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The Journal of Immunology, 2006, 176: 4147-4154.
Copyright © 2006 by The American Association of Immunologists

TLR3-Involved Modulation of Pregnancy Tolerance in Double-Stranded RNA-Stimulated NOD/SCID Mice1

Yi Lin2, Zhenhong Liang, Yijing Chen and Yaoying Zeng

Key Laboratory of Ministry of Education for Tissue Transplantation and Immunology, College of Life Science and Technology, Jinan University, Guangzhou City, China


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
This study aims to extend understanding of the relationship between TLR3-involved cell signaling and dsRNA-induced embryo resorption. Upon stimulation of dsRNA, the resorption rate of embryos was boosted dramatically in syngeneic mating BALB/c mice, but not significantly influenced in syngeneic mating NOD/SCID mice. Accordingly, there was an enhanced cell surface expression of TLR3 on placental CD45+ cells derived from BALB/c mice, concomitant with both increased percentages of CD45+CD80+ cells and CD8{alpha}+CD80+ cells in flow cytometric analysis. In addition, both increased IL-2 and decreased IL-10 expression could be observed in CD45+ cell group in the intracellular detection by flow cytometry. In contrast, no such trends were observed in NOD/SCID model, and its resorption rate of embryos was kept at a low level throughout pregnancy. Neutralizing Abs against TLR3 could abrogate the embryo rejection induced by dsRNA in BALB/c mice, and simultaneously could reduce the CD80+ percentage in the CD45+ cell group. These results indicate that the interaction between dsRNA and TLR3 may be involved in the mobilization of CD45+CD80+ and CD8{alpha}+CD80+ cells, followed by the up-regulation of IL-2 and down-regulation of IL-10 expression at the feto-maternal interface, and finally resulting in embryo rejection. The relatively low responsiveness of NOD/SCID mice may be one of the reasons why these mice appeared to be resistant to dsRNA-induced embryo resorption.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
The development of effective animal models is a key prerequisite for studies on the mechanisms of pregnancy tolerance. Bosma et al. (1) originally reported that C.B-17 mice homozygous for the SCID (scid/scid) mutation lack functional T and B lymphocytes. Although they are defective in cellular and humoral immune function, these mice display normal NK cell function (2). In contrast, the NOD/Lt strain is characterized by a functional deficit in NK cells (3). Shultz et al. (4) has established a murine model, NOD/LtSz-scid/scid (NOD/SCID for short), characterized by multiple defects in innate immunity as well as in T and B cell function, by backcrossing of the scid mutation onto the NOD/Lt strain background. Because NOD/SCID mouse has deficient T cell, B cell, and NK cell activity and has a sufficiently long lifespan (mean lifespan 8.5 mo) to allow dynamic observation of fertility features (4, 5), it can be used as a valuable model in the comparative studies of reproductive immunology, together with those normal fertile control mice.

Viral challenge of a vertebrate host leads to an intricate series of responses that orchestrate antiviral immunity. The success of this multifaceted system in overcoming viral encounters hinges on complex pathogen-host interactions. In particular, dsRNA, a nucleic acid associated with viral replication, is involved in numerous interactions contributing to antiviral mechanisms. Specifically, dsRNA is responsible for stimulating important protective responses, such as the induction of type 1 IFN and stimulation of dsRNA-activated protein kinase and oligoadenylate synthetase. Furthermore, the modulation and shaping of this overall immune response is facilitated through nucleic acid interactions with pattern recognition receptors such as TLR3 (6).

TLR is a family of innate pattern recognition receptors characterized by amino-terminal leucine-rich repeat domains and carboxyl-terminal Toll/IL-1 receptor signaling domains (7). TLR family members recognize unique pathogen-associated molecular patterns to initiate innate and shape adaptive immune responses (8, 9). Alexopoulou et al. (10) established that TLR3 is essential for cellular responses to dsRNA. Induction of TLR3 signaling via dsRNA activates transcription factors such as NF-{kappa}B and IFN regulatory factor 3, resulting in the production of proinflammatory and antiviral cytokines (10, 11, 12).

Polyinosinic-polycytidylic acid (poly(I:C))3 is a synthetic dsRNA capable of boosting the resorption rate of embryos (RR) in murine models (13). Although dsRNA is known as a virus-specific signature and a ligand for TLR3, largely uncharacterized multiple pathways associate with dsRNA-mediated embryo resorption. In this study, the RR was observed dynamically at several time points during pregnancy in both syngeneic mating BALB/c and NOD/SCID mice. dsRNA stimulation was performed in these mice at an early stage of gestation in the presence or absence of TLR3-blocking, using neutralizing Ab against this receptor. The expression pattern of cell surface TLR3, CD80, and CD8{alpha}, as well as intracellular IL-2 and IL-10 was evaluated by flow cytometry to investigate the characteristics of pregnancy tolerance in multiple immunodeficiency NOD/SCID mice and to clarify the potential mechanisms of dsRNA-induced embryo resorption.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 Disclosures
 References
 
Murine mating combination

BALB/c and NOD/SCID mice were purchased from Experimental Animal Center of Zhongshan University (Guangzhou City, China) and bred under specific pathogen-free conditions in the laboratory for immunodeficiency animals (Jinan University, Guangzhou, China). All experiments involving animals followed national guidelines for animal usage in research. Newborn pups from syngeneic BALB/c x BALB/c and NOD/SCID x NOD/SCID mating combinations were fed by their mother, respectively. Pup age was recorded. To investigate TLR3 expression in splenocytes, spleens were collected from BALB/c and NOD/SCID mice at different ages, ranging from 1 day to 10 wk. Splenocytes were isolated using methods described elsewhere, and CD45+ and TLR3+ cells were detected by flow cytometry using the method described in Isolation of mononuclear cells and flow cytometry.

In another design, mice were used at an age of 10–12 wk. Syngeneic NOD/SCID x NOD/SCID breeding scheme was established, while BALB/c x BALB/c mating combination was used as a nonimmunodeficiency control. The model of dsRNA stimulation was established by i.p. injection of poly(I:C) (gamma-irradiated, catalog no. P0913; Sigma-Aldrich, St. Louis, MO) at a dosage of 200 µg/20 g of body weight in a volume of 200 µl at gestational days 6.5 and 7.5, respectively (13, 14). The pregnant mice were killed at day 8.5, 12.5, 16.5, or 20.5 of gestation, and RR was calculated as follows: RR (%) = (number of resorbed embryo)/(number of total embryo) x 100 (14).

Pretreatment of TLR3 using neutralizing Abs against this receptor

TLR3 was blocked by multiple-injection of mAb against this receptor (catalog no. 24-9031-91; eBioscience, San Diego, CA). The injections were performed i.p. at a dilution of 1/10 in a volume of 200 µl on days 4.5, 5.5, and 6.5 of gestation, consecutively. Afterward, poly(I:C) administration was performed at gestational days 6.5 and 7.5, or instead, mice were given an equal volume of PBS at the same time points of pregnancy and served as a control. In this design, only the CD45+CD80+/CD45+ cell percentage was detected, restricted by the number of available NOD/SCID mice, with methods described in Isolation of mononuclear cells and flow cytometry.

Fluorescein-conjugated mAbs used in this study

The following Abs were purchased from BD Pharmingen, eBioscience, or Bethyl Laboratories: anti-CD45-PE and anti-CD45-FITC (clone 30-F11), anti-CD80-PE and anti-CD80-FITC (clone 16-10A1), anti-CD8{alpha}-PE (clone 53-6.7), anti-IL-2-PE (clone JES6-5H4), and anti-IL-10-PE (clone JES5-16E3). Rat anti-mouse-TLR3 staining was followed by FITC-conjugated goat anti-rat Ig (catalog no. A110-109F) to label TLR3+ cells indirectly.

Isolation of mononuclear cells and flow cytometry

Mononuclear cells were isolated from pooled placentas of each mouse using methods described previously (4, 14). In brief, hysterolaparotomy was performed to collect embryo-depleted placentas and associated decidual tissues including decidua basalis. The pooled placentas were carefully cut into small pieces and then collected in HBSS and filtered through a 50-µm pore size nylon mesh to obtain a mononuclear cell suspension. Mononuclear cells were purified by centrifugation of cell suspension on Ficoll-Hypaque density medium. Any contaminating RBCs that might have persisted in the single-cell suspension were eliminated by incubation with RBC lysis buffer twice at 37°C. Splenocytes mentioned above and placental mononuclear cells were incubated in the indicated mAb conjugates, respectively, for 30 min in a total volume of 50 µl of PBS containing 3% BSA. After two washes with PBS, these cells were resuspended in 1% paraformaldehyde. Immunostained cells were analyzed on a FACSCalibur flow cytometer using CellQuest software (BD Biosciences). A total of 10,000 cells was detected in each sample.

Negative control

Adequately designed gate thresholds were set to capture lymphoid cells using appropriate FITC- or PE-conjugated isotype control Abs (BD Pharmingen or eBioscience) (15, 16).

Intracellular detection of IL-2 and IL-10

The isolated placental mononuclear cells were stained with anti-CD45-FITC, washed with PBS, and resuspended with permeabilization buffer containing 0.1% saponin and 0.09% sodium azide (catalog no. 00-8333-56; eBioscience) for 1 h at 4°C. Then the cell suspension was repelleted, and the supernatant was flicked out. PE-conjugated anti-mouse IL-2 and IL-10 mAbs (mentioned in Fluorescein-conjugated mAbs used in this study) were added, respectively, at 106 cells per 0.25 µg, in a total volume of 50 µl of PBS. After incubation in the dark for 20 min, the cells were washed with PBS and then detected with flow cytometry (17).

Calculation of cell percentage

The percentage of TLR3+ cells in CD45+ cell group equals CD45+TLR3+ cell number/CD45+ cell number x 100. In addition, the following cell percentages were calculated similarly: CD45+CD80+ cells in the CD45+ cell group, CD8{alpha}+CD80+ cells in the CD8{alpha}+ cell group, and CD45+IL-2+ cells and CD45+IL-10+ cells in the CD45+ cell group.

Statistical analysis

Flow cytometry data were analyzed using Quad statistics (18). RR was analyzed using a {chi}2 test. The cell percentage was analyzed using Student’s t test, and the results were given as means ± SEM. Flow cytometric characterization of placenta cells was determined at day 12.5 of gestation.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 Disclosures
 References
 
Resorption rate of embryos

The RR was observed dynamically at several time points during pregnancy. As shown in Fig. 1, no significant difference was observed in the RR between BALB/c and NOD/SCID mice in the absence of dsRNA stimulus. However, upon dsRNA stimulation, the RR was boosted significantly from 4.8% to a level of around 22% at day 12.5 of gestation in BALB/c mice. In contrast, the RR merely was increased slightly in NOD/SCID mice detected at the same time point. Afterward, almost no more fetuses were resorbed in either BALB/c or NOD/SCID mice.


Figure 1
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  		FIGURE 1. Dynamic observation of RR during pregnancy. Upon dsRNA-stimulation, the RR was significantly boosted at day 12.5 of gestation in syngeneic mating BALB/c mice, and stayed at a relatively stable level after then. No such trend was observed in NOD/SCID mice. The RR steadily maintained at a low level throughout pregnancy, regardless of dsRNA stimulation.

 
In BALB/c mice pretreated with anti-TLR3 neutralizing Ab, the induction of dsRNA on embryo rejection was almost completely abrogated; the RR in these mice was 8.1%, not statistically different from that in control PBS group, but it was significantly lower than that in BALB/c mice without anti-TLR3 pretreatment. No such trends were observed in NOD/SCID mice. The RR in NOD/SCID mice stayed stable at a low level throughout pregnancy, regardless of the dsRNA stimulation or anti-TLR3 neutralization (Table I).


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  		Table I. Abrogation of dsRNA-induced embryo rejection with anti-TLR3 pretreatmenta

 
TLR3 expression in splenocytes from BALB/c and NOD/SCID mice at different ages

To detect the cell percentage accurately, both double-negative control and single CD45+ control were established (Fig. 2, A and B). In splenocytes, TLR3 expression on the surface of CD45+ cells could be detected even in neonatal BALB/c and NOD/SCID mice. In addition, the percentages of CD45+TLR3+ cells in the CD45+ cell group in these pups were not significantly different from those in weanling pups (3 wk old) or mature ones (10 wk old) (Fig. 2).


Figure 2
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  		FIGURE 2. The expression pattern of TLR3 in splenocytes isolated from mice at different ages. A, Double-negative control derived from costaining of FITC- and PE-conjugated isotype control Abs. B, Single CD45+ control derived from costaining of FITC-conjugated isotype Ab and anti-CD45-PE. C, Neonatal BALB/c (1 day old). D, Neonatal NOD/SCID. E, Three-week-old female BALB/c. F, Three-week-old female NOD/SCID. G, Ten-week-old virgin BALB/c. H, Ten-week-old virgin NOD/SCID.

 
TLR3 expression in placenta cells from NOD/SCID mice

TLR3 expression was also detectable in CD45+ placenta cells collected at embryonic day 12.5. The basal level expression of TLR3 on the surface of CD45+ placental cells isolated from BALB/c mouse was significantly higher than that from NOD/SCID mice (7.85 ± 0.56% vs 2.12 ± 0.16%, p < 0.01; Table II). The CD45+TLR3+ cell percentage was markedly increased in dsRNA-stimulated BALB/c mice compared with control PBS group. In contrast, lower responsiveness to dsRNA stimulus could be observed in pregnant NOD/SCID mice; the CD45+TLR3+ percentage was not significantly changed upon dsRNA stimulation in these NOD/SCID mice during pregnancy (Table II and Fig. 3).


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  		Table II. Flow cytometric analyses of placenta cells showing percentages of cell populationa

 

Figure 3
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  		FIGURE 3. Cell surface expression of TLR3 in placental cells isolated at day 12.5 of gestation from BALB/c and NOD/SCID mice. A–C, From BALB/c. D–F, From NOD/SCID mice. A and D, Double-negative control. B and E, Control PBS group. C and F, dsRNA-treated group.

 
CD45+CD80+cells in placentas from NOD/SCID mice

In the absence of anti-TLR3 pretreatment, the CD45+CD80+/CD45+ cell percentage could be increased to a level of around 22.5% in BALB/c mice upon dsRNA stimulation, which was notably higher than that of 6.8% in PBS-treated BALB/c ones (p < 0.01). However, in those BALB/c pretreated with neutralizing Ab against TLR3, dsRNA stimulus failed to boost the CD45+CD80+ percentage (Table I and Fig. 4, A–F).


Figure 4
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  		FIGURE 4. The CD45+CD80+ cell percentage in placental CD45+ cells from BALB/c and NOD/SCID mice upon dsRNA stimulation with or without anti-TLR3 pretreatment. Placentas were collected at day 12.5 of gestation. A–C, From BALB/c without anti-TLR3 pretreatment. D–F, From NOD/SCID mice without anti-TLR3 pretreatment. G–I, From BALB/c pretreated with anti-TLR3. J–L, From NOD/SCID pretreated with anti-TLR3. A, D, G, and J, Double-negative control. B, E, H, and K, Control PBS group. C, F, I, and L, dsRNA-stimulated group.

 
No such trends were observed in pregnant NOD/SCID mice; the CD45+CD80+ cell percentage was not significantly altered after dsRNA stimulation. Furthermore, no significant difference of CD45+CD80+ percentage was observed in pregnant NOD/SCID mice stimulated with dsRNA in the presence or absence of TLR3 blocking (Table I and Fig. 4, G–L).

CD8{alpha}+CD80+ cells in placentas from NOD/SCID mice

Upon dsRNA stimulation, the CD8{alpha}+CD80+/CD8{alpha}+ cell percentage was enhanced to around 24% in BALB/c mice, ~4-fold of the level in the control PBS group (Table II and Fig. 5). No statistically supported percentage increase was observed in NOD/SCID mice upon the same stimulation (p = 0.089, Table II and Fig. 5)


Figure 5
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  		FIGURE 5. CD8{alpha}+CD80+cells in placental CD8{alpha}+ cells isolated at day 12.5 of gestation. A–C, From BALB/c. D–F, From NOD/SCID. A and D, Double-negative control. B and E, Control PBS group. C and F, dsRNA-treated group.

 
Intracellular IL-2 expression in placenta CD45+ cells from NOD/SCID mice

Similarly, the intracellular IL-2-positive percentage in CD45+ cell population was increased significantly in BALB/c mice stimulated by dsRNA, from ~14% to 29% (p < 0.01, as shown in Table II and Fig. 6). In contrast, the CD45+IL-2+ cell percentage was unchanged in NOD/SCID mice after dsRNA stimulation (Table II and Fig. 6).


Figure 6
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  		FIGURE 6. Intracellular IL-2 expression in placental CD45+ cells detected at day 12.5 of gestation. A–C, From BALB/c. D–F, From NOD/SCID. A and D, Double-negative control. B and E, Control PBS group. C and F, dsRNA-treated group.

 
Intracellular IL-10 expression in placenta CD45+ cells from NOD/SCID mice

Upon dsRNA stimulation, the intracellular IL-10-positive percentage in CD45+ cell population was decreased both in BALB/c and NOD/SCID mice (p < 0.01, respectively). However, in BALB/c mice, the percentage after stimulation was around 20% (5.20% divided by 27.05%) of the level in control PBS group, while the percentage in dsRNA-treated NOD/SCID mice was around 65% (15.70% divided by 24.02%) of the level in control PBS group (Table II and Fig. 7).


Figure 7
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  		FIGURE 7. Intracellular IL-10 expression in placental CD45+ cells detected at day 12.5 of gestation. A–C, From BALB/c. D–F, From NOD/SCID. A and D, Double-negative control. B and E, Control PBS group. C and F, dsRNA-treated group.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
Disclosures
 References
 
Because mouse is characterized as a multiparous animal, murine abortion or failure of pregnancy tolerance could be evaluated by using RR. Generally, the RR is around 4–5% in normal fertile mice, representing the frequency of chromosome abnormalities (19). If a significantly elevated RR is observed, it means that many healthy embryos (i.e., with normal karyotype) have been rejected or killed, and it indicates the presence of imbalance in pregnancy tolerance.

In our previous observation, the RR at day 13.5 of gestation was ~5.8% in BALB/c x BALB/c mice, similar to the frequency of murine chromosome abnormalities. But such a level could be boosted to 29.0% by dsRNA induction (20). In our current study, the RR detected at day 12.5 (1 day earlier than our previous report) was around 4.8% without stimulation and elevated to 22.0% after dsRNA-administration, consistent with our earlier observation (20). Compared with those detected at day 8.5, the RR of syngeneic mating BALB/c mice was dramatically increased by day 12.5. However, almost no more embryos were resorbed after then in these syngeneic mating BALB/c mice, indicating that dsRNA may mainly induce embryo resorption at an earlier stage of gestation. In NOD/SCID mice, things were quite different. Suffering from multiple immunodeficits, these mice displayed a low RR around the level of 4–5%, and appeared to be resistant to dsRNA-induced embryo rejection throughout pregnancy.

It is not fully understood why dsRNA induces fetal resorption mainly at the earlier stage of pregnancy under conditions given in the present study. The possible reasons may include the following: on one hand, dsRNA injected at an earlier stage of pregnancy may have been eliminated by the mouse within several days and, therefore, no longer affects the fetuses later. On the other hand, elder fetuses are intrinsically less sensitive to dsRNA-induced abortion than younger ones, possibly through a variety of complex mechanisms (21). Although the mechanisms are not clarified, the potential reason is that the recognition and tolerance between mother and fetuses have been established more stably at the later stage of pregnancy. In addition, a previous report indicated that a certain level of Ig could be detected in sera from normal fetus, which has been confirmed to be synthesized by the fetus itself, but not derived from the maternal circulation, and has the specificity against viral and bacterial Ags, and subsequently decreases the risk of abortion (21).

As stated in a previous report, TLR3 responds specifically to dsRNA and initiates production of proinflammatory and antiviral cytokines (7). The effect of TLR3 ligation in endometrial epithelium could be significant because the endometrium is an important site for viral entry and infection. Additionally, the cytokine milieu plays an essential role in normal functions of the endometrium, such as uterine cycle progression, embryo implantation, and placenta development (7). In our present study, strong TLR3 expression was detected in splenocytes derived from both BALB/c and NOD/SCID mice at different ages, ranging from 1 day to 10 wk. The expression pattern of this receptor implies its crucial function in antiviral mechanisms.

However, to our knowledge, the status of TLR3 expression and the significance of TLR3 in the mechanism of pregnancy tolerance have not been examined in NOD/SCID mice. In addition, the significance of TLR3 interaction with dsRNA remains unclear in the mechanisms of poly(I:C)-induced embryo rejection. Some investigators suggest that inflammatory factors may be involved, which are secreted after the activation of macrophages, NK cells, or other cell populations (13). It is believed that cytokine imbalance and immune dysfunction are implicated in problems such as spontaneous abortion and endometriosis, but the trigger of these cytokine imbalances is not known (22, 23). As TLR3 activation impacts cytokine production, the recognition of dsRNA in the endometrial epithelium could be a critical event in endometrial dysfunction (24).

In the present study, TLR3 expression was also found on the surface of placental cells, consistent with previous reports (25). The basal level expression of TLR3 on the surface of CD45+ placenta cells isolated from BALB/c mice was remarkably higher than that from NOD/SCID mice (p < 0.01). Moreover, an obvious elevated CD45+TLR3+ cell percentage was detected in dsRNA-stimulated BALB/c mice compared with control PBS group. In contrast, rather low responsiveness to dsRNA stimulus could be observed in pregnant NOD/SCID mice. The CD45+TLR3+ percentage was not significantly changed upon dsRNA stimulation, concomitant with unchanged RR in the NOD/SCID mice during pregnancy. Such results imply that TLR3 is involved in the immune responses after exposure to dsRNA and has the potential to alter the cytokine milieu and influence the outcome of pregnancy. In contrast, the lower baseline level of TLR3 expression in placenta CD45+ cell population derived from NOD/SCID mice may be one of the reasons why these mice appeared less sensitive to poly(I:C)-induced embryo resorption.

Further supporting this assumption, it was observed in our present study that the increased RR at day 12.5 of gestation was concomitant with the strengthened CD80 expression on both CD45+ cells and CD8{alpha}+ cells isolated from pooled placentas, including decidua basalis. In contrast, no such elevation was detected in NOD/SCID mice. The pattern of CD80 expression in the latter model system displays its relatively low responsiveness to dsRNA stimulus.

What is the relationship between CD45+CD80+ cells and CD8{alpha}+CD80+ cells in pregnancy loss? Because CD45 is more extensively expressed on leukocytes as a leukocyte common Ag than CD8{alpha}, it is reasonable to believe that the CD45+CD80+ cell group is a larger population in which both CD8{alpha}+CD80+ and CD8{alpha}–CD80+ cell subsets are included.

Are these cells bystanders in pregnancy loss, or is the elevated expression of CD80 merely a result secondary to the pregnancy loss? Or instead, is there a causal relationship between the elevated proportion of these cells and embryo resorption? It is more likely that both CD45+CD80+ and CD8{alpha}+CD80+ cell subsets are correlated with the boosted embryo resorption, and it is reasonable to believe that in CD45+CD80+ cell population, CD8+CD80+ cells, but not CD8–CD80+ cells, are more important in murine pregnancy loss. Indeed, merely based on our observation, we cannot conclude that there is a causal relationship between these cell subsets and murine pregnancy loss. However, such a proposal is powerfully supported by previous reports; CD8+, but not CD8–, dendritic cells are probably more important in the activation of CTLs in vivo (26).

A recent report indicates that costimulatory molecules, including CD80, are involved in TLR-mediated cell signaling (27). Dendritic cells undergo a complex program of maturation after the engagement of TLRs with their specific ligands, while Toll-IL-1 receptor homology domain containing adapter molecule 1 functions as a TLR adapter, resulting in strong up-regulation of the surface expression of costimulatory molecules including CD80 (27). This leads to their extraordinary capability to process phagocytosed Ag and initiate a complete and competent immune response by stimulating T cells, resulting in highly efficient T cell activation (10, 28, 29), followed by up-regulated production of proinflammatory cytokines.

Upon dsRNA stimulation, increased expression of CD80 could be observed in our current study, and this effect was abrogated in the presence of previous TLR3 blocking with its neutralizing Ab. Meanwhile, the RR changed corresponding to the alteration of CD80+ cell percentage. Our observation, together with the previous reports mentioned here, may support the assumption that both CD45+CD80+ and CD8{alpha}+CD80+ cell subsets play a role in the course of embryo resorption, while CD8{alpha}+CD80+ cells may belong to a subset of the CD45+CD80+ cell population.

Our present study identified an increased intracellular-positive rate of IL-2 as well as a decreased rate of IL-10 in placenta cells isolated from dsRNA-stimulated BALB/c mice. Accordingly, increased RR could be expected. Because IL-2 is known to be abortifacient, while IL-10 can correct abortion in the abortion-prone CBA x DBA/2 mating combination much more effectively (30), it can be hypothesized that the engagement of dsRNA with TLR3 may cause the activation or mobilization of CD8{alpha}+CD80+ cells, followed by the up-regulation of IL-2 expression and down-regulation of IL-10 expression, and finally result in the rejection of healthy embryos in BALB/c mice.

There are a number of pathways by which inflammatory cytokines are induced or up-regulated (27). After TLR3 blocking, such cytokines can also be up-regulated if stimulated with other adequate stimuli. However, because TLR3 is specific for the recognition of poly(I:C), previous TLR3 blocking will surely abrogate the effect of poly(I:C) as an inducer of proinflammatory cytokines, unless there is still other stimulus not depending on TLR3.

The reason of the relatively low responsiveness of NOD/SCID mice to dsRNA stimulation remains unclear. It may be due to their poor status of T cell and NK cell functions at the feto-maternal interface. This has been partially demonstrated in our previous report which showed that both the percentages of CD45+CD3+ cells in CD45+ splenocytes and CD45+DX5+ cells in CD45+ placental cells were significantly lower than those in BALB/c mice, using DX5 as a common marker of NK cells (31). Although there is apparent T cell deficit, a certain level of CD8{alpha}+ cell percentage has been detected in splenocytes from NOD/SCID mice in a previous report (4), consistent with our current observation in placenta and decidua cells.

Certainly, it is very valuable to investigate TLR3-involved cell signaling in TLR3 gene knockout mice and then compare the results with wild-type ones. Unfortunately, such mice are not available in our laboratory at the present time. Therefore, in our research, TLR3 blocking was performed by multiple injections of mAb against this receptor, considering that such a design is more reliable than single-injection ones.

Were IL-2- and IL-10-positive CD45+ cells also TLR3 positive? This was not investigated in the present study. Further research was restricted mainly by the scale of NOD/SCID mice available in our laboratory. In the future, the identity of CD45+CD80+ and CD8{alpha}+CD80+ cells can be detected with more specific markers for dendritic cells and other cell subsets. Multicolor flow cytometric schemes can be designed, such as costaining of FITC-conjugated TLR3, PE-conjugated CD45, and allophycocyanin-conjugated IL-2 (catalog no. 17-7021-82) or allophycocyanin-conjugated IL-10 (catalog no. 17-7101-82; eBioscience). In addition, the expression pattern of CD80, intracellular IL-2, IL-10, and other cytokines can be identified after TLR3 blocking or CD80 blocking (32), to further confirm the relationship among dsRNA, TLR3, CD80, and local cytokines, aiming at gradually clarifying the detailed mechanisms of poly(I:C)-involved embryo resorption as well as pregnancy tolerance in rodent models and humans.


   Disclosures
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
References
 
The authors have no financial conflict of interest.


   Footnotes
 
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

1 This work was supported by the Postdoctoral Science Foundation of China (2002032244), the Nature and Science Foundation of Guangdong Province (4300213), and the Foundation for Outstanding Newcomers to Jinan University (51204067), China (to Y.L.). Back

2 Address correspondence and reprint requests to Dr. Yi Lin, Key Laboratory of Ministry of Education for Tissue Transplantation and Immunology, College of Life Science and Technology, Jinan University, Guangzhou City, 510632 China. E-mail addresses: yilinonline{at}21cn.com and yilinonline{at}gmail.com Back

3 Abbreviations used in this paper: poly(I:C), polyinosinic-polycytidylic acid; RR, resorption rate of embryo. Back

Received for publication September 13, 2005. Accepted for publication January 19, 2006.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 

  1. Bosma, G. C., R. P. Custer, M. J. Bosma. 1983. A severe combined immunodeficiency mutation in the mouse. Nature 301: 527-530. [Medline]
  2. Dorshkind, K., S. B. Pollack, M. J. Bosma, R. A. Phillips. 1985. Natural killer (NK) cells are present in mice with severe combined immunodeficiency (SCID). J. Immunol. 134: 3798-3801. [Abstract]
  3. Kataoka, S., J. Satoh, H. Fujiya, T. Toyota, R. Suzuki, K. Itoh, K. Kumagai. 1983. Immunologic aspects of the nonobese diabetic (NOD) mouse: abnormalities of cellular immunity. Diabetes 32: 247-253. [Abstract]
  4. Shultz, L. D., P. A. Schweitzer, S. W. Christianson, B. Gott, I. B. Schweitzer, B. Tennent, S. McKenna, L. Mobraaten, T. V. Rajan, D. L. Greiner, E. H. Leiter. 1995. Multiple defects in innate and adaptive immunologic function in NOD/LtSz-scid mice. J. Immunol. 154: 180-191. [Abstract]
  5. Christianson, S. W., D. L. Greiner, R. Hesselton, J. H. Leif, E. J. Wagar, I. B. Schweitzer, T. V. Rajan, B. Gott, D. C. Roopenian, L. D. Shultz. 1997. Enhanced human CD4+ T cell engraftment in beta2-microglobulin-deficient NOD-scid mice. J. Immunol. 158: 3578-3586. [Abstract]
  6. Karpala, A. J., T. J. Doran, A. G. D. Bean. 2005. Immune responses to dsRNA: implications for gene silencing technologies. Immunol. Cell Biol. 83: 211-216. [Medline]
  7. Jorgenson, R. L., S. L. Young, M. J. Lesmeister, T. D. Lyddon, M. L. Misfeldt. 2005. Human endometrial epithelial cells cyclically express Toll-like receptor 3 (TLR3) and exhibit TLR3-dependent responses to dsRNA. Hum. Immunol. 66: 469-482. [Medline]
  8. Akira, S.. 2003. Toll-like receptor signaling. J. Biol. Chem. 278: 38105-38108. [Free Full Text]
  9. Schnare, M., G. M. Barton, A. C. Holt, K. Takeda, S. Akira, R. Medzhitov. 2001. Toll-like receptors control activation of adaptive immune responses. Nat. Immunol. 2: 947-950. [Medline]
  10. Alexopoulou, L., A. C. Holt, R. Medzhitov, R. A. Flavell. 2001. Recognition of double-stranded RNA and activation of NF-{kappa}B by Toll-like receptor 3. Nature 413: 732-738. [Medline]
  11. Vaidya, S. A., G. Cheng. 2003. Toll-like receptors and innate antiviral responses. Curr. Opin. Immunol. 15: 402-407. [Medline]
  12. Doyle, S. E., R. O’Connell, S. A. Vaidya, E. K. Chow, K. Yee, G. Cheng. 2003. Toll-like receptor 3 mediates a more potent antiviral response than Toll-like receptor 4. J. Immunol. 170: 3565-3571. [Abstract/Free Full Text]
  13. Shimada, S., K. Iwabuchi, K. Watano, H. Shimizu, H. Yamada, H. Minakami, K. Onoe. 2003. Expression of allograft inflammatory factor-1 in mouse uterus and poly (I:C)-induced fetal resorption. Am. J. Reprod. Immunol. 50: 104-112.
  14. Lin, Y., Y. Zeng, J. Zhao, S. Zeng, J. Huang, Z. Feng, J. Di, M. Zhan. 2004. Murine CD45+CD86+ cells isolated from para-aortic lymph nodes in an abortion-prone model. J. Reprod. Immunol. 64: 133-143. [Medline]
  15. Clark, D. A., A. Keil, Z. Chen, U. Markert, J. Manuel, R. M. Gorczynski. 2003. Placental trophoblast from successful human pregnancies expresses the tolerance signaling molecule, CD200 (OX-2). Am. J. Reprod. Immunol. 50: 187-195. [Medline]
  16. Zenclussen, A. C., S. B. R. Stumpo, S. Olmos, K. Arias, I. M. Borel, M. E. Roux, R. A. Margni. 2003. Murine abortion is associated with enhanced interleukin-6 levels at the feto-maternal interface. Cytokine 24: 150-160. [Medline]
  17. Maino, V. C., L. J. Picker. 1998. Identification of functional subsets by flow cytometry: intracellular detection of cytokine expression. Cytometry 34: 207-215. [Medline]
  18. Arck, P. C., D. A. Ferrick, D. Steele-Norwood, P. J. Egan, K. Croitoru, S. R. Carding, J. Dietl, D. A. Clark. 1999. Murine T cell determination of pregnancy outcome. II. Distinct Th1 and Th2/3 populations of V{gamma}1+{delta}6+ T cells influence success and failure of pregnancy in CBA/J x DBA/2J matings. Cell. Immunol. 196: 71-79. [Medline]
  19. Clark, D. A., G. Chaouat, P. C. Arck, H. W. Mittruecker, G. A. Levy. 1998. Cytokine-dependent abortion in CBA x DBA/2 mice is mediated by the procoagulant fgl2 prothrombinase. J. Immunol. 160: 545-549. [Abstract/Free Full Text]
  20. Lin, Y., Y. Zeng, J. Di, S. Zeng. 2005. Murine CD200+CK7+ trophoblasts in a poly (I:C)-induced embryo resorption model. Reproduction 130: 529-537. [Abstract/Free Full Text]
  21. Adinolfi, M.. 1997. Ontogeny of human natural and acquired immunity. L. B. Olding, ed. Reproductive Immunology 1st Ed.76-86. Springer-Verlag, Berlin.
  22. von Wolff, M., C. J. Thaler, T. Strowitzki, J. Broome, W. Stolz, S. Tabibzadeh. 2000. Regulated expression of cytokines in human endometrium throughout the menstrual cycle: dysregulation in habitual abortion. Mol. Hum. Reprod. 6: 627-634. [Abstract/Free Full Text]
  23. Arici, A.. 2002. Local cytokines in endometrial tissue: the role of interleukin-8 in the pathogenesis of endometriosis. Ann. NY Acad. Sci. 955: 101-109. [Medline]
  24. Quayle, A. J.. 2002. The innate and early immune response to pathogen challenge in the female genital tract and the pivotal role of epithelial cells. J. Reprod. Immunol. 57: 61-79. [Medline]
  25. Klaffenbach, D., W. Rascher, M. Rollinghoff, J. Dotsch, U. Meissner, M. Schnare. 2005. Regulation and signal transduction of toll-like receptors in human chorioncarcinoma cell lines. Am. J. Reprod. Immunol. 53: 77-84.
  26. den Haan, J. M., S. M. Lehar, M. J. Bevan. 2000. CD8+ but not CD8– dendritic cells cross-prime cytotoxic T cells in vivo. J. Exp. Med. 192: 1685-1696. [Abstract/Free Full Text]
  27. Seya, T., H. Oshiumi, M. Sasai, T. Akazawa, M. Matsumoto. 2005. TICAM-1 and TICAM-2: Toll-like receptor adapters that participate in induction of type 1 interferons. Int. J. Biochem. Cell Biol. 37: 524-529. [Medline]
  28. Banchereau, J., R. M. Steinman. 1998. Dendritic cells and the control of immunity. Nature 392: 245-252. [Medline]
  29. Schnare, M., A. C. Holt, K. Takeda, S. Akira, R. Medzhitov. 2000. Recognition of CpG DNA is mediated by signaling pathways dependent on the adaptor protein MyD88. Curr. Biol. 10: 1139-1142. [Medline]
  30. Chaouat, G., N. Ledee-Bataille, S. Dubanchet, S. Zourbas, O. Sandra, J. Martel. 2004. Reproductive immunology 2003: reassessing the Th1/Th2 paradigm?. Immunol. Lett. 92: 207-214. [Medline]
  31. Lin, Y., Y. Chen, Y. Zeng, T. Wang, S. Zeng. 2005. Lymphocyte phenotyping and NK cell activity analysis in pregnant NOD/SCID mice. J. Reprod. Immunol. 68: 39-51. [Medline]
  32. Judge, T. A., Z. Wu, X. G. Zheng, A. H. Sharpe, M. H. Sayegh, L. A. Turka. 1999. The role of CD80, CD86, and CTLA4 in alloimmune responses and the induction of long-term allograft survival. J. Immunol. 162: 1947-1951. [Abstract/Free Full Text]

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