Maternal IL-33 critically regulates tissue remodeling and type 2 immune responses in the uterus during early pregnancy in mice

Abstract

The pregnant uterus is an immunologically rich organ, with dynamic changes in the inflammatory milieu and immune cell function underlying key stages of pregnancy. Recent studies have implicated dysregulated expression of the interleukin-1 (IL-1) family cytokine, IL-33, and its receptor, ST2, in poor pregnancy outcomes in women, including recurrent pregnancy loss, preeclampsia, and preterm labor. How IL-33 supports pregnancy progression in vivo is not well understood. Here, we demonstrate that maternal IL-33 signaling critically regulates uterine tissue remodeling and immune cell function during early pregnancy in mice. IL-33-deficient dams exhibit defects in implantation chamber formation and decidualization, and abnormal vascular remodeling during early pregnancy. These defects coincide with delays in early embryogenesis, increased resorptions, and impaired fetal and placental growth by late pregnancy. At a cellular level, myometrial fibroblasts, and decidual endothelial and stromal cells, are the main IL-33⁺ cell types in the uterus during decidualization and early placentation, whereas ST2 is expressed by uterine immune populations associated with type 2 immune responses, including ILC2s, Tregs, CD4⁺ T cells, M2- and cDC2-like myeloid cells, and mast cells. Early pregnancy defects in IL-33-deficient dams are associated with impaired type 2 cytokine responses by uterine lymphocytes and fewer Arginase-1⁺ macrophages in the uterine microenvironment. Collectively, our data highlight a regulatory network, involving crosstalk between IL-33-producing nonimmune cells and ST2⁺ immune cells at the maternal-fetal interface, that critically supports pregnancy progression in mice. This work has the potential to advance our understanding of how IL-33 signaling may support optimal pregnancy outcomes in women.

Keywords: IL-33; decidual vasculature; pregnancy; tissue remodeling; type 2 immunity.

Fig. 1.

Myometrial fibroblasts and decidual stromal and endothelial cells are the main IL-33⁺ cell types in the uterus in early pregnancy. (A) Image (20×) (Top) showing Citrine, CD31, and DAPI in an implantation site (IS) from an IL-33 Citrine fluorescent reporter dam at E7.5; the rectangle denotes the mesometrial pole region shown as a 40× image (Bottom) with Citrine⁺CD31⁺ vessels in white ellipses. (Scale bars, 200 μm [Top] and 50 μm [Bottom].) AMR, anti-mesometrial region; Dec, decidua; e, embryo; MP, mesometrial pole; MR, mesometrial region; Myo, myometrium. Representative of n = 3 dams, 2 or 3 ISs per dam. (B and C) Dot plots at E7.5 (B) and summary data (C) showing the percentage of Citrine⁺ cells among total live cells in the Myo (open circles, black bars) and Dec (closed circles, white bars) of ISs from IL-33 Citrine fluorescent reporter dams at E5.5, E7.5, and E9.5 (n = 3 to 8 dams, 7 to 11 pooled ISs per dam) by flow cytometry. B includes a dot plot of cells from WT nonreporter dams at E7.5, used to determine gates for Citrine⁺ cells. (D) Western blot of IL-33 in the Myo and Dec from ISs from a WT dam at E7.5. Representative of n = 3 dams, 3 pooled ISs per dam. FL, full-length form; M, mature form. (E, F, H, and I) Live Citrine⁺ cells in the Myo and Dec (n = 9 pooled ISs) at E7.5 were analyzed by scRNA-seq. Cells with detectable Il33 transcripts were selected and clustered by K-means clustering. (E and H) Uniform manifold approximation and projection (UMAP) shows unbiased clustering of Il33⁺ cells from Myo (E) or Dec (H). (F and I) Feature plots show expression of specified transcripts in individual Il33⁺ cells from Myo (F) or Dec (I). (G and J) Percentage of Podoplanin⁺CD31^–EpCAM^–CD45^–, CD31⁺CD45^– cells, and EpCAM⁺CD45^– cells among live Citrine⁺ cells in Myo (G) and Dec (J) at E5.5, E7.5, and E9.5 by flow cytometry (n = 3 to 8 dams, 7 to 11 pooled ISs per dam). Data points represent individual dams. Data are displayed as mean ± SEM.

Fig. 2.

Maternal IL-33 deficiency impairs implantation chamber formation, decidualization, and embryonic development in early pregnancy. (A) Reconstructed image of an implantation chamber from WT and IL-33 KO dams at E5.5 by confocal microscopy. Cytokeratin 8 (CK8, green) staining shows uterine epithelium. White lines indicate implantation chamber length. (B) Implantation chamber length in WT and IL-33 KO dams (n = 6 per group) at E5.5 by confocal microscopy as in A. (C) Average IS weight per dam from WT (n = 62) and IL-33 KO (n = 43) dams at E7.5. (D) Normalized Wnt4 and Fkbp5 transcript abundance in decidua from WT and IL-33 KO dams at E7.5 by qRT-PCR. Data points show two or three individual ISs from WT (n = 6) and IL-33 KO (n = 3) dams. (E) Reconstructed images of an embryo (white circles) within the implantation chamber from WT and IL-33 KO dams at E5.5 by confocal microscopy. Cytokeratin 8 (green) shows uterine epithelium encircling the chamber. Hoechst (white) shows nuclei. (F) Embryo volume in WT and IL-33 KO dams (n = 6 dams per group) at E5.5, measured by confocal microscopy after 3D reconstruction as in A. (G) Frequency of embryos at prestreak (Pre-S), primitive streak (PS), neural plate (NP), and headfold (HF) stages in WT (n = 18) and IL-33 KO (n = 19) dams at E7.5. (H) Images of uteri from WT and IL-33 KO dams at E12.5 showing resorptions in black circles. (I) Frequency of resorptions in WT (n = 13) and IL-33 KO (n = 8) dams at E12.5, identified as in H. Significance was determined by unpaired two-tailed Student’s t test (B, C, and F), unpaired two-tailed Mann–Whitney U test (D), or Fisher’s exact test (G and I) (*P < 0.05, **P < 0.01, ***P < 0.001). Data are displayed as mean ± SEM.

Fig. 3.

IL-33 KO dams exhibit abnormal uterine vasculature and impaired SpA remodeling in early pregnancy. (A) Representative 20× image (Top) of CD31, αSMA, and DAPI in an IS from a WT dam at E7.5 (representative of n = 8 dams). (Scale bar, 200 μm.) MP, mesometrial pole; CTR, central region; e, embryo; AMR, anti-mesometrial region. Image (40×) (Bottom) of CD31, αSMA, and DAPI in MP of decidua from a WT dam at E7.5 (representative of n = 5 dams). (B) Signal density of CD31⁺ vessels in the MP, CTR, and AMR of the decidua from WT (n = 8) and IL-33 KO (n = 8) dams at E7.5. Data points reflect individual dams. (C) Signal density of αSMA⁺ vessels in the MP of the decidua from WT and IL-33 KO dams (n = 5 dams per group) at E7.5, identified as in A, Bottom. (D) Images (40×) of H&E staining of SpAs (black arrowheads) from WT and IL-33 KO dams at E9.5. (Scale bars, 100 μm.) (E) SpAs in ISs from WT (n = 5, 2 ISs per dam) and IL-33 KO (n = 5, 2 ISs per dam) dams at E9.5 were assessed for vessel and lumen area, wall thickness, and vessel-to-lumen ratio. Significance was determined by unpaired two-tailed Student’s t test (*P < 0.05; ns, not significant). Data are displayed as mean ± SEM.

Fig. 4.

Maternal IL-33 deficiency results in poor pregnancy outcome parameters in late pregnancy. Pregnancy outcome parameters were measured at E18.5 from WT (n = 12) and IL-33 KO dams (n = 12) mated with WT sires or from WT dams (n = 9) mated with IL-33 KO sires. Bar graphs show summary data as follows. (A) Uterus weight. (B) Number of viable fetuses. (C) Frequency of resorptions. (D) Fetal weight. (E) Frequency of SGA fetuses, defined as weighing less than 10th percentile of fetuses from WT × WT pregnancies. (F) Placental diameter. Significance was determined by one-way ANOVA (A), Kruskal–Wallis test with post hoc t tests (B), Fisher’s exact test (C and E), or mixed-effects model analysis of repeated measures, with dam as subject (D and F) (*P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001; ns, not significant). Data are displayed as mean ± SEM.

Fig. 5.

Il1rl1 is expressed by type 2 immune cells in the uterus at E7.5. (A and D) UMAP of scRNA-seq data showing normalized Il1rl1 expression in individual live CD45⁺ cells purified from the myometrium (A) or decidua (D) of implantation sites from WT dams at E7.5 (n = 2 dams, 8 pooled ISs per dam). (B and E) Il1rl1⁺ cells in Myo (B) and Dec (E) were reclustered by K-means clustering and visualized by UMAP. (C and F) Heatmaps showing select differentially expressed genes (false discovery rate <0.5, fold >2 or <−2) among the distinct clusters of Il1rl1⁺ cells in the Myo (C) and Dec (F).

Fig. 6.

Maternal IL-33 promotes type 2 immune responses by uterine immune cells during early pregnancy. (A) Percentage of ILC2s, CD4⁺Foxp3⁺ T cells (Tregs), and CD4⁺Foxp3^– (Tconv) in the Dec and Myo among ST2⁺ lymphocytes from WT dams at E7.5 by flow cytometry (n = 5 dams, 6 to 9 pooled ISs per dam). (B) Representative flow plot (Left) showing the percentage of IL-5⁺ ILC2s from the myometrium of WT and IL-33 KO dams at E7.5 by flow cytometry. Summary data (Right) for WT (n = 10 dams, 7 to 11 pooled ISs per dam) and IL-33 KO (n = 8 dams, 7 to 12 pooled ISs per dam) from seven independent experiments, shown as fold change relative to average for the WT group in each experiment. (C) As in B, except showing IL-13. (D) As in C, except showing IL-13⁺CD4⁺ T cells from WT (n = 7 dams, 7 to 11 pooled ISs per dam) and IL-33 KO dams (n = 5, 7 to 12 pooled ISs per dam) at E7.5. Significance was determined by two-tailed Student’s t test. (E) Number of eosinophils in the decidua at E7.5 (Left) and E9.5 (Right) from WT dams (n = 8 to 17, 5 to 11 pooled ISs per dam) and IL-33 KO dams (n = 8 to 14, 6 to 10 pooled ISs per dam), shown as the number per IS. Significance was determined by unpaired two-tailed Mann–Whitney U test. (F) Number of Arginase-1⁺ macrophages in the decidua (Left) and myometrium (Right) from WT (n = 7 dams, 6 to 11 pooled ISs per dam) and IL-33 KO dams (n = 8 dams, 7 to 9 pooled ISs per dam) at E7.5. Shown as the number per IS or number per milligram of tissue. Significance was determined by unpaired two-tailed Student’s t test (*P < 0.05, **P < 0.01, ***P < 0.001). Data are displayed as mean ± SEM.