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: firstname.lastname@example.org
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
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443The Journal of Immunology