Journal: Inflammation

Article Title: Programmed death-ligand 1 (PD-L1) Modulates Chemokine Production Via the TLR4/TRAF6 Signaling Axis During LPS + IFN-γ-Induced Endotoxemia-mimicked Sepsis

doi: 10.1007/s10753-025-02394-2

Figure Lengend Snippet: PD-L1 mediates the regulation of chemokine biosynthesis in M1-polarized macrophages, as determined by RNA-seq. ( A ) PD-L1 NC /THP-1-M and PD-L1 HI /THP-1-M cells were treated with LPS (100 ng/mL) + IFN-γ (20 ng/mL) for 6 h and subjected to RNA sequencing analysis. The KEGG database was used to identify enriched signaling pathways whose activation significantly differed (up). Single-gene GSEA was conducted using the GSE57065 , GSE65682 , and GSE95233 datasets to identify the relationship between PD-L1 and chemokine signaling pathways (down). ( B ) Intersection analysis of upregulated genes between the PD-L1 HI /THP-1-M + LPS + IFN-γ group and PD-L1 NC /THP-1-M + LPS + IFN-γ group and between the PD-L1 NC /THP-1-M + LPS + IFN-γ group and PD-L1 NC /THP-1-M + PBS group via a Venn diagram, followed by further intersection with chemokine datasets, revealed three highly expressed PD-L1-mediated chemokines (CCL8, CXCL9, and CXCL10) under LPS + IFN-γ stimulation. ( C ) RNA sequencing analysis revealed the expression of CCL8, CXCL9 and CXCL10 upon PD-L1 overexpression. ( D ) PD-L1 NC /THP-1-M and PD-L1 HI /THP-1-M cells were treated with LPS + IFN-γ for 12 h, after which CCL8 and CXCL9 mRNA and protein levels were quantified ( n = 3 independent biological replicates). ( E ) Human monocytes were induced into macrophages, transfected with scramble siRNA and PD-L1 siRNA, and then treated with LPS + IFN-γ for 12 h. CCL8 and CXCL9 mRNA and protein levels were quantified ( n = 3 independent biological replicates). ( F ) PD-L1 NC /THP-1-M and PD-L1 KO /THP-1-M cells were treated with LPS + IFN-γ for 12 h, after which CCL8 and CXCL9 mRNA and protein levels were quantified ( n = 3 independent biological replicates). The data are presented as the mean ± SEM. Statistical analysis for D, E and F was performed by one-way ANOVA with LSD test for post hoc analyses. * p < 0.05

Article Snippet: CCL8 and CXCL9 concentrations in cell supernatants or plasma were quantified using ELISA kits (Boster Biological Technology; Wuhan, China) with absorbance measurements at 450 nm, following the manufacturer’s protocols.

Techniques: RNA Sequencing, Protein-Protein interactions, Activation Assay, Expressing, Over Expression, Transfection

Journal: Inflammation

Article Title: Programmed death-ligand 1 (PD-L1) Modulates Chemokine Production Via the TLR4/TRAF6 Signaling Axis During LPS + IFN-γ-Induced Endotoxemia-mimicked Sepsis

doi: 10.1007/s10753-025-02394-2

Figure Lengend Snippet: TLR4 acts as a critical upstream regulator of PD-L1 in modulating CCL8 and CXCL9 expression. ( A ) GSEA of RNA-seq data to investigate the relationship between PD-L1 expression and Toll-like receptor signaling pathway activation. ( B ) PD-L1 HI /THP-1-M and PD-L1 NC /THP-1-M cells were transfected with scramble siRNA or TLR4 siRNA, respectively, followed by 12 h of stimulation with LPS (100 ng/mL) + IFN-γ (20 ng/mL). The supernatants and cells were harvested for concurrent measurements of CCL8 and CXCL9 protein and mRNA levels via ELISAs and qRT‒PCR ( n =3 independent biological replicates). The data are presented as the mean ± SEM. Statistical analysis for B was performed by one-way ANOVA with LSD test for post hoc analyses. * p < 0.05

Article Snippet: CCL8 and CXCL9 concentrations in cell supernatants or plasma were quantified using ELISA kits (Boster Biological Technology; Wuhan, China) with absorbance measurements at 450 nm, following the manufacturer’s protocols.

Techniques: Expressing, RNA Sequencing, Activation Assay, Transfection

Journal: Inflammation

Article Title: Programmed death-ligand 1 (PD-L1) Modulates Chemokine Production Via the TLR4/TRAF6 Signaling Axis During LPS + IFN-γ-Induced Endotoxemia-mimicked Sepsis

doi: 10.1007/s10753-025-02394-2

Figure Lengend Snippet: PD-L1 binds to the TLR4 downstream molecule TRAF6 to regulate CCL8 and CXCL9 expression. ( A ) Venn diagram analysis of proteins coimmunoprecipitated with PD-L1 in THP-1-M cells stimulated with LPS (100 ng/mL) + IFN-γ (20 ng/mL) for 6 h via IP‒MS. After excluding proteins bound to the IgG control group, 289 PD-L1-specific interacting proteins were identified (up). Representative MS/MS spectrum showing peptide fragments of the TRAF6 protein identified in the immunoprecipitation complex (down). ( B ) THP-1-M (up) and human monocyte-derived (down) macrophages were treated with or without LPS+IFN-γ for 6 h and then harvested for Co-IP. TRAF6 protein levels were measured via Western blotting. ( C ) PD-L1 HI /THP-1-M and PD-L1 NC /THP-1-M cells were transfected with scramble siRNA or TRAF6 siRNA, respectively, followed by 12 hours of stimulation with LPS+IFN-γ. The supernatants and cells were harvested for concurrent measurements of CCL8 and CXCL9 protein and mRNA levels via ELISAs and qRT‒PCR ( n =3 independent biological replicates). ( D ) Structural models of full-length PD-L1 and TRAF6 predicted by AlphaFold (PAE plots were obtained from AlphaFold). ( E ) Molecular docking generic matching information. ( F ) A protein‒protein interaction cartoon model generated by PyMOL (left). Predicted binding sites between PD-L1 and TRAF6 (right). ( G ) PD-L1 NC /THP-1-M, PD-L1 HI /THP-1-M and PD-L1 Δ62-139 /THP-1-M cells were treated with LPS+IFN-γ for 12 h. The supernatants and cells were harvested for concurrent measurements of CCL8 and CXCL9 protein and mRNA levels via ELISAs and qRT‒PCR ( n =3 independent biological replicates). The data are presented as the mean ± SEM. Statistical analysis for C and G was performed by one-way ANOVA with LSD test for post hoc analyses. * p <0.05.

Article Snippet: CCL8 and CXCL9 concentrations in cell supernatants or plasma were quantified using ELISA kits (Boster Biological Technology; Wuhan, China) with absorbance measurements at 450 nm, following the manufacturer’s protocols.

Techniques: Expressing, Control, Tandem Mass Spectroscopy, Immunoprecipitation, Derivative Assay, Co-Immunoprecipitation Assay, Western Blot, Transfection, Generated, Binding Assay

Journal: Inflammation

Article Title: Programmed death-ligand 1 (PD-L1) Modulates Chemokine Production Via the TLR4/TRAF6 Signaling Axis During LPS + IFN-γ-Induced Endotoxemia-mimicked Sepsis

doi: 10.1007/s10753-025-02394-2

Figure Lengend Snippet: PD-L1 knockout attenuates septic mouse mortality and suppresses CCL8 and CXCL9 expression. ( A – C ) PD-L1 WT /mice and PD-L1 KO /mice were intraperitoneally injected with LPS (15 mg/kg) and IFN-γ (10 µg/kg) to induce endotoxemia-induced sepsis. ( B ) The survival rate was determined over a 60-hour observation period and is presented as a Kaplan‒Meier survival curve ( n = 15 mice per group). The results are presented as Kaplan–Meier survival curves. The results of the statistical analysis were analyzed with the log-rank test. * p < 0.05. ( C ) Peripheral blood was collected 12 h posttreatment for serum isolation and PBMC separation. CCL8 and CXCL9 protein levels in serum and their corresponding mRNA levels in PBMCs were subsequently analyzed ( n = 3 independent biological replicates). ( D ) Peritoneal macrophages (PMs) were extracted from PD-L1 wild-type and knockout mice and then treated with LPS (100 ng/mL) + IFN-γ (20 ng/mL) for 12 h. The cells were harvested for CCL8 and CXCL9 mRNA and protein detection by qRT‒PCR and ELISAs ( n = 3 independent biological replicates). (E) Peripheral blood monocytes were extracted from PD-L1 wild-type and knockout mice and then treated with LPS + IFN-γ for 12 h. The cells were harvested for CCL8 and CXCL9 mRNA and protein detection by qRT‒PCR and ELISAs ( n = 3 independent biological replicates). The data are presented as the mean ± SEM. Statistical analysis for C was performed by Student’s t test, and that for D and E was performed by one-way ANOVA with LSD test for post hoc analyses. * p < 0.05

Article Snippet: CCL8 and CXCL9 concentrations in cell supernatants or plasma were quantified using ELISA kits (Boster Biological Technology; Wuhan, China) with absorbance measurements at 450 nm, following the manufacturer’s protocols.

Techniques: Knock-Out, Expressing, Injection, Isolation