Journal: iScience

Article Title: Zinc-dependent histone deacetylases drive neutrophil extracellular trap formation and potentiate local and systemic inflammation

doi: 10.1016/j.isci.2021.103256

Figure Lengend Snippet:

Article Snippet: Staphylococcus aureus subsp. aureus Rosenbach , ATCC , Cat# 25904.

Techniques: Virus, Recombinant, Staining, Enzyme-linked Immunosorbent Assay, Software

Journal: Gut Microbes

Article Title: Gut microbiota dysbiosis exacerbates acute pancreatitis via Escherichia coli -driven neutrophil heterogeneity and NETosis

doi: 10.1080/19490976.2025.2606480

Figure Lengend Snippet: Enrichment of Escherichia coli during acute-phase AP correlates with increased serum cfDNA as a marker of NETs formation. (A) Metagenomic sequencing of fecal samples from AP patients revealed the top 10 bacterial species significantly altered between the acute phase (day of onset) and recovery phase (day 14), as determined by Wilcoxon rank-sum test. (B–C) qRT-PCR validation of intestinal E. coli abundance in healthy controls (HC) and AP patients (B), and in paired samples from AP patients during acute and recovery phases (C). (D–G) Violin plots comparing the relative abundance of common Gram-negative pathogens—including E. coli, Klebsiella pneumoniae, Pseudomonas aeruginosa , and Acinetobacter baumannii —between acute and recovery phases of AP. (H–I) Measurement of serum cfDNA in AP patients compared to healthy controls (H), and in paired acute versus recovery phase samples (I). (J) Correlation heatmap depicting the relationship between E. coli abundance and NETs- associated biomarkers (cfDNA and pro-inflammatory cytokines) in clinical samples. (K–L) Relative intestinal E. coli abundance and serum cfDNA levels in a murine cer-induced P model. (M–N) Correlation analyzes between intestinal E. coli levels and serum cfDNA (M) or IL-6 levels (N) in mice. Data are presented as mean ± SEM. Statistical significance was determined using an unpaired two-tailed Student’s t-test. * p <0.05, ** p <0.01, *** p <0.001; NS, not significant. Abbreviations: AP, acute pancreatitis; cfDNA, cell-free DNA; NETs, neutrophil extracellular traps; qRT-PCR, real-time quantitative PCR; HC, healthy controls; Conn, control; Cer, cerulein; IL-6, interleukin-6; SEM, standard error of mean.

Article Snippet: We further performed an in vivo study using GF mice monocolonized with a clinically relevant enterohemorrhagic E. coli strain (ATCC 43895) and subsequently subjected to cerulein-induced AP ( ).

Techniques: Marker, Sequencing, Quantitative RT-PCR, Biomarker Discovery, Two Tailed Test, Real-time Polymerase Chain Reaction, Control

Journal: Gut Microbes

Article Title: Gut microbiota dysbiosis exacerbates acute pancreatitis via Escherichia coli -driven neutrophil heterogeneity and NETosis

doi: 10.1080/19490976.2025.2606480

Figure Lengend Snippet: E. coli exacerbates cerulein-induced AP and promotes NETs formation in GF mice. (A) Schematic overview of the experimental protocol: GF mice were orally administered PBS or a pathogenic strain of E. coli (ATCC 43895) for one week, followed by hourly intraperitoneal injections of cerulein (100 μg/kg, 10 doses in total). Mice were sacrificed 24 h after the initial injection. (B) Representative H&E staining of pancreatic tissues (scale bar, 25 μm), with corresponding histological scoring for edema, inflammatory infiltration, and necrosis. (C) Immunohistochemical staining and quantification of neutrophils (MPO), macrophages (F4/80), and apoptotic cells (TUNEL assay) in pancreatic tissue sections (scale bars: MPO and F4/80, 25 μm; TUNEL, 50 μm). (D) Serum amylase and lipase levels as biochemical indicators of pancreatic injury. (E) Serum concentrations of inflammatory cytokines (TNF-α, IL-6, IL-1β) and LPS, reflecting systemic inflammatory responses and gut barrier disruption. (F) Flow cytometric analysis and quantification of CD11b + Ly6G + neutrophils (gated on CD45 + cells) in spleen and bone marrow. (G) Immunofluorescence staining and quantification of pancreatic tissue showing NETs formation (CitH3 + MPO + cells) (scale bar, 50 μm). (H) Serum levels of cfDNA measured by ELISA. (I) qRT-PCR analysis of pancreatic tissue expression of pro-inflammatory cytokines ( Il6, Tnfa, Il1β ), neutrophil chemotaxis and activation markers ( Cxcl1, Cxcl2, S100a8, S100a9 ), and genes associated with NETs formation ( Padi4, Nfkb, Nlrp3, Il17a ). Data are presented as mean ± SEM. Statistical significance was determined using an unpaired two-tailed Student’s t-test. * p <0.05, ** p <0.01, *** p <0.001. Abbreviations: GF, germ-free; Cer, cerulein; AP, acute pancreatitis; MPO, myeloperoxidase; CitH3, citrullinated histone H3; cfDNA, cell-free DNA; LPS, lipopolysaccharide; NETs, neutrophil extracellular traps; qRT-PCR, real-time quantitative PCR.

Article Snippet: We further performed an in vivo study using GF mice monocolonized with a clinically relevant enterohemorrhagic E. coli strain (ATCC 43895) and subsequently subjected to cerulein-induced AP ( ).

Techniques: Injection, Staining, Immunohistochemical staining, TUNEL Assay, Disruption, Immunofluorescence, Enzyme-linked Immunosorbent Assay, Quantitative RT-PCR, Expressing, Chemotaxis Assay, Activation Assay, Two Tailed Test, Real-time Polymerase Chain Reaction

Journal: Cell reports

Article Title: Cytosolic LPS-induced caspase-11 oligomerization and activation is regulated by extended synaptotagmin 1

doi: 10.1016/j.celrep.2023.112726

Figure Lengend Snippet: KEY RESOURCES TABLE

Article Snippet: EHEC , ATCC , 700927.

Techniques: Virus, Recombinant, Transfection, Enzyme-linked Immunosorbent Assay, Control, Software

Journal: iScience

Article Title: Zinc-dependent histone deacetylases drive neutrophil extracellular trap formation and potentiate local and systemic inflammation

doi: 10.1016/j.isci.2021.103256

Figure Lengend Snippet: Class I/II HDACs drive NET formation (A and B) Freshly purified human (A) or mouse (B) neutrophils were treated with the indicated HDAC inhibitors at different doses (10, 2, 0.4, 0.08 μM), or with GSK484 (10 μM), for 1 h and then stimulated with 1 μM PMA for 3 h. Cells were fixed and stained with anti-citrullinated histone H3 (CitH3) antibody. NETosis induction was measured as the level of anti-CitH3 fluorescent signal. PMA alone represents 100% of NET induction. “-”: Cells treated with the highest dose of the indicated drug but not activated with PMA. (C and D) Human neutrophils were treated, or not, with ricolinostat or GSK484 and then stimulated with 1 μM PMA. NETosis induction was measured as area of nuclei stained with DAPI. (C) 3D reconstruction of nuclei in each treatment (grid size 7.8 μm). (D) Quantification of DAPI signal from multiple fields of view (D) (n = 9 for unstimulated, not pretreated with any drug [NT] and ricolinostat, n = 7 for GSK484). (E and F) Human neutrophils were treated as in (C-D), and immunoblot analysis of the indicated proteins was performed. Relative quantification of band intensity was analyzed with ImageJ software (F). White violins plots depict the quantification of histone H3 acetylation; red violin plots depict the quantification of histone H3 citrullination. Immunoblot in E is representative of four independent experiments. Gasdermin D NT, gasdermin D N terminal; pMLKL, phospho-MLKL. (G) Human (left) or murine (right) neutrophils were treated, or not, with ricolinostat (10 μM) and then stimulated with 1 μM PMA for 3 h. ROS production was measured by luminescence with luminol-HRP. Histograms represent the total amount of ROS produced overtime. (H) Murine neutrophils were treated, or not, with ricolinostat (10 μM) and then stimulated with fluorescent E. coli at the indicated multiplicity of infection (MOI) for 1 h. The histogram represents the percentage of neutrophils positive for E. coli staining. Graphs show mean ± SEM. Statistics were calculated using one-way ANOVA (A, B, D) and two-way ANOVA (F, G, H) (∗p < 0.05; ∗∗p < 0.01; ∗∗∗∗p < 0.0001. n.s., not significant). Statistics refers to the comparison with neutrophils stimulated with PMA alone (A, B). Gray stars represent comparison of all experimental groups with the unstimulated control, and black stars represent comparison between drug-treated groups and cells treated with PMA only (NT) (D). See also Figure S1 .

Article Snippet: Escherichia coli (O111) , ATCC , Cat# BAA-2440.

Techniques: Purification, Staining, Western Blot, Quantitative Proteomics, Software, Produced, Infection, Comparison, Control

Journal: iScience

Article Title: Zinc-dependent histone deacetylases drive neutrophil extracellular trap formation and potentiate local and systemic inflammation

doi: 10.1016/j.isci.2021.103256

Figure Lengend Snippet:

Article Snippet: Escherichia coli (O111) , ATCC , Cat# BAA-2440.

Techniques: Virus, Recombinant, Staining, Enzyme-linked Immunosorbent Assay, Software

Journal: BMC Microbiology

Article Title: Influence of platelet-activating factor receptor (PAFR) on Brucella abortus infection: implications for manipulating the phagocytic strategy of B. abortus

doi: 10.1186/s12866-016-0685-8

Figure Lengend Snippet: Production of PAF linked to LPCAT2 activation is enhanced by B. abortus infection. (a) RAW264.7 cells pretreated with AG490 (75 μM) or CV3988 (1 μM), a PAFR antagonist, for 1 h were infected with live B. abortus for 5 min. The cells were infected with heat-killed B. abortus and live E. coli O157:H7 for 5 min. The levels of PAF secretion in the culture supernatants were quantified by PAF ELISA. Data represent the mean ± SD of triplicate trials from three independent experiments. Statistically significant differences from the untreated samples are indicated by asterisks (*, P < 0.05; ***, P < 0.001). (b) Cells pretreated with PAF (200 nM), a PAFR agonist, for 5 min and AG490 (75 μM), a JAK2 inhibitor, for 1 h were infected with B. abortus for 5 min. (c) Cells were also were infected with heat-killed B. abortus or live E. coli O157:H7 for 5 min. The cells were processed for immunoprecipitation with a LPCAT2 antibody and then were probed with a phosphoserine antibody. The membrane was then stripped and re-probed with LPCAT2 antibody. The images shown are representative of three independent experiments

Article Snippet: In all experiments, the contents of freshly thawed vials were cultured in Brucella broth (Becton Dickinson, Franklin Lakes, NJ) or Brucella agar without antibiotics for 3 days at 37 °C with aeration; whereas, enterohemorrhagic E. coli (EHEC) O157:H7 (ATCC 43894) cultures were grown in Luria-Bertani (LB) broth.

Techniques: Activation Assay, Infection, Enzyme-linked Immunosorbent Assay, Immunoprecipitation, Membrane

Journal: Cell reports

Article Title: Cytosolic LPS-induced caspase-11 oligomerization and activation is regulated by extended synaptotagmin 1

doi: 10.1016/j.celrep.2023.112726

Figure Lengend Snippet: (A) Kaplan-Meier survival curve of WT, ESyt1−/− and Casp-11−/− mice infected with 2 × 107 colonyforming units (CFUs) intraperitoneally (i.p.) with B. thailandensis. WT mice (n = 10), ESyt1−/− mice (n = 12), Casp-11−/− mice (n = 8). ***p < 0.001; *p < 0.05; 1og rank test.

Article Snippet: Burkholderia thailandensis , ATCC , 700388.

Techniques: Infection

Journal: Cell reports

Article Title: Cytosolic LPS-induced caspase-11 oligomerization and activation is regulated by extended synaptotagmin 1

doi: 10.1016/j.celrep.2023.112726

Figure Lengend Snippet: KEY RESOURCES TABLE

Article Snippet: Burkholderia thailandensis , ATCC , 700388.

Techniques: Virus, Recombinant, Transfection, Enzyme-linked Immunosorbent Assay, Control, Software