Fig 1: UDPG-P2Y14 pathway regulates STAT1 expression.a Stat1 siRNA transfected BMDMs were stimulated with IFN-?/LPS for 24 h, Nos2, Tnf, Il6, and Il1b expression was determined by real-time PCR. b Pgm1, Ugp2, Pygl or P2ry14 siRNA transfected BMDMs were stimulated with IFN-?/LPS ± UDPG for 24 h, Stat1 expression was determined by real-time PCR. c–f BMDMs were pretreated with inhibitor (GPI or 6AN) for 30 min or pre-transfected with siRNA (Pygl or G6pdx) for 24 h prior to stimulation with IFN-?/LPS for 24 or 36 h, STAT1, ZNF-148, IRF-1, and RARß expression was determined by western blot (c, d), RARß expression and location were analyzed by real-time PCR (e) and confocal microscope, scale bar, 10 µm (f). g–i P2ry14 siRNA or P2Y14-overexpression vectors (P2Y14-OE) transfected BMDMs were stimulated with IFN-?/LPS for 24 or 36 h, RARß expression and location were determined by real-time PCR (g, h) and confocal microscope, scale bar, 10 µm (i). j, k IFN-?/LPS stimulated BMDMs were treated with UDPG for 24 or 36 h, RARß and STAT1 expression was determined by real-time PCR (j) and western blot (k). l–q RARß-overexpression vectors (RARß-OE) or Rarb siRNA transfected BMDMs were stimulated with IFN-?/LPS for 24 or 36 h, Stat1, Nos2, Tnf, Il6, and Il1b expression was determined by real-time PCR (l, o), RARß, STAT1, iNOS, TNF, and IL-6 expression was determined by western blot (m, p) and ELISA (n, q). r Schematic representation of the promoter region on the upstream of the transcription start site of Stat1. BMDMs were pretreated with GPI or 6AN for 30 min prior to stimulation with IFN-?/LPS for 24 h, RARß enrichment around the promoter of Stat1 were analyzed by ChIP-PCR and IgG was used as a negative control. ChIP-qPCR were used to Stat1 quantitative detection and total genomic DNA was used as input. Data are presented as mean ± SEM of n = 3 biologically independent experiments (a, b, e, g, j, l, n, o, q) or n = 4 biologically independent experiments (h) or n = 12 from two independent experiments (r). P values were calculated using one-way ANOVA (a, b, e, g, j, o, q, and r) and two-tailed unpaired Student’s t-tests (h, l, and n).
Fig 2: Glycogen metabolism regulates macrophages.In inflammatory macrophages, glycogen is synthesized and then channeled through glycogenolysis to generate G6P and further through the pentose phosphate pathway to yield abundant NADPH, ensuring high levels of reduced glutathione for inflammatory macrophage survival. Glycogen metabolism also increases UDPG levels and the receptor P2Y14 in macrophages. The UDPG/P2Y14 signaling pathway not only upregulates the expression of STAT1 via activating RARß but also promotes STAT1 phosphorylation by downregulating phosphatase TC45.
Fig 3: UDPG regulates inflammatory macrophages via P2Y14 receptor.a–c Pgm1, Ugp2 or Gys1 siRNA transfected BMDMs were stimulated with IFN-?/LPS for 24 h, Nos2, Tnf, Il6 and Il1b expression was determined by real-time PCR. d Gys1, Pgm1 or Ugp2 siRNA transfected BMDMs were stimulated with IFN-?/LPS for 24 h, UDPG in supernatants was determined by ELISA. e IFN-?/LPS-stimulated BMDMs were treated with UDPG (0, 100 or 200 µM) for 24 or 36 h, Nos2, Tnf, Il6, and Il1b expression was determined by real-time PCR (left), iNOS, TNF and IL-6 expression was determined by western blot (middle) and ELISA (right). f, g Ugp2 or G6pdx siRNA transfected BMDMs were stimulated with IFN-?/LPS ± UDPG (200 µM) for 24 or 36 h, Nos2, Tnf, Il6, and Il1b expression was determined by real-time PCR (left), iNOS, TNF and IL-6 expression was determined by western blot (middle) and ELISA (right). h Pygl or G6pdx siRNA transfected BMDMs were stimulated with IFN-?/LPS for 24 h, UDPG in supernatants was determined by ELISA. i BMDMs were pretransfected with siRNA (Pygl or G6pdx) for 24 h prior to stimulation with IFN-?/LPS for 6 h and switched to 13C-glucose for 6 h, lysed cells were analyzed by LC-MS/MS to determine m + 6-labeled UDPG. j, k P2Y14 receptor expression in untreated, IFN-?/LPS- or IL-4-treated BMDMs was determined by real-time PCR (j) and western blot (k). l, m IFN-?/LPS-stimulated BMDMs were treated with UDPG (0, 100 or 200 µM) for 24 or 36 h, P2Y14 expression was determined by real-time PCR (l) and western blot (m). n–p P2ry14 siRNA transfected BMDMs were stimulated with or without IFN-?/LPS for 24 or 36 h, Nos2, Tnf, Il6, and Il1b expression was determined by real-time PCR (n), iNOS, TNF, and IL-6 expression was determined by western blot (o) and ELISA (p). Unless otherwise specified, n = 3 biologically independent experiments were performed. Data are presented as mean ± SEM. P values were calculated using one-way ANOVA. **p < 0.01, ****p < 0.0001.
Fig 4: Macrophage glycogen metabolism regulates sepsis in patients.a THP-1 cells were cultured with PMA (100 ng mL-1) for 3 days and differentiated into macrophages, followed with IFN-?/LPS or IL-4 stimulation for 24 h. NOS2, TNF, IL6 and IL1B expression was determined by real-time PCR, n = 3 biologically independent experiments. b–e PMA incubated THP-1 cells were pretreated for 30 min with GPI or 6AN prior to stimulation with IFN-?/LPS for 24 or 36 h, NOS2, TNF, IL6, and IL1B expression was determined by real-time PCR, n = 3 biologically independent experiments (b). UDPG in supernatants was determined by ELISA, n = 3 biologically independent experiments (c). RARß and STAT1 expression was determined by real-time PCR, n = 3 biologically independent experiments (d) and western blot (e). f, g PMA and IFN-?/LPS-stimulated THP-1 cells were treated with or without UDPG for 24 or 36 h, RARB, STAT1, NOS2, TNF, IL6, and IL1B expression was determined by real-time PCR, n = 3 biologically independent experiments (f), RARß and STAT1 expression was determined by western blot (g). h, i P2RY14 or RARB siRNA transfected THP-1 cells were stimulated with IFN-?/LPS for 24 h, STAT1, NOS2, TNF, IL6, and IL1B expression was determined by real-time PCR, n = 3 biologically independent experiments. j–l Blood samples from patients with sepsis (n = 25) and SIRS (n = 28) and healthy controls (n = 30) were collected. Intracellular glycogen levels in human peripheral blood CD14+ monocytes were determined by colorimetric assay (j). GYS1, PYGL, RARB, and STAT1 expression was determined in human peripheral blood CD14+ monocytes by real-time PCR (k, l). m, n Two sepsis patients’ CD14+ monocytes were isolated and cultured with M-CSF (20 ng mL-1) for 4 days, and then treated with GPI or 6AN respectively. UDPG in supernatants was determined by ELISA, n = 3 biologically independent experiments (m) and RARB, STAT1, NOS2, TNF, IL6, and IL1B expression was determined by real-time PCR (n), n = 3 biologically independent experiments. Data are presented as mean ± SEM. P values were calculated using one-way ANOVA (a–d and h–n) and two-tailed unpaired Student’s t-tests (f).
Fig 5: UDPG-P2Y14 pathway regulates STAT1 phosphorylation.a, b BMDMs were pretreated with GPI (20 or 50 µM) for 30 min or pretransfected with Pygl siRNA for 24 h prior to stimulation with IFN-?/LPS, followed by western blot analysis of STAT1 and its phosphorylation from 15 to 120 min after stimulation. c, d Pygl siRNA transfected BMDMs were treated with IFN-?/LPS ± UDPG, STAT1 and its phosphorylation was analyzed by two-photon confocal microscope, scale bar, 10 µm (c) and western blot (d). e BMDMs were pretransfected with Pygl siRNA for 24 h prior to stimulation with IFN-?/LPS, followed by western blot analysis of JAK1 and JAK2 from 15 to 120 min after stimulation. f, g BMDMs were pretreated with GPI alone or combined with MG132 (f) or Na3VO4 (g) for 30 min prior to stimulation with IFN-?/LPS, followed by western blot analysis of STAT1, JAK1, and JAK2 from 15 to 120 min after stimulation. h BMDMs were pretreated with GPI for 30 min prior to stimulation with IFN-?/LPS, the cytoplasmic and nuclear protein fractions were blotted for TC45, ß-actin (cytoplasmic marker) and Histone H3 (nuclear marker). i Ptpn2 siRNA transfected BMDMs were stimulated with IFN-?/LPS, STAT1, JAK1, JAK2, and TC45 were analyzed by western blot. j Pygl siRNA transfected BMDMs were stimulated with IFN-?/LPS ± UDPG, ERK, JNK, P38, JAK1, JAK2, and TC45 were analyzed from 15 to 120 min after stimulation by western blot. k, l IFN-?/LPS stimulated BMDMs were treated with UDPG alone or combined with U0126, SP600125 or SB203580 for 1 and 3 h, JAK1, JAK2, STAT1, and TC45 were analyzed by western blot (k). Nos2, Tnf, Il6, and Il1b expression was determined by real-time PCR (l). Data are presented as mean ± SEM of n = 3 biologically independent experiments. P values were calculated using one-way ANOVA.
Supplier Page from Abcam for Anti-P2Y14 antibody