of June 13, 2013.
This information is current as
Herpes Simplex Virus
Production by Microglial Cells in Response to
Proinflammatory Cytokine and Chemokine
Cutting Edge: TLR2-Mediated
Joseph M. Palmquist and James R. Lokensgard
Rajagopal N. Aravalli, Shuxian Hu, Timothy N. Rowen,
2005; 175:4189-4193; ;
, 15 of which you can access for free at:
cites 24 articles
is online at:
The Journal of Immunology
Information about subscribing to
Submit copyright permission requests at:
Receive free email-alerts when new articles cite this article. Sign up at:
Print ISSN: 0022-1767 Online ISSN: 1550-6606.
Immunologists All rights reserved.
Copyright © 2005 by The American Association of
9650 Rockville Pike, Bethesda, MD 20814-3994.
The American Association of Immunologists, Inc.,
is published twice each month by
The Journal of Immunology
by guest on June 13, 2013
Cutting Edge: TLR2-Mediated Proinflammatory Cytokine
and Chemokine Production by Microglial Cells in
Response to Herpes Simplex Virus1
Rajagopal N. Aravalli, Shuxian Hu, Timothy N. Rowen, Joseph M. Palmquist, and
James R. Lokensgard2
Recent studies indicate that TLRs are critical in generat-
ing innate immune responses during infection with
HSV-1. In this study, we investigated the role of TLR2
signaling in regulating the production of neuroimmune
mediators by examining cytokine and chemokine expres-
sion using primary microglial cells obtained from
TLR2?/?as well as wild-type mice. Data presented here
demonstrate that TLR2 signaling is required for the pro-
duction of proinflammatory cytokines and chemokines:
TNF-?, IL-1?, IL-6, IL-12, CCL7, CCL8, CCL9,
CXCL1, CXCL2, CXCL4, and CXCL5. CXCL9 and
CXCL10 were also induced by HSV, but their production
was not dependent upon TLR2 signaling. Because
TLR2?/?mice display significantly reduced mortality
and diminished neuroinflammation in response to brain
infection with HSV, the TLR2-dependent cytokines iden-
tified here might function as key players influencing viral
neuropathogenesis. The Journal of Immunology, 2005,
immune responses (1, 2). During the early stages of infection,
microglial cells act as key cellular mediators of neuroinflamma-
tory processes and contribute to the first line of defense before
lymphocyte infiltration into the brain by producing and secret-
ing a number of immune modulators. Secreted cytokines and
chemokines, in turn, activate immune responses of cells that
infiltrate the CNS to counter invading pathogens. Although
significant progress has been made in understanding neuroim-
mune responses to viral infections, the exact brain cell-specific
molecular mechanisms that lead to the production of proin-
flammatory immune mediators remain to be elucidated.
In recent years, it has become clear that TLRs, a class of pat-
tern recognition receptors, are critical in recognizing a wide
range of pathogens (3). These reports suggest that viruses may
icroglial cells, the resident macrophages of the
brain, are sensors of viral infection within the CNS
and play a pivotal role in generating innate neuro-
trigger inflammatory cytokine production via multiple TLRs
(8), and several studies have implicated TLRs as important
players during a number of herpesvirus infections, depending
upon the cell type. For example, HSV infection of dendritic
cells induces secretion of a number of cytokines and chemo-
dendritic cells produce IFN-? in response to HSV-1 and
HSV-2 infection via TLR9-dependent and -independent
pathways (6, 9–11). HSV-1 has also been shown to activate
IFN-producing cells in vitro through TLR9 (8). TLR9 rec-
ognizes abundant CpG motifs in HSV-1 DNA and initiates
In addition to TLR9, TLR2 has been demonstrated to play a
major role in the pathogenesis of HSV-induced encephalitis (5,
13). In support of the pivotal role for TLR2 in HSV neuro-
pathogenesis, infected TLR2?/?mice had significantly lower
mortality rates than wild-type mice, and they did not show a
neuroinflammatory infiltrate (13). Both HSV-1 and HSV-2
were shown to induce IL-6 and IL-8 expression in human pe-
ripheral-blood mononuclear cells in a dose-dependent manner
and to activate NF-?B. This cytokine response was found to be
mediated through TLR2 and was not dependent upon virus
Previous studies from our laboratory have demonstrated that
HSV-1 infection of primary human astrocytes and neurons
leads to robust virus growth and replication, but neither cell
type produced cytokines or chemokines in response to infec-
tion. In contrast, human microglia produced significant
amounts of TNF-?, IL-1?, CXCL-10, and CCL5 but did not
permit productive viral replication (14). Because microglial
cells produce inflammatory cytokines and chemokines in re-
sponse to HSV, the role of TLR2 signaling in the generation of
fection and to understand the role of TLR2 signaling in facili-
tating these responses, we studied the production and secretion
of proinflammatory immune mediators using isolated murine
Center for Infectious Diseases and Microbiology Translational Research, Department of
Medicine, University of Minnesota Medical School, Minneapolis, MN 55455
Received for publication June 22, 2005. Accepted for publication July 22, 2005.
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 United States Public Health Service Award MH-57617.
2Address correspondence and reprint requests to Dr. James R. Lokensgard, University of
Minnesota, 3-220 McGuire Translational Research Facility, 2001 6th Street SE, Minne-
apolis, MN 55455. E-mail address: firstname.lastname@example.org
Copyright © 2005 by The American Association of Immunologists, Inc.0022-1767/05/$02.00
by guest on June 13, 2013
microglial cells from wild-type and TLR2?/?mice. In this
study, we show for the first time that HSV infection of micro-
signaling is required for robust production of a number of ma-
jor proinflammatory cytokines.
Materials and Methods
Mice and virus
Wild-type C57BL/6 and TLR2?/?(Tlr2tm1Kir/J) mice were purchased from
The Jackson Laboratory. Purified microglial cell cultures were prepared as de-
scribed previously with minor modifications (15). A total of 1 ? 106cells/well
was used for the microarrays, and 2 ? 105cells/well were used for protein as-
says. Microglial cell cultures used in these experiments were ?99% pure, as
determined by MAC-1 Ab staining (Roche Applied Science). A highly neuro-
at a multiplicity of infection of 2. After adding the virus, culture plates were
incubated at 37°C for 5 h.
(OMM-11) (SuperArray) was used for our studies, and hybridization proce-
dures were performed per manufacturer’s instructions. Wild-type and
after 5 h postinfection (p.i.)3using the RNeasy Mini Kit (Qiagen). Chemilu-
ysis Suite software (SuperArray). Data are presented as relative induction of
each cytokine and chemokine, normalized to GAPDH, and are representative
of two independent experiments.
1 ?g of total RNA using SuperScript II Reverse Transcriptase (Invitrogen Life
Technologies) and oligo(dT)6–12primers (Sigma-Genosys). Quantitative real-
time PCR was performed with LightCycler 2.0 thermocycler (Roche Applied
Applied Science) following the manufacturer’s specifications. The PCR
conditions were as follows: 40 denaturation cycles of 95°C for 10 s, anneal-
ing at 58°C for 5 s, and elongation at 72°C for 10 s. The relative product
levels were quantified using the 2???CTmethod (16). Primer sequences are
available upon request. Data are presented as relative induction of each cy-
tokine and chemokine, normalized to ?-actin, and are representative of two
A sandwich ELISA-based system (17) was used to quantify cytokine and che-
mokine levels from microglial cell culture supernatants. In brief, purified rat
anti-mouse TNF-?, IL-6, and IL-12 Abs (BioSource International), CCL2 Ab
(BD Biosciences), and goat anti-mouse CXCL9 and CXCL10 Abs (R&D Sys-
tems) were coated onto microtiter plates at 2–4 ?g/ml. Plates (96-well) were
blocked with 1% BSA, and after adding 50 ?l each sample and standard cyto-
kines and chemokines plates were incubated for 2 h at 37°C. The wells were
washed with PBS/Tween 20 (0.5%) and incubated with primary Abs followed
wash, they were treated with streptavidin for color development and were read
at 450 nm. Data are presented as relative induction of each cytokine, and bars
represent the mean ? SD of triplicate samples, which are representative of at
least two (TLR2?/?) or three (wild-type) independent experiments.
At 4 h p.i., microglial cells were fixed with 4% paraformaldehyde for 20 min
and washed with PBS. They were then stained with mouse anti-ICP4 Ab (Ad-
vanced Biotechnologies) at a concentration of 10 ?g/ml. Vector M.O.M. im-
munodetection kit (Vector Laboratories) containing the secondary Ab and de-
the localization of ICP4 within the nucleus of infected microglial cells.
Expression of immune mediators by HSV-infected murine microglia
HSV-1 infection of human microglia results in the production
of proinflammatory cytokines and chemokines (14), and
IL-1?, TNF-?, CCL5, IL-6, IFN-?, and CXCL10 have been
shown to be up-regulated in other types of HSV-infected pri-
mary cells as well as cell lines (5, 13, 14, 18). To investigate the
role of TLR2 signaling in proinflammatory cytokine and che-
mokine production by microglia, we assessed the expression
from infected and uninfected microglial cells from both wild-
type and TLR2?/?mice at 5 h p.i. The array contained pro-
and anti-inflammatory cytokines and chemokines, their recep-
tors, and signaling pathway-related molecules. Interestingly,
TLR2 mediated the expression of a number of major cytokines
and chemokines in wild-type cells upon infection (Table I).
3Abbreviation used in this paper: p.i., postinfection.
Table I. Induced expression of proinflammatory cytokines, chemokines,
and related genes in HSV-infected C57BL/6 vs TLR2?/?microglial cells
at 5 h p.i.
GeneCommon Name Fold ?
Chemokine (C-X-C motif) ligand 2
Chemokine (C-C motif) ligand 9
Chemokine (C-C motif) ligand 7
IFN-? family, gene 2
Chemokine (C-X3-C) receptor 1
Chemokine (C-X-C motif) ligand 4
Chemokine (C-X-C motif) ligand 11
Chemokine (C-C motif) ligand 3
Chemokine (C-C motif) ligand 8
IL-6 signal transducer
NO synthase 2, inducible
RAS-related C3 botulinum substrate 1
IL-10 receptor, ?
Chemokine (C-C motif) receptor 3
Chemokine (C-C motif) ligand 19
Chemokine (C-X-C motif) ligand 1
Integrin ? M
Chemokine (C-C motif) ligand 17
Chemokine (C-C motif) receptor 2
Chemokine (C-X3-C motif) ligand 1
Chemokine (C-X-C motif) ligand 15
IL-1 receptor antagonist
Complement component 3
Chemokine (C-C motif) receptor 9
Chemokine (C-C motif) ligand 25
Chemokine (C-X-C motif) ligand 5
Chemokine (C-C motif) ligand 11
Chemokine (C-C motif) receptor 8
Macrophage migration inhibitory factor
Integrin ? 2
4190CUTTING EDGE: TLR2-MEDIATED PROINFLAMMATORY CYTOKINE PRODUCTION
by guest on June 13, 2013
These immune mediators, with the exception of TNF-?, were
not expressed in TLR2?/?cells showing that TLR2 signaling
expression of important inflammatory cytokines TNF-?, IL-
1?, IL-6, IL-12, CCL7, CCL8, CCL9, CXCL1, CXCL2,
CXCL4, and CXCL5, and the levels of others such as macro-
phage migration inhibitory factor were elevated in HSV-in-
fected wild-type cells when compared with infected TLR2?/?
cells (Table I). Taken together, these findings indicate that
TLR2 signaling was required for the production of major in-
flammatory cytokines and chemokines during the early stages
of HSV-1 infection.
Cytokines and chemokines induced by TLR2 signaling
To validate the differences identified in the microarray expres-
sion profiles of wild-type and TLR2?/?microglia and to con-
number of select proinflammatory cytokines and chemokines
using quantitative real-time PCR. As shown in Fig. 1, the ex-
was highly induced by HSV-1 in microglia from wild-type
mice, but was not detectable in HSV-infected microglia from
TLR2?/?mice, clearly demonstrating that the production of
these immune mediators required TLR2 signaling. Differences
in cytokine induction levels observed between microarray and
methods used. The expression of two other chemokines
(CXCL9 and CXCL10) and IL-18, which was not dependent
upon TLR2 signaling, was also validated (Fig. 1). Although
higher expression of IL-18 and CXCL10 was found in infected
wild-type cells, CXCL9 was expressed at a higher level in
response to HSV-1, possibly through other TLRs.
To further examine whether the expression patterns of se-
lected proinflammatory cytokines and chemokines identified
by array and real-time PCR studies correlated with protein lev-
els, ELISA were performed using supernatants of HSV-treated
and untreated microglial cells. For ELISA, 8-, 24-, and 48-h
time points were chosen because protein production is known
to appear after mRNA (14). ELISA clearly demonstrated the
induction of TNF-?, IL-6, IL-12, CCL2, CXCL9, and
CXCL10 in HSV-infected wild-type cells (Fig. 2A). HSV-in-
chemokines at the 8-h time point. This result also confirmed
that their expression is mediated through TLR2. Interestingly,
the production of chemokines CCL2, CXCL9, and CXCL10
was induced in both TLR2?/?and wild-type microglia. In ad-
dition, when ELISA were performed with LPS-stimulated un-
infected microglial cells, we observed no differences in the pro-
duction of TNF-? between wild-type and TLR2?/?cells (Fig.
2B). Thus, LPS-stimulated signaling through TLR4 is intact in
and chemokines by HSV-infected microglia. Uninfected
and infected microglia (5 h p.i.) from wild-type and
TLR2?/?mice were lysed, and RNA was isolated. Real-
cytokines and chemokines.
Expression of proinflammatory cytokines
4191The Journal of Immunology
by guest on June 13, 2013
Viral entry and immediate-early gene expression in TLR2?/?
viral glycoproteins (gB or gC or both) with cell surface glycos-
aminoglycan chains of heparin sulfate proteoglycans (19).
virions was found to be sufficient to trigger the production of
proinflammatory cytokines and IFN-stimulated genes in
PBMCs through TLR2-dependent pathway (4). To test
whether production of cytokines and chemokines seen in
mouse microglia was the effect of inefficient viral infection, we
examined HSV entry into microglia from wild-type and
TLR2?/?mice. Immunostaining for the immediate-early gene
ICP4 at 4-h p.i. showed that this viral protein was expressed
both in wild-type and TLR2?/?cells at similar levels (Fig. 3,
A–D). This result demonstrates that HSV can penetrate wild-
type as well as TLR2?/?cells with similar efficiencies and that
the reduced cytokine and chemokine expression by microglial
cells from TLR2?/?mice was truly due to the lack of TLR2
signaling and not a lack of viral penetration into these cells.
HSV infection of the CNS may result in severe focal, necrotiz-
ing encephalitis accompanied by swelling of the brain, leading
to the death of the infected individual. However, the precise
cellular and molecular mechanisms that cause mortality are un-
clear. It has recently been reported that wild-type adult mice
succumbed to infection when a very high dose (5 ? 109PFU)
of HSV was administered, whereas TLR2?/?mice had a lower
mortality rate in response to the same viral challenge (20). In
challenge with only 103PFU, whereas TLR2?/?animals sur-
vived at the same dose of HSV (13). Furthermore, pathological
assessment showed no signs of inflammatory infiltrates in the
brains of TLR2?/?mice as opposed to the wild-type mice, al-
though viral titers were the same in brains of both groups.
In the present study, we showed for the first time that TLR2
matory cytokines and chemokines including TNF-?, IL-1?,
IL-6, IL-12, CCL2, CCL7, CCL8, CCL9, CXCL1, CXCL4,
and CXCL5. These findings clearly demonstrate that TLR2 is
involved in generating neuroimmune responses through the
production of these important mediators. In another study us-
ing murine microglia, it was recently reported that Staphylococ-
cus aureus peptidoglycan induced TNF-?, IL-12B, CXCL2,
microglial cells. Uninfected wild-type microglia (A), HSV-infected wild-type
(D) were probed using Abs against ICP4 and stained following 4-h infection
HSV entry and expression of ICP4 in wild-type and TLR2?/?
murine microglia. A, ELISA were performed for cytokines and chemokines in
LPS-induced expression of TNF-? in infected wild-type and TLR2?/?
TLR2-mediated expression of cytokines and chemokines in
4192CUTTING EDGE: TLR2-MEDIATED PROINFLAMMATORY CYTOKINE PRODUCTION
by guest on June 13, 2013
TLR2 does mediate the production of a number of important
proinflammatory cytokines and chemokines in response to
tokines and chemokines, such as IL-18, CCL2, CXCL9, and
CXCL10, does not appear to be under control of TLR2.
It has recently been reported that the expression of the IFN-
?-inducible chemokines CXCL9, CXCL10, and CXCL11, as
with these reports indicating that some chemokines (e.g.,
CXCR3 ligands) are produced in response to HSV-1 infection,
but their expression is not dependent on TLR2.
In this study, we show that the robust production of proin-
flammatory cytokines seen in microglial cells was not mediated
produced by these cells. Furthermore, it has been reported that
a biphasic production of cytokines takes place in some infected
cell types, one that is dependent upon virus replication and one
that is not (24). To examine this possibility, we studied cyto-
16 h p.i., in addition to 5 h p.i., and no significant difference
was observed between the two time points (data not shown).
Thus, it appears that, at least in murine microglia, HSV-infec-
tion does not induce a second wave of cytokine production
other than that seen at early phase of infection.
In this study we focused on the early events during HSV-1
infection of microglial cells. HSV-1 was found to infect
TLR2 itself was highly elevated in response to viral infection of
wild-type microglia. This newly up-regulated TLR2 may pro-
mote additional expression of proinflammatory cytokines, fur-
ther amplifying the innate immune response. Thus, inhibition
of TLR2 signaling might prove to be an effective approach in
the treatment of overzealous neuroimmune responses seen dur-
ing HSV-induced encephalitis.
The authors have no financial conflict of interest.
1. Streit, W. J., R. E. Mrak, and W. S. T. Griffin. 2004. Microglia and neuroinflamma-
tion: a pathological perspective. J. Neuroinflammation 1: 14–17.
2. Rock, R. B., G. Gekker, S. Hu, W. S. Sheng, M. Cheeran, J. R. Lokensgard, and
P. K. Peterson. 2004. Role of microglia in central nervous system infections. Clin.
Microbiol. Rev. 17: 942–964.
3. Akira, S., and K. Takeda. 2004. Toll-like receptor signaling. Nat. Rev. Immunol. 4:
4. Compton, T., E. A. Kurt-Jones, K. W. Boehme, J. Belko, E. Latz, D. T. Golenbock,
and R. W. Finberg. 2003. Human cytomegalovirus activates inflammatory cytokine
responses via CD14 and Toll-like receptor 2. J. Virol. 77: 4588–4596.
5. Kurt-Jones, E. A., M. Chan, S. Zhou, J. Wang, G. Reed, R. Bronson, M. M. Arnold,
like receptor 2 contributes to lethal encephalitis. Proc. Natl. Acad. Sci. USA 101:
6. Lund, J., A. Sato, S. Akira, R. Medzhitov, and A. Iwasaki. 2003. Toll-like receptor
9-mediated recognition of herpes simplex virus-2 by plasmacytoid dendritic cells.
J. Exp. Med. 198: 513–520.
7. Tabeta, K., P. Georgel, E. Janssen, X. Du, K. Hoebe, K. Crozat, S. Mudd, L. Shamel,
S. Sovath, J. Goode, et al. 2004. Toll-like receptors 9 and 3 are essential components
of innate immune defense against mouse cytomegalovirus infection. Proc. Natl. Acad.
Sci. USA 101: 3516–3521.
8. Olson, J. K., and S. D. Miller. 2004. Microglia initiate central nervous system innate
and adaptive immune responses through multiple TLRs. J. Immunol. 173:
9. Krug, A., G. D. Luker, W. Barchet, D. A. Lieb, S. Akira, and M. Colonna. 2004.
Herpes simplex virus type 1 activates murine natural interferon-producing cells
through Toll-like receptor 9. Blood 103: 1433–1437.
10. Hochrein, H., B. Schlatter, M. O’Keeffe, C. Wagner, F. Schmitz, M. Schiemann,
S. Bauer, M. Suter, and H. Wagner. 2004. Herpes simplex virus type-1 induces IFN-
production via Toll-like receptor 9-dependent and -independent pathways. Proc.
Natl. Acad. Sci. USA 101: 11416–11421.
11. Malmgaard, L., J. Melchjorsen, A. G. Bowie, S. C. Mogensen, and S. R. Paludan.
2004. Viral activation of macrophages through TLR-dependent and -independent
pathways. J. Immunol. 173: 6890–6898.
12. Zheng, M., D. M. Klinman, M. Gierynska, and B. T. Rouse. 2002. DNA containing
CpG motifs induces angiogenesis. Proc. Natl. Acad. Sci. USA 99: 8944–8949.
and R. W. Finberg. 2005. The role of Toll-like receptors in herpes simplex infection
in neonates. J. Infect. Dis. 191: 746–748.
14. Lokensgard, J. R., S. Hu, W. Sheng, M. vanOijen, D. Cox, M. C-J. Cheeran, and
P. K. Peterson. 2001. Robust expression of TNF-?, IL-1?, RANTES, and IP-10 by
human microglial cells during nonproductive infection with herpes simplex virus.
J. Neurovirol. 7: 208–219.
15. Chao, C. C., T. W. Molitor, and S. Hu. 1993. Neuroprotective role of IL-4 against
activated microglia. J. Immunol. 151: 1473–1481.
16. Livak, K. J., and T. D. Schmittgen. 2001. Analysis of relative gene expression data
using real-time quantitative PCR and the 2???CTmethod. Methods 25: 402–408.
17. Peterson, P. K., S. Hu, J. Salak-Johnson, T. W. Molitor, and C. C. Chao. 1997. Dif-
ferential production of and migratory response to ? chemokines by human microglia
and astrocytes. J. Infect. Dis. 175: 478–481.
18. Mansur, D. S., E. G. Kroon, M. L. Nogueira, R. M. E. Arantes, S. C. O. Rodrigues,
S. Akira, R. T. Gazzinelli, and M. A. Campos. 2005. Lethal encephalitis in myeloid
differentiation factor 88-deficient mice infected with herpes simplex virus.
Am. J. Pathol. 166: 1419–1426.
20. Finberg, R. W., and E. A. Kurt-Jones. 2004. Viruses and Toll-like receptors. Microbes
Infect. 6: 1356–1360.
21. Kielian, T., N. Esen, and E. D. Bearden. 2005. Toll-like receptor 2 (TLR2) is pivotal
22. Sellner, J., F. Dvorak, Y. Zhou, J. Haas, R. Kehm, B. Wildemann, and
U. Meyding-Lamade. 2005. Acute and long-term alteration of chemokine mRNA
expression after anti-viral and anti-inflammatory treatment in herpes simplex virus
encephalitis. Neurosci. Lett. 374: 197–202.
23. Wickham, S., B. Lu, J. Ash, and D. J. Carr. 2005. Chemokine receptor deficiency is
associated with increased chemokine expression in the peripheral and central nervous
systems and increased resistance to herpetic encephalitis. J. Neuroimmunol. 162:
Suppression of proinflammatory cytokine expression by herpes simplex virus type 1.
J. Virol. 78: 5883–5890.
4193The Journal of Immunology
by guest on June 13, 2013