Journal: Oncotarget

Article Title: Lipid rafts promote liver cancer cell proliferation and migration by up-regulation of TLR7 expression

doi: 10.18632/oncotarget.11697

Figure Lengend Snippet: Immunohistochemistry showing ( A ) TLR7 was highly expressed on cell membrane and perinuclei only in malignant hepatocytes but as the scattered spots in cytoplasm of hepatocytes of Normal, CHB and LC. ( B ) Correlation of TLR7 and Ki-67 expression in HCC. Serial sections from the same paraffin block were stained with different antibodies. Hepatic expression of the proliferative marker Ki-67 was high in nuclei. Membranous TLR7 expression was also high in malignant hepatocytes. ( C ) Percentage of cases from the validation set demonstrating no (0), weak (1) or high (2) expression of TLR7. ( D ) Correlation of TLR7 staining with Ki-67 index in HCC. ( E ) Western blotting analysis of TLR7 in liver tissue lysates. Bands were observed at approximately 120 kDa and normalized by β-actin. There was a significant increase in the TLR7 expression of HCC group compared with Normal control. * P < 0.05, results were representative of three independent experiments.

Article Snippet: Mouse IgG 2A anti-Human TLR7 PE-conjugated monoclonal antibody and mouse IgG 2A PE-conjugated isotype control were purchased from R&D Systems, Inc. (Minneapolis, MN, USA).

Techniques: Immunohistochemistry, Membrane, Expressing, Blocking Assay, Staining, Marker, Biomarker Discovery, Western Blot, Control

Journal: Oncotarget

Article Title: Lipid rafts promote liver cancer cell proliferation and migration by up-regulation of TLR7 expression

doi: 10.18632/oncotarget.11697

Figure Lengend Snippet: Tissue clinical data and TLR7 staining

Article Snippet: Mouse IgG 2A anti-Human TLR7 PE-conjugated monoclonal antibody and mouse IgG 2A PE-conjugated isotype control were purchased from R&D Systems, Inc. (Minneapolis, MN, USA).

Techniques: Membrane

Journal: Oncotarget

Article Title: Lipid rafts promote liver cancer cell proliferation and migration by up-regulation of TLR7 expression

doi: 10.18632/oncotarget.11697

Figure Lengend Snippet: To evaluate the efficacy of the experiment, an isotype-matched control was used. At least 3000 cells were assessed to calculate the mean fluorescence intensity values (MFI). ( A ) Statistical analyses of TLR7 MFI. Data were shown as mean ± SEM. * P < 0.05 compared with the Normal group. ( B ) Expression of TLR7 using flow cytometry analysis. ( C ) The immune cells were identified and evaluated by flow cytometry for the further observation on TLR7-related immune cell infiltrates. NK cells and pDC were in association to TLR7 expression levels. CD3, for T cells; CD8, for cytotoxic T cells; CD19, for B cells; CD56, for NK cells or NKT cells; CD16, for granulocytes; CD14, for mononuclear phagocytes; CD11c, for pDC.

Article Snippet: Mouse IgG 2A anti-Human TLR7 PE-conjugated monoclonal antibody and mouse IgG 2A PE-conjugated isotype control were purchased from R&D Systems, Inc. (Minneapolis, MN, USA).

Techniques: Control, Fluorescence, Expressing, Flow Cytometry

Journal: Oncotarget

Article Title: Lipid rafts promote liver cancer cell proliferation and migration by up-regulation of TLR7 expression

doi: 10.18632/oncotarget.11697

Figure Lengend Snippet: ( A ) Lipid raft and non-raft microdomains were isolated using a non-detergent method and analyzed for caveolin-1 (lipid raft marker), flotillin-1 (lipid raft marker), clathrin (non-raft marker) and TLR7 distribution by western blotting. The graph showed the expression of caveolin-1, flotillin-1, clathrin and TLR7 in HCC relative to Normal tissue. * P < 0.05, lipid rafts compared with the non-rafts. ( B ) The co-expression of TLR7 and lipid rafts was analyzed by double immunohistochemistry. The co-expression of TLR7 and lipid rafts was located in the cell membrane only in HCC group but as the scattered spots in cytoplasm of live cells in Normal, CHB, LC groups. Interestingly, margination orientation of TLR7/Caveolin-1 or TLR7/Flotillin-1 from cytoplasm to membrane was observed in LC tissues. The microphotographs were magnified 1000 times.

Article Snippet: Mouse IgG 2A anti-Human TLR7 PE-conjugated monoclonal antibody and mouse IgG 2A PE-conjugated isotype control were purchased from R&D Systems, Inc. (Minneapolis, MN, USA).

Techniques: Isolation, Marker, Western Blot, Expressing, Immunohistochemistry, Membrane

Journal: Oncotarget

Article Title: Lipid rafts promote liver cancer cell proliferation and migration by up-regulation of TLR7 expression

doi: 10.18632/oncotarget.11697

Figure Lengend Snippet: ( A ) HepG2 cells were incubated with 10 mM MβCD or 1 μg/ml gardiquimod for 1 h. Cell lysates were harvested and the proteins were analyzed by immunoblotting with the indicated antibodies. ( B ) TLR7, MyD88, NFκB were quantified and normalized to β-actin. Data were presented as mean ± SEM; n = 3. Statistically significant differences between groups were indicated (* P < 0.05). ( C ) HepG2 cells were treated with 10 mM MβCD or 1 μg/ml gardiquimod for 1h and then lipid rafts (fractions 4–6) were pooled, and immunoprecipitated with anti-flotillin-1 polyclonal antibody, followed by immunoblotting with anti-TLR7 monoclonal antibody. Results were representative of three independent experiments. ( D ) Lipid rafts isolated as described in panel C were immunoprecipitated with anti-TLR7 monoclonal antibody, followed by immunoblotting with anti-flotillin-1 polyclonal antibody. Results were representative of three independent experiments.

Article Snippet: Mouse IgG 2A anti-Human TLR7 PE-conjugated monoclonal antibody and mouse IgG 2A PE-conjugated isotype control were purchased from R&D Systems, Inc. (Minneapolis, MN, USA).

Techniques: Incubation, Western Blot, Immunoprecipitation, Isolation

Journal: Oncotarget

Article Title: Lipid rafts promote liver cancer cell proliferation and migration by up-regulation of TLR7 expression

doi: 10.18632/oncotarget.11697

Figure Lengend Snippet: TLR7 immunohistochemistry scoring system

Article Snippet: Mouse IgG 2A anti-Human TLR7 PE-conjugated monoclonal antibody and mouse IgG 2A PE-conjugated isotype control were purchased from R&D Systems, Inc. (Minneapolis, MN, USA).

Techniques: Immunohistochemistry, Staining

Journal: bioRxiv

Article Title: Macrophage mediated recognition and clearance of Borrelia burgdorferi nelicits MyD88-dependent and -independent phagosomal signals that contribute to phagocytosis and inflammation

doi: 10.1101/593566

Figure Lengend Snippet: (A-C) Confocal images and colocalization analysis of internalized Bb with MyD88 (A), TLR2 (B) or TLR7 (C) in WT BMDMs after stimulation at MOI 10:1. White box indicates phagosome depicted in inset. Large inset in (B) shows coiling pseudopod formation around Bb on cell surface. Graph shows the intensity of each indicated pixel marker across the white line (distance on x-axis). Green is Bb, blue is MyD88 (A), TLR2 (B) or TLR7 , and red is actin.

Article Snippet: The next day, cells were stained with different combinations of anti-GFP (Thermo Scientific A-21311, 1:100), phalloidin conjugated with Alexa Fluor 647 (Biolegend 424205, 1:20), anti-MyD88 (Santa Cruz 11356, 1:100), anti-TLR2 (eBioscience 14-9021-82, 1:100), anti-TLR7 (R&D MAB7156, 1:100), anti-ASC (Santa Cruz 22514-R, 1:100) and anti-LAMP-1 (eBioscience 14-1071-82, 1:100).

Techniques: Marker

Journal: Immunity

Article Title: Excessive Polyamine Generation in Keratinocytes Promotes Self-RNA Sensing by Dendritic Cells in Psoriasis.

doi: 10.1016/j.immuni.2020.06.004

Figure Lengend Snippet: Figure 6. Polyamine-Mediated Self-RNA Sensing Requires Endosomal Tlr7 (A) ELISA quantification of IL-6 in WT and Tlr7-deficient BMDC cultures stimulated with the indicated reagents (n = 3). (B) qPCR detection of human 28S rRNA in endosomes isolated from mouse BMDCs treated with the indicated reagents (n = 7). The fold changes relative to the self-RNA group are presented. (C) Immunoblotting analysis of IkappaB-z expression in nuclear fractions of BMDCs treated with the indicated reagents for 4 h. (D and E) RNAscope of Krt14 combined with immunofluorescent labeling of Tlr7 and spermidine on DCs isolated from mouse skin treated with IMQ or untreated (UT). Scale bars, 100 mm (D) or 3 mm (E). (F) GSEA of genes in CLEC9A+WDFY4+ cDC1 and in CD1C+SIRPA+ cDC2 or moDC from human psoriatic skin relative to healthy skin with the genes induced in R848 stimulated DCs enriched (GSE2706). GSEA of skin DCs in lesional K5. Pp6fl/flskin relative to normal mouse skin with the genes induced in R848 stimulated DCs enriched (GSE2706). Data in (A–E) are representative of two independent experiments. *p < 0.05, **p < 0.01, two-tailed Student’s t test (mean ± SEM). See also Figure S7.

Article Snippet: Immunofluorescence was then performed using antibodies against Tlr7 (Novus cat. NBP2-27332, 1:50 dilution) and spermidine (Novus cat. NB100-1847, 1:50 dilution). e9 Immunity 53, 1–13.e1–e10, July 14, 2020

Techniques: Enzyme-linked Immunosorbent Assay, Isolation, Western Blot, Expressing, RNAscope, Labeling, Two Tailed Test

Journal: Nature immunology

Article Title: Genetic dissection of TLR9 reveals complex regulatory and cryptic proinflammatory roles in mouse lupus.

doi: 10.1038/s41590-022-01310-2

Figure Lengend Snippet: Fig. 4 | Effects of TLR9 and sex on TLR7 expression and distribution. a–d, Intracellular staining of TLR7 and TLR9 (ratio of TLR7 MFI over average TLR7 MFI of the corresponding male or female WT group) in FO (a,c) and MZ (b,d) B cells of Tlr9-mutant male or female BALB/c mice. Data points indicate individual mice (Tlr9K51E/K51E male, n = 7; female, n = 11; Tlr9 WT male, n = 7, female, n = 11; Tlr9P915H/P915H male, n = 7, female, n = 10; Tlr9+/− male, n = 3, female, n = 7; Tlr9−/− male, n = 7, female, n = 11), and bars indicate the mean ± s.e.m. of two or three experiments pooled (except from one experiment for Tlr9+/− males); *P < 0.05, **P < 0.01, ***P < 0.001 and ****P < 0.0001, one-way ANOVA with Tukey’s multiple comparisons test; female, Tlr9K51E/K51E versus Tlr9−/−, P = 0.0026; Tlr9−/− versus Tlr9P915H/P915H, P = 0.0251; Tlr9WT versus Tlr9−/−, P = 0.0003 (a); female, Tlr9K51E/K51E versus Tlr9−/−, P = 0.0170; Tlr9WT versus Tlr9P915H/

Article Snippet: Cells were stained at 50–100 million cells per ml overnight at 4 °C in 1× Perm/Wash buffer with fluorochrome-conjugated antibodies CD45R (clone RA3-6B2, 1:400), TLR7 (clone A94B10, 1:800), TLR9–biotin (clone Nar9, from K. Miyake67, biotin conjugation in-house, 1:600) and unconjugated EEA1 (polyclonal anti-rabbit, Novus, 1:100) or LAMP-1 (polyclonal anti-rabbit, Abcam, 1:100).

Techniques: Expressing, Staining, Mutagenesis

Journal: PLoS ONE

Article Title: Cholecalciferol decreases inflammation and improves vitamin D regulatory enzymes in lymphocytes in the uremic environment: A randomized controlled pilot trial

doi: 10.1371/journal.pone.0179540

Figure Lengend Snippet: Expression of TLR7, TLR9, IL-6, IFN-γ, VDR, CYP27b1 and CYP24a1 in B and T lymphocytes.

Article Snippet: After lysing, cells were permeabilized as described above to intracellular stain with 5 μL pf FITC-labeled IL-6 (BD Biosciences, San Diego, USA), 10 μL of PE-labeled TLR7 antibody (R&D Systems, Mineapolis, USA), 5 μL of APC-Cy7-labeled IFN-γ (eBiosciences, San Diego, USA) and 10 μL of APC-labeled TLR9 (BD Biosciences, San Diego, USA) antibodies.

Techniques: Expressing

Journal: PLoS ONE

Article Title: Cholecalciferol decreases inflammation and improves vitamin D regulatory enzymes in lymphocytes in the uremic environment: A randomized controlled pilot trial

doi: 10.1371/journal.pone.0179540

Figure Lengend Snippet: Effect of 25 and 1,25 vitamin D on TLR7 and TLR9 expression (MFI) in B lymphocytes (Figs A and B) and T lymphocytes (Figs C and D) in presence of healthy or uremic serum (US) and after CYP24 silencing (siRNA).

Article Snippet: After lysing, cells were permeabilized as described above to intracellular stain with 5 μL pf FITC-labeled IL-6 (BD Biosciences, San Diego, USA), 10 μL of PE-labeled TLR7 antibody (R&D Systems, Mineapolis, USA), 5 μL of APC-Cy7-labeled IFN-γ (eBiosciences, San Diego, USA) and 10 μL of APC-labeled TLR9 (BD Biosciences, San Diego, USA) antibodies.

Techniques: Expressing

Journal: Journal of the American Society of Nephrology : JASN

Article Title: Toll-like receptor-7 modulates immune complex glomerulonephritis.

doi: 10.1681/ASN.2005070714

Figure Lengend Snippet: Figure 1. Toll-like receptor 7 (TLR7) expression in MRLlpr/lpr

Article Snippet: Immunostaining was performed on either paraffin-embedded or frozen sections as described (8) using the following primary antibodies: Anti-mouse TLR7 (1:50, IMG581; Imgenex, San Diego, CA), anti-mouse ER-HR3 (1:50, monocytes/macrophages; DPC Biermann, Bad Nauheim, Germany), anti-mouse CD11c (1:50, clone HL3, BD Pharmingen, Heidelberg, Germany), anti-mouse CD3 (1:100, clone 500A2; BD Pharmingen), anti-mouse smooth muscle actin (1:100, myofibroblasts, clone 1A4; Dako, Carpinteria, CA), anti-mouse CCL5 (1:50, clone VL1; Peprotech, Rocky Hill, NJ), anti-mouse CCL2/MCP-1 (1:50, polyclonal; Santa Cruz Biotechnology, Santa Cruz, CA), anti-mouse IgG1 (1:100, clone H143.225.8; Dianova, Hamburg, Germany), anti-mouse IgG2a (1:100, clone R19–15; Dianova), and anti-mouse C3c (1:200, GAM/C3c/ FITC; Nordic Immunological Laboratories, Tilburg, Netherlands).

Techniques: Expressing

Journal: Journal of the American Society of Nephrology : JASN

Article Title: Toll-like receptor-7 modulates immune complex glomerulonephritis.

doi: 10.1681/ASN.2005070714

Figure Lengend Snippet: Figure 2. TLR7 immunostaining and uptake of labeled single- stranded RNA (ssRNA) in kidneys of MRLlpr/lpr mice. (A) An mTLR7-specific antibody was used on renal sections of 18-wk- old nephritic MRLlpr/lpr mice. A PE-labeled secondary anti- body was used for detection. Positive signals co-localized with ER-HR3–positive macrophages or CD11c-positive dendritic cells, both detected by a FITC-labeled secondary antibody. (B) Rhodamine-labeled ssRNA40 was injected intravenously into 18-wk-old MRLlpr/lpr mice, and renal tissue was harvested 2 h later. Fluorescence imaging of frozen sections showed uptake of ssRNA40 (red) into ER-HR3–positive macrophages (green). (C) Co-staining of rhodamine-labeled cells (red) for TLR7 (green) demonstrates uptake of RNA40 into TLR7-positive cells. Magnification, 530.

Article Snippet: Immunostaining was performed on either paraffin-embedded or frozen sections as described (8) using the following primary antibodies: Anti-mouse TLR7 (1:50, IMG581; Imgenex, San Diego, CA), anti-mouse ER-HR3 (1:50, monocytes/macrophages; DPC Biermann, Bad Nauheim, Germany), anti-mouse CD11c (1:50, clone HL3, BD Pharmingen, Heidelberg, Germany), anti-mouse CD3 (1:100, clone 500A2; BD Pharmingen), anti-mouse smooth muscle actin (1:100, myofibroblasts, clone 1A4; Dako, Carpinteria, CA), anti-mouse CCL5 (1:50, clone VL1; Peprotech, Rocky Hill, NJ), anti-mouse CCL2/MCP-1 (1:50, polyclonal; Santa Cruz Biotechnology, Santa Cruz, CA), anti-mouse IgG1 (1:100, clone H143.225.8; Dianova, Hamburg, Germany), anti-mouse IgG2a (1:100, clone R19–15; Dianova), and anti-mouse C3c (1:200, GAM/C3c/ FITC; Nordic Immunological Laboratories, Tilburg, Netherlands).

Techniques: Immunostaining, Labeling, Injection, Fluorescence, Imaging, Staining

Journal: Journal of the American Society of Nephrology : JASN

Article Title: Toll-like receptor-7 modulates immune complex glomerulonephritis.

doi: 10.1681/ASN.2005070714

Figure Lengend Snippet: Figure 4. TLR7 agonists activate ER-HR monocytes and CD11c dendritic cells that were isolated from MRLlpr/lpr

Article Snippet: Immunostaining was performed on either paraffin-embedded or frozen sections as described (8) using the following primary antibodies: Anti-mouse TLR7 (1:50, IMG581; Imgenex, San Diego, CA), anti-mouse ER-HR3 (1:50, monocytes/macrophages; DPC Biermann, Bad Nauheim, Germany), anti-mouse CD11c (1:50, clone HL3, BD Pharmingen, Heidelberg, Germany), anti-mouse CD3 (1:100, clone 500A2; BD Pharmingen), anti-mouse smooth muscle actin (1:100, myofibroblasts, clone 1A4; Dako, Carpinteria, CA), anti-mouse CCL5 (1:50, clone VL1; Peprotech, Rocky Hill, NJ), anti-mouse CCL2/MCP-1 (1:50, polyclonal; Santa Cruz Biotechnology, Santa Cruz, CA), anti-mouse IgG1 (1:100, clone H143.225.8; Dianova, Hamburg, Germany), anti-mouse IgG2a (1:100, clone R19–15; Dianova), and anti-mouse C3c (1:200, GAM/C3c/ FITC; Nordic Immunological Laboratories, Tilburg, Netherlands).

Techniques: Isolation

Journal: Journal of Biological Chemistry

Article Title: Epigenetic Regulation of Tumor Necrosis Factor α (TNFα) Release in Human Macrophages by HIV-1 Single-stranded RNA (ssRNA) Is Dependent on TLR8 Signaling

doi: 10.1074/jbc.m112.342683

Figure Lengend Snippet: FIGURE 3. Dependence of TNF release in human macrophages on TLR8 activation by HIV-1 ssRNA. A and B, intracellular expression of TLR7 and TLR8 in macrophages. THP-1 (A) and AM (B) were incubated with PE-conjugated anti-TLR7 or anti-TLR8 or isotype control antibody, and intracellular expression was determined by flow cytometry. Representative profiles were similar in three independent experiments (n 3 subjects for AM). C–E, functional silencing of human TLR8 leads to marked diminution of TNF release in human macrophages. C, flow cytometry analysis of human TLR7/8 after gene silencing with the use of TLR7 or TLR8 siRNA and siRNA or isotype control. A representative flow cytometry tracing shows representative results from one experiment repeated independently in three experiments. N.S., nonsilencing. D an E, THP-1 cells were pretreated with either TLR7 siRNA (D) or TLR8 siRNA (E) or nonsilencing control. Cells were differentiated with phorbol ester, challenged with HIV-1 ssRNA and incubated for 24 h. Cell free supernatant was assayed for TNF by ELISA. Results are representative of three independent experiments performed in triplicate; *, p 0.05 when compared with nonsilencing siRNA control. BLP served as positive control. US, unstimulated.

Article Snippet: Antibodies—Anti-TLR7-PE and anti-TLR8-PE antibodies were purchased from Imgenex, San Diego, CA.

Techniques: Activation Assay, Expressing, Incubation, Control, Flow Cytometry, Functional Assay, Enzyme-linked Immunosorbent Assay, Positive Control

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