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. 2019 Jan 10:9:3142.
doi: 10.3389/fimmu.2018.03142. eCollection 2018.

Galectin-9 Alleviates LPS-Induced Preeclampsia-Like Impairment in Rats via Switching Decidual Macrophage Polarization to M2 Subtype

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Galectin-9 Alleviates LPS-Induced Preeclampsia-Like Impairment in Rats via Switching Decidual Macrophage Polarization to M2 Subtype

Zhi-Hui Li et al. Front Immunol. .

Abstract

Dysfunction of decidual macrophages (DMs) is considered a critical event in the pathogenesis of pre-eclampsia (PE). T cell immunoglobulin mucin 3 (Tim-3) is an important negative regulatory molecule that induces immune tolerance by interacting with its ligand Galectin-9 (Gal-9) and thus modulating function of various immune cells, including macrophages. However, the regulatory effects of Tim-3/Gal-9 signaling on DMs polarization and its role in PE remain unclear. In this study, we established a PE-like rat model by administering 1.0 μg/kg lipopolysaccharide (LPS) to normal pregnant Sprague-Dawley rats via the tail vein at embryonic day 5 (E5). Apart from the pre-eclamptic manifestations, increased M1 subtype and decreased M2 subtype were observed at the maternal-fetal interface, as well as increased pro-inflammatory cytokines (TNF-α and IL-1β) and reduced anti-inflammatory cytokines (TGF-β and IL-10). Moreover, the expression of Tim-3 in DMs and that of Gal-9 at the maternal-fetal interface were reduced. After administration of recombinant Galectin-9 (rGal-9) protein, we found that liver and renal injuries and maternofetal placental functional deficiency, including inadequate trophoblast cells invasion, impaired spiral artery remodeling and fetal capillary development, were reversed. In addition, the polarization of DMs was inclined to M2 subtype, which was similar to the polarization of DMs in the control rats but contrary to the PE-like rats. Interestingly, at E9, the expression of Tim-3 in DMs and that of Gal-9 at the maternal-fetal interface were significantly increased in the rGal-9 protein intervention group. Taken together, our findings show that administration of rGal-9 protein can alleviate the PE-like rat manifestations induced by LPS. This finding may be related to the activation of the Tim-3/Gal-9 signaling pathway, which promotes DMs polarization dominantly shifting to M2 subtype. Moreover, upregulation of Tim-3 in DMs and Gal-9 at the maternal-fetal interface at E9 suggests that Tim-3/Gal-9 pathway may play some important roles in early pregnancy and even embryo development.

Keywords: Galectin-9; Tim-3; decidual macrophage; polarization; pre-eclampsia.

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Figures

Figure 1
Figure 1
Schematic diagram of the animal experimental design and the mean SBP, 24 h urinary protein, and fetal development in each group. (A) Presence of vaginal spermatozoa confirmed successful pregnancy and was designated E0. Normal pregnant rats were injected with LPS via the tail vein at E5 to establish a PE-like rat model. The control group was injected with saline at the same time. Gal-9 recombination protein was administered to the PE-like rats by intraperitoneal injection at E7, E12, and E16. (B) SBP was monitored at E0, 3, 6, 8, 10, 12, 14, 16, and 18 and (C) 24 h urinary protein was measured at E0, 3, 6, 9, 12, 15, and 18. Saline, n = 7; LPS, n = 7; LPS+Gal-9, n = 5. Data are expressed as the mean ± SEM. ΔΔp < 0.01 LPS+Gal-9 vs. saline group at E6; **p < 0.01 LPS vs. saline group on the corresponding E; ##p < 0.01 LPS vs. LPS+Gal-9 group on the corresponding E. (D–H) Fetal and placental development in each group. (D) Fetal length and (E) fetal wet weight in different group, **p < 0.01. (G) Embryo absorption (yellow arrow).
Figure 2
Figure 2
Morphological features of the liver and kidney in the different groups. H&E staining of liver (E) and kidney tissue (F–H) from a PE-like rat showed hepatocyte balloon degeneration and spotty necrosis (black short arrow, E), renal tubular epithelial cell collapsed and swollen (red long arrow, G), inflammatory cell infiltrates in the cortex (red short arrow, G) and interstitial tissue (blue short arrow, H). PAS staining of kidney tissue from the PE-like rats showed glomerular swelling and even atrophy and mesangial extracellular matrix expansion (blue long arrow, F). After Gal-9 recombination protein intervention, H&E and PAS staining of liver (I) and kidney tissues (J–L) were basically normal without obvious pathological changes, which was similar to normal pregnancy (A–D). Scale bars: 100 μm, 50 μm.
Figure 3
Figure 3
Trophoblast invasion and SA remodeling in different groups. (A) There were fewer trophoblasts (CK7 staining, red) and more vascular smooth muscle (α-SMA staining, green) in the LPS-treated group (white short arrow, b). In the Gal-9 intervention group, trophoblast invasion, and SA remodeling were reversed (white arrow, c), similar to the control pregnancy group (white arrow, a). Scale bars: 500 μm. (B) PAS staining of fibrinoids in the SA of the mesometrial triangle in different groups. In the control pregnancy group, the fibrinoid wall was obviously integrated (black arrow, a). In comparison, the fibrinoid wall almost disappeared in the LPS-treated group (black short arrow, b). In the Gal-9 intervention group, the fibrinoid wall was formed (blue arrow, c). SA, spiral artery. Scale bars: 100 μm.
Figure 4
Figure 4
Immunohistochemistry for laminin in the labyrinth of different groups. There were fewer branches from Cp (b), fewer fetal capillaries and narrowed lumen (B) in the labyrinth of LPS-treated group. In the Gal-9 intervention group, the branches from Cp increased (c) and the maternal-fetal vascular network fully developed (C), which was similar to the control pregnancy group (a and A). Cp, Chorionic plate; f, fetal vessel. Scale bars: 200 μm.
Figure 5
Figure 5
Gal-9 suppressed the polarization of DMs biased to M1 but promoted a shift to M2. (A) Immunofluorescence for CD68 and CCR7 in the decidua of different groups. The CCR7 expression on macrophages (CD68+CCR7+) in the decidua's of LPS-treated group was significantly increased. In the Gal-9 intervention group, M1 macrophages decreased, which was similar to the control pregnancy group. Scale bars: 100 μm. (B) Immunofluorescence for CD68 and Arg1 in the decidua of different groups. The Arg1 expression on macrophages (CD68+Arg1+) in the decidua's of LPS-treated group was significantly decreased. In the Gal-9 intervention group, M2 macrophages increased, especially at E9, which was similar to the control pregnancy group. Scale bars: 100 μm. (C) The percentages of M1 and M2 subsets in decidua were detected by FCM. A single-cell decidual suspension was obtained by enzymatic digestion. The total leukocyte population was identified in live cells by the expression of CD45, and then gated for identification of macrophage subsets by the expression of CD68 and CD163 (CD45+CD68+CD163 and CD45+CD68+CD163+ represent M1 and M2 subsets, respectively). Contour plots are representative of three independent experiments. (D,E) Comparison of the percentages of M1 and M2 subsets at E9 and E20 from different groups (data are presented as mean ± SEM). *p < 0.05, **p < 0.01, ***p < 0.001.
Figure 6
Figure 6
The mRNA expression levels of M1 and M2 markers, and pro-inflammatory and anti-inflammatory cytokines by qRT-PCR in different groups. (A,D) mRNA levels of iNOS (M1 subset marker) and Arg1 (M2 subset marker) in the placenta from the three groups. (B,C) mRNA levels of TNF-α and IL-1β (pro-inflammatory cytokines) and (E,F) mRNA levels of IL-10 and TGF-β (anti-inflammatory cytokines) in the placenta from the three groups. β-actin was used for normalization. Data are showed as the mean ± SEM, one placenta per rat, *p < 0.05, **p < 0.01, ***p < 0.001.
Figure 7
Figure 7
Immunofluorescence for CD68 (green) and Tim-3 (red) in the decidua of different groups. The expression of Tim-3 in DMs of the LPS-treated group at E20 was significantly decreased (B and b). In the LPS+Gal-9 treated group, the expression of Tim-3 was rescued (C and c, D and d), which was similar to the control (saline) group (A and a), especially at E9 (D and d). Pan macrophage marker CD68 is labeled in green, and Tim-3 labeled in red. DAPI labeled nuclei in blue. Scale bars: 100 μm.
Figure 8
Figure 8
Western blotting for CD68, CCR7, Arg1 and Tim-3 in the decidua's in different groups. (A) The protein levels of Tim-3 (33kDa), Arg1 (35kDa), CCR7 (43kDa), and CD68 (37kDa) in the placenta were determined by western blotting and normalized to the level of β-actin (42kDa). (B–E) The quantified relative band intensity was determined by ImageJ. The results are shown as the mean ± SEM of three separate experiments, *p < 0.05, **p < 0.01.
Figure 9
Figure 9
Immunohistochemistry and Western blotting for identification of Gal-9 at the implantation site of different groups. (A) Immunohistochemistry for Gal-9 at the implantation site of different groups. The pictures at the lower-left corner of each group are the negative controls. Scale bars: 100 μm. (B) The protein levels of Gal-9 (40 kDa) were determined by western blotting and normalized to the level of β-actin (42kDa). (C) The quantified relative band intensity was determined by ImageJ. The results are shown as the mean ± SEM of three separate experiments, *p < 0.05, **p < 0.01.
Figure 10
Figure 10
Tim-3-mediated harmonious dialogue at the maternal-fetal interface guarantees a smooth gestation by inducing M2 DM polarization. During normal pregnancy, the polarization of DMs is biased to M2 subsets to induce immune tolerance and promote trophoblast cell invasion and SA remodeling (A), which are essential for the establishment and maintenance of a successful pregnancy. Aberrant expression of Tim-3 in DMs lead to Tim-3/Gal-9 signal pathway dysfunction, mediating the polarization of macrophage-biased to M1 subsets. The immune tolerance at the maternal-fetal interface was broken. Trophoblast cell invasion insufficiency and SA remodeling impairment ultimately lead to PE (B). After exogenous administration of Gal-9, the expression of Tim-3 in DMs was reversed, and the polarization of DMs biased to M1 was suppressed but a shift to M2 was promoted (C), which contributed to inducing immune tolerance.

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