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Endocrinology, doi:10.1210/en.2007-1776
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Endocrinology Vol. 149, No. 9 4402-4412
Copyright © 2008 by The Endocrine Society

Expression Patterns and Functions of Toll-Like Receptors in Mouse Sertoli Cells

Hui Wu, Haikun Wang, Weipeng Xiong, Song Chen, Hongmei Tang and Daishu Han

Department of Cell Biology, School of Basic Medicine, Peking Union Medical College, Institute of Basic Medical Sciences, Chinese Academy of Medical Sciences, Beijing 100005, People’s Republic of China

Address all correspondence and requests for reprints to: Daishu Han, Ph.D., Department of Cell Biology, Peking Union Medical College and Chinese Academy of Medical Sciences, 5 Dong Dan San Tiao, Beijing 100005, People’s Republic of China. E-mail: dshan{at}ibms.pumc.edu.cn.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Toll-like receptors (TLRs) play crucial roles in mediating innate and adaptive immunity. Sertoli cells create a microenvironment that protects seminiferous tubules from autoantigens and invading pathogens. Here we examined the expression and potential function of TLR family in mouse Sertoli cells. RT-PCR, Western blotting, and flow cytometry were used to analyze gene expression. Immunofluorescence staining was used to determine activation of nuclear factor-{kappa}B. ELISA was used to detect secreted cytokines in culture medium. The phagocytosis assay was performed by Oil Red O staining for lipid droplets. We demonstrated that TLR2, TLR3, TLR4, and TLR5 are highly expressed; TLR6, TLR7, and TLR13 are expressed at relatively low level; and TLR1, TLR8, TLR9, TLR11, and TLR12 are not detected in mouse Sertoli cells. We focused our study on the roles of TLR2-TLR5 in Sertoli cells. Our data indicated that TLR2-TLR5 can be activated by their ligands in mouse Sertoli cells and subsequently increase expression of the inflammatory cytokines IL-1{alpha}, IL-6, and interferon-{alpha}, and -β. The augmented expression of the cytokines might be induced by activation of nuclear factor-{kappa}B. Notably, activation of TLR3 by its ligand, poly (I:C), specifically promoted phagocytosis of apoptotic spermatogenic cells by Sertoli cells. The TLR-induced Sertoli cell phagocytosis was found to be associated with the up-regulation of scavenger receptors. The results suggest that TLRs expressed in mouse Sertoli cells may play roles in defense against invasion of allo- and autoantigens in the seminiferous tubules.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
THE MAMMALIAN TESTIS is composed of the seminiferous tubules and the interstitium. In addition to the macrophages in the interstitium, which may construct the first line of testicular defense against pathogens from the bloodstream (1, 2), the Sertoli cells within the seminiferous tubules play a crucial role to maintain testis as an immune privileged site in which both allo- and autoantigens can be tolerated (3, 4). Infections of the testis are usually overcome, indicating that an active and efficient immune response against pathogens can be locally generated. In fact, Sertoli cells respond to inflammatory cytokines and lipopolysaccharide (LPS) by up-regulating the expression of intercellular adhesion molecule (ICAM)-1 and IL-6 (5, 6). Furthermore, in addition to the physical and trophic supports to developing germ cells, Sertoli cells endocytose and degrade apoptotic germ cells and residual bodies, which is necessary for healthy germ cells to proceed through spermatogenesis (7, 8, 9). Clearance of apoptotic germ cells and residual bodies by Sertoli cells in vivo may prevent potential autoimmune responses against spermatogenic cells induced by autoantigens. Hence, Sertoli cells possess the defense machinery against allo- and autoantigens. However, immunological function of Sertoli cells in the testis is poorly understood at molecular level.

Toll-like receptors (TLRs) play essential roles in activating signal transduction pathways leading to the killing and clearance of pathogens. To date, 10 distinct TLRs have now been identified in humans (10) and 13 in mice (11). TLRs recognize highly conserved, pathogen-coded molecular structures termed pathogen-associated molecular patterns (12). The respective ligands of most TLRs have been revealed (13). For example, TLR2, in association with TLR1 or TLR6, recognizes different bacterial components including peptidoglycan, lipopeptide, and lipoprotein (14, 15). TLR3 recognizes double-stranded RNA that is produced by many viruses during replication and also can be activated by synthetic double-stranded RNA analog, polyinosinic-polycytidylic acid [poly (I:C)] (16). TLR4 recognizes LPS, a major component of the outer membrane of Gram-negative bacteria (17, 18). TLR5 recognizes bacterial flagellin (19). TLR7 recognizes synthetic imidazoquinoline-like molecules, guanosine analogs, single-stranded RNA, and influenza virus (20, 21). TLR8 shows the highest homology to TLR7, whereas human TLR8 mediates the recognition of imidazoquinolines and single-stranded RNA; mouse TLR8 is thought to be nonfunctional (21). TLR9 recognizes bacterial and viral CpG DNA motifs and malaria pigment hemozoin (22, 23). TLR11 responds specifically to uropathogenic bacteria (24) and profilin-like molecule from the protozoan parasite infection (25). The ligands for TLR10, TLR12, and TLR13 have not been identified yet (26, 27).

TLRs are expressed by both immune cells, such as lymphocytes, dendritic cells and macrophages, and nonimmune cell types including epithelia cells of many tissues (28). The expression and function of TLRs on epithelia cells of various tissues, such as lung, kidney, small intestine, and the reproductive tracts, have been extensively investigated (24, 29, 30). More attention has been focused on mucosal surfaces that are in contact with an environment rich in microorganisms. In fact, the incidence of infection is low in this site despite the abundance of environmental microorganisms partial owing to TLRs-mediated immune responses. It has been demonstrated that intestinal epithelial cells express TLR1-TLR4, TLR6, and TLR9 and that gastric epithelial cells express TLR2, TLR4, and TLR5 (31, 32, 33). Human vaginal and cervical epithelial cell lines express TLR1-TLR6. As for upper reproductive tract, primary uterine epithelial cells express TLR1-TLR9 (34). Different immune molecules are produced upon stimulation of TLRs according to cell type. These previous studies indicate that the expression and function of TLRs would differ at different tissues and cell types.

Various studies have identified expression of TLRs in testis. Adult human testis expresses TLR2 and TLR4 at high levels and TLR5 and TLR6 at lower levels (35), and rat testis expresses TLR1-TLR10 (30). A recent study reported that mouse Sertoli cells express TLR2, TLR4, TLR5, and TLR6 (36), which can be activated by their agonists in Sertoli cells and may initiate testicular innate immune responses by inducing augmented secretion of the chemokine monocyte chemotactic protein-1 and increased ICAM-1 expression. This previous study focused mainly on the role of the TLR2/TLR6 complex and TLR5 and their ability to stimulate the expression of monocyte chemotactic protein-1 and ICAM-1. All these previous reports suggest that Sertoli cells may play a role in modulating locally the activity of immune competent cells. However, TLR-mediated immunological roles of Sertoli cells in testis are poorly understood. In the current study, we further investigate the expression and function of TLRs in mouse Sertoli cells. We focused on TLR2-TLR5 and their ability to activate nuclear factor-{kappa}B (NF-{kappa}B) and induce inflammatory cytokines, especially Sertoli cell phagocytotic activity mediated by TLR3. Our data expand previous understanding and provide novel insight into the function of TLRs in Sertoli cells.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Animals
Mice (C57 BL/6J) were maintained and breeding in the animal facility of Beijing Union Medical College (Beijing, China). All the measures taken for the mice were in accordance with approved guidelines (Guideline for the Care and Use of Laboratory Animals) established by the Chinese Council on Animal Care.

Isolation of Sertoli cells
The procedure for isolation of Sertoli cells was based on a previous description (37) with a modification. Briefly, 3-wk-old mice were anesthetized with CO2 and then killed by cervical dislocation. Decapsulated testes were incubated with 0.5 mg/ml collagenase (Sigma, St. Louis, MO) at room temperature for 15 min with gentle oscillation and then were filtered through 80-µm copper meshes to eliminate interstitial cells. The seminiferous tubules were resuspended in the collagenase at room temperature for 15 min to remove myoid cells. The tubules were then incubated with 0.5 mg/ml hyaluronidase (Sigma) for 15 min with gentle oscillation and pipetting. The cells were washed three times with F12/DMEM (GIBCO, Grand Island, NY) and cultured in F12/DMEM supplemented with sodium bicarbonate (1.2 mg/ml), penicillin (100 U/ml), streptomycin (100 mg/ml), and 10% fetal calf serum (GIBCO Life Technologies). The cells were maintained in 5% CO2 at 32 C for 48 h. The spermatogenic cells that did not adhere on culture dishes were collected for inducing spontaneous apoptosis. Those adhering to Sertoli cells were removed by treatment with a hypotonic solution [20 mM Tris (pH 7.4)] for 1 min. Twenty-four hours later, the Sertoli cells were collected for RNA extraction, flow cytometry, and Western blotting analysis or reseeded in plates for other experiments. The purity of Sertoli cells obtained by this approach was more 95% based on immunostaining for Wilms’ tumor nuclear protein 1 (a marker of Sertoli cells).

Preparation of apoptotic spermatogenic cells
Spermatogenic cells were prepared as a previous description (38). Briefly, at 48 h after culture of primary Sertoli cells, the spermatogenic cells that did not adhere on culture dishes were collected, and cultured for another 2 d to induce spontaneous apoptosis. Apoptotic rate of the spermatogenic cells was evaluated by a staining with dye mix of acridine orange (AO and ethidium bromide (EB; Sigma). The dye mix for AO/EB staining was 100 µg/ml AO and 100 µg/ml EB in PBS. Procedures were followed as described previously (39). Briefly, 2 x 105 cells were pelleted by centrifugation at 120 x g for 5 min. After washing with PBS once, the pellets were resuspended in 25 µl PBS. Subsequently, 2 µl AO/EB dye mixes were added to the suspension. The cells were viewed and counted under a fluorescence microscope (IX-71; Olympus, Tokyo, Japan). The test was done in triplicate, and 100 cells were counted in each test.

Isolation of macrophages
The resident peritoneal macrophages were isolated based on a previous approach (40). Briefly, 3-wk-old mice were anesthetized with CO2 and then killed by cervical dislocation. The peritoneal cavities were lavaged with 5 ml cold PBS. The peritoneal cavity cells were cultured in RPMI 1640 supplemented with 10% fetal calf serum on culture dishes in a humidified atmosphere containing 5% CO2 at 37 C. After 2 h, suspending cells were removed by washing with PBS, and the macrophages attached on dishes were collected for RNA extraction and flow cytometry analysis.

RT-PCR
Total RNA was isolated using TRIzol reagent according to the manufacturer’s instructions (Invitrogen, Carlsbad, CA). The RNA was treated with ribonuclease-free deoxyribonuclease to remove potential contamination of genomic DNA. Total RNA (0.5 µg) was reverse transcribed into cDNA in 20 µl of reverse transcriptase reaction mixture containing 2.5 µM random hexamers, 2 mM deoxynucleotide triphosphates, and 200 U Muloney murine leukemia virus reverse transcriptase (Promega, Madison, WI). PCRs (cycles of 94 C for 30 sec, annealing at 52–62 C for 30 sec, and extension at 72 C for 1 min) were performed for a number of cycles corresponding to the high end of the range in which a linear increase in products could be detected. The β-actin gene was used as the control of equal amounts of cDNA in the PCRs. The PCR products were subjected to electrophoresis in 1% agarose gels, and densitometric quantification of the bands between target genes and β-actin gene were analyzed using YLN2000 gel analysis system (Yalien, Beijing, China). The primers for PCR were shown in Table 1Go.


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  		TABLE 1. Specific primers used for RT-PCR

 
Real-time RT-PCR analysis was performed with Power SYBR Green PCR master mix kit (Applied Biosystems, Foster City, CA) using an ABI PRISM 7300 real-time cycler (Applied Biosystems). The mRNA levels of target genes were normalized to β-actin. The primers used in real-time PCR are listed in Table 2Go.


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  		TABLE 2. Primers used for real-time RT-PCR

 
Flow cytometry
Sertoli cells and macrophages were detached with 0.02% EDTA and washed with cold PBS plus 1% BSA. The cells were fixed and permeabilized using the Cytofix/Cytoperm kit (BD Biosciences, San Jose, CA) according to the manufacturer’s instructions. For detection of TLR3 expression, rat antimouse TLR3 polyclonal antibody and isotypic control (eBioscience, San Diego, CA) were used at 1:500 for 30 min incubation on ice. After three washes with PBS, the cells were incubated with biotin-conjugated mouse antirat IgG (0.5 µg per 106 cells in 0.2 ml PBS; eBioscience) for 15 min on ice, followed by a 15-min incubation with 1 µg/ml allophycocyanin-conjugated streptavidin (eBioscience). For detection of TLR2, TLR4, and TLR5 expression, either fluorescein isothiocyanate (FITC)-conjugated antimouse TLR2 (eBioscience), phycoerythrin-conjugated antimouse TLR4 (eBioscience) and FITC-conjugated antimouse TLR5 (Imgenex, San Diego, CA) monoclonal antibodies, or the appropriate isotypic controls were used at 0.5 µg per 106 cells for 30 min incubation on ice. After washing, cells were analyzed with a BD FACScanTO flow cytometer (BD Biosciences).

ELISA
The concentrations of IL-1{alpha}, IL-6, and interferon (IFN)-{alpha}/β secreted by Sertoli cells were determined using ELISA kits (Zhongshan Biotechnology Co., Beijing, China). The assays were performed according to manufacturer’s instructions. Briefly, Sertoli cells were cultured in 24-well plates at the density of 1 x 106 cells/well for 24 h. The cells were treated with inhibitors of TLR2–5, 50 µg/ml TLR2 antibody (eBioscience; 16-9024), 10 µg/ml TLR3 antibody (eBioscience; 24-9031), 10 µg/ml Polymixin B (InvivoGen, San Diego, CA; tlrl-pmb), a blocker of TLR4, and 10 µg/ml TLR5 antibody (Santa Cruz Biotechnology, Santa Cruz, CA; sc-30003) for 2 h. Then the cells were stimulated with agonists of TLR2–5 for additional 24 h. The supernatants were collected for measurement of the cytokines.

Immunofluorescence staining
Sertoli cells cultured on Lab-Tek chamber slides (Nunc, Naperville, IL) were fixed with cold methanol at –20 C for 2 min. The cells were treated with 0.3% Triton X-100 in PBS for 15 min at room temperature to increase cellular permeability. After blocking by preincubation with 10% normal goat serum in PBS at room temperature for 30 min, rabbit antimouse NF-{kappa}Bp65 and inhibitor of NF-{kappa}B (I{kappa}B)-{alpha} polyclonal antibodies (Santa Cruz Biotechnology) were applied at a dilution of 1:200 and incubated at 37 C in a moist chamber for 1 h. After three washes with PBS, the cells were incubated with the FITC-conjugated goat antirabbit IgG (Zhongshan) for 30 min. The cells were mounted with VECTASHIELD mounting medium (Vector Laboratories Inc., Burlingame, CA) after three washes with PBS and examined under a fluorescence microscope (IX-71; Olympus). Negative control cells were incubated with preimmune rabbit serum instead of primary antibodies. The assays were repeated three times, and 150 cells were counted for each assay.

Phagocytosis assay
Phagocytosis of apoptotic spermatogenic cells and residual bodies by Sertoli cells results in formation of lipid droplets in the Sertoli cells. Therefore, the lipid droplets in Sertoli cells were detected by Oil Red O (ORO) staining and used as a criterion to evaluate phagocytic ability of Sertoli cells. The procedure of phagocytosis assay was based on a previous protocol (41) with modifications. At 96 h after isolation, Sertoli cells were detached and reseeded in 24-well plates at 5 x 104 cells/well. Twenty-four hours later, the cells were washed three times with D-Hanks’ solution and cultured in different conditions: F12/DMEM medium containing 10% fetal calf serum, serum-free medium, and serum-free medium supplemented with different TLR ligands [200 ng/ml poly(I:C), 1 µg/ml LPS, 10 ng/ml flagellin, or 10 µg/ml zymosan (InvivoGen)]. Meanwhile, the apoptotic spermatogenic cells were added to the Sertoli cells at 5 x 105 cells/well. At 24 h after coculture, the lipid droplets in the Sertoli cells were detected by ORO staining. The area ratios of lipid droplets to nuclei of Sertoli cells were analyzed by image analyzer (Image-pro plus 6.0; Olympus) and used to assess phagocytic activity of Sertoli cells. A total of 150 Sertoli cells from three repeat wells were analyzed in each assay. The assay was done in triplicate. The mean values were presented in the results.

Assay for engulfment of Escherichia coli was performed to evaluate phagocytosis of microbial pathogens by Sertoli cells. E. coli TOP 10 were grown up in Luria-Betani (broth) medium. After inactivation by heating at 60 C for 1 h, E. coli were washed two times with PBS and labeled with 1 mg/ml FITC (Amresco Inc., Solon, OH) for 15 min in the dark. The 2 x 106 FITC-labeled E. colis in each well were added to the Sertoli cells cultured in 24-well plates. At 12 h after infection, the E. coli-FITC was removed by three washes with PBS, and the Sertoli cells were examined under a fluorescence microscope (IX-71; Olympus).

Uptake of yellow fluorescent-labeled latex beads of 3 µm (Polysciences Inc., Warrington, PA) was used as a measure of general phagocytic activity of Sertoli cells stimulated by poly (I:C). The procedure was based on a previous description (42). Briefly, Sertoli cells were seeded in a 24-well plate at 5 x 104 cells/well. The following day, Sertoli cells were washed two times in media without serum, and fluorescent latex beads (1 x 106 in 100 µl of culture medium) were added to the cultures. After 6 h coculture, Sertoli cells were washed with D-Hanks’ solution, and detached by incubating in 1 ml of D-Hanks’ containing 0.05% trypsin for 10 min at 32 C. The Sertoli cells were collected by low-speed centrifugation and washed twice with D-Hanks’ solution. The whole procedure can efficiently eliminate particles bound to the cells. Inhibition of actin involvement by 50 µg cytochalasin B (Sigma) in the phagocytic activity was used as control. The cells were counted under a fluorescent microscope (IX-71; Olympus). The ratio of the cells having internalized fluorescent beads expressed the phagocytic capacity of Sertoli cells. One hundred cells were counted in each test, and the results were presented as the mean value of three tests.

ORO staining
Sertoli cells cocultured with apoptotic spermatogenic cells were washed in PBS by pipetting for removing apoptotic cells and fixed with 10% formalin for 30 min. After a wash with PBS, the cells were stained with ORO (Sigma) solution (ORO-saturated solution in isopropanol-water, 3:2) for 15 min as a previous description (41). Then the cells were washed with 70% alcohol for 5 sec to remove background staining. Finally, the cells were rinsed in tap water, counterstained with Harris hematoxylin for 10 sec, and mounted in glycerol-PBS (9:1) for observation.

Western blotting
Total Sertoli cells lysates were prepared by lysing and scraping the cells off the culture plate with cell lysis buffer (BioDev-Technology, Beijing, China). Protein concentration was determined by using the microbicinchonic acid method (Pierce Biotechnology, Rockford, IL). Equal amounts of proteins were subject to SDS-PAGE and subsequently electrotransferred onto polyvinyl difluoride membranes (Millipore, Bedford, MA). After blocking with 5% nonfat dry milk in Tris-buffered saline (TBS) for 1 h, the electrotransferred membranes were incubated with first antibodies at 1:500 to 1:1000 dilutions at 4 C for overnight: goat anti-scavenger receptor class B type I (SR-B1) (Santa Cruz); goat anti-CD36 (Santa Cruz); rabbit anti-TLR2 (Imgenex); rat anti-TLR3 (eBioscience); rabbit anti-TLR4 (eBioscience); rat anti-TLR6 (Imgenex); rat anti-TLR7 (eBioscience); polyclonal antibodies; and mouse TLR5 monoclonal antibody (Imgenex). After washing with TBS, the membrane was incubated with appropriated peroxidase-conjugated affinipured second antibodies (Zhongshan) at room temperature for 1 h. After washing with TBS, antigen-antibody complex was visualized by using an enhanced chemiluminescence detection kit (Zhongshan).

Statistical analyses
Data are presented as mean ± SEM for n given samples. Student’s t tests were used to determine significance between groups of cell types or treatments (e.g. treatments with TLR blockers). One-way ANOVA tests with Bonferroni corrections were used to calculate significance for multiple comparisons of different treatments (e.g. treatments with different TLR ligands). All calculations were performed with SPSS version 11.0 statistic software (SPSS Inc., Chicago, IL). P < 0.05 was considered significant.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Expression of TLRs in mouse Sertoli cells
To examine expression pattern of TLRs in Sertoli cells, primary Sertoli cells were isolated from 3-wk-old C57 BL/6J mice, and the total RNA was extracted. By using RT-PCR, the expression of all 12 mouse TLRs at the mRNA level was examined in the primary Sertoli cells. The macrophages, which express most members of TLR family, were used as controls. As shown in Fig. 1Go, A and B, strong signals for TLR2, TLR4, and TLR5 were detected in Sertoli cells at a level comparable with macrophages, whereas TLR6 and TLR7 were expressed relatively weak by Sertoli cells, compared with macrophages. Notably, abundant TLR3 mRNA was detected in Sertoli cells but very weak in macrophages. The highest expressions of TLR2–5 in Sertoli cells were confirmed by real-time RT-PCR (Fig. 1CGo). TLR13 was also abundantly expressed in Sertoli cells, although it is lower than in macrophages. TLR8, TLR9, and TLR11 mRNA was not detected in Sertoli cells, but expressed by macrophages. TLR12 was not observed in both Sertoli cells and macrophages. Thymus cells were used as a positive control for TLR12. No homologous gene of human TLR10 has been identified in mouse (27). The results of Western blotting demonstrated the highest protein levels of TLR2–5 in Sertoli cells (Fig. 1DGo). The presence of TLR2-TLR5 proteins was further confirmed by flow cytometry analysis (Fig. 1EGo). Macrophages were used as controls for flow cytometry. Consistent with RT-PCR results, we did not detected TLR3 protein in macrophage.


Figure 1
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  		FIG. 1. Expression pattern of TLRs in mouse Sertoli cells. A, Representative images of RT-PCR for detection of TLR expression in mouse Sertoli cells and macrophages (M{varphi}). Electrophoresis referred to 32 cycles for TLRs and 22 cycles for β-actin are shown. B, The densitometry of the target genes and β-actin gene bands was presented for semiquantitative comparison of their expression levels between Sertoli cells and macrophages. C, Quantitative analysis on the expression of TLR2–7, 13 by real-time RT-PCR. D, Western blotting for detection of TLR2–7 at protein levels. E, The expression of TLR2–5 proteins on Sertoli cells was confirmed with FACS. The gray area represents the negative or isotypic control antibody fluorescence. The diagrams are representatives of three independent experiments. Macrophages were used as controls for all analysis on TLRs expression. Data are mean ± SEM for three experiments. *, P < 0.05; **, P < 0.01.

 
Activation of NF-{kappa}B in Sertoli cells by TLR ligands
Although various members of the TLR family are expressed in Sertoli cells, their functions in this cell type remain to be clarified. Therefore, we examined whether TLR2-TLR5, which were expressed relatively high in Sertoli cells, can be activated by their ligands. It is known that the activation of TLR2-TLR5 results in NF-{kappa}B activation (11). NF-{kappa}B is sequestered in the cytoplasm as its inactive form and migrates into the nucleus when it is activated. Here we used immunofluorescence staining for NF-{kappa}Bp65 to detect the translocation of NF-{kappa}B in Sertoli cells. To select optimal doses of agonists to active TLRs, the Sertoli cells were stimulated by different doses of zymosan, poly (I:C), LPS, and flagellin (the ligands of TLR2, TLR3, TLR4, and TLR5, respectively). Immunofluorescence staining was performed using polyclonal antibody against NF-{kappa}Bp65. As shown in Fig. 2AGo, all four ligands-induced translocation of NF-{kappa}Bp65 from cytoplasms to nuclei in a dose-dependent manner. With the selected doses of TLR ligands for highly positive effects, we performed time-dependent effects of the ligands. Compared with 0 min, when all nuclei were negative for NF-{kappa}Bp65, a certain proportion of Sertoli cell nuclei were positively stained at 30 min after stimulation (Fig. 2Go, B and C). Poly (I:C) and LPS induced more efficiently nuclei translocation of NF-{kappa}B, compared with flagellin and zymosan, at 30 min after stimulation, with about 30 vs. 15% NF-{kappa}B-positive nuclei (P < 0.05). However, at 1 h after stimulation, all four ligands induced almost 100% Sertoli cell nuclei positive for NF-{kappa}Bp65 (Fig. 2Go, B and C). It is known that NF-{kappa}B is activated through I{kappa}B degradation. To verify this process, I{kappa}B degradation in Sertoli cells was examined by immunofluorescence staining for I{kappa}B{alpha} after stimulation with TLR ligands. The results were shown in Fig. 2DGo. Before stimulation, Sertoli cells were positively stained for I{kappa}B{alpha}. The I{kappa}B{alpha}-positive signals were decreased at 30 min after stimulation and much weaker at 1 h. These observations indicate that TLR2-TLR5 can be activated by their ligands in Sertoli cells and thus result in NF-{kappa}B activation.


Figure 2
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  		FIG. 2. NF-{kappa}B activation and I{kappa}B degradation by TLR agonists. A, Dose-dependent efficiency of NF-{kappa}B translocation to nuclei of Sertoli cells. The Sertoli cells were stimulated with TLR ligands: poly (I:C) [p (I:C)], LPS, flagellin (Flag), and zymosan (Zymo). Immunofluorescence staining for NF-{kappa}B was performed using rabbit antimouse polyclonal antibody against NF-{kappa}Bp65 at 1 h after stimulation. B, Representative images of the translocation of NF-{kappa}Bp65 from cytoplasms to nuclei of Sertoli cells in a time-dependent manner after stimulation with TLR ligands. C, Quantitative analysis on the efficiency of NF-{kappa}B translocation at different times after stimulation. Data are mean ± SEM of three assays, *, P < 0.05 for comparison at 30 min after stimulation; ##, P < 0.01 for comparison at 1 h with 30 min after stimulation. D, Degradation of I{kappa}B in Sertoli cells stimulated by TLR ligands. The presence of I{kappa}B{alpha} was examined by immunofluorescence staining using specific antibody against I{kappa}B{alpha}. Preimmune rabbit serum was used to instead of the primary antibody for negative control (Ctrl). Bar, 20 µm.

 
Induction of inflammatory cytokines by TLR agonists in Sertoli cells
It is known that activation of TLRs by their ligands can trigger a common signaling pathway to up-regulate inflammatory cytokines such as IL-1, IL-6, IL-12, and TNF{alpha} (26). Particularly, TLR3, TLR4, TLR7, TLR8, and TLR9 induce antiviral responses by inducing type I IFN (IFN{alpha} and IFNβ). To detect whether stimulation of TLR2, TLR3, TLR4, and TLR5 in Sertoli cells can up-regulate these inflammatory cytokines, we performed semiquantitative RT-PCR to detect mRNA of IL-1{alpha}, IL-6, IL-12, TNF{alpha}, IFN{alpha}, and IFNβ before and after treatment of Sertoli cells by TLR ligands. The results were shown in Fig. 3Go, A and B. At 12 h after stimulation with TLR ligands, Sertoli cells expressed about 10-fold IL-6 and 5-fold IL-1{alpha} higher than controls. The four TLR ligands induced augmentation comparable at mRNA level for IL-6 and IL-1{alpha}. However, up-regulation of IFN{alpha} and IFNβ was observed only in Sertoli cells treated by poly (I:C) and LPS. The stimulation of Sertoli cells by zymosan and flagellin did not up-regulate IFN{alpha} and IFNβ. No IL-12 and TNF{alpha} was detected in Sertoli cells by RT-PCR before and after stimulation by TLR ligands (data not shown).


Figure 3
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  		FIG. 3. Up-regulation of inflammatory cytokines by TLR agonists. A, Representatives of RT-PCR results. Electrophoresis referred to 32 cycles for IL-1{alpha}, IL-6, IFN{alpha}, and IFNβ and 22 cycles for β-actin are shown. B, The densitometric quantitation of cytokines and β-actin bands. C, The cytokines levels in culture medium before and after inhibiting of TLRs were quantitatively measured with ELISA. The data are mean ± SEM of three experiments. *, P < 0.05; **, P < 0.01 for comparison with control (Ctrl). #, P < 0.05; ##, P < 0.01 for comparison between with and without inhibitors of TLRs.

 
To determine whether the induction of the cytokines by TLR ligands was specifically mediated by TLRs, we used antibodies to block TLR2, TLR3, and TLR5 chemical inhibitor to block TLR4 before the presence of TLR ligands. The secretory cytokines in medium were detected using ELISA. The production of the cytokines was significantly induced by ligands of TLR2–5, and the inductions were inhibited by the blockers of the TLRs (Fig. 3CGo).

Increased phagocytic ability of Sertoli cells by TLR3
Sertoli cells are professional phagocytes in the seminiferous tubules to remove apoptotic germ cells and residual bodies. A previous study demonstrated that TLRs increased macrophages phagocytosis of bacteria (43). Therefore, we asked whether TLRs play a role in regulating phagocytosis by Sertoli cells. To examine this effect, Sertoli cells were cocultured with apoptotic germ cells in serum-free medium (DMEM/F12) in the presence of TLR agonists. Apoptotic spermatogenic cells were obtained through spontaneous induction during culture in vitro. To assess apoptotic rate of the cells, we stained the cells with dye mix of AO/EB. After staining, the nuclei of apoptotic cells show a yellow fluorescence, the nuclei of dead cells show an orange color, and nuclei of living cells appear a green color (Fig. 4AGo). Based on the procedures, we found that about 75% of spermatogenic cells are apoptotic cells; 15% of them are dead and 10% of them remain living.


Figure 4
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  		FIG. 4. Effect of TLR ligands on the phagocytic activity of Sertoli cells. Sertoli cells were cultured under different conditions as described in Materials and Methods. A, The image of apoptotic spermatogenic cells after staining with AO/EB. The nuclei of apoptotic cells are yellow (open arrows), those of dead cells are orange (closed arrows), and those of living cells show green fluorescence (arrowheads). B, The images of lipid droplets in Sertoli cells after staining with ORO. C, Area ratios of the lipid droplets to nuclei of Sertoli cells were used to evaluate phagocytic ability of Sertoli cells. D, In serum-free medium, poly (I:C) stimulated phagocytic ability of Sertoli cells in a dose-dependent manner. E, Effect of poly (I:C) on the phagocytosis of latex beads (LB) by Sertoli cells. Each datum was presented as mean ± SEM of three experiments. *, P < 0.05; **, P < 0.01; #, P < 0.05. Bar, 20 µm.

 
Lipid droplet formation after phagocytosis was used as a criterion to evaluate phagocytotic ability of Sertoli cells. At 24 h after coculture with apoptotic spermatogenic cells, the Sertoli cells were stained by ORO. Figure 4BGo represents images of lipid droplets in Sertoli cells cocultured with apoptotic germ cells under different stimulation. Quantitative data were shown in Fig. 4CGo. Poly (I:C) significantly increased lipid droplet formation by 3-fold, compared with control. In contrast, the other TLR agonists, zymosan, LPS, and flagellin, did not increase the formation of lipid droplets in Sertoli cells after coculture with apoptotic spermatogenic cells. Notably, amount of lipid droplets in the Sertoli cells stimulated by poly (I:C) in serum-free medium was comparable with that in the Sertoli cells cultured in medium containing 10% fetal calf serum (FCS). However, FCS and poly (I:C) had no additive effect on the lipid droplet formation in Sertoli cells. These observations suggest that poly (I:C) stimulates the phagocytosis of apoptotic spermatogenic cells by Sertoli cells, and factors in serum distract the effect of poly (I:C). To substantiate the specificity of poly (I:C) effect on Sertoli cell phagocytosis, the Sertoli cells were preincubated with serum-free medium containing 10 µg/ml polyclonal antibody against TLR3 (eBioscience; 24–9031) for 2 h before presence of poly (I:C) in culture. With blocking of TLR3 using the antibody, the lipid droplet formation was reduced significantly in the Sertoli cells after coculture with apoptotic spermatogenic cells (Fig. 4Go, B and C).

To further verify the effect of poly (I:C)/TLR3 signaling on Sertoli cell phagocytosis, a dose-dependent effect of poly (I:C) on the lipid droplet formation in Sertoli cells cocultured with apoptotic germ cells was examined. At 24 h after coculture in the presence of different concentrations of poly (I:C) (0, 2, 20, 200, 2000 ng/ml), the Sertoli cells were stained by ORO. A dramatic increased lipid droplets appeared in the Sertoli cells cultured in serum-free medium containing 200 ng/ml poly (I:C) (Fig. 4DGo), and no further increase in the lipid droplets was observed in the presence of more poly (I:C) (2 µg/ml). These results confirm the effect of poly (I:C) on the phagocytosis of apoptotic germ cells by Sertoli cells.

To determine whether TLR3-mediated Sertoli cell phagocytosis is specific to apoptotic germ cells or a general phenomenon, we examined the effect of poly (I:C) on Sertoli cells to ingest latex beads. As shown in Fig. 4EGo, there were no difference in the ingestion of latex beads between Sertoli cells cultured in serum-free medium with and without poly (I:C). The percentages of Sertoli cells ingested latex beads were 27.2% and 28.3% under two culture conditions. In contrast, treatment with cytochalasin B resulted in a marked decrease in the phagocytosis of latex beads by Sertoli cells. In macrophages, TLRs activate signal transduction pathways leading to ingest pathogens such as bacteria. To determine whether TLR3 induces the phagocytosis of bacteria by Sertoli cells, we detected uptaking of fluorescence-labeled E. coli. Neither Sertoli cells cultured with poly (I:C) nor without poly (I:C) could ingest the inactivated bacteria (data not shown). These observations suggest that TLR3 specifically promotes the phagocytosis of apoptotic spermatogenic cells by Sertoli cells but does not affect general phagocytic ability of Sertoli cells.

Increased expression of phagocytic genes in Sertoli cells stimulated by poly (I:C)
To define the molecular mechanisms underlying the TLR3-induced phagocytic activity of Sertoli cells observed in Fig. 4Go, we analyzed the expression of phagocytic genes in Sertoli cells stimulated by poly (I:C). Primary Sertoli cells were treated with TLR ligands for 12 h and then subjected to the extraction of total RNAs. Two scavenger receptors (CD36, SR-B1) and one receptor tyrosine kinase (Mer) are known to be involved in the engulfment of apoptotic spermatogenic cells by Sertoli cells (44, 45, 46). Therefore, the expression of these three genes was examined by semiquantitative RT-PCR in Sertoli cells treated with TLR ligands. We found that both CD36 and SR-B1 were induced by poly (I:C) at 12 h after the treatment (Fig. 5AGo). Compared with controls, 2.5- and 2-fold increases in the expression of CD36 and SR-B1 were detected in the Sertoli cells stimulated by poly (I:C), whereas LPS, flagellin, and zymosan did not up-regulate the expression of CD36 and SR-B1 (Fig. 5BGo). In contrast, Mer was expressed consistently in the Sertoli cells treated by TLR ligands. To determine whether induction of SR-B1 and CD36 transcripts correlated with increased levels of proteins, we performed Western blotting using specific antibodies against SR-B1 and CD36. We found that poly (I:C) can significantly induce expression of these proteins (Fig. 5Go, C and D). These data suggest that TLR3 could promote phagocytosis of apoptotic spermatogenic cells through up-regulation of SR-B1 and CD36 by Sertoli cells.


Figure 5
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  		FIG. 5. Induction of phagocytic genes by poly (I:C). The primary Sertoli cells were stimulated with TLR agonists in serum-free medium for 12 h. A, Total RNA was extracted and subjected to RT-PCR for analysis of the expression of CD36, SR-B1, and Mer. Electrophoresis referred to 32 cycles for CD36, 25 cycles for SR-B1, 32 cycles for Mer, and 22 cycles for β-actin were shown. B, The densitometric quantitation of the target genes to β-actin bands. C, Whole-cell extracts were subjected to Western blotting to examine CD36 and SR-B1 proteins after stimulation with poly (I:C). D, The densitometry of the bands of CD36 and SR-B1 to β-actin. The data are mean ± SEM of three experiments. *, P < 0.05; **, P < 0.01.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
TLRs play a crucial role in host defense against invading pathogens by mediating innate and adaptive immunity. Sertoli cells, the only somatic cell type within the seminiferous tubules, are important in the maintenance of testis as an immune privilege site in which both invading pathogen and autoantigens can be tolerated (47). The mechanisms that Sertoli cells involve in the immunity against pathogens and autoantigens have not been adequately studied. We demonstrate here that TLRs expressed in Sertoli cells may play an important role for the protection of testis against allo- and autoantigens.

We have demonstrated that mouse Sertoli cells express TLR2, TLR3, TLR4, and TLR5 at relatively high levels and TLR6, TLR7, and TLR13 at lower levels and do not express TLR1 and TLR8-TLR12. These results are largely in agreement with those reported in adult human testis (35), with main expression of TLR2 and TLR4 and low level of TLR6 and in mouse testis (36), with main expression of TLR2, TLR4, and TLR5 and low level of TLR6. Both these previous studies did not mention the expression of other TLR members. It is the first time that we report the expression of TLR3, TLR7, and TLR13 in mouse Sertoli cells. In particular, we demonstrate that TLR3 is abundantly expressed in Sertoli cells but is very weak in macrophages. Discrepancy among previous studies and our present study could be explained, considering that different strain of murine, number of PCR cycles, and primer pairs were used at different studies. Moreover, we used isolated Sertoli cells with purity more than 95% that excluded other testicular cells including macrophages. Although TLR13 is abundantly expressed in Sertoli cells, its ligand has not been identified yet (26). Therefore, we focused our study on the activation and function of TLR2-TLR5 in Sertoli cells.

The production of inflammatory cytokines after activation of TLRs by their agonists is essential for antimicrobial responses. It has been known that TLRs trigger a common intracellular signaling pathway that results in the induction of inflammatory cytokines, such as IL-6, IL-1, IL-12, and TNF{alpha} as well as alternative pathways that induce appropriate responses against different types of pathogen. In particular, TLR3 and TLR4 induce antiviral responses by inducing IFNβ and multiple IFN{alpha} (26). In agreement with previous observations in other cells, we detected increased IL-1{alpha} and IL-6 by the all four TLR agonists and increased IFN{alpha} and IFNβ by poly (I:C) and LPS. The results suggest that the stimulation of TLRs in Sertoli cells induces the production of inflammatory cytokines that may mediate immune responses against pathogens in testis. We did not detect the expression of IL-12 and TNF{alpha} in Sertoli cells before and after stimulation of TLRs, suggesting a different expression pattern of the cytokines in different cell types.

TLRs trigger various conserved inflammatory pathways, culminating in the activation of NF-{kappa}B (48). Before stimulation, NF-{kappa}B is presented in the cytoplasm as an inactive form by interacting with I{kappa}B protein. Based on stimulation with TLR ligands, I{kappa}B is phosphorylated and degraded through ubiquitin pathway, and then NF-{kappa}B is activated and translocated into the nucleus to bind to the {kappa}B site. To determine whether this inflammatory pathway was triggered in Sertoli cells upon stimulation of TLRs by their agonists, we detected the degradation of I{kappa}B and translocation of NF-{kappa}B by immunofluorescence staining. Our results demonstrate the degradation of I{kappa}B and the translocation of NF-{kappa}B from cytoplasms to the nuclei after treatment of Sertoli cells by TLR ligands. The results confirmed that the TLRs signaling in Sertoli cells are functional.

Phagocytosis is critical for innate immunity. A previous study demonstrated that TLR ligands promoted bacterial phagocytosis by macrophages (43). Sertoli cells are professional phagocytes in the seminiferous tubule to remove apoptotic germ cells and residual bodies (49). Therefore, it is worthwhile to determine whether the TLRs regulate phagocytic function of Sertoli cells. To address this issue, we analyzed the phagocytosis of apoptotic germ cells, latex beads, and bacteria by Sertoli cells after treatment with TLR ligands. Uptake of E. coli by Sertoli cells was not observed before and after stimulation by TLR agonists, suggesting that Sertoli cells cannot be responsible for innate immune response by directly ingesting invading pathogens. Cells undergoing apoptosis are efficiently eliminated from the organism by phagocytosis, and this phenomenon is likely to be a part of self-defense mechanisms (50). During spermatogenesis, more than 70% of spermatogenic cells are estimated to undergo apoptosis under physiological conditions (51, 52). A great deal residual bodies are formed in later stage of spermatogenesis (7). The rapid elimination of apoptotic cells and residual bodies by Sertoli cells is necessary for the normal production of sperm (53). The mechanism of this process remains to be clarified. In this study, we demonstrate that TLR3, but not other TLRs, specifically promotes phagocytosis of apoptotic spermatogenic cells by Sertoli cells. In contrast, activation of TLR3 does not induce Sertoli cells to ingest latex beads and bacteria. This finding provides novel insight into the mechanism underlying the phagocytosis of apoptotic spermatogenic cells by Sertoli cells.

It has been known that the phagocytosis of apoptotic germ cells by Sertoli cells is mediated by CD36 (45), SR-B1 (44) and Mer (46). In the present study, we show that poly (I:C), but not other TLR agonists, increases significantly expression of CD36 and SR-B1 in Sertoli cells. Therefore, we speculate that TLR3 promotes Sertoli cell phagocytosis of apoptotic germ cells through augmentation of CD36 and SR-B1. Induction of scavenger receptor (SR) genes including SR-A, LOX-1, and MARCO by TLRs has been reported to enhance macrophage-mediated phagocytosis of bacteria (43). Despite induction of SR genes by TLRs, each of these SR genes displays differential induction kinetics. Thus, further studies will be required to understand additional mechanisms underlying TLR-induced SR genes expression, particularly in different cell types.

Although poly (I:C) increases significantly the phagocytosis of apoptotic spermatogenic cells by Sertoli cells cultured in serum-free medium, this effect cannot be observed in Sertoli cells cultured in medium containing 10% FCS. These observations suggest that TLR3 enhances the phagocytic ability of Sertoli cells in absence of serum. This functional pattern could be particular important for Sertoli cells because they are seldom reached by blood circulation in vivo owing to the blood-testis barrier and lacking blood vessels in the seminiferous epithelium. What are the sources of TLR3 agonist in testis? The biological ligand of TLR3 is double-strained RNA, which can be released from broken-down cells. It has been reported that the molecules released from damaged cells can be as activators of TLRs (54). It was estimated that most of spermatogenic cells undergo apoptosis in physiological condition. However, only a limited number of apoptotic cells are detected histochemically. It is probably due to rapid elimination of apoptotic cells by Sertoli cells through phagocytosis of them. Unexpectedly, only a few ingested particles were observed in Sertoli cells by ultrastructure studies on rat testis sections, which was explained by the hypothesis that Sertoli cells degraded phagosome rapidly (7). After all, no study could exclude the possibility that apoptotic cells are broken down before phagocytosis of them. If this is a case, double-strained RNA from the damaged germ cells can be a functional ligand of TLR3 to enhance Sertoli cells to ingest apoptotic cells or cell debris. We are now investigating this possibility.

In summary, we demonstrate the expression patterns of TLR family in Sertoli cells. Activation of TLR2-TLR5 by their ligands increases the production of inflammatory cytokines. Interestingly, TLR3 ligand specifically promotes phagocytosis of apoptotic spermatogenic cells by Sertoli cells. The data suggest that TLRs in Sertoli cells may play important roles in the protection of the seminiferous epithelium from invading pathogens and autoantigens.


   Footnotes
 
This work was supported by the National Basic Research Program of China (Grants 2006CB504001 and 2007CB947504) and the National Natural Science foundation of China (Grants 30570678).

Disclosure Statement: The authors have nothing to disclose.

First Published Online May 22, 2008

Abbreviations: AO, Acridine orange; EB, ethidium bromide; FCS, fetal calf serum; FITC, fluorescein isothiocyanate; I{kappa}B, inhibitor of NF-{kappa}B; ICAM, intercellular adhesion molecule; IFN, interferon; LPS, lipopolysaccharide; NF-{kappa}B, nuclear factor-{kappa}B; ORO, Oil Red O; poly (I:C), polyinosinic-polycytidylic acid; SR, scavenger receptor; SR-B1, SR class B type I; TBS, Tris-buffered saline; TLR, Toll-like receptor.

Received December 21, 2007.

Accepted for publication May 12, 2008.


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