Journal: Heliyon

Article Title: Sodium butyrate restricts neutrophils migration and NETs formation through reducing macrophage-derived CXCL16 in calculous cholecystitis

doi: 10.1016/j.heliyon.2024.e25189

Figure Lengend Snippet: Sodium butyrate reduced M1 macrophages polarization stimulated by LPS in RAW264.7 cells. (a) Immunofluorescent staining of CD86 and CD206 in LPS with or without Sodium butyrate (NaB) induced RAW264.7 mouse macrophages, Scale bar: 20 μm. (b) Quantitative analysis of fluorescence intensity for CD86 and CD206 in RAW264.7 cells. (c–d) qRT-PCR results for TNF-α and IL-10 mRNA levels after LPS stimulation with or without Sodium butyrate treatment. ** and *** denote P < 0.01 and P < 0.001 compared to the control group, & , && and &&& denote P < 0.05, P < 0.01 and P < 0.001 compared to the NaB (0) group, respectively.

Article Snippet: The cells were fixed and stained according to the above method for immunofluorescence assay of RAW264.7 cells with primary antibody of MPO (bioss, bs-4943R, 1:100).

Techniques: Staining, Fluorescence, Quantitative RT-PCR, Control

Journal: Heliyon

Article Title: Sodium butyrate restricts neutrophils migration and NETs formation through reducing macrophage-derived CXCL16 in calculous cholecystitis

doi: 10.1016/j.heliyon.2024.e25189

Figure Lengend Snippet: Sodium butyrate reduced exosomal CXCL16 secreted by LPS-induced RAW264.7 cells. (a) Schematic of the protocol for exosome collection. (b) EVs obtained from medium were observed by TEM. Scale bar 200 nm. (c) The distribution of EVs were obtained by NanoSight tracking analysis.(d–e) Westernblot analysis (d) of exosomal proteins (CD63, TSG101, HSP70 and CXCL16) collected from the same volume RAW264.7 cells culture supernatant and quantitative analysis (e). **and***denote P < 0.01 and P < 0.001 compared to the control group, & and && denote P < 0.05 and P < 0.01 compared to the NaB (0) group, respectively.

Article Snippet: The cells were fixed and stained according to the above method for immunofluorescence assay of RAW264.7 cells with primary antibody of MPO (bioss, bs-4943R, 1:100).

Techniques: Control

Journal: Heliyon

Article Title: Sodium butyrate restricts neutrophils migration and NETs formation through reducing macrophage-derived CXCL16 in calculous cholecystitis

doi: 10.1016/j.heliyon.2024.e25189

Figure Lengend Snippet: Sodium butyrate reduced neutrophil migration and NETs formation by inhibiting exosomal CXCL16 secretion. (a) Representative fluorescence images of neutrophil migration assay. Neutrophils were stained with calcein-AM and co-cultured with RAW264.7 cells exosomes in the presence of LPS, Sodium butyrate or CXCL16 antagonist/recombinant CXCL16, quantitative assessment of neutrophil migration across a permeable transwell chamber. Data represent mean ± SD (n = 5), ***p < 0.001 compared to control group, && and &&& denote p < 0.01 and p < 0.001 compared to LPS group. (c) CXCR6 protein levels were detected by Western blot. (d–e) Neutrophilic extracellular traps (NETs) were identified by MPO staining observed by confocal microscopy (green). Quantification of dsDNA and circulating NET structures in the supernatant of cultured PMNs using PicoGreen fluorescent dye and MPO-DNA-ELISA, respectively.** P < 0.01 compared to control group, & and && denote P < 0.05 and P < 0.01 compared to LPS group. $ denote P < 0.05 compared to LPS + NaB(H) group. (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.)

Article Snippet: The cells were fixed and stained according to the above method for immunofluorescence assay of RAW264.7 cells with primary antibody of MPO (bioss, bs-4943R, 1:100).

Techniques: Migration, Fluorescence, Staining, Cell Culture, Recombinant, Control, Western Blot, Confocal Microscopy, Enzyme-linked Immunosorbent Assay

Journal: Nature Cardiovascular Research

Article Title: Gasdermin D-dependent platelet pyroptosis exacerbates NET formation and inflammation in severe sepsis

doi: 10.1038/s44161-022-00108-7

Figure Lengend Snippet: a , Boxplots showing quantification of MPO–DNA and dsDNA (NET structures) in the plasma of HS ( n = 53) and patients with severe sepsis (with or without septic shock) ( n = 51). The boxes indicate the 25% quantile, median and 75% quantile. b , Scatterplots displaying correlations of platelet pyroptosis (activated caspase 1 positive platelets) with the levels of MPO–DNA (HS, n = 15; severe sepsis, n = 34) complexes and dsDNA (HS, n = 20; severe sepsis, n = 29) in the plasma of patients with severe sepsis (with or without septic shock) and HS ( r , correlation coefficient; n = 49). c , Immunofluorescence analysis showing S100A8/A9-induced pyroptotic platelets induced NET formation. Cells were stained with Hoechst for DNA (blue), anti-MPO for PMNs or NETs (green) and CD41 for platelets (red). Scale bars, 25 μm and 7.5 μm. d , Quantification of MPO–DNA and dsDNA in the supernatant of cells ( n = 6). e , Immunofluorescence analysis showing NET formation in PMNs treated with rmS100A8/A9-induced platelets (WT or Tlr4 −/− ) (n = 6). Cells were stained with Hoechst for DNA (blue), anti-MPO for PMNs or NETs (red) and CD41 for platelets (green). Scale bars, 25 μm and 7.5 μm. f , Bar graphs displaying the levels of MPO–DNA and dsDNA in the supernatant of cells treated with rmS100A8/A9-induced platelets ( Tlr4 −/− or WT) ( n = 6). g , Levels of ox-mtDNA in supernatant of S100A8/A9-induced pyroptotic platelets (for 4 h) determined using a General 8-OHdG ELISA Kit ( n = 5). h , Levels of ox-mtDNA in plasma from sepsis patients (sepsis, n = 11; severe sepsis, n = 17) and HS ( n = 13). i , Levels of ox-mtDNA in plasma of sham (n = 7) or CLP mice ( n = 9). Data were presented as mean ± s.d. Statistical analysis was conducted using two-tailed Mann–Whitney test ( a ), two-tailed Pearson’s correlation test ( b ), one-way ANOVA and Tukey’s multiple comparisons test ( d , f , g ), Kruskal–Wallis test and Dunn’s multiple comparisons test ( h ) and two-tailed unpaired t -test ( i ).

Article Snippet: MPO–DNA complexes and dsDNA in plasma of human/mice and medium supernatant were detected by anti-MPO polyclonal (biotin conjugated) primary antibody (Bioss, 1:500, bs-4943R-Biotin) with a Cell Death Detection enzyme-linked immunosorbent assay (ELISA) kit (Roche, 11774425001) and Quant-iT PicoGreen dsDNA reagent (Invitrogen, catalog no. P7581) according to the manufacturer’s protocol.

Techniques: Immunofluorescence, Staining, Enzyme-linked Immunosorbent Assay, Two Tailed Test, MANN-WHITNEY

Journal: Nature Cardiovascular Research

Article Title: Gasdermin D-dependent platelet pyroptosis exacerbates NET formation and inflammation in severe sepsis

doi: 10.1038/s44161-022-00108-7

Figure Lengend Snippet: a , S100A8/A9 released from NETs determined by ELISA ( n = 5). b , Representative immunofluorescence images of pyroptotic platelets incubated with PMA-treated murine PMNs (WT or S100a9 −/− ). Cells were stained with Hoechst for DNA (blue), anti-MPO for PMNs or NETs (cyan), CD41 for platelets (red) and caspase 1 for pyroptosis (green). Scale bars, 25 μm and 5 μm. c , Bar graphs displaying mitochondrial ROS production in platelets cocultured with PMA-treated murine PMNs (WT or S100a9 −/− ) using FACS analysis ( n = 5). d , Immunofluorescence analysis showing coexpression of CD41 (green), ASC (red) and NLRP3 (blue) in platelets cocultured with PMA-treated murine PMNs (WT or S100a9 −/− ) ( n = 5). Purple indicates overlap. Scale bars, 5 μm and 1 μm. e , Quantified results for the NLRP3 inflammasome are shown. f , Bar graphs displaying the caspase 1 activity of platelets cocultured with PMA-treated murine PMNs (WT or S100a9 −/− ) by FACS analysis ( n = 5). Data were presented as mean ± s.d. Statistical analysis was conducted using one-way ANOVA and Tukey’s multiple comparisons test ( a , c , e , f ).

Article Snippet: MPO–DNA complexes and dsDNA in plasma of human/mice and medium supernatant were detected by anti-MPO polyclonal (biotin conjugated) primary antibody (Bioss, 1:500, bs-4943R-Biotin) with a Cell Death Detection enzyme-linked immunosorbent assay (ELISA) kit (Roche, 11774425001) and Quant-iT PicoGreen dsDNA reagent (Invitrogen, catalog no. P7581) according to the manufacturer’s protocol.

Techniques: Enzyme-linked Immunosorbent Assay, Immunofluorescence, Incubation, Staining, Activity Assay

Journal: Nature Cardiovascular Research

Article Title: Gasdermin D-dependent platelet pyroptosis exacerbates NET formation and inflammation in severe sepsis

doi: 10.1038/s44161-022-00108-7

Figure Lengend Snippet: a , PMNs ( S100a9 -/- or WT) were incubated with 50 nM PMA to induced NET formation for 4 hours, and then incubated with platelets for another 4 hours. Representative immunofluorescence of PMNs incubated with platelets. Cells were stained with Hoechst for DNA (blue), anti-MPO for PMNs or NETs (cyan), CD41 for platelet (red) and activated caspase 1 for pyroptosis (green); scale bars: 25 μm and 5 μm. b , In the CLP-induced sepsis model, platelet counts in Gsdmd fl/fl PF4-Cre mice and littermate control Gsdmd fl/fl mice were assessed at 0, 2, 4, and 6 hours using a hematology analyzer (n = 5). Data was presented as mean ± SD. Two-way ANOVA and Tukey’s multiple comparisons test for b. Abbreviation is as follow: PLT, platelet; NS, not statistically significant; PMNs, polymorphonuclear neutrophils; PMA, phorbol myristate acetate; MPO, myeloperoxidase; Sham, sham-operated mice; CLP, CLP-induced sepsis mice; GSDMD, Gasdermin D.

Article Snippet: MPO–DNA complexes and dsDNA in plasma of human/mice and medium supernatant were detected by anti-MPO polyclonal (biotin conjugated) primary antibody (Bioss, 1:500, bs-4943R-Biotin) with a Cell Death Detection enzyme-linked immunosorbent assay (ELISA) kit (Roche, 11774425001) and Quant-iT PicoGreen dsDNA reagent (Invitrogen, catalog no. P7581) according to the manufacturer’s protocol.

Techniques: Incubation, Immunofluorescence, Staining

Journal: Nature Cardiovascular Research

Article Title: Gasdermin D-dependent platelet pyroptosis exacerbates NET formation and inflammation in severe sepsis

doi: 10.1038/s44161-022-00108-7

Figure Lengend Snippet: a , Immunofluorescence analysis showing NET formation induced by platelets from Gsdmd fl/fl PF4-Cre mice and Gsdmd fl/fl mice treated with S100A8/A9 ( n = 5). Cells were stained with Hoechst for DNA (blue), anti-MPO for PMNs or NETs (red) and CD41 for platelets (green). Scale bars, 25 μm and 7.5 μm. b , Bar graphs displaying the levels of MPO–DNA and dsDNA in supernatant of rmS100A8/A9-induced platelets from Gsdmd fl/fl PF4-Cre mice and Gsdmd fl/fl mice using PicoGreen fluorescent dye and MPO–DNA–ELISA, respectively ( n = 5). c – h , In the CLP-induced sepsis murine model, levels of NETs, heterodimer S100A8/A9 and proinflammatory cytokines in plasma of Gsdmd fl/fl PF4-Cre mice and Gsdmd fl/fl mice were detected using ELISA kits after 6 h. c , d , Bar graphs displaying plasma levels of MPO–DNA complexes ( c ) and dsDNA ( d ) in mice ( n = 5). e , Bar graphs showing plasma levels of heterodimer S100A8/A9 in mice ( Gsdmd fl/fl + sham, n = 7; Gsdmd fl/fl + CLP, n = 8; Gsdmd fl/fl PF4-Cre+sham, n = 8; Gsdmd fl/fl PF4-Cre+CLP, n = 6). f – h , Bar graphs showing plasma levels of IL-1β ( f ), TNF-α ( g ) and IL-6 ( h ) in mice ( n = 5). i , Survival analysis of Gsdmd fl/fl PF4-Cre mice and Gsdmd fl/fl mice in CLP-induced sepsis model for 7 d ( n = 11). Data were presented as mean ± s.d. Statistical analysis was conducted using one-way ANOVA and Tukey’s multiple comparisons test ( b – h ) and the log-rank (Mantel–Cox) test ( i ).

Article Snippet: MPO–DNA complexes and dsDNA in plasma of human/mice and medium supernatant were detected by anti-MPO polyclonal (biotin conjugated) primary antibody (Bioss, 1:500, bs-4943R-Biotin) with a Cell Death Detection enzyme-linked immunosorbent assay (ELISA) kit (Roche, 11774425001) and Quant-iT PicoGreen dsDNA reagent (Invitrogen, catalog no. P7581) according to the manufacturer’s protocol.

Techniques: Immunofluorescence, Staining, Enzyme-linked Immunosorbent Assay