Journal: Journal of Clinical and Translational Hepatology

Article Title: Single-cell Sequencing Reveals Neutrophil Extracellular Traps in Association with Endotheliopathy and Immunothrombosis in Hepatitis B Virus-related Acute-on-chronic Liver Failure

doi: 10.14218/JCTH.2025.00666

Figure Lengend Snippet: (A) Plasma concentrations of NETs markers, including MPO–DNA complexes, MPO, and NE, in HBV-ACLF, CHB, and HC groups. (B–C) Correlations between plasma MPO–DNA levels and INR and TAT (B), and LDH (C). (D) Correlations between plasma MPO–DNA levels and established prognostic scoring systems, including the COSSH-ACLF II and MELD-Na scores. (E) Comparison of NETs marker levels (MPO–DNA and MPO) between survivors and non-survivors within the HBV-ACLF cohort. (F) Kaplan–Meier survival curves of HBV-ACLF patients stratified by plasma MPO–DNA levels (≤2-fold vs >2-fold). *** P < 0.001, and **** P < 0.0001. NETs, neutrophil extracellular traps; HBV-ACLF, hepatitis B virus-related acute-on-chronic liver failure; MPO, myeloperoxidase; NE, neutrophil elastase; CHB, chronic hepatitis B; HC, healthy controls; INR, international normalized ratio; TAT, thrombin-antithrombin complex; LDH, lactate dehydrogenase; COSSH-ACLF II, Chinese Onset Study of Severe Hepatitis with Acute-on-Chronic Liver Failure II; MELD-Na, Model for End-Stage Liver Disease-Sodium.

Article Snippet: Specifically, the Human Myeloperoxidase ELISA Kit (RayBiotech, Cat. No. ELH-MPO) and the Human Neutrophil Elastase ELISA Kit (RayBiotech, Cat. No. ELH-NEUTRO-1) were used according to the manufacturers’ protocols.

Techniques: Clinical Proteomics, Comparison, Marker, Virus

Journal: Journal of Clinical and Translational Hepatology

Article Title: Single-cell Sequencing Reveals Neutrophil Extracellular Traps in Association with Endotheliopathy and Immunothrombosis in Hepatitis B Virus-related Acute-on-chronic Liver Failure

doi: 10.14218/JCTH.2025.00666

Figure Lengend Snippet: (A–C) Correlation analyses between plasma MPO–DNA complex levels and syndecan-1 (A), ICAM-1 (B), and sTM and vWF (C). (D) Immunofluorescence staining of endothelial cells (CD31), NETs (citrullinated histone H3, Cit-H3), and platelets (CD41) in liver tissues from patients with HBV-ACLF, CHB, and HC. Scale bars: 50 µm. (E) Neutrophils isolated from CHB patients were stimulated with PMA. NETs were collected and incubated with human aortic endothelial cells, with or without DNase I treatment. Syndecan-1 levels in supernatants and plasma clotting time of cells were measured after 6 h. ** P < 0.01, *** P < 0.001, and **** P < 0.0001. NETs, neutrophil extracellular traps; HBV-ACLF, hepatitis B virus-related acute-on-chronic liver failure; MPO, myeloperoxidase; ICAM-1, intercellular adhesion molecule-1; sTM, soluble thrombomodulin; vWF, von Willebrand factor; CHB, chronic hepatitis B; HC, healthy controls; PMA, phorbol 12-myristate 13-acetate; DNase I, deoxyribonuclease I.

Article Snippet: Specifically, the Human Myeloperoxidase ELISA Kit (RayBiotech, Cat. No. ELH-MPO) and the Human Neutrophil Elastase ELISA Kit (RayBiotech, Cat. No. ELH-NEUTRO-1) were used according to the manufacturers’ protocols.

Techniques: Clinical Proteomics, Immunofluorescence, Staining, Isolation, Incubation, Coagulation, Virus

Journal: Nature Communications

Article Title: Agonist signaling drives neutrophil subpopulations to promote/inhibit colorectal cancer liver metastasis

doi: 10.1038/s41467-025-67579-7

Figure Lengend Snippet: A Surface marker expression profiles of three neutrophil subsets. Created in BioRender. Guo, Z. (2025) https://BioRender.com/1ay5uxr . B DRUG-seq analysis of HDNs, M-LDNs, and IM-LDNs from the peripheral blood of CRC patients. Heatmap shows upregulated genes. C Comparison of three neutrophil subsets from the peripheral blood samples in this study with published human neutrophil states. D The scRNA-seq of 11 CRC tumor tissues (GSE302903), and UMAP plots displayed the six neutrophil subsets in the CRC tissues. E Comparisons among three neutrophil subsets from peripheral blood with six neutrophil subsets from the tumor tissues of patients with CRC. F The migration assay showed that HDNs exhibited the highest chemotaxis toward both CRC and healthy intestinal epithelial cells, M-LDNs showed partial chemotaxis toward CRC cells, whereas no chemotaxis was observed for IM-LDNs. n = 3, data were presented as mean ± SD, two-way ANOVA. G Phagocytosis assay showed M-LDNs exhibited higher phagocytic ability than HDNs, whereas IM-LDNs retained no phagocytic function. Scale bars, 200 μm. n = 9, data were presented as mean ± SD, one-way ANOVA. H The killing assay showed that HDNs were cytotoxic to CRC cells in a coculture environment, whereas M-LDN and IM-LDN did not show any cytotoxic effects. n = 3, data were presented as mean ± SD, two-way ANOVA. I The apoptosis detection showed that HDNs induced apoptosis in CRC cells in a coculture environment, whereas M-LDNs and IM-LDNs did not. n = 4, data were presented as mean ± SD, one-way ANOVA. J CFSE staining was used to evaluate the proliferation of T cells, and the fluorescence of the proliferating cells was reduced. M-LDNs inhibited T-cell proliferation, whereas HDNs and IM-LDNs did not. K CD8⁺T cells were pretreated with neutrophils and then co-cultured with CRC patient-derived organoids (PDOs). The killing assay showed that activated CD8 T were toxic to CRC PDOs. M-LDN and IM-LDN significantly inhibited the T-cell killing of CRC PDOs, whereas HDNs exhibited no such effect. n = 3, data were presented as mean ± SD, two-way ANOVA. L The H O content in the cell culture medium (CM) and the lysate of neutrophils were detected, M-LDNs released high H O into the CM. n = 3, data were presented as mean ± SD, two-way ANOVA. M , N The oxidative stress level analysis showed that M-LDNs mainly produced and released reactive oxygen species (ROS), whereas HDNs mainly produced and released mitochondrial ROS. O SYTOX Green was used to measure NET release to assess NETosis rate in neutrophils. M-LDNs were more prone to high rate of NETosis. Scale bars, 200 μm. P M-LDNs were more prone to release H₂O₂ into the CM when induced by PMA (25 ng/mL), ionomycin (5 μM), or LPS (100 ng/mL). n = 3, data were presented as mean ± SD, two-way ANOVA. Q NETosis was induced in HDNs and M-LDNs using PMA (25 ng/mL), ionomycin (5 μM), and LPS (100 ng/mL). Scale bars, 200 μm. n = 3, data were presented as mean ± SD, two-way ANOVA. R The content of granular constituents in the CM of HDN and M-LDN were analyzed using ELISA. HDNs can release more particles than M-LDNs. HDNs secreted NE, MPO, AZU, and CG, whereas M-LDNs mainly secreted MPO and AZU. n = 6, data were presented as mean ± SD, two-tailed unpaired T-test. S The electron microscope showed the intracellular structure. HDN was spherical, and the cytoplasmic particles were large, whereas M-LDN was oblong with smaller cytoplasmic particles. Data are representative of three independent experiments. Scale bars, 2 μm. T CRC cell line, SW620, was used to construct liver metastasis model in nude mice. HDN treatment inhibited liver metastases, whereas M-LDN promoted liver metastases. Data are representative of three independent experiments. Scale bars, 2000 μm. Stimuli concentrations as in Supplementary Data. . See also Figure – . Source data are provided as a file.

Article Snippet: Cytokine levels were measured using the Human Neutrophil Elastase enzyme-linked immunosorbent assay (ELISA) Kit (RayBiotech), Human Myeloperoxidase/MPO ELISA Kit (RayBiotech), Human Azurocidin/CAP37/HBP ELISA Kit (RayBiotech), Human Cathepsin G ELISA Kit (Abcam), Human IL-8/CXCL8 ELISA Kit (Abclonal), and Human CXCL5/ENA-78 ELISA Kit (abclonal), Human GM-CSF ELISA Kit (abclonal), Human IFN-gamma ELISA Kit (abclonal), and Human ARG1 ELISA Kit (abclonal), all according to the manufacturer’s instructions.

Techniques: Marker, Expressing, Comparison, Migration, Chemotaxis Assay, Phagocytosis Assay, Staining, Fluorescence, Cell Culture, Derivative Assay, Produced, Enzyme-linked Immunosorbent Assay, Two Tailed Test, Microscopy, Construct