* Your assessment is very important for improving the workof artificial intelligence, which forms the content of this project
Download Female sex hormones regulate the Th17 immune response to sperm
Survey
Document related concepts
Lymphopoiesis wikipedia , lookup
Monoclonal antibody wikipedia , lookup
Molecular mimicry wikipedia , lookup
Drosophila melanogaster wikipedia , lookup
Hygiene hypothesis wikipedia , lookup
DNA vaccination wikipedia , lookup
Immune system wikipedia , lookup
Polyclonal B cell response wikipedia , lookup
Adaptive immune system wikipedia , lookup
Adoptive cell transfer wikipedia , lookup
Cancer immunotherapy wikipedia , lookup
Innate immune system wikipedia , lookup
Immunocontraception wikipedia , lookup
Psychoneuroimmunology wikipedia , lookup
Transcript
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. For Permissions, please email: [email protected] 3284 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 3285 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. 3286 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. 3287 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, 3288 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. References Aref I, Reda M, Kandil O, El Tagi A. Postcoital recovery of sperm in Douglas pouch aspirates of infertile patients. Int J Gynaecol Obstet 1984;22:29 – 34. Asch RH. Laparoscopic recovery of sperm from peritoneal fluid, in patients with negative or poor Sims-Huhner test. Fertil Steril 1976;27:1111– 1114. Basten A. Birth control vaccines. Baillieres Clin Immunol Allergy 1988;2: 759 – 774. Berlier W, Cremel M, Hamzeh H, Levy R, Lucht F, Bourlet T, Pozzetto B, Delezay O. Seminal plasma promotes the attraction of Langerhans cells via the secretion of CCL20 by vaginal epithelial cells: involvement in the sexual transmission of HIV. Hum Reprod 2006;21:1135– 1142. Blum M, Pery J, Blum I. Antisperm antibodies in young oral contraceptive users. Adv Contracept 1989;5:41 – 46. Buelens C, Verhasselt V, De Groote D, Thielemans K, Goldman M, Willems F. Human dendritic cell responses to lipopolysaccharide and CD40 ligation are differentially regulated by interleukin-10. Eur J Immunol 1997;27:1848 – 1852. Cambi A, Netea MG, Mora-Montes HM, Gow NA, Hato SV, Lowman DW, Kullberg BJ, Torensma R, Williams DL, Figdor CG. Dendritic cell interaction with Candida albicans critically depends on N-linked mannan. J Biol Chem 2008;283:20590– 20599. Choi JM, Lebwohl B, Wang J, Lee SK, Murray JA, Sauer MV, Green PH. Increased prevalence of celiac disease in patients with unexplained infertility in the United States. J Reprod Med 2011;56:199– 203. Clarke GN. Etiology of sperm immunity in women. Fertil Steril 2009; 91:639 – 643. Conti HR, Shen F, Nayyar N, Stocum E, Sun JN, Lindemann MJ, Ho AW, Hai JH, Yu JJ, Jung JW et al. Th17 cells and IL-17 receptor signaling are essential for mucosal host defense against oral candidiasis. J Exp Med 2009;206:299 – 311. Lasarte et al. Cunningham DS, Fulgham DL, Rayl DL, Hansen KA, Alexander NJ. Antisperm antibodies to sperm surface antigens in women with genital tract infection. Am J Obstet Gynecol 1991;164:791 – 796. De Bernardis F, Lucciarini R, Boccanera M, Amantini C, Arancia S, Morrone S, Mosca M, Cassone A, Santoni G. Phenotypic and functional characterization of vaginal dendritic cells in a rat model of Candida albicans vaginitis. Infect Immun 2006;74:4282– 4294. Dunbar B, Patel M, Fahey J, Wira C. Endocrine control of mucosal immunity in the female reproductive tract: impact of environmental disruptors. Mol Cell Endocrinol 2012;354:85 – 93. Eschenbach DA, Thwin SS, Patton DL, Hooton TM, Stapleton AE, Agnew K, Winter C, Meier A, Stamm WE. Influence of the normal menstrual cycle on vaginal tissue, discharge, and microflora. Clin Infect Dis 2000; 30:901 – 907. Escribese MM, Kraus T, Rhee E, Fernandez-Sesma A, Lopez CB, Moran TM. Estrogen inhibits dendritic cell maturation to RNA viruses. Blood 2008; 112:4574– 4584. Fernandez S, Molina IJ, Romero P, Gonzalez R, Pena J, Sanchez F, Reynoso FR, Perez-Navero JL, Estevez O, Ortega C et al. Characterization of gliadin-specific Th17 cells from the mucosa of celiac disease patients. Am J Gastroenterol 2011;106:528 – 538. Fichorova RN, Boulanov ID. Anti-seminal plasma antibodies associated with infertility: I. Serum antibodies against normozoospermic seminal plasma in patients with unexplained infertility. Am J Reprod Immunol 1996;36: 198 – 203. Fichorova RN, Dimitrova E, Nakov L, Tzvetkov D, Penkov R, Taskov H. Detection of antibodies toward epididymal sperm antigens—an obligatory step in evaluation of human immunologic infertility? Am J Reprod Immunol 1995;33:341 – 349. Fidel PL Jr, Cutright J, Steele C. Effects of reproductive hormones on experimental vaginal candidiasis. Infect Immun 2000;68:651– 657. Friberg J, Confino E, Suarez M, Gleicher N. Chlamydia trachomatis attached to spermatozoa recovered from the peritoneal cavity of patients with salpingitis. J Reprod Med 1987;32:120 – 122. Gillgrass AE, Fernandez SA, Rosenthal KL, Kaushic C. Estradiol regulates susceptibility following primary exposure to genital herpes simplex virus type 2, while progesterone induces inflammation. J Virol 2005;79: 3107 – 3116. Gorgens A, Leibold W, Klug E, Schuberth HJ, Martinsson G, Zerbe H. Inseminate components are modulating the chemotactic activity of uterine polymorphonuclear granulocytes (PMN) of mares. Anim Reprod Sci 2005;89:308– 310. Iijima N, Linehan MM, Saeland S, Iwasaki A. Vaginal epithelial dendritic cells renew from bone marrow precursors. Proc Natl Acad Sci USA 2007;104: 19061 – 19066. Kaushic C, Zhou F, Murdin AD, Wira CR. Effects of estradiol and progesterone on susceptibility and early immune responses to Chlamydia trachomatis infection in the female reproductive tract. Infect Immun 2000;68:4207– 4216. Keane FE, Ison CA, Taylor-Robinson D. A longitudinal study of the vaginal flora over a menstrual cycle. Int J STD AIDS 1997;8:489 – 494. Kim JS, Smith-Garvin JE, Koretzky GA, Jordan MS. The requirements for natural Th17 cell development are distinct from those of conventional Th17 cells. J Exp Med 2011;208:2201 – 2207. Kovats S. Estrogen receptors regulate an inflammatory pathway of dendritic cell differentiation: mechanisms and implications for immunity. Horm Behav 2012;62:254 – 262. Kyurkchiev D, Ivanova-Todorova E, Hayrabedyan S, Altankova I, Kyurkchiev S. Female sex steroid hormones modify some regulatory properties of monocyte-derived dendritic cells. Am J Reprod Immunol 2007;58:425 – 433. Estradiol diminishes Th17 response to sperm LeBlanc DM, Barousse MM, Fidel PL Jr. Role for dendritic cells in immunoregulation during experimental vaginal candidiasis. Infect Immun 2006;74:3213 – 3221. Liang J, Sun L, Wang Q, Hou Y. Progesterone regulates mouse dendritic cells differentiation and maturation. Int Immunopharmacol 2006;6:830– 838. Liang SC, Long AJ, Bennett F, Whitters MJ, Karim R, Collins M, Goldman SJ, Dunussi-Joannopoulos K, Williams CM, Wright JF et al. An IL-17F/A heterodimer protein is produced by mouse Th17 cells and induces airway neutrophil recruitment. J Immunol 2007;179:7791 – 7799. Maathuis JB. Investigation of peritoneal fluid obtained by laparoscopy from gynecological patients. J Reprod Med 1974;12:248 – 249. Mao A, Paharkova-Vatchkova V, Hardy J, Miller MM, Kovats S. Estrogen selectively promotes the differentiation of dendritic cells with characteristics of Langerhans cells. J Immunol 2005;175:5146– 5151. Mazumdar S, Levine AS. Antisperm antibodies: etiology, pathogenesis, diagnosis, and treatment. Fertil Steril 1998;70:799 – 810. Moldoveanu Z, Huang WQ, Kulhavy R, Pate MS, Mestecky J. Human male genital tract secretions: both mucosal and systemic immune compartments contribute to the humoral immunity. J Immunol 2005; 175:4127– 4136. Monteleone I, Sarra M, Del Vecchio Blanco G, Paoluzi OA, Franze E, Fina D, Fabrizi A, MacDonald TT, Pallone F, Monteleone G. Characterization of IL-17A-producing cells in celiac disease mucosa. J Immunol 2010; 184:2211– 2218. Murphy KM, Stockinger B. Effector T cell plasticity: flexibility in the face of changing circumstances. Nat Immunol 2010;11:674– 680. Nagy B, Sutka P. Demonstration of antibodies against Candida guilliermondii var. guilliermondii in asymptomatic infertile men. Mycoses 1992; 35:247– 250. Naz RK. Antisperm contraceptive vaccines: where we are and where we are going? Am J Reprod Immunol 2011;66:5– 12. Naz RK, Menge AC. Antisperm antibodies: origin, regulation, and sperm reactivity in human infertility. Fertil Steril 1994;61:1001 – 1013. Paharkova-Vatchkova V, Maldonado R, Kovats S. Estrogen preferentially promotes the differentiation of CD11c+ CD11b(intermediate) dendritic cells from bone marrow precursors. J Immunol 2004;172:1426–1436. Politch JA, Tucker L, Bowman FP, Anderson DJ. Concentrations and significance of cytokines and other immunologic factors in semen of healthy fertile men. Hum Reprod 2007;22:2928– 2935. Reis e Sousa C. Dendritic cells in a mature age. Nat Rev Immunol 2006; 6:476 – 483. Relloso M, Esponda P. In-vivo transfection of the female reproductive tract epithelium. Mol Hum Reprod 2000;6:1099 – 1105. Relloso M, Aragoneses-Fenoll L, Lasarte S, Bourgeois C, Romera G, Kuchler K, Corbi AL, Munoz-Fernandez MA, Nombela C, Rodriguez-Fernandez JL et al. Estradiol impairs the Th17 immune response against Candida albicans. J Leukoc Biol 2012;91:159 – 165. Remes Lenicov F, Rodriguez Rodrigues C, Sabatte J, Cabrini M, Jancic C, Ostrowski M, Merlotti A, Gonzalez H, Alonso A, Pasqualini RA et al. Semen promotes the differentiation of tolerogenic dendritic cells. J Immunol 2012;189:4777– 4786. Robertson SA, Guerin LR, Moldenhauer LM, Hayball JD. Activating T regulatory cells for tolerance in early pregnancy—the contribution of seminal fluid. J Reprod Immunol 2009;83:109 – 116. Robertson SA, Prins JR, Sharkey DJ, Moldenhauer LM. Seminal fluid and the generation of regulatory T cells for embryo implantation. Am J Reprod Immunol 2013;69:315 – 330. Rozeboom KJ, Troedsson MH, Molitor TW, Crabo BG. The effect of spermatozoa and seminal plasma on leukocyte migration into the uterus of gilts. J Anim Sci 1999;77:2201– 2206. 3291 Sabatte J, Faigle W, Ceballos A, Morelle W, Rodriguez Rodrigues C, Remes Lenicov F, Thepaut M, Fieschi F, Malchiodi E, Fernandez M et al. Semen clusterin is a novel DC-SIGN ligand. J Immunol 2011;187:5299– 5309. Sallusto F, Schaerli P, Loetscher P, Schaniel C, Lenig D, Mackay CR, Qin S, Lanzavecchia A. Rapid and coordinated switch in chemokine receptor expression during dendritic cell maturation. Eur J Immunol 1998; 28:2760– 2769. Schuberth HJ, Taylor U, Zerbe H, Waberski D, Hunter R, Rath D. Immunological responses to semen in the female genital tract. Theriogenology 2008;70:1174 – 1181. Segerer SE, Muller N, van den Brandt J, Kapp M, Dietl J, Reichardt HM, Rieger L, Kammerer U. Impact of female sex hormones on the maturation and function of human dendritic cells. Am J Reprod Immunol 2009;62:165 – 173. Sharkey DJ, Macpherson AM, Tremellen KP, Robertson SA. Seminal plasma differentially regulates inflammatory cytokine gene expression in human cervical and vaginal epithelial cells. Mol Hum Reprod 2007;13:491– 501. Sharkey DJ, Tremellen KP, Jasper MJ, Gemzell-Danielsson K, Robertson SA. Seminal fluid induces leukocyte recruitment and cytokine and chemokine mRNA expression in the human cervix after coitus. J Immunol 2012a; 188:2445– 2454. Sharkey DJ, Macpherson AM, Tremellen KP, Mottershead DG, Gilchrist RB, Robertson SA. TGF-beta mediates proinflammatory seminal fluid signaling in human cervical epithelial cells. J Immunol 2012b;189:1024 – 1035. Stone SC. Peritoneal recovery of sperm in patients with infertility associated with inadequate cervical mucus. Fertil Steril 1983;40:802– 804. Suarez SS, Pacey AA. Sperm transport in the female reproductive tract. Hum Reprod Update 2006;12:23 – 37. Templeton AA, Cooper I, Kelly RW. Prostaglandin concentrations in the semen of fertile men. J Reprod Fertil 1978;52:147 – 150. Thompson HL, Wilton JM. Interaction and intracellular killing of Candida albicans blastospores by human polymorphonuclear leucocytes, monocytes and monocyte-derived macrophages in aerobic and anaerobic conditions. Clin Exp Immunol 1992;87:316–321. Uemura Y, Liu TY, Narita Y, Suzuki M, Matsushita S. 17 Beta-estradiol (E2) plus tumor necrosis factor-alpha induces a distorted maturation of human monocyte-derived dendritic cells and promotes their capacity to initiate T-helper 2 responses. Hum Immunol 2008;69:149 – 157. Ulcova-Gallova Z. Ten-year experience with antispermatozoal activity in ovulatory cervical mucus and local hydrocortisone treatment. Am J Reprod Immunol 1997;38:231 – 234. Wilcox AJ, Weinberg CR, Baird DD. Timing of sexual intercourse in relation to ovulation. Effects on the probability of conception, survival of the pregnancy, and sex of the baby. N Engl J Med 1995;333:1517– 1521. Witkin SS, Chaudhry A. Circulating interferon-gamma in women sensitized to sperm: new mechanisms of infertility. Fertil Steril 1989;52:867 – 869. Yang WC, Kwok SC, Leshin S, Bollo E, Li WI. Purified porcine seminal plasma protein enhances in vitro immune activities of porcine peripheral lymphocytes. Biol Reprod 1998;59:202– 207. Yang L, Hu Y, Hou Y. Effects of 17beta-estradiol on the maturation, nuclear factor kappa B p65 and functions of murine spleen CD11c-positive dendritic cells. Mol Immunol 2006a;43:357 – 366. Yang L, Li X, Zhao J, Hou Y. Progesterone is involved in the maturation of murine spleen CD11c-positive dendritic cells. Steroids 2006b; 71:922– 929. Yokochi T, Ikeda H, Inoue Y, Kimura Y, Ito H, Fujii Y, Kato N. Characterization of autoantigens relevant to experimental autoimmune orchitis (EAO) in mice immunized with a mixture of syngeneic testis homogenate and Klebsiella O3 lipopolysaccharide. Am J Reprod Immunol 1990;22:42 – 48.