TLR9 Contributes to Antiviral Immunity during
Simone Guggemoos,* Doris Hangel,†Svetlana Hamm,†Antje Heit,†Stefan Bauer,‡
and Heiko Adler2*
The human gammaherpesviruses Kaposi’s sarcoma-associated herpesvirus and EBV cause important infections. As pathogenetic
studies of the human infections are restricted, murine gammaherpesvirus 68 serves as a model to study gammaherpesvirus
pathogenesis. TLRs are a conserved family of receptors detecting microbial molecular patterns. Among the TLRs, TLR9 recog-
nizes unmethylated CpG DNA motifs present in bacterial and viral DNA. The aim of this study was to assess the role of TLR9 in
gammaherpesvirus pathogenesis. Upon stimulation with murine gammaherpesvirus 68, Flt3L-cultured bone marrow cells (den-
dritic cells) from TLR9?/?mice secreted reduced levels of IL-12, IFN-?, and IL-6, when compared with dendritic cells from
wild-type mice. Intranasal infection of TLR9?/?and wild-type mice did not reveal any differences during lytic and latent infection.
In contrast, when infected i.p., TLR9?/?mice showed markedly higher viral loads both during lytic and latent infection. Thus,
we show for the first time that TLR9 is involved in gammaherpesvirus pathogenesis and contributes to organ-specific
immunity. The Journal of Immunology, 2008, 180: 438–443.
nasopharyngeal carcinoma (1). Human herpesvirus-8 (also called
Kaposi’s sarcoma-associated herpesvirus), a gamma 2-herpesvi-
rus, is associated with lymphoproliferative disorders and Kaposi’s
sarcoma (2). In vivo studies of gammaherpesvirus pathogenesis
have been limited to clinical investigation of the infection because
of the restricted host range of these viruses. Recently, the murine
gammaherpesvirus 68 (MHV-68)3has been established as a mouse
model for the study of gammaherpesvirus pathogenesis (3–8).
MHV-68 is a natural pathogen of wild rodents (9) and is capable
of infecting laboratory mice. The nucleotide sequence of MHV-68
is similar to EBV and even more closely related to Kaposi’s sar-
coma-associated herpesvirus (10). In particular, MHV-68 is very
useful to study the role of immunity in gammaherpesvirus infec-
iseases caused by gammaherpesviruses continue to be a
challenge for human health. The prototypic gamma
1-herpesvirus, EBV, is associated with lymphomas and
Host immune responses play a pivotal role in the control of
gammaherpesvirus infection and in pathogenesis. Whereas the
adaptive immune response during gammaherpesvirus infection has
been an area of intensive research, surprisingly little is known
about the role of innate immunity in the control of gammaherpes-
virus infection (16, 17). The TLR system is responsible for the
primary recognition of infectious agents leading to the initiation of
the innate and adaptive immune response (18, 19). Recently, a
number of viruses, for example, HSV, CMV, respiratory syncytial
virus, influenza A virus, and vesicular stomatitis virus, have been
shown to activate cells via TLR family members (20–22). The
activation of TLRs by viruses might lead to antiviral immune re-
sponses but viruses may also use these pathways to enhance their
own replication (20). The important role of TLRs in antiviral im-
mune responses is also mirrored by viral immune evasion strate-
gies used against TLRs (22).
In a very recent study, it has been shown that EBV particles
induce NF-?B activation in transfected human embryonic kidney
cells and chemokine secretion by primary monocytes in a TLR2-
dependent manner (23). The authors did not show whether intra-
cellular TLRs like TLR9 also play a role after uptake of virus.
TLR9 recognizes unmethylated CpG DNA motifs that are present
in bacterial and viral DNA (19). Accordingly, it has been shown
that TLR9 is required for IFN-? production in response to DNA
viruses including murine CMV (MCMV) and HSV (19, 21, 22).
There are some hints that gammaherpesviruses might also interact
with TLR9. EBV-stimulated human plasmacytoid dendritic cells
(DCs) promote the activation of NK cells and CD3?T cells. This
activation was dependent on cell-to-cell contact and was partially
linked to TLR9 signaling (24). MHV-68 can induce IL-12 pro-
duction in macrophages and DCs (25). HSV-1-induced IL-12
production during infection is mediated by TLR9 (26).
Thus, we considered TLR9 as a potential candidate to be acti-
vated by gammaherpesvirus infection and wanted to study its role
in particular in vivo after MHV-68 infection. We demonstrate that
TLR9 mediates the production of inflammatory cytokines by Flt3
ligand-cultured bone marrow cells (FL-DCs) in response to
MHV-68 infection. By infection of TLR9?/?mice, we show that
*Institute of Molecular Immunology, Clinical Cooperation Group Hematopoietic Cell
Transplantation, GSF-National Research Center for Environment and Health, Mu-
nich, Germany;†Institute of Medical Microbiology, Immunology and Hygiene,
Technical University, Munich, Germany; and‡Institute of Immunology, Univer-
sity Marburg, Marburg, Germany
Received for publication June 21, 2007. Accepted for publication October 16, 2007.
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.
1This work was supported by grants from the Deutsche Forschungsgemeinschaft
(DFG; Ad121/2-1, 2-2, and 2-4) and the Bundesministerium fu ¨r Bildung und For-
schung (NGFN-2, FKZ 01GS0405) to H.A., and the DFG Schwerpunktprogramm
1110 “Innate Immunity” to S.B.
2Address correspondence and reprint requests to Dr. Heiko Adler, Institute of Mo-
lecular Immunology, Clinical Cooperation Group Hematopoietic Cell Transplanta-
tion, GSF-National Research Center for Environment and Health, Marchioninistrasse
25, D-81377 Munich, Germany. E-mail address: email@example.com
3Abbreviations used in this paper: MHV-68, murine gammaherpesvirus 68; MCMV,
murine CMV; DC, dendritic cell; FL-DC, Flt3L-cultured bone marrow cell; i.n., in-
tranasal; BHK, baby hamster kidney cell; wt, wild type; pDC, plasmacytoid DC;
MOI, multiplicity of infection; gB, glycoprotein B; p.i., postinfection.
Copyright © 2007 by The American Association of Immunologists, Inc. 0022-1767/07/$2.00
The Journal of Immunology
TLR9 is involved in the antiviral immune response to MHV-68
infection. In the absence of TLR9 expression, lytic virus titers in
the lung 6 days after intranasal (i.n.) infection were not affected but
were increased in the spleen after i.p. infection. Similarly, in the
absence of TLR9, the latent virus load in the spleen 17 days after
infection was increased after i.p. infection but not after i.n. infec-
tion. Thus, we provide for the first time genetic evidence for an
interaction of a gammaherpesvirus with TLR9. We demonstrate
that TLR9 plays an important role in gammaherpesvirus immunity
both during lytic infection and latency amplification and contrib-
utes to organ-specific immunity.
Materials and Methods
Cell culture and virus stocks
Baby hamster kidney cells (BHK-21) were maintained in Glasgow’s mod-
ified Eagle’s medium (Biochrom) supplemented with 5% FCS, 5% tryp-
tose-phosphate broth, 100 U/ml penicillin, 100 ?g/ml streptomycin, and 2
mM L-glutamine. NIH3T3 cells were grown in DMEM high glucose (Cell
Concepts) supplemented with 10% FCS, 2 mM L-glutamine, 100 U/ml
penicillin, and 100 ?g/ml streptomycin. Human embryonic kidney 293
cells were maintained in DMEM with 10% FCS, 2 mM L-glutamine, 100
U/ml penicillin, and 100 ?g/ml streptomycin. MHV-68 stocks were prop-
agated and viral titers were determined by plaque assay on BHK-21 cells
as described (27). Briefly, 10-fold dilutions were incubated on BHK-21
cells for 90 min at 37°C. After removing the inoculum, cells were incu-
bated for 5 days at 37°C with fresh medium containing 1.5% methylcel-
lulose. After 4–5 days, cells were stained with 0.1% crystal violet solution
to determine the number of plaques.
Generation and stimulation of FL-DCs
To obtain FL-DCs, bone marrow cells from wild-type (wt) and knockout
mice were cultured with 35 ng/ml human recombinant Flt3L (R&D Sys-
tems) for 8 days as described (28). FL-DCs contain a mixture of plasma-
cytoid DCs (pDCs) and myeloid DCs (28), and pDCs are known to rec-
ognize viruses via TLRs. FL-DCs were stimulated with the TLR4-ligand
LPS (Sigma-Aldrich), the TLR7/8-ligand R848 (GLSynthesis), the TLR9-
ligand CpG-oligodeoxynucleotide 2216 (gggggacgatcgtcgggggg; Ref. 29)
(TIB MOLBIOL) and MHV-68 (multiplicity of infection (MOI) of 1 and
0.1, respectively) for 24 h. Afterward, supernatants were harvested and
stored at ?80°C for cytokine determinations.
TLR9?/?mice, backcrossed to C57BL/6 mice for 10 generations, were
bred under standard pathogen-free conditions in the animal facility of the
Institute of Medical Microbiology, Immunology and Hygiene (Technical
University Munich). Age-matched C57BL/6 mice (wt controls) were pur-
chased from Charles River Laboratories. All mice were housed in individually
ventilated cages during the MHV-68 infection period.
Infection of mice and analysis of tissue
wt and knockout mice were anesthetized using ketamine/xylazine and in-
fected i.n. with 5 ? 104PFU of MHV-68 in 30 ?l of sterile PBS. Alter-
natively, mice were infected i.p. with 5 ? 105PFU in 250 ?l of sterile PBS.
To assess lytic viral replication, lungs were harvested on day 6 after i.n.
infection and spleens on day 6 after i.p. infection. Viral titers in organ
homogenates were determined as described previously. The detection limit
of the assay is 50 PFU/organ as determined by spiking uninfected organs
with known amounts of virus (30). On day 17 after either i.n. or i.p. in-
fection, spleens were harvested; single splenocyte suspensions were pre-
pared and analyzed in the ex vivo limiting dilution reactivation assay as
described (30). Briefly, serial 3-fold dilutions of infected mouse spleno-
cytes were plated on monolayers of 1 ? 104low-passage NIH3T3 cells/
well in 96-well tissue culture plates. Twenty-four wells were plated per
dilution (starting with 1.5 ? 105cells/well). NIH3T3 cells were screened
microscopically for a viral cytopathic effect for up to 3 wk. To differentiate
between latently infected cells and infectious virus in the samples, serial
3-fold dilutions of spleen cells were plated before or after mechanical dis-
ruption of viable cells (by two freeze-thaw cycles). No infectious virus was
detected in samples of mechanically disrupted cells (data not shown). Fre-
quencies of reactivating cells were calculated on the basis of the Poisson
distribution by determining the cell number at which 63.2% of the wells
scored positive for CPE. All animal experiments were in compliance with
protocols approved by the local animal care and use committee.
Measurement of latent viral load by quantitative real-time PCR
Viral load in the spleens of infected mice was determined by quantitative
real-time PCR using the ABI 7300 Real Time PCR System (Applied Bio-
systems). DNA was extracted from spleen cells using the QIAmp DNA
Mini kit (Qiagen) and quantified by UV spectrophotometry. Amplification
of 100 ng of DNA per reaction was performed with TaqMan universal PCR
master mix and universal cycling conditions (Applied Biosystems). Using
primers and probes as described (31), a 70-bp region of the MHV-68 gly-
coprotein B (gB) gene was amplified and viral DNA copy number was
quantified. A standard curve was created using known amounts of a plas-
mid containing the HindIII-N fragment of MHV-68 encompassing the gB
gene. The murine ribosomal protein L8 (rpl8) was amplified in parallel and
used to normalize for input DNA between samples. The primer and probe
sequences for L8 were as follows: forward: 5?-CATCCCTTTGGAGGT
GGTA-3?; reverse: 5?-CATCTCTTCGGATGGTGGA-3? and probe: 5?-
ACCACCAGCACATTGGCAAACC-3?. A standard curve for rpl8 was
generated by serial 10-fold dilution of a plasmid containing rpl8 (RZPD
clone IRAVp968B01123D6). The data are presented as viral genome copy
numbers relative to the copy number of L8. The quantification limit was set
at 50 copies per sample, according to published recommendations (32).
IL-12 p40/p70 (BD Pharmingen), IL-6 (BD Pharmingen), and IFN-? (PBL
Biomedical Laboratories) in supernatants of FL-DCs were determined by
ELISA as recommended by the manufacturers.
For FACS analysis, 106cells were suspended in 100 ?l of FACS buffer
(PBS, 2% FCS). Nonspecific binding of Abs to FCR was blocked by in-
cubating cells with 1 ?g of the anti-CD16/CD32 mAb 2.4G2 (BD Pharm-
ingen). After 5 min at 4°C, the relevant mAbs were added at a concentra-
tion of 0.5 ?g/106cells and cells were incubated for 30 min at 4°C. The
following mAbs from BD Pharmingen were used: FITC-conjugated anti-
mouse CD45R/B220 (RA3-6B2), FITC-conjugated anti-mouse CD3 mo-
lecular complex (17A2), FITC-conjugated anti-mouse CD4 (L3T4)
(GK1.5), FITC-conjugated anti-mouse CD8a (Ly-2) (53-6.7), FITC-con-
jugated anti-mouse CD14 (rmC5-3), PE-conjugated anti-mouse Ly-6A/E
(Sca-1) (E13-161.7), allophycocyanin-conjugated anti-mouse NK1.1
(PK136), FITC-conjugated rat IgG2a, ? isotype standard (R35-95), and
FITC-conjugated rat IgG2b, ? isotype standard (A95-1). After staining,
cells were washed twice, resuspended in 500 ?l of 0.5% paraformaldehyde
in PBS and analyzed on a FACSCalibur using CellQuest software (BD
Histopathological analysis of lung tissue
Lungs of mice were harvested on day 6 after intranasal infection and fixed
in 10% formalin in PBS. For histopathological analysis, organs were em-
bedded in paraffin, sections were cut, stained with H&E and analyzed by
If not otherwise indicated, data were analyzed by Student’s t test.
TLR9 mediates the production of inflammatory cytokines by
FL-DCs in response to MHV-68
To investigate a potential role of TLR9 in gammaherpesvirus-host
cell interaction, we stimulated FL-DCs, generated from either wt
or TLR9?/?mice, with MHV-68 (MOI ? 0.1). After 24 h, the
supernatants were analyzed for IL-12 p40/p70 and IFN-? by
ELISA. As positive control, a type A CpG-oligodeoxynucleotide
(2216) was used for stimulation. Although FL-DCs generated from
wt mice produced IL-12 in response to both MHV-68 and CpG-
DNA, the production of IL-12 was abolished in the absence of
TLR9 in either case (Fig. 1A). Similarly, the production of IFN-?
in response to CpG-DNA was abolished in the absence of TLR9.
In contrast, the production of IFN-? in response to MHV-68 was
reduced but not completely abolished in the absence of TLR9 (Fig.
1B). To further define the role of TLR9 in the production of in-
flammatory cytokines by FL-DCs in response to MHV-68, we ex-
amined the production of IL-6, a cytokine produced at high levels
439The Journal of Immunology
during MHV-68 infection (33). FL-DCs generated from wt mice
produced IL-6 in response to both MHV-68 and CpG-DNA. In the
absence of TLR9, the production of IL-6 was abolished in response
to CpG-DNA and significantly reduced in response to MHV-68
(Fig. 1C). The induction of IL-6 by MHV-68 in FL-DCs generated
from wt mice was dose-dependent (MOI of 1 and 0.1, respec-
tively). In control cultures, FL-DCs generated from both wt or
TLR9?/?mice produced IL-6 in response to LPS (TLR4 ligand)
and R848 (TLR7/8 ligand). In addition, heat-inactivated MHV-68
(1 h, 65°C) was used as control. Heat denaturation, which leads to
the disruption of the viral envelope and thereby prevents infection,
prevented the induction of IL-6. Taken together, these results
strongly suggested a role of TLR9 in gammaherpesvirus-host cell
Lack of TLR9 affects both lytic and latent MHV-68 after i.p.
but not after intranasal infection
To analyze the role of TLR9 in gammaherpesvirus pathogenesis,
we infected C57BL/6 and TLR9?/?mice with MHV-68. In a first
series of experiments, mice were infected i.n. with 5 ? 104PFU of
MHV-68. Lytic viral replication in the lungs was determined by
plaque assay on day 6 postinfection (p.i.), the time point at which
viral titers usually reach a peak. Comparable viral titers were ob-
served in both groups of mice (Fig. 2). In addition, histopatholog-
ical analysis of lung tissue revealed no obvious differences either
(data not shown). The establishment of latency in the spleen is
associated with a marked splenomegaly and an increase in the
number of latently infected B cells which peaks around 2–3 wk p.i.
controls after intranasal infection are similar. Mice were infected i.n. with
5 ? 104PFU of MHV-68. On day 6 p.i., lungs were harvested and viral
titers determined by plaque titration on BHK-21 cells. Titers of individual
mice (n ? 5), compiled from two independent experiments, and mean
values are shown.
Lung titers of MHV-68-infected TLR9?/?mice and wt
TLR9?/?mice after i.n. infection. Mice (C57BL/6 and TLR9?/?) were
infected i.n. with 5 ? 104PFU of MHV-68. Spleens were harvested on day
17 p.i. A, The number of splenocytes reactivating virus was determined by
an ex vivo reactivation assay. Shown are means ? SD of two individual
experiments (in each experiment, cells pooled from two or three mice per
group were analyzed). B, DNA from splenocytes was isolated and analyzed
for viral genomes using quantitative real-time PCR. The data are presented
as viral genome copy number (gB) per 1000 copies L8. Data shown are
compiled from two independent experiments. Each symbol represents an
individual mouse (n ? 5), and the horizontal lines indicate the means.
Latent MHV-68 infection in the spleen is not altered in
virus titers in the spleen after i.p. infection. wt and TLR9?/?mice were
infected i.p. with 5 ? 105PFU of MHV-68. Viral titers in the spleen were
quantified 6 days after infection by plaque assay on BHK-21 cells. In A,
titers of individual mice (n ? 9) and means are illustrated. Data shown are
compiled from three independent experiments. In TLR9?/?mice, titers of
MHV-68 were significantly higher (p ? 0.00024; unpaired Student’s t
test). B, DNA was isolated from splenocytes on day 6 p.i. and assayed for
viral genomes by quantitative PCR. The data are presented as viral genome
copy number (gB) per 1000 copies L8. Data shown are compiled from four
independent experiments. Each symbol represents an individual mouse
(n ? 11), and the horizontal lines indicate the means. TLR9?/?spleen cells
harbored significantly higher numbers of viral genomes compared with wt
controls (p ? 0.0059; unpaired Student’s t test).
The absence of TLR9 expression results in increased lytic
by FL-DCs in response to MHV-68. FL-DCs from TLR9?/?mice and wt
controls were stimulated for 24 h as indicated. Supernatants were assayed
for IL-12 p40/p70 (A), IFN-? (B), and IL-6 (C) by ELISA. Data shown in
A and B are from a representative experiment (means ? SD from dupli-
cates) which has been repeated twice with similar results. Data shown in C
are means ? SD derived from two (for LPS and CpG 2216) or three (for
medium, R848, and MHV-68) individual experiments, each performed in
duplicates. The difference in the production of IL-6 in response to MHV-68
between the TLR9?/?and wt group was significant (p ? 0.0156 using
two-way ANOVA); ?, not detectable.
TLR9 mediates the production of inflammatory cytokines
440 TLR9 AND GAMMAHERPESVIRUS PATHOGENESIS
Thus, spleens of infected mice were harvested 17 days p.i. to as-
sess the role of TLR9 during latent infection. The number of la-
tently infected cells reactivating virus, as determined by an ex vivo
reactivation assay, was comparable between wt and TLR9?/?
mice. In wt mice, the frequency of reactivating splenocytes was 1
in 6,145 cells. The corresponding number in TLR9?/?mice was 1
in 5,837 (Fig. 3A). Preformed infectious virus was not detected in
spleens harvested 17 days p.i. (data not shown). Consistent with
the reactivation data, spleens of infected wt and TLR9?/?mice
harbored similar amounts of viral genomes as determined by quan-
titative real-time PCR (Fig. 3B). Thus, both lytic and latent
MHV-68 infection were not altered in TLR9?/?mice after i.n.
infection. In a second series of experiments, mice were infected
i.p. with 5 ? 105PFU of MHV-68. On day 6 p.i., spleens were
harvested and analyzed. As shown in Fig. 4, i.p. infection resulted
in significantly higher lytic viral titers in the spleens of TLR9?/?
mice compared with wt mice, as determined both by plaque assay
(Fig. 4A) and quantitative real-time PCR (Fig. 4B). To assess the
role of TLR9 during latency amplification after i.p. infection,
spleens of MHV-68-infected mice were harvested 17 days p.i. and
analyzed both by ex vivo reactivation assay and quantitative real-
time PCR. The number of spleen cells reactivating virus was sig-
nificantly higher in TLR9?/?mice than in wt controls (frequency
of reactivation: 1 in 40,124 in TLR9?/?mice and 1 in 88,878 in
wt mice) (Fig. 5A). In accordance with these findings, the viral
genomic load in spleens of knockout mice was significantly higher
than in wt mice (Fig. 5B). Preformed infectious virus was not
detected in spleens harvested 17 days after i.p. infection (data not
In this study, using MHV-68 as a model, we demonstrated that
TLR9 plays an important role in gammaherpesvirus immunity both
during lytic infection and latency amplification. We could show
that TLR9 mediates the production of inflammatory cytokines by
FL-DCs in response to MHV-68 infection in vitro. Although the
production of IL-12 was abolished in the absence of TLR9, some
residual production of IFN-? and IL-6 was observed. These results
suggest that MHV-68 may, as shown for other viruses, also engage
mechanisms other than TLR9 to induce cytokine secretion. For
example, HSV-1 can activate both TLR2 and TLR9 (26, 34). For
MCMV, both TLR9/MyD88-dependent and -independent pro-
cesses for IFN-? release have been described (35). MHV-68 in-
fection induces a number of cytokines, for example, IFN-?/IFN-?,
IFN-?, IL-6, IL-10, and IL-12 (25, 33, 36). The type I IFNs have
been shown to play a key role in the control of early (37) as well
as latent MHV-68 infection (38). MHV-68-induced IL-12 func-
tions to limit the viral burden but also contributes to virus-medi-
ated splenomegaly (25). Although the cellular sources of the
MHV-68-induced IFN-?/IFN-? have not yet been analyzed in de-
tail, DCs have been shown to be a source for MHV-68-induced
IL-12 (25). MHV-68-induced IL-10 increases the viral load but
limits the virus-induced splenomegaly (39). IL-6 appears to be not
essential for the development of an effective immune response to
MHV-68 (40). IL-10 and IL-6 have been shown to be produced
both by T cells and non-T cells (33).
The absence of TLR9 expression resulted in increased lytic vi-
rus titers in the spleen after i.p. infection but not in the lung after
i.n. infection. Similarly, in TLR9-deficient mice, the latent virus
load in the spleen 17 days after infection was increased after i.p.
but not after i.n. infection. Thus, the role of TLR9 in gammaher-
pesvirus immunity seems to depend on the route of infection and
to be organ specific. The natural route of MHV-68 infection is
unknown but intranasal infection is believed to reproduce mucosal
infection which is characteristic of natural herpesvirus transmis-
sion (15). After i.n. infection, primary lytic replication takes place
in lung epithelial cells. Virus is then transported to lymphoid tis-
sue, most likely by infected DCs. Infected B cells from the medi-
astinal lymph nodes then traffic to the spleen and other lymphoid
organs and establishment of lifelong latency takes place (5, 15).
The establishment of latency in the spleen is associated with a
strong increase in the number of latently infected B cells (41). In
addition to B lymphocytes which are the major reservoir harboring
latent MHV-68 (42), macrophages (43), DCs (44), and lung epi-
thelial cells (45) have also been shown to harbor latent virus. In
contrast to i.n. infection, i.p. infection seeds lytic virus directly to
the spleen and thus allows splenocytes to be infected by direct lytic
spread (46). As a consequence, TLR9-expressing cells may come
in direct contact with lytic virus in the spleen after i.p. infection but
not after i.n. infection, providing a possible explanation as to why
the effect of TLR9 on latency amplification is apparent only after
i.p. but not after i.n., infection. With regard to lytic replication, we
again observed an effect of TLR9 only in the spleen but not in the
lung. Clearly, in this case, lytic virus is present in both organs and
thus could interact with TLR9-expressing cells. However, it has
been shown by Northern blot analysis that TLR9 is much stronger
expressed in the spleen than in the lung and a variety of other
tissues (47). In addition, it has recently been demonstrated that
both myeloid DCs and pDCs in the lung show no detectable ex-
pression of TLR9 while both subsets in the spleen express TLR9
(48). Consequently, CpG oligonucleotides exerted differential ef-
fects on lung and spleen DCs when administered to mice (48).
Similarly, in our study, the absence of TLR9 resulted in differential
effects in the lung and spleen. In TLR9-deficient mice, lytic virus
replication in the lung was undistinguishable from wt mice, which
would be consistent with the above-mentioned fact that TLR9 ex-
pression was undetectable in lung DCs of wt mice. In contrast,
absence of TLR9 in the spleen, an organ where TLR9 is regularly
expressed in DCs of wt mice, resulted in significantly higher lytic
virus titers in TLR9-deficient mice. Although differences in TLR9
expression between lung and spleen might explain our results,
deficient mice 17 days after i.p. infection. wt and TLR9?/?mice were
infected i.p. with 5 ? 105PFU of MHV-68. To address latent infection,
spleens were harvested 17 days p.i., and spleen cells reactivating virus were
quantified by an ex vivo reactivation assay. In A, means ? SEM of three
independent experiments are depicted. To calculate significance, frequen-
cies of reactivation events were statistically analyzed by paired t test over
all cell dilutions. Reactivation was significantly higher in splenocytes from
TLR9?/?mice, when compared with wt (p ? 0.003; paired Student’s t
test). B, The number of viral genomes in TLR9?/?spleen cells was also
significantly increased (p ? 0.0432; unpaired Student’s t test). The data are
presented as viral genome copy number (gB) per 1000 copies L8. Data
shown are compiled from three independent experiments. Each symbol
represents an individual mouse (n ? 8), and the horizontal lines indicate
The latent virus load is increased in the spleen of TLR9-
441 The Journal of Immunology
there are also other possibilities which can be envisaged: depen-
dent on the route of infection (i.n. vs i.p.), different cell types in the
spleen may be infected. However, it has been demonstrated that
establishment and maintenance of gammaherpesvirus latency are
independent of the infective dose and route of infection (49). In
contrast, analyses of MHV-68 mutants have shown that viral genes
such as M2 play a role specifically after i.n. but not after i.p.
infection. This suggests that the requirements for the establishment
of latency are affected by the route of infection (50). Thus, it might
be possible that subtle differences in the cell types infected in the
spleen by different routes of infection may account for our results.
This deserves further studies.
It has been hypothesized that the biological significance of the
absence of TLR9 in lung DCs may be a protective measure that has
evolved to protect the lung from the development of diseases that
are associated with high cytokine (IL-6) production such as pul-
monary fibrosis (48). Thus, it is tempting to speculate that the same
applies to MHV-68 lung infection, namely that the “physiological”
absence of TLR9 in lung DCs may prevent an overwhelming in-
nate immune response, for example, inflammatory cytokine pro-
duction, and thereby limits pulmonary disease while having only
little effect on virus replication. Histopathological analysis of lung
tissue revealed indeed no obvious differences between infected wt
and TLR9-deficient mice. It has very recently been suggested that
innate immunity functions in an organ-specific fashion designed to
sustain organ physiology, for example, by different expression pro-
files of pattern-recognition receptors between organs (51). Sup-
porting observations in this direction have been made with HSV-1
and West Nile virus. In the case of HSV-1, TLR2-mediated in-
duction of inflammatory cytokines in the brain of infected mice
was not protective but associated with lethal encephalitis. As a
result, TLR2-deficient mice showed reduced mortality when com-
pared with wt mice (34). In the case of West Nile virus, TLR3-
induced inflammatory responses contribute to pathogenesis rather
than to protection by triggering a breakdown of the blood-brain
barrier. As a consequence, TLR3-deficient mice survive an other-
wise lethal infection because of reduced virus entry into the brain
(52). In contrast, activation of TLRs is often associated with pro-
tective antiviral innate immune responses (20, 22). For example,
both TLR3- and TLR9-deficient mice are more susceptible to
MCMV infection (35, 53). MyD88, a key intermediate of multiple
TLR-signaling pathways, is essential for the induction of type I
IFN, the production of neutralizing Abs and protection of mice
from lethal infection after i.n. but not after i.v. infection with vesicular
stomatitis virus (54). Thus, TLR activation may either reduce or ex-
acerbate disease, depending on the pathogen and the location of the
In summary, we provide for the first time genetic evidence for
an interaction of a gammaherpesvirus with TLR9. We demonstrate
that TLR9 plays an important role in gammaherpesvirus immunity
both during lytic infection and latency amplification, and that
TLR9 contributes to organ-specific immunity.
We are grateful to Dr. B. Adler for critical reading of the manuscript and
to Dr. R. Kammerer for providing reagents.
The authors have no financial conflict of interest.
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442TLR9 AND GAMMAHERPESVIRUS PATHOGENESIS