Journal: Cell Communication and Signaling : CCS

Article Title: Spinster homolog 2/S1P signaling ameliorates macrophage inflammatory response to bacterial infections by balancing PGE 2 production

doi: 10.1186/s12964-024-01851-z

Figure Lengend Snippet: Spns2/S1P deficiency enhances AA metabolism through p38-MAPK mediated cPLA 2 activation (A) The volcano plot illustrates the identification of 98 distinct metabolites between WT and Spns2 −/− PMs. AA and its derivatives are enriched in Spns2 −/− PMs. (B) KEGG enrichment analysis indicates elevated activities of glycerophospholipid metabolism and AA metabolism in Spns2 −/− PMs. (C) The heat map of the differential metabolites highlights the accumulation of AA derivatives and lysophospholipids in Spns2 −/− PMs. N = 6 biological replicates ( A to C ). ( D , E ) Deficient Spns2/S1P signaling enhances p38-MAPK mediated cPLA 2 activation. N = 3 biological replicates. Data are presented as mean ± s.e.m. (E) . P values were determined by one-way ANOVA with Sidak’s correction for multiple comparisons (E) . * P < 0.05; ** P < 0.01; *** P < 0.001; n.s., not significant

Article Snippet: Spns2 −/− models were treated with 1 ml of 10 μM S1P (Cayman Chemical, 62570), 1 μM EP2 antagonist PF-04418948 (TargetMOI, T3306) [ ], or 1 μM EP4 antagonist ONO-AE3-208 (TargetMOI, TQ0290) [ ] immediately after infection.

Techniques: Activation Assay

Journal: Cell Communication and Signaling : CCS

Article Title: Spinster homolog 2/S1P signaling ameliorates macrophage inflammatory response to bacterial infections by balancing PGE 2 production

doi: 10.1186/s12964-024-01851-z

Figure Lengend Snippet: Altered AA metabolism promotes PGE 2 production in Spns2 −/− PMs (A) The flow diagram illustrating AA metabolism reveals a significant elevation in the gene expression of Ptges , encoding mPGES-1, in Spns2 −/− PMs. TPM, transcripts per kilobase of exon model per million mapped reads. (B) The volcano plot of up-regulated genes in Spns2 −/− PMs highlights the significant alteration in Ptges expression. N = 3 biological replicates ( A and B ). ( C ) Spns2 −/− PMs release elevated levels of PGE 2 under resting conditions. N = 5 biological replicates. Data are presented as mean ± s.e.m. ( A and C ). P values were determined by unpaired t -test. * P < 0.05; ** P < 0.01; *** P < 0.001; n.s., not significant

Article Snippet: Spns2 −/− models were treated with 1 ml of 10 μM S1P (Cayman Chemical, 62570), 1 μM EP2 antagonist PF-04418948 (TargetMOI, T3306) [ ], or 1 μM EP4 antagonist ONO-AE3-208 (TargetMOI, TQ0290) [ ] immediately after infection.

Techniques: Gene Expression, Expressing

Journal: Cell Communication and Signaling : CCS

Article Title: Spinster homolog 2/S1P signaling ameliorates macrophage inflammatory response to bacterial infections by balancing PGE 2 production

doi: 10.1186/s12964-024-01851-z

Figure Lengend Snippet: Overproduction of PGE 2 impairs MAS activity and mitochondrial dynamics (A) Gene expression of E-type prostanoid receptors in resting PMs. TPM, transcripts per kilobase of exon model per million mapped reads. N = 3 biological replicates. (B) Inhibition of EP4 with ONO-AE3-208, but not EP2 with PF-04418948, elevates the protein levels of MAS components Slc25a12 and Slc25a13 in Spns2 −/− PMs. N = 3 biological replicates. (C) EP4 activation contributes to the downregulation of Slc25a12 and Slc25a13 in WT PMs exposed to either PGE 2 or Spns2 inhibitor SLF1081851. N = 3 biological replicates. ( D , E ) Flow cytometry analysis of overlaid Δψm probed by MitoTracker™ Red (MT, D) and mitochondrial mass probed by CytoFix™ MitoRed (CF, E). MFI, mean fluorescent intensity. (F) Inhibition of EP4 restores the average Δψm (calculated by the ratio of MT/CF) in Spns2 −/− PMs. N = 3 biological replicates ( D to F) . (G) EP4 inhibition modulates the expression of mitochondrial dynamics-related proteins, promoting mitochondrial fusion in Spns2 −/− PMs. N = 3 biological replicates. (H) Transmission electron microscopy reveals that EP4 inhibition facilitates mitochondrial fusion in Spns2 −/− PMs. Scale bar = 1 μm. Arrowheads indicate fused (red) and fragmented (green) mitochondrial morphology. N = 3 biological replicates. Data are presented as mean ± s.e.m. P values were determined by unpaired t -test (A) and one-way ANOVA with Sidak’s correction for multiple comparisons ( B to H) . * P < 0.05; ** P < 0.01; *** P < 0.001; n.s., not significant

Article Snippet: Spns2 −/− models were treated with 1 ml of 10 μM S1P (Cayman Chemical, 62570), 1 μM EP2 antagonist PF-04418948 (TargetMOI, T3306) [ ], or 1 μM EP4 antagonist ONO-AE3-208 (TargetMOI, TQ0290) [ ] immediately after infection.

Techniques: Activity Assay, Gene Expression, Inhibition, Activation Assay, Flow Cytometry, Expressing, Transmission Assay, Electron Microscopy

Journal: Cell Communication and Signaling : CCS

Article Title: Spinster homolog 2/S1P signaling ameliorates macrophage inflammatory response to bacterial infections by balancing PGE 2 production

doi: 10.1186/s12964-024-01851-z

Figure Lengend Snippet: Excessive EP4 activation impairs mitochondrial respiration and increases oxidative stress in Spns2 −/− PMs (A) Blocking EP4 with ONO-AE3-208 increases the oxygen consumption rates (OCR) in Spns2 −/− PMs. (B) Quantitative analysis of basal respiration, maximal respiration, ATP production, and proton leakage reveal the restoration of mitochondrial respiration following EP4 blockade. N = 4 biological replicates ( A and B) . (C) EP4 inhibition reduces intracellular lactate levels in Spns2 −/− PMs. N = 12 biological replicates. (D) Flow cytometry analysis reveals a decrease in MitoSOX™ Red-probed mtROS generation in ONO-AE3-208-treated Spns2 −/− PMs. N = 3 biological replicates. (E) Total intracellular ROS probed by CellROX ® Orange remains comparable among each group. N = 3 biological replicates. (F) EP4 inhibition diminishes the activities of total superoxide dismutase (SOD) and catalase, indicating alleviated oxidative stress in Spns2 −/− PMs. N = 6 biological replicates. Data in the panels A , B , D , and E are presented as mean ± s.e.m. In panels C and F , the central bands represent the median values, the boxes represent the distance between the third and the first quartile, and the whiskers represent the ranges between the minimum and maximum values. P values were determined by one-way ANOVA with Sidak’s correction for multiple comparisons. * P < 0.05; ** P < 0.01; *** P < 0.001; n.s., not significant

Article Snippet: Spns2 −/− models were treated with 1 ml of 10 μM S1P (Cayman Chemical, 62570), 1 μM EP2 antagonist PF-04418948 (TargetMOI, T3306) [ ], or 1 μM EP4 antagonist ONO-AE3-208 (TargetMOI, TQ0290) [ ] immediately after infection.

Techniques: Activation Assay, Blocking Assay, Inhibition, Flow Cytometry

Journal: Cell Communication and Signaling : CCS

Article Title: Spinster homolog 2/S1P signaling ameliorates macrophage inflammatory response to bacterial infections by balancing PGE 2 production

doi: 10.1186/s12964-024-01851-z

Figure Lengend Snippet: PGE 2 contributes to the early-phase hyperinflammation during bacterial infections ( A , B ) Flow cytometry analysis shows reduced mtROS generation probed by MitoSOX™ Red (A ) and decreased total intracellular ROS probed by CellROX ® Orange (B) in ONO-AE3-208-treated Spns2 −/− PMs at 3-h post-LPS challenge. N = 3 biological replicates ( A and B ). (C) EP4 blockade reduces the gene expression of inflammatory cytokines within 3-h post-LPS challenge due to the suppression of the lactate-ROS axis. Notably, EP2 blockade also attenuates the early-phase hyperinflammation, possibly via a mechanism independent of the lactate-ROS axis. Both EP2 and EP4 blockade partially restore the suppressed gene expression of inflammatory cytokines in Spns2 −/− PMs after 6-h post-LPS challenge. N = 3 biological replicates. (D) Schematic of the in vivo experiments using heat-killed E. coli -induced peritoneal infection models. (E, F) Both EP2 and EP4 inhibition alleviate hyperinflammation (E) and significantly improve survival rates (F) in Spns2 −/− sepsis models triggered by intraperitoneal infection with heat-killed E. coli . N = 6 biological replicates for cytokine measurement. N = 6 to 8 biological replicates for survival analysis. Data are presented as mean ± s.e.m. ( A , B , and E ) and percentage (F) . P values were determined by one-way ANOVA with Sidak’s correction for multiple comparisons ( A , B , and E ) and log-rank test adjusted by the Bonferroni method (F) . * P < 0.05; ** P < 0.01; *** P < 0.001; n.s., not significant. # indicates P value is less than the Bonferroni-corrected threshold

Article Snippet: Spns2 −/− models were treated with 1 ml of 10 μM S1P (Cayman Chemical, 62570), 1 μM EP2 antagonist PF-04418948 (TargetMOI, T3306) [ ], or 1 μM EP4 antagonist ONO-AE3-208 (TargetMOI, TQ0290) [ ] immediately after infection.

Techniques: Flow Cytometry, Gene Expression, In Vivo, Infection, Inhibition

Journal: Cell Communication and Signaling : CCS

Article Title: Spinster homolog 2/S1P signaling ameliorates macrophage inflammatory response to bacterial infections by balancing PGE 2 production

doi: 10.1186/s12964-024-01851-z

Figure Lengend Snippet: Excessive PGE 2 production induces immunosuppression as infection progresses (A) Spns2 −/− PMs exhibit significantly elevated gene expression of Ptges compared to WT PMs before and after the LPS challenge. TPM, transcripts per kilobase of exon model per million mapped reads. N = 3 biological replicates. (B) Spns2 −/− PMs release higher levels of PGE 2 than WT PMs within 6-h post-LPS challenge. N = 6 biological replicates. (C) Gene expression of E-type prostanoid receptors in PMs at 3-h post-LPS challenge. N = 3 biological replicates. (D) Blockade of both EP2 and EP4 enhances TNFα and IL-6 release by Spns2 −/− PMs within 12-h post-LPS challenge. N = 4 biological replicates. (E) Schematic of the in vivo experiments using CLP models. (F) Survival curves from CLP models demonstrate that partial recovery of the inflammatory response induced by either EP2 or EP4 blockade improves the survival of Spns2 −/− rats. N = 7 to 12 biological replicates. (G) The levels of serum pro-inflammatory cytokines measured at 36-h post-infection indicate that EP2 or EP4 inhibition is effective but insufficient to overcome immunosuppression in Spns2 −/− CLP models. N = 4 biological replicates. (H) Colony-forming units (CFU) counts in livers and spleens at 36-h post-infection reveal higher bacterial loads in EP2- and EP4-inhibited Spns2 −/− CLP models. N = 5 to 6 biological replicates. Data are presented as mean ± s.e.m. ( A to D , G , and H ) and percentage (F) . P values were determined by unpaired t -test ( A to C ), one-way ANOVA with Sidak’s correction for multiple comparisons ( D , G , and H ), and log-rank test adjusted by the Bonferroni method (F) . * P < 0.05; ** P < 0.01; *** P < 0.001; n.s., not significant. # indicates P value is less than the Bonferroni-corrected threshold

Article Snippet: Spns2 −/− models were treated with 1 ml of 10 μM S1P (Cayman Chemical, 62570), 1 μM EP2 antagonist PF-04418948 (TargetMOI, T3306) [ ], or 1 μM EP4 antagonist ONO-AE3-208 (TargetMOI, TQ0290) [ ] immediately after infection.

Techniques: Infection, Gene Expression, In Vivo, Inhibition

Journal: Cell Communication and Signaling : CCS

Article Title: Spinster homolog 2/S1P signaling ameliorates macrophage inflammatory response to bacterial infections by balancing PGE 2 production

doi: 10.1186/s12964-024-01851-z

Figure Lengend Snippet: Spns2/S1P signaling impacts mitochondrial functions through coordinated activation of multiple S1P receptors (A) All five S1PRs are transcriptionally detectable in PMs. N = 3 biological replicates. (B) Inhibition of individual S1PRs in WT PMs increases the gene expression of Ptges , while activation of S1PR2 and S1PR4 in Spns2 −/− PMs may slightly reduce Ptges expression. N = 4 biological replicates. (C) In WT PMs, blocking individual S1PRs reduces the fluorescence intensity of MitoTracker™ Red, indicating diminished ΔΨm. (D) S1PR3 blockade significantly increases the fluorescence intensity of CytoFix™ MitoRed, indicating increased mitochondrial mass, while blockade of other receptors may cause a slight increase in mitochondrial mass. (E) All treatments result in reduced average Δψm (calculated by the ratio of MT/CF) in WT PMs. N = 3 biological replicates ( C to E ). ( F , G ) In Spns2 −/− PMs, activation of S1PR3 and S1PR5 increases MitoTracker™ Red fluorescence intensity (F) , but none of the treatments affect mitochondrial mass (G) . (H) Only S1PR3 activation partially restores the average Δψm in Spns2 −/− PMs. N = 3 biological replicates ( F to H ). (I) All the antagonists increase mtROS generation in WT PMs, especially with S1PR2 and S1PR4 blockade. N = 3 biological replicates. (J) In Spns2 −/− PMs, activation of S1PR2 and S1PR4 attenuates oxidative stress. N = 3 biological replicates. Data are presented as mean ± s.e.m. P values were determined by unpaired t -test (A) and one-way ANOVA with Sidak’s correction for multiple comparisons ( B to J ). * P < 0.05; ** P < 0.01; *** P < 0.001; n.s., not significant

Article Snippet: Spns2 −/− models were treated with 1 ml of 10 μM S1P (Cayman Chemical, 62570), 1 μM EP2 antagonist PF-04418948 (TargetMOI, T3306) [ ], or 1 μM EP4 antagonist ONO-AE3-208 (TargetMOI, TQ0290) [ ] immediately after infection.

Techniques: Activation Assay, Inhibition, Gene Expression, Expressing, Blocking Assay, Fluorescence

Journal: Cell Communication and Signaling : CCS

Article Title: Spinster homolog 2/S1P signaling ameliorates macrophage inflammatory response to bacterial infections by balancing PGE 2 production

doi: 10.1186/s12964-024-01851-z

Figure Lengend Snippet: Schematic illustration of how Spns2/S1P signaling modulates mitochondrial functions and inflammatory response through PGE 2 production in macrophages

Article Snippet: Spns2 −/− models were treated with 1 ml of 10 μM S1P (Cayman Chemical, 62570), 1 μM EP2 antagonist PF-04418948 (TargetMOI, T3306) [ ], or 1 μM EP4 antagonist ONO-AE3-208 (TargetMOI, TQ0290) [ ] immediately after infection.

Techniques: