partial recovery due to the lack ofMyD88 molecule (Fig. 7, C and D). These results demonstratethat MyD88 adaptor molecule may be involved in the subver-sion of CD4� and CD8� T cell responses by JEV infection.

To test if defective T cell responses in response to JEV infectionresult in greater susceptibility to secondary microbial infection,C57BL/6 mice that were previously infected with JEV were chal-lenged with zosteriform infection of HSV-1 strain 17 on the day7 p.i. Despite that JEV induced no morbidity and mortality, clinicalsigns of HSV zosteriform infection progressed at a much fasterrate in JEV-infected mice than in mock-infected mice (Fig. 7E).Furthermore, 66% of HSV-challenged mock-infected mice sur-vived for 17 days p.i., while only 22% of JEV-infected mice sur-vived for the same length of time ( p � 0.041). MyD88-deficientmice that received JEV infection showed enhanced susceptibilityto HSV-1 zosteriform infection, but this enhanced susceptibilitywas not significantly different from that of mock-infected MyD88-deficient mice. On the other hand, the susceptibility of MyD88-deficient mice to HSV-1 zosteriform infection was greater than inwild-type mice, as supported by a previous report (46). Takentogether, these results suggest that JEV infection can cause im-

paired CD4� and CD8� T cell responses in MyD88-dependentand -independent manners, which result in the generation of de-fective antiviral immune responses.

DiscussionDCs and macrophages play important roles in conferring antiviralimmunity during the initial stages of viral infection. Recognition ofviral infection by DCs and macrophages through TLR and/or non-TLR sensors induces immediate production of inflammatory cy-tokines that subsequently provide early antiviral immunity (6–8).In addition to activation of innate effector cells, signals from TLRsand/or non-TLRs result in maturation of macrophages and DCs,thereby leading to T cell priming. Thus, signals of innate receptorsare necessary to translate innate immunity into Ag-specific re-sponses of the adaptive immune system (6–12). However, in-creased levels of inflammatory mediators can initiate irreversibleimmune responses and lead to cell death (29). Therefore, a directviral cytopathic response and both direct and indirect immunolog-ical responses can contribute to CNS degeneration through JEV-infected cell exclusion by macrophages and CTLs, secretion ofcytokines and chemokines, and activation of microglia (30–33).Although DCs are critical to the priming of antiviral adaptive im-mune responses, few studies have been conducted to evaluate viralinfection of DCs and their role in JEV infection. The results of thepresent study suggest that JEV-infected DCs undergo modulationthat differs from macrophages with respect to cytokine productionand phenotypic changes. Furthermore, signaling through the

FIGURE 7. Reduced induction of CD4� and CD8� T cell responses in JEV-infected mice depends on MyD88 adaptor molecule. A, Reduced responsesof CD4� T cells in JEV-infected mice. After infecting C57BL/6 (Wild-type) and MyD88-deficient (MyD88 KO) mice with JEV (103 TCID50), mice wereimmunized intramuscularly with HSV-1 (106 PFU/mouse) 7 days after JEV infection. After 14 days, the responses of the purified CD4� T cells to HSV-1Ag stimulation were evaluated by proliferation using a viable cell ATP bioluminescence assay (left) and enumeration of the IFN-�-producing cells usingELISPOT (right). B, Suppressed in vivo CTL killing activity of Ag-specific CD8� T cells in JEV-infected mice. JEV- and mock-infected (C57BL/6 andMyD88-deficient) mice were immunized with HSV-1 seven days p.i., and the activity of in vivo CTL was assessed 14 days later. The bars in graph denotethe mean SD of specific lysis (%) observed from four mice per group. C, IFN-�-producing CD8� T cells in response to the immunodominant gB498–505

(SSIEFARL) peptide of HSV-1. Fourteen days after immunization of JEV-infected mice with HSV-1, mice were reimmunized with HSV-1 to induce recallresponse. The number of IFN-�-producing CD8� T cells were then determined by intracellular cytokine staining 5 days later. The dot plot represents oneof four mice per group, and the percentages seen in the upper right quadrant show the means SD. D, The total number of SSIEFARL-specific CD8�

T cells determined by intracellular cytokine staining. Data represent the mean SD of four mice per group. E, Susceptibility of JEV-infected mice againstHSV-1 zosteriform infection. C57BL/6 and MyD88-deficient mice (n � 9) were infected with zosteriform of HSV-1 strain 17 seven days after beinginfected i.p. with JEV. The graphs show the proportion of surviving mice on different days p.i.

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MyD88 adaptor molecule was found to have a certain role in caus-ing subversion of primary CD4� and CD8� T cell responses. Wealso demonstrated that IL-10 produced by JEV-infected DCs wascapable of mediating the reduced priming of CD8� T cells but notCD4� T cells. Therefore, our results demonstrate that JEV inducesthe functional impairment of DCs that leads to poor responses ofCD4� and CD8� T cells through MyD88-dependent and -inde-pendent pathways.

During viral infection, DCs undergo maturation that is mani-fested by up-regulation of Ag presentation, costimulatory mole-cules, and cytokine secretion (6, 7). Our study revealed that JEVinfection of macrophages followed the classical pathway of up-regulation of tested costimulatory molecules and cytokine produc-tion (Figs. 2 and 3), but that JEV-infected DCs failed to up-regu-late some costimulatory molecules. Of more interest, JEV-infectedDCs produced the antiinflammatory cytokine IL-10, which was notdetected in JEV-infected macrophages (Fig. 2). IL-10 blocksproinflammatory cytokine production, costimulation, MHC class IIexpression, and chemokine production (47, 48). IL-10 is also re-leased with, or after, the secretion of proinflammatory cytokines inan effort to maintain homeostasis (49). Indeed, IL-10 was releasedfrom JEV-infected DCs along with the proinflammatory cytokinesIL-6, IL-12, and TNF-�. Because the cytokine milieu in the mi-croenvironment in which T cell and DC contact occurs is an im-portant determinant of T cell outcome, alteration of the DC-pro-duced cytokine profile may be an effective strategy for theprevention of T cell activation during viral infection (50). Neu-tralizing Ab assays of IL-10 consistently demonstrated that IL-10produced by JEV-infected DCs functioned as a mediator of thesuppression of T cell activation, at least in CD8� T cells (Fig. 6).Furthermore, it was observed that MyD88 adaptor molecule me-diated the production of IL-10 by JEV-infected DCs (Fig. 4),which indicates that MyD88 molecule may play a role in subver-sion of CD8� T cell responses through release of regulatory sol-uble factors such as IL-10. However, the incorporation of IL-10Ab failed to recover suppressed CD4� T cell responses, even whenthe expression of MHC class II and CD40 molecules was partiallyrecovered. Therefore, the unknown pathway by which JEV-in-fected DCs showed impaired CD4� T cell responses requires fur-ther study. Recent studies have revealed that excessive amounts ofIL-6 and other proinflammatory cytokines have deleterious effectson T cell responses (51), suggesting that IL-10, which is known toinhibit DC maturation, may act synergistically with IL-6 and causefurther down-regulation of the functions by which DCs prime Tcells.

To further define the signal pathway that leads to the productionof pro- and antiinflammatory cytokines in JEV-infected DCs andmacrophages, we determined the levels of cytokine production us-ing MyD88-deficient DCs and macrophages. The incomplete in-hibition of cytokine production in the MyD88-deficient DCs andmacrophages may occur as a result of NF-�B and MAPK alsobeing activated by the MyD88-independent pathway, and the in-tersection between the MyD88-dependent and -independent path-ways is thought to be TRAF6 (20, 21). A few viruses are knownto activate p38 MAPK and augment IL-10 production by host cells(52). Since the inhibition of p38 MAPK completely abrogated cy-tokine production by DCs in response to JEV infection (Fig. 4D),activation of p38 MAPK through MyD88-dependent and possibly-independent signals may be critical to the production of cytokineby DCs following JEV infection. To support our finding regardingthe role that the MyD88 molecule plays in modulation of DC func-tion by JEV, we used a HSV-1 challenge model to demonstratethat JEV infection induced subversion of CD4� and CD8� T cellresponses in a MyD88-dependent manner (Fig. 7). The results sug-

gested that impairment of DC function by JEV infection contrib-uted to the reduction of CD4� and CD8� T cell responses, asshown in Fig. 5. Additionally, MyD88-deficient DCs were foundto have a comparable proliferation of CD4� and CD8� T cellsafter JEV infection, when demonstrated by the relative prolifera-tion to maximum proliferation induced by mock-infected DCs(data not shown). Although MyD88-deficient DCs themselvesshowed poor proliferation of primary CD4� and CD8� T cellswhen compared with wild-type DCs, JEV infection may impairDC functions and induce subverted T cell responses, thereby lead-ing to enhanced susceptibility to secondary microbial infections byviruses such as HSV zosteriform. JEV has a positive-sense, single-stranded RNA genome that could trigger signal through severalTLR molecules such as TLR3 and TLR7 (8). When we examinedthe production of cytokines by TLR2- or TLR3-deficient DCs fol-lowing JEV infection, the significantly reduced production of cy-tokines in response to JEV was shown, but complete inhibition wasnot induced (supplemental Fig. 1). Therefore, it is possible thatJEV may synthesize an array of several agonists that can triggerTLR and/or non-TLR sensors.

Besides alteration of cytokine profile, JEV infection elicited im-pairment of DC maturation, as evidenced by the phenotypic acti-vation markers. Clear down-regulation of CD40 and MHC class IIlevels with marginal changes in CD80 and CD86 levels was ob-served in JEV-infected DCs. In contrast, macrophages elicited theclassical maturation by JEV infection, as proved by the enhancedexpression of tested costimualtory molecules. Moreover, theMyD88 adaptor molecule appeared to contribute to such alterationof phenotypic markers in JEV-infected DCs and macrophages(Fig. 4B). Low expression of MHC class II and CD40 moleculeswill prevent DCs from their crucial interaction with CD4� Th cellsin a process called licensing, which is a step that is necessary toenable DCs to adequately prime CD8� T cells (53). Virusesachieve down-regulation of MHC molecules by blocking traffick-ing to the cell membrane (54, 55), increasing destruction by ubiq-uitination (56), and preventing biosynthesis (57). Additionally, vi-ruses such as murine cytomegalovirus down-regulate MHC class IImolecules through IL-10 production (58). It is likely that IL-10produced by DCs after JEV infection is involved in down-regula-tion of MHC class II levels since neutralization of IL-10 by mAbrescued the expression level of MHC class II. Interestingly, MHCclass I was up-regulated by JEV infection in a manner similar tothat observed in response to other flaviviruses (45), even thoughJEV-infected DCs showed poor proliferation of CD8� T cells.Investigation using an IL-10-neutralizing Ab revealed that IL-10may cause impaired responses in CD8� T cells, even when thereis enhanced expression of MHC class I molecule. Truly JEV in-duces a paralytic state in DCs that is coordinated with altered cy-tokine profile and phenotype marker, thereby predisposing the hostto other viral infections.

JEV infection of DCs has not been well characterized. Weexamined morphological changes in DCs and macrophages fol-lowing JEV infection. There were no changes observed in mac-rophages, but morphological changes such as rounding and de-tachment from culture surfaces were observed in DCs, eventhough cell death through apoptosis did not follow. Also, it waslikely that JEV did not replicate its RNA in DCs as efficientlyas in macrophages, and infectious progeny viruses were notreleased from DCs. Since T cell activation occurs after the en-gagement of TCRs with DCs (1, 2), the functional impairmentof DCs by JEV may also be secondary to morphologicalchanges that result in disarranged cell architecture. Further-more, since it was unlikely that all plated DCs and macrophageswere infected by JEV (Fig. 1E), some soluble factors released

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from JEV-infected cells may indirectly affect functions such asT cell priming in neighboring cells. In conclusion, JEV infec-tion of DCs elicited early immune responses. These responseswere characterized by the immediate production of pro- andantiinflammatory cytokines through MyD88-dependent and -in-dependent pathways that resulted in p38 MAPK activation.IL-10 and reduced costimulation is likely incapable of provid-ing adequate signals to initiate T cell priming. The findingspresented herein suggest that imbalanced activation and mod-ulation of both DCs and macrophages by JEV contribute toimmunopathological degeneration of the CNS and prevent ad-equate signals from initiating antiviral adaptive immunity,thereby leading to boost virus survival and dissemination inthe body.

DisclosuresThe authors have no financial conflicts of interest.

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