Journal: Frontiers in Immunology

Article Title: Up-Regulation of TLR7-Mediated IFN-α Production by Plasmacytoid Dendritic Cells in Patients With Systemic Lupus Erythematosus

doi: 10.3389/fimmu.2018.01957

Figure Lengend Snippet: Up-regulation of TLR7-mediated IFN-α production by pDCs in SLE patients. (A) Representative flow cytometry plots showing TLR7/9-mediated IFN-α production in Lin − HLA-DR + CD11c − CD123 + pDCs from healthy control subjects and SLE patients. (B) TLR7/9-mediated IFN-α production in pDCs of each group. (C) Representative flow cytometry plots showing TLR7/9 expression in pDCs of healthy control subjects and SLE patients. (D) TLR7/9 expression in pDCs of each group. Horizontal lines represent the mean value of each group. * p < 0.05, ** p < 0.01, compared to the control (Mann-Whitney's U -test). Numbers in brackets on the abscissa represent the number of subjects of each group.

Article Snippet: After fixation and permeabilization with Fixation/Permeabilization buffer (e-Biosciences), the cells were stained with PE-conjugated anti-IFN-α2b (clone 7N4-1), PE-conjugated anti-IFN-α (clone LT27:295 recognize the majority of the IFN-α subtypes, but not IFN-α2b), and FITC-conjugated anti-IFN-β (clone MMHB-1) for intracellular cytokine or PE-conjugated anti-TLR7 (clone 4G6) and APC-conjugated anti-TLR9 (clone eB72-1665) for intracellular TLR.

Techniques: Flow Cytometry, Expressing, MANN-WHITNEY

Journal: Frontiers in Immunology

Article Title: Up-Regulation of TLR7-Mediated IFN-α Production by Plasmacytoid Dendritic Cells in Patients With Systemic Lupus Erythematosus

doi: 10.3389/fimmu.2018.01957

Figure Lengend Snippet: TLR7-mediated IFN-α production correlates with disease activity in SLE patients. Correlation between TLR7/9-mediated IFN-α production and SLEDAI or anti-dsDNA Ab titer. Statistical analysis by the Spearman's correlation coefficient.

Article Snippet: After fixation and permeabilization with Fixation/Permeabilization buffer (e-Biosciences), the cells were stained with PE-conjugated anti-IFN-α2b (clone 7N4-1), PE-conjugated anti-IFN-α (clone LT27:295 recognize the majority of the IFN-α subtypes, but not IFN-α2b), and FITC-conjugated anti-IFN-β (clone MMHB-1) for intracellular cytokine or PE-conjugated anti-TLR7 (clone 4G6) and APC-conjugated anti-TLR9 (clone eB72-1665) for intracellular TLR.

Techniques: Activity Assay

Journal: Frontiers in Immunology

Article Title: Up-Regulation of TLR7-Mediated IFN-α Production by Plasmacytoid Dendritic Cells in Patients With Systemic Lupus Erythematosus

doi: 10.3389/fimmu.2018.01957

Figure Lengend Snippet: TLR7/9 response of pDCs is regulated by the priming effects of types I and II IFNs in PBMCs of healthy subjects. (A) Representative flow cytometry plots showing TLR7/9-mediated IFN-α production in pDCs after pre-treatment with each cytokine. (B) TLR7/9-mediated IFN-α production in pDCs after pre-treatment with each cytokine. (C) TLR7/9 expression in pDCs after pre-treatment with each cytokine. (D) TLR7/9-mediated IFN-α production in pDCs after pre-treatment with different doses of IFN-α and IFN-γ. Data are mean ± S.E.M. of 4 independent experiments. * p < 0.05, ** p < 0.01, compared to pre-treatment with media (Student's t -test).

Article Snippet: After fixation and permeabilization with Fixation/Permeabilization buffer (e-Biosciences), the cells were stained with PE-conjugated anti-IFN-α2b (clone 7N4-1), PE-conjugated anti-IFN-α (clone LT27:295 recognize the majority of the IFN-α subtypes, but not IFN-α2b), and FITC-conjugated anti-IFN-β (clone MMHB-1) for intracellular cytokine or PE-conjugated anti-TLR7 (clone 4G6) and APC-conjugated anti-TLR9 (clone eB72-1665) for intracellular TLR.

Techniques: Flow Cytometry, Expressing

Journal: Frontiers in Immunology

Article Title: Up-Regulation of TLR7-Mediated IFN-α Production by Plasmacytoid Dendritic Cells in Patients With Systemic Lupus Erythematosus

doi: 10.3389/fimmu.2018.01957

Figure Lengend Snippet: TLR7 response was quickly up-regulated by IFN-α, but down-regulation of TLR9 response by IFN-γ was required long time. PBMCs were pre-treated with IFN-α and IFN-γ for 2, 12, and 24 h, followed by stimulation with TLR7/9 agonist for 5 h. TLR7/9-mediated IFN-α production in pDCs after pre-treatment with each condition. Data are mean ± S.E.M. of 3 independent experiments. * p < 0.05, ** p < 0.01, compared to pre-treatment with media (Student's t -test).

Article Snippet: After fixation and permeabilization with Fixation/Permeabilization buffer (e-Biosciences), the cells were stained with PE-conjugated anti-IFN-α2b (clone 7N4-1), PE-conjugated anti-IFN-α (clone LT27:295 recognize the majority of the IFN-α subtypes, but not IFN-α2b), and FITC-conjugated anti-IFN-β (clone MMHB-1) for intracellular cytokine or PE-conjugated anti-TLR7 (clone 4G6) and APC-conjugated anti-TLR9 (clone eB72-1665) for intracellular TLR.

Techniques:

Journal: Frontiers in Immunology

Article Title: Up-Regulation of TLR7-Mediated IFN-α Production by Plasmacytoid Dendritic Cells in Patients With Systemic Lupus Erythematosus

doi: 10.3389/fimmu.2018.01957

Figure Lengend Snippet: Both IFN-α and IFN-γ have direct priming effect on purified pDCs. (A) Representative flow cytometry plots showing TLR7/9-mediated IFN-α production by purified pDCs after pre-treatment with IFN-α and IFN-γ. (B) TLR7/9-mediated IFN-α production by purified pDCs after pre-treatment with IFN-α and IFN-γ. Data are mean ± S.E.M. of 3 independent experiments.

Article Snippet: After fixation and permeabilization with Fixation/Permeabilization buffer (e-Biosciences), the cells were stained with PE-conjugated anti-IFN-α2b (clone 7N4-1), PE-conjugated anti-IFN-α (clone LT27:295 recognize the majority of the IFN-α subtypes, but not IFN-α2b), and FITC-conjugated anti-IFN-β (clone MMHB-1) for intracellular cytokine or PE-conjugated anti-TLR7 (clone 4G6) and APC-conjugated anti-TLR9 (clone eB72-1665) for intracellular TLR.

Techniques: Purification, Flow Cytometry

Journal: Frontiers in Immunology

Article Title: Up-Regulation of TLR7-Mediated IFN-α Production by Plasmacytoid Dendritic Cells in Patients With Systemic Lupus Erythematosus

doi: 10.3389/fimmu.2018.01957

Figure Lengend Snippet: Increased localization of TLR7 in late endosome and lysosome by IFN-α. (A) Representative images showing TLR7 (red) and indicated endosomal maturation markers (green) in pDCs pre-treated with or without IFN-α. White arrows indicate robust co-localization of TLR7 with Rab7 and LAMP1. (B) Quantification of co-localization between TLR7 and EEA1, Rab7, and LAMP-1. Data shows 10 cells per each condition in one of three independent experiments.

Article Snippet: After fixation and permeabilization with Fixation/Permeabilization buffer (e-Biosciences), the cells were stained with PE-conjugated anti-IFN-α2b (clone 7N4-1), PE-conjugated anti-IFN-α (clone LT27:295 recognize the majority of the IFN-α subtypes, but not IFN-α2b), and FITC-conjugated anti-IFN-β (clone MMHB-1) for intracellular cytokine or PE-conjugated anti-TLR7 (clone 4G6) and APC-conjugated anti-TLR9 (clone eB72-1665) for intracellular TLR.

Techniques:

Journal: Journal of immunology (Baltimore, Md. : 1950)

Article Title: Age-specific Adjuvant Synergy: Dual TLR7/8 and Mincle Activation of Human Newborn Dendritic Cells Enables Th1-polarization

doi: 10.4049/jimmunol.1600282

Figure Lengend Snippet: MoDCs were stimulated with a concentration range of TLR7/8-agonist (R848 or VTX-294, 1, 5, 10, 25 and 50 μM), Mincle-agonist (TDB, 1, 5, 10, 50 and 100 μg/ml), or both (A–D) or with a TLR4-agonist (MPLA or GLA-AF, 10, 50, 100, 500 and 1000 ng/ml), Dectin-1-agonist (Zymosan or BGP, 1, 5, 10, 50 and 100 μg/ml), or both (E–H) (n=5–7). Blue and red stars indicate statistically significant differences between combination and the CLR agonist alone or TLR agonist alone, respectively. The Loewe definition of additivity was applied to determine whether the agonists act synergistically (D<1), additive (D=1) or antagonistically (D>1). Degree of synergy (1/D) was compared between age groups (I). (Mean+SEM, unpaired Student’s t-test, *p<0.05, **p<0.01 and ***p<0.001).

Article Snippet: MoDCs were resuspended in PBS/0.5% Human Serum Albumin (HSA, Octapharma USA, Inc., Hoboken, NJ, USA) and stained at 4°C with any of the following fluorescent antibodies: anti-CD14-V450 (clone MϕP9), anti-HLA-DR-PE.Cy7 (clone G46-6), anti-CD80-PE.Cy7 (clone L307.4), anti-CD83-APC (clone HB15e) and anti-CD209-V450 (clone DCN46) were purchased from BD Biosciences (San Jose, CA, USA), anti-TLR4-FITC (clone 76B357.1), anti-TLR7-PE (clone 4G6) and anti-TLR8-PE (clone 44C143) were purchased from Thermo Scientific (Wilmington, DE, USA).

Techniques: Concentration Assay

Journal: Nature Communications

Article Title: The role of platelets in mediating a response to human influenza infection

doi: 10.1038/s41467-019-09607-x

Figure Lengend Snippet: Influenza mediates C3 release from platelets through TLR7. a C3 in plasma from healthy donors ( n = 14) and influenza-infected patients ( n = 18). The graphs represent the average ± SD; significance was assessed by Mann–Whitney U test, * p < 0.0001. b C3 release from human platelets that express TLR7 (assessed by qPCR) had been isolated from healthy donors and mixed with influenza strain WSN/33 at a proportion of 1 pfu to 100 platelets ( n = 4, 3F, 1M); p < 0.0001, F = 27.49, df = 3. c C3 release from human platelets that do not express TLR7 (assessed by qPCR) treated as in ( b ). Graph is representative of n = 3 different blood draws; p = 0.2813, F = 1.833, df = 3. d C3 release from human platelets from the same donors as in ( b ) treated with the TLR7 agonist loxoribine 1 mM in the presence or absence of TLR7 inhibitor IRS661; p = 0.0086, F = 7.989, df = 3. b – d Data in graphs are represented as average ± SD; significance was assessed using ANOVA followed by Bonferroni multiple comparison test and star symbol (*) indicates p < 0.05. Source data for ( a – d ) are provided as a file. e Confocal images of permeabilized isolated platelets from influenza-infected patient stained for flu-FITC; TLR7-APC; lysosomal marker CD63-BV421. f Confocal images of isolated healthy human platelets incubated with WSN/33 for 30 min (1 pfu to 10 platelets) and stained as in ( e ). e , f Representative images of platelets from n = 4 (2M, 2F) different donors are shown

Article Snippet: The following antibodies (in 100 μL of staining solution) were used throughout this study: anti-human : 10 μL CD41-FITC or 8 μL CD41-APC (clone HIP8, eBioscience, CA, USA, cat# 11-0419 and cat# 17-0419), 5 μL CD66b-APC (clone G10F5, eBioscience, cat# 17-0666), 5 μL MPO-FITC (clone MPO455-8E6, eBioscience, cat# 11-1299), 2 μL Histone H4-AF647 (clone 31830, Abcam, MA, USA, cat# ab197515, also recognizes mouse), 2 μL Histone H3 (Abcam, cat# ab1791), followed by FITC-conjugated Goat Anti-Rabbit IgG H&L secondary antibody (Abcam, cat# ab6717); 2 μL TLR7-APC (clone 4G6, Novus Biologicals, cat# NBP2-25274APC), 5 μL LAMP-1/CD107a -DyLight 405 (clone 5E7, Novus Biologicals, cat# NBP2-52721V), 5 μL CD63-BV421 (clone H5C6, Biolegend, cat #353029); anti-mouse: 10 μL CD41-FITC (clone MWReg30, eBioscience, cat# 11-0411), 5 μL Ly6G-APC (clone RB6-8C5, eBioscience, cat# 17-5931); and 2 μL Influenza A-NP-FITC (Abcam, cat# ab20921), 2 μL Influenza B-NP-FITC (Invitrogen, cat# MA1-7306).

Techniques: Clinical Proteomics, Infection, MANN-WHITNEY, Isolation, Comparison, Staining, Marker, Incubation

Journal: Nature Communications

Article Title: The role of platelets in mediating a response to human influenza infection

doi: 10.1038/s41467-019-09607-x

Figure Lengend Snippet: Role of platelet GM-CSF and C3 in neutrophil-DNA release. a – c Isolated human platelets and neutrophils were incubated together or by themselves for 30 min (at constant rotation and 37 °C) in the presence of TLR agonists [TLR7 (Loxo)—1 mM; TLR2 (Pam 3 CSK 4 , PAM)—10 μg/μL] or thrombin (IIa)—0.05 U/mL. GM-CSF release from a platelets ( p = 0.8903, F = 0.2071, df = 3), b neutrophils ( p = 0.7255, F = 0.4422, df = 3), and c platelets and neutrophils incubated together was measured by ELISA ( p = 0.0200, F = 4.116, df = 3). The graphs represent the average fold change for each individual of n = 6 (3F; 3M) ± SD. d Confocal images of isolated human neutrophils treated with C3 (30 ng/mL) and neutrophils treated with C3 in the presence of GM-CSF (25 ng/mL). Neutrophils were treated in HEPES-modified Tyrode’s buffer (0.04 × 10 5 neutrophils/μL) for 30 min at 37 °C, and constant rotation (aggregometer). At the end, cells were fixed and stained [CD41-FITC-platelets (green); H4-AF637-histone (red); DAPI-DNA (blue)]. Neutrophil-DNA aggregates were visualized by confocal microscopy. Images are representatives of n = 4 different donors. C3-treated neutrophils from healthy donors release their DNA and form large aggregates. e Quantitation of ( d ). The graph ( n = 4, 2F, 2M) is represented as average ( p < 0.0001, F = 71.42, df = 3) ± SD. Significance in ( a – c , e ) was assessed using ANOVA followed by Bonferroni multiple comparison test and star symbol (*) indicates p < 0.05. Source data are provided as a file

Article Snippet: The following antibodies (in 100 μL of staining solution) were used throughout this study: anti-human : 10 μL CD41-FITC or 8 μL CD41-APC (clone HIP8, eBioscience, CA, USA, cat# 11-0419 and cat# 17-0419), 5 μL CD66b-APC (clone G10F5, eBioscience, cat# 17-0666), 5 μL MPO-FITC (clone MPO455-8E6, eBioscience, cat# 11-1299), 2 μL Histone H4-AF647 (clone 31830, Abcam, MA, USA, cat# ab197515, also recognizes mouse), 2 μL Histone H3 (Abcam, cat# ab1791), followed by FITC-conjugated Goat Anti-Rabbit IgG H&L secondary antibody (Abcam, cat# ab6717); 2 μL TLR7-APC (clone 4G6, Novus Biologicals, cat# NBP2-25274APC), 5 μL LAMP-1/CD107a -DyLight 405 (clone 5E7, Novus Biologicals, cat# NBP2-52721V), 5 μL CD63-BV421 (clone H5C6, Biolegend, cat #353029); anti-mouse: 10 μL CD41-FITC (clone MWReg30, eBioscience, cat# 11-0411), 5 μL Ly6G-APC (clone RB6-8C5, eBioscience, cat# 17-5931); and 2 μL Influenza A-NP-FITC (Abcam, cat# ab20921), 2 μL Influenza B-NP-FITC (Invitrogen, cat# MA1-7306).

Techniques: Isolation, Incubation, Enzyme-linked Immunosorbent Assay, Modification, Staining, Confocal Microscopy, Quantitation Assay, Comparison

Journal: Nature Communications

Article Title: The role of platelets in mediating a response to human influenza infection

doi: 10.1038/s41467-019-09607-x

Figure Lengend Snippet: Platelet-TLR7 solely mediates neutrophil-DNA release through C3. Platelets and neutrophils were isolated from human blood. Each population was pretreated for 15 min with a TLR7 agonist (1 mM Loxoribine, Loxo) and then incubated with the other population for 30 min. All experiments were carried out at an approximately physiological ratio of 50 platelets:1 neutrophil. a Confocal microscopy of the incubated cells. Cells were stained with (CD41-FITC-platelets; CD66b-APC-neutrophils; DAPI-DNA) and visualized with a confocal microscope. b Quantitation of the confocal images in ( a ), p = 0.017, F = 6.624; df = 2. c DNA release from neutrophils in the presence of platelets pretreated with a C3 inhibitor, compstatin (0.088 mg/mL) for 10 min and then stimulated with Loxo for 30 min ( p = 0.0033, F = 11.51, df = 2). d Assessment of neutrophil-DNA release by confocal images of isolated platelets and neutrophils treated with influenza WSN/33 in the presence or absence of the TLR7 inhibitor IRS661. e Quantitation of the confocal images in ( d ), p < 0.0001; F = 61.07, df = 3. In all cases data in the graphs are represented as the average ± SD. Statistical significance was measured by ANOVA followed by a Bonferroni follow-up test of n = 4 (2F and 2M, with the exception of ( e ), where we used 3F and 1M), star symbol (*) indicates p < 0.05. Source data for all graphs are provided as a file

Article Snippet: The following antibodies (in 100 μL of staining solution) were used throughout this study: anti-human : 10 μL CD41-FITC or 8 μL CD41-APC (clone HIP8, eBioscience, CA, USA, cat# 11-0419 and cat# 17-0419), 5 μL CD66b-APC (clone G10F5, eBioscience, cat# 17-0666), 5 μL MPO-FITC (clone MPO455-8E6, eBioscience, cat# 11-1299), 2 μL Histone H4-AF647 (clone 31830, Abcam, MA, USA, cat# ab197515, also recognizes mouse), 2 μL Histone H3 (Abcam, cat# ab1791), followed by FITC-conjugated Goat Anti-Rabbit IgG H&L secondary antibody (Abcam, cat# ab6717); 2 μL TLR7-APC (clone 4G6, Novus Biologicals, cat# NBP2-25274APC), 5 μL LAMP-1/CD107a -DyLight 405 (clone 5E7, Novus Biologicals, cat# NBP2-52721V), 5 μL CD63-BV421 (clone H5C6, Biolegend, cat #353029); anti-mouse: 10 μL CD41-FITC (clone MWReg30, eBioscience, cat# 11-0411), 5 μL Ly6G-APC (clone RB6-8C5, eBioscience, cat# 17-5931); and 2 μL Influenza A-NP-FITC (Abcam, cat# ab20921), 2 μL Influenza B-NP-FITC (Invitrogen, cat# MA1-7306).

Techniques: Isolation, Incubation, Confocal Microscopy, Staining, Microscopy, Quantitation Assay

Journal: Nature Communications

Article Title: The role of platelets in mediating a response to human influenza infection

doi: 10.1038/s41467-019-09607-x

Figure Lengend Snippet: Platelet-TLR7mediates release of MPO from neutrophils. Isolated human platelets and neutrophils were incubated together or by themselves for 30 min, at 37 °C and constant rotation, in the presence of TLR agonists [TLR7 (Loxo)—1 mM; TLR2 (Pam)—10 μg/mL] or thrombin (IIa)—0.05 U/mL. Myeloperoxidase (MPO) release from neutrophils was measured in the a absence ( p = 0.7206, F = 0.4493, df = 3) and b presence of platelets by ELISA ( p = 0.0002, F = 10.4, df = 3). The graphs represent the average fold change for each individual of n = 6 (3F; 3M) ± SD. Source data are provided as a file. Significance was measured by ANOVA followed by Bonferroni follow-up test; in all cases star symbol (*) indicates p < 0.05

Article Snippet: The following antibodies (in 100 μL of staining solution) were used throughout this study: anti-human : 10 μL CD41-FITC or 8 μL CD41-APC (clone HIP8, eBioscience, CA, USA, cat# 11-0419 and cat# 17-0419), 5 μL CD66b-APC (clone G10F5, eBioscience, cat# 17-0666), 5 μL MPO-FITC (clone MPO455-8E6, eBioscience, cat# 11-1299), 2 μL Histone H4-AF647 (clone 31830, Abcam, MA, USA, cat# ab197515, also recognizes mouse), 2 μL Histone H3 (Abcam, cat# ab1791), followed by FITC-conjugated Goat Anti-Rabbit IgG H&L secondary antibody (Abcam, cat# ab6717); 2 μL TLR7-APC (clone 4G6, Novus Biologicals, cat# NBP2-25274APC), 5 μL LAMP-1/CD107a -DyLight 405 (clone 5E7, Novus Biologicals, cat# NBP2-52721V), 5 μL CD63-BV421 (clone H5C6, Biolegend, cat #353029); anti-mouse: 10 μL CD41-FITC (clone MWReg30, eBioscience, cat# 11-0411), 5 μL Ly6G-APC (clone RB6-8C5, eBioscience, cat# 17-5931); and 2 μL Influenza A-NP-FITC (Abcam, cat# ab20921), 2 μL Influenza B-NP-FITC (Invitrogen, cat# MA1-7306).

Techniques: Isolation, Incubation, Enzyme-linked Immunosorbent Assay

Journal: Nature Communications

Article Title: The role of platelets in mediating a response to human influenza infection

doi: 10.1038/s41467-019-09607-x

Figure Lengend Snippet: TLR7 stimulation in vivo leads to DNA release from Ly6G positive cells. WT and TLR7 KO mice were injected intraperitoneally with a TLR7 agonist, or were intranasally infected with influenza (PR8 strain, 40,000 pfu in 30 µL). Blood was collected by cardiac puncture at 24 h and immediately fixed (red blood cells were lysed at the same time); Ly6G is predominantly expressed by murine neutrophils. a Representative images of DNA release from Ly6G-positive cells (at 24 h post-Loxo stimulation) resolved by confocal microscopy. b Quantitation of DNA release from Ly6G-positive cells in blood of mice ( n = 4/group) at 24 h after agonist stimulation ( p = 0.016, df = 6). c Representative images of DNA release (at 24 h post influenza infection) resolved by confocal microscopy. Pictures showing Ly6G-highly positive origin of the released DNA are included in Supplementary Fig. . d Quantitation of the DNA release from Ly6G-positive cells in blood of mice ( n = 4/group) at 24 h post-infection ( p < 0.001, df = 6). In all cases, the bar represents 10 μm and values in the bar graphs represent the average ± SD; star symbol (*) indicates p < 0.05. Significance was assessed by unpaired t -test (two-tail value). Source data are provided as a file

Article Snippet: The following antibodies (in 100 μL of staining solution) were used throughout this study: anti-human : 10 μL CD41-FITC or 8 μL CD41-APC (clone HIP8, eBioscience, CA, USA, cat# 11-0419 and cat# 17-0419), 5 μL CD66b-APC (clone G10F5, eBioscience, cat# 17-0666), 5 μL MPO-FITC (clone MPO455-8E6, eBioscience, cat# 11-1299), 2 μL Histone H4-AF647 (clone 31830, Abcam, MA, USA, cat# ab197515, also recognizes mouse), 2 μL Histone H3 (Abcam, cat# ab1791), followed by FITC-conjugated Goat Anti-Rabbit IgG H&L secondary antibody (Abcam, cat# ab6717); 2 μL TLR7-APC (clone 4G6, Novus Biologicals, cat# NBP2-25274APC), 5 μL LAMP-1/CD107a -DyLight 405 (clone 5E7, Novus Biologicals, cat# NBP2-52721V), 5 μL CD63-BV421 (clone H5C6, Biolegend, cat #353029); anti-mouse: 10 μL CD41-FITC (clone MWReg30, eBioscience, cat# 11-0411), 5 μL Ly6G-APC (clone RB6-8C5, eBioscience, cat# 17-5931); and 2 μL Influenza A-NP-FITC (Abcam, cat# ab20921), 2 μL Influenza B-NP-FITC (Invitrogen, cat# MA1-7306).

Techniques: In Vivo, Injection, Infection, Confocal Microscopy, Quantitation Assay

Journal: Nature Communications

Article Title: The role of platelets in mediating a response to human influenza infection

doi: 10.1038/s41467-019-09607-x

Figure Lengend Snippet: Platelets contribute to C3 and Ly6G-DNA release in vivo. Platelets were eliminated from male mice with antiplatelet antibody CD42 (αPlt) and compared to control IgG. At 24 h post elimination, mice were infected with the PR8 strain of influenza (as in Fig. ). a Confocal microscopy of blood showing DNA release in murine blood 3–4 days post-infection. Images are representative of n = 4 mice/group. b Quantitation of the confocal images in ( a ). Graph is a representative of 4 mice/group ( p = 0.0003, F = 14.26, df = 3). c C3 levels in murine plasma at the same time as in ( a ). The graph represents the average levels ± SD, n = 4 mice/group, with the exception of IgG+sal, where n = 3 mice were used ( p = 0.0415, F = 4.733, df = 3). Significance was measured by ANOVA followed by Bonferroni follow-up test; in all cases star symbol (*) indicates p < 0.05. d Gene expression levels of influenza RNA in isolated murine platelets 12 days post-infection ( n = 4 of IgG+flu; n = 4 of αPlt+flu). The graph represents average expression ± SD; significance was calculated by two-tailed unpaired t -test, p = 0.0715, df = 6. Of note, Mann–Whitney non-parametric t -test gave p = 0.0286. Source data are provided as a file. Abbreviations: IgG—control antibody; αPlt—antiplatelet CD42b antibody; Sal—phosphate buffered saline: e Proposed mechanism of platelet-mediated neutrophil-DNA release during influenza infection. During influenza infection, virions cross into the circulation and become engulfed by platelets. Influenza virions lead to the release of complement C3 from platelets in a platelet-TLR7-dependent manner. C3 in turn activates neutrophils to release their DNA and leads to the formation of platelet–neutrophil aggregates that can circulate freely in blood. Aggregates of this nature can increase the risk for thrombosis and potentially lead to unstable coronary syndrome when there is vessel stenosis or inflamed endothelium

Article Snippet: The following antibodies (in 100 μL of staining solution) were used throughout this study: anti-human : 10 μL CD41-FITC or 8 μL CD41-APC (clone HIP8, eBioscience, CA, USA, cat# 11-0419 and cat# 17-0419), 5 μL CD66b-APC (clone G10F5, eBioscience, cat# 17-0666), 5 μL MPO-FITC (clone MPO455-8E6, eBioscience, cat# 11-1299), 2 μL Histone H4-AF647 (clone 31830, Abcam, MA, USA, cat# ab197515, also recognizes mouse), 2 μL Histone H3 (Abcam, cat# ab1791), followed by FITC-conjugated Goat Anti-Rabbit IgG H&L secondary antibody (Abcam, cat# ab6717); 2 μL TLR7-APC (clone 4G6, Novus Biologicals, cat# NBP2-25274APC), 5 μL LAMP-1/CD107a -DyLight 405 (clone 5E7, Novus Biologicals, cat# NBP2-52721V), 5 μL CD63-BV421 (clone H5C6, Biolegend, cat #353029); anti-mouse: 10 μL CD41-FITC (clone MWReg30, eBioscience, cat# 11-0411), 5 μL Ly6G-APC (clone RB6-8C5, eBioscience, cat# 17-5931); and 2 μL Influenza A-NP-FITC (Abcam, cat# ab20921), 2 μL Influenza B-NP-FITC (Invitrogen, cat# MA1-7306).

Techniques: In Vivo, Control, Infection, Confocal Microscopy, Quantitation Assay, Clinical Proteomics, Gene Expression, Isolation, Expressing, Two Tailed Test, MANN-WHITNEY, Saline