Rapamycin prevents spontaneous abortion by triggering decidual stromal cell autophagy-mediated NK cell residence

Abstract

Deficiency in decidualization has been widely regarded as an important cause of spontaneous abortion. Generalized decidualization also includes massive infiltration and enrichment of NK cells. However, the underlying mechanism of decidual NK (dNK) cell residence remains largely unknown. Here, we observe that the increased macroautophagy/autophagy of decidual stromal cells (DSCs) during decidualization, facilitates the adhesion and retention of dNK cells during normal pregnancy. Mechanistically, this process is mediated through activation of the MITF-TNFRSF14/HVEM signaling, and further upregulation of multiple adhesion adhesions (e.g. Selectins and ICAMs) in a MMP9-dependent manner. Patients with unexplained spontaneous abortion display insufficient DSC autophagy and dNK cell residence. In addition, poor vascular remodeling of placenta, low implantation number and high ratio of embryo loss are observed in NK cell depletion mice. In therapeutic studies, low doses of rapamycin, a known autophagy inducer that significantly promotes endometrium autophagy and NK cell residence, and improves embryo absorption in spontaneous abortion mice models, which should be dependent on the activation of MITF-TNFRSF14/HVEM-MMP9-adhension molecules axis. This observation reveals novel molecular mechanisms underlying DSCs autophagy-driven dNK cell residence, and provides a potential therapeutic strategy to prevent spontaneous abortion.Abbreviations: ACTA2/αSMA: actin alpha 2, smooth muscle; ATG: autophagy-related; ATG5^over ESC: ATG5-overexpressed ESCs; BTLA: B and T lymphocyte associated; CDH1: cadherin 1; CDH5: cadherin 5; CXCL12: C-X-C motif chemokine ligand 12; dNK: decidual NK; DIC: decidual immune cell; DSC: decidual stromal cell; EOMES: eomesodermin; ESC: endometrial stromal cell; FCGR3A/CD16: Fc fragment of IgG receptor IIIa; HUVEC: human umbilical vein endothelial cell; ICAM: intercellular cell adhesion molecule; ILC: innate lymphoid cell; ITGB1: integrin subunit beta 1; ITGA2: integrin subunit alpha 2; IPA: Ingenuity Pathway Analysis; KIR2DL1: killer cell immunoglobulin like receptor, two Ig domains and long cytoplasmic tail 1; KLRD1/CD94: killer cell lectin like receptor D1; KLRK1/NKG2D: killer cell lectin like receptor K1; MAP1LC3B/LC3B: microtubule associated protein 1 light chain 3 beta; 3-MA: 3-methyladenine; MITF: melanocyte inducing transcription factor; MiT-TFE: microphthalmia family of bHLH-LZ transcription factors; MMP9: matrix metalloproteinase 9; MTOR: mechanistic target of rapamycin kinase; NCAM1/CD56: neural cell adhesion molecule 1; NCR2/NKp44: natural cytotoxicity triggering receptor 2; NK: natural killer; KLRB1/NK1.1: killer cell lectin like receptor B1; NP: normal pregnancy; PBMC: peripheral blood mononuclear cell; PECAM1/CD31: platelet and endothelial cell adhesion molecule 1; pNK: peripheral blood NK; PRF1/Perforin: Perforin 1; PTPRC/CD45: protein tyrosine phosphatase receptor type C; Rapa: rapamycin; rh-TNFSF14/LIGHT: recombinant human TNFSF14/LIGHT; SA: spontaneous abortion; SELE: selectin E; SELP: selectin P; SELL: selectin L; siATG5 DSCs: ATG5-silenced DSCs; siTNFRSF14/HVEM DSCs: TNFRSF14/HVEM-silenced DSCs; TBX21/T-bet: T-box transcription factor 21; SQSTM1/p62: sequestosome 1; TNFRSF14/HVEM: TNF receptor superfamily member 14; TNFSF14/LIGHT: TNF superfamily member 14; uNK: uterine NK; UIC: uterine immune cell; USC: uterine stromal cell; VCAM1: vascular cell adhesion molecule 1; VIM: vimentin.

Keywords: Autophagy; MITF; MMP9; NK cells; TNFRSF14/HVEM; abortion; decidual stromal cells; early pregnancy; residence.

Figure 1.

Decidualization is accompanied by enhanced autophagy and NK cell enrichment. (A) Primary cultured ESCs of secretory phase (n = 4) and DSCs (n = 5) were examined by transmission electron microscopy for the number of autophagic structures, including autophagosomes and autolysosomes (indicated by the red triangle). The quantity statistics were shown in (B). (C) The expression of autophagy-related proteins LC3-I, LC3-II and SQSTM1 in ESCs of secretory phase (n = 6) and DSCs (n = 6) was detected by western blotting assays. (D) The ratio of LC3-II to LC3-I was quantified. (E) After C57 pregnant mice were treated with PBS (n = 9) or 3-MA (n = 9, 100 mg/kg, twice a week), the number of embryos implanted and the embryo resorption rate of pregnant mice at the gestation of day 13.5 were quantified in (F). (G) In vitro cell adhesion assays were performed to analyze the adhesion of PKH67-labeled ESCs (n = 5) or DSCs (n = 5) to PKH26-labeled dNK cells. The number of adhered dNK cells was counted in (H). (I) RT-PCR was used to detect the expression levels of adhesion-related genes (SELE, SELP, SELL, VCAM1, ICAM2 and ICAM5) in ESCs (n = 5) and DSCs (n = 6). (J) Flow cytometry was used to detect the proportion and number of NK cells in the uterus of estrus (n = 8) and pregnant mice (n = 6) at the gestation of day 7.5. The statistical graph was displayed in (K). Data were presented as mean ± SEM or median and quartile and analyzed by t test. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001, NS: no significance

Figure 2.

DSC autophagy promotes NK cells residence in decidua. (A) Adhesion assays were performed on ATG5^over ESCs (n = 6) or control ESCs (n = 6, GFP green fluorescence) and PKH-67-labeled dNK cells. The number of attached dNK cells was counted in (B). (C) Adhesion assays were performed on siATG5 ESCs (n = 5) or control ESCs (n = 5, GFP green fluorescence) and PKH67-labeled dNK cells. The number of adherent dNK cells was counted in (D). (E) The differential protein expression profile of ATG5^over ESCs and control ESCs from protein microarray assay was shown, and differential expression of adhesion-related proteins were selected. (F) The expression levels of a series of adhesion molecules in DSCs of control group (n = 6) and 3-MA treatment group (n = 6, 10 mM, 48 h) were detected by flow cytometry. (G–J) C57 pregnant mice were injected intraperitoneally with PBS (n = 6) or 3-MA (n = 6, 100 mg/kg, twice a week), and the uterus of mice at the gestation of day 7.5 was collected. (G,H) The expression level of adhesion molecules on VIM⁺ USCs (uterine stromal cells) was evaluated by flow cytometry. (I,J) The proportion and absolute number of CD3⁻ KLRB1⁺ NK cells in PTPRC⁺ uterine immune cells (UICs) was also detected by flow cytometry at the gestation of day 7.5. Data were presented as mean ± SEM or median and quartile and analyzed by t test. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001

Figure 3.

NK cell autophagy does not regulate its adhesion ability and cytotoxic activity-related molecules expression. (A) The autophagy structures in pNK (n = 6) and dNK cells (n = 6) were photographed using a transmission electron microscope. The number of autophagy structures was counted in (B). (C) The adhesion of dNK cells pre-treated with 3-MA (10 mM, 48 h, n = 7) or vehicle (1‰ PBS, n = 9) to DSCs was evaluated by in vitro adhesion assays. The number of adhered dNK cells was counted in (D). The expression of adhesion molecules (E) or functional molecules (F) on dNK cells treated with 3-MA (10 mM, 48 h, n = 7) or vehicle (1‰ PBS, n = 7) was analyzed by flow cytometry. Data were presented as mean ± SEM or median and quartile and analyzed by t test. *P < 0.05, *P < 0.01, NS: no significance

Figure 4.

DSC autophagy-mediated NK cell residence is dependent on the MITF-TNFRSF14 pathway. The transcription level or protein of TNFRSF14 in ESCs (n = 4) and DSCs (n = 4) were compared by RT-PCR (A) and western blotting (B). The transcription or protein level of TNFRSF14 in ATG5^over (n = 4) and control ESCs (n = 4) was detected by RT-PCR (C) or western blotting (D). (E) RT-PCR was performed to detect the level of TNFRSF14 in vehicle (1‰ DMSO) or rapamycin-treated (2 µM, 48 h) control and siTNFRSF14 DSCs (n = 6 per group). (F) The adhesion ability of vehicle or rapamycin-treated DSCs and/or siTNFRSF14 DSCs (GFP green fluorescence) to PKH-67 labeled dNK cells was evaluated by in vitro cell adhesion assays (n = 6 per group). The statistical graph of the number of dNK cells adhered to each group was shown in (G). (H) The expression of adhesion-related genes in vehicle (1‰ DMSO), or rapamycin-treated (2 µM, 48 h) control and/or siTNFRSF14 DSCs (n = 6 per group) was detected by RT-PCR. (I) The transcription levels of MITF in ESCs (n = 5) and DSCs (n = 6) were detected by RT-PCR. (J) The expression level of MITF in siATG5 DSCs (n = 8) and control DSCs (n = 8) was analyzed by RT-PCR. (K) The protein level of TNFRSF14 in ATG5^over ESCs (n = 4) or control ESCs (n = 4) was detected by western blotting. (L) Schematic diagram of the prediction of the binding region of the transcription factor MITF and wild-type or mutated TNFRSF14 promoter region. (M) Dual luciferase reporter assays were performed in HEK-293 T cells to verify the combination of MITF and wild-type or mutated TNFRSF14 promoter region. Data were presented as mean ± SEM or median and quartile and analyzed by t test or ANOVA. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001, NS: no significance

Figure 5.

TNFRSF14 enhances adhesion ability of DSC through MMP9. (A) MMP9 levels in vehicle or rapamycin-treated control or siTNFRSF14 DSCs (n = 6 per group) were analyzed by RT-PCR. (B) The transcription level of MMP9 in ESCs (n = 5) and DSCs (n = 6) was analyzed by RT-PCR. (C) The MMP9 transcription levels in ATG5^over ESCs (n = 4), control ESCs (n = 4), siATG5 DSCs (n = 8) and control DSCs (n = 8) were analyzed by RT-PCR. (D) The level of MMP9 protein in ATG5^over (n = 3) and control ESCs (n = 3) was detected by western blotting. (E) After treatment with different concentrations (0, 1, 10, 100, 1000 µM) of edaravone, the expression of MMP9 in DSCs (n = 6) was evaluated by western blotting. (F) The expression of adhesion molecules in DSCs (n = 6) after 1 mM edaravone treatment was analyzed by flow cytometry. (G) After treatment with C57 pregnant mice by vehicle (1% DMSO) (n = 5) or edaravone (n = 5, 2 mg/kg, daily). The embryo resorption rates of the two groups were compared (H) and the weight of the embryo and placenta was recorded (I) at the gestation of day 13.5. At the gestation of day 7.5, the proportion of PTPRC⁺ immune cells (J,K) in the uterus, the proportion and number of CD3⁻ KLRB1⁺ NK cells (J,L) and the expression of adhesion molecules on VIM⁺ USCs (M,N) of pregnant mice were analyzed by flow cytometry (n = 7 per group). Data were presented as mean ± SEM or median and quartile and analyzed by t test or ANOVA. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001, NS: no significance

Figure 6.

Patients with unexplained spontaneous abortion display insufficient DSC autophagy and dNK residence. (A) The transcriptional levels of autophagy-related genes (ATG5, MAP1LC3B and STSQT1) in DSCs of normal early pregnant women (NP, n = 6) and patients with unexplained spontaneous abortion (SA, n = 4) were detected by RT-PCR. (B) The mRNA levels of MITF, TNFRSF14 and MMP9 in DSCs of NP group (n = 6) and SA group (n = 4) were detected by RT-PCR. (C) The adhesion ability of NP group or SA group DSCs to dNK cells from NP or SA group (n = 6 per group) was evaluated by in vitro adhesion assays. The statistical graph of the number of adherent dNK cells was presented in (D). (E) The adhesion molecules on the surface of DSCs in NP (n = 6) or SA (n = 6) group were analyzed by flow cytometry. The statistical graph was shown in (F). (G) The density of NCAM1⁺ NK cells in NP (n = 6) or SA (n = 6) tissues was observed by immunofluorescence. The number of NK cells in the decidua was counted in (H). (I,J) After C57 pregnant mice were treated with KLRB1 neutralizing antibody (n = 7, 15 mg/kg, every other day) or isotype control IgG antibody (n = 7, 15 mg/kg, every other day) by intraperitoneal injection, the depletion degree of uterine NK cells in pregnant mice at the gestation of day 7.5 was verified by flow cytometry. (K,L) The number of implanted embryos and the embryo resorption rate of pregnant mice at the gestation of day 13.5 were recorded and counted. Data were presented as mean ± SEM or median and quartile and analyzed by t test or ANOVA. *P < 0.05, **P < 0.01, ***P < 0.001

Figure 7.

Rapamycin prevents pregnancy loss by promoting DSC autophagy and NK cell residence in decidua. After ESCs were treated with rapamycin (2 µM, 48 h), the transcriptional levels of MITF (n = 6), TNFRSF14 (n = 4) and MMP9 (n = 5) (A) and adhesion-related genes (n = 5) (B) were detected by RT-PCR. (C,D) At the gestation of day 13.5, embryo resorption rates were counted in normal pregnant mice (CBA/J♀×BABL/c♂, n = 6), spontaneous aborted mice (CBA/J♀×DBA/2♂) treated with vehicle (1% DMSO) (n = 4), or spontaneous aborted mouse models treated with rapamycin (n = 6, 0.04 mg/kg, three times a week). (E,F) Flow cytometry was used to detect the proportion and number of uterine CD3⁻ITGA2⁺ NK cells in PTPRC⁺ immune cells (n = 9) at the gestation of day 7.5. (G,H) The expression levels of adhesion molecules on VIM⁺ UCSs cells in the uterus of pregnant mice were also analyzed by flow cytometry (n = 9) at the gestation of day 7.5. Data were presented as mean ± SEM or median and quartile and analyzed by t test or ANOVA. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001, NS: no significance

Figure 8.

Schematic roles of rapamycin in preventing spontaneous abortion by triggering DSC autophagy-mediated NK residence during early pregnancy. Decidualization and establishment of normal pregnancy are accompanied by enhanced autophagy of DSCs and enrichment of dNK cells. High level of autophagy in DSCs increases the expression of the transcription factor MTIF. MITF enters into nucleus, binds to the promoter region of TNFRSF14 and positively regulates its transcription. TNFSF14, the TNFRSF14 ligand, is expressed on dNK cells. The TNFSF14-TNFRSF14 signal should contribute to the increases of adhesion molecules and the adhesion ability by upregulation of MMP9 expression. The strong adhesion of DSCs facilitates the localization and enrichment of NK cells in decidua. NK cells with sufficient density in decidua are beneficial to vascular remodeling, placental development, embryo growth, and maintenance of pregnancy. Spontaneous abortion of early pregnancy is associated with low levels of DSC autophagy, inactivation of the MITF-TNFRSF14-MMP9 axis, and insufficient NK cell residence. Autophagy inhibitor 3-MA or MMP9 inhibitor edaravone increases the risk of spontaneous abortion. The autophagy inducer rapamycin releases the inhibitory effect of MTOR on autophagy by inhibiting MTOR, and initiates the enhancement of DSC adhesion associated with MITF-TNFRSF14-MMP9 axis and more dNK cell residence, and prevents spontaneous abortion