Journal: Journal of Advanced Research

Article Title: HDAC2 enhances the antimicrobial activity of neutrophils by promoting the formation of neutrophil extracellular traps (NETs) in sepsis

doi: 10.1016/j.jare.2025.08.041

Figure Lengend Snippet: HDAC2 deficiency or inhibition compromises the antimicrobial efficacy, but improves anti-inflammatory responses in the mice with sepsis. (A-D) Effects of the HDAC2-knockout or inhibition on the survival of mice with CLP-induced or LPS-induced sepsis. Kaplan-Meier curves for the HDAC2-knockout or inhibition by SAHA-treated mice with or CLP-induced or LPS-induced sepsis (n = 20 mice). (E-H) Serum IL-6 and IL-1β levels were determined in the sham or CLP-induced or LPS-induced septic mice treated with or without SAHA (25 mg/kg). These inflammatory parameters were measured by ELISA. Data are shown as mean ± SEM. *p < 0.05; n = 5 mice per group. (I and J) Serum AST, ALT, Urea and Crea levels were measured in sham or CLP-induced or LPS-induced septic mice treated with or without SAHA. Blood was collected by orbital sampling, and these biochemical parameters were measured by the Automatic Biochemistry Analyzer. Data are shown as mean ± SEM. **p < 0.01; n = 5 mice per group.

Article Snippet: For mouse models of CLP-induced sepsis with HDAC2 inhibition, the animals received an intraperitoneal injection of SAHA (25 mg/kg; MCE, HY-10221) after 0.5 h CLP modeling; For mouse models of LPS-induced sepsis with HDAC2 inhibition, the mice were injected intraperitoneally with LPS (10 mg/kg; Sigma, L4130, derived from E. coli O111:B4) after 1 h injection of SAHA.

Techniques: Inhibition, Knock-Out, Enzyme-linked Immunosorbent Assay, Sampling

Journal: Journal of Advanced Research

Article Title: HDAC2 enhances the antimicrobial activity of neutrophils by promoting the formation of neutrophil extracellular traps (NETs) in sepsis

doi: 10.1016/j.jare.2025.08.041

Figure Lengend Snippet: HDAC2 is required for NET formation. (A and B) Phagocytic uptake and intracellular killing in neutrophils were detected by flow-based assay and colon-formation assay. (A) Phagocytic uptake in neutrophils was detected by flow-based assay. Neutrophils were isolated from peripheral blood of HDAC2 KO or HDAC2 WT mice, and they were co-incubated with E. coli -GFP for two hours. (B) Intracellular killing in neutrophils was detected by colon-formation assay. Blood was taken from the tail vein of the mice, then applied to the pretreated solid MHA medium and incubated at 37 °C for 24 h. The number of bacterial clones was calculated. n = 5 mice per group. (C-D) NETs were detected in the neutrophils from peripheral blood of HDAC2 KO or HDAC2 WT mice by immunofluorescence. Representative data showing MPO-positive cells defined as positive NETs. Data are presented as the means ± SEM. of independent experiments. *P < 0.05, **P < 0.01 (two-tailed Student’s t -test). (E) NETs were detected in the neutrophils from peripheral blood of HDAC2 KO or HDAC2 WT mice by a flow cytometry-based assay. Representative flow data showing positive cells in global Histone H3 citrullination of H3R2, H3R8 or H3R17 sites, defined as positive NETs. (F) NETs markers were detected by western blotting in the neutrophils from HDAC2 WT mice after stimulation with LPS (50 μg/ml) and SAHA (5 μM). Representative data showing H3-Cit expression defined as NETs. The H3 protein was used for western blot loading controls.

Article Snippet: For mouse models of CLP-induced sepsis with HDAC2 inhibition, the animals received an intraperitoneal injection of SAHA (25 mg/kg; MCE, HY-10221) after 0.5 h CLP modeling; For mouse models of LPS-induced sepsis with HDAC2 inhibition, the mice were injected intraperitoneally with LPS (10 mg/kg; Sigma, L4130, derived from E. coli O111:B4) after 1 h injection of SAHA.

Techniques: Tube Formation Assay, Isolation, Incubation, Clone Assay, Immunofluorescence, Two Tailed Test, Flow Cytometry, Western Blot, Expressing

Journal: Journal of Advanced Research

Article Title: HDAC2 enhances the antimicrobial activity of neutrophils by promoting the formation of neutrophil extracellular traps (NETs) in sepsis

doi: 10.1016/j.jare.2025.08.041

Figure Lengend Snippet: HDAC2 promotes NETs formation through the acetylation-citrullination pathway. (A–C) Representative images for LPS (50 μg/ml)-stimulated primary neutrophils from HDAC2 WT and HDAC2 KO mice. (A) Immunostaining for histone H3K18 acetylation (green) was performed with DNA counterstained with Hoechst 33342 (blue) and corresponding MFI of quantitative data (E). Scale bars, 20 μm. (B) Immunostaining for histone H3R17 methylation (green) was performed with DNA counterstained with Hoechst 33342 (blue) and corresponding MFI of quantitative data (F). Scale bars, 20 μm. (C) Immunostaining for histone H3R17 citrullination (green) was performed with DNA counterstained with Hoechst 33342 (blue) and corresponding MFI of quantitative data (G). Scale bars, 20 μm. (D) Histone H3R17 citrullination was regulated by H3R17 methylation inhibitor (EZM2302) and HDAC2 inhibitor (SAHA), and immunostaining for histone H3R17 citrullination (green) was performed with DNA counterstained with Hoechst 33342 (blue), the corresponding MFI of quantitative data (H). Scale bars, 20 μm. (I) H3K18 acetylation, H3R17 methylation and H3R17 citrullination were detected by western blotting in the neutrophils from HDAC2 WT and HDAC2 KO mice after stimulation with LPS (50 μg/ml). The H3 protein was used for western blot loading controls. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

Article Snippet: For mouse models of CLP-induced sepsis with HDAC2 inhibition, the animals received an intraperitoneal injection of SAHA (25 mg/kg; MCE, HY-10221) after 0.5 h CLP modeling; For mouse models of LPS-induced sepsis with HDAC2 inhibition, the mice were injected intraperitoneally with LPS (10 mg/kg; Sigma, L4130, derived from E. coli O111:B4) after 1 h injection of SAHA.

Techniques: Immunostaining, Methylation, Western Blot

Journal: Journal of Advanced Research

Article Title: HDAC2 enhances the antimicrobial activity of neutrophils by promoting the formation of neutrophil extracellular traps (NETs) in sepsis

doi: 10.1016/j.jare.2025.08.041

Figure Lengend Snippet: Protective effects of HDAC2 inhibition and histone methylation inhibition in CLP-induced septic mice (A) Kaplan-Meier curves of CLP-induced septic mice treated with or without SAHA and EZM2302 (n = 20 mice). (B and C) Serum MPO, IL-6 and IL-1β levels were measured with mouse ELISA kit in the CLP-induced septic mice treated with or without SAHA and EZM2302. (D) Serum AST, ALT, Urea and Crea levels were measured in the CLP-induced septic mice treated with or without SAHA and EZM2302. Blood was collected by orbital sampling, and serum biochemical parameters were measured by the Automatic Biochemistry Analyzer. The experiments were performed in quintuplicate, data are presented as the means ± SEM. of independent experiments. *P < 0.05, **P < 0.01 (two-tailed Student’s t -test). (n = 5 mice). (E and F) Histology of lungs, kidneys and livers from mice treated with or without SAHA and EZM2302. (E) The Histology with H&E staining for lungs, kidneys and livers. (F) Cell apoptosis in lungs, kidneys and livers was stained with cleaved-caspase 3. The mice were treated with EZM2302 (25 mg/kg) two hours prior to cecal ligation and puncture (CLP), and SAHA was administered after CLP surgery. SAHA, a HDAC2 inhibitor; EZM2302, a methylation inhibitor.

Article Snippet: For mouse models of CLP-induced sepsis with HDAC2 inhibition, the animals received an intraperitoneal injection of SAHA (25 mg/kg; MCE, HY-10221) after 0.5 h CLP modeling; For mouse models of LPS-induced sepsis with HDAC2 inhibition, the mice were injected intraperitoneally with LPS (10 mg/kg; Sigma, L4130, derived from E. coli O111:B4) after 1 h injection of SAHA.

Techniques: Inhibition, Methylation, Enzyme-linked Immunosorbent Assay, Sampling, Two Tailed Test, Staining, Ligation