Ovarian insufficiency and early pregnancy loss induced by activation of the innate immune system

Abstract

We describe a murine model of early pregnancy failure induced by systemic activation of the CD40 immune costimulatory pathway. Although fetal loss involved an NK cell intermediate, it was not due to lymphocyte-mediated destruction of the fetus and placenta. Rather, pregnancy failure resulted from impaired progesterone synthesis by the corpus luteum of the ovary, an endocrine defect in turn associated with ovarian resistance to the gonadotropic effects of prolactin. Pregnancy failure also required the proinflammatory cytokine TNF-alpha and correlated with the luteal induction of the prolactin receptor signaling inhibitors suppressor of cytokine signaling 1 (Socs1) and Socs3. Such links between immune activation and reproductive endocrine dysfunction may be relevant to pregnancy loss and other clinical disorders of reproduction.

Figure 1

Decreased serum progesterone concentrations following systemic CD40 ligation precedes overt embryo resorption. Mice either were treated with control rat IgG or agonistic anti-CD40 antibodies (FGK45) once on E4 and sacrificed 24 hours later on E5, or were treated daily on E4–7 and sacrificed on E8. (A) Serum progesterone concentrations. Data represent mean ± SEM of six mice per group. Progesterone concentrations in FGK45-treated mice were significantly reduced compared with those in control mice. *P < 0.001. (B–E) Implantation sites on E5 from mice treated with rat IgG (B and D) or FGK45 (C and E). (B and C) Whole-mount preparations of uteri, with arrowheads indicating two implantation sites. (D and E) Paraffin sections of implantation sites stained with anti-CD45 antibodies to visualize all leukocytes (red), and counterstained with DAPI to visualize all cell nuclei (blue). The mesometrial pole of each implantation site is oriented toward the top of the panel. A recently implanted embryo can be seen in the center of each section (arrowhead). Immunostaining is representative of two to three mice per group, encompassing a total of 15–20 implantation sites in each group. Scale bar: 0.5 mm.

Figure 2

Postreceptor prolactin signaling defects and luteal insufficiency following systemic CD40 ligation. (A and B) Histological appearance of corpora lutea from mice on E8 following daily E4–7 injections of rat IgG (A) or anti-CD40 antibodies (FGK45) (B). Paraffin sections stained with H&E are shown. (C) Whole-ovary mRNA expression levels determined by real-time RT-PCR. Ovaries were collected from mice that either had been treated with control rat IgG or anti-CD40 antibodies (FGK45) once on E4 and sacrificed 24 hours later on E5 (n = 4 mice per group; upper panel), or had been treated daily on E4–7 and sacrificed on E8 (n = 3 mice per group; lower panel). Data represent mean ± SD. On both E5 and E8, 20α-HSD and LHR mRNA levels were increased and decreased, respectively (fold changes are indicated by brackets), whereas 3β-HSD and prolactin receptor (Prlr) mRNA levels were decreased on E5 only. *P < 0.02; **P < 0.005; ***P < 0.001. (D) Serum prolactin and LH concentrations. Prolactin and LH concentrations in FGK45-treated mice on E5 were significantly elevated compared with those in control mice (#P < 0.05; data represent mean ± SEM of 12 mice per group), but there was no change in prolactin concentrations on E8 (n = 6 mice per group). The limit of detection for LH was 0.2 ng/ml.

Figure 3

Rescue of CD40 ligation–induced pregnancy failure by exogenous progesterone. (A–H) Whole-mount uteri (A, C, E, and G) and implantation-site histology (B, D, F, and H) from mice on E8 following daily treatments on E4–7. Paraffin sections of implantation sites were stained with anti-CD45 antibodies to visualize all leukocytes (red), and counterstained with DAPI (blue). Only mice treated with FGK45 and sesame seed oil vehicle (C and D) showed abortion, leukocytic infiltration into the uterine stroma, and occasional cell debris in the uterine lumen (D, arrowhead). The CD45 staining intensity in these sections tended to be greater than that at viable implantation sites, because they do not contain the high numbers of CD45^dim uterine NK cells that are present in the decidua. Immunostaining is representative of two mice per group, encompassing a total of about 12 implantation sites in each group. Scale bar: 0.5 mm.

Figure 4

Systemic immune activation by CD40 ligation is unaffected by progesterone administration. Mice were sacrificed on E8 after daily treatment on E4–7 with rat IgG or FGK45 plus either concurrent progesterone injection (which produced no abortion with either rat IgG or FGK45) or concurrent sesame seed oil vehicle injection (which produced abortion with FGK45 only). (A) Elevated splenocyte numbers following FGK45 treatment in both groups. *P < 0.005. Data represent mean ± SD for three to four mice per group. (B–D) Three-color flow cytometric analysis of splenocytes to determine cell size and surface activation marker expression. In all panels, shaded histograms show data from rat IgG–treated mice, while open histograms show data from FGK45-treated mice. Data represent three mice per group. (B and C) Lymphocyte activation. Increased forward scatter (FSC), indicating increased cell size (B), and increased CD69 expression, indicating cell activation (C), were seen in B cells, NK cells, and a fraction of T cells following FGK45 treatment in both groups. (D) Dendritic cell activation. Increased CD80, CD86, and MHC class II expression, indicating cell activation, was seen on splenic CD11c⁺ cells following FGK45 treatment in both groups.

Figure 5

Involvement of TNF-α in anti-CD40–induced pregnancy failure. (A) Elevated TNF-α and IL-6 serum concentrations 24 hours after treatment with anti-CD40 antibodies (FGK45). Data represent mean ± SEM of five mice per group. The limits of detection were 31.1 pg/ml TNF-α and 54.7 pg/ml IL-6. ND, not detectable. (B and C) NF-κB p65 distribution in E4 corpora lutea visualized by immunostaining. Whereas the p65 distribution was cytoplasmic in mice treated 24 hours earlier on E4 with control rat IgG (B), it assumed a nuclear distribution when mice were treated with FGK45 (C). Data represent immunostained ovaries from three mice per group. Scale bar: 50 μm.

Figure 6

Ovarian mRNA expression of IL-6, IκBα, Socs1, and Socs3 upon systemic CD40 ligation. Whole-ovary mRNA expression levels were determined by real-time RT-PCR. Ovaries were collected from mice on E4 12 hours after treatment with rat IgG or FGK45 (n = 4 mice per group); with FGK45 plus control hamster IgG or FGK45 plus TNF-α–neutralizing mAb’s (n = 5 mice per group); or with rat IgG plus progesterone or FGK45 plus progesterone (n = 4 mice per group). Ovaries were collected on E8 12 hours after injection of rat IgG or FGK45 (n = 3 mice per group). Data represent mean ± SEM. On E4, IL-6, IκBα, Socs1, and Socs3 mRNA levels were increased following FGK45 injection (*P < 0.05; **P < 0.005). These increases were inhibited by TNF-α blockade but not prevented by concurrent progesterone treatment. On E8, FGK45 injection led to upregulated mRNA expression of IL-6 and IκBα, but not Socs1 or Socs3. For ease of graphical representation, transcript levels for IL-6, IκBα, and Socs3 were multiplied by 1,000, 2, and 5, respectively.