Journal: bioRxiv

Article Title: Macrophage extracellular traps promote maladaptive cardiac remodelling and heart failure via PAD4-dependent mechanisms

doi: 10.64898/2026.03.15.711858

Figure Lengend Snippet: A , Schematic overview of the experimental design. WT-BMT and PAD4KO-BMT mice received either IgG isotype control or anti-Ly6G antibody (100 μg per mouse) by intraperitoneal injection every 3 days for 28 days, as indicated. B , Peripheral blood cell counts, and neutrophil and monocyte fractions (n=4-5). Neutrophils were determined by CD45 high Gr-1 high CD11b low and monocytes were determined by CD45 high Gr-1 low CD11b high by FACS analysis. C , Echocardiographic assessment 28 days after TAC (n=6 in each group). D , Representative fluorescent immunohistochemistry images of left ventricular tissue stained for neutrophil elastase (NE, green), citrullinated histone H3 (CitH3, red), and troponin I (yellow), with nuclear counterstaining by DAPI (blue). Arrowheads indicate NET-positive cells (NE + CitH3 + ). Scale bars, 50 μm. E , Quantification of the numbers of neutrophils (NE + ) and NETs (NE + CitH3 + ) per myocardial tissue area (n=4). F , Representative left ventricular sections stained for CD68 (green), CitH3 (red), troponin I (yellow) and DAPI (blue). Arrowheads indicate MET-positive cells (CD68 + CitH3 + ). Scale bars, 50 μm. G , Quantification of CD68 + macrophages and METs (CD68 + CitH3 + ) per tissue area (n=3-4). All data are presented as mean ± SEM. *P < 0.05 versus the corresponding IgG-treated group, † P < 0.05 versus IgG-treated WT-BMT mice, and ‡ P < 0.05 versus anti-Ly6G-treated WT-BMT mice as determined by one-way analysis of variance with Tukey’s post hoc analysis.

Article Snippet: The sections were incubated with primary antibodies against citrullinated histone H3 (ab5103, Abcam), troponin I (ab188877, Abcam), CD68 (MCA1957, Bio-Rad) or neutrophil elastase (MAB4517, R&D Systems), followed by appropriate secondary antibodies, including Donkey Anti-Rat IgG H&L, Alexa Fluor 594 (ab150156, Abcam), Donkey anti-Goat IgG H&L, Alexa Fluor 488 (ab150129, Abcam), Donkey anti-Rabbit IgG (H+L) Highly Cross-Adsorbed Secondary Antibody, Alexa Fluor 647 (A31573, Thermo Fisher Scientific) or Donkey Anti-Rabbit IgG H&L Alexa Fluor 750 (ab175731, Abcam), and then mounted with a DAPI-containing mounting medium.

Techniques: Control, Injection, Immunohistochemistry, Staining

Journal: bioRxiv

Article Title: Macrophage extracellular traps promote maladaptive cardiac remodelling and heart failure via PAD4-dependent mechanisms

doi: 10.64898/2026.03.15.711858

Figure Lengend Snippet: A , Representative fluorescence images of mouse cardiomyocyte-derived exophers (tdTomato). Scale bars, 20 μm. B , Electron microscopy images of the exophers. The boxed area in the upper panel is enlarged and highlighted in the lower panel. Scale bars, 1 μm and 500 nm. C , Mitochondrial DNA (mtDNA) copy number of the cardiac exophers. D , Representative immunofluorescence images of BMDMs from WT or PAD4 knockout mice after exposure to cardiac exophers. At 24 h after stimulation, the cells were stained for CD68 (green), CitH3 (magenta), and DAPI (blue). Exophers were identified by endogenous tdTomato fluorescence (red). Scale bars, 10 μm. E , Venn diagram illustrating the number of proteins identified in NET and MET components. Peripheral neutrophils and BMDMs were stimulated with mtDNA and purified components were analysed by liquid chromatography–tandem mass spectrometry. F , Schematic experimental protocol. BMDMs from WT mice were stimulated with mtDNA. Primary mouse cardiac fibroblasts were incubated with the conditioned medium (CM) for 24 h in the presence or absence of a TLR4 inhibitor. G , Representative immunofluorescence images of cardiac fibroblasts stained with anti-αSMA antibody (magenta), wheat germ agglutinin (green) and DAPI (blue). Scale bars, 100 μm. H , Quantitative analysis of relative αSMA-signal intensity. Data are expressed as a relative ratio to vehicle from 5 independent experiments. All data are presented as mean ± SEM. I , The proposed model of METs and heart failure.

Article Snippet: The sections were incubated with primary antibodies against citrullinated histone H3 (ab5103, Abcam), troponin I (ab188877, Abcam), CD68 (MCA1957, Bio-Rad) or neutrophil elastase (MAB4517, R&D Systems), followed by appropriate secondary antibodies, including Donkey Anti-Rat IgG H&L, Alexa Fluor 594 (ab150156, Abcam), Donkey anti-Goat IgG H&L, Alexa Fluor 488 (ab150129, Abcam), Donkey anti-Rabbit IgG (H+L) Highly Cross-Adsorbed Secondary Antibody, Alexa Fluor 647 (A31573, Thermo Fisher Scientific) or Donkey Anti-Rabbit IgG H&L Alexa Fluor 750 (ab175731, Abcam), and then mounted with a DAPI-containing mounting medium.

Techniques: Fluorescence, Derivative Assay, Electron Microscopy, Immunofluorescence, Knock-Out, Staining, Purification, Liquid Chromatography, Mass Spectrometry, Incubation

Journal: Frontiers in Immunology

Article Title: Sustained STING-IRF7 signaling aggravates LPS-induced endometrial inflammation via excessive neutrophil extracellular traps generation

doi: 10.3389/fimmu.2025.1671848

Figure Lengend Snippet: STING deficiency suppressed NETs formation in endometritis. (A) Representative images of ELA2 (red) in LPS-treatment Tmem173 gt mice endometrial tissues or control for 24 h at 400 × magnification (scale bar = 25 μm). The DNA was staining with DAPI. (B) Quantification of ELA2-positive cells in the endometrial tissues (n=10). One-way ANOVA test was applied with *** P <0.001 (WT vs. Tmem173 gt in LPS group). (C) Representative images of citH3 (green) in LPS-treatment Tmem173 gt mice endometrial tissues or control for 24 h at 400 × magnification (scale bar = 25 μm). The DNA was staining with DAPI. (D) Quantification of citH3 positive cells in the endometrial tissues (n=10). One-way ANOVA test was applied with ** P <0.01 (WT vs. Tmem173 gt in LPS group). (E) Representative immunohistochemical staining of ELA2 in endometrial tissues of chronic endometritis or control at 400 × magnification (scale bar = 25 μm). (F) The area of ELA2 were quantified (n=8). One-way ANOVA test was applied with ** P <0.01 (WT vs. Tmem173 gt in LPS group). (G) Representative immunohistochemical staining of MPO in endometrial tissues of chronic endometritis or control at 400 × magnification (scale bar = 25 μm). (H) The area of MPO were quantified (n=8). One-way ANOVA test was applied with ** P <0.01 (WT vs. Tmem173 gt in LPS group). (I) Representative immunoblots of citH3, ELA2 and MPO in LPS-treatment Tmem173 gt mice endometrial tissues or control endometrium tissues. (J) MPO concentration in endometrial tissues of LPS-treatment Tmem173 gt mice endometrial tissues or control (n=6). One-way ANOVA test was applied with * P <0.05 (WT vs. Tmem173 gt in PBS group), * P <0.05 (WT vs. Tmem173 gt in LPS group). (K) Detection of NETs formed by flow cytometry in mice bone marrow neutrophils stimulated with STING agonis cGAMP and or STING inhibitor H-151. (L) Quantification of citH3 + cells on neutrophils stimulated with STING agonis cGAMP and or STING inhibitor H-151 by flow cytometry. Representative flow data showing citH3 + cells defined as NETs. One-way ANOVA test was applied with ** P <0.001, * P <0.05.

Article Snippet: After blocking with 5% non-fat milk for 1 h, cells were incubated with the following antibodies: IL-1β (1:1000, 26048-1-AP, Proteintech, China), citH3 (1:1000, AB281584, Abcam, USA), ELA2 (1:1000, 27642-1-AP, Proteintech, China) and MPO (1:1000, 22225-1-AP, Proteintech, China), CD11b (MAC-1) (1:1000, AB133357, Abcam, MA, USA), IRF7(1:1000, Cat. no. 22392-1-AP, Proteintech, Wuhan, China), CEBPB (1:1000, D155298; BBI, China), and VDR (1:1000, D151709, BBI, China), and GAPDH (1:1000, 60004-1-AP; Proteintech, China) overnight at 4°C and washed in Tris-buffered saline (TBST) in triplicate for 5 min.

Techniques: Control, Staining, Immunohistochemical staining, Western Blot, Concentration Assay, Flow Cytometry

Journal: Frontiers in Immunology

Article Title: Sustained STING-IRF7 signaling aggravates LPS-induced endometrial inflammation via excessive neutrophil extracellular traps generation

doi: 10.3389/fimmu.2025.1671848

Figure Lengend Snippet: Neutrophil NETs formation in chronic endometritis patients. (A) Representative immunohistochemical staining of STING, IRF7, LCN2 and MC4R in endometrial tissues of chronic endometritis or control at 400 × magnification (scale bar = 100 μm). (B) Representative immunohistochemical staining of ELA2 in endometrial tissues of chronic endometritis or control at 400 × magnification (scale bar = 25 μm). (C) The area of ELA2 was quantified (n=6). Student’s t test was applied with ** P <0.01. (D) Representative immunofluorescence image of citH3 in endometrial tissues of chronic endometritis or control at 400 × magnification (scale bar = 25 μm). The staining color of citH3 was green. The DNA was staining with DAPI. (E) The mean of citH3 were quantified (n=6). Student’s t test was applied with * P <0.05. (F) Representative immunofluorescence image of MPO in endometrial tissues of chronic endometritis or control at 400 × magnification (scale bar = 25 μm). The staining color of MPO was red. The DNA was staining with DAPI. (G) The mean of MPO were quantified (n=6). Student’s t test was applied with *** P <0.001.

Article Snippet: After blocking with 5% non-fat milk for 1 h, cells were incubated with the following antibodies: IL-1β (1:1000, 26048-1-AP, Proteintech, China), citH3 (1:1000, AB281584, Abcam, USA), ELA2 (1:1000, 27642-1-AP, Proteintech, China) and MPO (1:1000, 22225-1-AP, Proteintech, China), CD11b (MAC-1) (1:1000, AB133357, Abcam, MA, USA), IRF7(1:1000, Cat. no. 22392-1-AP, Proteintech, Wuhan, China), CEBPB (1:1000, D155298; BBI, China), and VDR (1:1000, D151709, BBI, China), and GAPDH (1:1000, 60004-1-AP; Proteintech, China) overnight at 4°C and washed in Tris-buffered saline (TBST) in triplicate for 5 min.

Techniques: Immunohistochemical staining, Staining, Control, Immunofluorescence