Journal: FASEB journal : official publication of the Federation of American Societies for Experimental Biology

Article Title: Extracellular CIRP and TREM-1 axis promotes ICAM-1-Rho-mediated NETosis in sepsis

doi: 10.1096/fj.202000482R

Figure Lengend Snippet: Inhibition of Rho activation reduces NET formation in BMDN. A, BMDN (2.5 × 106) were stimulated with PBS or rmCIRP in presence of ICAM-1 neutralizing Ab for 120 minutes. After stimulating the BMDN with rmCIRP, cells were lysed in lysis buffer and Rho activity was determined by GTP pull down process, followed by western blot assays using anti-Rho Ab. A fraction of each lysate was retained to determine the total Rho and β-actin by western blot using rabbit anti-Rho and β-actin Abs. Representative western blots for active Rho, total Rho, and β-actin are shown. Active Rho in each sample was normalized to total Rho expression and the mean values of PBS-treated group was standardized as one for comparison. Experiment was repeated twice. Data are expressed as mean ± SD (n = 5–6 samples/group). The groups were compared by one-way ANOVA and SNK method (*P < .05 vs PBS; #P < .05 vs rmCIRP). B, C, A total of 106 BMDN were stimulated with rmCIRP for 4 hours in presence of vehicle control (PBS) or Rho inhibitor. After stimulation with rmCIRP, the neutrophils were surface-stained with APC-rat anti-mouse Ly-6G Ab, FITC-mouse MPO Ab, and rabbit anti-histone H3 (CitH3) Ab followed by staining with PE-donkey anti-rabbit IgG. NETs (MPO+citH3+ neutrophils) were assessed by flow cytometry. B, Representative dot plots of the NETs+ BMDN are shown. C, Bar diagram showing the frequencies of NETs+ neutrophils in the lungs are shown. Data are expressed as mean ± SE (n = 4 samples/group). The groups were compared by one-way ANOVA and SNK method (*P < .05 vs PBS; #P < .05 vs vehicle + rmCIRP)

Article Snippet: APC-rat anti-mouse Ly-6G Ab (clone 1A8; Biolegend, San Diego, CA), anti-TREM-1 Ab (clone: 174021, R&D systems, Minneapolis, MN), anti-phosphotyrosine Ab (Cat. No.: 05–321; clone: 4G10; EMD Millipore), anti-DAP12 Ab (Cell Signaling Technology), anti-pSyk Ab (Abcam), anti-Syk Ab (Cell Signaling Technology), anti-β-actin Ab (Sigma-Aldrich, St Louis, MO), anti-ICAM-1 Ab (clone: 3E2, BD Biosciences, San Jose, CA), anti-MPO Ab (clone: mAb 8F4; Hycult Biotech, Uden, The Netherlands), anti-NE Ab (Biotin, Cat. No.: ab 79962, Abcam), anti-DNA-POD Ab (cell death detection kit, Cat. No.: 11 544 675 001, Roche Diagnostics, GmbH, Mannheim, Germany), FITC-mouse anti-MPO Ab (clone: 2D4; Abcam), rabbit anti-histone H3 (CitH3) Ab (Cat. No 5103: Abcam), PE-donkey anti-rabbit IgG (clone: Poly4064; Biolegend), rabbit polyclonal IgG (clone: Poly29108, Biolegend), and FITC-mouse IgG1 (B11/5, ab91356, Abcam).

Techniques: Inhibition, Activation Assay, Lysis, Activity Assay, Western Blot, Expressing, Staining, Flow Cytometry

Journal: FASEB journal : official publication of the Federation of American Societies for Experimental Biology

Article Title: Extracellular CIRP and TREM-1 axis promotes ICAM-1-Rho-mediated NETosis in sepsis

doi: 10.1096/fj.202000482R

Figure Lengend Snippet: ICAM-1 deficient neutrophils or blockade of TREM-1 in WT neutrophils with LP17 produce less NETs following rmCIRP stimulation. A total of 106 BMDN isolated from WT or ICAM-1−/− mice were stimulated with rmCIRP for 4 hours. After stimulation with rmCIRP, isolated NETs were loaded in (A) MPO:DNA and (B) NE:DNA coated plates and assessed by ELISA. C, A total of 106 BMDN isolated from WT or ICAM-1−/− mice were stimulated with rmCIRP for 4 hours. After stimulation with rmCIRP, the neutrophils were surface-stained with APC-rat anti-mouse Ly-6G Ab, FITC-mouse MPO Ab, and rabbit anti-histone H3 (CitH3) Ab followed by staining with PE-donkey anti-rabbit IgG. NETs (MPO+citH3+ neutrophils) were assessed by flow cytometry. D, E, After 4 hours of injection of rmCIRP intratracheally (i.t.) in WT and ICAM-1−/− mice lungs were perfused and harvested. Single cell suspensions of lung tissues were surface-stained with APC-rat anti-mouse Ly-6G Ab, FITC-mouse anti-MPO Ab, and rabbit anti-histone H3 (CitH3) Ab followed by staining with PE-donkey anti-rabbit IgG and NETs were determined by flow cytometry. D, The frequencies and (E) numbers of NET forming neutrophils in lungs are shown. Experiments were repeated at least three times. Data are expressed as mean ± SE or SD (n = 5–14 samples/group). The groups were compared by one-way ANOVA and SNK method (*P < .05 vs WT PBS; #P < .05 vs WT rmCIRP). F, A total of 106 BMDN/mL in 24-well plate were treated with PBS or rmCIRP in the presence of vehicle (PBS), LP17 or scramble. After 4 hours of rmCIRP stimulation, NETs were collected from the cells and assessed NE contents by ELISA. Experiments were repeated at least three times. Data are expressed as mean ± SD (n = 6–11 samples/group). The groups were compared by one-way ANOVA and SNK method (*P < .05 vs PBS-treated group; #P < .05 vs vehicle (PBS) + rmCIRP-treated group; †P < .05 vs scramble + rmCIRP-treated group)

Article Snippet: APC-rat anti-mouse Ly-6G Ab (clone 1A8; Biolegend, San Diego, CA), anti-TREM-1 Ab (clone: 174021, R&D systems, Minneapolis, MN), anti-phosphotyrosine Ab (Cat. No.: 05–321; clone: 4G10; EMD Millipore), anti-DAP12 Ab (Cell Signaling Technology), anti-pSyk Ab (Abcam), anti-Syk Ab (Cell Signaling Technology), anti-β-actin Ab (Sigma-Aldrich, St Louis, MO), anti-ICAM-1 Ab (clone: 3E2, BD Biosciences, San Jose, CA), anti-MPO Ab (clone: mAb 8F4; Hycult Biotech, Uden, The Netherlands), anti-NE Ab (Biotin, Cat. No.: ab 79962, Abcam), anti-DNA-POD Ab (cell death detection kit, Cat. No.: 11 544 675 001, Roche Diagnostics, GmbH, Mannheim, Germany), FITC-mouse anti-MPO Ab (clone: 2D4; Abcam), rabbit anti-histone H3 (CitH3) Ab (Cat. No 5103: Abcam), PE-donkey anti-rabbit IgG (clone: Poly4064; Biolegend), rabbit polyclonal IgG (clone: Poly29108, Biolegend), and FITC-mouse IgG1 (B11/5, ab91356, Abcam).

Techniques: Isolation, Enzyme-linked Immunosorbent Assay, Staining, Flow Cytometry, Injection

Journal: FASEB journal : official publication of the Federation of American Societies for Experimental Biology

Article Title: Extracellular CIRP and TREM-1 axis promotes ICAM-1-Rho-mediated NETosis in sepsis

doi: 10.1096/fj.202000482R

Figure Lengend Snippet: ICAM-1−/− mice produce less NETs in the lungs in sepsis. Sepsis was induced in WT and ICAM-1−/− mice by cecal ligation and puncture (CLP). After 4 hours of CLP, lung tissues were harvested and single cell suspensions were prepared. Cells were surface-stained with APC-rat anti-mouse Ly-6G Ab, FITC-mouse anti-MPO Ab, and rabbit anti-histone H3 (CitH3) Ab followed by staining with PE-donkey anti-rabbit IgG and NETs were determined by flow cytometry. A, Representative dot plots of the NETs+ cells in the lungs are shown. B, The frequencies and (C) numbers of NET forming neutrophils in lungs are shown. Experiments were repeated at least three times. Data are expressed as mean ± SE (n = 6 mice/group). The groups were compared by one-way ANOVA and SNK method (*P < .05 vs WT sham; #P < .05 vs WT CLP)

Article Snippet: APC-rat anti-mouse Ly-6G Ab (clone 1A8; Biolegend, San Diego, CA), anti-TREM-1 Ab (clone: 174021, R&D systems, Minneapolis, MN), anti-phosphotyrosine Ab (Cat. No.: 05–321; clone: 4G10; EMD Millipore), anti-DAP12 Ab (Cell Signaling Technology), anti-pSyk Ab (Abcam), anti-Syk Ab (Cell Signaling Technology), anti-β-actin Ab (Sigma-Aldrich, St Louis, MO), anti-ICAM-1 Ab (clone: 3E2, BD Biosciences, San Jose, CA), anti-MPO Ab (clone: mAb 8F4; Hycult Biotech, Uden, The Netherlands), anti-NE Ab (Biotin, Cat. No.: ab 79962, Abcam), anti-DNA-POD Ab (cell death detection kit, Cat. No.: 11 544 675 001, Roche Diagnostics, GmbH, Mannheim, Germany), FITC-mouse anti-MPO Ab (clone: 2D4; Abcam), rabbit anti-histone H3 (CitH3) Ab (Cat. No 5103: Abcam), PE-donkey anti-rabbit IgG (clone: Poly4064; Biolegend), rabbit polyclonal IgG (clone: Poly29108, Biolegend), and FITC-mouse IgG1 (B11/5, ab91356, Abcam).

Techniques: Ligation, Staining, Flow Cytometry

Journal: PLoS Pathogens

Article Title: Lung macrophage scavenger receptor SR-A6 (MARCO) is an adenovirus type-specific virus entry receptor

doi: 10.1371/journal.ppat.1006914

Figure Lengend Snippet: A) Exogenous expression of human SR-A6 in L-929 cells (low CAR expression) promotes binding of HAdV-C5 to the cells. L-929 cells were transfected with a plasmid directing the synthesis of human SR-A6 from the cytomegalovirus major immediate early promoter and transfected cells were identified by immunostaining with anti-human SR-A6 antibody PLK1. Alexa-Fluor488-labeled HAdV-C5 particles were added to transfected L-929 cells at 4°C for 60 min. Fixed samples were imaged by confocal microscopy and virus particles associated with PLK1-positive and PLK1-negative cells were scored from maximum projections of confocal stacks. The plot shows number of virus particles per cell, one dot representing one cell. Horizontal bars represent mean values. Number of cells analyzed is indicated. The difference between PLK1-positive and -negative cells was statistically highly significant (P<0.0001, Kolmogorov-Smirnov test). The right-hand panel shows a representative image as a maximum projection of confocal stacks. In the overlay panel virus particles are shown in green, SR-A6-positive cells in red and nuclei (DAPI) in blue. Scale bar = 10 μm. B) Exogenous expression of human SR-A6 in HDF-TERT cells boosts HAdV-C5-mediated gene transduction. SR-A6 was expressed in the cells from a plasmid that directed synthesis of the protein from a bi-cistronic SR-A6-IRES-Tomato mRNA. Transfected HDF-TERT cells were infected with two different amounts of HAdV-C5_dE1_GFP, and nuclear GFP signals were scored at 30 h post infection (pi) by microscopy. Non-transfected cells or cells transfected with an empty vector were used as controls. The mean nuclear GFP intensities of Tomato-positive and Tomato-negative cells are shown as Tukey box plots. The difference between Tomato-positive and Tomato-negative cells in the SR-A6 transfection was statistically highly significant with a P-value <0.0001 (Kolmogorov-Smirnov test), whereas the difference in empty vector transfections did not reach P<0.0001 significance levels. Over 300 Tomato-positive cells and more than 5000 Tomato-negative cells were scored for each sample. C) Mouse SR-A6 expression in HDF-TERT cells leads to higher HAdV-C5_dE1_GFP infection efficiency than human SR-A6 expression. Both mouse (mSR-A6) and human (hSR-A6) proteins were expressed from bi-cistronic SR-A6-IRES-Tomato mRNAs. The mean nuclear GFP intensities of Tomato-positive and Tomato-negative cells are shown as Tukey box plots. Over 500 Tomato-positive cells and more than 4500 Tomato-negative cells were scored for each sample.

Article Snippet: Transfected cells were identified by staining for surface SR-A6 using anti-human SR-A6 antibody PLK1 (HM2208, Hycult Biotech) and Alexa-Fluor 594-conjugated secondary anti-mouse antibodies as described [ ].

Techniques: Expressing, Binding Assay, Transfection, Plasmid Preparation, Immunostaining, Labeling, Confocal Microscopy, Transduction, Infection, Microscopy