Journal: Advanced Science

Article Title: Metabolic Reprogramming Driven by Trophoblasts and Decidual XCR1 + PMN‐MDSC Crosstalk Controls Adverse Outcomes Associated With Advanced Maternal Age

doi: 10.1002/advs.202513370

Figure Lengend Snippet: Impaired expression of the XCL1–XCR1 axis is linked to reduced dPMN‐MDSC abundance and function in advanced maternal age (AMA). (A) Incidence of pregnancy complications in healthy pregnancies (HP) and AMA ( n = 200). (B–D) Representative flow cytometry plots and statistical analysis of LOX1 + PMN‐MDSCs among CD45 + cells (peripheral blood, PB, n = 30. decidua, DE, HP, n = 23. AMA, n = 28). (E) Enriched signaling pathways of differentially expressed genes (DEGs) in neutrophils between the HP and AMA groups. (F) Uniform Manifold Approximation and Projection (UMAP) visualizing the neutrophil subclusters and proportion of each cluster (HP, n = 4. AMA, n = 3). (G) XCR1 expression in neutrophil cluster_1. (H) XCR1 expression pattern overlaid on the pseudotime trajectory of neutrophil clusters. (I) Dynamic changes in XCR1 expression along the inferred development path of neutrophil clusters. (J) mRNA expression level of XCR1 in decidual PMN‐MDSCs (dPMN‐MDSCs) ( n = 4 replicates). (K–M) Representative flow cytometry plots (K), percentages of XCR1 + cells in PMN‐MDSCs (L, HP, n = 19. AMA, n = 13), and statistical analysis of XCR1 + PMN‐MDSCs (M, n = 17). (N) Distribution of XCR1 expression among human decidua. (O–Q) mRNA expression levels (O, n = 4 replicates), percentages (P, n = 18), and secretion levels (Q, n = 4) of XCL1 in trophoblasts. (R) Distribution of XCL1 expression among human decidua. Data are presented as mean ± SEM. Each dot represents a single individual. ns, not significant; * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001. Statistical significance was determined using a Student's t‐ test (C, D, J, L, and O–Q), Mann–Whitney test (C, D, and M), or Wilcoxon test (G).

Article Snippet: Xcr1 −/− females were created by crossing Xcr1 −/− males with Xcr1 +/− females (Shanghai Model Organisms Center, Inc).

Techniques: Expressing, Flow Cytometry, Protein-Protein interactions, MANN-WHITNEY

Journal: Advanced Science

Article Title: Metabolic Reprogramming Driven by Trophoblasts and Decidual XCR1 + PMN‐MDSC Crosstalk Controls Adverse Outcomes Associated With Advanced Maternal Age

doi: 10.1002/advs.202513370

Figure Lengend Snippet: Weakened expression of the XCL1–XCR1 axis leads to FGR in AMA mice by diminishing dPMN‐MDSC count and function. (A) Cross strategies of different mouse models. (B–F) Representative image, maternal weight gain, embryo number per litter ( n = 10), placental weight, and fetal indicators ( n = 76, 58, and 48, respectively) in each group. (G–I) Representative flow cytometry and statistical analysis of dPMN‐MDSCs in CD45 + cells ( n = 10). (J–L) Representative flow cytometry (J), percentages of XCR1 + cells in PMN‐MDSCs (K, n = 8), and statistical analysis of XCR1 + PMN‐MDSCs (L, n = 14). (M) mRNA expression levels of Xcr1 in PMN‐MDSCs ( n = 4 replicates). (N) Percentages of XCR1 + cells in dPMN‐MDSCs across gestational stages ( n = 4). (O) Xcr1 expression among murine decidua at embryonic day E14. (P) Xcl1 expression among murine decidua. (Q) Percentages of XCL1 + cells in trophoblasts across gestational stages ( n = 4). (R–T) mRNA expression levels (R, n = 4 replicates), percentages (S, n = 6), and secretion levels (T, n = 4) of XCL1 in trophoblasts. Data are presented as mean ± SEM. Each dot represents a single mouse. ns, not significant; * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001. Statistical significance was determined using a Student's t‐ test (K, M, and R–T), Mann–Whitney test (K and L), or one‐way ANOVA (C–F, H, I, N, and Q). Post‐hoc analyses were performed using Tukey's test (C, E, F, H, N, and Q) or Dunnett's T3 test (D, F, and I).

Article Snippet: Xcr1 −/− females were created by crossing Xcr1 −/− males with Xcr1 +/− females (Shanghai Model Organisms Center, Inc).

Techniques: Expressing, Flow Cytometry, MANN-WHITNEY

Journal: Advanced Science

Article Title: Metabolic Reprogramming Driven by Trophoblasts and Decidual XCR1 + PMN‐MDSC Crosstalk Controls Adverse Outcomes Associated With Advanced Maternal Age

doi: 10.1002/advs.202513370

Figure Lengend Snippet: Decidual XCR1 + PMN‐MDSCs are essential for fetal development. (A) Cross strategies of 8–12‐week‐old males and females with different genotype backgrounds. (B–F) Representative image, maternal weight gain, embryo number per litter ( n = 4), placental weight, and fetal growth indicators ( n = 30, 29, and 16, respectively) in different groups. (G–I) Representative flow cytometry and statistical analysis of dPMN‐MDSCs in CD45 + cells ( n = 4). (J) Percentages of XCR1 + cells in dPMN‐MDSCs ( n = 4). (K) 24–32‐week‐old AMA mice were injected with splenic neutrophils from non‐pregnant wild‐type mice, dPMN‐MDSCs from pregnant Xcr1 +/+ or Xcr1 −/− mice intravenously (i.v.) at E13.5. (L–P) Representative image, maternal weight gain, embryo number per litter ( n = 4), placental weight, and fetal growth indicators ( n = 18, 32, and 19, respectively) across the groups. (Q) Proportions of dPMN‐MDSCs in CD45 + cells ( n = 4). Data are presented as mean ± SEM. Each dot represents a single mouse. ns, not significant; * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001. Statistical significance was determined using one‐way ANOVA (C–F, H–J, and M–Q). Post‐hoc analyses were performed using Tukey's test (C–F, H–J, and M–Q) or Dunnett's T3 test (P).

Article Snippet: Xcr1 −/− females were created by crossing Xcr1 −/− males with Xcr1 +/− females (Shanghai Model Organisms Center, Inc).

Techniques: Flow Cytometry, Injection

Journal: Advanced Science

Article Title: Metabolic Reprogramming Driven by Trophoblasts and Decidual XCR1 + PMN‐MDSC Crosstalk Controls Adverse Outcomes Associated With Advanced Maternal Age

doi: 10.1002/advs.202513370

Figure Lengend Snippet: XCL1 effectively improves FGR phenotypes in AMA mice but not in pregnant Xcr1 −/− mice. (A) 24–32‐week‐old AMA and 8–12‐week‐old pregnant Xcr1 −/− mice were injected daily with PBS or 0.2 µg recombinant mouse XCL1 protein (rXCL1) intraperitoneally (i.p.) from E13.5 to E16.5. (B–H) Representative image, maternal weight gain, embryo number per litter ( n = 6), placental weight, and fetal growth indicators ( n = 32, 50, 53, 38, and 32, respectively) between the groups. (I) Proportions of dPMN‐MDSCs in CD45 + cells ( n = 6). (J–L) Mean fluorescence intensity (MFI) of osteopontin (OPN), osteoglycin (OGN), and pleiotrophin (PTN) in dPMN‐MDSCs ( n = 6). (M) Percentages of XCR1 + cells in dPMN‐MDSCs ( n = 6). Data are presented as mean ± SEM. Each dot represents a single mouse. ns, not significant; * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001. Statistical significance was determined using a Student's t‐ test (C–M) or Mann–Whitney test (C, E, G, I, and M).

Article Snippet: Xcr1 −/− females were created by crossing Xcr1 −/− males with Xcr1 +/− females (Shanghai Model Organisms Center, Inc).

Techniques: Injection, Recombinant, Fluorescence, MANN-WHITNEY

Journal: Advanced Science

Article Title: Metabolic Reprogramming Driven by Trophoblasts and Decidual XCR1 + PMN‐MDSC Crosstalk Controls Adverse Outcomes Associated With Advanced Maternal Age

doi: 10.1002/advs.202513370

Figure Lengend Snippet: Attenuated XCL1–XCR1 expression that causes FGR is related to impaired FOXO1 expression and activity. (A, B) Enriched signaling pathways of DEGs in neutrophil cluster_1 between the HP and AMA groups (A) and the transcriptional factor in the FoxO signaling pathway (B). (C) mRNA expression level of FOXO1 in human dPMN‐MDSCs ( n = 4 replicates). (D–F) mRNA expression level (D, n = 4 replicates) and MFI of FOXO1 (E, n = 8) and phosphorylated FOXO1 (pFOXO1) (F, n = 8) in dPMN‐MDSCs. (G) Western blot analysis of whole‐cell lysate (WCL), cytoplasmic (C), and nuclear (N) distributions of FOXO1 expression in dPMN‐MDSCs of HP and AMA mice (replicated twice). (H) Western blot analysis of phosphorylated (p)‐ and total (t)‐AKT1 in dPMN‐MDSCs (replicated three times). (I–K, M–O) mRNA expression levels (I, M, n = 4 replicates) and MFI of FOXO1 (J, N, n = 6) and pFOXO1 (K, O, n = 6) in dPMN‐MDSCs. (L, P) Western blot analysis of p‐ and t‐AKT1 in dPMN‐MDSCs (replicated three times). (Q) 8–12‐week‐old Foxo1 fl/fl and Foxo1 fl/fl S100a8 cre females mated with 8–12‐week‐old males. (R–U) Maternal weight gain, embryo number per litter ( n = 6), placental weight, and fetal growth indicators ( n = 53 and 39, respectively) between the groups. (V, W) Statistical analysis of dPMN‐MDSCs in CD45 + cells ( n = 6). Data are presented as mean ± SEM. Each dot represents a single individual or mouse. ns, not significant; * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001. Statistical significance was determined using a Student's t ‐test (C–F, I, J, M, N, and R–W), Mann–Whitney test (K and O), or Wilcoxon test (B).

Article Snippet: Xcr1 −/− females were created by crossing Xcr1 −/− males with Xcr1 +/− females (Shanghai Model Organisms Center, Inc).

Techniques: Expressing, Activity Assay, Protein-Protein interactions, Western Blot, MANN-WHITNEY

Journal: Advanced Science

Article Title: Metabolic Reprogramming Driven by Trophoblasts and Decidual XCR1 + PMN‐MDSC Crosstalk Controls Adverse Outcomes Associated With Advanced Maternal Age

doi: 10.1002/advs.202513370

Figure Lengend Snippet: Activated FOXO1 promotes the transcription of OXPHOS‐related targets in decidual XCR1 + PMN‐MDSCs. (A) Venn diagram showing FOXO1 downstream target selection and IGV visualization showing the target binding peaks. (B–D) mRNA expression levels of Lnma , Plaur , and Casp9 in decidual XCR1 + PMN‐MDSCs across groups ( n = 4 replicates). (E) Dual‐luciferase reporter assay after transfection with the indicated plasmids ( n = 3 replicates). (F) Relative luciferase activity of the Lmna , Plaur , and Casp9 promoters upon FOXO1 overexpression or knockdown ( n = 3 replicates). (G–I) Enrichment of FOXO1 in the target promoters of decidual XCR1 + PMN‐MDSCs across groups ( n = 4 replicates). (J–M) Basal oxygen consumption rate (OCR), ATP production, and maximal respiratory capacity of decidual XCR1 + PMN‐MDSCs across groups ( n = 3 replicates). (N–Q) Basal OCR, ATP production, and maximal respiratory capacity of decidual XCR1 + PMN‐MDSCs from pregnant Foxo1 fl/fl and Foxo1 fl/fl S100a8 cre mice ( n = 3 replicates). (R, S) MFI of FOXO1 and pFOXO1 in dPMN‐MDSCs from AMA mice after treatment with adenoviral vector (AdV)‐XCL1 conditioned supernatant, transfection with AdV‐XCR1, or both ( n = 4). (T–V) mRNA expression levels of Lmna , Plaur , and Casp9 in dPMN‐MDSCs under different treatments ( n = 4 replicates). (W–Y) mRNA expression levels of Lmna , Plaur , and Casp9 in dPMN‐MDSCs from AMA and pregnant Xcr1 –/– mice after transfection with either AdV‐control (AdV‐Ctrl) or AdV‐FOXO1 ( n = 4 replicates). Data are presented as mean ± SEM. Each dot represents a single mouse. ns, not significant; * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001. Statistical significance was determined using a Student's t‐ test (C–I, K–M, and O–Y) or Mann–Whitney test (B, C, and G–I).

Article Snippet: Xcr1 −/− females were created by crossing Xcr1 −/− males with Xcr1 +/− females (Shanghai Model Organisms Center, Inc).

Techniques: Selection, Binding Assay, Expressing, Luciferase, Reporter Assay, Transfection, Activity Assay, Over Expression, Knockdown, Plasmid Preparation, Control, MANN-WHITNEY

Journal: Advanced Science

Article Title: Metabolic Reprogramming Driven by Trophoblasts and Decidual XCR1 + PMN‐MDSC Crosstalk Controls Adverse Outcomes Associated With Advanced Maternal Age

doi: 10.1002/advs.202513370

Figure Lengend Snippet: OXPHOS activation rescues developmental delays associated with impaired decidual XCR1 + PMN‐MDSCs. (A) 24–32‐week‐old AMA mice were subjected to the following treatments: daily injection with the control vehicle or 2.0 mg/kg Oltipraz from E13.5 to E16.5 or the transfer of decidual XCR1 + PMN‐MDSCs isolated from Oltipraz‐treated AMA donors at E13.5. (B–E) Maternal weight gain (B, n = 6, 6, and 4, respectively), embryo number per litter (C, n = 6, 6, and 4, respectively), and fetal growth indicators (D, E, n = 38, 46, and 35, respectively) in the respective indicated groups. (F) Percentages of dPMN‐MDSCs in CD45 + cells ( n = 6, 6, and 4, respectively). (G) Percentages of XCR1 + cells in dPMN‐MDSCs ( n = 6, 6, and 4, respectively). (H–J) MFI of FOXO1 (H), pFOXO1 (I), and pAKT1 (J) in dPMN‐MDSCs ( n = 6, 6, and 4, respectively). (K) mRNA expression levels of Lmna , Plaur , and Casp9 in dPMN‐MDSCs ( n = 4 replicates). (L) 8–12‐week‐old pregnant Xcr1 −/− mice were subjected to the following treatments: daily injection with the control vehicle or 2.0 mg/kg Oltipraz from E13.5 to E16.5 or the transfer of dPMN‐MDSCs isolated from Oltipraz‐treated pregnant Xcr1 −/− donors at E13.5. (M–P) Maternal weight gain (M, n = 4), embryo number per litter (N, n = 4), and fetal growth parameters (O, P, n = 23, 30, and 29, respectively). (Q) Percentages of dPMN‐MDSCs in CD45 + cells ( n = 4). (R) Percentages of XCR1 + cells in dPMN‐MDSCs ( n = 4). S–U) MFI of FOXO1 (S), pFOXO1 (T), and pAKT1 (U) in dPMN‐MDSCs ( n = 4). V) mRNA expression levels of Lmna , Plaur , and Casp9 in dPMN‐MDSCs ( n = 4 replicates). Data are presented as mean ± SEM. Each dot represents a single mouse. ns, not significant; * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001. Statistical significance was determined using one‐way ANOVA (B–K and M–V). Post‐hoc analyses were performed using Tukey's test (B–K, O–Q, and S–V), Dunn's test (E and N), or Dunnett's T3 test (M and R).

Article Snippet: Xcr1 −/− females were created by crossing Xcr1 −/− males with Xcr1 +/− females (Shanghai Model Organisms Center, Inc).

Techniques: Activation Assay, Injection, Control, Isolation, Expressing

Journal: Advanced Science

Article Title: Metabolic Reprogramming Driven by Trophoblasts and Decidual XCR1 + PMN‐MDSC Crosstalk Controls Adverse Outcomes Associated With Advanced Maternal Age

doi: 10.1002/advs.202513370

Figure Lengend Snippet: Metabolic imbalance mediated by reduced FOXO1 activity in decidual XCR1 + PMN‐MDSCs is associated with FGR in cases of AMA. (A, B) MFI of FOXO1 (A, n = 15) and pFOXO1 (B, n = 9) in human dPMN‐MDSCs. (C) Western blot analysis of whole‐cell lysate (WCL), cytoplasmic (C), and nuclear (N) distributions of FOXO1 expression in decidual XCR1 + PMN‐MDSCs (replicated twice). (D) Western blot analysis of p‐ and t‐AKT1 in decidual XCR1 + PMN‐MDSCs (replicated three times). (E) LMNA , PLAUR , and CASP9 expression levels in neutrophil cluster_1. (F) mRNA expression levels of the indicated genes in decidual XCR1 + PMN‐MDSCs ( n = 4 replicates). (G) Enrichment of FOXO1 to the promoters of LMNA , PLAUR , and CASP9 in decidual XCR1 + PMN‐MDSCs ( n = 4 replicates). (H–K) Basal OCR, ATP production, and maximal respiratory capacity of decidual XCR1 + PMN‐MDSCs ( n = 3 replicates). (L–N) Correlations between dPMN‐MDSC (L, n = 51), XCL1 + trophoblast (M, n = 36), decidual XCR1 + PMN‐MDSC (N, n = 38) levels and biparietal diameter (BPD), head circumference (HC), abdominal circumference (AC), femur length (FL), and humerus length (HL). Data are presented as mean ± SEM. Each dot represents a single individual. ns, not significant; * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001. Statistical significance was determined using a Student's t‐ test (A, F, and I–K), Mann–Whitney test (B and G), Wilcoxon test (E), or Pearson's correlation coefficient (L–N).

Article Snippet: Xcr1 −/− females were created by crossing Xcr1 −/− males with Xcr1 +/− females (Shanghai Model Organisms Center, Inc).

Techniques: Activity Assay, Western Blot, Expressing, MANN-WHITNEY