Regulatory T Cells Are Locally Induced during Intravaginal Infection of Mice with Neisseria gonorrhoeae

Abstract

Neisseria gonorrhoeae is a gram-negative diplococcus that in human beings produces gonorrhea. Much clinical evidence has led to the conclusion
that gonococcus has important mechanisms to evade host immune functions; however, these mechanisms are only now beginning
to be elucidated. In this study, we determined that the BALB/c mouse is a good animal model to study gonococcus infection
and examined the immune response against the bacteria. We determined that after intravaginal inoculation of mice with Neisseria gonorrhoeae, the bacteria reached and invaded the upper female reproductive tissues and elicited a T-cell-specific immune response associated
with a very weak humoral response, altogether resembling gonococcus infection and disease in women. Remarkably, in the draining
lymph nodes of the genital tracts of infected mice, we found an increase of regulatory T lymphocytes, namely, transforming
growth factor β1-positive CD4+ T cells and CD4+ CD25+ Foxp3+ T cells. Altogether, results indicate that N. gonorrhoeae induces regulatory T cells, which might be related to the local survival of the pathogen and the establishment of a chronic
asymptomatic infection.

Full text

INFECTION AND IMMUNITY, Dec. 2008, p. 5456–5465 Vol. 76, No. 12
0019-9567/08/$08.00⫹0 doi:10.1128/IAI.00552-08
Copyright © 2008, American Society for Microbiology. All Rights Reserved.
Regulatory T Cells Are Locally Induced during Intravaginal Infection
of Mice with Neisseria gonorrhoeae
䌤
Mo´nica Imarai,
1
* Enzo Candia,
1
Carolina Rodriguez-Tirado,
1
Javier Tognarelli,
1
Mirka Pardo,
1
Tomas Pe´rez,
1
Daniel Valde´s,
1
Sebastia´n Reyes-Cerpa,
1
Pablo Nelson,
1
Claudio Acuna-Castillo,
1
and Kevin Maisey
1,2
Laboratorio de Inmunología, Departamento de Biología, Facultad de Química y Biología, Universidad de Santiago de
Chile, Alameda 3363, Correo 40, Casilla 33, Santiago, Chile,
1
and Unidad de Investigacio´n y Desarrollo,
Macrocap S.A., Santiago, Chile
2
Received 6 May 2008/Returned for modification 18 June 2008/Accepted 19 September 2008
Neisseria gonorrhoeae is a gram-negative diplococcus that in human beings produces gonorrhea. Much
clinical evidence has led to the conclusion that gonococcus has important mechanisms to evade host immune
functions; however, these mechanisms are only now beginning to be elucidated. In this study, we determined
that the BALB/c mouse is a good animal model to study gonococcus infection and examined the immune
response against the bacteria. We determined that after intravaginal inoculation of mice with Neisseria
gonorrhoeae, the bacteria reached and invaded the upper female reproductive tissues and elicited a T-cell-
specific immune response associated with a very weak humoral response, altogether resembling gonococcus
infection and disease in women. Remarkably, in the draining lymph nodes of the genital tracts of infected mice,
we found an increase of regulatory T lymphocytes, namely, transforming growth factor ␤1-positive CD4
ⴙ
T
cells and CD4
ⴙ
CD25
ⴙ
Foxp3
ⴙ
T cells. Altogether, results indicate that N. gonorrhoeae induces regulatory T
cells, which might be related to the local survival of the pathogen and the establishment of a chronic
asymptomatic infection.
Gonorrhea is a sexually transmitted disease produced by the
gram-negative diplococcus Neisseria gonorrhoeae. In women,
infection affects the cervix and may spread to the uterus and
oviduct, inducing endometritis and pelvic inflammatory dis-
ease. Strikingly, about 50% of the cases proceed without symp-
toms, inducing damage mainly in the fallopian tube, while in
men infection occurs with distinctive clinical symptoms (11,
21). The hallmark of humoral immune response against N.
gonorrhoeae is the extremely low levels of antigonococcal an-
tibodies found in serum and secretions of the human (male and
female) during infection (19, 20). Antibodies are directed
against several major membrane molecules, such as the pilus-
and opacity-associated outer membrane proteins (Pil and Opa,
respectively), the porin protein (Por), and the lipooligosaccha-
ride (5, 19, 20, 38, 39, 55). Although some of these have
bactericidal activity, they are not protective and seem to be
blocked by outer membrane protein 3 (RmP)-specific immu-
noglobulin Gs (IgGs) (38).
The highly asymptomatic infection in women and the poor
immune response related to gonococcus prime and multiple
challenges might actually be related to mechanisms of immune
evasion acquired by the bacteria to constrain the immune
response. Moreover, the presence of a significant, although
weak, humoral immune response during gonococcus infection
(19) suggests that the bacteria might stimulate a regulatory
type of immune response. One of these mechanisms might be
the induction of noninflammatory responses dominated by
Th2-type cytokines and the activation of regulatory T cells
(Tregs), which in turn would contribute to the suppression of
most of the mechanisms of protection against intracellular
pathogens (2, 40). Tregs are CD4
⫹
T lymphocytes involved in
the induction of suppressor responses; it has been determined
experimentally that they show several phenotypes. One of the
major characteristics is related to the presence of transforming
growth factor ␤1 (TGF-␤1) (52), and subgroups can be distin-
guished because of the expression of CD25 and the Foxp3
transcription factor. CD4
⫹
CD25
⫹
T cells correspond to a
subgroup expressing Foxp3, which originate in the thymus and
are called natural Tregs, while the CD4
⫹
CD25
⫺
T cells are
induced at the periphery after antigenic stimulation in the
presence of a distinct cytokine environment (28, 31, 50, 52).
TGF-␤1 blocks T-cell proliferation, inhibits Th1, Th2, and
CTL differentiation, and moreover induces Foxp3 expression
in Tregs (27).
Overall, primary cell and organ culture systems have been
successfully developed to examine the initial phase of gono-
coccal pathogenesis (9, 10, 13, 29, 48) where several gonococ-
cus membrane components, such as Pil and Opa, are highly
relevant (30). However, the mechanisms explaining why female
infection occurs in a high number of cases without inflamma-
tory signs, with low levels of antibody induction, and with no
disease resolution responses are not understood. Understand-
ing pathogenesis at this level has been greatly hindered due to
the ethical considerations associated with human research
work and the lack of an animal model of experimental infec-
tion (9). Only recently, a murine model introduced by Ann
Jerse has allowed studies of protection against gonococcus
infection (23, 37).
* Corresponding author. Mailing address: Laboratorio de Inmu-
nología, Departamento de Biología, Facultad de Química y Biología,
Universidad de Santiago de Chile, Alameda 3363, Correo 40, Casilla
33, Santiago, Chile. Phone: 56-2-718 1164. Fax: 56-2-681 2108. E-mail:
monica.imarai@usach.cl.
䌤
Published ahead of print on 29 September 2008.
5456

In this study, we have further investigated the mouse exper-
imental model of N. gonorrhoeae infection and demonstrated
that the bacteria reach and invade the upper female reproduc-
tive tissues (uterus and oviduct), resembling gonococcus infec-
tion and disease in women. The murine model allowed us to
determine that infection elicits a T-cell-specific immune re-
sponse associated with a weak humoral response. In addition,
the local response includes the induction of regulatory TGF-
␤1
⫹
T cells which, acting to suppress the activation of the
immune system, would support the occurrence of infection.
Altogether, results indicate that N. gonorrhoeae induces regu-
latory mechanisms of immunity which, in turn, might explain
the local survival of the pathogen and the establishment of a
chronic asymptomatic infection in women.
MATERIALS AND METHODS
Bacteria and culture conditions. The following three different variants of N.
gonorrhoeae strain P9, kindly provided by Myron Christodoulides (University of
Southampton, United Kingdom), were used in this study: P9-13 (Pil
⫺
Opa
a⫹
),
P9-16 (Pil
⫺
Opa
b⫹
), and P9-17 (Pil
⫹
Opa
b⫹
) (8). Bacteria were routinely grown
on GC agar (Becton Dickinson, Maryland) supplemented with 1% IsoVitaleX
(Becton Dickinson) for 18 h at 37°C in 5% CO
2
. Gonococcal variants containing
a red shift mutant green fluorescent protein (GFP) plasmid were grown in GC
agar containing ampicillin (5 ␮g/ml). Analysis of colony morphology under a
stereomicroscope and Western blotting for the detection of Pil and Opa were
routinely performed to discard phenotypic variability. Monoclonal antibodies
against Pil and Opa were kindly provided by Mumtaz Virji (University of Bristol,
United Kingdom) and Mark Achtman (Max Planck Institute, Berlin, Germany),
respectively.
Animals. BALB/c mice were obtained from the Institute of Public Health
(Santiago, Chile), housed under regulated light and temperature conditions, and
sacrificed by cervical dislocation. Research was conducted in accordance with
institutional guidelines and with the International Guiding Principles for
Biomedical Research Involving Animals of the Society for the Study of
Reproduction.
Mouse uterine cell cultures. Cultures were prepared from mouse uteri as
previously described (17). Briefly, uterine horns were washed in RPMI 1640
medium (GIBCO Invitrogen Co., Carlsbad, CA) containing 100 U/ml penicillin
and 100 ␮g/ml streptomycin (GIBCO Invitrogen Co.). Organs were cut into
small pieces and then transferred to a plate containing 0.25% trypsin and 2.5%
pancreatin (Life Technologies, Grand Island, NY) and incubated for 60 min at
4°C followed by 60 min at 20°C. Organs were transferred to a sterile tube
containing 15 ml of cold Hanks’ balanced salt solution (GIBCO Invitrogen Co.).
Digested uteri were then vortexed and the released epithelial cells were recov-
ered and transferred to a clean tube. The procedure was repeated three times
and cells in suspension were collected, centrifuged for 8 min at 180 ⫻g, and
resuspended in RPMI 1640 supplemented with 10% fetal bovine serum (Hy-
Clone Laboratories, Inc., Logan, UT), 1 mM L-glutamine, 100 ␮g/ml streptomy-
cin, and 100 U/ml penicillin. Uterine cells were cultured at 37°C in 5% CO
2
until
they reached 90% confluence. Positive immunostaining with a mouse anticyto-
keratin monoclonal antibody (Chemicon International, Temecula, CA) and neg-
ative staining with antivimentin antibody (Chemicon International) confirmed
the epithelial origin of these cells. Typically, cultures had more than 95% epi-
thelial cells.
Infection of primary uterine cell cultures. Gonococcal isolates were taken
from frozen stocks and cultured on GC agar plates at 37°C in a 5% CO
2
-air
atmosphere. Bacteria were then scraped from confluent culture plates and re-
suspended in 1 ml of Dulbecco’s modified Eagle’s medium without phenol red
(GIBCO Invitrogen Co.). The concentration was estimated by comparison as 1
optical density unit at 600 nm, corresponding to 3.2 ⫻10
9
CFU/ml. Cells were
infected with N. gonorrhoeae fluorescence variants at a multiplicity of infection of
10 and cultured at 37°C in 5% CO
2
until they reached 90% confluence. To
determine the uptake of bacteria, epithelial cells were treated with 100 ␮g/ml of
gentamicin for 60 min at 37°C to kill extracellular bacteria. Cells were then
diluted in phosphate-buffered saline (PBS) containing 1% saponin and incubated
for 15 min at 37°C. A tenfold dilution series was prepared and 100-␮l portions
were spread on GC plates. The internalization of bacteria in epithelial cells was
evident by the recovery of colonies composed of gram-negative, oxidase-positive
diplococci after 18 h of growth at 37°C.
Mouse experimental infection. Experimental infection of BALB/c mice was
performed as previously described (23) with some modifications. Briefly, 7 days
before bacterium inoculation, four groups of five female BALB/c mice (6 to 8
weeks old) were daily injected with 3 ␮g of cetrorelix acetate (subcutaneous
administration). On day 4, half of the groups were additionally inoculated daily
with 300 ng of estradiol [estra-1,3,5(10)-triene-3,17-beta-diol; Sigma] until the
end of the experiment. On day 7, one estradiol-treated group and one control
group were intravaginally inoculated with 10
8
CFU N. gonorrhoeae fluorescence
variants suspended in Dulbecco’s modified Eagle’s medium without phenol red
(GIBCO Invitrogen Co.). The other two groups were treated with medium. The
procedure was performed using a sterile syringe connected to flexible tubing
which allowed instillation. Tubing was inserted into the vagina, first dorsally and
then cranially, until the cervix was reached. Fifty-microliter portions of bacterial
suspension were inoculated in the mice. At 1, 3, and 5 days postinoculation, the
genital tracts of infected and control mice were removed. Uteri were weighed to
assess the effectiveness of estradiol treatment.
Microscopy. For microscopic analysis, cells were cultured on coverslips and
infected as described above. After the indicated times, cells were fixed with 1%
paraformaldehyde in PBS. In the cases of vaginas, uteri, and oviducts, tissues
were fixed in 4% paraformaldehyde in PBS for 1 h before sequential transfer to
10% sucrose in PBS for 1 h, 20% sucrose in PBS for 1 h, and 30% sucrose in PBS
overnight. Organs were mounted in embedding compound (Cryo-M-Bed; Bright
Instrument Co. Ltd., Huntingdon, United Kingdom) and frozen at ⫺20°C. Slices
of5to10␮m were cut using a Bright Starlet cryostat at ⫺20°C. Fixed cultured
cells and tissue sections were counterstained using a solution of 1 ␮g/ml pro-
pidium iodide in PBS and mounted in a solution of PBS containing 10% (vol/vol)
1,4-diazobicyclo[2.2.2] octane (Dabco; Sigma) and 90% (vol/vol) glycerol. Anal-
ysis of interactions of N. gonorrhoeae with uterine cells was performed by con-
focal microscopy (Zeiss LSM510). Briefly, confocal z-plane slices were obtained
and orthogonal views and three-dimensional images were generated from iso-
lated cells and tissues. For transmission electron microscopy analysis, cells were
fixed in 1% glutaraldehyde in PBS (pH 7.4) and dehydrated for embedding in
epoxy resin. Ultrathin sections were stained with uranyl acetate and lead citrate.
Control and infected samples were viewed with a Philips EM-200 transmission
electron microscope.
ELISA. An enzyme-linked immunosorbent assay (ELISA) was performed for
serum titration. Briefly, whole-protein extracts were prepared by freeze-thawing
N. gonorrhoeae strain P9-17 in water, and protein levels were determined by the
Bradford assay. Extracts prepared from Escherichia coli JM109 were used as a
negative control. Microtiter plates (Falcon; Becton Dickinson Co., Santiago,
Chile) were activated with 10 ␮g of protein per well and incubated overnight at
4°C for 24 h. After the washing, sera collected from infected (n⫽4) and control
(n⫽4) mice were added to plates (at 1:20, 1:50, 1:100, 1:200, and 1:400
dilutions). Specific antibodies to gonococcus were detected with goat anti-mouse
IgG-alkaline phosphatase conjugate (Sigma) and p-nitrophenyl phosphate
(Sigma). Each mouse serum sample was assayed in duplicate.
T-cell antigen proliferation assays. To determine specific T-cell responses,
mice were intravaginally infected as described above and then boosted intraperi-
toneally on day 15. Seven days after the boost (day 22), mice were sacrificed and
the spleens and lymph nodes (renal, iliac, and caudal) were removed (34). CD4
⫹
T cells were isolated by negative selection using antibody-coated magnetic beads
(Dynal; Invitrogen). Isolated T cells (1 ⫻10
5
cells/well) from treated and control
mice were cultured with mitomycin-treated autologous splenocytes (2 ⫻10
4
cells/well) previously pulsed with 1 or 10 ␮g of whole-protein extract of N.
gonorrhoeae. Experiments were carried out in 96-well plates in triplicate. As a
positive control, T cells were stimulated with phytohemagglutinin (PHA) (10
␮l/ml; GIBCO Invitrogen Co.). Plates were incubated at 37°C and 5% CO
2
for
72 h and then pulsed with 1 ␮Ci methyl-[
3
H]thymidine (Amersham Biosciences,
Buckinghamshire, United Kingdom) for the final 18 h before harvesting. The
amount of incorporated radioactivity was measured using a liquid scintillation
counter (Tri-Carb 2100 TR; Packard). Data were expressed as experimental
minus control counts per minute.
Leukocyte isolation. A previously described protocol was followed (24).
Briefly, uteri dissected from mice (four animals/experiment) were placed in
sterile ice-cold Hanks’ balanced salt solution (GIBCO, Grand Island, NY),
weighed, and then transferred to a mixture of pancreatin (GIBCO), trypsin
(Sigma, St. Louis, MO), and DNase (Worthington, Lakewood, NJ). Under sterile
conditions, uterine tissues in the enzyme mixture were cut into small pieces,
transferred to six-well culture plates, and incubated first for1hat4°Candthen
for an additional hour at room temperature with gentle shaking. Cells were
recovered from the supernatant and were pooled for each experiment.
Immunofluorescence staining and analysis. For TGF-␤1 detection, leukocytes
(5 ⫻10
5
cells/ml) were incubated with anti-TGF-␤1 antibody (1:100; Santa Cruz
VOL. 76, 2008 T-CELL IMMUNE RESPONSE AGAINST GONOCOCCUS 5457

Biotechnology) for1hat4°C. After being washed with PBS, cells were incubated
with a fluorescein isothiocyanate-conjugated polyclonal anti-rabbit IgG (1:200;
Santa Cruz Biotechnology) for1hat4°C. For CD11b detection, cells were
incubated for 30 min on ice with 2 ml of RPMI-10% fetal bovine serum with 10%
heat-inactivated normal mouse serum to reduce the nonspecific binding of an-
tibodies. After incubation, 10
6
cells/ml were labeled with phycoerythrin-conju-
gated anti-mouse CD11b (Santa Cruz Biotechnology, Inc.) for1hat4°C. For
analysis of CD4
⫹
CD25
⫹
Foxp3
⫹
T lymphocytes, 1 ⫻10
6
cells were labeled
using PE-Cy5.5-conjugated anti-CD4 antibody (clone GK1.5; eBioscience, San
Diego, CA) and fluorescein isothiocyanate-conjugated anti-CD25 antibody
(clone PC61.5; eBioscience). After being washed, cells were resuspended in 200
␮l of Fix/Perm buffer (eBioscience) and left at 4°C for 30 min. After being
washed, cells were incubated with phycoerythrin-conjugated anti-mouse Foxp3
(clone FJK-16S; eBioscience, San Diego, CA) for 30 min. Isotypic controls were
routinely included in all experiments. Labeled cells were analyzed on a FACScan
flow cytometer (Becton Dickinson) with CellQuest software.
Mixed lymphocyte reaction (MLR). Fresh T cells isolated from the lymph
nodes and spleens of control and infected BALB/c mice (H-2
d
) were first labeled
with 5 ␮M carboxyfluorescein diacetate succinimidyl ester (CFSE). T-cell re-
sponders (10
5
) were incubated with spleen cells (5 ⫻10
5
) isolated from C57BL/6
(H-2
b
) mice in 200 ␮l of RPMI at 37°C. After 5 days of culture in 96-well
round-bottom plates, cells were washed and fixed in 1% paraformaldehyde, and
CFSE dilution was assessed by flow cytometry.
RESULTS
N. gonorrhoeae infects isolated murine uterine cells. Previ-
ously, it had been extensively described that N. gonorrhoeae is
a strictly human pathogen; nevertheless, recent reports indi-
cated that the bacteria are also able to colonize the mouse
genital female tract. Before initiating studies of infection in
vivo, our first aim was to demonstrate that the bacteria attach,
bind, and invade murine genital tract cells. Isolated mouse
uterine epithelial cells were chosen as targets for in vitro stud-
ies. Cells were infected with N. gonorrhoeae fluorescent (GFP)
strain P9, variant P9-17 (Pil
⫹
Opa
b
⫹
), at a multiplicity of
infection of 10 bacteria per cell. A simultaneous detection of
GFP-expressing gonococci and the red-stained nucleus on 0.4-
␮m-thickness cross-sectional images by confocal microscopy
showed that, after 24 h of incubation, gonococci were found
associated with the epithelial cells, with a significant number
inside the cells surrounding the nucleus (Fig. 1A). Similar
results were obtained for variants P9-16 and P9-13. The intra-
cellular localization of the bacteria was confirmed by transmis-
FIG. 1. (A) Confocal photomicrograph of serial sections (0.4 ␮m) shows the presence of N. gonorrhoeae (P9-17) around the nuclei of uterine
epithelial cells. Bacteria appear green, and the cell nuclei are red. (B) Uterine cells; control cultures stained with propidium iodide. (C and D)
Transmission electron microscopy images of isolated uterine epithelial cells infected with N. gonorrhoeae (variant P9-16) show multiple cytoplasmic
bacteria enclosed in membrane-bound vesicles (arrowheads). Bars, 20 ␮m (B); 1 ␮m (C); 0.1 ␮m (D).
5458 IMARAI ET AL. INFECT.IMMUN.

sion electron microscopy, as micrographs exhibited N. gonor-
rhoeae P9-16 enclosed in membrane-bound vesicles of the
mouse uterine cells (Fig. 1C and D). Similar structures were
not found in control cells (not shown). In addition, an average
of 3.5 ⫻10
4
intracellular gonococci per well (24-well plate) was
recovered from infected uterine epithelial cells after selective
antibiotic killing of extracellular bacteria. Colonies had the
expected morphology, i.e., variants exhibited small opaque col-
onies with sharp edges, which correspond to Pil
⫹
Opa
⫹
gono-
cocci. Much higher numbers of bacteria were observed under
fluorescent microscopy, indicating that the major portion of
gonococci appear attached to the uterine cell, exactly as pre-
viously described for the infection of human endometrial and
oviductal cells (8). Results demonstrated that N. gonorrhoeae is
able to attach and invade the uterine epithelial, thus unequiv-
ocally identifying murine cells as a target of gonococcus infec-
tion.
N. gonorrhoeae invades the upper female reproductive tract
of the mouse. Once it was determined that N. gonorrhoeae is
able to colonize isolated uterine epithelial cells, the next step
was to corroborate that the bacteria colonize the upper repro-
ductive tract in mice, as seen for women. Because estradiol
seems to be crucial for gonococcus infection in mice (23), we
inoculated bacterial strain P9-17 expressing GFP (10
8
CFU)
FIG. 2. Confocal images of uterine sections of mice intravaginally infected with GFP-expressing N. gonorrhoeae (variant P9-17). The cell nuclei
are stained with propidium iodide. (A) Three days after inoculation. Bar, 50 ␮m. (B) Five days after inoculation. Bar, 20 ␮m. (C) Orthogonal views
of a midplane zsection; height, 1.3 ␮m. (D) Negative control. Arrows denote fluorescent bacteria.
VOL. 76, 2008 T-CELL IMMUNE RESPONSE AGAINST GONOCOCCUS 5459

into the vaginas of estradiol-treated BALB/c mice. Uteri were
removed after several days postinoculation, processed as de-
scribed in Materials and Methods, and analyzed by confocal
microscopy. Fluorescent bacteria were found in the uteri of all
inoculated mice (n⫽25), particularly in the epithelial and
subepithelial tissues of the organs. On day 1 after inoculation,
gonococci were mostly observed in the epithelium and occa-
sionally in the stroma, while on day 3, most of the bacteria were
present in the stroma (Fig. 2A). On day 5, the bacteria were
distributed throughout the stroma but also in the most external
tissues of the uterus (Fig. 2B). An orthogonal view of the
infected uterus (midplane zsection; height, 1.3 ␮m,) confirmed
the bacterial localization within the depths of the tissue (Fig.
2C). As the bacteria were also found in the epithelial and
subepithelial tissues of the vaginas and the oviducts, overall
these results indicate that intravaginal inoculation of N. gonor-
rhoeae allows the bacteria to reach, attach to, and invade the
mucosal tissues of the lower and upper organs of the mouse
genital tract. Further experiments were performed to evaluate
the contribution of estradiol in allowing infection. The exper-
iments were repeated several times with different mice at ran-
dom stages of the reproductive cycle, and surprisingly, infec-
tion was also observed for all mice tested. Moreover, infection
seemed to be persistent, because the bacteria were detected as
late as 22 days postinoculation; however, as is the case for
women, no clinical signs of the disease were apparent in
the mice.
Immune response. After corroborating that gonococcus in-
fects the mouse genital tract, as it does in humans, we wanted
to establish whether the bacteria also induce an antigen-spe-
cific response in mice. To evaluate the humoral immune re-
sponse, sera from infected and control animals obtained at day
22 after treatment were tested using ELISA against a whole-
protein extract of the P9-17 bacterial variant. Very low anti-
body titers in response to N. gonorrhoeae were detected for two
out of the four infected mice (Fig. 3), while as expected, no
detectable antibody was observed in serum samples of control
mice. To examine the T-cell response, CD4
⫹
T lymphocytes
isolated from uterus-draining lymph nodes from infected and
control mice were examined for the ability to proliferate in
response to a whole-protein extract of the bacteria (variant
P9-17). The results show that T cells from infected BALB/c
mice exhibited a dose-dependent T-cell-proliferative response
to gonococcus protein extract (Fig. 4). These findings indicate
that N. gonorrhoeae induces a local antigen-specific T-cell re-
sponse associated with a low- or no-antibody response during
experimental infection in the mouse.
Tregs increase in infected animals. As mentioned previ-
ously, specific stimulation of Tregs might explain the weak
antigonococcal humoral immune response and the absence of
protective immunity. Thus, we evaluated whether the BALB/c
T-cell response to gonococcus also involves the stimulation of
Tregs. As the major phenotype of Tregs is the synthesis of
TGF-␤1, we first quantified the percentage of TGF-␤1-produc-
ing T cells locally induced 22 days after infection. In six inde-
pendent experiments, the percentages of CD4
⫹
TGF-␤1
⫹
T
cells isolated from the regional lymph nodes of infected mice
(Fig. 5C, top right, and F) were twofold higher than those of
cells of the control group of mice (Fig. 5B, top right, and F).
No changes in the CD4
⫹
TGF-␤1
⫹
T lymphocytes were ob-
served for T cells isolated from spleens of the same animals
(Fig. 5D and E, top right, and F). Interestingly, a strong shift
in CD4
⫺
TGF-␤1
⫹
splenocytes from infected (Fig. 5E) versus
noninfected (Fig. 5D) mice occurred. This effect was not ob-
served in the regional lymph nodes, and the cells might corre-
spond to a different type of regulatory cells. In addition, we
examined the stimulated T cells in four independent experi-
ments to determine the presence of CD25 and Foxp3, addi-
tional molecular markers of Tregs. Results revealed that the
percentage of CD4
⫹
CD25
⫹
Foxp3
⫹
T cells detected in the
local lymph nodes of the group of infected animals was higher
FIG. 4. Proliferative response of isolated CD4
⫹
T cells (LT) from
infected mice to a protein extract of N. gonorrhoeae. T cells were
stimulated with PHA or added to BALB/c splenocytes (antigen-pre-
senting cells [APC]) pretreated with 1 or 10 ␮g of whole-protein
extract of N. gonorrhoeae P9-17 (Ngo). Proliferation was measured by
[
3
H]thymidine incorporation. Each bar represents the mean ⫾stan-
dard error of the mean (SEM) from triplicates. Data were expressed as
experimental minus control counts per minute. Two independent ex-
periments were done with similar results. Asterisks and double aster-
isks indicate Pvalues of ⬍0.05 and ⬍0.0001, respectively, versus values
for LT plus APC only by analysis of variance.
FIG. 3. Analysis of serum response to N. gonorrhoeae by ELISA.
Mouse sera were assayed on a whole-protein extract of the P9-17
bacterial variant in a reciprocal dilution series starting at 1:50, and
binding was detected with an alkaline phosphatase anti-mouse conju-
gate. Black symbols represent infected mice (n⫽4) and open symbols
control mice (n⫽4).
5460 IMARAI ET AL. INFECT.IMMUN.

than that of the control group (P⫽0.005) (Fig. 6A and B, top
right, and E). Once again, no statistically significant differences
were found when cells in the spleens of the animals were
quantified (Fig. 6C and D, top right, and E). Then, we tested
the suppressive function of Tregs in an allogeneic MLR. Only
cells from the lymph nodes of infected BALB/c mice, where
Tregs have been expanded by gonococcus infection, showed
suppression in the MLR stimulated by C57BL/6 spleen cells,
FIG. 5. Flow cytometric analysis of TGF-␤1-producing CD4
⫹
T cells. (A to E) Representative experiment. (A) Control of autofluorescence. (B) Cells
isolated from the regional lymph nodes of control mice. (C) Cells isolated from the regional lymph nodes of infected mice. (D) Splenocytes of control mice.
(E) Splenocytes of infected mice. The percentage of TGF-␤1
⫹
CD4
⫹
T cells is shown in the top right panel of each figure. (F) Percentages of TGF-␤1
⫹
CD4
⫹
T cells in lymph nodes and spleens of infected (white bars) and control (black bars) BALB/c mice. Bars represent means ⫾SEM from six independent
experiments. Lymph nodes of infected mice showed a significantly higher percentage of TGF-␤1-producing CD4
⫹
T cells than did the lymph nodes of the control
group. No differences were found in spleens. *,Pvalue of ⬍0.02 by the Mann-Whitney U test.
VOL. 76, 2008 T-CELL IMMUNE RESPONSE AGAINST GONOCOCCUS 5461

while cells from the lymph nodes of control mice and from the
spleens of infected mice, where no T-regulatory expansion was
observed, showed a regular response to allogeneic C57BL/6
stimulation (Fig. 7). Overall, these results indicate that gono-
coccus infection of the murine genital tract induces a signifi-
cant stimulation of Tregs as part of the local immune response.
TGF-␤1
ⴙ
cells infiltrate the uterine tissues. We further
investigated whether a local immune response was also accom-
panied by a uterine infiltration of TGF-␤1
⫹
cells. Because N.
gonorrhoeae in vitro induces the expression of TGF-␤1 in mac-
rophages (unpublished data), cells isolated from the uteri of
infected and control animals were labeled with anti-TGF-␤1
and anti-CD11b (macrophage marker) antibodies. Data from a
flow cytometric analysis show that infected animals have a
moderate but significant increase of CD11b
⫹
TGF-␤1
⫹
cells in
the uteri (Fig. 8C and D, top right), which indicates that gono-
coccus induces a mucosal infiltration of macrophages whose
phenotype would favor the differentiation of Tregs. Interest-
FIG. 6. Flow cytometric detection of regulatory CD4
⫹
T cells. (A to D) Representative experiment; results are expressed as the percentage of
CD25
⫹
or CD25
⫺
T cells expressing FoxP3 in the gated CD4
⫹
T-cell population of local lymph nodes of control BALB/c mice (A), local lymph
nodes of infected BALB/c mice (B), spleens of control mice (C), and spleens of infected mice (D). (E) Percentages of CD4
⫹
CD25
⫹
Foxp3
⫹
T
cells in lymph nodes and spleens of infected (white bars) and control (black bars) mice. Bars represent means ⫾SEM of four independent
experiments. Lymph nodes of infected mice showed a significantly higher percentage of CD4
⫹
CD25
⫹
Foxp3
⫹
T cells than did the lymph nodes
of the control group. No differences were found in spleens. *,Pvalue of ⬍0.05 by the Mann-Whitney U test.
5462 IMARAI ET AL. INFECT.IMMUN.

ingly, in addition to being associated with the infiltration of
macrophages, infection is also associated with the infiltration
of a great number of CD11b
⫺
TGF-␤1
⫹
cells (Fig. 8E), which
might correspond to Tregs.
DISCUSSION
In this study, we established that N. gonorrhoeae invades the
murine mucosa of the upper genital tract, results that confirm
and extend the extensive mouse work developed by Ann Jerse
and collaborators (23, 37, 43). Moreover, we demonstrated
that infection induces a weak humoral immune response asso-
ciated with a local increase of TGF-␤1
⫹
CD4
⫹
Tregs.
Until now, it has been widely accepted that N. gonorrhoeae is
a strictly human pathogen, which calls the validity of the use of
mice as an animal model of experimental infection into ques-
tion. This is reinforced by the fact that most of human recep-
tors for attachment and invasion do not seem to be expressed
in the mouse (30). However, we demonstrated here that N.
gonorrhoeae is able to infect murine epithelial cells isolated
from the uterus; the uptake of gonococcus by murine uterine
epithelial cells was demonstrated by means of three indepen-
dent assays, a gentamicin resistance test, confocal multiple
scan analysis, and transmission electron microscopy. More-
over, in vivo, we showed that GFP-expressing gonococci are
able to reach the upper genital organs and to invade uterine
tissues in all infected animals. Because in the experiments
there were no sources of green fluorescence other than that of
the bacteria, the uterus is normally sterile, and colonies of
gonococcus were recovered from the uterine cells of infected
mice, we are confident that the 1- to 2-␮m fluorescent spots
seen in tissues by confocal microscopy correspond to bacteria.
Therefore, although mice are not natural hosts for N. gonor-
rhoeae, results confirmed that gonococcus infects the lower
genital tract of BALB/c mice (23) and demonstrated for the
first time that the bacteria not only reach the upper genital
tract but also invade the upper murine mucosae, as occurs in
humans.
Analyses performed to characterize the immune response of
BALB/c mice showed that once gonococcus colonizes the fe-
male genital tract, bacterial antigens are detected by the im-
mune system, as revealed by the ability of CD4
⫹
T lymphocytes
to respond to the bacterial extract and by the presence of
specific antibodies in serum. However, as has been observed
for humans, antibodies that recognize N. gonorrhoeae were
FIG. 7. Geometric mean fluorescence of CFSE-labeled T cells. Re-
sponder cells were isolated from the lymph nodes and spleens of
control (white bars) and infected (black bars) BALB/c mice. For MLR,
stimulators were obtained from C57BL/6 spleens. CSFE-labeled re-
sponder cells were incubated with stimulator cells (ratio of 1:5) for 5
days and CFSE dilution was assessed by flow cytometry at the end of
the experiment. Bars represent geometric means (G mean) ⫾SEM
from nine independent experiments. Only cells from the lymph nodes,
where Tregs have been expanded by gonococcus infection, showed
suppression to the MLR. ⴱ,Pvalue of ⬍0.0001 by the Kruskal-Wallis
test.
FIG. 8. Flow cytometry analysis of CD11b
⫹
TGF-␤1
⫹
cells infil-
trating genital tract tissues following infection with N. gonorrhoeae.
(A) Autofluorescence control. (B) Secondary antibody control. (C and
D) CD11b
⫹
TGF-␤1
⫹
cells from control (C) and infected (D) tissues.
(E) White bars, TGF-␤1
⫹
cells from control groups; black bars, TGF-
␤1
⫹
cells from infected mice.
VOL. 76, 2008 T-CELL IMMUNE RESPONSE AGAINST GONOCOCCUS 5463

detected at low levels in the infected mice. Gonococcal acti-
vation of CD4
⫹
T cells has also been described for humans, as
gonococcal pilus interaction with CD4
⫹
T cells induces the
activation and proliferation of lymphocytes and stimulates the
secretion of interleukin-10 (IL-10) (36). In contrast, it has also
been shown that N. gonorrhoeae Opa proteins mediate binding
to CEACAM-1 expressed by CD4
⫹
T cells and suppress the
activation and proliferation of naive lymphocytes (4, 25). Al-
though not fully comparable, this does not seem to occur in
mice, since PHA-stimulated naive T cells from control mice
showed proliferation (not shown).
The analysis of the nature of the CD4
⫹
T cells stimulated by
gonococci during the experimental infection leads us to ob-
serve that infection induces TGF-␤1
⫹
CD4
⫹
T-cell responders
in the mucosal lymph nodes, including a subset of CD25
⫹
Foxp3
⫹
Tregs. In particular, these Tregs showed a small but
significant increase in number which is similar to that observed
after infection with parasites (15, 47, 53). Interestingly, the
induction of this type of immunity did not occur at the systemic
level. Even more, the regulatory activity was confirmed in vitro
in an allogeneic MLR, indicating that N. gonorrhoeae might
induce this type of response to avoid the host mechanisms of
protection. The results support the idea that suppression is
induced at least in part by TGF-␤1, which either in a cell-
surface-bound or a secreted form inhibits the immune re-
sponse at a variety of levels, i.e., inhibits IL-2 production,
IL-12-dependent cell activation, and Th1 development, among
other responses (27, 46). The source of TGF-␤1 was not dem-
onstrated in this study; nevertheless, epithelial and stromal
cells of the reproductive organs of the mouse and human,
which are targets of gonococcus infection, express high levels
of TGF-␤1 and other molecules involved in conditioning im-
mune privilege sites (6, 17, 22, 51). Because TGF-␤1 also has
a role in the induction of Tregs (54) and, moreover, because
CD4
⫹
CD25
⫹
peripheral T cells can be converted to Foxp3
⫹
Tregs by stimulation via the T-cell receptor in the presence of
TGF-␤1 (7, 12), we believe that the cytokine milieu found in
the reproductive tract subsidizes the induction of Tregs by N.
gonorrhoeae. Moreover, antigen-presenting cells, such as mac-
rophages and dendritic cells, regularly present in the reproduc-
tive tissues (16, 44) might also contribute to Treg differentia-
tion, as they may produce TGF-␤1 after infection. Actually, we
detected an increase of CD11b
⫹
macrophages infiltrating the
reproductive mucosae of infected mice, which is consistent
with such a role. Moreover, a high number of CD11b
⫺
TGF-
␤1
⫹
cells, which seem to be T cells by morphology criteria, also
infiltrated the mucosal reproductive tissues during infection,
suggesting that Tregs also play an important role at the mu-
cosal level.
If extrapolated to humans, the results would indicate that
gonococcus infection in women might also be related to a
similar Treg induction, explaining in part the lack of protective
immune response. In fact, there are various examples of eva-
sion through pathogen-induced modulation of the immune
response; one of these has been reported for the human filarial
infection, where the parasite also induces an imbalance toward
the Th2 response, which is at the same time accompanied by a
diminished production of inflammatory factors and an increase
of anti-inflammatory components, including Tregs (1). In hu-
man beings, N. gonorrhoeae can induce other highly efficient
ways to overcome immune defense mechanisms, resulting in
disease or chronic infection (14). Previous studies have focused
attention on antigenic variation (49), epitope mimicry (18, 33),
and phagosome subversion (3) as molecular mechanisms of
immune evasion. Moreover, recent studies have explored a
putative role of T cells as a determinant of successful gono-
coccus infection. As mentioned above, Opa proteins from
gonococci have the ability to inhibit CD4
⫹
T-cell proliferation,
which will prevent adaptive immune response. What is more
interesting is the gonococcus-dependent induction of IL-10
(36), a cytokine that it is also involved in the differentiation of
Tregs (Tr1 type) (26, 41). A study of infected patients will shed
light on the mechanism of pathogenesis and the presumed role
of Tregs.
Altogether, the results showed that the infection of mice
with N. gonorrhoeae induces a tolerant type of response which
may correspond to a form of immune evasion that has not been
previously studied for gonorrhea. Most studies describing
these evasion mechanisms have been reported for parasite
infection and chronic diseases caused by viruses (14, 32, 35, 42,
45). In those cases, immune regulation seems to favor the
persistence of infection, which becomes evident when Tregs
are depleted, and the disease is soon controlled by the immune
system and the pathogen cleared. We are currently investigat-
ing the effect that the depletion of Tregs may have on the
development of gonorrhea; moreover, studies of human sub-
jects are currently in progress to investigate the role of these
regulatory cells during infection in women.
ACKNOWLEDGMENTS
This work was supported by grants 1020354 from the Fondo Nacio-
nal de Desarrollo Científico y Tecnolo´gico (FONDECYT), 020743IB
and 020540AC from the Direccio´n de Investigaciones Científicas y
Tecnolo´gicas (DICYT), and IPA06 from the Programa Bicentenario
en Ciencia y Tecnología (PBCT).
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Editor: B. A. McCormick
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