STAT4 Regulates Antiviral Gamma Interferon Responses and
Recurrent Disease during Herpes Simplex Virus 2 Infection
Alexandra Svensson,aPetra Tunbäck,bInger Nordström,aAndrey Shestakov,aLeonid Padyukov,cand Kristina Erikssona
Department of Rheumatology & Inflammation Research, Sahlgrenska Academy, University of Gothenburg, Gothenburg,aDepartment of Dermatovenerology, Sahlgrenska
University Hospital, Gothenburg,band Rheumatology Unit, Department of Medicine, Karolinska Institutet, Stockholm,cSweden
increased in vitro secretion of IFN-? in response to the virus. Mice that lacked STAT4 had impaired HSV-2-specific IFN-? pro-
infections in the world. Up to 25% of the Swedish population is
seropositive for HSV-2 (5, 11, 31), while the prevalence in some
sub-Saharan African countries is as high as 80% (47). The clinical
effects of HSV-2 infection range from no symptoms (asymptom-
atic infection) to severe and recurrent episodes of genital lesions
and ulcers. Although the mechanisms underlying these different
disease outcomes are not known, strong gamma interferon
(IFN-?) responses (10, 37), previous HSV-1 infection (22), high
levels of mannan-binding lectin (13), specific HLA alleles (23),
and variants of both the TLR2 gene (6) and the gene encoding
mannose-binding lectin 2 (34) increase the likelihood of an
Efficient HSV-2-specific acquired immunity requires CD4?T
cells and IFN-? production. Mice that lack CD4?T cells, IFN-?,
or the Th1-inducing transcription factor T-bet are unable to
mount a protective immune response to HSV-2 following vacci-
nation (14, 15, 40). However, the treatment of mice with IFN-?
circumvents the need for CD4?T cells, which implies that the
main function of CD4?T cells in HSV-2-specific immunity is to
produce IFN-? (14). The situation is similar in humans, as HSV-
specific IFN-? responses compared with asymptomatic HSV-2
carriers (10, 37).
IFN-? secretion and Th1 differentiation are induced and
acterized by the transcription factors T-bet and STAT4 (20, 42).
Mice that lack either T-bet or STAT4 have impaired IFN-? and
that lack both T-bet and STAT4 have no Th1 responses, even
under Th1-polarizing conditions (12, 20, 43). It is generally be-
activation, which leads to T-bet expression. In addition to IFN-?
secretion, T-bet induces the expression of the interleukin-12 (IL-
respond to IL-12 and to induce further IFN-? production via
enital herpes infection caused by herpes simplex virus 2
(HSV-2) is one of the most common sexually transmitted
STAT4 signaling (17, 18, 45). The secreted IFN-? binds to the
IFN-? receptor and signals via Stat1 so as to amplify the expres-
sion of T-bet, thereby stabilizing the Th1 polarization (24).
Given that STAT4 is important for Th1 immunity, mice that
are deficient for STAT4 are highly susceptible to infections (19).
STAT4 deficiency is associated with increased parasitic burden
and decreased IFN-? responses to pathogens such as Leishmania
major and Toxoplasma gondii (9, 38). Studies of viral infections,
i.e., with lymphocytic choriomeningitis virus (LCMV), influenza
virus, and HSV-1, have revealed diverse roles for STAT4 depend-
ing on the type of infection and the immune mediators that are
involved in the antiviral response (2, 3, 7, 16, 29). As a conse-
a decreased production of inflammatory cytokines (e.g., tumor ne-
crosis factor alpha [TNF-?]) during autoimmune disease and are
thereby resistant to the development of Th1-mediated autoimmune
diseases, such as experimental autoimmune encephalomyelitis,
animal models of infectious diseases, little is known about its role
on correlations between variations in the STAT4 gene and the
incidence of autoimmune diseases. These diseases include rheu-
matoid arthritis, systemic lupus erythematosus (SLE), and pri-
mary Sjögrens syndrome, for which single nucleotide polymor-
phisms (SNPs) in the STAT4 gene have been associated with the
disease incidence in several different populations (21). Further-
Received 17 April 2012 Accepted 15 June 2012
Published ahead of print 20 June 2012
Address correspondence to Kristina Eriksson, Kristina.Eriksson@microbio.gu.se.
Copyright © 2012, American Society for Microbiology. All Rights Reserved.
September 2012 Volume 86 Number 17 Journal of Virology p. 9409–9415jvi.asm.org
severity; a polymorphism in the STAT4 gene has been associated
with a more severe disease outcome in SLE patients (44).
The present study was undertaken to evaluate the role of
purpose, DNA samples from 143 patients with recurrent HSV-2
infection and 85 asymptomatic carriers of HSV-2 were screened
for variations in 7 SNPs of the STAT4 gene. The SNPs were sub-
sequently correlated with HSV-2 disease severity and the levels of
HSV-2-induced IFN-? production by isolated peripheral blood
mononuclear cells (PBMC). To study in greater detail the role of
STAT4 during HSV-2 infection, mice that lack STAT4 were vac-
cinated and/or infected with HSV-2 and their abilities to mount a
that a variation in the human STAT4 gene is associated with in-
ease. In addition, we show that STAT4 is required for adequate
HSV-2-specific IFN-? responses and for the induction of steriliz-
ing immunity in mice.
MATERIALS AND METHODS
Human sample collection. (i) HSV-2-infected individuals. DNA sam-
ples were collected from 228 HSV-2-infected individuals (male, 57%; fe-
male, 43%) who were recruited from the sexually transmitted disease
(STD) clinics at Sahlgrenska University Hospital, Borås Hospital, and
Uddevalla Hospital, Sweden. The average age of the subjects was 39 years
for men (range, 20 to 70 years) and 37 years for women (range, 20 to 68
years). Permission for this study was granted by the Ethics Committee of
the University of Gothenburg, and all patients gave informed consent.
HSV-2 infection was confirmed serologically by enzyme-linked immu-
nosorbent assay (ELISA) (see below). The patients were divided into two
groups based on clinical status.
The symptomatic HSV-2 infection group comprised 143 patients: 77
males (54%; average age, 38 years; range, 23 to 70 years) and 66 females
(46%; average age, 38 years; range, 23 to 68 years) who had a typical
history of recurrent genital herpes. Symptomatic HSV-2 infection was
confirmed by PCR, and serology and history clinical recurrence were re-
year to ensure the presence of clinical disease. Overall, 113 of the 143
The asymptomatic HSV-2 infection group comprised 85 patients: 54
males (64%; average age, 40 years; range, 21 to 66 years) and 31 females
(36%; average age, 34 years; range, 20 to 52 years) who were seropositive
for HSV-2 without any signs of clinical disease. Asymptomatic patients
were recruited from an ongoing screening study of HSV-2 infection in
visitors to the STD clinics and among the partners of the HSV-2-infected
patients. All the subjects were provided with detailed information about
the clinical spectrum of herpes and interviewed about genital symptoms.
the above information admitted to having genital symptoms were ex-
cluded from the study.
Three individuals (all symptomatic) were on immunosuppressive
treatment (for rheumatoid arthritis, Bechterew’s disease, and CNS vasc-
ent in the study population were hepatitis B (one symptomatic individ-
ual), diabetes (two symptomatic individuals), allergic asthma (one
symptomatic individual), multiple sclerosis (one symptomatic individ-
ual), and lichen sclerosis (one asymptomatic individual).
(ii) Control subjects. For the control group, 162 healthy HSV-2-neg-
ska University Hospital. This group consisted of 54% males and 46%
females with an average age of 40 and 41 years, respectively (ranges, 20 to
63 and 20 to 65 years, respectively). All individuals were screened for
HSV-2 infection by ELISA. All the individuals were routinely screened
(and found to be negative) for blood-derived contaminating diseases,
including hepatitis A and B, HIV-1 and -2, and human T-lymphotropic
virus I (HTLV-I) and -II.
nous blood using the salting-out method. Genotyping was performed on
SNPs [rs7574865 (T¡G), rs4853543 (A¡G), rs7572482 (A¡G),
rs13017460 (A¡G), rs7601754 (G¡A), rs3024896 (C¡T), and
rs6752770 (A¡G)] using TaqMan allelic discrimination (premade or
customized Applera assays) at the Core Facility of the Sahlgrenska Acad-
on the following criteria: (i) they are commonly detected in Caucasians
(minor allelic frequency ? 0.20, according to the Hap-Map-CEU north-
ELISA for detection of HSV-2-specific antibodies. Plasma samples
from HSV-2-uninfected individuals were screened for anti-HSV-2 glyco-
protein G (gG) antibodies using an HSV-2 ELISA kit according to the
manufacturer’s manual (HerpesSelect2 ELISA IgG; Focus Technologies),
and plasma samples from HSV-2-infected individuals were screened for
mgG-2-specific antibodies using an ELISA, as previously described (46).
Virus and antigen preparation. HSV-2 strain 333 and the attenuated
strain Lyons (35) were obtained as described previously (40). For antigen
IFN-? responses of human PBMC. Freshly isolated PBMC (1 ? 106
medium (Lonza, Verviers, Belgium) supplemented with 1% L-glutamine,
in the presence or absence of UV-inactivated HSV-2 (corresponding to
4 ? 105PFU/ml). Culture supernatants were collected after 48 h and
stored at ?20°C until assayed for IFN-? using a human IFN-? ELISA
DuoSet kit (R&D Systems) according to the manufacturer’s instructions.
Mice. For this study, 6-to-8-week-old female STAT4?/?and wild-
were maintained under standard conditions of temperature and light in
the animal facilities at the Department of Rheumatology and Inflamma-
Animals Ethics Committee in Gothenburg, Sweden.
Genital HSV-2 infection. Mice were pretreated with Depo-Provera
(Pharmacia) and then vaccinated and infected as previously described
(40). Mice were examined daily for vaginal inflammation, neurologic ill-
ness, and death. The severity of diseases was graded as follows: 0, healthy;
tion (hair loss, small nonpurulent lesions); 3, severe and purulent genital
lesions and/or generally bad condition; 4, hind-limb paralysis; and 5,
Virus quantification. Vaginal washings were collected 2 days postin-
fection and stored at ?70°C until further use. Spinal cords were collected
and homogenized 7 days (unvaccinated mice) or 14 days (vaccinated
mice) after infection and stored at ?70°C until further use. DNA was
extracted in a MagNA Pure LC robot (Roche Diagnostics, Mannheim,
Germany) using a MagNA Pure DNA Isolation kit according to the man-
ufacturer’s instructions. The number of HSV-2 DNA copies was deter-
in terms of genome equivalents (geq).
as previously described (40).
Cytokine responses of mouse splenocytes. Assays of IFN-? produc-
tion from CD4?spleen cells and IL-12 production from spleen cells were
performed using a cell ELISA that utilizes murine IFN-? and IL-12 Duo-
Set ELISA kits (R&D Systems), as previously described (40). CD4?T-cell
activation was performed with CD8-depleted splenocyte cultures in the
Svensson et al.
jvi.asm.orgJournal of Virology
culture supernatants from HSV-2-activated spleen cells were also ana-
lyzed using a Mouse Th1/Th2 10plex Ready-to-Use FlowCytomix Multi-
plex kit (eBioscience) and a FACSCanto flow cytometer (BD).
significant at P ? 0.05. The calculations were conducted using online
software. Hardy-Weinberg tests, haplotype association tests, and permu-
tation tests were performed using Haploview software. Bonferroni’s cor-
rection for multiple testing was used when appropriate. All the allele and
genotype frequencies were found to be in Hardy-Weinberg equilibrium.
were performed using the nonparametric Mann-Whitney U test and
GraphPad Prism ver. 5 software. Differences were considered significant
at P ? 0.05.
Genetic variations in STAT4 are associated with HSV-2 disease
pared seven different SNPs (rs7574865, rs4853543, rs7572482,
rs13017460, rs7601754, rs3024896, and rs6752770) of the human
STAT4 gene in asymptomatic and symptomatic HSV-2-infected
individuals, as well as in healthy controls. We did not detect any
infection when comparing HSV-2-infected individuals and
healthy controls (data not shown). However, for one of the SNPs,
rs7572482, which is located in an intron close to the 5= untran-
scribed region (UTR), the minor A allele variant correlated with
the incidence of symptomatic infection (Table 1) (P ? 0.0184).
However, this difference was not significant after Bonferroni’s
correction for multiple testing. We also investigated whether the
minor A allele of rs7572482 correlated with the need for antiviral
treatment. The A allele distribution was 45% in HSV-2-infected
individuals who required acyclovir treatment, compared with
33% in HSV-2-infected individuals who did not require antiviral
treatment (P ? 0.02).
To assess further the role of STAT4 variations in HSV-2 infec-
tion, we analyzed the haplotype frequencies of the seven STAT4
SNPs. Of the seven STAT4 haplotypes with ?4% frequency in at
least one of the groups, the CAATAGG combination was signifi-
cantly more common in asymptomatically infected individuals
than in individuals with symptomatic HSV-2 infection and in
healthy controls. The CAATAGG haplotype frequencies were 4%
and 12% in symptomatically and asymptomatically infected indi-
viduals, respectively (P ? 0.0029), and remained significant after
100,000 permutations (P ? 0.012) (Table 2).
Given that previous HSV-1 infection correlates with asymp-
tomatic infection, we compared the frequencies of HSV-1 sero-
positivity in the asymptomatic and symptomatic HSV-2-infected
individuals but found no statistically significant differences.
The G allele of STAT4 rs7572482 is associated with stronger
ciation of STAT4 variations with HSV-2 infection and the neces-
sity for STAT4 in Th1-mediated immunity (20), we examined
secretion in PBMC from HSV-2-infected individuals (both
asymptomatic and symptomatic) who had different STAT4
rs7572482 genotypes, representative for the CAATAGG haplo-
type. The levels of HSV-2-specific IFN-? production were higher
in the PBMC from individuals who were homozygous for the
major G allele than in those from individuals who were heterozy-
gous or homozygous for the minor A allele (P ? 0.04; Fig. 1).
was more than 2-fold higher in the cells from HSV-2-infected
individuals with the GG genotype than in individuals with the AA
genotype (Fig. 1). We and others have previously reported that
asymptomatic HSV-2 infection is associated with higher T-cell
recall IFN-? responses, compared with symptomatic HSV-2 in-
TABLE 1 Allele frequencies in symptomatic and asymptomatic HSV-2-
infected individuals for seven SNPs of the STAT4 genea
% (na) allele distribution
an, number of chromosomes.
TABLE 2 Haplotype frequencies in symptomatic and asymptomatic
HSV-2-infected individuals for the seven SNPs of the STAT4 genea
% (nb) frequency
aData were estimated using Haploview 4.2 software in the following order: rs3024896,
rs13017460, rs7601754, rs7574865, rs6752770, rs4853543, and rs7572482. Only
haplotypes with frequency ? 4% are presented.
bn, number of chromosomes.
FIG 1 Increased HSV-2-specific IFN-? secretion in individuals homozygous
for the major allele variant of the STAT4 SNP rs7572482. PBMC from HSV-
2-infected individuals (66 asymptomatic and 111 symptomatic) were stimu-
lated with UV-inactivated HSV-2 for 48 h and then analyzed for IFN-? secre-
tion. The data shown represent the individual levels (circles) and the median
levels (lines) of secreted IFN-? from HSV-2-infected individuals with the AA
(n ? 26), AG (n ? 84), or GG (n ? 67) genotype at rs7572482. *, P ? 0.05
STAT4 Regulates HSV-2 Disease Severity
September 2012 Volume 86 Number 17 jvi.asm.org 9411
fection (10, 37). However, we could not confirm this observation
in the current patient cohort (data not shown).
Impaired viral clearance in the genital tracts of vaccinated
infection, naïve and vaccinated STAT4-deficient and wild-type
were examined daily for genital inflammation and neurologic ill-
were measured. Initially, we measured disease development dur-
ing primary infection. No major differences between the
STAT4?/?and wild-type mice were observed after genital inocu-
lation of HSV-2. Neither disease development nor the survival
rates were affected by STAT4 deficiency (data not shown). The
STAT4?/?and wild-type mice had comparable levels of HSV-2
DNA in vaginal washes obtained 2 days postinfection (Fig. 2A)
and in the central nervous system (CNS) on day 7 postinfection
Subsequently, we challenged the vaccinated mice and assessed
the viral loads and disease development. The viral load in the
genital tract 2 days postinfection was significantly higher in the
vaccinated STAT4?/?mice than in the wild-type mice (Fig. 2C).
At this time point, we did not detect any viral DNA in the vaginal
washes from wild-type mice whereas the median amount of viral
DNA/ml. Two weeks after the HSV-2 challenge, the vaccinated
mice were sacrificed to measure the virus content in the CNS.
DNA (data not shown). Nevertheless, the STAT4?/?mice were
protected against terminal illness to the same extent as all the
wild-type mice, and all the STAT4?/?mice survived the HSV-2
Impaired HSV-2-specific Th1 responses in mice that lack
STAT4. To assess the role of STAT4 in virus-specific CD4?T-cell
responses, we measured the DTH and cytokine responses of the
STAT4?/?and wild-type mice 4 weeks after HSV-2 vaccination.
The mice that lacked STAT4 had impaired HSV-2-specific CD4?
response was evaluated by measuring DTH footpad swelling 48 h
after injection of inactivated HSV-2. The HSV-2-specific inflam-
matory responses were significantly lower in the STAT4?/?mice
tion in spleen cell cultures that were depleted of CD8?T cells was
measured using a cell ELISA. We found that the levels of HSV-2-
specific IFN-? secretion from the CD4?T cells of the STAT4?/?
mice were negligible compared to the high levels seen in the cells
from the wild-type mice (Fig. 3B). In addition, we screened the
Th2, and Th17 cytokines. We confirmed that the IFN-? levels
were significantly reduced in the STAT4-deficient mice (Fig. 3C).
However, there were no significant differences in the levels of the
other Th1 cytokines (Fig. 3C) or Th2 cytokines (Fig. 3D) between
any of the culture supernatants (data not shown).
In the present report, we provide evidence to implicate STAT4 in
HSV-2-specific immunity in both humans and mice. We show
that a variation in the human STAT4 gene is associated with
asymptomatic HSV-2 infection and increased HSV-2-specific
IFN-? secretion. In addition, we show that HSV-2 does replicate
in the genital tracts of vaccinated STAT4-deficient mice, most
likely due to their impaired HSV-2-specific IFN-? responses.
Genetic variations in the human STAT4 gene have previously
been associated with Th1-mediated inflammatory diseases, such
as rheumatoid arthritis, SLE, and Sjögrens syndrome (21). The
STAT4 variations are associated with an infectious disease. In our
study population, which consisted of 143 symptomatic and 85
asymptomatic HSV-2-infected individuals, we found that one of
the haplotypes of the human STAT4 gene, consisting of seven
SNPs from the same haplotype block, was associated with en-
hanced IFN-? responses and asymptomatic HSV-2 infection.
Therefore, STAT4 represents, together with certain HLA alleles
HSV-2 infection. However, the effect size of these associations is
very modest and can serve only as an indication that the gene is
important for the expression of a specific phenotype. This also
implies that more-profound sequence changes in this immune-
(A and B) and vaccinated (C) wild-type mice (filled circles) and STAT4?/?
mice (empty circles) were challenged intravaginally with HSV-2. The levels of
HSV-2 DNA were measured in vaginal fluids obtained 48 h after HSV-2 inoc-
ulation (A and C) and in spinal cords 7 days after HSV-2 inoculation (B). The
data shown represent the log10values for HSV-2 DNA copy numbers (geq;
genome equivalents/milliliter) for each individual (circle) and the median for
each group (line) obtained in two independent experiments with five mice/
group. **, P ? 0.01 (Mann-Whitney test).
Svensson et al.
jvi.asm.org Journal of Virology
We were not able to correlate genetic variations in the STAT4
gene with susceptibility to HSV-2 infection. However, genetic
variations in both innate and adaptive immune-regulating genes
may affect susceptibility to HSV-2 infection; SNPs in the genes
that encode the Th1-inducing transcription factor T-bet, TBX21
(39), and in the TLR3 gene (41) are risk factors for HSV-2 acqui-
sition. Thus, the host repertoire of several immune-regulating
genes may influence both susceptibility to and the severity of
The roles of STAT4 in human cells and during human infec-
tions have not been extensively studied. Similar to murine cells,
human CD4?T cells secrete IFN-? upon IL-12 stimulation and
STAT4 activation (4, 27). However, sufficient IFN-? secretion is
expression (32). In the present report, we show that a genetic
variation in the human STAT4 gene affects the in vitro levels of
IFN-? produced in response to HSV-2 recall activation. The ma-
specific IFN-? secretion in vitro, as well as with asymptomatic
HSV-2 infection. This supports the results of previous studies, in
which increased IFN-? responses were associated with symptom-
free HSV-2 infection (8, 10, 37). Taken together, our data suggest
HSV-2 disease severity, possibly by modulating virus-specific
The significance of IFN-? and CD4?T-cell responses in
HSV-2 immunity has been well described in murine models of
genital HSV-2 infection, in which IFN-? (14, 25, 30), and T-bet
expression (39) are critical for viral clearance and resistance to
infection in the genital tract. Here, we show that vaccinated
STAT4-deficient mice, in contrast to wild-type mice, do not de-
velop sterilizing immunity to HSV-2 in the genital tract. The in-
creased local viral load in STAT4?/?mice is associated with im-
responses in vivo and reduced IFN-? secretion in vitro. However,
even though we detected considerable amounts of viral DNA in
the virus did not reach the CNS. This implies that STAT4 is im-
portant for the vaccine-induced prevention of vaginal viral repli-
cation, whereas it plays a marginal role in preventing the virus
from spreading to the CNS. This notion differs from the conclu-
sions drawn from studies using the closely related HSV-1. Allen
and colleagues have shown in a model of ocular HSV-1 infection
that vaccinated STAT4-deficient mice resolve the infection to the
same extent as wild-type mice (2). This discrepancy could be due
to the different routes of infection. Moreover, the validity of our
HSV-2-specific IFN-? production. Therefore, we propose that
STAT4 is important for HSV-2-specific IFN-? responses in both
humans and mice.
None of the analyzed polymorphisms of the STAT4 gene were
FIG 3 Impaired HSV-2-specific IFN-? and DTH responses in STAT4?/?mice. Wild-type mice (filled circles) and STAT4?/?mice (open circles) were
vaccinated intravaginally with HSV-2 Tk- and analyzed 4 weeks later for virus-specific T-cell responses. (A) DTH responses were elicited by injection of
thickness of the experimental and control footpads after 48 h. (B) HSV-2-specific IFN-? secretion from spleen cell populations depleted of CD8?T cells was
inactivated HSV-2, as described in Materials and Methods. The data shown represent the levels of (A) footpad swelling and (B to D) cytokine secretion (in
picograms/milliliter) for individual animals (circles) and the median value (line) for experiments with five animals/group. *, P ? 0.05; **, P ? 0.01 (Mann-
STAT4 Regulates HSV-2 Disease Severity
September 2012 Volume 86 Number 17 jvi.asm.org 9413
wise, STAT4 deficiency did not affect either the viral load or dis-
ease progression in primary HSV-2 infection in mice. This is
somewhat different from previous findings obtained using a
model of ocular HSV-1 infection. Allen et al. found that STAT4-
deficient animals did not differ from wild-type animals with re-
spect to survival or disease development, despite having higher
levels of viral replication in the eye (2). Banerjee et al. have re-
ported that STAT4?/?mice are more susceptible to HSV-1-in-
duced encephalitis, although in their hands the animals also suc-
cumbed earlier to the infection (3). It is interesting that the two
Th1 transcription factors STAT4 and T-bet play different roles in
HSV-2 infection in both humans and mice. T-bet appears to be
more important during primary infection (39, 40), while STAT4
controls viral reactivation. This is probably linked to the func-
tional differences between these two transcription factors. While
T-bet expression is required during the early phase of Th1 differ-
Th1 phenotype by amplification of both T-bet and IFN-? expres-
sion (33). Thus, the importance of STAT4 appears to be in the
long-time maintenance of the HSV-2-specific IFN-? responses
that are required to control viral reactivation.
In summary, we show that STAT4 regulates HSV-2-specific
the human STAT4 gene are associated with increased IFN-? syn-
thesis and asymptomatic HSV-2 infection, while vaccinated mice
deficient in STAT4 have impaired HSV-2-specific IFN-? re-
certain genetic variations in STAT4 affect HSV-2 replication and
disease severity by altering the IFN-?-mediated antiviral immune
This work was supported by the Swedish Science Council, the Västra
Götaland Region through LUA/ALF, Torsten and Ragnar Söderberg’s
Foundation, Inga-Lill and Arne Lundberg’s Foundation, and Wilhelm
and Martina Lundgren’s Research Foundation.
We thank Christin Grimhag at the Sahlgrenska University Hospital
for collecting the blood samples and Maria Bergquist for help with the
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