Download Murine gammaherpesvirus-68 productively infects immature

Journal of General Virology (2007), 88, 1896–1905
DOI 10.1099/vir.0.82931-0
Murine gammaherpesvirus-68 productively infects
immature dendritic cells and blocks maturation
Romana Hochreiter,1 Catherine Ptaschinski,1 Steven L. Kunkel2
and Rosemary Rochford1
Correspondence
Rosemary Rochford
[email protected]
Received 19 February 2007
Accepted 1 March 2007
1
Department of Microbiology and Immunology, SUNY Upstate Medical University, 750 East Adams
Sweet, Syracuse, NY, USA
2
Department of Pathology, University of Michigan, Ann Arbor, MI, USA
Many viruses have evolved mechanisms to evade host immunity by subverting the function of
dendritic cells (DCs). This study determined whether murine gammaherpesvirus-68 (cHV-68)
could infect immature or mature bone-marrow-derived DCs and what effect infection had on DC
maturation. It was found that cHV-68 productively infected immature DCs, as evidenced by
increased viral titres over time. If DCs were induced to mature by exposure to LPS and then
infected with cHV-68, only a small percentage of cells was productively infected. However,
limiting-dilution assays to measure viral reactivation demonstrated that the mature DCs were
latently infected with cHV-68. Electron microscopy revealed the presence of capsids in the
nucleus of immature DCs but not in mature DCs. Interestingly, infection of immature DCs by
cHV-68 did not result in upregulation of the co-stimulatory molecules CD80 and CD86 or
MHC class I and II, or induce cell migration, suggesting that the virus infection did not induce
DC maturation. Furthermore, cHV-68 infection of immature DCs did not result in elevated
interleukin-12, an important cytokine in the induction of T-cell responses. Finally,
lipopolysaccharide and poly(I : C) stimulation of cHV-68-infected immature DCs did not induce
increases in the expression of co-stimulatory molecules and MHC class I or II compared with
mock-treated cells, suggesting that cHV-68 infection blocked maturation. Taken together, these
data demonstrate that cHV-68 infection of DCs differs depending on the maturation state of
the DC. Moreover, the block in DC maturation suggests a possible immunoevasion strategy by
cHV-68.
INTRODUCTION
Dendritic cells (DCs) play a key role in the initiation of a
primary T-cell response (Banchereau et al., 2000; Hart,
1997) and also function in the maintenance of adaptive
immune responses. Several features of myeloid DCs
contribute to their function. For example, DCs are very
efficient at antigen uptake and processing and are able to
present antigen for both MHC class I and class II pathways
(Banchereau et al., 2000; Banchereau & Steinman, 1998).
Of equal importance is their capacity to migrate through
tissue as part of the peripheral immune surveillance (Caux
et al., 2000). Induction of T-cell immunity against microbial pathogens is thought to occur following the recruitment of immature DCs from the periphery to lymphoid
tissues and their subsequent differentiation into mature
DCs (Kelsall et al., 2002). Numerous phenotypic and functional features distinguish immature from mature DCs.
Immature DCs phagocytose pathogens; this ability is lost
following maturation. Maturation of DCs is associated
with increases in expression of the co-stimulatory molecules CD80 (B7.1) and CD86 (B7.2), upregulation of MHC
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class I and II (Ardavin, 2003) and expression of cytokines
such as interleukin (IL)-12 (Trinchieri et al., 2003).
Maturation also results in the upregulation of the chemokine receptor CCR7 directing the DCs to the lymph nodes
where they prime a T-cell response (Forster et al., 1999;
Martin-Fontecha et al., 2003). Because of the primary role
of DCs in the initiation of adaptive immunity, viruses have
evolved numerous strategies to subvert DC function
(reviewed by Moll, 2003). One strategy observed in several
viral infections is the inhibition of DC maturation
(Engelmayer et al., 1999; Moutaftsi et al., 2002; Pollara
et al., 2003; Salio et al., 1999; Smith et al., 2005).
Two important human pathogens, the gammaherpesviruses Epstein–Barr virus (EBV) and Kaposi’s sarcoma
herpesvirus (KSHV) infect DCs (Li et al., 2002;
Rappocciolo et al., 2006). EBV can infect precursor myeloid cells and interferes with DC development, thus preventing the differentiation of sufficient numbers of DCs (Li
et al., 2002). Patients with Kaposi’s sarcoma have been
reported to have functionally impaired DCs (Stebbing et al.,
2003), but whether this is due to infection with KSHV is
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Gammaherpesvirus infection of DCs
unknown. More recently, KSHV was shown to bind to DCSIGN on myeloid DCs ex vivo, and infection of DCs
inhibits endocytosis and antigen presentation (Rappocciolo
et al., 2006). Although these studies suggest that infection
of DCs could be an integral part of the gammaherpesvirus
pathogenesis, there are limitations in the study of EBV and
KSHV pathogenesis.
An amenable model system to study gammaherpesvirus
pathogenesis utilizes murine gammaherpesvirus-68 (cHV68). cHV-68-infected DCs can be isolated from lymphoid
compartments (e.g. spleen and mediastinal lymph node)
and from lung (Flano et al., 2000, 2003, 2005; Marques
et al., 2003). More recently, Flano et al. (2005) demonstrated that bone-marrow-derived DCs could be infected
with cHV-68. In their study, both productive and latent
infection of DCs was detected following ex vivo infection,
but they did not distinguish immature from mature DCs in
their analysis (Flano et al., 2005). An important question
that remains is whether cHV-68 preferentially infects
immature DCs or mature DCs, or whether cHV-68 is
capable of infecting both.
In this study, we tested whether cHV-68 infection of bonemarrow-derived DCs was dependent on the maturation
state of the DCs and whether infection resulted in phenotypic changes. We found that cHV-68 productively infected
immature DCs and blocked maturation. In contrast, bonemarrow-derived DCs driven to maturation following lipopolysaccharide (LPS) stimulation were latently infected with
cHV-68. In addition, we found that cHV-68 infection of
bone-marrow precursor cells drastically reduced the development of DCs. These data suggest that, like the other members
of the herpesvirus family, gammaherpesviruses target DCs as
one potential strategy to subvert host immunity.
METHODS
Cell lines and viruses. Owl monkey kidney (OMK) cells (ATCC
CRL-1556) and NIH 3T3 cells (ATCC CRL-1658) were maintained in
culture as described previously (Rochford et al., 2001). MEF-1 cells
(ATCC CRL-2214) were maintained in DMEM containing 4.5 g
glucose l21, 1 mM sodium pyruvate, 10 % fetal bovine serum (FBS),
4 mM glutamine, 100 IU penicillin ml21 and 100 mg streptomycin ml21. cHV-68 expressing enhanced green fluorescent protein
(cHV-eGFP) was a generous gift of Dr Ren Sun (UCLA, Los Angeles,
CA, USA) and the generation of this recombinant virus has been
described previously (Wu et al., 2001). Generation of virus stocks and
determination of viral titre by plaque assay were carried out as
described previously (Cardin et al., 1996), with the exception that
some plaque assays were done on MEF-1 cells instead of NIH 3T3
cells. To UV-inactivate virus, 1 ml of virus stock was placed in a 24well plate and exposed to UV light for three 5 min exposures in a
Stratalinker (Strategene). Plaque assays on virus stocks following UV
exposure indicated that the virus had been inactivated (data not
shown). For mock infection, uninfected OMK cells were processed
exactly as if they had been virus infected (Rochford et al., 2001). The
resulting supernatant was used for all mock infections.
cells were flushed out of the tibia and femur of C57Bl/6 mice. After
ammonium chloride lysis of red blood cells, 26106 bone-marrow
cells were plated on non-cell-culture-treated 100 mm Petri dishes in
RPMI 1640 supplemented with 10 % heat-inactivated FBS, 2 mM
glutamine, 50 mM 2-mercaptoethanol, 100 IU penicillin ml21, 100 mg
streptomycin ml21 and 10 ng granulocyte–macrophage colony-stimulating factor (GM-CSF) ml21. GM-CSF was prepared from the
culture supernatant of X63.AG-rGMCSF cells (a gift of S. Dewhurst,
University of Rochester, NY, USA) (Zal et al., 1994). Cells were fed on
days 3 and 6 by adding 50 % fresh medium containing GM-CSF. By
day 9, cells had a typical purity of 80–90 % CD11c+ CD11b+ myeloid
DCs (data not shown). For infections, DCs were infected with cHVeGFP at an m.o.i. of 5 for 1 h. Unbound virus was removed by washing and cells were replated for 24–48 h. After transfer to new plates,
the immature phenotype of the adherent population was verified by
their low expression of CD80, CD86 and MHC II, as well as by high
uptake of FITC–dextran (data not shown). In maturation experiments,
non-adherent cells (mature) were removed prior to the addition of the
toll-like receptor (TLR) ligands LPS 026 : B6 (0.1–1 mg ml21) or
poly(I : C) (20 mg ml21) (Sigma) onto the adherent, immature DCs. In
some experiments, immature DCs harvested at day 8 of culture were
matured with 0.1–1 mg LPS 026 : B6 ml21 or poly(I : C), 24 h prior to
infection with cHV-eGFP at an m.o.i. of 5 for 1 h.
Ex vivo limiting-dilution reactivation assays and plaque assays
of infected DCs. Measurement of the frequency of viable cells that
could reactivate from latency was done by a limiting-dilution reactivation assay essentially as described previously (van Dyk et al., 2000;
Weck et al., 1996), with the exception that serial threefold dilutions of
cells were carried out. To detect pre-formed virus, a duplicate aliquot
of cells was disrupted by three rounds of freeze-thawing. To determine viral titres in infected DCs, cells were infected for 1 h, washed
and plated in 12-well plates. Supernatant and cells were harvested at
various times post-infection (p.i.), cells were disrupted by three
rounds of freeze-thawing, and plaque assays were carried out as
described previously (Cardin et al., 1996). The yield of virus for each
time point was calculated as number of p.f.u. per input number of
DCs in each well.
Electron microscopy. Immature and mature DCs were infected with
cHV-68 for 1 h at an m.o.i. of 5. After 48 h, DCs were harvested and
centrifuged. The resulting pellets were fixed overnight in 2.5 %
phosphate-buffered glutaraldehyde, post-fixed in 1 % OsO4, dehydrated in a graded ethanol series followed by propylene oxide and
then embedded in Araldite 502 epoxy resin. Ultrathin sections were
stained with uranyl acetate and Reynold’s lead citrate before examination using a Tecnai BioTWIN 12 transmission electron microscope
(FEI Co.). At least 30 cells were scanned in each preparation.
Immunofluorescence staining and flow cytofluorimetric analyses. All methods, media formulations, reagents and strategies for
single-colour and multi-colour immunofluorescence staining and cell
sorting have been detailed elsewhere (Hobbs et al., 1993), with the
exception that FC-Block (BD-Pharmingen) was added. Multi-colour
immunofluorescent-stained cells were analysed with an LSRII flow
cytometer (Becton Dickinson). Live cells were collected by gating on
forward–side scatter characteristics. Data acquisition was performed
with FACSDIVA software (BD Biosciences) and data were further
analysed with FLOWJO (Tree Star Inc.) and WINMDI 2.8 software (The
Scripps Research Institute). Anti-CD11c, anti-CD80, anti-CD86,
anti-MHC class I (H-2Kb), anti-MHC class II (I-A/I-E) and antiCD11b monoclonal antibodies (mAbs) were obtained from BD
PharMingen.
Cytokine ELISA. Supernatants of DC cultures were harvested at
Culture of DCs. Bone-marrow-derived myeloid DCs were generated
by a method adapted from Lutz et al. (1999). Briefly, bone-marrow
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24 h p.i. and stored at 280 uC until analysis. The p40 subunit of
IL-12 (IL-12p40) was quantified by sandwich ELISA using the BD
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R. Hochreiter and others
OptEIA system (BD Pharmingen) according to the manufacturer’s
instructions. IL-10 was detected by a sandwich ELISA technique as
described previously (Wen et al., 2006).
Transwell migration assay. DC migration was determined by
measuring cells migrating through a polycarbonate filter (5 mm pore
size) in 24-well Transwell chambers (Costar Corning). cHV-eGFP-
infected DCs were harvested at 24 h p.i. and tested for migration
towards CCL19 chemokine (Peprotech), a CCR7 ligand. The lower
chambers of the Transwell plates were filled with 600 ml DMEM
supplemented with 1 % BSA and 10 mM HEPES with CCL19
(100 ng ml21). DCs (56105) were added in 100 ml DMEM/BSA/
HEPES into the upper chamber and cells were incubated at 37 uC for
3 h. Input cells as well as migrated cells were stained with phycoerythrin (PE)-conjugated anti-CD86 and analysed by flow cytometry.
RESULTS
The maturation state of DCs affects their
susceptibility to productive cHV-68 infection
To investigate the susceptibility of DCs of different maturation states to cHV-68 infection, we first isolated and
cultured DCs from C57BL/6 bone marrow. After 8 days of
culture, the ex vivo-expanded DCs were left untreated
(immature) or treated with LPS to induce maturation.
After 24 h, cells were infected with a recombinant cHV-68
that expressed eGFP under control of the human cytomegalovirus (HCMV) promoter (cHV-eGFP). Cells were
harvested 24 h later and stained with allophycocyanin
(APC)-conjugated anti-CD11c mAb (a DC cell-surface
marker) and PE-conjugated anti-CD86 mAb (a maturation
marker). In a representative set of over ten experiments,
greater than 70 % of cells were immature CD11c+ CD86lo
DCs. Of these immature cells, 70 % were infected, as indicated by the percentage of CD11c+ cells that were GFP
positive (Fig. 1a). In contrast, DCs driven to maturation by
treatment with LPS and then infected with cHV-eGFP
appeared to be refractory to cHV-eGFP infection, as
indicated by the very low number of GFP-expressing cells
(Fig. 1a).
To determine whether the immature DCs were productively infected with cHV-68, we performed plaque assays
on both cell-associated virus and supernatant harvested
from immature DCs and mature DCs at various times p.i.
Wells were infected in duplicate and the mean titre in p.f.u.
per 106 input DCs from a representative experiment is
shown (Fig. 1b). We observed that the supernatant virus
titre in the infected immature DCs increased between 8 and
24 h p.i., but reached a plateau by 48 h p.i. The cellassociated virus titre reached its peak at 24 h p.i. and then
declined. In contrast, following infection of mature DCs,
virus titres did not increase significantly from the input
levels, suggesting that they were not productively infected.
Cell viability of both mature and immature DCs was
slightly lower than mock-infected cells at 24 h p.i. and
began to decline by 48 h p.i. (Fig. 1c). However, even at
48 h p.i., there was still greater than 70 % cell viability of
infected DCs.
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Although less than 8 % of cells were GFP positive following
infection of mature DCs, it remained possible that GFP was
not expressed if the cells were latently infected. To test this
possibility, we performed a limiting-dilution reactivation
assay to determine whether there were latently infected
mature DCs. In this assay, both viable cells and cells that
had been mechanically disrupted to release pre-formed
virus were plated onto susceptible MEFs and the percentage of cells showing cytopathic effect (CPE) was calculated
to determine the frequency of infected cells. As shown in
Fig. 1(d), for immature DCs, there was no difference in the
percentage of wells showing CPE when live cells were
plated compared with cell lysates, indicating that the
infected immature DCs were productively infected and
there were no latently infected cells. In contrast, we
observed a difference in the frequency of wells showing
CPE between wells that received live cells (1 : 3.5) versus
disrupted cells (1 : 9). This meant that the majority of
mature DCs were latently infected with cHV-68.
To compare cHV-68 infection of immature and mature
DCs further, electron microscopy studies were performed.
Immature and mature DCs were infected for 48 h, and cells
were washed and then fixed before being processed for
electron microscopy. As shown in Fig. 2(a, b), nucleated
capsids were clearly seen in the nucleus of infected immature DCs. Interestingly, we also observed infected DCs
being phagocytosed by other DCs in the culture (Fig. 2c).
Higher magnification of the phagocytosed infected DCs
revealed that the capsids appeared to be empty in contrast
to the capsids from infected but non-phagocytosed DCs
(compare Fig. 2b and d). No capsids were observed in the
nucleus of mature DCs (data not shown). Taken together,
the data presented demonstrate that the maturation state
of the DCs affects their ability to sustain a productive
infection (immature DCs) or a latent infection (mature
DCs).
cHV-68 infection of immature DCs blocks
maturation
Immature DCs are characterized by their ability to phagocytose and have relatively low-level expression of costimulatory molecules (CD80 and CD86), as well as MHC
class I and II. In contrast, mature DCs lack the ability to
phagocytose but have high-level expression of MHC class I
and II as well as co-stimulatory molecules. To determine
whether cHV-68 infection of immature DCs induced
their maturation, DCs infected with cHV-eGFP or mock
infected for 24 h were harvested and stained with APCconjugated anti-CD11c and PE-conjugated antibodies
specific for MHC class I and II, co-stimulatory molecules
(CD80 and CD86) and CD11b (highly expressed on myeloid DCs). Cells were gated on CD11c+ cells, and GFP
expression and histograms for individual surface markers
are shown in Fig. 3(a). As a control, immature DCs were
treated with LPS to verify that all of the cells were
susceptible to a known DC maturation signal. After 24 h of
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Journal of General Virology 88
Gammaherpesvirus infection of DCs
Fig. 1. cHV-68 infection of immature and mature DCs. (a) Immature DCs or DCs matured for 24 h with 100 ng LPS ml”1 were
infected with cHV-eGFP and harvested 24 h later. Cells were stained with APC-conjugated CD11c and PE-conjugated CD86
mAbs. Cells were gated on CD11c+ (top panel) and analysed for expression of CD86 (maturation marker) and GFP (virus
infection) as shown in the bottom panel. (b) Immature DCs or DCs matured for 24 h with 100 ng LPS ml”1 were infected with
cHV-eGFP. At 1.5, 4, 8, 24, 48 and 72 h p.i., supernatants (filled bars) and cell lysates [i.e. cell-associated virus; empty bars]
were harvested and virus titres were determined by plaque assays. The number of p.f.u. is reported based on the input number
of DCs per well. (c) Immature DCs or DCs matured for 24 h with 100 ng LPS ml”1 were infected or not with cHV-eGFP (filled
bars, mock infection; shaded bars, cHV-68; empty bars, UV-inactivated cHV-68). After 24, 48 or 72 h p.i., cell viability was
determined by a trypan blue exclusion assay. (d) Limiting-dilution reactivation assay. Immature (upper panel) and LPS-matured
(lower panel) DCs were infected for 24 h at an m.o.i. of 5. Cells were harvested and plated on MEFs in limiting dilution. In
parallel, a separate aliquot of cells was disrupted by freeze-thawing and plated on MEFs. The results are shown as the
percentage of wells positive for CPE relative to the value scored positive for viral CPE 3 weeks after plating. Twenty-four wells
were plated per cell dilution in each experiment. The results shown are representative experiments from a minimum of three
different experiments. X, Total; &, lysate.
LPS treatment, we observed a significant increase in DC
maturation markers. In contrast, MHC class II and CD80
and CD86 levels remained significantly lower in the
infected GFP+ cells, suggesting that viral infection did
not induce differentiation. No changes in MHC class I
expression were observed following infection. To test that
binding of virus particles induced DC maturation, DCs
were infected with cHV-68 that had been inactivated by UV
irradiation. Increases in CD86 and MHC II were observed
following infection with UV-inactivated virus, suggesting
that inhibition of maturation by cHV-68 requires virus
replication. In Fig. 3(b), the mean fluorescence intensity
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from six to eight experiments is shown. These data showed
that infection of immature DCs did not upregulate CD80,
CD86 or MHC class II compared with LPS-matured DCs or
DCs infected with UV-inactivated virus.
cHV-68 infection of DCs inhibits chemotaxis
In addition to upregulation of co-stimulatory molecules,
maturation of DCs is also characterized by changes in the
expression of the chemokine receptors (Sallusto et al.,
1998; Vecchi et al., 1999). Maturing DCs downregulate
CCR1, CCR5 and CXCR1 and upregulate CXCR4, CCR4
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R. Hochreiter and others
To assess the effect of cHV-68 on the expression of IL-12,
we infected DCs with cHV-eGFP or treated the cells with
UV-inactivated virus or mock lysate. After 24 h, IL-12p40
and IL-10 were measured in the cell-culture supernatants
by ELISA (Fig. 3d). Significantly lower levels of IL-12 were
observed in the cHV-eGFP-infected wells compared with
cells treated with UV-inactivated virus or mock infected. In
contrast, both mock- and cHV-eGFP-infected DCs produced IL-10, although the level of IL-10 in the infected cells
was not significantly higher than in the mock-infected cells.
No IL-10 was observed in the DCs infected with UVinactivated virus.
cHV-68 infection of DCs blocks LPS and
poly(I : C)-induced maturation
Fig. 2. Visualization of cHV-68-infected DCs. Immature DCs were
infected with cHV-68 at an m.o.i. of 5, and at 48 h p.i. cells were
harvested and visualized under an electron microscope as
described in Methods. The images in (b) and (d) represent the
boxed insets in (a) and (c), respectively. Bars, 2 mm (a, c); 0.5 mm
(b, d).
and CCR7. In particular, CCR7, which responds to the
chemokines CCL19 and CCL21, is important for directing
DCs to the lymphoid organs (Martin-Fontecha et al., 2003;
Sallusto et al., 1998). To test whether infected DCs were
responsive to CCL19, we performed a Transwell migration
assay to measure the capacity of infected DCs to migrate.
DCs were infected with cHV-eGFP for 24 h and the
adherent population was harvested and placed in the upper
chamber of a Transwell. Cells were routinely greater than
80 % viable at the time of transfer. The lower chamber
contained the CCR7 ligand CCL19 (100 ng ml21). Only a
small percentage of infected DCs was able to migrate
towards CCL19 (Fig. 3c). Even stimulation with LPS for
24 h after infection did not increase the number of migrating cHV-68-infected cells (data not shown). This suggested that cHV-68 infection of DCs not only inhibited the
upregulation of co-stimulatory molecules, but also prevented the induction of DC migration in infected cells.
cHV-68 infection of DCs inhibits IL-12 expression
but not IL-10 expression
Activation and maturation of DCs causes secretion of
IL-12, a potent stimulator of gamma interferon (IFN-c)
and inducer of Th1 responses (Lamont & Adorini, 1996).
1900
DCs can be induced to mature via stimulation with signalling through TLRs. To determine whether cHV-68 infection impaired DC maturation induced by LPS (a TLR4
ligand) or poly(I : C) (a TLR3 ligand), immature DCs were
infected with cHV-eGFP or mock infected. After 24 h,
non-adherent cells were removed and discarded and the
ability of the immature, adherent cells to mature was
tested by adding 1 mg LPS ml21, 20 mg poly(I : C) ml21 or
medium alone for an additional 24 h (Fig. 4). After a total
of 48 h of infection, cells were stained with appropriate
antibodies and flow cytometry was carried out to assess
changes in DC phenotype. As expected, we observed
increased levels of CD80, CD86 and MHC class II in
response to LPS and poly(I : C) in mock-infected cells,
demonstrating the susceptibility of ex vivo-generated,
bone-marrow-derived DCs to maturation signals. However, the majority of infected DCs (GFP+ DCs) showed
limited responsiveness to LPS or to poly(I : C) stimulation
as measured by the low-level expression of CD80, CD86
and MHC class II. Together, these data suggested that
cHV-68 infection not only failed to induce DC maturation,
but also blocked maturation induced by the TLR ligands
LPS and poly(I : C).
Supernatant from mature DCs blocks productive
infection of MEFs
To test whether mature DCs express a factor that blocks
lytic infection, we treated immature DCs with LPS to
induce maturation or left them untreated. After 24 h, the
supernatant was harvested from untreated and LPSmatured DCs. MEFs, which are very sensitive to productive
cHV-68 infection, were then incubated with the supernatant for 1 or 24 h, infected for 1 h, and incubated for
6 days to determine the viral titre. We found that there was
a significantly lower viral titre following infection of MEFs
that had been pre-treated with the supernatant from
mature DCs compared with MEFs that were treated with
supernatant from immature DCs or were incubated with
medium alone (Fig. 5). This suggested that the mature DCs
secreted a soluble protein that actively inhibited lytic
replication.
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Gammaherpesvirus infection of DCs
Fig. 3. Phenotype and function of cHV-68-infected immature DCs. (a) Immature DCs were infected with cHV-eGFP or
UV-inactivated virus, or were mock infected or treated with LPS. Twenty-four hours later, adherent cells were harvested and
stained with APC-conjugated anti-CD11c mAb and PE-conjugated anti-CD80, -CD86, -MHC class I or -MHC class II. Shown
is a representative FACS analysis, gated on live cells positive for CD11c and GFP for cHV-eGFP-infected cells. (b) Mean
fluorescence intensity (MFI) of UV-inactivated virus-treated and GFP+ (infected) DCs from multiple experiments were
determined at 24 h p.i. and normalized to the MFI of mock-infected cells. The graph shows the mean±SEM of six to eight
independent experiments. GFP+ DCs expressed significantly lower levels of CD80, CD86 and MHC class II compared with
UV-inactivated virus-treated DCs. *P,0.05 (paired Student’s t-test). (c) DCs that had been infected for 24 h with cHV-eGFP
were seeded in the upper chamber of a Transwell and allowed to migrate towards the lower chamber, which contained the
CCR7 ligand CCL19 (100 ng ml”1). After 3 h, input cells and migrated cells were stained with PE-conjugated anti-CD86 mAb
and analysed by flow cytometry. Circled cells represent immature DCs. (d) Immature DCs were infected with cHV-eGFP or
UV-inactivated virus, or were mock infected. Supernatants were harvested at 24 h p.i., and IL-12 and IL-10 levels were
determined by ELISA. Results are shown as the mean±SEM of at least four experiments per treatment. cHV-68-infected cells
produced significantly lower amounts of IL-12p40 compared with mock-infected and UV-inactivated virus-treated DCs.
*P,0.05 (paired Student’s t-test).
cHV-68 infection of precursor DCs blocks
development
The immature DCs used in the previous studies were
derived following isolation of bone-marrow cells and 9
days growth ex vivo. To determine whether cHV-68 infection blocked DC development, freshly isolated bonemarrow cells were infected with cHV-68 or UV-inactivated
cHV-68 or mock-infected for 1 h, and then cultured in
standard culture medium supplemented with GM-CSF to
drive DC differentiation. At 9 days p.i., we analysed the
cells for expression of CD11c and CD11b by flow cytometry (Fig. 6a). The majority of cells that were mock
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infected or infected with UV-inactivated cHV-68 expressed
high levels of CD11c and CD11b (84.6 and 93.4 %, respectively), indicative of immature DCs. In contrast, following cHV-68 infection, only 36.8 % of cells were CD11chi
CD11bhi. Interestingly, the majority of the cells were
CD11clo CD11bhi, indicating that they were more typical of
granulocytes, a population not seen in the mock-infected
or UV-inactivated virus-treated cultures. Analysis of the
cell yield also revealed that cHV-68 infection resulted in a
significant reduction in the total cell yield (Fig. 6b). Thus,
infection of bone-marrow cells with cHV-68 depleted
immature DCs and promoted outgrowth of granulocytes.
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R. Hochreiter and others
Fig. 4. Effect of cHV-68 infection on the expression of maturation
markers induced by LPS or poly(I : C) stimulation. Immature DCs
were infected with cHV-eGFP at an m.o.i. of 5 or were mockinfected. Twenty-four hours later, the medium was replaced and
cells were left untreated or stimulated with 1 mg LPS ml”1 or 20 mg
poly(I : C) ml”1 for an additional 24 h. Cells were harvested and
stained with APC-conjugated anti-CD11c mAb and PE-conjugated anti-CD80, -CD86 and -MHC class I, -MHC class II mAbs
or isotype controls (not shown). For analysis, cells were gated on
CD11c and GFP. Histograms show CD11c+ GFP+ cells (shaded
histogram) in comparison with CD11c+ mock-infected cells (black
line). Similar results were obtained in two additional experiments.
DISCUSSION
Herpesviruses have evolved a variety of mechanisms to
escape the immune response in order to establish life-long
latency in the host. Because of their central role in initiating
antiviral immune responses, DCs have been targeted by
viruses from several different viral families (reviewed by
Tortorella et al., 2000). In this study, we have reported
that cHV-68 can infect immature DCs and block their
Fig. 5. Effects of supernatant (SN) from mature DCs on virus
replication. MEFs were incubated with supernatant harvested from
immature DCs, DCs induced to mature for 24 h with LPS or
medium for 1 or 24 h prior to infection with cHV-68 at an m.o.i. of
5. Cells were then incubated for 6 days and virus titre was
determined by plaque assay. Results are shown as the mean±SEM
of three experiments. A significantly lower titre was found in mature
supernatant-treated MEFs compared with mock or immature
supernatant-treated MEFs at 24 h. *P,0.01 (paired Student’s
t-test).
1902
Fig. 6. Infection of DC precursors. Bone-marrow cells were
infected with cHV-eGFP at an m.o.i. of 5, or were treated with
mock lysate or UV-inactivated virus. (a) After 9 days of culture in
GM-CSF, non-adherent cells were harvested and stained with
APC-conjugated anti-CD11c mAb and PE-conjugated antiCD11b mAb and analysed by flow cytometry. (b) Cell yield was
determined by counting cells using a haemocytometer. Cell yield in
mock-treated cultures was set at 100 % and the cell numbers in
the cHV-68-infected cultures were normalized to that value. The
graph shows the mean±SEM of three independent experiments.
maturation, suggesting that this is another strategy used to
subvert host immunity.
Previous studies have found that bone-marrow-derived
DCs can be infected with cHV-68 and that both latently
and lytically infected cells are detected in the cultures
(Flano et al., 2005). Our studies confirmed these results
and extended them by demonstrating that lytic infection
was predominantly restricted to the immature DCs, whilst
LPS-matured DCs were latently infected. The different
patterns of viral infection in immature DCs versus mature
DCs is concordant with observations from in vivo infection
of mice, where lung DCs, which are predominantly immature, harbour lytic infections, whereas DCs isolated from
lymph nodes, which are predominantly mature, harbour
latent infections (Flano et al., 2005). We initially utilized a
recombinant virus, cHV-eGFP, where GFP was under the
control of the HCMV promoter, to distinguish infected
from uninfected DCs in our culture. However, limitingdilution reactivation assays revealed that the mature DCs,
whilst being GFP negative, were indeed latently infected
with the virus. Electron microscopy also confirmed the
absence of capsids in mature DCs. Thus, the HCMV promoter was suppressed in the latently infected cells. Whether
this was due to the cellular environment or to the insertion
site in the viral genome remains to be determined. None
the less, the demonstration that a latent infection is
established following infection of LPS-matured DCs opens
up a new tool to examine cHV-68 latency in an ex vivo
model. Currently, the only model system for studying
cHV-68 latency ex vivo is the S11 B-cell line derived from
tumour cells isolated from a cHV-68-infected mouse
(Usherwood et al., 1996).
We observed that if immature DCs were infected with
cHV-68 for 24 h and then treated with LPS, maturation
was blocked. This block was not seen following infection of
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Journal of General Virology 88
Gammaherpesvirus infection of DCs
immature DCs with replication-deficient, UV-inactivated
virus. This observation differs from previous studies by
Flano et al. (2005) who did not observe this block in
maturation following LPS stimulation of infected cells.
However, in their study, they treated DCs with LPS concurrent with cHV-68 infection. Together, these results
support the idea that a viral protein(s) is necessary to block
maturation, possibly through blocking maturation signals.
One possibility is that cHV-68 infection results in loss of
TLR-4, which is essential for LPS responsiveness (Poltorak
et al., 1998). We have observed that TLR4 expression is
maintained in DCs following cHV-68 infection (C.
Ptaschinski, R. Hochreiter & R. Rochford, unpublished
observations), suggesting that the block must occur further
downstream in the LPS signalling pathway. Furthermore,
a similar block in DC maturation was observed after
stimulation with poly(I : C), a TLR3 ligand. In contrast
to TLR4 signalling, which can be MyD88-dependent and
-independent, TLR3 signalling occurs only via an MyD88independent pathway (Takeda & Akira, 2004). This again
argues for a cHV-68-mediated inhibitory effect of DC
maturation on a broader range, rather than interference
with single TLR ligand expression or inhibition early in the
signalling cascade. A general inhibitory effect on protein
synthesis after cHV-68 infection was not observed up to
48 h p.i., as MHC class I, CD11b and CD11c expression
were not affected. Infection of immature DCs and subsequent inhibition of maturation has also been reported for
herpes simplex virus type 1 (HSV-1) (Pollara et al., 2003;
Salio et al., 1999), HCMV (Moutaftsi et al., 2002), murine
cytomegalovirus (Andrews et al., 2001), human herpes
virus 6 (HHV-6) (Smith et al., 2005) and vaccinia virus
(Engelmayer et al., 1999), suggesting that this is a common
viral strategy for immune evasion. HSV-1 and HCMV have
been shown to interfere with the IFN signalling JAK/STAT
pathway (Miller et al., 1998; Yokota et al., 2004).
As DCs are exquisitely sensitive to maturation signals, ex
vivo culture of bone-marrow-derived DCs routinely results
in a mixed population of cells that contain both phenotypically immature and mature DCs. By using the cHV-68
recombinant virus expressing eGFP, we were able to
determine that cHV-68 preferentially productively infects
immature DCs, characterized by low-level expression of
co-stimulatory molecules, low-level expression of both
MHC class I and II, and an inability to migrate towards
CCL19. In the infected immature DCs, we did observe a
small fraction of GFP-positive cells expressing higher levels
of CD86 (albeit not as high as in the mature DCs). We
hypothesize that these were cells that had already received
the signal for maturation before infection and thus
productive infection was not blocked. This would fit in
with the data of Flano et al. (2005), who found that LPS
treatment simultaneous with infection did not block
maturation. Our data also suggest that maturation of
DCs decreases the susceptibility to productive cHV-68
infection. One possibility for decreased susceptibility is the
production of IFNs that may induce an antiviral state in
http://vir.sgmjournals.org
LPS-matured DCs, reducing virus replication. This model
is supported by our observation that treatment of MEFs
with the supernatant from LPS-matured DCs but not from
immature DCs prior to cHV-68 infection significantly
reduced the viral yield.
In order to activate T cells, DCs need to provide three
signals: presentation of antigenic peptide in the context of
MHC molecules, high-level expression of co-stimulatory
molecules and secretion of cytokines (Lipscomb & Masten,
2002). Together, these signals not only induce T-cell proliferation but also drive CD4 T cells down the Th1 or Th2
differentiation pathways, as well as inducing T regulatory
cells (Maldonado-Lopez & Moser, 2001; Maldonado-Lopez
et al., 1999). Importantly, secretion of IL-12 by DCs is
critical for the induction of Th1 differentiation, as well as
for proliferation and enhanced cytotoxic activity of natural
killer cells (Trinchieri, 2003). We observed that DCs
infected with cHV-68 produced only very low levels of
IL-12 compared with LPS-stimulated DCs or DCs infected
with UV-inactivated virus. This suggests that cHV-68infected DCs would have limited function in inducing a
Th1 response. An inability to secrete IL-12 following CD40
ligation is also observed in measles virus-infected DCs
as well as in HHV-6-infected DCs after LPS and IFN-c
stimulation (Smith et al., 2005), suggesting that this might
be a common immunoevasion strategy (Fugier-Vivier et al.,
1997). Flano et al. (2005) observed that, after an additional
activation signal (LPS stimulation), cHV-68-infected DCs
produced elevated levels of IL-10, which was found to be
responsible for the limited proliferation of allogeneic T
cells. In contrast, in the absence of LPS stimulation, Flano
et al. (2005) failed to detect IL-10 with or without cHV-68
infection in DC cultures after 96 h p.i. In our study, we
were unable to detect significant differences in IL-10
production between mock-treated and cHV-68-infected
DCs at 24 h p.i. It is possible that differences in IL-10 levels
would be more apparent by 96 h p.i. We also observed that
no IL-10 was detected in immature DCs infected with
UV-inactivated virus. It is possible that the higher IL-12
levels induced could counteract IL-10 production in these
cells.
In addition to the inhibitory effects on DC maturation and
activation, cHV-68 infection also had a dramatic effect on
the generation of DCs from bone-marrow precursor cells.
Inhibition of DC development from bone-marrow cells has
been observed after lymphocytic choriomeningitis virus
clone 13 and measles virus infection (Hahm et al., 2005;
Sevilla et al., 2004). Also, the more closely related human
gammaherpesvirus EBV has been found to inhibit DC
development by inducing apoptosis in monocyte precursors (Guerreiro-Cacais et al., 2004; Li et al., 2002).
However, Li et al. (2002) demonstrated that the apoptosispromoting effect of EBV is restricted to the early stages
of DC differentiation and is independent of viral gene
expression (Li et al., 2002). This finding contradicts our
results where we observed that UV-inactivated virus had no
inhibitory effect on DC differentiation.
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1903
R. Hochreiter and others
In summary, we found that cHV-68 productively infects
immature DCs, whilst LPS-matured DCs are susceptible to
latent infection. Importantly, cHV-68 failed to induce DC
maturation following infection and infected cells could not
be induced to mature fully following LPS or poly(I : C)
treatment. Interference of DC maturation may be mediated
by a viral protein(s) that inhibits signalling pathways
involved in DC maturation. Certain viruses such as EBV,
human T cell leukemia virus I, herpesvirus saimiri and
KSHV can alter STAT activity to increase the virus persistence, replication and oncogenic potential (Chen et al.,
2001; Lund et al., 1997; Migone et al., 1995; Weber-Nordt
et al., 1996). Studies are under way to determine whether
this is the critical point in DC maturation blocked by cHV68 infection.
Flano, E., Kim, I. J., Moore, J., Woodland, D. L. & Blackman, M. A.
(2003). Differential gamma-herpesvirus distribution in distinct anato-
mical locations and cell subsets during persistent infection in mice.
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ACKNOWLEDGEMENTS
The authors thank Nancy Fiore and Pamela Lincoln for excellent
technical assistance and Maureen Barcza and Joyce Qui from the
Department of Pathology at SUNY Upstate Medical University for
transmission electron microscopy preparation and imaging. This
work was supported by the Hendricks Foundation and Department of
Microbiology and Immunology, SUNY Upstate Medical University. The authors thank Drs Ren Sun (UCLA) and Steven Dewhurst
(University of Rochester) for generous sharing of reagents.
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