Journal: iScience

Article Title: Bacterial pore-forming toxin pneumolysin drives pathogenicity through host extracellular vesicles released during infection

doi: 10.1016/j.isci.2024.110589

Figure Lengend Snippet: PLY-EVs induce dendritic cell maturation and inflammatory cytokine release upon internalization (A) Confocal microscopy images showing the internalization of CFSE-labelled PLY (0.1) and naive EVs (green) by THP-1-monocyte-derived DCs at 24 h post-treatment. Scale bars, 25 μm. (B) Flow cytometry histograms ( N = 3) to quantify the DC uptake of CFSE-labeled PLY(0.5)EVs and naive EVs. (C) Dose-dependent uptake of PLY (0.1, 0.5) EVs by DCs. (D) Phase-contrast microscopy images of immature day 5 DCs coincubated with PLY (0.1, 0.5) EVs and naive EVs for 24 h. Arrows indicate matured DCs (magnified in inset). Scale bars, 50 μm. Images are representative of three independent experiments. (E–G) Flow cytometry histograms ( N = 3) to quantify the expression levels of (E) CD80, (F) CD86, and (G) CD83 on THP-1-monocyte-derived DCs treated with PLY(0.5) and naive EVs. (H and I) Flow cytometry histograms ( N = 2) showing the expression levels of DC maturation marker CD83 at 96 h post-incubation of primary human monocytes with (H) PLY(0.5) and naive EVs and (I) naive EVs pre-treated with recombinant PLY protein (naive EVs+rPLY). (J and K) Cytokine ELISA showing the levels of secreted TNF-α from (J) DCs treated with PLY (0.1) EVs or naive EVs alone ( N = 3) for 24 h and (K) DCs pre-treated with PLY (0.1,0.5) or naive EVs for 24 h followed by subsequent infection with S. pneumoniae , T4R strain ( N = 2). Recombinant PLY (0.5 μg/mL) was used as positive control. All data are represented as mean ± SEM. ∗ p < 0.05, ∗∗ p < 0.005, and ∗∗∗ p < 0.001 by one-way ANOVA with Tukey’s multiple comparisons test. n.s., not significant. See also Figures S6–S9 .

Article Snippet: Mouse TNF-α DuoSet ELISA kit , R & D Systems , DY410-05.

Techniques: Confocal Microscopy, Derivative Assay, Flow Cytometry, Labeling, Microscopy, Expressing, Marker, Incubation, Recombinant, Enzyme-linked Immunosorbent Assay, Infection, Positive Control

Journal: iScience

Article Title: Bacterial pore-forming toxin pneumolysin drives pathogenicity through host extracellular vesicles released during infection

doi: 10.1016/j.isci.2024.110589

Figure Lengend Snippet: Adoptive transfer of EVs from infected mice drives inflammation and pathology in a PLY-dependent manner (A) C57BL/6 mice were intranasally administered with 4 × 10 6 CFU of serotype 4 strain, T4 or the isogenic PLY mutant strain, T4Δply. At day 4 post-infection, EVs isolated from BALF were labeled and administered to healthy recipient mice at 35 μg/mice. The EV retention in murine respiratory tract was imaged by IVIS imaging and immune infiltration into lungs, and cytokine levels in BALF was measured. (B) Bacterial load in murine BALF ( N = 5 mice/group) upon infection with T4 and T4Δply strains was measured by CFU dilution assay. ∗∗ in (B) indicates p < 0.01 by Mann-Whitney test. (C) Quantification of relative total EV protein content from mice ( N = 3 mice/group) infected with T4 and T4Δply strains by BCA protein assay. PBS-treated mice served as control. ∗ and ∗∗ in (C) indicates p < 0.05 and p < 0.005, respectively, by unpaired t test. (D) IVIS imaging of mice intranasally administered with Nile-red-labeled EVs isolated from mice infected with T4 (EVs-T4) or T4Δply (EVs-T4Δply). EVs from PBS-treated mice (naive EVs) served as control. ROI intensity values indicate the total flux (photons/sec) recorded from the given region showing higher intensity of EVs from T4-infected mice in the respiratory tract. The color scale (photons/sec/cm 2 ) indicates the relative intensities of individual signals. (E and F) Flow cytometry analysis of inflammatory macrophages (F4/80 + ) and neutrophils (Ly6G + ) in BALF of mice ( N = 6 mice/group) administered with EVs from infected or untreated mice at 18 h. (G) TNF-α levels in the BALF of mice ( N = 5 mice/group) treated with EVs isolated from infected or untreated mice were measured post-sacrifice at 18 h by ELISA. ∗∗ and ∗∗∗ in (G) indicates p < 0.01 and p < 0.001, respectively, by unpaired t test. (H) Hematoxylin and eosin (H&E) staining of mouse lungs ( N = 6 mice/group) at 18 h post-administration of EVs from infected or PBS-treated mice. Mice treated with EVs from T4-infected mice showed tissue microlesions (MLEs) and immune cell infiltration in the alveolar interstitium indicative of PLY-induced tissue damage (magnified in the inset). BR, bronchiole; MLE, microlesions. Scale bars, 200 μm. Blind histopathological scoring was performed based on presence or absence of cellularity in alveolar interstitium and lesions. A score of “0” was given when no lesions were found, and a score of “1” was given to tissue showing increasing cellularity and lesions. Mouse BALF flow cytometry and histology data are representative of three independent experiments. All data are represented as mean ± SEM. See also Figure S12 .

Article Snippet: Mouse TNF-α DuoSet ELISA kit , R & D Systems , DY410-05.

Techniques: Adoptive Transfer Assay, Infection, Mutagenesis, Isolation, Labeling, Imaging, Dilution Assay, MANN-WHITNEY, Bicinchoninic Acid Protein Assay, Control, Flow Cytometry, Enzyme-linked Immunosorbent Assay, Staining

Journal: iScience

Article Title: Bacterial pore-forming toxin pneumolysin drives pathogenicity through host extracellular vesicles released during infection

doi: 10.1016/j.isci.2024.110589

Figure Lengend Snippet:

Article Snippet: Mouse TNF-α DuoSet ELISA kit , R & D Systems , DY410-05.

Techniques: Virus, Mutagenesis, Isolation, Recombinant, Modification, Saline, Labeling, Staining, Electron Microscopy, Lysis, Western Blot, Buffer Exchange, Bicinchoninic Acid Protein Assay, Enzyme-linked Immunosorbent Assay, Clone Assay, Software, Membrane

Journal: Frontiers in Immunology

Article Title: Increased neutrophil extracellular trap formation in oligoarticular, polyarticular juvenile idiopathic arthritis and enthesitis-related arthritis: biomarkers for diagnosis and disease activity

doi: 10.3389/fimmu.2024.1436193

Figure Lengend Snippet: Demographic, clinical, and laboratory characteristics.

Article Snippet: To visualize whether TNF-α-induced NET formation was inhibited by TNF-α antagonists, neutrophils were seeded in the same way as described above and pretreated with a humanized anti-TNF-α antibody (adalimumab, Humira, Abbvie) (4 μg/mL, equivalent to the average serum concentration in the human body) for 15 minutes prior to TNF-α stimulation.

Techniques:

Journal: Frontiers in Immunology

Article Title: Increased neutrophil extracellular trap formation in oligoarticular, polyarticular juvenile idiopathic arthritis and enthesitis-related arthritis: biomarkers for diagnosis and disease activity

doi: 10.3389/fimmu.2024.1436193

Figure Lengend Snippet: Neutrophils derived from the peripheral blood of JIA patients increase NET formation in vitro , either spontaneously or in response to TNF-α or PMA. (A) Representative images of NETs released by neutrophils derived from at least 10 patients with JIA and 6 HCs spontaneously or in response to PMA or TNF-α. NETs were double immunostained for extracellular DNA (DAPI; blue) and MPO (MPO antibody; green), and NETs were identified by the presence of extracellular DNA stained with SYTOX-Green. Images were captured at 20× magnification; the scale bars represent 100 µm. (B) In a quantitative analysis of the above experiments, the percentages of NET-occupied areas in the total area in the JIA group were markedly higher than those in the HC group. (C–F) Quantitative assessment of NET formation in the o-JIA, p-JIA, and ERA groups by microplate assays. Neutrophils isolated from patients with o-JIA, p-JIA, and ERA and healthy controls were incubated for 6 hours under the conditions of (C) no intervention, (D) PMA (30 nM) stimulus, or (E) the addition of TNF-a (100 ng/ml). (F) The amounts of NETs were compared among the three subtypes of o-JIA, p-JIA, and ERA, respectively. The results are expressed as the fluorescence intensity of DNA in NETs. One-way ANOVA or the Kruskal−Wallis test was used to compare three or more groups. Student’s independent-sample t-test was used to compare the two groups. The bar graphs show the mean ± SEM or median with IQR. *p<0.05, **p<0.01, ***p<0.001, ns, nonsignificant; spontan, spontaneous.

Article Snippet: To visualize whether TNF-α-induced NET formation was inhibited by TNF-α antagonists, neutrophils were seeded in the same way as described above and pretreated with a humanized anti-TNF-α antibody (adalimumab, Humira, Abbvie) (4 μg/mL, equivalent to the average serum concentration in the human body) for 15 minutes prior to TNF-α stimulation.

Techniques: Derivative Assay, In Vitro, Staining, Isolation, Incubation, Fluorescence

Journal: Frontiers in Immunology

Article Title: Increased neutrophil extracellular trap formation in oligoarticular, polyarticular juvenile idiopathic arthritis and enthesitis-related arthritis: biomarkers for diagnosis and disease activity

doi: 10.3389/fimmu.2024.1436193

Figure Lengend Snippet: Associations of cf-DNA levels with immune-related parameters in the entire cohort of patients with JIA. (A) The concentrations of cf-DNA were strongly correlated with the number and percentage of neutrophils and CD19+ B cells, the expression of inflammatory markers (TNF-α, ESR, and hs-CRP), the levels of IgG, complement C3 and C4 in peripheral blood, but no correlation with IL-6 level. R values of Spearman or Pearson’s rank correlation and p values of their null hypothesis are shown. (B) ESR and TNF-α are independent variables by the multiple linear regression analysis.

Article Snippet: To visualize whether TNF-α-induced NET formation was inhibited by TNF-α antagonists, neutrophils were seeded in the same way as described above and pretreated with a humanized anti-TNF-α antibody (adalimumab, Humira, Abbvie) (4 μg/mL, equivalent to the average serum concentration in the human body) for 15 minutes prior to TNF-α stimulation.

Techniques: Expressing

Journal: Frontiers in Immunology

Article Title: Increased neutrophil extracellular trap formation in oligoarticular, polyarticular juvenile idiopathic arthritis and enthesitis-related arthritis: biomarkers for diagnosis and disease activity

doi: 10.3389/fimmu.2024.1436193

Figure Lengend Snippet: Effect of an anti-TNF-a antibody (adalimumab) on NET formation. Neutrophils isolated from JIA patients and HCs were pretreated with adalimumab (4 μg/ml) prior to the addition of TNF-α. (A) Representative images of TNF-α induced NETs and minimal NET formation after adalimumab intervention. The third column is an image of spontaneous NET formation as a negative control. (B) The percentage of the NET-occupied area under TNF-α stimulation was significantly decreased in the presence of adalimumab (n=18, 41.22% ± 2.351 vs. 20.39% ± 1.683, p < 0.0001). (C–F) The levels of NET release were measured via microplate assays to further evidence the ability of adalimumab to inhibit NET generation. (C) DPI can effectively inhibit PMA-induced NETosis. (D) Adalimumab (n=27) and DPI (n=16) effectively inhibited TNF-α-induced NET formation (n=27). (E) Comparison of the inhibitory effects of adalimumab and DPI on TNF-α-induced NET formation. (F) The inhibitory rate of adalimumab in NETosis by neutrophils from JIA (n=18) and HCs (n=9) under TNF-α stimulation. (G) The effect of TNF-α inhibitors on cf-DNA concentration and JADAS27 in JIA patients. The data were analyzed with paired-sample t test or unpaired-sample t test. The bar graphs show the mean ± SEM. *** p <0.001, **** p <0.0001, ns, nonsignificant.

Article Snippet: To visualize whether TNF-α-induced NET formation was inhibited by TNF-α antagonists, neutrophils were seeded in the same way as described above and pretreated with a humanized anti-TNF-α antibody (adalimumab, Humira, Abbvie) (4 μg/mL, equivalent to the average serum concentration in the human body) for 15 minutes prior to TNF-α stimulation.

Techniques: Isolation, Negative Control, Comparison, Concentration Assay

Journal: Frontiers in Immunology

Article Title: Increased neutrophil extracellular trap formation in oligoarticular, polyarticular juvenile idiopathic arthritis and enthesitis-related arthritis: biomarkers for diagnosis and disease activity

doi: 10.3389/fimmu.2024.1436193

Figure Lengend Snippet: ROS levels were measured ex vivo using a FACSCalibur flow cytometer. (A) The exposure of neutrophils to TNF-α increased in the production of ROS. (B) Baseline ROS levels in neutrophils derived from JIA patients were higher than those in HC-derived neutrophils (n = 20; p = 0.0074). The paired-sample t test was used to analyze all the data. * p < 0.05, ** p <0.01.

Article Snippet: To visualize whether TNF-α-induced NET formation was inhibited by TNF-α antagonists, neutrophils were seeded in the same way as described above and pretreated with a humanized anti-TNF-α antibody (adalimumab, Humira, Abbvie) (4 μg/mL, equivalent to the average serum concentration in the human body) for 15 minutes prior to TNF-α stimulation.

Techniques: Ex Vivo, Flow Cytometry, Derivative Assay

Journal: Frontiers in Immunology

Article Title: Increased neutrophil extracellular trap formation in oligoarticular, polyarticular juvenile idiopathic arthritis and enthesitis-related arthritis: biomarkers for diagnosis and disease activity

doi: 10.3389/fimmu.2024.1436193

Figure Lengend Snippet: The expression levels of NET-associated signaling components. (A) The phosphorylation of PI3K and AKT in neutrophils purified from JIA patients and HCs before and after TNF-α stimulation (p-PI3K n = 10, p = 0.0008; p-AKT, n = 6, p = 0.0313). The paired-sample t test and Wilcoxon test were used to analyze the data. (B) Comparison of phosphorylated PI3K and AKT protein levels in neutrophils derived from JIA patients and HCs after TNF-α stimulation (n = 5, p = 0.7340; n = 3, p = 0.3386, respectively). (C) Phosphorylation of PI3K (15 HCs vs. 18 JIA patients, p = 0.0222) and AKT (n = 10 p = 0.0135) and the protein expression of MPO (n = 15, p = 0.0012) in neutrophils derived from JIA patients and HCs. (D) MAPK-CDK and ERK1/2 phosphorylation was assessed (6 HCs vs. 7 JIA patients p = 0.0239). (A, C, D) Representative western blots. The Mann−Whitney U test was used to compare two groups. Stim, stimulated; unstim, unstimulated. The bar graphs show the mean ± SEM. * p < 0.05, ** p < 0.01, *** p < 0.001, ns, nonsignificant.

Article Snippet: To visualize whether TNF-α-induced NET formation was inhibited by TNF-α antagonists, neutrophils were seeded in the same way as described above and pretreated with a humanized anti-TNF-α antibody (adalimumab, Humira, Abbvie) (4 μg/mL, equivalent to the average serum concentration in the human body) for 15 minutes prior to TNF-α stimulation.

Techniques: Expressing, Phospho-proteomics, Purification, Comparison, Derivative Assay, Western Blot, MANN-WHITNEY