Journal: International journal of biological sciences

Article Title: PD-L1 promotes GSDMD-mediated NET release by maintaining the transcriptional activity of Stat3 in sepsis-associated encephalopathy.

doi: 10.7150/ijbs.79913

Figure Lengend Snippet: Figure 1. Neutrophils accumulate in the hippocampus and contribute to the progression of SAE. (A and B) Representative immunoblots and quantification of Ly6G levels in the hippocampus at 24 hours after operation. (C and D) Representative FACS plots and quantification of neutrophils (Ly6G+CD11b+) measured by flow cytometry in blood at 24 hours after operation. (E and F) Freezing to context and freezing to tone examined at 24 hours after operation. (G) The BBB permeability of hippocampus evaluated by Evans blue extravasation at 24 hours after operation. (H) Representative TUNEL (green) and DAPI (blue) immunofluorescence staining in the hippocampus. Scale bar indicates 20 μm. Higher magnification images are shown at the right row of figures-scale bar indicates 10 μm. (I) The quantitative results of the percentage of TUNEL-positive area in the total area of the image (whole microscopic field) in the hippocampus. (J) Representative IBA-1 (red) and DAPI (blue) immunofluorescence staining in the hippocampus. Scale bar indicates 20 μm. Higher magnification images are shown at the right row of figures-scale bar indicates 10 μm. (K) The quantitative results of the percentage of IBA-1 positive area in the total area of the image (whole microscopic field) in the hippocampus. The values are presented as mean ± SD (n=6 for Fig. B, D, G, I and K; n=8 for Fig. E and F; **P<0.01, ***P<0.001, ****P<0.0001, ns=not significant, one-way analysis of variance).

Article Snippet: Mice blood neutrophils were isolated by positive selection magnetic cell separation (MACS) using the Neutrophil Isolation Kit (130-097-658, Miltenyi Biotec, Bergisch Gladbach, Germany) according to instructions.

Techniques: Western Blot, Flow Cytometry, Permeability, TUNEL Assay, Immunofluorescence, Staining

Journal: International journal of biological sciences

Article Title: PD-L1 promotes GSDMD-mediated NET release by maintaining the transcriptional activity of Stat3 in sepsis-associated encephalopathy.

doi: 10.7150/ijbs.79913

Figure Lengend Snippet: Figure 2. Sepsis induces neutrophil releasing NETs and NETs digestion with DNase I attenuates the development of SAE. (A) Representative immunofluorescence images of isolated peripheral blood neutrophils at 24 hours after operation. Neutrophils are stained with SYTOX Orange (red) and Cit-H3 (green). Arrows indicate NETs. Scale bar indicates 10 μm. (B) Quantification of the percentage of Cit-H3-positive neutrophils. (C) Levels of plasma cfDNA are measured at 24 hours after operation. (D and E) Representative immunoblots of NETs appearance (D) and quantification of the Cit-H3 levels (E) in the hippocampus at 24 hours after operation. (F) Representative immunofluorescence images of Cit-H3 (green) and MPO (red) staining with blue DAPI nuclear staining in hippocampus. Neutrophils express MPO (red) and NET forming neutrophils also express Cit-H3 (green). Cyan fluorescence represents the colocalization of Cit-H3 with DNA. The white arrows point to neutrophils with NETs and the red arrows to neutrophils without NETs. Scale bar indicates 20 μm. Higher magnification images are shown at the right row of figures-scale bar indicates 10 μm. (G) Total NETs score of each group. (H and I) Freezing to context and freezing to tone examined at 24 hours after operation. (J) The BBB permeability of hippocampus evaluated by Evans blue extravasation at 24 hours after operation. (K) Representative TUNEL (green) and DAPI (blue) immunofluorescence staining in the hippocampus. Scale bar indicates 20 μm. Higher magnification images are shown at the right row of figures-scale bar indicates 10 μm. (L) The quantitative results of the percentage of TUNEL positive area in the total area of the image (whole microscopic field) in the hippocampus. (M) Representative IBA-1 (red) and DAPI (blue) immunofluorescence staining in the hippocampus. Scale bar indicates 20 μm. Higher magnification images are shown at the right row of figures, and the scale bar indicates 10 μm. (N) The quantitative results of the percentage of IBA-1 positive area in the total area of the image (whole microscopic field) in the hippocampus. The values are presented as mean ± SD (n=6; *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001, ns=not significant, one-way analysis of variance).

Article Snippet: Mice blood neutrophils were isolated by positive selection magnetic cell separation (MACS) using the Neutrophil Isolation Kit (130-097-658, Miltenyi Biotec, Bergisch Gladbach, Germany) according to instructions.

Techniques: Immunofluorescence, Isolation, Staining, Clinical Proteomics, Western Blot, Fluorescence, Permeability, TUNEL Assay

Journal: International journal of biological sciences

Article Title: PD-L1 promotes GSDMD-mediated NET release by maintaining the transcriptional activity of Stat3 in sepsis-associated encephalopathy.

doi: 10.7150/ijbs.79913

Figure Lengend Snippet: Figure 3. GSDMD regulates NET release and GSDMD deficiency in neutrophils attenuates the progression of SAE. (A) Representative immunofluorescence images of isolated peripheral blood neutrophils at 24 hours after operation. Neutrophils are stained with SYTOX Orange (red) and Cit-H3 (green). Arrows indicate NETs. Scale bar indicates 10 μm. (B) Quantification of the percentage of Cit-H3-positive neutrophils. (C) Levels of plasma cfDNA are measured at 24 hours after operation. (D and E) Representative immunoblots of NETs appearance (D) and quantification of the Cit-H3 levels (E) in the hippocampus at 24 hours after operation. (F) Representative immunofluorescence images of Cit-H3 (green) and MPO (red) staining with blue DAPI nuclear staining in hippocampus. Neutrophils express MPO (red) and NET forming neutrophils also express Cit-H3 (green). Cyan fluorescence represents the colocalization of Cit-H3 with DNA. The white arrows point to neutrophils with NETs and the red arrows to neutrophils without NETs. Scale bar indicates 20 μm. Higher magnification images are shown at the right row of figures-scale bar indicates 10 μm. (G) Total NETs score of each group. (H and I) Freezing to context and freezing to tone examined at 24 hours after operation. (J) The BBB permeability of hippocampus evaluated by Evans blue extravasation at 24 hours after operation. (K) Representative TUNEL (green) and DAPI (blue) immunofluorescence staining in the hippocampus. Scale bar indicates 20 μm. Higher magnification images are shown at the right row of figures-scale bar indicates 10 μm. (L) The quantitative results of the percentage of TUNEL positive area in the total area of the image (whole microscopic field) in the hippocampus. (M) Representative IBA-1 (red) and DAPI (blue) immunofluorescence staining in the hippocampus. Scale bar indicates 20 μm. Higher magnification images are shown at the right row of figures-scale bar indicates 10 μm. (N) The quantitative results of the percentage of IBA-1 positive area in the total area of the image (whole microscopic field) in the hippocampus. The values are presented as mean ± SD (n=6; *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001, ns=not significant, one-way analysis of variance).

Article Snippet: Mice blood neutrophils were isolated by positive selection magnetic cell separation (MACS) using the Neutrophil Isolation Kit (130-097-658, Miltenyi Biotec, Bergisch Gladbach, Germany) according to instructions.

Techniques: Immunofluorescence, Isolation, Staining, Clinical Proteomics, Western Blot, Fluorescence, Permeability, TUNEL Assay

Journal: International journal of biological sciences

Article Title: PD-L1 promotes GSDMD-mediated NET release by maintaining the transcriptional activity of Stat3 in sepsis-associated encephalopathy.

doi: 10.7150/ijbs.79913

Figure Lengend Snippet: Figure 4. PD-L1 can regulate GSDMD expression in septic neutrophils. (A to C) Representative immunoblots and quantification of PD-L1 and GSDMD levels in the neutrophils from healthy subjects and septic patients. (D to F) Representative immunoblots and quantification of PD-L1 and GSDMD levels in the neutrophils from septic patients at 21 hours after PD-L1 siRNA treatment. (G) The GSDMD mRNA levels of neutrophils from septic patients at 21 hours after PD-L1 siRNA treatment. (H) Representative immunofluorescence images of isolated peripheral blood neutrophils from healthy subjects and septic patients. Neutrophils are stained with SYTOX Orange (red) and Cit-H3 (green). Arrows indicate NETs. Scale bar indicates 10 μm. (I) Quantification of the percentage of Cit-H3-positive neutrophils. (J) cfDNA levels of supernatant of cultured neutrophils are measured at 21 hours after PD-L1 siRNA treatment. (K) The nucleus is extracted from neutrophils from healthy subjects and septic patients. Representative immunoblot of PD-L1 in neutrophil nucleus. (L) Representative images of neutrophils PD-L1 from healthy subjects and septic patients by confocal microscopy. Neutrophils are stained with PD-L1 (green) and DAPI (blue). Scale bar indicates 10 μm. Higher magnification images are shown at the right row of figures-scale bar indicates 5 μm. The values are presented as mean ± SD (n=6; *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001, ns=not significant, 2-tailed Student’s t test for 4B, 4C, 4E, and 4F; one-way analysis of variance for 4G, 4I, 4J).

Article Snippet: Mice blood neutrophils were isolated by positive selection magnetic cell separation (MACS) using the Neutrophil Isolation Kit (130-097-658, Miltenyi Biotec, Bergisch Gladbach, Germany) according to instructions.

Techniques: Expressing, Western Blot, Immunofluorescence, Isolation, Staining, Cell Culture, Confocal Microscopy

Journal: International journal of biological sciences

Article Title: PD-L1 promotes GSDMD-mediated NET release by maintaining the transcriptional activity of Stat3 in sepsis-associated encephalopathy.

doi: 10.7150/ijbs.79913

Figure Lengend Snippet: Figure 5. nPD-L1 forms a complex with p-Y705-Stat3 to transcriptionally activate GSDMD expression in neutrophils from septic patients. (A and B) Representative immunoblots and quantification of p-Y705-Stat3 level in the neutrophils from healthy subjects and sepsis patients. (C) Immunoprecipitation (IP) and western blot analysis of the PD-L1/p-Y705-Stat3 interaction in neutrophils from healthy subjects and septic patients. (D) Representative images of neutrophils PD-L1/p-Y705-Stat3 interaction from healthy subjects and septic patients by confocal microscopy. Neutrophils are stained with p-Y705-Stat3 (green), PD-L1 (red) and DAPI (blue). Scale bar indicates 10 μm. Higher magnification images are shown at the right row of figures, and the scale bar indicates 5 μm. (E and F) The nucleus is extracted from septic neutrophils at 24 hours after treating with DMSO or inhibitor HO-3867 (20 μM). Representative immunoblots and quantification of PD-L1 level in neutrophil nucleus. (G and H) Representative immunoblots and quantification of GSDMD level in the neutrophils from septic patients at 24 hours after treating with DMSO or inhibitor HO-3867 (20 μM). (I) The GSDMD mRNA levels of neutrophils from septic patients at 24 hours after treating with DMSO or inhibitor HO-3867 (20 μM). (J) Sequential ChIP-PCR analysis of the interactions between p-Y705-Stat3 and the GSDMD promoter in septic neutrophils. The values are presented as mean ± SD (n=6; **P<0.01, ****P<0.0001, ns=not significant, 2-tailed Student’s t test for 5B; one-way analysis of variance for 5F, 5H, and 5I).

Article Snippet: Mice blood neutrophils were isolated by positive selection magnetic cell separation (MACS) using the Neutrophil Isolation Kit (130-097-658, Miltenyi Biotec, Bergisch Gladbach, Germany) according to instructions.

Techniques: Expressing, Western Blot, Immunoprecipitation, Confocal Microscopy, Staining

Journal: International journal of biological sciences

Article Title: PD-L1 promotes GSDMD-mediated NET release by maintaining the transcriptional activity of Stat3 in sepsis-associated encephalopathy.

doi: 10.7150/ijbs.79913

Figure Lengend Snippet: Figure 6. Genetic deletion of neutrophil PD-L1 reduces the expression of GSDMD in neutrophils in vivo and attenuates the release of NETs in CLP mice. (A and B) Representative FACS plots and quantification of GSDMD+ neutrophils measured by flow cytometry in blood at 24 hours after operation. (C) Representative immunofluorescence images of isolated peripheral blood neutrophils at 24 hours after operation. Neutrophils were stained with GSDMD (green) and DAPI (blue). Scale bar indicates 20 μm. Arrows indicate GSDMD+ neutrophils. (D) Quantification of the percentage of GSDMD-positive neutrophils. (E) Representative immunofluorescence images of isolated peripheral blood neutrophils at 24 hours after operation. Neutrophils are stained with SYTOX Orange (red) and Cit-H3 (green). Arrows indicate NETs. Scale bar indicates 10 μm. (F) Quantification of the percentage of Cit-H3-positive neutrophils. (G) Levels of plasma cfDNA are measured at 24 hours after sham or CLP surgery. (H and I) Representative immunoblots of NETs appearance (H) and quantification of the Cit-H3 levels (I) in the hippocampus at 24 hours after operation. (J) Representative immunofluorescence images of Cit-H3 (green) and MPO (red) staining with blue DAPI nuclear staining in hippocampus. Neutrophils express MPO (red) and NET forming neutrophils also express Cit-H3 (green). Cyan fluorescence represents the colocalization of Cit-H3 with DNA. The white arrows point to neutrophils with NETs and the red arrows to neutrophils without NETs. The scale bar indicates 20 μm. Higher magnification images are shown at the right row of figures-scale bar indicates 10 μm. (K) Total NETs score of each group. The values are presented as mean ± SD (n=6; ****P<0.0001, ns=not significant, one-way analysis of variance).

Article Snippet: Mice blood neutrophils were isolated by positive selection magnetic cell separation (MACS) using the Neutrophil Isolation Kit (130-097-658, Miltenyi Biotec, Bergisch Gladbach, Germany) according to instructions.

Techniques: Expressing, In Vivo, Flow Cytometry, Immunofluorescence, Isolation, Staining, Clinical Proteomics, Western Blot, Fluorescence

Journal: International journal of biological sciences

Article Title: PD-L1 promotes GSDMD-mediated NET release by maintaining the transcriptional activity of Stat3 in sepsis-associated encephalopathy.

doi: 10.7150/ijbs.79913

Figure Lengend Snippet: Figure 7. Genetic knockout of Neutrophil PD-L1 attenuates the progression of SAE. (A and B) Freezing to context and freezing to tone examined at 24 hours after operation. (C) The BBB permeability of hippocampus evaluated by Evans blue extravasation at 24 hours after operation. (D) Representative TUNEL (green) and DAPI (blue) immunofluorescence staining in the hippocampus. Scale bar indicates 20 μm. Higher magnification images are shown at the right row of figures-scale bar indicates 10 μm. (E) The quantitative results of the percentage of TUNEL positive area in the total area of the image (whole microscopic field) in the hippocampus. (F) Representative IBA-1 (red) and DAPI (blue) immunofluorescence staining in the hippocampus. Scale bar indicates 20 μm. Higher magnification images are shown at the right row of figures-scale bar indicates 10 μm. (G) The quantitative results of the percentage of IBA-1 positive area in the total area of the image (whole microscopic field) in the hippocampus. (H) Schematic illustration of the main findings: Sepsis-induced NETosis contributes to hippocampus-dependent memory impairment, and increases BBB permeability, neuronal apoptosis, and microglia activation in the hippocampus region. The NET release is promoted by the cleavage of GSDMD, which is transcriptionally regulated by the nuclear translocation of a PD-L1/p-Y705-Stat3 complex. Together, PD-L1/Stat3/GSDMD is essential for NET production and development of sepsis-associated encephalopathy. The values are presented as mean ± SD (n=6; *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001, one-way analysis of variance).

Article Snippet: Mice blood neutrophils were isolated by positive selection magnetic cell separation (MACS) using the Neutrophil Isolation Kit (130-097-658, Miltenyi Biotec, Bergisch Gladbach, Germany) according to instructions.

Techniques: Knock-Out, Permeability, TUNEL Assay, Immunofluorescence, Staining, Activation Assay, Translocation Assay

Journal: Advanced Science

Article Title: Activated Platelet–Released Heat Shock Protein 90α Triggers Autophagy‐Dependent Neutrophil Extracellular Trap Formation and Amplifies Sepsis

doi: 10.1002/advs.202515933

Figure Lengend Snippet: eHSP90α facilitated sepsis‐induced thrombosis by neutrophils. (A) Plasma HSP90α levels of sham and CLP‐operated mice in the absence or presence of anti‐PLTs treatment ( n = 5). (B) Plasma TAT levels of sham and CLP‐operated mice in the absence or presence of anti‐HSP90α treatment ( n = 5). (C) Plasma fibrinogen levels of sham and CLP‐operated mice in the absence or presence of anti‐HSP90α treatment ( n = 5). (D) HE staining and IHC staining of CD41 in livers and lungs of sham and CLP‐operated mice in the absence or presence of anti‐HSP90α treatment ( n = 5). HE staining statistical analysis for thrombus area in mice livers and lungs was shown ( n = 5). IHC staining statistical analysis for platelets in mice livers and lungs is shown ( n = 5). Scale bars: 200 µm. (E) Bar graphs displaying the levels of TNF‐α, IL‐6, and IL‐1β in plasma from each group. (F) Plasma TAT and fibrinogen levels of CLP‐operated mice, CLP‐operated mice treated with recombinant HSP90α and/or anti‐Ly6G antibody ( n = 5). (G) HE staining in livers and lungs of CLP‐operated mice, CLP‐operated mice treated with recombinant HSP90α and/or anti‐Ly6G antibody ( n = 5). HE staining statistical analysis for thrombus area in mice livers and lungs was shown ( n = 5). (H) Bar graphs displaying the levels of TNF‐α and IL‐6 in plasma from each group. All data are presented as the mean ± SD. Statistical analysis was conducted using one‐way ANOVA and Holm‐Šídák's multiple comparisons test (A–H). * P < 0.05, ** P < 0.01, *** P < 0.001. Each data point represents one mouse (A–H). For A–H, three independent experiments were performed.

Article Snippet: For NET formation in peripheral neutrophils, isolated neutrophils were cultured in 1640 medium (Procell, PM150110A) supplemented with 10% fetal bovine serum (Procell, 164220) for 4 h in a CO 2 incubator.

Techniques: Clinical Proteomics, Staining, Immunohistochemistry, Recombinant

Journal: Advanced Science

Article Title: Activated Platelet–Released Heat Shock Protein 90α Triggers Autophagy‐Dependent Neutrophil Extracellular Trap Formation and Amplifies Sepsis

doi: 10.1002/advs.202515933

Figure Lengend Snippet: Sepsis platelets promote NET formation via eHSP90α. (A) NET markers MPO‐DNA were measured in plasma samples from HD ( n = 10) and sepsis patients ( n = 10) by ELISA. (B) Immunofluorescence and quantification analysis of NETs in neutrophils from HD ( n = 10) and sepsis patients ( n = 10). Scale bars: 50 µm. Cells were stained with DAPI for DNA (blue) and anti‐MPO for PMNs or NETs (green). (C) Boxplots showing quantification of HSP90α in the plasma of HD ( n = 10) and sepsis patients ( n = 10). (D) Evaluate the correlation between serum HSP90α and MPO‐DNA concentration in sepsis patients ( n = 10). (E) Immunofluorescence and quantification analysis of NETs in neutrophils treated with HD PPP ( n = 10), sepsis PPP ( n = 10) and anti‐HSP90α preincubated sepsis PPP ( n = 10). Cells were stained with DAPI for DNA (blue) and anti‐MPO for PMNs or NETs (green). Scale bars: 50 µm. (F,G) Immunofluorescence and quantification analysis of NETs following in vitro co‐culture of neutrophils with HD PLTs ( n = 5), sepsis PLTs ( n = 5) and anti‐HSP90α preincubated sepsis PLTs ( n = 5). Scale bars: 25 µm. (H) Quantification of MPO‐DNA in the supernatant of neutrophils from each group ( n = 5). (I) Immunofluorescence and quantification analysis of NET‐forming neutrophils following recombinant HSP90α stimulation ( n = 4). Scale bars: 50 µm. All data are presented as the mean ± SD. Statistical analysis was conducted using an unpaired two‐tailed t‐test (A–C), one‐way ANOVA and Holm‐Šídák's multiple comparisons test (E–I) and two‐tailed Pearson's correlation test (D) * P < 0.05, ** P < 0.01, *** P < 0.001. Each data point represents one human sample (A–D) or one experiment well (E–I). For E–I, three independent experiments were performed. PPP, platelet‐poor plasma; PLTs, platelets.

Article Snippet: For NET formation in peripheral neutrophils, isolated neutrophils were cultured in 1640 medium (Procell, PM150110A) supplemented with 10% fetal bovine serum (Procell, 164220) for 4 h in a CO 2 incubator.

Techniques: Clinical Proteomics, Enzyme-linked Immunosorbent Assay, Immunofluorescence, Staining, Concentration Assay, In Vitro, Co-Culture Assay, Recombinant, Two Tailed Test

Journal: Advanced Science

Article Title: Activated Platelet–Released Heat Shock Protein 90α Triggers Autophagy‐Dependent Neutrophil Extracellular Trap Formation and Amplifies Sepsis

doi: 10.1002/advs.202515933

Figure Lengend Snippet: eHSP90α promotes autophagy‐mediated NET formation. (A) Representative images of transmission electron microscope following recombinant HSP90α stimulation ( n = 5). Scale bar: 1 µm. Red arrowheads denote autolysosomes. Autophagic vesicles (autophagosome/autolysosome) were quantified per cell. (B) Immunoblot and quantification analysis for LC3B and p62 in neutrophils incubated with recombinant HSP90α (10 µg/mL) ( n = 3) in absence or presence of anti‐HSP90α antibody (10 µg/mL) ( n = 3), 3‐MA (5 mM) ( n = 3) and bafilomycin A1 (1 µM) ( n = 3). (C,D) Representative immunofluorescence microscopy and quantification of LC3B positive cells in neutrophils incubated with recombinant HSP90α (10 µg/mL) ( n = 4) in absence or presence of anti‐HSP90α antibody (10 µg/mL) ( n = 4), 3‐MA (5 mM) ( n = 4) and bafilomycin A1 (1 µM) ( n = 4). Scale bars: 50 µm. The positive cells were defined as cells with more than five LC3 dots. (E,F), Immunofluorescence and quantification analysis of NETs in neutrophils incubated with recombinant HSP90α (10 µg/mL) ( n = 5) in absence or presence of anti‐HSP90α antibody (10 µg/mL) ( n = 5), 3‐MA (5 mM) ( n = 5) and bafilomycin A1 (1 µM) ( n = 5). Cells were stained with DAPI for DNA (blue) and anti‐MPO for PMNs or NETs (green). Scale bars: 50 µm. (G) Quantification of MPO‐DNA in the supernatant of neutrophils from each group ( n = 5). All data are presented as the mean ± SD. Statistical analysis was conducted using one‐way ANOVA and Holm‐Šídák's multiple comparisons test (A–G). * P < 0.05, ** P < 0.01, *** P < 0.001. Each data point represents one neutrophil (A) or one experiment well (B–G). For A–G, three independent experiments were performed. LC3B, microtubule associated protein 1 light chain 3 beta.

Article Snippet: For NET formation in peripheral neutrophils, isolated neutrophils were cultured in 1640 medium (Procell, PM150110A) supplemented with 10% fetal bovine serum (Procell, 164220) for 4 h in a CO 2 incubator.

Techniques: Transmission Assay, Microscopy, Recombinant, Western Blot, Incubation, Immunofluorescence, Staining

Journal: Advanced Science

Article Title: Activated Platelet–Released Heat Shock Protein 90α Triggers Autophagy‐Dependent Neutrophil Extracellular Trap Formation and Amplifies Sepsis

doi: 10.1002/advs.202515933

Figure Lengend Snippet: eHSP90α exerts its function through the TLR4‐MyD88 signaling axis. (A) The immunoprecipitation with anti‐HSP90α and anti‐TLR4 antibody was performed in neutrophils. (B) Immunofluorescence analysis of colocalization of FITC‐HSP90α and TLR4 in neutrophils. Control: unlabeled HSP90α. (C) Immunoblot and quantification analysis for LC3B and p62 in neutrophils incubated with recombinant HSP90α (10 µg/mL) ( n = 3) in absence or presence of TLR4 inhibitor (30 µ m ) ( n = 3), MyD88 inhibitor (1 µ m ) ( n = 3), 3‐MA (5 m m ) ( n = 3) and bafilomycin A1 (1 µ m ) ( n = 3). (D) The immunoprecipitation with anti‐MyD88 and anti‐Beclin 1 antibody was performed in neutrophils incubated with or without recombinant HSP90α (10 µg/mL). (E) Immunoblot and quantification analysis of p‐Beclin 1 and Beclin 1 in neutrophils incubated with recombinant HSP90α (10 µg/mL) ( n = 5) in absence or presence of anti‐HSP90α antibody (10 µg/mL) ( n = 5), TLR4 inhibitor (30 µ m ) ( n = 5), MyD88 inhibitor (1 µM) ( n = 5). (F,G) Immunofluorescence and quantification analysis of NETs in neutrophils incubated with recombinant HSP90α (10 µg/mL) ( n = 5) in absence or presence of TLR4 inhibitor (30 µM) ( n = 5) and MyD88 inhibitor (1 µM) ( n = 5). Cells were stained with DAPI for DNA (blue) and anti‐MPO for PMNs or NETs (green). Scale bars: 50 µm. (H) Quantification of MPO‐DNA in the supernatant of neutrophils from each group ( n = 5). All data are presented as the mean ± SD. Statistical analysis was conducted using one‐way ANOVA and Holm‐Šídák's multiple comparisons test (C, E–H). * P < 0.05, ** P < 0.01, *** P < 0.001. Each data point represents one experiment well (C, E, G, H). For A–H, three independent experiments were performed. LC3B, microtubule associated protein 1 light chain 3 beta.

Article Snippet: For NET formation in peripheral neutrophils, isolated neutrophils were cultured in 1640 medium (Procell, PM150110A) supplemented with 10% fetal bovine serum (Procell, 164220) for 4 h in a CO 2 incubator.

Techniques: Immunoprecipitation, Immunofluorescence, Control, Western Blot, Incubation, Recombinant, Staining

Journal: Journal of Experimental & Clinical Cancer Research : CR

Article Title: Neutrophil-macrophage crosstalk via NETs–IL-17/VEGF/S100A9 axis promotes hepatocellular carcinoma progression

doi: 10.1186/s13046-025-03618-x

Figure Lengend Snippet: The levels of NETs increase in HCC patients, especially in advanced and metastatic stages. A A heatmap illustrating the differential expression patterns of NETs-related genes in normal and HCC samples based on TCGA database analysis. B Box plots depicting the expression differences of NETs-related genes between normal and HCC samples from the TCGA database. C , D Representative images of HE (Hematoxylin and eosin) and IF staining for CD66b (C) and CitH3 (D) in tissue sections from HCs and HCC patients. The mean IOD of CD66b or CitH3 was quantified (Image Pro Plus) from three random regions per section (HC, n = 5; HCC, n = 5) and shown as fold change relative to HCs. E Representative images of IF staining for CitH3 in peripheral blood neutrophils from HC and HCC patient F Western blot analysis of CitH3 expression in peripheral blood neutrophils from HCs ( n = 8) and HCC ( n = 8) patients, with band intensities normalized to β-actin and shown as fold change relative to controls G ELISA analysis of serum MPO-DNA levels in HCs and HCC patients. H ELISA analysis of serum MPO-DNA levels in HCC patients at different TNM stages (I–IV). I ELISA analysis of serum MPO-DNA levels in HCC patients with and without metastasis. J ELISA analysis of serum MPO-DNA levels in HCC patients with intrahepatic metastasis and extrahepatic metastasis. K Western blot analysis of CitH3 expression in peripheral blood neutrophils from HCC patients with ( n = 6) and without ( n = 6) extrahepatic metastasis. Band intensities were normalized to β-actin and quantified relative to the non-metastasis group White and black scale bars: 50 μm. Data are presented as median (IQR) (G-J) and other data are presented as mean ± SD. * p < 0.05, ** p < 0.01, *** p < 0.001. Student’s t test (C, D, F, K), Mann–Whitney U test (B, G, I, J), Kruskal–Wallis test (H)

Article Snippet: Neutrophils were isolated from the peripheral blood of healthy volunteers and HCC patients using the Human Peripheral Blood Neutrophil Isolation Kit (P9040, Solarbio, China), according to the manufacturer’s instructions.

Techniques: Quantitative Proteomics, Expressing, Staining, Western Blot, Enzyme-linked Immunosorbent Assay, MANN-WHITNEY

Journal: Journal of Experimental & Clinical Cancer Research : CR

Article Title: Neutrophil-macrophage crosstalk via NETs–IL-17/VEGF/S100A9 axis promotes hepatocellular carcinoma progression

doi: 10.1186/s13046-025-03618-x

Figure Lengend Snippet: Activation of IL-17R/NF-κB signaling by NETs-attached IL-17 is responsible for NETs-induced M2 macrophage polarization. A Schematic illustration of the transcriptome sequencing protocol for THP1-M0 under three treatment conditions. The volcano plot shows significantly DEGs for Group B vs. A, Group C vs. A, and Group C vs. B. B KEGG pathway enrichment analysis of DEGs was conducted using the DAVID tool. C FCM analysis of the proportion of CD68 + CD206 + M2 derived from THP-1-M0 following 48-hour co-culture of HuH-7 cells with NETs and (or) AMG827. Quantification of the fold change in the proportion from three independent experiments is shown in the right panel. D Western blot analysis of IL-10 and VEGF expression in cell lysates following the same treatments as in C. Densitometric values were normalized to β-actin and compared to the control group, with quantification shown in the right panel E Western blot analysis of IL-17 expression in extracted and purified NETs from peripheral blood neutrophils of HCs ( n = 8) and HCC patients ( n = 8). F ELISA analysis of IL-17 levels carried by NETs standardized to 50 ng/µL in HCs ( n = 8) and patients with HCC ( n = 8). G Western blot analysis of IL-17 expression in peripheral blood neutrophils from HCs ( n = 8) and HCC ( n = 8) patients. Densitometric values were normalized to β-actin and shown as a fold change relative to HCs. H Representative IF images of IL-17 and NE staining in peripheral blood neutrophils with (lower panel) or without PMA stimulation (upper panel) for 4 h from HCs and HCC patients. I Western blot analysis was conducted to assess the expression levels of p-65 and p-p65(S536) in THP-1-M0 following 48 h co-culture of HuH-7 cells with NETs and (or) AMG827. Densitometric values were normalized to β-actin and shown as fold changes relative to control in the right panel. J FCM was used to analyze the proportion of CD68 + CD206 + M2 derived from THP-1-M0 following 48-hour co-culture of HuH-7 cells with NETs and (or) BAY11-7082. Quantification of the fold change in the proportion from three independent experiments is shown in the right panel. K IF analysis of CD206 + M2 macrophage from the above co-culture systems of J treatment for 48 h. L ELISA analysis of secretion levels of M2d-related cytokines VEGF, IL-10 and TGF-β, in the culture supernatant from the above co-culture systems of J treatment for 48 h White scale bars: 20 μm. Data are presented as mean ± SD. Ns, not significant. * p < 0.05, ** p < 0.01, *** p < 0.001. One-way ANOVA followed by the Newman-Keuls multiple comparison test (C, D, I, J, L); Student’s t test (F, G)

Article Snippet: Neutrophils were isolated from the peripheral blood of healthy volunteers and HCC patients using the Human Peripheral Blood Neutrophil Isolation Kit (P9040, Solarbio, China), according to the manufacturer’s instructions.

Techniques: Activation Assay, Sequencing, Derivative Assay, Co-Culture Assay, Western Blot, Expressing, Control, Purification, Enzyme-linked Immunosorbent Assay, Staining, Comparison

Journal: Thrombosis Journal

Article Title: Complement C5a induces the generation of neutrophil extracellular traps by inhibiting mitochondrial STAT3 to promote the development of arterial thrombosis

doi: 10.1186/s12959-022-00384-0

Figure Lengend Snippet: C5a promoted arterial thrombosis by triggering neutrophils to release NETs. A , B H&E staining of thrombus, cross-sections ( A ) and longitudinal sections ( B ). Scale bar = 100 µm. C Quantification of the thrombus size in cross-sections by H&E staining ( n = 5 each). D Thrombus weight ( n = 5 each). E The blood flow velocity in the LICA was dramatically decreased in the FeCl 3 -induced thrombus mice compared with the sham mice, and this change was reversed by PMX53. F Quantification of the blood flow velocity ( n = 4 each). G Immunofluorescence staining of cross-sections of the LCCA for CitH3 (red), Ly6g (green) and DAPI (blue) 6 h after FeCl 3 exposure. Thirty minutes before FeCl 3 exposure, PMX53 was administered by intraperitoneal injection. Scale bar = 75 µm. H Quantification of NET formation capacity shown as the ratio of NETs/neutrophil in LCCA cross-sections subjected to immunofluorescence staining (NaCl group = 3, PMX53 group = 4). I Representative images of immunofluorescence stainings of NET formation for DNA (SYTOX green), CitH3 (red), Ly6g(blue) in vitro after stimulation of C5a, and NET formation was attenuated by PMX53. Scale bar = 75 µm. J Quantification of NET formation capacity in vitro shown as the percentage of NET release, which was assessed by immunofluorescence staining ( n = 3 each). (K) NETosis was measured using a plate reader assay. NET release is expressed as percentage of total DNA ( n = 3 each). Data are presented as mean ± SD

Article Snippet: Mouse neutrophils were isolated from the bone marrow of tibias and femurs from healthy C57BL/6 J mice using the Neutrophil Isolation Kit (Solarbio, China) following the manufacturer’s instructions.

Techniques: Staining, Immunofluorescence, Injection, In Vitro

Journal: Thrombosis Journal

Article Title: Complement C5a induces the generation of neutrophil extracellular traps by inhibiting mitochondrial STAT3 to promote the development of arterial thrombosis

doi: 10.1186/s12959-022-00384-0

Figure Lengend Snippet: Interactions among C5a, mitochondrial STAT3 and NETs. A , B Western blot analysis was performed to test the expression levels of mitochondrial STAT3 and p-STAT3 (Ser 727 ) in neutrophil-like cells cocultured with C5a, compared with the control. VDAC, a marker of mitochondria, was used as a loading control for mitochondria ( n = 5 each). C NET release in response to buffer or AG490 was measured using a plate reader assay ( n = 3 each). D Representative images of immunofluorescence staining for DNA (SYTOX green), CitH3 (red) and Ly6g (blue) in vitro after stimulation with AG490 showing the presence of NETs. Scale bar = 100 µm. E Quantification of NET formation capacity shown as the percentage of NET release in vitro after stimulation with buffer or AG490, as assessed by immunofluorescence staining. ( n = 3 each). Data are presented as mean ± SD

Article Snippet: Mouse neutrophils were isolated from the bone marrow of tibias and femurs from healthy C57BL/6 J mice using the Neutrophil Isolation Kit (Solarbio, China) following the manufacturer’s instructions.

Techniques: Western Blot, Expressing, Control, Marker, Immunofluorescence, Staining, In Vitro

Journal: Thrombosis Journal

Article Title: Complement C5a induces the generation of neutrophil extracellular traps by inhibiting mitochondrial STAT3 to promote the development of arterial thrombosis

doi: 10.1186/s12959-022-00384-0

Figure Lengend Snippet: The reduction in arterial thrombotic burden induced by PMX53 was abolished by AG490 in vivo. A , B H&E staining of thrombus, cross-sections A and longitudinal sections B . The thrombus area was reduced by PMX53, and AG490 abolished this effect. A Scale bar = 100 µm; B Scale bar = 200 µm. C Quantification of the thrombus size in cross-sections by H&E staining ( n = 5 each). D Thrombus weight ( n = 5 each). E Blood flow velocity in the LICA. AG490 reversed the increased in blood flow induced by PMX53. F Quantification of the blood flow velocity ( n = 4 each). G Immunofluorescence staining of cross-sections of the LCCA for CitH3 (red), Ly6g (green) and DAPI (blue) 6 h after FeCl 3 exposure. Scale bar = 75 µm. H Quantification of NET formation capacity shown as the ratio of NETs/neutrophil in LCCA cross-sections subjected to immunofluorescence staining (NaCl group = 3; PMX53 group = 4; PMX53 + AG490 group = 3; AG490 group = 4). Data are presented as mean ± SD

Article Snippet: Mouse neutrophils were isolated from the bone marrow of tibias and femurs from healthy C57BL/6 J mice using the Neutrophil Isolation Kit (Solarbio, China) following the manufacturer’s instructions.

Techniques: In Vivo, Staining, Immunofluorescence

Journal: Thrombosis Journal

Article Title: Complement C5a induces the generation of neutrophil extracellular traps by inhibiting mitochondrial STAT3 to promote the development of arterial thrombosis

doi: 10.1186/s12959-022-00384-0

Figure Lengend Snippet: Visual summary: the effect of complement C5a in arterial thrombosis. In arterial thrombosis, C5a chemotactically attracts neutrophils to migrate towards the culprit site and triggers the release of NETs, which contribute to thrombosis by promoting coagulation and stabilizing clots. C5a-induced promotion of NET release is dependent on Mito-ROS production. C5a induces the Mito-ROS production by inhibiting mitochondrial STAT3 activity

Article Snippet: Mouse neutrophils were isolated from the bone marrow of tibias and femurs from healthy C57BL/6 J mice using the Neutrophil Isolation Kit (Solarbio, China) following the manufacturer’s instructions.

Techniques: Coagulation, Activity Assay