Journal: Blood Vessels, Thrombosis & Hemostasis

Article Title: Epigenetic metabolite lipid nanoparticles alleviate venous thrombosis via bone marrow reprogramming

doi: 10.1016/j.bvth.2025.100086

Figure Lengend Snippet: Immunomodulatory effects of ITA-LNP in vitro and in vivo. (A) Immunoblot probing for IL-1β in BMDMs treated with 100 ng/mL LPS in the presence of various doses of ITA-LNPs. (B) Schematic of the experiments in C57BL/6 mice subjected to LPS-induced endotoxemia. (C) Kaplan-Meier survival analysis after LPS-induced (15 mg/kg) endotoxemia in C57BL/6 mice pretreated with Ctrl/ITA-LNPs or anti–IL-1β mAb. (D) Rectal temperature measured in the mice from panel C before and 6 hours after LPS injections. (E-F) The levels of IL-1β and IL-6 in the serum from mice of a separate cohort injected as in panel B but with a sublethal dose of 8 mg/kg LPS; n = 10 to 15 mice per group. (G) NETs in primary human neutrophils incubated for 5 hours in vitro with 50 μM LNPs as indicated and in the presence of 100 nM PMA followed by immunofluorescence analysis; n = 6 for Ctrl-LNP and ITA-LNP without PMA; n = 8 for Ctrl-LNP with PMA; n = 9 for ITA-LNP with PMA; and n = 4 for PBS groups with or without PMA. (H) Cytokine analysis in supernatants from experiments in panel G. Statistical analysis is through pairwise t test with Holm post hoc test . N.d., not detected; PBS, phosphate-buffered saline; ROI, region of interest; T, temperature.

Article Snippet: C57BL/6 male mice (10 weeks old, n = 15 per group) were IV injected with 50 mg/kg ITA- or control (Ctrl)-LNPs or 50 mg/kg anti-mouse IL-1β monoclonal antibody (clone 7E3, InvivoGen mil1b-mab9-10).

Techniques: In Vitro, In Vivo, Western Blot, Injection, Incubation, Immunofluorescence, Saline

Journal: Blood Vessels, Thrombosis & Hemostasis

Article Title: Epigenetic metabolite lipid nanoparticles alleviate venous thrombosis via bone marrow reprogramming

doi: 10.1016/j.bvth.2025.100086

Figure Lengend Snippet: ITA-LNPs alleviate venous thrombosis in IVC ligation model. (A) Schematic of the experiments. WT mice IV pretreated (50 mg/kg, bolus) with ITA- or Ctrl-LNPs were subjected to IVC ligation surgery followed by the isolation of the blood clots; n = 6 for Ctrl-LNP and n = 8 for ITA-LNP. (B) Gross pathology with clot weight quantification. (C) Hematoxylin and eosin staining of blood clot sections from these experiments. (D) Immunofluorescence staining of the same sections using Ly6G/C mAb (Red) and nuclei (blue). (E) MPO (top, alkaline phosphatase immunohistochemistry) and IL-1β (bottom, immunofluorescence) staining in the same clots. (F) Quantification of the staining in panel E. For each biological replicate (n = 6 for Ctrl-LNP and n = 8 for ITA = LNP), 3 to 4 histopathological locations at different depths (100 μm apart) were analyzed. (G) NET staining in the same sections using triple-positive identification through colocalization of the signals from MPO (green), H4Cit3 (red), and DNA (DAPI; blue). (H) Quantification of the staining in panel G. Quantification was accomplished using 2 to 3 different histopathological locations and the same aforementioned biological replicates. Statistical analysis is through a 2-tailed t test. DAPI, 4′,6-diamidino-2-phenylindole; H4Cit3, citrullinated histone H4; MPO, myeloperoxidase; WT, wild type.

Article Snippet: C57BL/6 male mice (10 weeks old, n = 15 per group) were IV injected with 50 mg/kg ITA- or control (Ctrl)-LNPs or 50 mg/kg anti-mouse IL-1β monoclonal antibody (clone 7E3, InvivoGen mil1b-mab9-10).

Techniques: Ligation, Isolation, Staining, Immunofluorescence, Immunohistochemistry

Journal: Scientific Reports

Article Title: Deletion of PTHrP nuclear localization sequence and carboxyl terminus leads to lung dysplasia

doi: 10.1038/s41598-025-08163-3

Figure Lengend Snippet: Deletion of PTHrP NLS and C-terminus promoted production of inflammatory cytokines in the lungs. ( A ) Representative IL-1β immunohistochemical micrographs (×400 magnification, scale bar = 25 μm). ( B ) Representative IL-6 immunohistochemical micrographs (×400 magnification, scale bar = 25 μm). ( C ) Representative TNF-α immunohistochemical micrographs (×400 magnification, scale bar = 25 μm). ( D ) Statistical results of the percentage of IL-1β-positive area in WT and KI mice. ( E ) Statistical results of the percentage of IL-6-positive area in WT and KI mice. ( F ) Statistical results of the percentage of TNF-α-positive area in WT and KI mice. ( G ) Western blot results of IL-1β, IL-6, TNF-α, and p65 proteins in lung tissues, with β-actin serving as the loading control. ( H ) Statistical comparison of IL-1β, IL-6, TNF-α, and p65 protein expression levels. ( A - H ) Data are from three independent biological replicates ( n = 3 WT and 3 KI mice). Adjusted P-value = Original P-value × Number of multiple comparisons (Bonferroni correction) adjusted p = raw p ×7 * p < 0.05, ** p < 0.01, *** p < 0.001.

Article Snippet: Goat Anti-Type I Collagen-UNLB (1:200, Southern Biotech, USA), monoclonal mouse Anti-Actin Antibody (1:200, Santa Cruz Biotechnology, USA), polyclonal rabbit TGF-beta 1 antibody (1:100, ABclonal, China), Smad2 monoclonal rabbit antibody (1:50, Cell Signaling Technology, USA), monoclonal mouse anti-proliferating cell nuclear antigen (PCNA) (1:400, Dako, Denmark), polyclonal rabbit Anti-Caspase-3 antibody (1:300, abcam, USA), monoclonal mouse Anti-IL-1β Antibody (1:200, Santa Cruz Biotechnology, USA), monoclonal mouse Anti-IL-6 Antibody (1:200, Santa Cruz Biotechnology, USA), monoclonal mouse Anti-TNFα Antibody (1:200, Santa Cruz Biotechnology, USA), monoclonal mouse Anti-DNA/RNA Damage antibody (1:400, abcam, USA), monoclonal rabbit Anti-Chk2 antibody (1:400, abcam, USA) and monoclonal rabbit Anti-gamma H2A.X (phospho S139) antibody (1:400, abcam, USA) were used in this study.

Techniques: Immunohistochemical staining, Western Blot, Control, Comparison, Expressing

Journal: Frontiers in Immunology

Article Title: IL-1beta expressing neutrophil extracellular traps in Legionella pneumophila infection

doi: 10.3389/fimmu.2025.1573151

Figure Lengend Snippet: Neutrophils phagocytose opsonized L. pneumophila . (A) HC neutrophils cocultured with either non-opsonized (serum-free conditions) L. pneumophila (mean ± SD; 0.0% ± 0, n=3) or serum-opsonized L. pneumophila (mean ± SD; 84.19% ± 12.45, n=3. P = 0.0072), (confocal microscopy; blue: DAPI/DNA, green: MPO, red: L. pneumophila ). Images were acquired with a Leica TCS SP8 confocal microscope using a 63x objective. Scale bar, 20 μm. (B) Quantification of phagocytosis using Imaris v 9.3.1. Data from 3 independent experiments in panel (B) are presented as means ± SDs (**p <0.01). (C) 3D reconstruction of neutrophils infected with opsonized and non-opsonized L. pneumophila . Scale bar, 10 μm.

Article Snippet: Immunolabeling was conducted with the following antibodies: a rabbit anti-citrullinated H3 (R2+R8+R17) (1/500 dilution; Abcam) polyclonal Ab, a goat anti-myeloperoxidase (MPO)-specific mAb (1/150 dilution; R&D systems) and a mouse anti-IL-1β antibody (1/200 dilution; OriGene).

Techniques: Confocal Microscopy, Microscopy, Infection

Journal: Frontiers in Immunology

Article Title: IL-1beta expressing neutrophil extracellular traps in Legionella pneumophila infection

doi: 10.3389/fimmu.2025.1573151

Figure Lengend Snippet: Neutrophils produce ROS in response to L. pneumophila in a phagocytosis-dependent manner. ROS production in neutrophils from (A) HC neutrophils (mean ROS MFI ± SD; 12.45 ± 2.654, n=7) cocultured with non-opsonized L. pneumophila in serum-free conditions (mean ROS MFI ± SD; 10.72 ± 1.703, n=7. p = 0.0903) and (B) HC neutrophils (mean ROS MFI ± SD; 3.835 ± 0.9617, n=10) cocultured with opsonized L. pneumophila (mean ROS MFI ± SD; 7.819 ± 2.190, n=10. p = 0.002). Data from 7 independent experiments in panel (A) and 10 independent experiments in panel (B) are presented as means ± SDs (n.s., not significant, p >0.05; **p <0.01).

Article Snippet: Immunolabeling was conducted with the following antibodies: a rabbit anti-citrullinated H3 (R2+R8+R17) (1/500 dilution; Abcam) polyclonal Ab, a goat anti-myeloperoxidase (MPO)-specific mAb (1/150 dilution; R&D systems) and a mouse anti-IL-1β antibody (1/200 dilution; OriGene).

Techniques:

Journal: Frontiers in Immunology

Article Title: IL-1beta expressing neutrophil extracellular traps in Legionella pneumophila infection

doi: 10.3389/fimmu.2025.1573151

Figure Lengend Snippet: L. pneumophila induces NETs in neutrophils in vitro in a ROS-independent manner. (A) NETs in HC peripheral blood neutrophils (mean ± SD; 4.4% ± 1.647, n=10), either treated with ionomycin (mean ± SD; 30.6% ± 4.742, n=10, p < 0.0001) or cocultured with L. pneumophila (mean ± SD; 22% ± 3.801, n=10. p < 0.0001) (confocal microscopy; blue: DAPI/DNA, green: MPO, red: citH3). Images were acquired with a Leica TCS SP8 confocal microscope using a 20x objective. Scale bar, 100 μm. (B) Quantification of NET-releasing neutrophils from (A) and (C) Quantification of NET releasing neutrophils with MPO/DNA complex ELISA. (D) NETs in HC peripheral blood neutrophils cocultured with L. pneumophila in the presence (mean ± SD; 15.5% ± 4.123, n=4. p > 0.9999) or absence (mean ± SD; 14% ± 3.651, n=4.) of DPI. Images were acquired with a Leica TCS SP8 confocal microscope using a 20x objective. Scale bar, 100 μm. (E) Quantification of NET-releasing neutrophils from (D) Data from 10 independent experiments in panel (B) , from 6 independent experiments in panel (C) and 4 independent experiments in panel (E) are presented as means ± SDs (n.s., not significant, p >0.05; **p <0.01; ****p <0.0001).

Article Snippet: Immunolabeling was conducted with the following antibodies: a rabbit anti-citrullinated H3 (R2+R8+R17) (1/500 dilution; Abcam) polyclonal Ab, a goat anti-myeloperoxidase (MPO)-specific mAb (1/150 dilution; R&D systems) and a mouse anti-IL-1β antibody (1/200 dilution; OriGene).

Techniques: In Vitro, Confocal Microscopy, Microscopy, Enzyme-linked Immunosorbent Assay

Journal: Frontiers in Immunology

Article Title: IL-1beta expressing neutrophil extracellular traps in Legionella pneumophila infection

doi: 10.3389/fimmu.2025.1573151

Figure Lengend Snippet: L. pneumophila induces IL-1β expressing NETs in vitro . (A) NETs in HC peripheral blood neutrophils, either treated with ionomycin or cocultured with L. pneumophila (confocal microscopy; blue: DAPI/DNA, red: citH3, magenta: MPO, green: IL−1β). Images were acquired with a Leica TCS SP8 confocal microscope using a 20x objective. Scale bar, 20 μm. (B) Western Blot analysis of NET proteins derived from healthy controls after treatment with ionomycin or coculture with L. pneumophila .

Article Snippet: Immunolabeling was conducted with the following antibodies: a rabbit anti-citrullinated H3 (R2+R8+R17) (1/500 dilution; Abcam) polyclonal Ab, a goat anti-myeloperoxidase (MPO)-specific mAb (1/150 dilution; R&D systems) and a mouse anti-IL-1β antibody (1/200 dilution; OriGene).

Techniques: Expressing, In Vitro, Confocal Microscopy, Microscopy, Western Blot, Derivative Assay