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. 2019 Jan 2;129(1):422-436.
doi: 10.1172/JCI99945. Epub 2018 Dec 10.

Sialic acid is a critical fetal defense against maternal complement attack

Markus Abeln  1 , Iris Albers  1 , Ulrike Peters-Bernard  1 , Kerstin Flächsig-Schulz  1 , Elina Kats  1 , Andreas Kispert  2 , Stephen Tomlinson  3 , Rita Gerardy-Schahn  1 , Anja Münster-Kühnel  1 , Birgit Weinhold  1
Affiliations

Sialic acid is a critical fetal defense against maternal complement attack

Markus Abeln et al. J Clin Invest. .

Abstract

The negatively charged sugar sialic acid (Sia) occupies the outermost position in the bulk of cell surface glycans. Lack of sialylated glycans due to genetic ablation of the Sia-activating enzyme CMP-sialic acid synthase (CMAS) resulted in embryonic lethality around day 9.5 post coitum (E9.5) in mice. Developmental failure was caused by complement activation on trophoblasts in Cmas-/- implants and was accompanied by infiltration of maternal neutrophils at the fetal-maternal interface, intrauterine growth restriction, impaired placental development, and a thickened Reichert's membrane. This phenotype, which shared features with complement receptor 1-related protein Y (Crry) depletion, was rescued in E8.5 Cmas-/- mice upon injection of cobra venom factor, resulting in exhaustion of the maternal complement component C3. Here we show that Sia is dispensable for early development of the embryo proper but pivotal for fetal-maternal immune homeostasis during pregnancy, i.e., for protecting the allograft implant against attack by the maternal innate immune system. Finally, embryos devoid of cell surface sialylation suffered from malnutrition due to inadequate placentation as a secondary effect.

Keywords: Complement; Embryonic development; Glycobiology; Immunology; Reproductive Biology.

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Conflict of interest statement

Conflict of interest: The authors have declared that no conflict of interest exists.

Figures

Figure 1
Figure 1. Cmas–/– embryos lack sialylation.
(A) Lectin-binding epitopes for MAA detecting terminal α2,3-linked Sia and for PNA detecting terminal galactose. Symbol nomenclature is according to ref. . (B) Sagittal paraffin sections of uteri with E6.5 and E8.5 implants were costained with the lectins MAA (red) and PNA (green). Insets show TGC lining the fetal-maternal interface. The area of the EPC is indicated by brackets. MAA-positive cells in Cmas–/– (inset, magnified) are maternal leukocytes. Asterisks indicate RM, arrows mark the apical side of the ectodermal epithelia, and arrowheads indicate mesenchymal cells. MAA specificity was confirmed by neuraminidase treatment of sagittal paraffin sections prior to lectin staining. Neuraminidase releases Sia from glycans and thereby depletes MAA-binding epitopes. Simultaneously, removal of Sia exposes underlying galactose to PNA binding. Scale bars: 50 μm (insets). Nuclei were stained with DAPI and are shown in white. Representative images from n = 3 (E6.5 control and Cmas–/–, E8.5 Cmas–/–) and n = 5 (E8.5 control) embryos within the uterus.
Figure 2
Figure 2. Cmas–/– mice exhibit IUGR and extraembryonic developmental deficits.
(A) H&E-stained sagittal paraffin sections of uteri at E6.5, E7.5, and E8.5. Insets show mesenchymal ME cells migrating from posterior to anterior. H, heart; S, somite. Scale bars: 12.5 μm (insets). Images are representative of at least 3 embryos within the uterus for each genotype and time point. (B) Mean fetal size as measured by the sum of the areas of the amniotic cavity, exocoelomic cavity, ectoplacental cavity, and embryo proper (μm2/105). For a schematic overview of the measured areas, see Supplemental Figure 5. E6.5 (control, n = 4; Cmas–/–, n = 5), E7.5 (control, n = 4; Cmas–/–, n = 4), E8.5 (control, n = 6; Cmas–/–, n = 7). Error bars indicate SD. Statistical analyses were performed by Student’s t test (*P < 0.05). (C) Immunohistochemical detection of cytokeratin-8 as a marker for trophoblast cells on sagittal paraffin sections of E8.5 uteri. TGC and the CP are indicated by arrows, and internal trophoblasts of the EPC are represented by arrowheads. The border of the EPC is marked by dotted lines. Images are representative of n = 3 embryos for each genotype. (D) Collagen IV and laminin indirect immunofluorescence staining on sagittal paraffin sections of uteri at E8.5 to visualize RM (arrow) and parietal endoderm (arrowheads). Nuclei stained with DAPI are shown in white. Images are representative of n = 3 embryos for each genotype.
Figure 3
Figure 3. Infiltration of the fetal-maternal interface of Cmas–/– animals by maternal neutrophils.
(A) Gr-1 (neutrophils) immunohistochemical staining and quantification of Gr-1–positive cells surrounding fetal tissues of sagittal paraffin sections of uteri at E6.5 to E8.5. Insets show Gr-1–positive cells at the EPC and in the vicinity of fetal trophoblasts at the antimesometrial pole. E6.5 (control, n = 4; Cmas–/–, n = 5), E7.5 (control, n = 4; Cmas–/–, n = 4), and E8.5 (control, n = 5; Cmas–/–, n = 5). Error bars indicate SD. Scale bars: 12.5 μm (insets). (B) DBA lectin (dNK cells) immunohistochemical staining and quantification of sagittal paraffin sections of uteri at E6.5 to E8.5. Mean number of DBA lectin-positive cells in the decidua basalis. E6.5 (control, n = 4; Cmas–/–, n = 5), E7.5 (control, n = 8; Cmas–/–, n = 5), E8.5 (control, n = 5; Cmas–/–, n = 3). Error bars indicate SD. Staining of VE was only observed in control implants. (C) F4/80 (macrophages) immunohistochemical staining and quantification of sagittal paraffin sections of uteri from E6.5 to E8.5. Statistical analysis of the number of F4/80-positive cells surrounding fetal tissues. E6.5 (control n = 4; Cmas–/–, n = 4), E7.5 (control n = 6; Cmas–/–, n = 5), E8.5 (control n = 4; Cmas–/–, n = 5). Scale bars: 25 μm (insets). Error bars indicate SD. All statistical analyses were performed by Student’s t test (**P < 0.01; ***P < 0.001).
Figure 4
Figure 4. Depletion of maternal neutrophils does not rescue the Cmas–/– phenotype.
(A) Neutrophils were depleted by intraperitoneal injection of 500 μg anti-Ly6G antibody (1A8, BioXCell) into pregnant mice at E4.5. Ly6G FACS analysis of whole blood from untreated and anti-Ly6G–injected pregnant mice at day E8.5. (B) Ly6G (neutrophils) immunohistochemical staining of sagittal paraffin sections of embryos within the uterus. Pregnant females were either treated with the isotype antibody (2A3, BioXCell) as a negative control or with anti-Ly6G for neutrophil depletion. Representative images of control (n = 6) and Cmas–/– (n = 4) embryos. (C) Mean fetal size of control and Cmas–/– embryos from anti-Ly6G–treated mother mice, as measured by the sum of the areas of the amniotic cavity, exocoelomic cavity, ectoplacental cavity, and embryo proper (μm2/105) (control, n = 6; Cmas–/–, n = 4). For a schematic overview of the measured areas, see Supplemental Figure 5. Error bars indicate SD. Statistical analyses were performed by Student’s t test (**P < 0.01). (D) Immunohistochemical detection of cytokeratin-8 to visualize trophoblast cells on sagittal paraffin sections of E8.5 uteri from Ly6G-treated mother mice. Representative images of control (n = 6) and Cmas–/– (n = 4) embryos. (E) Collagen IV indirect immunofluorescence staining on sagittal paraffin sections of uteri at E8.5 from Ly6G-treated mother mice to visualize RM (arrow) and parietal endoderm (arrowheads). Representative images of control (n = 6) and Cmas–/–(n = 4) embryos. Nuclei stained with DAPI are shown in white.
Figure 5
Figure 5. Cmas–/– trophoblast cells exhibit increased deposition of complement C3.
(A) C3 immunohistochemical staining of sagittal paraffin sections of uteri at E8.5. Insets show TGCs. Scale bars in the insets: 25 μm. C3 staining of VE (arrowhead) was observed only in control embryos. Representative images of control (n = 16) and Cmas–/– (n = 6) embryos within the uterus. (B) C3 immunohistochemical staining of sagittal paraffin sections of uteri at E8.5 from anti-Ly6g–treated mother mice. Insets show TGCs without C3 staining on control tissue, but extensive staining at the TGC cell surface. Representative images of control (n = 6) and Cmas–/– (n = 4) embryos within the uterus. Scale bars: 50 μm (insets).
Figure 6
Figure 6. CVF decomplements the maternal serum and rescues the inflammatory phenotype of Cmas–/– implants.
(A) C3 Western blot analysis. Serum of PBS- or CVF-treated pregnant mice at E8.5 was separated by SDS-PAGE and immunostained with anti-C3 antibody. C3 protein was only detectable in PBS-treated mice, but was depleted in CVF-treated pregnant mice. Anti-albumin staining was used as loading control. (B) C3 immunohistochemical staining of sagittal paraffin sections of E8.5 embryos within the uterus of PBS- or CVF-treated mice. In PBS-treated mothers, C3 reactivity was restricted to the EPC in control implants, but was expanded to the entire fetal-maternal interface in Cmas–/– embryos, with strong staining at the surface of TGCs. In implants of CVF-treated mothers, the C3 reactivity was abolished irrespective of the genotype. Insets show fetal TGCs. (C) Ly6G immunohistochemical staining for neutrophils on sagittal paraffin sections of E8.5 uteri from PBS- or CVF-treated mice. Ly6G-positive cells are sparsely distributed in proximity of control embryos of PBS-treated mothers. In contrast, the entire fetal-maternal boundary of Cmas–/– implants is infiltrated with Ly6G-positive cells in PBS-treated mice. CVF treatment does not change the phenotype of controls but reverts Ly6G staining of Cmas–/– implants to that of controls. (D) Quantification of Ly6G-positive cells (neutrophils) on sagittal paraffin sections of E8.5 uteri of PBS- or CVF-treated pregnant mice. Error bars indicate SD. Statistical analyses were performed by ANOVA with Newman-Keuls post test (***P < 0.001). Images are representative of experiments of 3 PBS-treated pregnant mice with n = 5 control and n = 3 Cmas–/– embryos, and of 3 CVF-treated pregnant mice with n = 5 control and n = 4 Cmas–/– embryos (B–D).
Figure 7
Figure 7. Maternal decomplementation rescues defects in extraembryonic tissues and growth restriction of Cmas–/– mice.
Pregnant Cmas+/– mice were treated at E4.5 and E6.5 either with PBS (n = 3) or CVF (n = 3) to deplete maternal C3. E8.5 sagittal uteri paraffin sections (A and B). (A) The reduced size of the EPC and lack of a CP in Cmas–/– embryos of PBS-treated mothers was reverted to the control phenotype upon CVF treatment, as visualized by immunohistochemical cytokeratin-8 staining. The lack of CEBPB reactivity (indirect immunofluorescence) in Cmas–/– embryos of PBS-treated mothers was restored upon CVF treatment. PNA reactivity documenting the loss of cell surface sialylation was maintained in Cmas–/– embryos of PBS- and CVF-treated mothers, indicating that the asialo phenotype was not influenced by CVF. Representative images of experiments with PBS-treated mice: control, n = 5; Cmas–/–, n = 3 embryos; CVF treated mice: control, n = 5; Cmas–/–, n = 4 embryos. (B) Collagen IV indirect immunofluorescence (red). Thickened RM (arrow) in Cmas–/– embryos of PBS-treated mothers was converted to the control phenotype upon CVF treatment. Parietal endoderm is marked by arrowheads. Nuclei shown in white were stained with DAPI. (C) Quantification of RM thickness measured on collagen IV immunofluorescence images at the anti-mesometrial pole (PBS: control, n = 6; Cmas–/–, n = 3 embryos; CVF: control, n = 5; Cmas–/–, n = 4 embryos). (D) Mean of fetal size as measured by the sum of the areas of the amniotic cavity, exocoelomic cavity, ectoplacental cavity, and embryo proper in (μm2/105) (PBS: control, n = 5; Cmas–/–, n = 4 embryos; CVF: control, n = 5; Cmas–/–, n = 3 embryos); a schematic of the areas is shown in Supplemental Figure 5. Statistical analyses by 1-way ANOVA with Newman-Keuls post test (*P < 0.05; **P < 0.01; ***P < 0.001). Error bars indicate SD (C and D).
Figure 8
Figure 8. Cmas–/– trophoblast cells activate the alternative pathway.
Immunohistochemical analyses of complement components (A) C1q, (B) C4d, (C) properdin, and (D) C9 on sagittal paraffin-embedded sections of E8.5 embryos within the uterus. The positive C9 staining in both genotypes (upper insets) most likely reflects C9 in the fluid phase in the lumen of decidual blood vessels. Scale bars in all insets: 20 μm. All experiments shown are representative images of control (n = 12) and Cmas–/– (n = 3) embryos.
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