Fig 2: Effects of NOS2 inhibition by aminoguanidine (AG, 1 mM) or COX2 inhibition by indomethacin (Indo, 100 µM) on 4T1 phenotype along hypoxic gradients.a Spatial distribution of NOS2, COX2, and HIF1α imaged by widefield immunofluorescence microscopy of 7-day old untreated 4T1 disks (control), AG-treated disks, and Indo-treated disks. b NOS2, COX2, and HIF1α distributions in 7-day old untreated 4T1 disks (control, N = 3), AG-treated disks (N = 4 for NOS2, COX2, and HIF1α), and Indo-treated disks (N = 4). c Spatial distribution of DAF and TMRE fluorescence imaged by confocal microscopy of 7-day old untreated 4T1 disks (control), AG-treated disks, and Indo-treated disks. d DAF, TMRE, and ATP5A distributions in 7-day old untreated 4T1 disks (control, N = 4), AG-treated disks (N = 4), and Indo-treated disks (N = 4). “Normalized Distance” refers to the relative distance from the center to the edge of a disk. MFI mean fluorescence intensity normalized to the first point of the untreated control. Magnified areas of normoxic (green squares) and hypoxic (red squares) cells are displayed below each image. Scale bars = 500 µm, inset scale bars = 50 µm. Data plotted as mean ± SEM.

Fig 3: Upregulation of ALOX15 in human Tregs.Naive CD4+ T lymphocytes (Th0) were isolated from healthy volunteers, differentiated to Tregs (see methods for details) and the expression of lipid mediator biosynthetic enzymes was determined (A) using quantitative real-time PCR (B–F) using fluorescently labeled antibodies and flow cytometry and compared to the expression observed in naïve CD4+ T-cells (Th0). Protein expression in the two cell types was then normalized against the signal obtain for the respective isotype. (G) Representative histogram plots depicting the expression of ALOX and COX2 enzymes in natural Tregs isolated from human peripheral blood. (H) Temporal regulation of ALOX15 and FOXP3 during Treg differentiation was determined using fluorescently labeled antibodies and flow cytometry. Results are mean ± s.e.m. of n = 4–5 donors from two distinct experiments and expressed for A as fold change from Th0 (2-??CT) and for B–H as mean fluorescence intensity units (MFI); *p < 0.05, **p < 0.01, One sample t test for A or Unpaired t test with Welch’s correction for C–F; For G ***p < 0.001, 1-way ANOVA with Tukey multiple comparisons test.

Fig 4: IL-27 maintains normal pregnancy in a uterine COX-2+ macrophage-dependent manner.(A) Expression of COX-2 in uMφs of WT (mated with male Il27ra−/− mice, n = 8) or Il27ra−/− pregnant mice (mated with male WT mice, n = 8) was detected by FCM. (B) Expression of COX-2 in uMφs of saline (1%, n = 8) or 2-DG (50 mg/kg per day, n = 8)–treated pregnant mice was detected by FCM. (C) Expression of differentiation molecules in uMφs of WT (n = 8) or Ptgs2−/− pregnant mice (n = 8) was detected by FCM. (D to F) Photograph (red arrow: absorption point) of uterus, the CRL of embryos, or absorption rates from WT (n = 8) or Ptgs2−/− pregnant mice (n = 8). (G) Differential proteins of supernatants between IL-27RA+ dMφs and IL-27RA− dMφs were evaluated by the proteomic microarray. (H to J) Transcription levels of decidualization-related genes in uterus, depth of CK7+ trophoblast infiltration into uterus, and embryo absorption rates of Il27ra−/− pregnant mice (mated with male WT mice, n = 8) adopted with WT or Ptgs2−/− macrophages. Data were presented as means ± SEM or median and quartile and analyzed by t test, Mann-Whitney U test, or one-way ANOVA test. *P < 0.05, **P < 0.01, and ***P < 0.001.

Fig 5: The FBP–IL-27–COX-2 regulatory axis in decidua is insufficient in patients with unexplained RSA.(A) The mRNA levels of FBP1 and PFK1 between DSCs from normal pregnant women (n = 10) or patients with unexplained RSA (n = 10) were detected by RT-PCR. (B and C) Expression of FBP1 and PFK1 between decidua tissues from normal pregnant women (n = 12) or patients with unexplained RSA (n = 12) were detected by immunohistochemistry. (D) The FBP levels in the blood plasma from nonpregnant (NP) or pregnant (P) normal women or patients with unexplained RSA (n = 10 per group) were detected. (E) The mRNA level of IL-27 between DSCs from normal pregnant women (n = 10) or patients with unexplained RSA (n = 10) was detected by RT-PCR. (F and G) Expression of IL-27 between decidua tissues from normal pregnant women (n = 20) or patients with unexplained RSA (n = 20) was detected by immunohistochemistry. (H and I) The levels of COX-2 (H), CD80, CD86, INF-γ, CD206, CD209, and IRF4 (I) in dMφs from normal pregnant women (n = 12) or patients with unexplained RSA (n = 12) were detected by FCM. Data were presented as means ± SEM and analyzed by t test or one-way ANOVA test. *P < 0.05, **P < 0.01, and ***P < 0.001.