Journal: Cellular and Molecular Life Sciences: CMLS

Article Title: Knockout of the C3a receptor protects against renal ischemia reperfusion injury by reduction of NETs formation

doi: 10.1007/s00018-023-04967-6

Figure Lengend Snippet: Abolition of C3aR blocks the formation of NETs during I/R. A Immunofluorescence staining of kidney sections from WT, C3aRA, and C3aR KO mice after 45 min of ischemia and 24 h of reperfusion. DAPI (blue), myeloperoxidase (MPO, red), CitH3 (green). Colocalization of MPO and CitH3 (yellow) with DNA (DAPI, blue) indicates of NETs. Scale bar: 25 μm. B Immunohistochemical (IHC) images showing PAD4-positive cells in kidney sections. Scale bars: 10 μm. C Representative images and quantitative analysis of CitH3 in whole kidney lysates, as assessed by Western blotting. * P < 0.5, ns, no significant

Article Snippet: For the NETs disruption experiments, PAD4 inhibitor GSK 484 (4 mg/kg, MCE, cat# HY-100514/CS-0019553, powder dissolved in PBS and 2% DMSO), DNase I (20 mg/kg, Roche, cat# 11284932001, powder dissolved in PBS and 2% DMSO), NEi (2.5 mg/kg, GW311616 , MCE, cat# HY-15891/CS-3126, powder dissolved in PBS and 5% DMSO) or vehicle (2% DMSO) was administered by intraperitoneal injection 24 h and 2 h before surgery and every 12 h thereafter.

Techniques: Immunofluorescence, Staining, Immunohistochemical staining, Western Blot

Journal: Cellular and Molecular Life Sciences: CMLS

Article Title: Knockout of the C3a receptor protects against renal ischemia reperfusion injury by reduction of NETs formation

doi: 10.1007/s00018-023-04967-6

Figure Lengend Snippet: The proposed signaling pathways of NETs formation following AKI. Following I/R, a large amount of C3 and a large number of neutrophils infiltrate into the interstitial space. C3a binds to C3aR on the surface of neutrophils, activates the neutrophils, and induces the release of NETs via the ERK/ROS/PAD4 pathway

Article Snippet: For the NETs disruption experiments, PAD4 inhibitor GSK 484 (4 mg/kg, MCE, cat# HY-100514/CS-0019553, powder dissolved in PBS and 2% DMSO), DNase I (20 mg/kg, Roche, cat# 11284932001, powder dissolved in PBS and 2% DMSO), NEi (2.5 mg/kg, GW311616 , MCE, cat# HY-15891/CS-3126, powder dissolved in PBS and 5% DMSO) or vehicle (2% DMSO) was administered by intraperitoneal injection 24 h and 2 h before surgery and every 12 h thereafter.

Techniques: Protein-Protein interactions

Journal: Advanced Science

Article Title: MMP9 High Neutrophils are Critical Mediators of Neutrophil Extracellular Traps Formation and Myocardial Ischemia/Reperfusion Injury

doi: 10.1002/advs.202415205

Figure Lengend Snippet: MMP9 High neutrophils accounted for NET formation and MI/RI. A) Violin plot of NET formation scores for each cluster. B) Heatmap of marker genes for NET formation associated with the 5 neutrophil clusters. C) The concentrations of MPO/DNA‐NETs (Mann‐Whitney test) in serum from healthy donors ( n = 12) and MI/RI patients ( n = 20). D) Spearman correlation analysis of MMP9 High neutrophils with serum MPO/DNA‐NETs ( n = 10). E) Representative fluorescence images of NETs stained for DNA (DAPI, blue), citH3 (citH3, green), and myeloperoxidase (MPO, red) of human neutrophils from healthy donors or MI/RI patients treated with LPS, *200, scale bar 180 µm; *400, scale bar 100 µm. F) The concentrations of MPO/DNA‐NETs (unpaired t ‐test with Welch's correction) in the neutrophil culture supernatants ( n = 5). G) Representative fluorescence images of NET formation in MI/RI tissue sections stained for DAPI (blue), MPO (green), Ly6G (red), *200, scale bar 50 µm; *400, scale bar 20 µm. H) The concentrations of MPO/DNA‐NETs (unpaired t ‐test) in serum from control mice ( n = 5) and MI/RI mice ( n = 10). I) Representative images of echocardiography of the mice undergoing MI/RI with saline or GSK484 treatment. J) LVEF (top, unpaired t ‐test) and LVFS (bottom, unpaired t ‐test) of MI/RI + saline mice ( n = 9) and MI/RI + GSK484 mice ( n = 9). K) Representative FACs images of neutrophil clustering in blood and heart of MI/RI + saline mice and MI/RI + GSK484 mice. L) Proportion of MMP9 High neutrophils (top left, Mann‐Whitney test) and IFIT1 High neutrophils (top right, unpaired t ‐test) in the blood of MI/RI + saline mice ( n = 9) and MI/RI + GSK484 mice ( n = 9). The proportion of MMP9 High neutrophils (bottom left, unpaired t ‐test) and IFIT1 High neutrophils (bottom right, unpaired t ‐test) in the heart of MI/RI + saline mice ( n = 9) and MI/RI + GSK484 mice ( n = 9). M) Pearson correlation analysis of MMP9 High neutrophils proportion in blood with LVEF ( n = 18) (left). Pearson correlation analysis of MMP9 High neutrophils proportion in heart with LVEF ( n = 18) (right). N) Plasma concentration of MMP9 (left, unpaired t ‐test) and MPO/DNA‐NETs (right, unpaired t ‐test) of MI/RI + saline mice ( n = 10) and MI/RI + GSK484 mice ( n = 5). All data was displayed as median with interquartile range or mean ± SEM. NS, not significant, p > 0.05; * p < 0.05; ** p < 0.01; *** p < 0.001; **** p < 0.0001.

Article Snippet: GSK484‐treated mice were injected intraperitoneally with 4mg kg −1 GSK484 (S7803, Selleck) per mouse as previously described.

Techniques: Marker, MANN-WHITNEY, Fluorescence, Staining, Control, Saline, Clinical Proteomics, Concentration Assay

Journal: Advanced Science

Article Title: MMP9 High Neutrophils are Critical Mediators of Neutrophil Extracellular Traps Formation and Myocardial Ischemia/Reperfusion Injury

doi: 10.1002/advs.202415205

Figure Lengend Snippet: GSK484, DB2313, or CST7 CKO can decrease the proportion of MMP9 High neutrophils and weaken their function in vitro and in vivo. A) Representative FACs images of neutrophil clustering of neutrophils from the blood of healthy donors. B) Proportion of MMP9 High neutrophils (left, one‐way ANOVA test with Tukey's multiple comparisons test) and IFIT1 High neutrophils (right, Kruskal‐Wallis's test with Dunn's multiple comparisons test) in neutrophil from the blood of healthy donors (control + vehicle) ( n = 7), control + LPS+ vehicle ( n = 8), control + LPS + GSK484 ( n = 8), and control + LPS + DB2313 ( n = 4). C) FACs analysis of phagocytosis of E. coli fluorescent bioparticles in neutrophil subsets and reactive oxygen species production. D, E) Relative protein levels of MMP9 (one‐way ANOVA test with Tukey's multiple comparisons test) of neutrophils ( n = 4). F) Relative mRNA levels of MMP9 (left, one‐way ANOVA test with Tukey's multiple comparisons test) and IFIT1 (right, one‐way ANOVA test with Tukey's multiple comparisons test) of neutrophils ( n = 6). G) Representative image of left ventricular tissue sections stained with Evans blue and 2,3,5‐triphenyl tetrazolium chloride at 12 h after MI/RI to delineate the area at risk (AAR, red) and the infarcted area (IR, white) (scale bar, 1 mm). H) The ratios of AAR/LV (left, one‐way ANOVA test with Tukey's multiple comparisons test) and IR/AAR (right, one‐way ANOVA test with Tukey's multiple comparisons test) were compared ( n = 4). I) Relative mRNA levels of CSF1R (top left, unpaired t ‐test), E2F1 (top right, unpaired t ‐test with Welch's correction), CALM1 (bottom left, unpaired t ‐test with Welch's correction), and SLC2A3 (bottom right, unpaired t test with Welch's correction) of neutrophils ( n = 6). All data was displayed as median with interquartile range or mean ± SEM. NS, not significant, p > 0.05; * p < 0.05; ** p < 0.01; *** p < 0.001; **** p < 0.0001.

Article Snippet: GSK484‐treated mice were injected intraperitoneally with 4mg kg −1 GSK484 (S7803, Selleck) per mouse as previously described.

Techniques: In Vitro, In Vivo, Control, Staining

Journal: Advanced Science

Article Title: MMP9 High Neutrophils are Critical Mediators of Neutrophil Extracellular Traps Formation and Myocardial Ischemia/Reperfusion Injury

doi: 10.1002/advs.202415205

Figure Lengend Snippet: Differentiation of IFIT1 High neutrophils to MMP9 High neutrophils accelerate during MI/RI by SPI1/CST7 pathway and exacerbate MI/RI by NET formation and degranulation. Cluster 5 (IFIT1 High ) neutrophils differentiate into Cluster 1 & 4 (MMP9 High ) neutrophils and Cluster 3 (IL‐1β High ) neutrophils, respectively. MMP9 High neutrophils mainly account for NET formation. During MI/RI, this differentiation process is accelerated and MMP9 High neutrophils can exacerbate MI/RI by forming NETs. The differentiation process of IFIT1 High neutrophils to MMP9 High neutrophils is mainly regulated by the transcription factor SPI1 and its downstream marker gene CST7 . Administration of GSK484 (PADI4 inhibitor), DB2313 (SPI1 inhibitor), or depletion of CST7 can reduce MMP9 High neutrophils and NET formation, thus ameliorating MI/RI. NETs, neutrophil extracellular traps; MI/RI, myocardial ischemia/reperfusion injury.

Article Snippet: GSK484‐treated mice were injected intraperitoneally with 4mg kg −1 GSK484 (S7803, Selleck) per mouse as previously described.

Techniques: Marker

Journal: Journal for Immunotherapy of Cancer

Article Title: CD276 regulates the immune escape of esophageal squamous cell carcinoma through CXCL1–CXCR2 induced NETs

doi: 10.1136/jitc-2023-008662

Figure Lengend Snippet: CD276 mediates tumor–neutrophil interactions via CXCL1/CXCR2. (A, B) Circle plots showing the differential interaction number (A) and strength (B) between the tumor cell and immune cell clusters based on CellChat analysis. (C) Dot plot showing the expression of receptor–ligand pairs between tumor cell and immune cell clusters in WT and cKO groups. (D) Violin plot showing the expression of Cxcr2 in different clusters. (E) Experimental strategy for the treatment by anti-Ly6G antibodies and representative esophagi image in mice treated with IgG control (IgG), cKO, anti-Ly6G, or cKO+anti-Ly6G group. (F, G) Quantification of lesion area (F) and lesion number (G) of IgG control (IgG), cKO, anti-Ly6G, or cKO+anti-Ly6G group. Data are presented as mean±SD (n=8). (H) Quantification of ESCC tumor grade in IgG control (IgG), cKO, anti-Ly6G, or cKO+anti-Ly6G group. (I, J) Quantification of percentage of Ki67 + (I) and Caspase3 + (J) cells in IgG control (IgG), cKO, anti-Ly6G, or cKO+anti-Ly6G group. (K, L) Quantification of percentage of CD8 + (K) and NCR1 + (L) cells in IgG control (IgG), cKO, anti-Ly6G, or cKO+anti-Ly6G group. (M) Quantification of percentage H3cit + cells of ESCC in control (CTL) and cKO group. (N) Experimental strategy for the treatment by GSK484 inhibitors and representative esophagi image in mice treated with control or GSK484 group. (O, P) Quantification of lesion area (O) and lesion number (P) of the control and GSK484 group. Data are presented as mean±SD (n=8). (Q) Quantification of ESCC tumor grade in the control and GSK484 group. Scale bar, 100 µm. (R) Quantification of percentage H3cit + cells of ESCC in the IgG and anti-CXCL1 group. ESCC, esophageal squamous cell carcinoma; NK, natural killer; 4NQO, 4-nitroquinoline 1-oxide; WT, wild type.

Article Snippet: For the blockade treatment, antibodies and inhibitors anti-CXCL1 (CXCL1 monoclonal antibody (Invitrogen, Cat# MA5-23745, 8 mg/kg body weight)), anti-Ly6G (InVivoMAb anti-mouse Ly6G, BioXcell Cat#BE0075-1, 500 µg/mouse), anti-NK1.1 (InVivoPlus anti-mouse NK1.1, BioXcell Cat#BP0036, 100 µg/mouse), GSK484 (GLPBIO, Cat#GC19184, 4 mg/kg body weight), anti-CD8 (InVivoPlus anti-mouse CD8α, Bioxcell Cat#BP0061, 100 µg/mouse), and IgG control (InVivoMAb rat IgG1 isotype control, BioXcell Cat#BE0088, 10 mg/kg body weight) were used.

Techniques: Expressing, Control