Role of invariant natural killer T cells in lipopolysaccharide-induced pregnancy loss

Cellular Immunology 286 (2013) 1–10

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Role of invariant natural killer T cells in lipopolysaccharide-induced pregnancy loss Liping Li a, Lijuan Shi b, Xinying Yang b, Lihua Ren a, Jing Yang a, Yi Lin c,⇑ a

Department of Obstetrics and Gynecology, Guangzhou Medical University Affiliated Guangzhou First People’s Hospital, Guangzhou 510180, China Department of Obstetrics and Gynecology, Renji Hospital, School of Medicine, Shanghai Jiaotong University, Shanghai 200001, China c Bio-X Institutes, Shanghai Jiao Tong University, Shanghai 200240, China b

a r t i c l e

i n f o

Article history: Received 30 October 2012 Accepted 25 October 2013 Available online 6 November 2013 Keywords: Inflammation Invariant natural killer T cells Lipopolysaccharide Pregnancy loss Toll-like receptor

a b s t r a c t We aimed to investigate the role of invariant natural killer T (iNKT) cells in infection-associated pregnancy loss. Wild-type (WT) C57BL/6 mice and iNKT cell-deficient Ja18 / mice were treated with lipopolysaccharide (LPS). Embryo resorption rates (ERRs), decidual costimulatory molecule and activation molecule expression, and cytokine production were determined. WT and Ja18 / mice were adoptively transferred with purified iNKT cells. ERRs, decidual costimulatory molecule and activation molecule expression, and cytokine production were assessed. LPS-treated Ja18 / mice showed markedly reduced ERRs, decreased CD40, CD80, CD86, and CD69 expression, and reduced Th1 cytokine production at the maternal-fetal interface compared with WT mice. ERRs, expression of CD40, CD80, CD86, and CD69, and Th1 cytokine production in LPS-injected Ja18 / mice following iNKT cell adoptive transfer were remarkably upregulated compared with control mice that did not receive adoptively transferred iNKT cells. Our results suggest that iNKT cells play an important role in LPS-induced pregnancy loss. Ó 2013 Elsevier Inc. All rights reserved.

1. Introduction Many factors are thought to be involved in recurrent spontaneous abortion. Besides chromosomal and structural abnormalities, inflammation processes are an important trigger of miscarriage [1,2]. The overwhelming majority of mid-trimester pregnancy loss cases are associated with ascending infection from the lower genital tract [3,4], such as chorioamnionitis is a common cause of second trimester pregnancy loss [4]. An efficient defense against invading pathogenic microorganisms is achieved through coordination of a complex network of both innate and acquired immune responses. The first step in the elimination of a pathogenic bacteria or viruses is reliable detection. Toll-like receptors (TLRs) are the major class of signaling receptor, which recognize conserved structures of microbes called pathogen-associated molecular patterns (PAMPs) in bacteria, viruses, fungi, and parasites [5,6]. TLRs are present not only on uterine leukocytes, but also on trophoblast cells, thus implying active crosstalk between the placenta and local immunity [7,8]. TLRs play an essential role in immune recognition, cell signal transduction, upstream events of immune response cascades, and inflammation-induced pregnancy loss [9,10]. Despite a growing association between TLRs and inflammation-induced pregnancy loss, the ⇑ Corresponding author. Fax: +86 21 50390610. E-mail addresses: [email protected], [email protected] (Y. Lin). 0008-8749/$ - see front matter Ó 2013 Elsevier Inc. All rights reserved. http://dx.doi.org/10.1016/j.cellimm.2013.10.007

precise mechanisms of TLR-mediated inflammation at the maternal-fetal interface are largely unknown. Natural killer T (NKT) cells constitute a highly conserved T lymphocyte subpopulation that was originally identified as an unusual T cell population co-expressing T cell receptors (TCRs) and some receptors characteristic of NK cells. Most NKT cells express an invariant TCR a chain (Va14-Ja18 in mice and Va24-Ja18 in humans), and are referred to as invariant NKT (iNKT) cells or type I NKT cells [11]. In contrast with conventional T cells, which recognize peptide antigens presented by major histocompatibility complex (MHC) class I or II proteins, iNKT cells recognize glycolipid antigens presented by the MHC class I-like antigen presenting molecule, CD1d [12]. The hallmark of iNKT cell activation is the rapid and robust secretion of a variety of cytokines immediately following TCR engagement [13,14]. iNKT cells directly recognize a-linked glycosphingolipids and diacylglycerol antigens that are expressed by bacteria such as Sphingomonas, Ehrlichia, and Borrelia burgdorferi in a CD1d-dependent manner [15–18]. Furthermore, LPS-positive bacteria (such as Salmonella and Escherichia coli) activate TLR4-expressing antigen presenting cells (APCs) through LPS and can indirectly induce iNKT cell activation through presentation of self-glycolipids in conjunction with IL-12 co-stimulation, or by an IL-12/IL-18-dependent, CD1d-independent mechanism [19,20]. These results suggest that a vast array of microorganisms might be able to induce iNKT cell activation indirectly through APC

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stimulation. Therefore, iNKT cells at the maternal-fetal interface may be activated by self-lipid antigens in conjunction with soluble factors from dendritic cells (DCs) upon TLR signaling, and play a role in inflammation-induced pregnancy loss. Conventional T cells do not express the invariant Va14-Ja18 antigen receptor [21], indicating their selective use in Va14 NKT cells. Thus, disruption of the invariant Va14-Ja18 receptor results in the selective loss of Va14 NKT cells, leaving other types of lymphoid cells, including T, B, and NK cells, intact [22,23]. Ja18 / mice, which are fertile and healthy in appearance [24], have no changes in the total lymphocyte populations compared with wild-type (WT) mice, with the exception of a complete loss of the Va14 NKT cell population [22,23,25]. The potential involvement of iNKT cells in the pathogenesis of infection-associated pregnancy loss remains to be determined. In our present study, we used an adoptive transfer system in Ja18 / mice to elucidate the role that iNKT cells may play in LPS-induced pregnancy loss. 2. Materials and methods 2.1. Animals Eight-week-old female and male WT C57BL/6 (B6) mice and iNKT cell-deficient Ja18 / mice on a C57BL/6 background were purchased from The Jackson Laboratory (Bar Harbor, Maine) and subsequently maintained under pathogen-free conditions in the Laboratory Animal Facility of Guangzhou Medical College. Animals were acclimated in our facility for at least 2 weeks before use in these experiments. All animal procedures followed the guidelines of the Chinese Council for Animal Care. Syngeneic B6  B6 and Ja18 /  Ja18 / mating combinations were established. Each female mouse was co-caged with one male. The point at which a vaginal plug was detected was designated as day 0 gestation.

When single or clumped cells were observed under a microscope, the released cells were separated from undigested tissue pieces by filtering through a 50 lm-pore nylon mesh. Mononuclear cells were purified with Ficoll-Hypaque density medium (density, 1.077 ± 0.002 g/ml) by centrifugation at 800  g for 20 min at 22 °C. Any contaminating red blood cells in decidual single-cell suspensions were eliminated by incubation with red cell lysis buffer (GenMed, Arlington, MA, USA) [25]. 2.4. Flow cytometry analysis Cells (106) in 50 ll PBS were incubated with PE/Cy5-conjugated CD45 (0.25 lg), PE-conjugated CD11c (0.25 lg), FITC-conjugated CD3 (0.5 lg), allophycocyanin/Cy7-conjugated CD19 (0.5 lg), allophycocyanin-conjugated CD49b (0.5 lg), and PE/Cy7-conjugated CD69 (0.25 lg) for 30 min at 4 °C. In some cases, cells (106) in 50 ll PBS were incubated with PE/Cy5-conjugated CD45 (0.25 lg), PE-conjugated CD11c (0.25 lg), FITC-conjugated CD40 (0.5 lg), allophycocyanin-conjugated CD80 (0.5 lg), and PE/Cy7conjugated CD86 (0.5 lg) for 30 min at 4 °C. After washing twice with PBS, cells were fixed in 10 g/l paraformaldehyde. Immunostained cells were analyzed on a FACSCanto flow cytometer using FACSDiva software (BD Biosciences, San Jose, CA, USA). Ten thousand cells were detected in each sample. Isotype controls for non-specific staining were established using matched fluorescence-labeled isotype control Abs. All of the fluorescence-labeled Abs and isotype controls were purchased from BioLegend (San Diego, CA, USA). CD45+ cells were gated. The percentages of CD11c+CD69+ cells in the CD11c+ cell population, CD3+CD69+ cells in the CD3+ cell population, CD19+CD69+ cells in the CD19+ cell population, and CD49b+CD69+ cells in the CD49b+ cell population were calculated. In other cases, the percentages of CD11c+CD40+ cells in the CD11c+ cell population, CD11c+CD80+ cells in the CD11c+ cell population, and CD11c+CD86+ cells in the CD11c+ cell population were calculated in the same way.

2.2. LPS-induced abortion model The average gestation period for mice ranged from 19 to 21 days. To induce embryo loss, on day 9 gestation, pregnant Ja18 / mice or B6 mice were injected intraperitoneally (i.p.) with 200 ll LPS (Sigma, Saint Louis, MO, USA) saline solution (50 lg/kg body weight) taking care to ensure that none of the solution entered the amniotic cavity. Pregnant WT mice and Ja18 / mice injected i.p. with 200 ll PBS served as negative controls. Ten female mice were used in each group. Animals were observed frequently (at least four times every day) for any signs of morbidity (piloerection, decreased movement, and vaginal bleeding). Pregnant mice were sacrificed on gestational day 12. At this stage of gestation, feto-placental units undergoing resorption can be clearly distinguished from their viable counterparts on the basis of size and the presence of extensive tissue wasting and hemorrhage. The number of implantation sites together with the number of resorbed and viable embryos per mouse was counted. The embryo resorption rate (ERR) was calculated as: ERR (%) = number of resorbed embryos/number of total embryos  100 [23,26]. 2.3. Isolation of decidual mononuclear cells For decidual tissue harvest, the uterine horns of pregnant mice were opened longitudinally, and the whole placental and decidual units were separated individually from the respective embryo and its implantation site. After washing with cold PBS, pooled decidual tissues were cut into small pieces (1 mm3). Selected tissue was digested three times with 1 mg/ml Dispase II (Roche, Mannheim, Germany) at 37 °C for 20 min in each cycle in a shaking water bath.

2.5. Cytokine assays Decidual mononuclear cells were cultured in a 5% CO2 atmosphere. After 24 h, culture supernatants were collected and stored at 80 °C for cytokine assays. The levels of mouse cytokines, including IFN-c, IL-12p70, IL-4, and IL-10 were assessed using commercially available ELISA kits from BD Pharmingen (San Diego, CA, USA). All assays were conducted according to the manufacturer’s instructions. For intracellular cytokine analysis, isolated decidual mononuclear cells (106 cells/ml) from PBS or LPS-treated WT mice were added with 50 ng/ml phorbol myristate acetate (PMA) (Sigma, Saint Louis, MO, USA), 500 ng/ml ionomycin (Sigma, Saint Louis, MO, USA) and 5 lg/ml brefeldin A (Sigma, Saint Louis, MO, USA), and cultured at 37 °C in 5% CO2 for 4 h. Stimulated cells were incubated with PE-Cy7-conjugated CD11c (0.25 lg), FITC-conjugated CD3 (0.5 lg), allophycocyanin-Cy7-conjugated CD49b (1.0 lg) and PE-Cy5-conjugated F4/80 (1.0 lg) for 30 min at 4 °C in PBS containing 2% fetal calf serum and 0.01% NaN3. After washing twice with PBS, cells were fixed in 10 g/l paraformaldehyde for 10 min. Cells were resuspended in PBS plus 2% fetal calf serum and 0.1% saponin (permeabilization buffer) and incubated with PE-conjugated anti-IL-12 (1.0 lg) and allophycocyanin-conjugated antiIFN-c (1.0 lg) in permeabilization buffer. CD11c+, CD3+, CD49b+, and F4/80+ cells were gated and screened for intracellular IL-12 and IFN-c production. Isotype controls were established using matched fluorescence-labeled isotype control Abs to avoid nonspecific staining. All of the fluorescence-labeled Abs and isotype controls were purchased from BioLegend (San Diego, CA, USA).

L. Li et al. / Cellular Immunology 286 (2013) 1–10

2.6. Isolation of spleen iNKT cells Single-cell suspensions were prepared from spleens using a cell strainer followed by RBC lysis. iNKT cells were isolated from female B6 mouse spleens by labeling with PE-conjugated a-galactosylceramide (a-GalCer)-loaded CD1d tetramer. The tetrameric CD1d/aGalCer complexes bind to the TCR of iNKT cells. iNKT cells were sorted with anti-PE-MicroBeads, according to the manufacturer’s protocol (Miltenyi Biotec, Teterow, Germany). Because of unspecific binding of the tetramer to B cells, B cells were depleted prior to iNKT cell enrichment by using B220 MicroBeads. In brief, single-cell suspension was prepared. Dead cells were removed by using the Dead Cell Removal Kit. The splenic single-cell suspension was passed through a 30 lm nylon mesh to remove cell clumps which may clog the column. The cells were labeled with B220 MicroBeads and passed through a LD column and an autoMACS™ Separator. The unlabeled B cell-depleted fraction was collected. FcR Blocking Reagent was added to prevent Fc receptor-mediated antibody labeling. The B cell-depleted fraction was labeled with PE-conjugated a-GalCer-loaded CD1d tetramer, washed and tetramer+ iNKT cells enriched by using anti-PE-Microbeads, two MS Columns and an autoMACS™ Separator. The percentage of iNKT cells in the enriched fraction was determined by labeling with FITC-conjugated CD3e and PE-conjugated a-GalCer/CD1d tetramer. The purity of sorted iNKT cells routinely exceeded 97%, as determined by flow cytometry [27].

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on d 9 gestation, and the rates of embryo resorption were analyzed. As shown in Table 1, there were no significant differences in the mean implantation sites per mouse for WT and Ja18 / mice treated with PBS or LPS. However, the number of viable embryos per mouse for WT mice treated with LPS was lower than that for PBS-treated WT or Ja18 / mice. Moreover, ERRs in WT mice treated with LPS were significantly higher than in WT or Ja18 / mice treated with PBS. In contrast, ERRs in Ja18 / mice treated with LPS were markedly lower than in WT mice, with no difference observed in ERRs in WT or Ja18 / mice injected with PBS. 3.2. Comparison of CD40, CD80, and CD86 expression by decidual DCs To explore the mechanism underlying the decreased ERR in iNKT cell-deficient Ja18 / mice, we examined the expression levels of costimulatory molecules CD40, CD80, and CD86 on decidual DCs from WT and Ja18 / mice. No significant differences were detected in expression levels of these markers on decidual DCs in PBS-treated WT and Ja18 / mice. However, expression levels of these markers on decidual DCs from LPS-injected WT mice were notably higher than those from PBS-injected WT or Ja18 / mice. The expression levels of CD40, CD80, and CD86 on decidual DCs from LPS-treated Ja18 / mice were remarkably reduced in comparison with those from LPS-treated WT mice (Fig. 1). 3.3. Comparison of CD69 expression by decidual DCs, T, B, and NK cells

2.7. Adoptive transfer of pure iNKT cells Va14 NKT cells accumulate in the liver, spleen, and bone marrow and reach steady-state levels by approximately 3 weeks of age [28]. BrdUrd incorporation studies have shown that peripheral Va14 NKT cells are a slowly dividing population [28]. However, the NKT cell pool is labile because of NKT cell redistribution, differentiation, and homeostatic expansion to allow the maintenance of peripheral NKT cell homeostasis [29]. In this study, we used an adoptive transfer system to assess the role of iNKT cells in infection-associated pregnancy loss. For iNKT cell reconstitution of Ja18 / mice, 2  105 purified iNKT cells or PBS were injected intravenously (i.v.) into the tail vein of Ja18 / mice 3 d prior to LPS or PBS injection. Pregnant mice were sacrificed on d 12 gestation. ERRs were calculated, decidual mononuclear cells were isolated and cultured for 24 h, and the levels of IFN-c, IL-12p70, IL-4, and IL-10 in the culture supernatants were assayed using commercially available ELISA kits from BD Pharmingen (San Diego, CA, USA) according to the manufacturer’s instructions. Costimulatory molecule expression on decidual DCs and decidual immune cell activation were also examined by flow cytometry. 2.8. Statistical analysis All statistical analyses were performed using SPSS 19.0 software. Data were determined by one-way ANOVA with Bonferroni test or independent-samples t test. Ten female mice were used in each group in induced pregnancy loss experiments. In molecule and cytokine expression assays, experiments were repeated independently eight times. Results were presented as means ± SD. A P value of <0.05 was considered to indicate statistical significance between analyzed groups.

To further examine the possible role of iNKT cells on decidual immune cell activation, we analyzed expression of the early activation marker CD69 on decidual DCs, T, B, and NK cells identified by expression of the relevant surface markers CD11c, CD3, CD19, and CD49b, respectively. There were no striking differences in CD69 expression by decidual DCs, T cells, and NK cells from PBS-treated WT mice and Ja18 / mice. The expression levels of CD69 on decidual DCs, T cells, and NK cells were markedly upregulated from LPSinjected WT mice compared with PBS-injected WT mice or Ja18 / mice. CD69 expression levels on decidual DCs, T cells, and NK cells were notably decreased from LPS-treated Ja18 / mice in comparison with LPS-treated WT mice. However, there were no significant differences in the expression levels of CD69 on decidual B cells among PBS-treated or LPS-treated WT and Ja18 / mice (Fig. 2). 3.4. Comparison of cytokine levels in the culture supernatants of decidual mononuclear cells To investigate the functions of iNKT cells in inflammation-induced embryo resorption, the levels of Th1 and Th2 cytokines in the culture supernatants of decidual mononuclear cells from WT mice and Ja18 / mice were measured. There were no significant differences in the concentrations of IFN-c, IL-12p70, IL-4, and IL10 between PBS-treated WT and Ja18 / mice. However, the concentrations of the Th1 cytokines IFN-c and IL-12p70 in the culture supernatants of decidual mononuclear cells from LPS-injected WT mice were apparently upregulated in comparison with those from PBS-treated WT or Ja18 / mice. The concentrations of IFN-c and IL-12p70 in the culture supernatants from LPS-treated Ja18 / mice were markedly reduced compared with LPS-treated WT mice. In contrast, the levels of IL-4 and IL-10 in the culture supernatants showed no marked differences among PBS-treated or LPS-treated WT and Ja18 / mice (Fig. 3).

3. Results 3.1. Comparison of ERRs between WT and Ja18

/

mice

To elucidate the intrinsic role of iNKT cells in LPS-induced pregnancy loss, WT, and Ja18 / mice were injected i.p. with LPS or PBS

3.5. Comparison of intracellular cytokine levels of decidual lymphocyte subsets To further explore which decidual lymphocyte subsets increased the production of the Th1 cytokines after LPS treatment.

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Table 1 Comparison of embryo resorption rates (ERRs) between wild-type and Ja18

a b c d e

Group

Mice

A B C D

WT Ja18 WT Ja18

P < 0.05 P < 0.05 P < 0.01 P < 0.01 P < 0.01

versus versus versus versus versus

group group group group group

/

/

/

mice.

Stimulator

N

Implantation sites per mouse

Viable embryos per mouse

ERR (%)

PBS PBS LPS LPS

10 10 10 10

8.4 ± 1.1 8.6 ± 1.1 8.4 ± 1.3 8.2 ± 1.3

7.2 ± 1.2 7.8 ± 1.4 6.1 ± 1.0a,b 7.3 ± 1.3

8.4 ± 5.9 9.4 ± 5.1 25.8 ± 6.7c,d,e 11.1 ± 6.5

A. B. A. B. D.

Fig. 1. Comparison of CD40, CD80, and CD86 expression on decidual DCs. Decidual mononuclear cells were incubated with anti-CD45, anti-CD11c, anti-CD40, anti-CD80 and anti-CD86 mAbs and isotype controls as indicated. CD45+ cells were gated and the percentages of decidual CD40+ cells in the CD11c+ cell population (A), CD80+ cells in the CD11c+ cell population (B), and CD86+ cells in the CD11c+ cell population (C) were calculated. Data represent means ± SD (n = 8 per group).

Fig. 2. Comparison of CD69 expression on decidual DC, T, B, and NK cell populations. Decidual CD45+ cells were gated, and the percentages of CD11c+CD69+ cells in the CD11c+ cell population (A), CD3+CD69+ cells in the CD3+ cell population (B), CD19+CD69+ cells in the CD19+ cell population (C), and CD49b+CD69+ cells in the CD49b+ cell population (D) were calculated. Values represent means ± SD (n = 8 per group).

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Fig. 3. Comparison of cytokine levels in the culture supernatants of decidual mononuclear cells. Decidual mononuclear cells were isolated and cultured as described in the methods. Concentrations of IFN-c (A), IL-12p70 (B), IL-4 (C) and IL-10 (D) in the culture supernatants were determined by ELISA. Results are shown as means ± SD (n = 8 per group).

Fig. 4. Comparison of intracellular cytokine levels in the decidual lymphocyte subsets. Decidual mononuclear cells from PBS and LPS-treated mice were isolated and cultured as described in the methods. Frequencies of IL-12 (A and B) and IFN-c (C and D) in the decidual DCs, T cells, NK cells, and macrophages were determined by flow cytometry. Data are shown as representative flow cytometry outcomes (%) (A and C) and means ± SD (n = 8 per group) (B and D). PBS, PBS-treated WT mice. LPS, LPS-injected WT mice.

The levels of intracellular IL-12 and IFN-c of decidual DCs, T cells, NK cells, and macrophages from PBS or LPS-treated WT mice were analyzed. As shown in Fig. 4A and B, the intracellular levels of IL-12 in decidual DCs and macrophages from LPS-treated WT mice were significantly increased in comparison with PBS-treated WT mice. In addition, the levles of intracellular IFN-c in decidual T and NK cells from LPS-treated WT mice were markedly elevated in comparison with PBS-treated WT mice (Fig. 4C and D).

3.6. ERRs in Ja18

/

mice after adoptive transfer of iNKT cells

To better understand the mechanisms by which iNKT cells affect infection-associated pregnancy loss, in vitro isolated iNKT cells were adoptively transferred to iNKT cell-deficient Ja18 / mice, and pregnancy outcome, costimulatory molecule expression, immune cell activation, and cytokine levels at the maternal-fetal interface were analyzed.iNKT cells were isolated from mouse

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spleen using a PE-conjugated CD1d tetramer loaded with a-GalCer and anti-PE Microbeads after depletion of B cells using B220 MicroBeads. The purity of isolated iNKT cells routinely exceeded 97%, as determined by flow cytometry (Fig. 5). As shown in Table 2, the implantation sites per mouse for Ja18 / mice treated with PBS or LPS were not significantly different with or without adoptive transfer of iNKT cells. However, the

number of viable embryos per mouse for LPS-treated Ja18 / mice in the presence of adoptively transferred iNKT cell was reduced in comparison with LPS-treated Ja18 / mice in the absence of the adoptive transfer, and PBS-treated Ja18 / mice with or without iNKT cell adoptive transfer, although this reduction did not reach the level of statistical significance. Moreover, ERRs for LPS-treated Ja18 / mice following iNKT cell adoptive transfer significantly in-

Fig. 5. Comparison of the frequencies of iNKT cells with or without purification. Spleen mononuclear cells were incubated with fluorescence-conjugated anti-CD3e, antiCD1d/a-GalCer, and isotype control Abs. The percentages of CD1d/a-GalCer+ cells in the CD3e+ cell population were calculated. (A) Spleen iNKT cells without purification. (B) Spleen iNKT cells with purification. (C) Double negative control. Data are the mean values of eight mice per group. Numbers in the upper right corner of each group represent the frequencies of iNKT cells. Table 2 Comparison of embryo resorption rates (ERRs) in Ja18

a b c

Group

Mice

E F G H

Ja18 Ja18 Ja18 Ja18

/ / / /

PBS PBS LPS LPS

/

mice with or without adoptive transfer of iNKT cells.

Adoptive transfer

N

Implantation sites per mouse

Viable embryos per mouse

ERR (%)

No Yes No Yes

10 10 10 10

8.1 ± 1.4 8.0 ± 1.2 8.2 ± 1.3 8.4 ± 1.4

7.2 ± 1.2 7.1 ± 0.9 7.2 ± 1.3 6.4 ± 0.8

11.0 ± 6.5 10.8 ± 6.2 11.3 ± 4.5 22.8 ± 7.1a,b,c

P < 0.01 versus group E. P < 0.01 versus group F. P < 0.01 versus group G.

Fig. 6. Comparison of CD40, CD80, and CD86 expression on decidual DCs in Ja18 / mice with or without adoptive transfer of iNKT cells. Ja18 / mice were transferred with or without purified iNKT cells as indicated in Section 2. Expression of CD40 (A), CD80 (B), and CD86 (C) on decidual CD11c+ cells was calculated. Results are shown as means ± SD (n = 8 per group). PBS, PBS-injected Ja18 / mice without iNKT cell adoptive transfer. PBS + iNKT, PBS-injected Ja18 / mice with iNKT cell adoptive transfer. LPS, LPS-injected Ja18 / mice without iNKT cell adoptive transfer. LPS + iNKT, LPS-injected Ja18 / mice with iNKT cell adoptive transfer.

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creased compared with that for LPS-treated Ja18 / mice in the absence of the adoptive transfer, and for PBS-treated Ja18 / mice with or without adoptive transfer of iNKT cells. 3.7. Costimulatory molecule expression on decidual DCs in Ja18 mice after adoptive transfer of iNKT cells

/

CD40, CD80, and CD86 expression levels on decidual DCs from LPS-treated Ja18 / mice following iNKT cell adoptive transfer

significantly increased compared with LPS-treated Ja18 / mice in the absence of the adoptive transfer, and PBS-treated Ja18 / mice with or without the adoptive transfer of iNKT cells (Fig. 6). 3.8. Decidual immune cell activation in Ja18 transfer of iNKT cells

/

mice after adoptive

As shown in Fig. 7, the expression levels of CD69 on decidual DCs, T cells, and NK cells were remarkably upregulated from LPS-

Fig. 7. Comparison of decidual immune cell activation in Ja18 / mice with or without adoptive transfer of iNKT cells. Ja18 / mice were transferred with or without isolated iNKT cells as described in Section 2. CD69 expression on the decidual CD11c+ cell population (A), CD3+ cell population (B), CD19+ cell population (C), and CD49b+ cell population (D) was measured. Results are shown as means ± SD (n = 8 per group). PBS, PBS-injected Ja18 / mice without iNKT cell adoptive transfer. PBS + iNKT, PBS-injected Ja18 / mice with iNKT cell adoptive transfer. LPS, LPS-injected Ja18 / mice without iNKT cell adoptive transfer. LPS + iNKT, LPS-injected Ja18 / mice with iNKT cell adoptive transfer.

Fig. 8. Comparison of cytokine levels in the culture supernatants of decidual mononuclear cells in Ja18 / mice with or without adoptive transfer of iNKT cells. Ja18 / mice were transferred with or without iNKT cells as detailed in Section 2. The concentrations of IFN-c (A), IL-12p70 (B), IL-4 (C) and IL-10 (D) in the culture supernatants of decidual mononuclear cells were determined by ELISA. Results are shown as means ± SD (n = 8 per group). PBS, PBS-injected Ja18 / mice without iNKT cell adoptive transfer. PBS + iNKT, PBS-injected Ja18 / mice with iNKT cell adoptive transfer. LPS, LPS-injected Ja18 / mice without iNKT cell adoptive transfer. LPS + iNKT, LPS-injected Ja18 / mice with iNKT cell adoptive transfer.

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treated Ja18 / mice following iNKT cell adoptive transfer in comparison with LPS-treated Ja18 / mice in the absence of the adoptive transfer, and PBS-treated Ja18 / mice with or without adoptive transfer of iNKT cells. However, there were no significant differences in the expression levels of CD69 on decidual B cells among PBS-treated or LPS-treated Ja18 / mice with or without iNKT cell adoptive transfer. 3.9. Cytokine levels of decidual mononuclear cells in Ja18 adoptive transfer of iNKT cells

/

mice after

The concentrations of the Th1 cytokines IFN-c and IL-12p70 in the culture supernatants of decidual mononuclear cells from LPSinjected Ja18 / mice following iNKT cell adoptive transfer were remarkably higher than those from LPS-injected Ja18 / mice in the absence of the adoptive transfer, and PBS-injected Ja18 / mice with or without iNKT cell adoptive transfer. In contrast, no notable differences were observed in the concentrations of the Th2 cytokines IL-4 and IL-10 among PBS-treated or LPS-treated Ja18 / mice with or without iNKT cell adoptive transfer (Fig. 8). 4. Discussion The presence of iNKT cells in both the mouse and the human decidua [23,26], and the expression of CD1d on both the villous and extravillous trophblasts [30,31], indicates that iNKT cells have an immunoregulatory role at the maternal-fetal interface. Moreover, murine iNKT cells accumulate in the decidua during pregnancy and provoke abortion upon stimulation with a-GalCer, a specific ligand for Va14 NKT cells by perforin-dependent killing and the secretion of TNF-a and IFN-c [23]. Similarly, it has been demonstrated that a-GalCer efficiently induces pregnancy loss during early pregnancy in a perforin-dependent manner, and pregnancy loss during mid-gestation by a perforinindependent, cytokine-dependent mechanism [26]. Therefore, we hypothesize that decidual iNKT cells are activated through indirect recognition of pathogen-related molecules such as bacterial LPS upon TLR stimulation at the maternal-fetal interface [19,20], subsequently producing massive amounts of cytokines that play a role in infection-associated pregnancy loss. In this study, we investigated the effects of iNKT cell deficiency on LPS-induced pregnancy loss. Ito et al. [23] and Boyson et al. [26] investigated the role of iNKT cells in miscarriage by using a-GalCer, the exogenous marine sponge glycosphingolipid that does not exist in vertebrates. In our current study, LPS was used to establish a murine model mimicking infection-associated pregnancy loss. LPS is an important component of the cell wall of Gram-negative bacteria that are commonly present in the normal vaginal flora of pregnant women. Pregnant mice were administered LPS (50 lg/kg body weight) on gestational d 9 with the aim of establishing a mid-trimester infection-associated pregnancy loss model [3,4]. This dose of LPS was chosen because it resulted in an elevated rate of embryo resorption comparable to that in the CBA/J  DBA/2 abortion-prone mouse model [32], with no maternal mortality or obvious morbidity. In this study, we first showed that the number of viable embryos per mouse was significantly increased and ERRs remarkably decreased for LPS-treated Ja18 / mice compared with LPS-treated WT mice. These data indicate that depletion of iNKT cells downregulated LPS-induced embryo resorption.iNKT cell adoptive transfer experiments were conducted using 2  105 cells based on the observation that approximately 105 NKT cells promote rejection of OVA-expressing skin grafts [33], and reduce the bacterial burden in the liver and spleen of the hosts after enteric L. monocytogenes infection [34]. The iNKT cells were adoptively transferred 3 d prior

to LPS injection based on the observation that adoptive transfer results in homeostatic expansion of Va14+ NKT cells, and approximately 90% of cells divide in the recipient mice by 3 d after transfer [29]. Uterine DCs are crucial for decidua formation during embryo implantation [35]. Nevertheless, these cells possess the capacity to contribute to proinflammatory responses upon pathogenic activation. Driven by pathogens and inflammatory signals, DCs undergo a complex maturation process, which not only leads to enhanced expression of costimulatory molecules and increased formation of stable MHC/peptide complexes but also to secretion of cytokines that modulate T cell activation and expansion, as well as synthesis of chemokines and chemokine receptors, and regulation of T cell and DC trafficking [36]. In the present study, we demonstrated that iNKT cells deficiency reduced the expression of the costimulatory molecules CD40, CD80, and CD86 by decidual DCs and the number of activated decidual DCs, suggesting that iNKT cells have an impact on infection-associated decidual DC maturation and activation. Productive activation of T cells occurs after concomitant engagement of the TCR with antigen presented on DCs in association with MHC molecules and the delivery of costimulatory signals resulting from the interaction of CD80, CD86 with CD28, and CD40 with CD40L on the cell surface of T cells [37]. As expected, decreased expression of the costimulatory molecules CD40, CD80, CD86, and the early activation marker CD69 on decidual DCs was consistent with downregulated CD69 expression on decidual T cells. In our study, the absence of iNKT cells reduced the number of activated T cells in decidua indicating that iNKT cells play a role in LPS-stimulated T cell activation. Accumulating evidence indicates that uterine NK (uNK) cells contribute to successful implantation and maintenance of pregnancy [38,39]. However, these cells become cytotoxic in nature in the presence of certain pathogens [40,41]. In our study, the number of activated NK cells in the decidua of LPS-treated Ja18 / mice was remarkably lower than that in LPS-treated WT mice, demonstrating that iNKT cells have an effect on infection-mediated decidual NK cell activation. Healthy pregnancy is the result of a tightly regulated system of crosstalk between the mother and the fetus. Implantation and parturition are specifically characterized by states of inflammation [42,43]. In contrast, the gestation period, comprising decidualization, placentation, and fetal development, requires uterine quiescence guided by high levels of progesterone and the production of anti-inflammatory cytokines from both maternal and fetal cells [44,45]. Abnormal cytokine production may initiate and intensify the cascade of inflammatory cytokine production involved in adverse pregnancy outcomes. In our experiments, the levels of IL12 in decidual DCs and macrophages and IFN-c in decidual T and NK cells were significantly increased after LPS treatment, suggesting that LPS stimulation inceases IL-12 production in APCs and may further promote T and NK cells IFN-c secretion. The levels of the Th1 cytokines IFN-c and IL-12p70 in the culture supernatants of decidual mononuclear cells from LPS-treated Ja18 / mice were obviously decreased compared with LPS-treated WT mice, whereas concentrations of the Th2 cytokines IL-4 and IL-10 were not significantly different. Our results suggest that activation of iNKT cells help to shift the Th1/Th2 balance to a Th1 predominant state. These results are consistent with those reported by Ito et al. [23], indicating that the Th1 cytokines IFN-c and TNF-a are essential for the Va14 NKT cell-mediated abortion. In contrast to previous studies [23,26], we used an in vitro iNKT cell adoptive transfer system to further confirm the role of iNKT cells in LPS-induced pregnancy loss. Expression levels of costimulatory molecules and CD69 and the concentrations of IFN-c and IL-12p70 at the maternal-fetal interface from LPS-injected Ja18 /-

L. Li et al. / Cellular Immunology 286 (2013) 1–10

mice following iNKT cell adoptive transfer were notably increased in comparison with LPS-injected Ja18 / mice in the absence of adoptive transfer. These data further support a role for iNKT cells in promoting DC maturation and immune cell activation, and shifting Th1/Th2 balance in pregnancy to a Th1 predominant state. As expected, the ERRs for LPS-treated Ja18 / mice following iNKT cell adoptive transfer were markedly elevated compared with those without iNKT cell adoptive transfer. In contrast to previous studies [23,26], we did not explore the role of perforin in iNKT cell-mediated pregnancy loss. While iNKT cells are capable of cytotoxic activity through the expression of perforin and Fas ligand, their primary effector function is to modulate immune responses by producing large amounts of cytokines [46]. Furthermore, transactivated NK cells following iNKT cell activation are also likely to mediate direct contact and lysis of embryonic trophoblasts in a perforin-dependent manner. Collectively, our results suggest that iNKT cells play a role in LPSinduced pregnancy loss by promoting decidual DC maturation and activation, decidual T cell and NK cell activation, and proinflammatory cytokine production. In future studies we will investigate whether iNKT cells also play such a role in infection-associated pregnancy loss in humans.

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Acknowledgments [23]

This study was supported by Grants from the National Basic Research Program of China (2013CB967404), the National Natural Science Foundation of China (81200478, 31171439), the National Funds for Distinguished Young Scientists of China (81125004), Guangdong Medical and Scientific Research Fund (A2012493) and Guangzhou Medical Science and Technology Projects (20121A011020).

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