Correspondence: Masahisa Watarai, The United Graduate School of Veterinary Science, Yamaguchi University, 1677-1 Yoshida, Yamaguchi 753-8515, Japan. Tel.: +81 83 933 5831; fax: +81 83 933 5831; e-mail: email@example.com
During pregnancy, maternal immune function is strictly controlled and immune tolerance is induced. Trophoblast giant (TG) cells exhibit phagocytic activity and show macrophage-like activity against microorganisms in the placenta. However, details of molecular receptors and mechanisms for uptake by TG cells have not been clarified. In this study, we investigated the involvement of the mannose receptor, C type 1 (MRC1), in the uptake of the abortion-inducible bacterium Listeria monocytogenes and abortion-uninducible bacteria Bacillus subtilis and Escherichia coli by TG cells differentiated from a mouse trophoblast stem cell line in vitro. Knockdown of MRC1 inhibited the uptake of all of these bacteria, as did the blocking of MRC1 by MRC1 ligands. The uptake of bacteria by MRC1 delayed the maturation of phagolysosomes. These findings suggest that MRC1 plays an important role in the uptake of various bacteria by TG cells and may provide an opportunity for those bacteria to escape from phagosomes.
Trophoblast giant (TG) cells are placental cells that establish direct contact with endometrial tissues during implantation (Muntener & Hsu, 1977). TG cells also show macrophage-like activity against microorganisms (Amarante-Paffaro et al., 2004; Albieri et al., 2005). We previously reported that TG cells utilize their phagocytic activity to ingest intracellular bacteria such as Brucella abortus and Listeria monocytogenes that cause abortion in pregnant animals (Watanabe et al., 2009). However, details of molecular receptors and mechanisms for uptake by TG cells have not been clarified. In this study we examined the receptors important for uptake of bacteria using a TG cell line differentiated from a trophoblast stem (TS) cell line in vitro.
In mammals, the first line of the defense mechanism includes antigen recognition by pattern recognition receptors (PRRs) such as Toll-like receptor (TLR), scavenger receptor (SR), and mannose receptor (MR) (Areschoug & Gordon, 2009; Gazi & Martinez-Pomares, 2009; Kumar et al., 2009). In a previous study, we demonstrated that TLR2 and class B scavenger receptor type 1 (SR-B1) play important roles in the uptake of abortion-inducible bacteria such as L. monocytogenes and B. abortus by TG cells (Watanabe et al., 2010). However, knockdown of TLR2 and SR-B1 failed to reduce completely the uptake of L. monocytogenes and B. abortus by TG cells. In addition, the uptake of abortion-uninducible bacteria such as Escherichia coli was independent of those receptors. These findings indicated that common receptors are involved in the uptake of both abortion-inducible and abortion-uninducible bacteria in TG cells.
In this study, we focused on the mannose receptor, C type 1 (MRC1). MRC1 is a PRR that recognizes mannose, fucose, and N-acetylglucosamine sugar residues on the surface of microorganisms (Largent et al., 1984). To clarify whether MRC1 is a common receptor for uptake of abortion-inducible and abortion-uninducible bacteria, we investigated the involvement of MRC1 in the uptake of abortion-inducible gram-positive bacterium (L. monocytogenes) and abortion-uninducible gram-positive and -negative bacteria (Bacillus subtilis and E. coli, respectively) by TG cells. Knockdown of MRC1 inhibited the uptake of all of these bacteria. Blocking of MRC1 by MRC1 ligands also reduced the uptake of those bacteria, suggesting that MRC1 plays a fundamental role in the uptake of various bacteria by TG cells.
Materials and methods
Listeria monocytogenes EGD, E. coli DH5α, E. coli JM109, and B. subtilis 168 were used in this study. Bacterial strains were maintained as frozen glycerol stocks. Listeria monocytogenes EGD was cultured in brain heart infusion (BHI) broth (Becton Dickinson, Franklin Lakes, NJ) or on BHI broth containing 1.5% agar (Wako, Osaka, Japan). Escherichia coli DH5α, E. coli JM109, and B. subtilis 168 were cultured in Luria–Bertani (LB) broth (MO BIO Laboratories, Inc., Carlsbad, CA) or LB broth containing 1.5% agar.
A mouse TS cell line was a gift from Dr. Tanaka (Tanaka et al., 1998; Watanabe et al., 2008). TS cells were cultured in mixed medium (TS medium : mouse embryonic fibroblast-conditioned medium = 3 : 7) containing 25 ng mL−1 fibroblast growth factor 4 (TOYOBO, Osaka, Japan) and 1 μg mL−1 heparin (Sigma, St. Louis, MO) as described in our previous study (Watanabe et al., 2008). TS medium was prepared by adding 20% fetal bovine serum (FBS), 1 mM sodium pyruvate, 100 μM β-mercaptoethanol, and 2 mM l-glutamine to RPMI 1640 medium. To induce differentiation of TS cells to TG cells, TS cells were cultured in the TS medium alone for 3 days at 37 °C, 5% CO2. RAW 264.7 cells were cultured in RPMI 1640 containing 10% FBS. TG or RAW 264.7 cells were seeded (1–2 × 105 per well) in 48-well or 12-well (4–8 × 105 per well) tissue culture plates.
TG or RAW 264.7 cells were washed twice with phosphate-buffered saline (PBS) and lysed in lysis buffer (ice-cold PBS containing 1% Triton X-100, 1 mM sodium orthovanadate, 2 mM phenylmethylsulfonyl fluoride, 100 mM sodium fluoride, and 1× Halt Protease Inhibitor Cocktail Kit (Thermo Fisher Science, Rockford, IL) at 4 °C for 30 min, and sonicated for 10 s, three times. The cell lysates were centrifuged (16 000 g, 4 °C, 20 min) and supernatants collected. Protein concentrations were determined using Bio-Rad Protein Assay (Bio-Rad, Richmond, CA). After separating 300 ng of each protein by SDS-PAGE with 10% polyacrylamide gels, the proteins were transferred onto polyvinylidene difluoride (PVDF) membranes (Millipore, Billerica, MA). After blocking with 5% nonfat dry milk in Tris-buffered saline (TBS) at room temperature for 2 h, membranes were incubated with anti-mouse MRC1 rat monoclonal antibody (1 : 200; R&D Systems Inc., Minneapolis, MN) or anti-mouse β-actin antibody (Sigma) at 4 °C overnight. After washing with TBS containing 0.02% Tween 20, membranes were incubated with horseradish peroxidase-conjugated secondary antibody (0.01 μg mL−1) at room temperature for 1 h and immunoreactions were visualized using the enhanced chemiluminescence detection system (GE Healthcare Life Science, Little Chalfont, UK).
RNA isolation and reverse transcription (RT)-PCR
Total RNA of TG cells was isolated using the RNAeasy Plus Mini Kit (Qiagen, Hilden, Germany). Purified RNA samples were stored at −80 °C prior to use. The RNA was quantified by absorption at 260 nm using the SmartSpec3000 spectrophotometer (Bio-Rad). RT-PCR was carried out using the SuperScript One-Step RT-PCR with Platinum Taq (Invitrogen, Carlsbad, CA). The sequences of primers were as follows. MRC1: 5′-GCAAATGGAGCCGTCTGTGC-3′ and 5′-CTCGTGGATCTCCGTGACAC-3′ (Bhatia et al., 2011), β-actin: 5′-TGGAATCCTGTGGCATCCATGAAAC-3′ and 5′-TAAAACGCAGCTCAGTAACAGTCCG-3′ (Nomura et al., 2002).
Efficiency of bacterial internalization and replication
Bacterial internalization assays were performed in a similar manner to that in our previous study (Watanabe et al., 2008). Bacterial strains were deposited onto TG cells cultured in 48-well plates with TS medium by centrifugation (150 g, 10 min, room temperature). To measure bacterial internalization efficiency after 30 min of incubation at 37 °C, the cells were washed once with TS medium and then incubated in a medium containing gentamicin (30 μg mL−1) for 30 min. The cells were then washed three times with PBS and lysed with cold distilled water. Colony-forming unit values were determined by serial dilution on BHI or LB plates. Recombinant interferon-γ (IFN-γ) (Cedarlane Laboratories, ON, Canada) was added 24 h before infection. Mannan from Saccharomyces cerevisiae (Sigma), d-mannose (Sigma), l-fucose (Sigma), or d-galactose (Sigma) were added 15 min before infection.
Small interfering RNA (siRNA) experiment
The siRNA duplexes used for silencing mouse MRC1 (target sequence: 5′-CAGCATGTGTTTCAAACTGTA-3′) and AllStars Negative Control siRNA were purchased from Qiagen. TG cells were transiently transfected using Lipofectamine RNAiMAX (Invitrogen) with or without siRNAs at a final concentration of 36 nM.
Measurement of phagolysosome maturation
Bacterial internalization assays were performed in a similar manner to that in our previous study (Watanabe et al., 2008). Escherichia coli JM109 was introduced with pAcGFP1 (E. coli GFP+). Plasmid pAcGFP1 was purchased from Clontech (Mountain View, CA). Escherichia coli GFP+ was deposited onto TG cells on coverslips by centrifugation (150 g, 10 min, room temperature), incubated at 37 °C for 30 min, and then incubated in TS medium containing gentamicin (30 μg mL−1) for 0.5, 1, 2, and 4 h. The samples were washed twice with PBS. For vital staining of lysosomes, cells were incubated with LysoTracker red (Molecular Probes, Eugene, OR) at 37 °C for 30 min. Cells were washed with PBS and fixed with 4% paraformaldehyde. Fluorescent images were obtained by the FluoView FV100 confocal laser scanning microscope (Olympus, Tokyo, Japan).
Total RNA was extracted from TS or TG cells using TRIzol reagent (Invitrogen). The RNA was quantified by absorbance at 260 nm and the purity was assessed by the 260/280 nm ration. genechip (Mouse Genome 430 2.0) and kits for cDNA synthesis, biotin-labeled cRNA synthesis, fragmentation of cRNA, and hybridization were obtained from Affymetrix (Santa Clara, CA). All experiments were performed according to the manufacturer's recommendations. The results were obtained from three genechips.
Statistical analyses were performed using Student's t-test. Statistically significant differences compared with control are indicated by asterisks (*P <0.01, **P <0.05). Data are the averages of triplicate samples from three identical experiments and the error bars represent standard deviations.
MRC1 is involved in bacterial uptake by TG cells
In a previous study, we demonstrated that TLR2 and SR-B1 play a central role in the uptake of the abortion-inducible bacteria L. monocytogenes and B. abortus (Watanabe et al., 2010). However, knockdown of these receptors failed to block the uptake completely. In addition, the uptake of abortion-uninducible bacteria such as E. coli was independent of TLR2 and SR-B1. These results indicated that other receptors were involved in the uptake of those bacteria. Since the uptake of E. coli was observed at the same levels in TG cells and immature TS cells, PRRs expressed at the same levels in TG and TS cells were searched from the data of microarray analysis (Table 1). MRC1 was one of the PRRs expressed in TG and TS cells. To examine whether MRC1 is expressed in TG cells, the expression of MRC1 in TG cells was measured by immunoblotting and RT-PCR (Fig. 1a and b). MRC1 was expressed in TG cells at levels comparable to those of a MRC1-positive cell, RAW264.7. To examine the influence of MRC1 on the uptake of bacteria, the expression of MRC1 in TG cells was blocked with MRC1-specific siRNA (Fig. 1a and b). Uptake of the abortion-inducible bacterium L. monocytogenes by TG cells was reduced significantly by siRNA (Fig. 1c). On the other hand, no reduction was observed in L. monocytogenes internalization in TG cells transfected with AllStars Negative Control siRNA. Uptake of abortion-uninducible bacteria such as E. coli and B. subtilis by TG cells was also decreased in a similar manner to that of L. monocytogenes. These results suggest that MRC1 is a receptor for the uptake not only of abortion-inducible bacteria in TG cells but of abortion-uninducible bacteria.
Table 1. Microarray analysis of TS and TG cells
CD209e antigen (DC-SIGN)
CD302 antigen (DCL-1)
MRC1 ligands inhibit bacterial uptake by TG cells
It was reported that microbial phagocytic activity via MRC1 is allayed by MRC1 ligands (Miller et al., 2008; Macedo-Ramos et al., 2011). To confirm the involvement of MRC1 in the uptake of bacteria by TG cells, TG cells were treated with MRC1 ligands and infected with various bacteria. The MRC1 ligands mannan (0, 3, 15, and 30 mg mL−1), d-mannose (0, 50, 100, and 200 mM), and l-fucose (0, 50, 100, and 200 mM) were selected. Treatment of TG cells with each ligand reduced the uptake of L. monocytogenes, E. coli, and B. subtilis in a dose-dependent manner (Fig. 2). In contrast, treatment with d-galactose (0, 50, 100, and 200 mM), a sugar not recognized by MRC1, showed no significant effect on uptake of those bacteria. These findings suggest that TG cells recognize specific sugar chains on the bacterial surface and ingest them using MRC1.
IFN-γ promotes bacterial uptake by TG cells
It has been reported that the expression of cell surface receptors is regulated by IFN-γ (Faure et al., 2001). Indeed, we demonstrated that IFN-γ augments the uptake of L. monocytogenes by TG cells (Watanabe et al., 2010). Next, we evaluated whether IFN-γ would affect the uptake of L. monocytogenes, E. coli, and B. subtilis by TG cells. TG cells were treated with IFN-γ for 24 h (0, 200, 400, and 1000 units mL−1) and internalization efficiency was measured. IFN-γ treatment increased the efficiency of L. monocytogenes uptake by TG cells in a concentration-dependent manner (Fig. 3b). Unexpectedly, IFN-γ failed to increase the uptake of E. coli and B. subtilis. To estimate the influence of IFN-γ on cell surface receptors, the levels of mRNA in MRC1 were measured by RT-PCR. IFN-γ treatment decreased the transcription levels of MRC1 (Fig. 3a). These findings suggest that decreased MRC1 expression levels inhibit the augmentation of E. coli and B. subtilis uptake by TG cells.
Maturation of phagolysosomes
To examine whether the bacteria internalized through MRC1 are killed by the phagocytic activity of TG cells, phagolysosome maturation was measured using LysoTracker red. Since L. monocytogenes shows strong cytotoxicity in TG cells and it is difficult to measure the maturation of phagolysosome, the maturation was observed using E. coli. In TG cells, 20–40% of internalized E. coli were colocalized with LysoTracker red at 0.5–4 h post-infection (Fig. 4a and b). In contrast, in MRC1-knockdown TG cells, colocalization was slightly but efficiently increased. The same effect was observed when TG cells were treated with IFN-γ, and colocalization of E. coli and LysoTracker red was slightly augmented (Fig. 4c). These results indicate that the uptake of E. coli by MRC1 delays the maturation of phagolysosomes.
During pregnancy, maternal immune function is strictly controlled and immune tolerance is induced (Warning et al., 2011). There is little information about the mechanisms by which the fetus is protected from infectious microorganisms under the condition of immunosuppression at the fetal–maternal interface. TG cells are present in the placental labyrinth zone and play important roles in acquiring nutrition and space for embryonic attachment and development in the endometrium, which are key to a successful pregnancy (Welsh & Enders, 1987; Bevilacqua & Abrahamsohn, 1988, 1994; Amarante-Paffaro et al., 2004; Hu & Cross, 2010). TG cells also exhibit phagocytic activity toward foreign antigens in a similar manner to macrophages (Welsh & Enders, 1987; Amarante-Paffaro et al., 2004; Albieri et al., 2005). However, the molecular mechanisms of recognition and uptake of bacteria in TG cells remain unclear.
We previously reported that TLR2 and SR-B1 were involved in the uptake of abortion-inducible intracellular bacteria such as B. abortus and L. monocytogenes by TG cells (Watanabe et al., 2010). On the other hand, TLR2 and SR-B1 were not involved in the uptake of the abortion-uninducible bacterium E. coli. The uptake of E. coli was observed in TG cells and immature TS cells at the same levels (Watanabe et al., 2010). Knockdown of TLR2 and SR-B1 resulted in partial reduction of uptake by TG cells. These results suggest the existence of other receptors that recognize and ingest various bacteria in TG and TS cells. Uptake of bacteria is controlled by a variety of receptors such as the TLR family, the SR family, MR, DEC-205-associated C-type lectin-1 (DCL-1), and dendritic cell-specific ICAM grabbing non-integrin (DC-SIGN) (Hsu et al., 1996; Faure et al., 2001; Kerrigan & Brown, 2009). Among these receptors, DCL-1 and DC-SIGN were not expressed in TG cells (Table 1). In the present study we focused on MRC1, since MRC1 was expressed in both TG and TS cells (Table 1, Fig. 1a and b). MRC1 is expressed on lymphatic and hepatic epithelia, kidney mesangial cells and retinal pigment epithelium (Shepherd et al., 1991; Lew et al., 1994; Linehan et al., 1999). This expression is also observed in a subpopulation of murine dendritic cells (McKenzie et al., 2007). MRC1, a C-type lectin, is known as a PRR. MRC1 recognizes mannose, fucose, and N-acetylglucosamine sugar residues on the surface of these microbes and is involved in the uptake of Mycobacterium tuberculosis, Klebsiella pneumoniae, and Streptococcus pneumoniae (Ezekowitz et al., 1991; Marodi et al., 1991; Schlesinger, 1993; O'Riordan et al., 1995; Chakraborty et al., 2001; Zamze et al., 2002). We demonstrated in this study that the inhibition of MRC1 by siRNA or MRC1 ligands decreases the uptake of both of abortion-inducible and abortion-uninducible bacteria (Figs 1c and 2), indicating that MRC1 is an important receptor for the recognition and uptake of various bacteria by TG cells.
IFN-γ is one of the key factors related to infectious abortion. In our previous study, we demonstrated that anti-IFN-γ antibody decreased the induction of abortion caused by B. abortus in mouse (Kim et al., 2005) and that IFN-γ enhanced the uptake of L. monocytogenes (Fig. 3b) and B. abortus by TG cells (Watanabe et al., 2010). It was reported that IFN-γ controls cell surface receptors (Faure et al., 2001). However, TLR2 and SR-B1 expression in TG cells was not regulated by IFN-γ (Watanabe et al., 2010), suggesting that IFN-γ facilitates the internalization step of bacteria, but not the binding step. It was reported that IFN-γ promoted bacterial internalization through extracellular-regulated kinase (ERK) 1/2 pathway in gut epithelial cells (Smyth et al., 2012). Although the expression of MRC1 is decreased by IFN-γ (Fig. 3a), L. monocytogenes may bind to TG cells through TLR2 or SR-B1, followed by up-regulation of uptake by IFN-γ. In contrast, the uptake of E. coli and B. subtilis by TG cells was not augmented by IFN-γ (Fig. 3b). It was reported that IFN-γ decreased MRC1 expression in murine macrophage (Harris et al., 1992). Indeed, the transcription level of MRC1 in TG cells was also decreased by IFN-γ treatment (Fig. 3a). Although the expression of MRC1 was decreased by IFN-γ, the uptake of E. coli and B. subtilis was not impaired. These results imply that IFN-γ augments the internalization step of these bacteria, but the augmentation is inhibited by the reduction in MRC1 expression, since these bacteria are thought to be bound to TG cells mainly through MRC1.
Knockdown of MRC1 or IFN-γ treatment increased phagosomal maturation (Fig. 4), indicating that internalization of bacteria by MRC1 delays the maturation of phagolysosomes. This finding is consistent with other reports that the uptake of M. tuberculosis and Mycobacterium avium by MR limits phagosome maturation in murine bone marrow-derived macrophages and human macrophages (Kang et al., 2005; Sweet et al., 2010). For abortion-inducible bacteria such as L. monocytogenes and B. abortus, escaping from phagolysosomes is key to their pathogenicity in the placenta (O'Callaghan et al., 1999; Foulongne et al., 2000; Boschiroli et al., 2001, 2002; Bakardjiev et al., 2005; Le Monnier et al., 2007). Internalization to TG cells by MRC1 followed by a delay in phagolysosomal maturation may result in the escape of these bacteria. Unfortunately, L. monocytogenes exhibits strong cytotoxicity and it was impossible to identify the relationship between phagolysosome maturation and MRC1 for L. monocytogenes infection. In addition, there is little information about signal transductions downstream of MR, since it lacks any known motifs on its cytoplasmic tail and therefore is presumed to interact with unknown signaling proteins. Further study is needed to clarify the relationship between infectious abortion and MRC1.
Overall, our findings in this study suggest that MRC1 is a fundamental receptor that plays an important role in the recognition and uptake of various bacteria. Although there are increasing numbers of reports maintaining that bacterial infection causes abortion or sterility in animals and humans (Low & Donachie, 1997; Bourke et al., 1998; Boschiroli et al., 2001; Lecuit, 2007), there is little information on how the fetus is protected from infectious microorganisms. Therefore, our finding may help to reveal the general mechanism of the placental immune system.
The authors declare that they have no conflicts of interest.