Download Female sex hormones regulate the Th17 immune response to sperm

Human Reproduction, Vol.28, No.12 pp. 3283– 3291, 2013
Advanced Access publication on September 24, 2013 doi:10.1093/humrep/det348
ORIGINAL ARTICLE Reproductive biology
Female sex hormones regulate the
Th17 immune response to sperm and
Candida albicans
S. Lasarte 1, D. Elsner 1, M. Guı́a-González 1, R. Ramos-Medina 2,
S. Sánchez-Ramón2, P. Esponda 3, M.A. Muñoz-Fernández 1,
and M. Relloso 1,*
1
Laboratorio InmunoBiologı́a Molecular, Hospital General Universitario Gregorio Marañón and Instituto de Investigación Sanitaria Gregorio
Marañón, Dr. Esquerdo 46, 28007 Madrid, Spain, 2Departamento de Inmunologı́a Clinica, Hospital General Universitario Gregorio Marañón and
Instituto de Investigación Sanitaria Gregorio Marañón, Dr. Esquerdo 46, 28007 Madrid, Spain and 3Centro de Investigaciones Biológicas, CSIC,
Ramiro de Maeztu 9, 28040 Madrid, Spain
*Correspondence address. Instituto de Investigación Sanitaria del Hospital Gregorio Marañón, Laboratorio de Inmunobiologı́a Molecular,
C/Dr. Esquerdo, 46, 28007 Madrid, Spain. Tel: +34-915868565; Fax: +34-915868018; E-mail: [email protected]
Submitted on February 6, 2013; resubmitted on July 27, 2013; accepted on August 6, 2013
study question: What role do female sex hormones play in the antisperm immune response?
summary answer: We found that sperm induce a Th17 immune response and that estradiol down-regulates the antisperm Th17
response by dendritic cells.
what is known already: Estradiol down-regulates the immune response to several pathogens and impairs the triggering of dendritic
cell maturation by microbial products.
study design, size, duration: Ex vivo and in vivo murine models of vaginal infection with sperm and Candida albicans were used to
study the induction of Th17 and its hormonal regulation.
participants/materials, setting, methods: We analyzed the induction of Th17 cytokines and T cells in splenocytes
obtained from BALB/c mice challenged with sperm and C. albicans. For the in vivo vaginal infection models, we used ovariectomized mice
treated with vehicle, estradiol or progesterone, and we assessed the effect of these hormones on the immune response in the lymph nodes.
main results and the role of chance: Th17 cytokines and T cells were induced by sperm antigens in both ex vivo and in vivo
experiments. Estrus levels of estradiol down-regulated the Th17 response to sperm and C. albicans in vivo.
limitations, reasons for caution: This study was conducted using murine models; whether or not the results are applicable to
humans is not known.
wider implications of the findings: Our results describe an adaptive mechanism that reconciles immunity and reproduction
and further explains why unregulated Th17 could be linked to infertility and recurrent infections.
study funding/competing interest(s): This work was supported by research grants from the Instituto de Salud Carlos III
(ISCIII) (PI10/00897) and Fundación Mutua Madrileña to M.R. M.R. holds a Miguel Servet contract from the ISCIII (CP08/00228). M.A.M.-F.
was supported by (ISCIII) INTRASALUD PI09/02029. We have no conflicts of interest to declare.
trial registration number: Not required.
Key words: estradiol / spermatozoa / Th17 / dendritic cells
Introduction
The mucosal immune system of the female reproductive tract (FRT)
must be uniquely prepared to maintain the balance between the
presence of commensal microbiota, sexually transmitted pathogens
and allogeneic spermatozoa. Semen is a foreign material for the female
FRT and, upon contact with the mucosa, it induces a series of immunological reactions to eliminate it. For instance, insemination is followed by
& The Author 2013. Published by Oxford University Press on behalf of the European Society of Human Reproduction and Embryology. All rights reserved.
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an influx of neutrophils to the mucosa of the FRT (Rozeboom et al., 1999;
Gorgens et al., 2005; Schuberth et al., 2008). In humans, semen was
shown to induce a local recruitment of leukocytes, detected only after
coitus without a condom (Sharkey et al., 2012a). Moreover, the presence
of antisperm antibodies in females has been widely reported (Clarke,
2009). However, the passage of sperm through the FRT must be regulated in order to maximize the probability of fertilization. In some
species, sperm may be stored for days (horses) or even months (bats)
before the arrival of the oocyte. In humans, fertilization occurs when
intercourse takes place up to 5 days before ovulation (Wilcox et al.,
1995). Female sex hormones regulate ovulation and have a potent
effect on the mucosal immune system of the FRT and the survival of
microbiota (Dunbar et al., 2012). Thus, a transient increase in vaginal
microbiota is observed during the follicular phase (proestrus) of the
ovarian cycle (Keane et al., 1997; Eschenbach et al., 2000). This increase
peaks during the ovulatory phase (estrus) and decreases during the luteal
phase (diestrus) (Fidel et al., 2000).
Dendritic cells (DCs) are professional antigen-presenting cells in the
mucosa of the FRT (De Bernardis et al., 2006; LeBlanc et al., 2006)
that sample the environment in search of antigens and danger signals.
Upon contact with exogenous ligands or inflammatory cytokines, DCs
migrate to draining lymph nodes, thereby promoting T-cell activation
and polarization (Buelens et al., 1997; Sallusto et al., 1998; De Bernardis
et al., 2006). DCs primed with exogenous materials can polarize CD4+
T cells to induce a highly efficient immune response that is specific to each
kind of pathogen (Murphy and Stockinger, 2010). For example, intracellular pathogens promote Th1 responses, and extracellular fungi and bacteria are very efficient at promoting Th17 responses (Conti et al., 2009).
The designated CD4+ Th17 T-cell lineage is characterized by production of the proinflammatory cytokines, IL-17A and IL-22, and the lineagespecific transcription factor, RORC (RAR-related orphan receptor
gamma or ROR-gt) (Kim et al., 2011). IL-17 is required for host
defense against fungal infections (Conti et al., 2009), because IL-17
recruits neutrophils to the site of infection through induction of granulocyte colony-stimulating factor; neutrophil-induced chemokines (Liang
et al., 2007) and neutrophils are the cells that clear fungal infections
(Thompson and Wilton, 1992).
Sperm are considered weakly immunogenic because they induce low
IFN-g levels (Witkin and Chaudhry, 1989), and the association between
antibodies against sperm and infertility is controversial (Clarke, 2009;
Naz, 2011). Therefore, most research on factors associated with
infertility/fertility has investigated seminal plasma-induced immune
responses and cytokines (Politch et al., 2007; Remes Lenicov et al.,
2012; Robertson et al., 2013). On the other hand, an association has
been established between celiac disease in female patients and infertility
(Choi et al., 2011), and it is well documented that these patients have
higher numbers of Th17 cells (Fernandez et al., 2011) and higher levels
of IL-17A expression (Monteleone et al., 2010). Similarly, male and
female fungal infections are closely linked with infertility, which also generate high numbers of Th17 cells and high levels of IL-17A expression
(Nagy and Sutka, 1992; Ulcova-Gallova, 1997). Furthermore, a strong association has been observed between Chlamydia antibodies and antisperm antibodies (Blum et al., 1989). We reasoned that the Th17
response could be likened to antisperm immunity. Therefore, we investigated the Th17 antisperm response and found that sperm-primed DCs
induce a Th17 response in the same way as do Candida albicans antigens.
Furthermore, we hypothesized that female sex hormones can modulate
Lasarte et al.
the induction of the Th17 antisperm response to allow sperm to survive
during ovulation. We observed that pretreatment with estradiol (E2)
diminished the Th17 response, whereas pretreatment with diestrus hormones (progesterone [P] and lower E2) restored the pathogen-host
equilibrium.
Methods
Mice, sperm collection and sperm
immunization
Experiments were performed using 4- to 6-week-old female BALB/c (H-2d)
mice and male CD1 Crl:CD1(ICR) (outbred) mice. The animals were maintained under specific pathogen-free conditions in the Animal Facility of IiSGM.
The vasa deferentia were removed and transferred to a 35-mm petri dish in
phosphate-buffered saline (PBS). Sperm were expelled carefully from the vas
deferens with a set of sterile fine-tipped forceps. Sperm were allowed to
‘swim out’ from the tissues for 10 min at room temperature before being
washed in PBS. Females were immunized by peritoneal injection [5 times
with 500 ml of PBS containing 5 to 1 × 106 sperm mixed with adjuvant
(200 ng of lipopolysaccharide (LPS) Sigma-Aldrich, USA)] every 20 days
(Basten, 1988; Yokochi et al., 1990).
C. albicans: strain, culture and immunization
protocol
The SC5314 C. albicans (ATCC MYA-2876) strain was grown on Sabouraud
dextrose chloramphenicol agar plates (Conda, Spain) overnight at 308C prior
to the experiments. Before staining, cells were collected, washed and labeled
with Oregon Green 488 or FITC (Relloso et al., 2012). Females were immunized (peritoneal injection) twice with 500 ml of PBS containing 5 × 105 yeast
every 20 days.
Splenocytes, spleen DCs and CD41 T-cell
purification
Five days after the last immunization, the spleens were dissected and the splenocytes were isolated. Five million cells/ml were plated on a 24-well plate on
RPMI 1640 without phenol red medium [10% heat-inactivated fetal calf
serum (FCS), 1 mM sodium pyruvate, 1% amino acids, 2 mM L-glutamine,
50 mM 2-ME and 15 mM HEPES] complemented with amphotericin B
(Duchefa Biochemie, Netherlands) at 0.09 mg/ml (Relloso et al., 2012). Splenocytes were treated with 17b-E2 (E2) or E2 and P (Calbiochem, Germany)
and dissolved in ethanol at physiological range concentrations (PaharkovaVatchkova et al., 2004; Mao et al., 2005) for 3 h. Control cells were
treated with hormone-free ethanol (vehicle). Splenocytes were pulsed
with C. albicans at an MOI of 10 or with 1 sperm for every 5 splenocytes.
After 20 – 24 h, the medium was removed and the cytokine concentration
was measured using enzyme-linked immunosorbent assay (ELISA) (Quantikine R&D Systems, USA). Splenic DCs were purified using a DC isolation kit
(CD11c MicroBeads, Miltenyi Biotec, Germany). CD4+ spleen T cells were
purified using the CD4+ T Cell Isolation Kit (Miltenyi Biotec, Germany) following the manufacturer’s instructions, and the live cells were counted in a
hemocytometer using trypan blue exclusion.
Ovariectomization and hormonal treatment
Four-week-old mice were bilaterally ovariectomized under anesthesia
(Relloso and Esponda, 2000) and given 2 weeks to recover. Females were
then injected subcutaneously with 0.01 mg of E2 or E2 and P (Calbiochem,
Germany) dissolved in 50 ml of sesame oil (Sigma-Aldrich, USA) every 4
days (Day 22, 1, 4 and 7). Control animals were injected with sesame oil
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Estradiol diminishes Th17 response to sperm
without hormone (vehicle). Hormone treatment was based on a previous
report, and the dose was sufficient to maintain hormone concentrations in
female mice (Fidel et al., 2000; LeBlanc et al., 2006; Relloso et al., 2012).
To test the effect of P, bilaterally ovariectomized were treated with E2 on
Day 22 and treated with E2 or E2 and P or vehicle at Day 1 after the infection.
Mice were sacrificed and samples were analyzed at Day 3.
Ethical approval
Procedures involving animals and their care complied with national and international laws and policies. The IiSGM Animal Care and Use Committee
approved all the procedures.
Results
Vaginal infection and insemination
At 72 h after the first hormonal treatment (Day 22), mice were inoculated
with 2 × 106 of SC5314 C. albicans blastoconidia or 5 × 106 sperm in 20 ml
of PBS into the vagina (Day 0). Samples were analyzed on the indicated day.
Single cell suspension from lymph nodes
Inguinal lymph nodes (ILNs) were aseptically removed, and tissues were disrupted by forcing them through a 70-mm cell strainer (BD Biosciences) in PBS.
Cells were stained for flow cytometry analysis with anti-CD3, anti-B220,
anti-MHC II and anti-CD11c antibodies (BD Pharmingen).
RORC staining
Cells were stained for flow cytometry analysis with anti-CD3 (BD Pharmingen, USA), anti-CD4 (BD Pharmingen, USA) and anti-RORC antibody
(eBioscience, USA). We used the BD Cytofix/Cytoperm Fixation/
Permeabilization kit (BD Biosciences, USA) following the manufacturer’s
instructions.
Flow cytometry
Cellular phenotypic analysis was carried out by indirect immunofluorescence. The antibodies used for cell surface staining were obtained from BD
Pharmingen (USA) and included CD3 (clone 17A2), CD4 (clone GK1.5),
CD11c (clone HL3), CD45R (B220; clone RA3 – 6B2) and MHC II (clone
2G9). All incubations were in the presence of 50 mg/ml mouse IgG to
prevent binding through the Fc portion of the antibodies. The same
isotype control antibody was always included as a negative control. Dead
cells were excluded by staining with 0.125 mg/ml of 7-amino-actinomycin
D (Sigma, USA). Flow cytometry was performed with a Gallios device
(Beckman Coulter, USA) and data were analyzed using the Flowjo software
(Tree Star, Inc., USA). Cells were counted using Flow-Count fluorospheres
(Beckman Coulter, USA) following the manufacturer’s instructions.
Vaginal fungal burden and PAS staining
After mice were inoculated in the vagina with 2 × 106 of C. albicans, vaginal
tissue was excised under sterile conditions and the fungal burden was analyzed. Vaginal samples were weighed under sterile conditions and homogenized in 0.5 ml of sterile water, and 1 –10 serial dilutions were plated on
YED agar media to assay the C. albicans concentration per milligram of
organ (in CFU). For the histology analysis, we performed the PAS staining.
Tissue was fixed in 10% formalin and embedded in paraffin. Sections of
5 mm were incubated in 0.5% of periodic acid, washed, incubated with
Schiff reagent for 15 min and stained with Mayer’s hematoxylin solution.
Statistical analysis
The test used to determine significance between the treatments in each experiment can be found in the figure legends. GraphPad Prism 5 (GraphPad
Software, Inc., USA) was used to determine statistical significance.
A P-value of ,0.05 was considered significant.
Sperm induce a Th17 immune response
Extracellular pathogens are very efficient at promoting Th17 responses,
which are characterized by production of the cytokines, IL-17A and
IL-22, and recruitment of neutrophils to the site of infection (Liang
et al., 2007). An influx of neutrophils is also observed after insemination
(Schuberth et al., 2008). Therefore, we hypothesized that sperm
could induce a Th17 immune response. To investigate the role of
Th17 in antisperm immunity, we performed a sperm mixed with LPS
challenge into the peritoneal cavity to mimic the human physiological
route of female immunization (Friberg et al., 1987; Suarez and Pacey,
2006). We chose LPS because it is an IFN-g/Th1 inducer and in our
experiments, splenocytes from LPS-challenged mice produced IFN-g
(Th1) when they were pulsed with LPS (Supplementary data, Fig. S1A).
We also used seminal plasma-free sperm to avoid the effect of semen
cytokines on the outcome of experiments (Politch et al., 2007; Remes
Lenicov et al., 2012).
Mice were challenged with PBS or sperm or sperm mixed with LPS as
an adjuvant. Splenocytes were isolated, plated and pulsed with seminal
plasma-free sperm, and the cytokine content in the medium was determined. We detected a strong increase in IL-17A production in the cultures of splenocytes from mice challenged with sperm mixed with LPS.
Our experiment indicated that the adjuvant is necessary for correct immunization of the mice (Supplementary data, Fig. S1B). The limulus amebocyte lysate assay (GenScript, USA) showed that endotoxin was
undetectable in our sperm preparations, which were applied to pulse
the splenocytes (Supplementary data, Fig. S1C). Therefore, splenocytes
released IL-17A as a result of the presence of sperm in the culture. We
also detected significant production of IL-22 and IFN-g but not of IL-4,
IL-10 or TGF-b (Fig. 1A). We compared the cytokine expression
profile with C. albicans, a well-known Th17 inducer. We challenged the
mice by injecting C. albicans into the peritoneal cavity to ensure
the same means of delivery in both cases. We detected a similar
range of IL-17A and IL-22 (Fig. 1B). However, IFN-g secretion which
was very low or undetected in sperm-pulsed splenocytes, was robust
and consistent in C. albicans-pulsed splenocytes.
To test the specificity of the antisperm immune response, mice were
challenged several times with PBS, C. albicans, LPS or sperm mixed with
LPS (Fig. 1C –G). Splenocytes were then isolated and pulsed with PBS,
LPS, C. albicans or seminal plasma-free sperm without LPS, and cytokine
IL-17A content in the media was determined using ELISA. We detected
release of IL-17A in all the cultures pulsed with either LPS or C. albicans,
regardless of the original challenge. However, only sperm-challenged
splenocytes produced significant amounts of IL-17A when they were
pulsed with sperm (Fig. 1C –F). Therefore, we can conclude that
several encounters with sperm mixed with microbial products in the
peritoneal cavity are necessary to produce an antisperm-specific adaptive immune response. Furthermore, the fact that sperm antigens
induce a Th17 response sheds new light on the female antisperm
immune response.
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Lasarte et al.
of IL-17A and IL-22 by the splenocytes after sperm pulsing, whereas diestrus levels of E2 and/or P had no effect on the release of cytokines
(Fig. 2A). We obtained similar results in the same experiments performed with C. albicans-pulsed splenocyte cultures (Fig. 2B). Therefore,
E2 reduces secretion of IL-17 and IL-22 in response to sperm and in
C. albicans antigens. Next, we investigated the presence of Th17 positive T cells by RORC expression detection in CD4 cells, and detected
a significant reduction in RORC+ cells in sperm-pulsed cultures
(Fig. 3A and B) and C. albicans-pulsed cultures (Fig. 3C) after treatment with estrus levels of E2. Our data suggest that the estrus E2 concentrations weaken the Th17 response. Therefore, the effect of E2 on
the Th17 response could enable sperm antigens to survive and at
the same time diminish the defense against pathogens. Furthermore,
diestrus hormones could restore the Th17 response to maintain the
equilibrium.
E2 delays the vaginal-induced Th17
immune response
Figure 1 Sperm induce the Th17 response and are regulated by E2.
(A) Splenocytes from 5 times sperm/LPS-challenged mice were isolated and sperm-pulsed. (B) Splenocytes from twice C. albicanschallenged mice were pulsed with C. albicans. Cytokine production
was measured in the media using ELISA. A representative experiment
taken from five independent tests is shown. Data are expressed as
mean + SD. *P , 0.05 (Mann – Whitney test) between the challenged
and non-challenged cultures. Splenocytes from (C) PBS-challenged,
(D) C. albicans-challenged, (E) sperm/LPS-challenged and (F) LPSchallenged mice were isolated and pulsed with PBS, LPS, C. albicans or
sperm. Cytokine production was measured in the media using ELISA.
Each circle represents a mouse. Data are expressed as mean + SEM
and are a combination of two separate experiments *P , 0.05,
**P , 0.01 and ***P , 0.005 (Mann – Whitney test) between the challenged and PBS-pulsed cultures. SEM, standard error of the mean;
SD, standard deviation.
E2 impairs the Th17 antisperm immune
response
After ovulation, a normal ovarian cycle has a window of vulnerability
during which females are more susceptible to infection, since the
immune response is weakened to allow sperm survival (Dunbar et al.,
2012). It has been demonstrated that E2 diminishes the pathogeninduced immune response (Kaushic et al., 2000; Gillgrass et al., 2005;
Escribese et al., 2008; Relloso et al., 2012); however, the effect of E2
on antisperm immunity is unknown. In order to test the hormones controlling the Th17-induced antisperm immune response, we treated splenocytes with estrus levels of E2 (10210 M) or diestrus levels of E2
(10211 M) and/or diestrus levels of P (1028 M) and pulsed them with
sperm. Estrus levels of E2 treatment significantly reduced the production
To further investigate the in vivo effect of female hormones on the Th17
response, we set up C. albicans vaginal infections in hormonally treated
ovariectomized mice (Fig. 4A). Three days after the inoculation, vaginal
tissue was examined to detect C. albicans by periodic acid Schiff staining
(PAS) and CFU counts. We did not detect C. albicans in vehicle- or in
P-treated animals (Supplementary data, Fig. S2A and B). However,
E2-treated mice showed numerous filaments of C. albicans, and we
detected the same number of CFUs at Day 3 as at 1 h (Supplementary
data, Fig. S2C). Therefore, E2-treated mice were not able to reduce
the infection. To further investigate the role of P, we pretreated the
mice with E2 and after the infection, we treated them with vehicle, E2
or P. Two days after the second hormone treatment, the tissue was
examined to detect C. albicans. We detected a higher number of cells
in E2-treated mice after infection than in the vehicle-treated mice.
However, E2-pretreated mice treated with P after the infection were
able to clear the fungal infection, since filaments or CFUs were not
detected (Supplementary data, Fig. S2D and E). Our data are consistent
with those of a previous report (Fidel et al., 2000), thus validating our
vaginal infection model.
DCs are responsible for triggering the
Th17 immune response
After contact with exogenous antigens or products, DCs migrate from
the vagina to the draining lymph nodes (LeBlanc et al., 2006). Therefore,
we studied their kinetics of entry into the ILNs after infection. As shown
in Fig. 4B, DCs recruited from vehicle- and P-treated mice reached their
maximum count at Day 3 after infection. However, DCs from E2-treated
mice were at their maximum count at Day 5. Therefore, E2 treatment
induced a delay in recruitment kinetics. To investigate the role of P, we
pretreated the animals with E2, P or vehicle and 4 days later we
treated them with E2, P or vehicle (Fig. 4A). Higher numbers of DCs
were detected in P-treated and vehicle-treated animals after infection
compared with those treated with E2. However, E2-pretreatment and
P treatment after infection revealed increased DC numbers in the
ILNs (Fig. 4C). These experiments suggest that treatment with E2
reduces the ability of the DCs to migrate to the ILNs but that treatment
with P reverts that effect. Next, we analyzed inflammation in the ILNs.
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Estradiol diminishes Th17 response to sperm
Figure 2 E2 reduces Th17 cytokine expression. (A) Splenocytes from sperm-challenged mice were isolated and treated with vehicle, estrus E2
(10210 M), diestrus E2 (10211 M) or P (1028 M) and pulsed with sperm. (B) Splenocytes from C. albicans-challenged mice were treated with hormones
and pulsed with C. albicans. Cytokine production was measured in the media using ELISA. A representative experiment taken from four independent
tests is shown. Data are expressed as mean + SD. *P , 0.05 and **P , 0.005 (Mann – Whitney test) between the vehicle and the hormone treatment.
E2, estradiol; Pg, progesterone; E, estrus; and D, diestrus.
We detected significant differences in T-cell numbers at Day 3 after infection in vehicle- and P-pretreated animals compared with non-infected
animals. In contrast, E2-treated animals showed significant differences in
T-cell numbers at Day 5 (Fig. 5A), an observation that is consistent with
DC recruitment in the ILNs (Fig. 4B). Moreover, when E2-pretreated
animals were treated with P after infection, the T-cell count recovered
(Fig. 5B). Furthermore, we detected lower RORC+ CD4 T-cell expansion in the ILNs of the E2-pretreated animals, but when E2-pretreated
animals were treated with P after infection, the RORC+ T-cell count
recovered (Fig. 5B).
We also investigated the in vivo role of female hormones in the Th17
antisperm response. We inoculated sperm into the vagina of ovariectomized mice treated with E2, Pg or vehicle (Fig. 4A). We did not detect
significant differences in T-cell numbers in the ILNs of the mice
that were vaginally inoculated with sperm alone (data not shown). In contrast, when mice were injected with sperm mixed with LPS in the peritoneal cavity a week before the hormone treatment and vaginal
inoculation, E2-treated animals showed a significant reduction in T-cell
counts at Day 3 compared with P- and vehicle-treated mice (Fig. 5C).
Moreover, we detected lower RORC+ T-cell expansion in the ILNs
of the E2-pretreated animals than in the Pg- and vehicle-treated mice
(Fig. 5D). Our data show that E2-treated mice present a delayed
immune response to foreign material in the vagina, thus allowing
sperm to survive. This could explain the increased survival of
C. albicans. Therefore, we hypothesize that DCs could have a central
role in this process, because E2-treated mice showed delayed kinetics
of entry for DCs in the draining lymph nodes and, as a result, diminished
T-cell activation and polarization.
E2 impairs triggering of the Th17 antisperm
immune response by DCs
To investigate the effect of E2 on T-cell activation by DC, we treated
CD11c+ spleen DCs with hormones and pulsed them with seminal
plasma-free sperm. Next, we added CD4+ T cells obtained from
sperm-challenged mice. We detected lower levels of IL-17 and IL-22
in the sperm-pulsed co-culture supernatants after treatment with
estrus levels of E2 compared with that after treatment with vehicle, P
or diestrus levels of E2 (Fig. 6A). In line with our previous observations,
we detected low levels of IFN-g in the sperm-pulsed co-cultures, in contrast to the C. albicans-pulsed co-cultures (Fig. 6B). However, reductions
in the expression of Th17 cytokines were consistent in both the spermand C. albicans-pulsed E2-treated cells. Our data suggest that spermpulsed DCs are potent Th17 inducers. Furthermore, during ovulation,
the E2 concentration down-regulates DC antigen presentation and cellular polarization toward the antisperm and anticandida Th17.
Discussion
The female reproductive mucosa recognizes semen as a foreign material
and induces reactions to eliminate it (Sharkey et al., 2007). The generation of immune responses against sperm during ovulation must be controlled in order to allow the sperm to survive and fertilize. Therefore,
there may be a window of vulnerability after ovulation during which
females are more susceptible to infection (Dunbar et al., 2012; Relloso
et al., 2012). Sperm are considered weakly immunogenic because they
induce low IFN-g levels (Clarke, 2009; Naz, 2011). As a result,
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Figure 3 E2 reduces Th17 T-cell counts. (A and B) Splenocytes from
sperm-challenged mice were isolated and treated with vehicle, estrus E2
(10210 M), diestrus E2 (10211 M) or P (1028 M) and pulsed with sperm.
(B) Splenocytes from C. albicans-challenged mice were treated with hormones and pulsed with C. albicans. Splenocytes were assayed for RORC
expression by gating on the CD4+/CD3+ cells. (A) Representative
flow cytometry plots. Numbers are the percentage of cells that are positive. (B and C) Average of the percentage of cells that are RORC+ in
three experiments. Data represent the mean + SD. *P , 0.05
(Mann – Whitney test) between the vehicle and the hormone treatment.
E2, estradiol; Pg, progesterone; E, estrus; and D, diestrus.
researchers have focused on seminal plasma-induced immune responses
to identify factors associated with infertility/fertility (Fichorova and
Boulanov, 1996; Yang et al., 1998; Moldoveanu et al., 2005). However,
antisperm immune responses have been documented (Witkin and
Chaudhry, 1989; Fichorova et al., 1995; Ulcova-Gallova, 1997), and
sperm have even been sought as vaccine candidates (Naz, 2011).
Although the nature of the antisperm immune response remains
unknown (Clarke, 2009), we demonstrate that sperm can induce a
Th17 immune response, thus shedding new light on female antisperm
immunity.
To investigate the role of Th17 in antisperm immunity, we performed a
sperm challenge using sperm mixed with LPS injected into the peritoneal
cavity. Given that human fallopian tubes open into the peritoneal cavity,
sperm can be found in the peritoneal cavity of women (Maathuis, 1974;
Asch, 1976; Stone, 1983; Aref et al., 1984; Suarez and Pacey, 2006), together with microbes and microbial products (Friberg et al., 1987) that
probably were in the vagina. It has been suggested that this could be
one of the ways females acquire antisperm immunity (Cunningham
et al., 1991; Naz and Menge, 1994; Mazumdar and Levine, 1998). We
used intraperitoneal injections of sperm combined with LPS as an adjuvant to mimic the human physiological route of female immunization.
We did not use fungal adjuvant, because it could lead to a Th17 response,
and we did not use Freund’s adjuvant, because it can affect mouse reproduction (Naz, 2011) and is made out of Mycobacterium tuberculosis
Lasarte et al.
Figure 4 E2 delays the entry kinetics of DCs into the inguinal lymph
nodes after vaginal infection. (A) Ovariectomized mice were treated
with hormones every 4 days. Vaginal inoculation (VI) with C. albicans
was performed 72 h after the first hormone treatment. (B) Number
of DCs (CD11c+ MHC IIhi) in the inguinal lymph nodes by flow cytometry. (C) Number of DCs at Day 3 after infection by flow cytometry. The
black +/2 represent hormone pretreatment. The gray +/2 represent hormone treatment after infection. Each circle represents a
mouse. Data are expressed as mean + SEM and are from a combination
of two or three separate experiments (n ¼ 4 to n ¼ 6 mice per condition). *P , 0.05 (Mann– Whitney test) between the vehicle and the
hormone treatment. E2, estradiol; Pg, progesterone.
products, which can also lead to a Th17 response. We chose LPS
because it is a bacterial product that promotes general activation to
the immune system; it has been described as an IFN-g/Th1 inducer,
and our experiments corroborated it.
DCs are present on the mucosal surfaces of the sites at which sperm
have to coexist with commensal microbiota and pathogens (Iijima et al.,
2007), while semen deposition results in DC recruitment (Berlier et al.,
2006) in the same fashion as C. albicans (De Bernardis et al., 2006;
LeBlanc et al., 2006; Iijima et al., 2007), since DC membrane molecules,
such as CD209 (also called DC-sign), are able to recognize both semen
(Sabatte et al., 2011) and C. albicans (Cambi et al., 2008). When they encounter foreign products and/or inflammatory cytokines, DCs initiate a
continuous process called maturation, which is completed during
pathogen-specific immune responses induced by DCs (Reis e Sousa,
2006). The mechanisms that prevent antisperm immune responses by
seminal plasma include induction of regulatory T cells (Robertson
et al., 2009, 2013), regulation of expression of cytokines (Sharkey
et al., 2007) such as TGF-b (Sharkey et al., 2012b) and prostaglandin
(Templeton et al., 1978), and even induction of the tolerogenic DC
phenotype (Remes Lenicov et al., 2012). In addition, we and others
have reported that E2-treated DCs fail to mature and promote an
immune response during pregnancy (Yang et al., 2006a; Kyurkchiev
et al., 2007; Uemura et al., 2008; Segerer et al., 2009) and during
estrus (Escribese et al., 2008; Kovats, 2012; Relloso et al., 2012), thus
explaining the window of vulnerability after ovulation during which
females are more susceptible to infection (Dunbar et al., 2012).
3289
Estradiol diminishes Th17 response to sperm
Figure 6 E2 mediates DC-mediated T-cell activation. Spleen DCs
were treated with vehicle, estrus E2 (10210 M), diestrus E2 (10211 M)
or P (1028 M), pulsed with (A) sperm or (B) C. albicans and co-cultured
with CD4+ cells from (A) sperm- or (B) C. albicans-challenged mice.
Cytokine production was measured in the media using ELISA. Data
are expressed as mean + SD. A representative experiment taken
from two independent tests is shown. *P , 0.05 (Mann– Whitney
test) between the vehicle and the hormone treatment. E2, estradiol;
Pg, progesterone; E, estrus; and D, diestrus.
Figure 5 E2 mediates in vivo T-cell activation. Ovariectomized mice
were treated with hormones every 4 days. Mice were inoculated with
C. albicans in the vagina 72 h after hormone treatment. (A) Number
of CD3+ T cells in the inguinal lymph nodes by flow cytometry.
(B) Number of CD3+ T cells and number of CD3+/CD4+/
RORC+ cells at Day 3 of infection by flow cytometry. The black
+/2 represent hormone pretreatment. The gray +/2 represent
hormone treatment after infection. (C and D) Ovariectomized mice
were challenged with sperm/LPS a week before hormone treatment.
Sperm were inoculated into the vagina 72 h after hormone treatment.
(C) Number of CD3+ T cells in the inguinal lymph nodes by flow cytometry. (D) Number of CD3+/CD4+/RORC+ cells in the inguinal
lymph nodes by flow cytometry. Data are expressed as mean + SEM.
Data are from a combination of two or three separate experiments.
n ¼ 4 to n ¼ 6 mice per condition. *P , 0.05 (Mann– Whitney test)
between the pulsed and non-pulsed animals or the hormone treatments. E2, estradiol; Pg, progesterone; and C.a, C. albicans.
Our data suggest that sperm-pulsed DCs are potent inducers of Th17.
Furthermore, the E2 concentration during ovulation down-regulates
DC antigen presentation and delays T-cell activation and polarization
toward the antisperm Th17 immune response, while diestrus hormones
restore the equilibrium. Moreover, the effect of E2 on Th17 is not sperm
antigen-specific, because the anti-C. albicans immune response is also
diminished by E2. The mechanism that we propose is consistent with
our in vivo data. E2-treated mice showed a delay in DC recruitment
kinetics and T-cell activation in draining lymph nodes. Furthermore,
E2-treated mice had lower numbers of CD4+/RORC+ T-cell levels
and reduced induction of IL-17A and IL-22. As a result, survival of
foreign material in the vagina improved. Therefore, a diminished Th17 response would enable the survival of allogeneic sperm, which thus acquire
the ability to fertilize during estrus (with high E2 concentrations). During
diestrus (with low E2 and high P concentrations), DCs recover their
antigen presentation efficiency (Liang et al., 2006) and T cell stimulation
efficiency (Yang et al., 2006b). In vivo evidence supporting this hypothesis
is that mice treated with P cleared the infection, and when P was administered after E2, the inhibitory effect of E2 on induction of Th17 was
reversed.
In conclusion, while the role of fungal infections and Th17-related diseases in female infertility is well documented (Ulcova-Gallova, 1997;
Choi et al., 2011), regulation of the Th17 response in antisperm immunity
is unknown. Our data show that sperm induce expression of Th17 cells
and cytokines and that E2 treatment diminishes it, since E2-treated DCs
are less efficient at triggering the adaptive immune response (Relloso
et al., 2012). Diestrus hormones restore the equilibrium; in the case of
hormone imbalance, pathogens such as C. albicans could use this diminished immune system reaction to grow and promote infection. However,
a strong Th17 response that is not controlled during estrus could lead to
infertility. Diagnosis of the implication of the Th17 immune response in
infertility or recurrent candidiasis could be greatly improved if more
were known about hormonal regulation of the balance between reproduction and infection.
Supplementary data
Supplementary data are available at http://humrep.oxfordjournals.org/.
3290
Acknowledgements
The authors thank the units of confocal microscopy, flow cytometry
and statistical analysis, as well as the animal facility of the IiSGM, for
help and support. We are grateful to F. Sánchez-Cobos for technical
help and to R. Madrid, V. Briz, M. Martı́nez-Bonet and R. González-Dosal
for corrections and comments.
Authors’ roles
M.R. conceived the study. S.L., D.E., M.G.-G., R.R.-M. and M.R. carried
out the experiments. M.R. wrote the manuscript and S.S.-R., P.E. and
M.A.M.-F. revised the manuscript. All the authors approved the submitted version.
Funding
This work was supported by research grants from the Instituto de
Salud Carlos III (ISCIII) (PI10/00897) and Fundación Mutua Madrileña
to M.R. M.R. holds a Miguel Servet contract from the ISC III (CP08/
00228). M.A.M.-F. was supported by (ISCIII) INTRASALUD PI09/
02029.
Conflict of interest
None declared.
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