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Functional Modulation of Dendritic Cells and
Macrophages by Japanese Encephalitis Virus
through MyD88 Adaptor
Molecule-Dependent and -Independent
Pathways
Abi G. Aleyas, Junu A. George, Young Woo Han, M. M.
Rahman, Seon Ju Kim, Sang Bae Han, Byung Sam Kim,
Koanhoi Kim and Seong Kug Eo
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Copyright © 2009 by The American Association of
Immunologists, Inc. All rights reserved.
Print ISSN: 0022-1767 Online ISSN: 1550-6606.
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J Immunol 2009; 183:2462-2474; Prepublished online 27
July 2009;
doi: 10.4049/jimmunol.0801952
http://www.jimmunol.org/content/183/4/2462
The Journal of Immunology
Functional Modulation of Dendritic Cells and Macrophages by
Japanese Encephalitis Virus through MyD88 Adaptor
Molecule-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*
M
acrophages and dendritic cells (DCs)4 are major players 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 in
vivo as potent APCs and play crucial roles in the enhancement and
regulation of cell-mediated immune reactions (1– 4). Since DCs
express various costimulatory and adhesion molecules, they can
efficiently activate naive T cells in primary responses. Upon encounters with pathogens, immature DCs undergo maturation processes that are characterized by the production of proinflammatory
*Laboratory of Microbiology, College of Veterinary Medicine and Bio-Safety Research Institute, Chonbuk National University, Jeonju, Republic of Korea; †College of
Pharmacy, Chungbuk National University, Cheonju, Republic of Korea; ‡Immunomodulation 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 page
charges. This article must therefore be hereby marked advertisement in accordance
with 18 U.S.C. Section 1734 solely to indicate this fact.
1
This work was supported by Grant RTI05-03-02 from the Regional Technology
Innovation Program of the Ministry of Commerce, Industry, and Energy (MOCIE), a
research 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 of
Microbiology, College of Veterinary Medicine and Bio-Safety Research Institute,
Chonbuk National University, Jeonju City 561-756, Republic of Korea. E-mail address: [email protected]
4
Abbreviations used in this paper: DC, dendritic cell; bmDC, bone marrow-derived
DC; bmM␾, bone marrow-derived macrophage; gB, glycoprotein B; JEV, Japanese
encephalitis 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
www.jimmunol.org/cgi/doi/10.4049/jimmunol.0801952
cytokines (TNF-␣, IL-12, and IL-6), up-regulation of costimulatory molecules (CD40, CD80, and CD86), alteration of chemokine
receptors (CCR2, CCR5, and CCR7), and enhanced Ag presentation (6, 7). Because these events are crucial in the development of
optimal antiviral responses, many viruses target these events to
prevent the development of antiviral immunity and boost viral
survival.
The recognition mechanisms that initiate and control innate reactions to viruses remain poorly understood. Accumulating evidence suggests that membrane-bound cell-surface or intracellular
TLRs form part of the surveillance system (8). Indeed, several of
13 recognized mammalian TLRs are known to be involved in the
recognition of viral components (8). Additionally, cytosolic nonTLR dsRNA sensors, including protein kinase R, melanoma differentiation-associated gene 5 (MDA5), and retinoic acid-inducible 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 the
main adaptor molecule to activate the downstream signaling pathway. Activation of this pathway, in turn, leads to the activation of
NF-␬B and MAPKs, such as stress-activated protein kinase/JNK
and p38, which subsequently leads to the production of inflammatory cytokines (13, 14). Furthermore, the MyD88-independent or
-dependent pathway results in production of IFN-␣/␤ in response
to stimulation of TLR3, TLR4, TLR7, TLR8, and TLR9 (15, 16).
TLR3 and TLR4 can signal via Toll/IL-1R domain-containing
adaptor-inducing IFN-␤ (TRIF), which induces IFN-␤ gene transcription partly through IFN regulatory factor 3 (IRF3) activation
(17). TLR7, TLR8, and TLR9 activate some IFN-␣ genes through
formation of the MyD88-TNFR-associated factor 6 (TRAF6)-IFN
regulatory factor 7 (IRF7) complex (18, 19). However, NF-␬B and
MAPK are also activated by the MyD88-independent pathway.
The point of intersection between the MyD88-dependent and -independent pathways is thought to be TRAF6 (20, 21).
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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 of
acute and epidemic viral encephalitis, are available. In this study, we investigated the interaction of JEV with DCs and macrophages. JEV replicated its viral RNA in both cells with different efficiency, and JEV infection of macrophages followed the classical
activation 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. Of
more interest, along with production of proinflammatory cytokines, DCs infected by JEV released antiinflammatory cytokine
IL-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 CD4ⴙ
and CD8ⴙ T cell responses. We also found that IL-10 released from JEV-infected DCs led to a reduction in the priming of CD8ⴙ
T cells, but not CD4ⴙ T cells. Taken together, our data suggest that JEV induces functional impairment of DCs through MyD88dependent and -independent pathways, which subsequently leads to poor CD4ⴙ and CD8ⴙ T cell responses, resulting in boosting
viral survival and dissemination in the body. The Journal of Immunology, 2009, 183: 2462–2474.
The Journal of Immunology
Materials and Methods
Animals
C57BL/6 mice (H-2b), 5 to 6 wk old, were purchased from Koatech. OT-I
and OT-II mice, which are transgenic for the V␣2/V␤5 TCR that recognizes
the H-2Kb-restricted peptide (OVA257–264, SIINFEKL) and the I-Ab-restricted peptide (OVA323–339, ISQAVHAAHAEINEAGR) of chicken
OVA, 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 Committee on the Care of Laboratory Animal Resources, Chonbuk National
University. The animal facility of the Chonbuk National University is fully
accredited 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 titer
of the virus in clarified brain lysates (HBSS-10% BSA) was determined by
a cytopathic assay using Vero cells (CCL81; American Type Culture Collection). Similarly, brain lysates from healthy mice were prepared and used
to inoculate a control group of mock-infected mice. HSV-1 strain 17 was
grown in Vero cells using DMEM supplemented with 2% FBS, penicillin
(100 U/ml), and streptomycin (100 U/ml). Virus stocks were concentrated
by centrifugation at 50,000 ⫻ g, titrated by a plaque assay, and then stored
in aliquots at ⫺80°C until needed.
Abs and peptides
The following mAbs were obtained from eBioscience or BD Biosciences
for flow cytometric analysis and other experiments: FITC-anti-CD40 (3/
23), CD80 (16-10A1), CD86 (GL1), MHC class II (25-9-17), MHC class
I (28-14.8), PE-anti-CD4 (GK1.5), CD8␣ (53-6.7), CD11b (M1/70), and
CD11c (N418). Anti-mouse IL-10 Ab (JES5-2A5) was used to neutralize
the IL-10 activity. The mAb against the nonstructural protein 1 (NS1) of
JEV 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) of
HSV-1 gB were chemically synthesized at Peptron. JEV-specific primers
for the detection of viral RNA (JEV10,564 –10,583 forward, 5⬘-CCC TCA
GAA CCG TCT CGG AA-3⬘ and JEV10,862–10,886 reverse, 5⬘-CTA TTC
CCA 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 previously described (36) with some modifications. Briefly, bone marrow cells
from femurs and tibiae were cultured in RPMI 1640 supplemented with 2
ng/ml GM-CSF and 10 ng/ml IL-4. On days 5 and 8 the culture was replenished with 5 ml of fresh media containing cytokines. Cells were harvested 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-derived
macrophages (bmM␾) were prepared by culturing bone marrow cells in
DMEM containing 30% conditioned culture media of L929 cells (37).
bmM␾ was harvested by using trypsin digestion following a 7-day incubation. The prepared bmM␾ was composed of ⬎85% F4/80⫹ cells that
consisted 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 after
JEV infection and then blocked with PBS containing 10% healthy mouse
serum for 1 h at 4°C. Following quenching by endogenous peroxidase with
0.2% H2O2 in methanol, infected cells were stained by overnight incubation at 4°C with a HRP-conjugated mAb against JEV E protein (38). After
rinsing with PBS the color was developed with 0.3% H2O2 and 3,3⬘-diaminobenzidine tetrahydrochloride. JEV E protein expression in infected
cells was then checked and captured using a light microscope equipped
with digital imaging equipment.
Quantitative SYBR Green-based real-time PCR for viral
replication
Relative levels of viral RNA in JEV-infected cells or the spleens of mice
were determined by conducting quantitative real-time PCR analysis on a
Mini Opticon system (Bio-Rad Laboratories) using a DyNAmo SYBR
Green qPCR kit (Finnzymes) following reverse transcription of total RNA
isolated from infected samples. The reaction mixture contained 2 ␮l of
template cDNA, 10 ␮l of 2⫻ Master Mix, 1.5 mM MgCl2, and 100 nM
primers at a final volume of 20 ␮l. The reactions were denatured at 95°C
for 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 temperature
was increased from 65°C to 95°C at a rate of 1°C/min, and the fluorescence
was measured every 15 s to construct a melting curve. A control sample
that contained no template DNA was run with each assay, and all determinations were performed at least in duplicate to ensure reproducibility.
The authenticity of the amplified product was determined by melting curve
analysis. The relative ratio of viral RNA in the infected samples to uninfected samples was determined. All data were analyzed using the Opticon
Monitor version 3.1 analysis software (MJ Research).
Cytokine ELISA/ELISPOT
Sandwich ELISA was used to determine the levels of cytokines in the
culture supernatants. The ELISA plates were coated with IL-2 (JES61A12), IL-4 (11B11), IL-6 (MP5-20F3), IL-10 (JES5-16E3), IL-12p70
(C18.2), IFN-␥ (R4-6A2), and TNF-␣ (1F3F3D4) anti-mouse Abs purchased from eBioscience and BD Bioscience, and then incubated overnight
at 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 h
at 37°C. The culture supernatant and standards for recombinant cytokine
proteins (PeproTech) were added to the plates and incubated for 2 h at
37°C. The plates were then washed again and the biotinylated IL-2 (JES65H4), 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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Japanese encephalitis virus (JEV), which is a mosquito-borne
member of the genus Flavivirus, such as West Nile and dengue
viruses, is responsible for most acute and epidemic cases of viral
encephalitis (22, 23). Approximately 60% of the world population
inhabits JEV endemic areas, and the virus is continuing to spread
to previously unaffected regions due to global warming (24 –26). It
is estimated that 30,000 –50,000 cases of JEV occur each year,
resulting in 10,000 –15,000 deaths, although this number may be
underestimated (22, 23). Additionally, 30 – 60% of surviving patients suffer from serious long-term neuropsychiatric sequelae (27,
28). However, the pathogenesis of JEV-associated disease in humans and in mice has not been completely elucidated. In many
cases, the virus is not directly involved in the destruction of brain
tissue, but instead causes indirect damage through cell-mediated
immune responses (29). Activated inflammatory cells secrete cytokines such as IL-1 and TNF-␣, which can cause apoptosis of
neuronal cells. Similarly, JEV infection leads to the production of
high levels of cytokines such as macrophage-derived chemokine
factor, TNF-␣, and IL-8 in the serum and cerebrospinal fluid (30 –
33). Increased levels of such cytokines may play protective roles
against infection or initiate irreversible immune responses. JEV
multiplies in macrophages and DCs of the periphery, which causes
initial viremia before entry into the CNS (34). Therefore, most
studies that have been conducted to evaluate the pathogenesis of
JEV infection have examined the interaction of the virus with macrophages and CNS cells (30 –33). These CNS cells include microglia and astrocytes, which are major contributors to the production
of inflammatory cytokines and CNS degeneration.
However, there is presently little information describing the interaction between JEV and DCs, which play a major role in immune responses. Therefore, in this study we have compared the
interaction of JEV with macrophages and DCs. Our data demonstrate that JEV infection induces differing modulations of cytokine
production and phenotypes in both DCs and macrophages. We also
defined certain roles of the MyD88 adaptor molecule in the cytokine production by DCs and macrophages and in the initiation of
primary CD4⫹ and CD8⫹ T cell responses. Additionally, we
gained insight into the contribution of IL-10 to primary immune
responses following JEV infection. We suggest that imbalanced
activation and modulation of macrophages and DCs in JEV infection are critical events that determine the immunopathological outcomes in the CNS and inadequate immune responses.
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and subsequent incubation with peroxidase-conjugated streptavidin
(eBioscience) for 1 h. Color development was then performed by the addition of a substrate (ABTS) solution. Cytokine concentrations were determined with an automated ELISA reader and SoftMax Pro4.3, according
to comparisons with two concentrations of standard cytokine proteins.
The IFN-␥-producing CD4⫹ T cells in response to HSV-1 Ag was enumerated by ELISPOT (39). Briefly, CD4⫹ T cells purified from HSVimmunized mice were stimulated with HSV Ag-pulsed APCs on IFN-␥
capture Ab-coated 96-well ELISPOT plates (Millipore). After 72 h of incubation, the plates were washed and biotinylated IFN-␥ Ab was added and
incubated for 2 h. After washing, streptavidin-alkaline phosphatase was
added and the mixture was incubated for an additional 30 min. Spots
were visualized by adding a 5-bromo-4-chloro-3-indolyl phosphate/tetra-NBT substrate solution (Promega) and counted 24 h later using a
stereomicroscope.
Intracellular cytokine staining and flow cytometric analysis
Proliferation of Ag-specific CD4⫹ and CD8⫹ T cells
The proliferation of CD4⫹ and CD8⫹ T cells was assessed by measuring
the viable cell ATP bioluminescence (40). Briefly, Ag-specific CD4⫹ and
CD8⫹ T cells were purified from OT-II and OT-I mice, respectively, using
a MACS LS column (Miltenyi Biotec) according to the manufacturer’s
instructions. The purified CD4⫹ and CD8⫹ T cells (5 ⫻ 105 cells/ml) were
then cultured together with stimulator cells at different ratios. JEV-infected
DCs were used as stimulator cells following pulsing with cognate antigenic
peptides (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 some
experiments to neutralize the IL-10 bioactivity. The culture was incubated
for 3 days at 37°C in a humidified 5% CO2 incubator. Replicate cultures
were transferred to V-bottom 96-well culture trays that were subsequently
centrifuged to collect the cells. The proliferated cells were then evaluated
using a Vialight cell proliferation assay kit (Cambrex Bio Science) according 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). To
control for Ag specificity, peptide-unpulsed syngeneic splenocytes were
labeled with a lower concentration of CFSE (0.25 ␮M). A 1:1 mixture of
each target cell population was then injected i.v. into mice for evaluation.
Splenocytes were then collected from recipient mice 24 h after adoptive
transfer of target cells and analyzed by flow cytometry. Each population
was distinguished by its respective fluorescence intensity. The percentage
killing of target cells in immunized animals was calculated using the following equation: ratio ⫽ (percentage CFSElow/percentage CFSEhigh). Percentage specific lysis was determined as: [1 ⫺ (ratio of naive/ratio of
immunized)]/100.
Zosteriform infection of herpes simplex virus
A zosteriform challenge experiment was performed as described by Gierynska et al. (42). Briefly, the left flank area was depilated before challenge using a combination of hair clipping and a depilatory chemical. The
animals were then anesthetized with Avertin (2,2,2-tribromoethanol) and
2-methyl-2-butanol (Sigma-Aldrich), and a total of five scarifications were
made on an ⬃4-cm2 area of the left flank region. A total dose of 10 ␮l of
HSV-1 strain 17 (containing 1 ⫻ 106 PFU) were then applied to the scarifications, after which the area was gently massaged. The animals were
inspected daily for the development of zosteriform ipsilateral lesions, general behavioral changes, encephalitis, and mortality.
Statistical analysis
Where specified, the data were analyzed for statistical significance using a
Student’s t test. A p value of ⬍0.05 was considered significant. KaplanMeier curves were also generated for mice that survived the zosteriform
challenge with HSV-1. The p values were then computed using the ␹2
method. The survival rates of the two groups were considered to be significantly different if the two-sided p value was ⬍0.05.
Results
DCs and macrophages are permissible for viral replication
of JEV
JEV can multiply in murine macrophages without causing cytopathic changes (43). However, few reports regarding interaction of
DCs with JEV are available so far. To examine the interaction of
DCs and macrophages with JEV, bmDCs and bmM␾ were morphologically observed following JEV infection. As shown in Fig.
1A, bmDCs infected with JEV showed morphological changes,
including rounding and detachment from the culture surface at
48 h postinfection (p.i.). Conversely, there was no apparent change
in JEV-infected bmM␾. When immunohistochemical staining of
the infected cells was conducted to identify the biosynthetic expression of viral proteins, both bmDCs and bmM␾ were found to
be permissible for the expression of viral E protein (Fig. 1B). To
further identify the interaction of both bmDCs and bmM␾ with
JEV, the titers of infectious progeny viruses in the culture supernatants of both cell types were determined daily by cytopathic
assays using Vero cells. bmM␾ induced the productive release of
infectious progeny viruses with levels that peaked at 4 –5 days p.i.,
while the productive release of infectious virus in bmDCs was not
detected in cytopathic assays (Fig. 1C). However, quantitative real-time PCR indicated that JEV viral RNA was capable of replicating in both cells, even though the viral RNA levels of bmDCs
were markedly lower than those of bmM␾ (Fig. 1D). To evaluate
the ratio of infected bmDCs and bmM␾, we determined the percentage of JEV-infected bmDCs and bmM␾ using mAb against
JEV NS1 protein, which is largely retained within infected cells
and involved in RNA replication (44). As shown in Fig. 1E, ⬎50%
of bmM␾ were infected with JEV, but fewer bmDCs were found
to be infected. Furthermore, to examine in vivo replication of JEV,
we determined viral RNA load in splenocytes of JEV-infected
mice. The viral RNA load was found to peak at 7 days p.i., after
which it declined (Fig. 1F). Additionally, splenic DCs (CD11chigh
cells) and macrophages (F4/80high cells) were observed to be infected with JEV, as confirmed by intra- and extracellular staining
with NS1 Ab (Fig. 1G). The expression of NS1 protein peaked at
3 days p.i., and splenic F4/80high macrophages showed more effective expression of NS1 protein than did splenic CD11chigh DCs.
However, the difference of JEV NS1 expression in CD11chigh DC
subsets (CD11chighCD8␣⫹ and CD11chighCD8␣⫺) was not observed (data not shown). Taken together, theses results indicate
that both DCs and macrophages can be infected with JEV, but that
there are differences in permissiveness of viral replication.
The profiles of cytokine production in DCs and macrophages
infected with JEV
DCs and macrophages play an important role in primary defenses
by generating and regulating adaptive immunity (1– 4). Viruses
must evolve ways to modulate DC and macrophage function that
enable them to evade detection and elimination by hosts. We examined the pattern of pro- and antiinflammatory cytokines produced by JEV-infected bmDCs and bmM␾ to investigate JEV
modulation on DC and macrophage function. As shown in Fig. 2A,
bmDCs produced high levels of IL-6, IL-10, IL-12, and TNF-␣ in
response to JEV infection. The production of IL-6, IL-10, and
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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 for
30 min with properly diluted mAbs. After staining, the cells were washed
twice 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-h
stimulation of synthetic peptide in the presence of 2 ␮M monensin (SigmaAldrich). For the intracellular IFN-␥ staining, cells were stained for surface
marker, washed, permeabilized, and stained with PE-conjugated antiIFN-␥. Following fixation, cells were resuspended in PBS and then analyzed using FACSCalibur equipped with the CellQuest program (BD Biosciences) and WinMDI 2.8 software.
FUNCTIONAL IMPAIRMENT OF DCs BY JEV
The Journal of Immunology
2465
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 detectable
only after 24 h p.i. The peak levels of IL-10 were observed at 24 h
p.i., after which they declined to a level that was higher than that
of mock-infected bmDCs. With regard to JEV-infected bmM␾,
cytokine production showed a similar pattern to that observed in
bmDCs. Importantly, however, IL-10 production was not detected
at any of the time points (Fig. 2A). Additionally, JEV-infected
bmM␾ produced greater amounts of TNF-␣ than did bmDCs,
whereas higher amounts of IL-6 and IL-12 were produced in JEVinfected bmDCs than bmM␾. Moreover, the amount of cytokines
produced after infection of both bmDCs and bmM␾ with different
doses of JEV varied depending on the infection doses (Fig. 2B). To
determine whether viral replication is required for the production
of cytokines by bmDCs and bmM␾, we infected bmDCs and
bmM␾ with equivalent doses of live and UV-irradiated/heat-inactivated JEV. As shown in Fig. 2C, JEV inactivated by UV irradiation and heat failed to induce the production of any cytokines,
which indicates that viral replication was necessary for cytokine
induction. Therefore, these results suggest that the interaction of
JEV with DCs and macrophages may differ, leading to the production of divergent cytokines.
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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 of
JEV 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 of
infectious progeny virus in the supernatants of infected bmDCs and bmM␾. The supernatants of JEV-infected bmDCs and bmM␾ were harvested on the
indicated days p.i., after which infectious viral titers were quantified by cytopathic assays using Vero cells (n ⫽ 3). D, Viral RNA levels of JEV-infected
bmDCs and bmM␾. The total RNA extracted from JEV-infected bmDCs and bmM␾ harvested on the indicated days p.i. was used to quantify JEV RNA
by 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 with
JEV. The percentage of infected bmDCs and bmM␾ was determined by flow cytometric analysis at the indicated time using mAb against NS1 protein of
JEV (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-infected
mice (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 viral
RNA relative to those in naive mice. G, The expression of NS1 in splenic DCs and macrophages of mice infected with JEV. Splenocytes of JEV-infected
mice (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 was
determined 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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FUNCTIONAL IMPAIRMENT OF DCs BY JEV
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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 and
macrophages. DCs (bmDCs) and macrophages (bmM␾) derived from bone marrow cells were infected with JEV Beijing-1 strain (5 ⫻ 105 TCID50/ml), after which
the cytokine levels in culture supernatants harvested at the indicated times p.i. were determined by ELISA. B, Dependence of cytokine production on infection
doses. 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, Viral
replication is required for the production of cytokines by bmDCs and bmM␾. Bone marrow-derived cells were infected with live JEV (5 ⫻ 105 TCID50/ml) or
equivalent 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 were
then quantified by ELISA at 24 h p.i. Data represent the means ⫾ SD from wells evaluated in quadruplicate. n.d., Not detected.
The Journal of Immunology
2467
Differential phenotypic modulation of DCs and macrophages
infected with JEV
Costimulatory molecules expressed on APCs are also critical to
the optimal development of adaptive immune responses. Costimulatory molecules are up-regulated after activation of APCs
upon pathogen exposure, which enables APCs to provide adequate immune responses. Therefore, alteration of costimulatory
molecules expressed on APCs may be a major target of viral
immune evasion. To determine whether JEV induced alteration
of costimulatory activation markers in both DCs and macrophages, we examined the expression levels of CD40, CD80,
CD86, MHC class I, and MHC class II molecules in JEV-infected DCs and macrophages by flow cytometric analysis. As
shown in Fig. 3A, JEV infection resulted in differential modulation of costimulatory molecules in both bmDCs and bmM␾.
Specifically, CD40 and MHC class II expression in bmDCs was
profoundly down-regulated by JEV infection, while CD80 and
CD86 showed enhanced expression (Fig. 3A). The enhanced
expression of MHC class I molecule was also observed in JEVinfected bmDCs, as seen in other flaviviral infection (45). In
contrast, after JEV infection bmM␾ showed clearly enhanced
expression of all CD40, CD80, CD86, and MHC classes I and
II molecules that were evaluated in this study (Fig. 3A). Moreover, when we examined the number of splenic DC subsets and
macrophages in mice infected with JEV, the significantly reduced number of splenic CD11chighCD8␣⫹ DC subset was observed at 7 days p.i., the time when viral RNA load was found
to peak. However, there was no change in the number of plasmacytoid DC (CD11chighB220⫹) and macrophages (F4/
80highCD11b⫹) (supplemental Table I).5 Also, JEV infection
induced similar in vivo modulation of costimulatory molecules
in splenic CD11chigh DCs and F4/80high macrophages. With the
exception of CD80, CD86, and MHC class I molecules, the
expression of CD40 and MHC class II molecules was consistently reduced in splenic CD11chigh DCs, which followed the
pattern observed with in vitro experiments (Fig. 3B). In contrast, splenic F4/80high macrophages showed enhanced expression of all tested activation markers following JEV infection
(Fig. 3B). These results suggest that JEV induces differential
alteration of the expression of costimulatory activation markers
in DCs and macrophages.
5
The online version of this article contains supplemental material.
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FIGURE 3. Phenotypic changes in DCs and macrophages infected with JEV. A, Phenotypic changes of bmDCs and bmM␾. DCs (bmDCs) and
macrophages (bmM␾) derived from bone marrow cells were infected with JEV Beijing-1 strain (5 ⫻ 105 TCID50/ml) and used to stain activation phenotypic
markers (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 phenotypic
markers. 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 the
indicated molecule in cells gated on CD11chigh and F4/80high cells is representative of four mice, and the values in histograms are the average of relative
MFI levels obtained from each mouse.
2468
FUNCTIONAL IMPAIRMENT OF DCs BY JEV
The cytokine production and phenotypic alteration of
JEV-infected DCs is partially dependent on MyD88
and p38 MAPK molecules
APCs have a range of innate receptors, including TLRs and
non-TLRs, that recognize pathogens (8). To examine the role
that TLRs play in the induction of cytokines by DCs and macrophages following JEV infection, we used DCs and macrophages derived from mice that lacked the MyD88 molecule,
which acts as an adaptor molecule in signal transduction from
all TLRs except TLR3. As shown in Fig. 4A, bmDCs prepared
from MyD88-deficient mice showed reduced production of
IL-6, IL-10, IL-12, and TNF-␣ following JEV infection, when
compared with bmDCs from wild-type mice. Similarly, JEVinfected bmM␾ showed reduced production of inflammatory
cytokines in the absence of the MyD88 adaptor molecule. These
findings suggest that cytokine production from JEV-infected
DCs and macrophages is partially dependent on signal transduction by MyD88 adaptor molecule. In particular, IL-6, IL-10,
and IL-12 production was markedly reduced in the absence of
MyD88 adaptor molecule, whereas TNF-␣ production by
MyD88-deficient bmDCs was not significantly reduced (Fig.
4A). Furthermore, bmDCs and bmM␾ derived from MyD88deficient mice also showed altered phenotypes of costimulatory
molecules following JEV infection (Fig. 4B). However, MyD88deficient bmDCs and bmM␾ failed to show more apparent changes
in any of the tested comstimulatory molecules in response to JEV
infection when compared with wild-type DCs. Consistent with
these results, the production of cytokines by splenic CD11chigh
DCs and F4/80high macrophages was found to depend on MyD88
adaptor molecule when splenic DCs and macrophages purified
from wild-type and MyD88-deficient mice were used for JEV infection (Fig. 4C). These findings indicate that MyD88 adaptor
molecule and possibly TLRs play a role in shaping innate and
adaptive immune responses against JEV. Moreover, these results
suggest that other MyD88-independent pathways contribute to
functional modulation of DCs and macrophages following JEV
infection, since the complete disappearance of cytokine production
and the expression of costimulatory molecules in MyD88-deficient
cells was not observed. To further characterize the signal transduction involved in the production of cytokines by JEV-infected
DCs, we used several inhibitors of MAPK, including p38, ERK,
JNK, and MEK-1. Of these inhibitors, treatment with p38 MAPK
inhibitor (SB203580) resulted in a marked reduction of IL-6, IL10, IL-12, and TNF-␣ production by wild-type bmDCs following
JEV infection (Fig. 4D), which suggests that the p38 MAPK pathway plays a pivotal role in cytokine production by JEV infection.
Taken together, these results demonstrate that the MyD88-dependent signal pathway may be involved in cytokine production by
JEV-infected DCs along with other MyD88-independent cellular
signal pathways.
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FIGURE 4. Involvement of MyD88 adaptor molecule and p38 MAPK in the production of cytokine by DCs and macrophages infected with JEV. A, The
production of cytokines from JEV-infected DCs and macrophages is partially dependent on the MyD88 adaptor molecule. bmDCs and bmM␾ prepared from
wild-type (C57BL/6) and MyD88-deficient (MyD88 KO) mice were infected with JEV Beijing-1 strain (5 ⫻ 105 TCID50/ml). The cytokine levels in culture
supernatants of the infected bmDCs were quantified by ELISA at 24 h p.i. B, The phenotypic changes of MyD88-deficient DCs and macrophages following
JEV infection. The expression levels of activation phenotypic markers (CD40, CD80, CD86, MHC classes I and II) were determined by flow cytometric
staining 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 of
cytokines produced by splenic CD11chigh DCs and F4/80high macrophages of wild-type and MyD88-deficient mice were determined by ELISA 24 h
following JEV infection. D, Dependence of cytokine production by JEV-infected DCs on p38 MAPK. DCs derived from bone marrow cells of C57BL/6
mice were infected with JEV in the presence or absence of p38 inhibitor (SB203580; 500, 50, and 5 ␮M). The cytokine levels in the culture supernatant
of infected DCs were determined by ELISA at 24 h p.i. ⴱ, p ⬍ 0.05; ⴱⴱ, p ⬍ 0.01; ⴱⴱⴱ, p ⬍ 0.001 compared with the levels of wild-type DCs infected
with JEV; n.d., not detected.
The Journal of Immunology
2469
JEV-infected DCs show defects in the priming of Ag-specific
CD4⫹ and CD8⫹ T cells
DCs are key players in the generation of adaptive T cell responses
through presentation of cognate Ags to T cells using MHC molecules. The quality and quantity of T cell responses is determined
not only by the level of Ag presented but also by the costimulatory
signals stimulated by the interaction of costimulatory molecules on
APCs with their ligands and the cytokine milieu. In a view of the
altered phenotype and cytokine production that occur in response
to infection, JEV-infected DCs may have important effects on T
cell priming and proliferation and the subsequent generation of
effector functions. To explore the ability of JEV-infected DCs to
prime Ag-specific CD4⫹ and CD8⫹ T cells, we used a TCR-transgenic model system of OT-II and OT-I mice from which we isolated a homogeneous population of naive Ag-specific CD4⫹ and
CD8⫹ T cells. As shown in Fig. 5, A and B, the primary proliferation and cytokine production of CD4⫹ and CD8⫹ T cells were
assessed after JEV-infected bmDCs that had been pulsed with cog-
nate peptide were cocultured with CD4⫹ and CD8⫹ T cells purified from OT-II and OT-I mice, respectively. CD4⫹ and CD8⫹ T
cells primed with JEV-infected bmDCs showed significantly less
proliferation than did those that were primed with mock-infected
bmDCs. The differences were more apparent when the ratio of
DCs to T cells was low, due to increased competition for DCs.
Similarly, CD4⫹ and CD8⫹ T cells primed with JEV-infected bmDCs were found to produce reduced amounts of the cytokines
IL-2, IL-4, and IFN-␥ (Fig. 5, A and B). To further confirm the
defective ability of JEV-infected DCs to prime CD4⫹ and CD8⫹
T cells, the ability of splenic CD11chigh DCs from JEV-infected
mice to support CD4⫹ and CD8⫹ T cell proliferation was compared with that of splenic CD11chigh DCs from uninfected mice
(Fig. 5C). Splenic CD11chigh DCs from JEV-infected mice were
found to have a decreased ability to induce the proliferation of
Ag-specific CD4⫹ and CD8⫹ T cells. Therefore, these results indicate that JEV induces defective T cell responses through modulation of the function of DCs.
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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) T
cells 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 prime
CD4⫹/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- and
mock-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 different
ratios. The levels of cytokine IFN-␥, IL-4, and IL-2 in culture supernatant were determined by ELISA at the indicated time. C, Splenic CD11chigh DCs
obtained from JEV-infected mice have a reduced ability to stimulate CD4⫹ and CD8⫹ T cells. CD4⫹ and CD8⫹ T cells were purified from corresponding
OT-II and OT-I mice and stimulated with splenic CD11chigh DCs purified from mice that were previously infected with JEV (103 TCID50). The proliferation
of CD4⫹ and CD8⫹ T cells was assessed by a bioluminescence assay following 72 h of incubation. The means ⫾ SD of RLUs from wells evaluated in
quadruplicate are shown. ⴱ, p ⬍ 0.05; ⴱⴱ, p ⬍ 0.01; ⴱⴱⴱ, p ⬍ 0.001 compared with mock-infected group.
2470
FUNCTIONAL IMPAIRMENT OF DCs BY JEV
CD8, but not CD4, T cells primed by JEV-infected DCs show
reduced proliferation in an IL-10-dependent manner
JEV infection induces the reduced CD4⫹ and CD8⫹ T cell
responses depending on MyD88 molecules
To test the involvement of MyD88 molecule in defective priming of CD4⫹ and CD8⫹ T cells by JEV-infected DCs, when we
examined the capacity of MyD88-deficient DCs to prime CD4⫹
and CD8⫹ T cells after JEV infection, MyD88-deficient DCs
showed higher proliferation of CD4⫹ and CD8⫹ T cells than
did wild-type DCs (data not shown), which indicates that
MyD88 adaptor molecule may mediate impairment of JEV-infected DCs to prime CD4⫹ and CD8⫹ T cells. Therefore, to
determine the involvement of MyD88 adaptor molecule in in
vivo defective CD4⫹ and CD8⫹ T cell responses in JEV-infected mice, we investigated the generation of CD4⫹ and CD8⫹
T cell responses using an HSV-1 challenge model. As shown in
Fig. 7, wild-type and MyD88-deficient mice were immunized
with HSV-1 seven days after JEV infection because viral RNA
load and reduced numbers of DC subsets were found to peak at
this time. The responses of CD4⫹ and CD8⫹ T cells were then
evaluated. Mice infected with JEV had significantly decreased
HSV-specific proliferation of purified CD4⫹ T cells and 2-fold
fewer IFN-␥-producing CD4⫹ T cells in response to HSV-1
antigenic stimulation than did the mock-infected group (Fig.
7A). Such suppressed responses of CD4⫹ T cells by JEV infection were observed to be more obvious in wild-type mice
than those in MyD88-deficient mice, which indicated that the
MyD88 adaptor molecule plays a role in generation of Ag-specific responses following JEV infection. It is also interesting to
note that MyD88-deficient mice showed reduced responses of
CD4⫹ T cells when compared with wild-type mice. Similarly,
mice that received JEV infection showed markedly suppressed
CD8⫹ T cell responses when HSV-specific CD8⫹ T cells were
FIGURE 6. Neutralization of IL-10 rescues the suppressed proliferation
of 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 cells
with JEV-infected bmDCs in the presence of IL-10 neutralizing Ab. DCs
derived from bone marrow cells of wild-type C57BL/6 mice were infected
with JEV Beijing-1 strain (5 ⫻ 105 TCID50/ml) and then pulsed with cognate 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⫹ and
CD8⫹ T cells was assessed by using a bioluminescence assay following a
96-h incubation with treated bmDCs in the presence of IL-10 neutralizing
Ab. The means ⫾ SD of RLUs from wells evaluated in quadruplicate are
shown. C, Phenotypic change in bmDCs following JEV infection in the
presence of IL-10-neutralizing Ab. DCs were infected with JEV in the
presence or absence of IL-10-neutralizing Ab and used for flow cytometric
staining of the surface activation markers (CD40, CD80, CD86, MHC
classes I and II) 24 h later. The bars in graph show the average ⫾ SD of
relative MFI obtained from each treated group (n ⫽ 4). ⴱⴱ, p ⬍ 0.01; ⴱⴱⴱ,
p ⬍ 0.001 compared with JEV plus isotype-treated group.
observed by in vivo CTL killing activity (Fig. 7B). JEV infection resulted in an average of 27% lysis of specific targets in
spleen, whereas 97% lysis of the targets was observed in mockinfected mice. In the absence of MyD88 adaptor molecule, such
differences of CD8⫹ T cell responses between JEV- and
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As with costimulatory molecules on the surface of DCs and soluble mediators in the microenvironment, several factors determine
the optimal priming of CD4⫹ and CD8⫹ T cells by DCs. Conversely, the presence of soluble factors such as IL-10 may exert
negative influences on the priming of CD4⫹ and CD8⫹ T cells.
Furthermore, since DCs may function as an IL-10 source in JEV
infection through MyD88-dependent and -independent pathways,
we employed the neutralization of IL-10 biological activity with an
IL-10-neutralizing Ab to investigate the role that IL-10 plays in
impaired priming of CD4⫹ and CD8⫹ T cells by JEV-infected
DCs. The incorporation of neutralizing IL-10 Ab in the coculture
media of purified CD4⫹ T cells and JEV-infected bmDCs induced
no significant changes in the suppression of the proliferation of
CD4⫹ T cells (Fig. 6A). However, suppression of the proliferation
of CD8⫹ T cells by JEV-infected bmDCs was reversed by the
incorporation of neutralizing IL-10 Ab (Fig. 6B). Therefore, this
suggests that IL-10 from JEV-infected DCs mediates reduced
priming of CD8⫹ T cells, but not CD4⫹ T cells. IL-10 acts on
APCs and causes down-regulation of their activation status. To test
if the incorporated IL-10 Ab affected the altered phenotype of
JEV-infected bmDCs, we examined the phenotypic modulation of
JEV-infected bmDCs in the presence of neutralizing IL-10 Ab. As
shown in Fig. 6C, the neutralization of IL-10 activity led to partial
recovery of the expression of CD40 and MHC class II molecules
on infected bmDCs. Taken together, these results suggest that
IL-10 produced by JEV-infected DCs can mediate impaired responses of CD8⫹ T cells. However, the reduced priming of CD4⫹
T cells by JEV-infected DCs may occur through unknown
pathways.
The Journal of Immunology
2471
mock-infected mice were significantly reduced, and MyD88deficient mice mounted comparable HSV-specific CD8⫹ T cell
responses to wild-type mice (Fig. 7B). Consistent with these
results, JEV-infected mice were found to have a decreased number of IFN-␥-producing CD8⫹ T cells following stimulation
with immunodominant epitope of HSV-1 (gB498 –505, SSIEFARL), which resulted in partial recovery due to the lack of
MyD88 molecule (Fig. 7, C and D). These results demonstrate
that MyD88 adaptor molecule may be involved in the subversion of CD4⫹ and CD8⫹ T cell responses by JEV infection.
To test if defective T cell responses in response to JEV infection
result in greater susceptibility to secondary microbial infection,
C57BL/6 mice that were previously infected with JEV were challenged with zosteriform infection of HSV-1 strain 17 on the day
7 p.i. Despite that JEV induced no morbidity and mortality, clinical
signs of HSV zosteriform infection progressed at a much faster
rate in JEV-infected mice than in mock-infected mice (Fig. 7E).
Furthermore, 66% of HSV-challenged mock-infected mice survived for 17 days p.i., while only 22% of JEV-infected mice survived for the same length of time ( p ⫽ 0.041). MyD88-deficient
mice that received JEV infection showed enhanced susceptibility
to HSV-1 zosteriform infection, but this enhanced susceptibility
was not significantly different from that of mock-infected MyD88deficient mice. On the other hand, the susceptibility of MyD88deficient mice to HSV-1 zosteriform infection was greater than in
wild-type mice, as supported by a previous report (46). Taken
together, these results suggest that JEV infection can cause im-
paired CD4⫹ and CD8⫹ T cell responses in MyD88-dependent
and -independent manners, which result in the generation of defective antiviral immune responses.
Discussion
DCs and macrophages play important roles in conferring antiviral
immunity during the initial stages of viral infection. Recognition of
viral infection by DCs and macrophages through TLR and/or nonTLR sensors induces immediate production of inflammatory cytokines that subsequently provide early antiviral immunity (6 – 8).
In addition to activation of innate effector cells, signals from TLRs
and/or non-TLRs result in maturation of macrophages and DCs,
thereby leading to T cell priming. Thus, signals of innate receptors
are necessary to translate innate immunity into Ag-specific responses of the adaptive immune system (6 –12). However, increased levels of inflammatory mediators can initiate irreversible
immune responses and lead to cell death (29). Therefore, a direct
viral cytopathic response and both direct and indirect immunological responses can contribute to CNS degeneration through JEVinfected cell exclusion by macrophages and CTLs, secretion of
cytokines and chemokines, and activation of microglia (30 –33).
Although DCs are critical to the priming of antiviral adaptive immune responses, few studies have been conducted to evaluate viral
infection of DCs and their role in JEV infection. The results of the
present study suggest that JEV-infected DCs undergo modulation
that differs from macrophages with respect to cytokine production
and phenotypic changes. Furthermore, signaling through the
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FIGURE 7. Reduced induction of CD4⫹ and CD8⫹ T cell responses in JEV-infected mice depends on MyD88 adaptor molecule. A, Reduced responses
of CD4⫹ T cells in JEV-infected mice. After infecting C57BL/6 (Wild-type) and MyD88-deficient (MyD88 KO) mice with JEV (103 TCID50), mice were
immunized 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-1
Ag stimulation were evaluated by proliferation using a viable cell ATP bioluminescence assay (left) and enumeration of the IFN-␥-producing cells using
ELISPOT (right). B, Suppressed in vivo CTL killing activity of Ag-specific CD8⫹ T cells in JEV-infected mice. JEV- and mock-infected (C57BL/6 and
MyD88-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 denote
the 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 recall
response. The number of IFN-␥-producing CD8⫹ T cells were then determined by intracellular cytokine staining 5 days later. The dot plot represents one
of 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 against
HSV-1 zosteriform infection. C57BL/6 and MyD88-deficient mice (n ⫽ 9) were infected with zosteriform of HSV-1 strain 17 seven days after being
infected i.p. with JEV. The graphs show the proportion of surviving mice on different days p.i.
2472
gested that impairment of DC function by JEV infection contributed to the reduction of CD4⫹ and CD8⫹ T cell responses, as
shown in Fig. 5. Additionally, MyD88-deficient DCs were found
to have a comparable proliferation of CD4⫹ and CD8⫹ T cells
after JEV infection, when demonstrated by the relative proliferation to maximum proliferation induced by mock-infected DCs
(data not shown). Although MyD88-deficient DCs themselves
showed poor proliferation of primary CD4⫹ and CD8⫹ T cells
when compared with wild-type DCs, JEV infection may impair
DC functions and induce subverted T cell responses, thereby leading to enhanced susceptibility to secondary microbial infections by
viruses such as HSV zosteriform. JEV has a positive-sense, singlestranded RNA genome that could trigger signal through several
TLR molecules such as TLR3 and TLR7 (8). When we examined
the production of cytokines by TLR2- or TLR3-deficient DCs following JEV infection, the significantly reduced production of cytokines in response to JEV was shown, but complete inhibition was
not induced (supplemental Fig. 1). Therefore, it is possible that
JEV may synthesize an array of several agonists that can trigger
TLR and/or non-TLR sensors.
Besides alteration of cytokine profile, JEV infection elicited impairment of DC maturation, as evidenced by the phenotypic activation markers. Clear down-regulation of CD40 and MHC class II
levels with marginal changes in CD80 and CD86 levels was observed in JEV-infected DCs. In contrast, macrophages elicited the
classical maturation by JEV infection, as proved by the enhanced
expression of tested costimualtory molecules. Moreover, the
MyD88 adaptor molecule appeared to contribute to such alteration
of phenotypic markers in JEV-infected DCs and macrophages
(Fig. 4B). Low expression of MHC class II and CD40 molecules
will prevent DCs from their crucial interaction with CD4⫹ Th cells
in a process called licensing, which is a step that is necessary to
enable DCs to adequately prime CD8⫹ T cells (53). Viruses
achieve down-regulation of MHC molecules by blocking trafficking to the cell membrane (54, 55), increasing destruction by ubiquitination (56), and preventing biosynthesis (57). Additionally, viruses such as murine cytomegalovirus down-regulate MHC class II
molecules through IL-10 production (58). It is likely that IL-10
produced by DCs after JEV infection is involved in down-regulation of MHC class II levels since neutralization of IL-10 by mAb
rescued the expression level of MHC class II. Interestingly, MHC
class I was up-regulated by JEV infection in a manner similar to
that observed in response to other flaviviruses (45), even though
JEV-infected DCs showed poor proliferation of CD8⫹ T cells.
Investigation using an IL-10-neutralizing Ab revealed that IL-10
may cause impaired responses in CD8⫹ T cells, even when there
is enhanced expression of MHC class I molecule. Truly JEV induces a paralytic state in DCs that is coordinated with altered cytokine profile and phenotype marker, thereby predisposing the host
to other viral infections.
JEV infection of DCs has not been well characterized. We
examined morphological changes in DCs and macrophages following JEV infection. There were no changes observed in macrophages, but morphological changes such as rounding and detachment from culture surfaces were observed in DCs, even
though cell death through apoptosis did not follow. Also, it was
likely that JEV did not replicate its RNA in DCs as efficiently
as in macrophages, and infectious progeny viruses were not
released from DCs. Since T cell activation occurs after the engagement of TCRs with DCs (1, 2), the functional impairment
of DCs by JEV may also be secondary to morphological
changes that result in disarranged cell architecture. Furthermore, since it was unlikely that all plated DCs and macrophages
were infected by JEV (Fig. 1E), some soluble factors released
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MyD88 adaptor molecule was found to have a certain role in causing subversion of primary CD4⫹ and CD8⫹ T cell responses. We
also demonstrated that IL-10 produced by JEV-infected DCs was
capable of mediating the reduced priming of CD8⫹ T cells but not
CD4⫹ T cells. Therefore, our results demonstrate that JEV induces
the functional impairment of DCs that leads to poor responses of
CD4⫹ and CD8⫹ T cells through MyD88-dependent and -independent pathways.
During viral infection, DCs undergo maturation that is manifested by up-regulation of Ag presentation, costimulatory molecules, and cytokine secretion (6, 7). Our study revealed that JEV
infection of macrophages followed the classical pathway of upregulation of tested costimulatory molecules and cytokine production (Figs. 2 and 3), but that JEV-infected DCs failed to up-regulate some costimulatory molecules. Of more interest, JEV-infected
DCs produced the antiinflammatory cytokine IL-10, which was not
detected in JEV-infected macrophages (Fig. 2). IL-10 blocks
proinflammatory cytokine production, costimulation, MHC class II
expression, and chemokine production (47, 48). IL-10 is also released with, or after, the secretion of proinflammatory cytokines in
an effort to maintain homeostasis (49). Indeed, IL-10 was released
from JEV-infected DCs along with the proinflammatory cytokines
IL-6, IL-12, and TNF-␣. Because the cytokine milieu in the microenvironment in which T cell and DC contact occurs is an important determinant of T cell outcome, alteration of the DC-produced cytokine profile may be an effective strategy for the
prevention of T cell activation during viral infection (50). Neutralizing Ab assays of IL-10 consistently demonstrated that IL-10
produced by JEV-infected DCs functioned as a mediator of the
suppression of T cell activation, at least in CD8⫹ T cells (Fig. 6).
Furthermore, it was observed that MyD88 adaptor molecule mediated the production of IL-10 by JEV-infected DCs (Fig. 4),
which indicates that MyD88 molecule may play a role in subversion of CD8⫹ T cell responses through release of regulatory soluble factors such as IL-10. However, the incorporation of IL-10
Ab failed to recover suppressed CD4⫹ T cell responses, even when
the expression of MHC class II and CD40 molecules was partially
recovered. Therefore, the unknown pathway by which JEV-infected DCs showed impaired CD4⫹ T cell responses requires further study. Recent studies have revealed that excessive amounts of
IL-6 and other proinflammatory cytokines have deleterious effects
on T cell responses (51), suggesting that IL-10, which is known to
inhibit DC maturation, may act synergistically with IL-6 and cause
further down-regulation of the functions by which DCs prime T
cells.
To further define the signal pathway that leads to the production
of pro- and antiinflammatory cytokines in JEV-infected DCs and
macrophages, we determined the levels of cytokine production using MyD88-deficient DCs and macrophages. The incomplete inhibition of cytokine production in the MyD88-deficient DCs and
macrophages may occur as a result of NF-␬B and MAPK also
being activated by the MyD88-independent pathway, and the intersection between the MyD88-dependent and -independent pathways is thought to be TRAF6 (20, 21). A few viruses are known
to activate p38 MAPK and augment IL-10 production by host cells
(52). Since the inhibition of p38 MAPK completely abrogated cytokine 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 cytokine
by DCs following JEV infection. To support our finding regarding
the role that the MyD88 molecule plays in modulation of DC function by JEV, we used a HSV-1 challenge model to demonstrate
that JEV infection induced subversion of CD4⫹ and CD8⫹ T cell
responses in a MyD88-dependent manner (Fig. 7). The results sug-
FUNCTIONAL IMPAIRMENT OF DCs BY JEV
The Journal of Immunology
from JEV-infected cells may indirectly affect functions such as
T cell priming in neighboring cells. In conclusion, JEV infection of DCs elicited early immune responses. These responses
were characterized by the immediate production of pro- and
antiinflammatory cytokines through MyD88-dependent and -independent pathways that resulted in p38 MAPK activation.
IL-10 and reduced costimulation is likely incapable of providing adequate signals to initiate T cell priming. The findings
presented herein suggest that imbalanced activation and modulation of both DCs and macrophages by JEV contribute to
immunopathological degeneration of the CNS and prevent adequate signals from initiating antiviral adaptive immunity,
thereby leading to boost virus survival and dissemination in
the body.
Disclosures
The authors have no financial conflicts of interest.
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