JPT2 Affects Trophoblast Functions and Macrophage Polarization and Metabolism, and Acts as a Potential Therapeutic Target for Recurrent Spontaneous Abortion

Abstract

Recurrent spontaneous abortion (RSA) is a pregnancy-related condition with complex etiology. Trophoblast dysfunction and abnormal macrophage polarization and metabolism are associated with RSA; however, the underlying mechanisms remain unknown. Jupiter microtubule-associated homolog 2 (JPT2) is essential for calcium mobilization; however, its role in RSA remains unclear. In this study, it is found that the expression levels of JPT2, a nicotinic acid adenine dinucleotide phosphate-binding protein, are decreased in the villous tissues of patients with RSA and placental tissues of miscarried mice. Mechanistically, it is unexpectedly found that abnormal JPT2 expression regulates trophoblast function and thus involvement in RSA via c-Jun N-terminal kinase (JNK) signaling, but not via calcium mobilization. Specifically, on the one hand, JPT2 deficiency inhibits trophoblast adhesion, migration, and invasion by inhibiting the JNK/atypical chemokine receptor 3 axis. On the other hand, trophoblast JPT2 deficiency contributes to M1 macrophage polarization by promoting the accumulation of citrate and reactive oxygen species via inhibition of the JNK/interleukin-6 axis. Self-complementary adeno-associated virus 9-JPT2 treatment alleviates embryonic resorption in abortion-prone mice. In summary, this study reveals that JPT2 mediates the remodeling of the immune microenvironment at the maternal-fetal interface, suggesting its potential as a therapeutic target for RSA.

Keywords: jupiter microtubule‐associated homolog 2; macrophage metabolism; macrophage polarization; recurrent spontaneous abortion; trophoblast.

Figure 1

Jupiter microtubule‐associated homolog 2 (JPT2) expression is decreased in recurrent spontaneous abortion (RSA). A,B) Calcium and nicotinic acid adenine dinucleotide phosphate (NAADP) concentrations in the villous tissues of the normal pregnancy (healthy control [HC]) and RSA groups were measured via calcium assay and enzyme‐linked immunosorbent assay (ELISA), respectively (each group: n = 30). C) Schematic diagram of the animal experimental protocol. CBA/J females were mated with BALB/c males to establish NP or with DBA/2 males to establish an abortion‐prone pregnancy (AP). Mice were euthanized on day 11.5 of gestation, and embryo resorption rates were calculated. D) Black arrow indicates the embryo resorption. Scale bar, 0.5 cm. E) Determination of embryo resorption rates in the NP and AP groups on gestation day 11.5 (each group: n = 8). F,G) Calcium and NAADP concentrations in the placental tissues of mice in the NP and AP groups were measured via calcium assay and ELISA, respectively (each group: n = 8). H–M) Protein levels and statistical results of LSM12 and JPT2 in the villous tissues of HC (n = 10) and patients with RSA (n = 10) and placental tissues of NP mice (n = 8) and AP mice (n = 8) were determined via western blotting. N–Q) Representative immunohistochemistry (IHC) images and statistical results of JPT2 expression in the villous tissues of HC (n = 10) and patients with RSA patients (n = 10) and placental tissues of NP mice (n = 8) and AP mice (n = 8). Scale bar, 50 µm. R–T) Pregnant mice were randomly divided into four groups (NP, NP+NAADP (0.181 mg kg⁻¹), AP, and AP+NAADP (0.181 mg kg⁻¹) groups) and subsequently assayed for placental calcium concentration and protein levels of JPT2 (each group: n = 8). R) Calcium assay kit was used to measure the placental calcium concentration. Protein levels of JPT2 in the placenta of pregnant mice were determined via western blotting, and the statistical results are shown in (S) and (T) (each group: n = 8). Error bars indicate the standard deviation (SD) of the mean. Student's t‐test was used to assess the differences between two groups, and one‐way analysis of variance (ANOVA) was used to compare the differences among multiple groups. **P < 0.01, ***P < 0.001, and ****P < 0.0001; ns, not significant.

Figure 2

JPT2 regulates the trophoblast adhesion, migration, and invasion. A) Volcano plot shows the differentially expressed genes (DEGs) between the sh‐Ctrl and sh‐JPT2 group HTR8 cells. B) Gene ontology (GO) bubbles showing the functional annotation of differential gene enrichment in (A). GO:0007155: cell adhesion; GO:0022610: biological adhesion; GO:0030334: regulation of cell migration. C,D) Cell adhesion of JPT2‐knockdown or ‐overexpressed HTR8 and JEG‐3 cells. E,F) Migration and quantitative values of HTR8 cells were examined via wound healing assays. Scale bar, 200 µm. G,H) Invasive ability and quantitative values of HTR8 cells were determined via transwell assays. Scale bar, 200 µm. I–K) Protein bands and quantitative values of E‐cadherin and vimentin protein levels in JPT2‐knockdown or ‐overexpressed HTR8 cells. I) Representative protein blot images. J) Quantitative values of E‐cadherin protein levels. K) Quantitative values of vimentin protein levels. L) Heatmap showing the differential expression of genes enriched in “cell adhesion,” “biological adhesion,” and “regulation of cell migration” in (B). M,N) Protein images and quantitative values of ACKR3 protein levels in JPT2‐knockdown or ‐overexpressed HTR8 cells. O–W) Effects of ACKR3 overexpression on trophoblast adhesion, migration, and invasion. O,P) Cell adhesion of HTR8 and JEG‐3 cells. Q,R) Migration ability and quantitative values of HTR8 cells. Scale bar, 200 µm. S,T) Invasive ability and quantitative values of HTR8 cells. Scale bar, 200 µm. U) Representative protein blot images of E‐cadherin and vimentin protein levels in HTR8 cells. V) Quantitative values of E‐cadherin protein levels. W) Quantitative values of vimentin protein levels. Data are represented as the mean ± SD of at least three independent experiments, with each data point representing an independent experiment. Error bars indicate the SD of the mean. One‐way ANOVA was used to compare the differences among multiple groups. *P < 0.05, **P < 0.01, ***P < 0.001, and ****P < 0.0001.

Figure 3

JPT2‐deficient trophoblast‐derived conditional medium promotes M1 polarization and reactive oxygen species (ROS) accumulation in macrophages. A) Co‐localization of CD68 and CD86 or CD68 and CD206 in the decidual tissues of patients in the HC and RSA groups (each group: n = 10). Scale bar, 50 µm. B,C) mRNA expression levels of M1 (CD86, IL‐1β, and tumor necrosis factor [TNF]‐α) and M2 (CD206, CD163, and IL‐10) macrophage markers were measured in macrophages after intervention with the conditioned medium derived from HTR8 and JEG‐3 cells via qPCR. D) Representative fluorescence images of CD68 and CD86 in macrophages treated with the conditioned medium of HTR8 and JEG‐3 cells. Scale bar, 50 µm. E,F) Flow cytometry and representative fluorescence images showing the fluorescence intensity of dichlorofluorescein (DCF) in macrophages treated with the conditioned medium of HTR8 cells. Scale bar, 200 µm. G) Kyoto encyclopedia of genes and genomes (KEGG) pathway enrichment analysis of HTR8 cells in the sh‐Ctrl and sh‐JPT2 groups. H) Heatmap showing the differential expression of genes enriched in cytokine–cytokine receptor‐associated differential genes. Left panel: cytokine–cytokine receptor‐associated differential genes. Right panel: selected cytokines and chemokines. I,J) Differential expression of cytokines and chemokines selected from (H) in JP2‐knocked down HTR8 and JEG‐3 cells were determined via qPCR. K) ELISA revealed the expression levels of IL‐6 in JPT2‐knocked down HTR8 and JEG‐3 cell supernatants. L–O) Protein expression levels of IL‐6R in macrophages after intervention with the conditioned medium of HTR8 and JEG‐3 cells. Representative protein blot images are shown in (L) and (N). Statistical values of IL‐6R protein levels are shown in (M) and (O). P,Q) Macrophages were treated with the supernatants of HTR8 and JEG‐3 cells with or without IL‐6 (50 ng mL⁻¹), and mRNA expression levels of M1 (CD86, IL‐1β, and TNF‐α) and M2 (CD206, CD163, and IL‐10) macrophage markers were determined via qPCR. R) Representative fluorescence images of CD68 and CD86 and quantitative values of the percentage of CD86‐positive cells after treatment of macrophages with the supernatants of HTR8 cells with or without IL‐6 (50 ng mL⁻¹). Scale bar, 50 µm. S,T) Flow cytometry and representative fluorescence images show the fluorescence intensity of DCF in macrophages treated with the conditioned medium of HTR8 cells with or without IL‐6 (50 ng mL⁻¹). Scale bar, 200 µm. Data are represented as the mean ± SD of at least three independent experiments, with each data point representing an independent experiment. Error bars indicate the SD of the mean. Student's t‐test was used to assess the differences between two groups, and one‐way ANOVA was used to compare the differences among multiple groups. *P < 0.05, **P < 0.01, ***P < 0.001, and ****P < 0.0001.

Figure 4

c‐Jun N‐terminal kinase (JNK) signaling is critical for JPT2‐mediated regulation of trophoblast functions and promotion of M1 polarization and ROS accumulation in macrophages. A) KEGG pathway enrichment analysis of HTR8 cells in the sh‐Ctrl and sh‐JPT2 groups. B–H) Representative protein bands and statistical values of p‐p38, p38, phospho‐extracellular signal‐regulated kinase (p‐ERK), ERK, p‐JNK, and JNK in JPT2‐knocked down or ‐overexpressed HTR8 and JEG‐3 cells. I–P) Changes in the migration and invasion of trophoblasts after treatment with the JNK activator, anisomycin (10 µm). I–L) Migration ability and quantitative values of HTR8 and JEG‐3 cells. Scale bar, 200 µm. M–P) Invasive ability and quantitative values of HTR8 and JEG‐3 cells. Scale bar, 200 µm. Representative protein bands and statistical values of ACKR3 in HTR8 and JEG‐3 cells after treatment with the JNK activator, anisomycin (10 µm), are shown in (Q–T). After the pretreatment of trophoblasts with JNK activator anisomycin (10 µm), the supernatant of trophoblasts was collected and used to treat macrophages. U) Representative fluorescence images of CD68 and CD86 and quantitative values of the percentage of CD86‐positive cells in macrophages. Scale bar, 50 µm. V,W) Flow cytometry and representative fluorescence images showing the fluorescence intensity of DCF in macrophages treated with the conditioned medium of HTR8 cells. Scale bar, 200 µm. X,Y) ELISA revealed the expression levels of IL‐6 in the supernatants of HTR8 and JEG‐3 cells after the treatment of trophoblasts with the JNK activator, anisomycin (10 µm). Data are represented as the mean ± SD of at least three independent experiments, with each data point representing an independent experiment. Error bars indicate the SD of the mean. One‐way ANOVA was used to compare the differences among multiple groups. *P < 0.05, **P < 0.01, ***P < 0.001, and ****P < 0.0001; ns, not significant.

Figure 5

JPT2‐deficient trophoblasts promote M1 polarization and ROS accumulation by enhancing the citrate production in macrophages. A) Metabolic pathway bubble diagram showing the pathways of differential metabolite enrichment. B) Citric acid levels in macrophages. C) Citric acid levels in macrophages after intervention with HTR8 cell‐derived conditioned medium were determined via citric acid content assay. D–G) Protein expression of citrate synthase (Cs) in macrophages after intervention with the HTR8 and JEG‐3 cell‐derived conditioned medium. Representative protein bands of Cs are shown in (D) and (F). Statistical results of protein expression of Cs are shown in (E) and (G). H–J) After treatment of macrophages with empty vector (EV) and sh‐Cs plasmids, the macrophages were intervened with the culture supernatants of HTR8 and JEG‐3 cells. H) Representative fluorescence images of CD68 and CD86 in macrophages. Scale bar, 50 µm. I,J) Quantitative values of the percentage of CD86‐positive cells. K–N) Flow cytometry and representative fluorescence images showing the fluorescence intensity of DCF in macrophages treated with the conditioned medium of HTR8 cells. Scale bar, 200 µm. O–R) Intervention of macrophages with the conditioned medium of HTR8 and JEG‐3 cells with or without IL‐6 (50 ng mL⁻¹). Protein expression levels of Cs in macrophages were determined via western blotting. Data are represented as the mean ± SD of at least three independent experiments, with each data point representing an independent experiment. Error bars indicate the SD of the mean. Student's t‐test was used to assess the differences between two groups, and one‐way ANOVA was used to compare the differences among multiple groups. *P < 0.05, **P < 0.01, ***P < 0.001, and ****P < 0.0001.

Figure 6

Macrophages educated by JPT2‐deficient trophoblasts inhibit the trophoblast proliferation, adhesion, migration, and invasion and promote trophoblast apoptosis. A) Schematic diagram of the cell treatment process. THP‐1 cells were converted into M0 macrophages by phorbol 12‐myristate 13‐acetate (PMA). The supernatants (with or without IL‐6 [50 ng mL⁻¹]) of trophoblasts from the sh‐Ctrl and sh‐JPT2 groups were used to culture M0 macrophages or fresh trophoblasts. The educated macrophages were cultured with the fresh medium, and the supernatant of M0 macrophages was used to culture the fresh trophoblasts. Finally, the proliferation, adhesion, migration, and invasion of these fresh trophoblasts were determined. B,C) Cell adhesion of HTR8 and JEG‐3 cells. D–G) Proliferation of HTR8 and JEG‐3 cells was assessed via the 5‐ethynyl‐2′‐deoxyuridine (EdU) assay. Scale bar, 100 µm. H–K) Apoptosis of HTR8 and JEG‐3 cells was assessed via TdT‐mediated dUTP nick‐end labeling (TUNEL) staining. Scale bar, 200 µm. L–O) Migration ability of HTR8 and JEG‐3 cells was assessed via wound healing assays. Scale bar, 200 µm. P–S) Representative images and quantitative values of invasion of HTR8 and JEG‐3 cells. Scale bar, 200 µm. Data are represented as the mean ± SD of at least three independent experiments, with each data point representing an independent experiment. Error bars indicate the SD of the mean. One‐way ANOVA was used to compare the differences among multiple groups. **P < 0.01, ***P < 0.001, and ****P < 0.0001; ns, not significant.

Figure 7

JPT2 treatment alleviates embryo loss in a mouse miscarriage model. A) Representative protein bands and statistical values of JPT2 in the placenta of pregnant mice treated with scAAV‐JPT2 or scAAV‐NC (each group: n = 8). B,C) Embryo resorption rates of each group of pregnant mice were measured at day 11.5 of gestation (each group: n = 8). B) Black arrows indicate embryo resorption. Scale bar, 0.5 cm. C) Statistical results of embryo resorption rate (each group: n = 8). D) Representative fluorescence images of E‐cadherin (E‐cad) at the placental interface of mice. Scale bar, 50 µm. E) Quantitative values of E‐cadherin mean fluorescence intensity (MFI; each group: n = 8). F,G) Representative fluorescence images and quantitative values of vimentin (Vim) at the placental interface of mice (each group: n = 8). Scale bar, 50 µm. H,I) Representative fluorescence images and quantitative values of the percentage of F4/80‐positive cells at the placental interface of mice (each group: n = 8). Scale bar, 50 µm. J,K) Representative flow cytometry results and quantitative values of CD206 in the decidua macrophages of mice (each group: n = 8). L,M) Representative flow cytometry results and statistical values of CD86 in the decidua macrophages of mice (each group: n = 8). N,O) Immunostaining and statistical results of ACKR3 at the placental interface of mice (each group: n = 8). Scale bar, 50 µm. P,Q) Immunostaining statistical results for IL‐6R in the decidua macrophages of mice (each group: n = 8). Scale bar, 50 µm. R–U) Representative protein bands and statistical values of p‐p38, p38, p‐ERK, ERK, p‐JNK, and JNK at the placental interface of mice (each group: n = 8). Error bars indicate the SD of the mean. Student's t‐test was used to assess the differences between two groups, and one‐way ANOVA was used to compare the differences among multiple groups. ***P < 0.001 and ****P < 0.0001; ns, not significant.

Figure 8

Activation of JNK signaling is vital for RSA treatment via JPT2. A) Pregnant mice received JNK inhibitor (JNK‐IN‐8, 20 mg kg⁻¹) injections on days 8.5 and 10.5 of gestation, mice were euthanized on day 11.5 of gestation, and embryo resorption rates were calculated (each group: n = 8). Scale bar, 0.5 cm. B) Immunostaining ACKR3 at the placental interface of mice (each group: n = 8). Scale bar, 50 µm. C) Immunostaining of IL‐6R in the decidual macrophages of mice (each group: n = 8). Scale bar, 50 µm.

Figure 9

JPT2 deficiency inhibits trophoblast functions via the JNK signaling pathway and reprograms macrophage polarization, leading to RSA. On the one hand, JPT2 deficiency inhibited trophoblast adhesion, migration, and invasion by inhibiting the JNK/ACKR3 axis. On the other hand, the lack of JPT2 in trophoblasts contributed to M1 macrophage polarization by promoting the accumulation of citrate and ROS via inhibition of the JNK/IL‐6 axis, which subsequently affected the trophoblast functions. Cs: Citrate synthase.