Journal: bioRxiv

Article Title: CLEC5A and TLR2 are critical in SARS-CoV-2-induced NET formation and lung inflammation

doi: 10.1101/2022.02.01.478701

Figure Lengend Snippet: (a&b) Neutrophils (4 × 10 5 /ml) from healthy donors were incubated with SARS-CoV-2 (MOI = 1) with or without autologous platelets (4 × 10 6 /ml) for 5 h at 37 °C. The scale bar is 10 μm. The detailed structure of SARS-CoV-2-induced NET formation was observed under a confocal microscope (Leica). NET formation was visualized by fluorescent staining of DNA (blue), histone (green), and MPO (red) (a) . NETs level was measured by MetaMorph software and presented as Cit-H3 area (mm 2 ) (b) . (c) Human neutrophils (4 × 10 5 /ml) were pretreated with anti-hCLEC5A mAb (3E12A2, 100 μg/ml), anti-TLR2 mAb (# MAB2616, 100 μg/ml), or combination of both antibodies for 30 min at room temperature, followed by incubation with SARS-CoV-2 (MOI = 0.1 and 1) in the presence or absence of platelets (4 × 10 6 /ml) for 5 h and 20 h. The level of NET formation was determined by histone area (μm 2 ). (d) Neutrophils (4 × 10 5 /ml) from WT, clec5a -/- tlr2 -/- , and clec5a -/- tlr2 -/- mice were incubated with SARS-CoV-2 (MOI = 1) in the presence or absence of WT platelets (4 × 10 6 /ml) for 5 h at 37 °C. (e) Human neutrophils were pre-treated with anti-hCLEC5A mAb (3E12A2, 100 μg/ml), anti-TLR2 mAb (# MAB2616, 100 μg/ml), or combination of both antibodies for 30 min at room temperature, followed by incubation with SARS-CoV-2 spike pseudotyped virus (MOI = 0.1) in the presence or absence of autologous platelets (4 × 10 6 /ml) for 3 h. Data are mean ± SEM and repeats of 3 to 5 independent experiments. *p<0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001 (Student’s t-test).

Article Snippet: For blocking assay, neutrophils were preincubated with isotype (100 μg/ml), anti-CLEC5A mAb (100 μg/ml, clone 3E12A2), anti-TLR2 mAb (100 μg/ml, R&D system), or a mixture of anti-CLEC5A mAb and anti-TLR2 mAb for 30 min at room temperature before incubation with SARS-CoV-2.

Techniques: Incubation, Microscopy, Staining, Software, Virus

Journal: bioRxiv

Article Title: CLEC5A and TLR2 are critical in SARS-CoV-2-induced NET formation and lung inflammation

doi: 10.1101/2022.02.01.478701

Figure Lengend Snippet: (a) EVs from healthy controls (HCs-EVs, n=5) and COVID-19 patients (COVID19-EVs, n=5) were harvested by ultracentrifugation, then lysed in RIPA solution before subjected to mass spectrometry analysis. Proteins expressed in COVID-19 EVs, but not in HCs EVs, were further analyzed using the QIAGEN Ingenuity Pathway Analysis (QIAGEN IPA) software. Proteins which were expressed in all the COVID19-EVs were displayed. (b&c) HCs-EVs (n=10) and COVID19-EVs (n=10) were analyzed by flow cytometry, and markers highly activated in COVID-19 platelets were expressed as a heat map (b) or by mean fluorescence intensity (c) . (d) Neutrophils were pre-incubated with anti-CLEC5A mAb (3E12A2, 100 μg/ml), anti-TLR2 mAb (# MAB2616, 100 μg/ml), or both anti-CLEC5A mAb (3E12A2, 100 μg/ml) and anti-TLR2 mAb (# MAB2616, 100 μg/ml), for 30 min at room temperature, followed by incubation with EVs (1 μg/ml) from COVID-19 patients (n=6) at 37°C for 3 h. Data are mean ± sd and repeats of at least three independent experiments. * p <0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001 (Student’s t -test).

Article Snippet: For blocking assay, neutrophils were preincubated with isotype (100 μg/ml), anti-CLEC5A mAb (100 μg/ml, clone 3E12A2), anti-TLR2 mAb (100 μg/ml, R&D system), or a mixture of anti-CLEC5A mAb and anti-TLR2 mAb for 30 min at room temperature before incubation with SARS-CoV-2.

Techniques: Mass Spectrometry, Software, Flow Cytometry, Fluorescence, Incubation

Journal: bioRxiv

Article Title: CLEC5A and TLR2 are critical in SARS-CoV-2-induced NET formation and lung inflammation

doi: 10.1101/2022.02.01.478701

Figure Lengend Snippet: C57BL/6 mice (WT) (n=3) and clec5a -/- tlr2 -/- mice (n=3)were inoculated with AAV-hACE2 for 14 days, followed by intranasal inoculation of SARS-CoV-2 (8 × 10 4 PFU/per mice). Tissues were collected at 3 days and 5 days post-infection. (a) The level of proinflammatory cytokines and chemokines were measured by real-time PCR and presented as fold change (compared to AAV-hACE2 uninfected mice/mock). ( b-d) NET structure and thrombus were detected by Hoechst. 33342 (blue), anti-MPO antibody (green), anti-citrullinated histone H3 (red), anti-CD42b antibody (yellow) (b) , and images were captured by a confocal microscope and subjected to determine the area of MPO (c) and CD42b (d) using MetaMorph TM software. (e) Cell infiltrated to lung. Interstitial macrophage (interstitial MΦ) was defined as CD11b + CD64 + F4/80 + cells; monocyte-derived dendritic cell (DC)/macrophage (MΦ) was defined as CD11b + CD64 + Ly6C + ; Ly6C + monocyte was defined as Ly6C + . The cell number of each cell population was calculated using the multiple fluorescent staining image and analyzed by software MetaMorph TM , and the data was presented as cell number/ per 664225 (815 × 815). Scale bar is 200 μm. Data are represented as mean ± SEM. * p <0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001 (Student’s t -test).

Article Snippet: For blocking assay, neutrophils were preincubated with isotype (100 μg/ml), anti-CLEC5A mAb (100 μg/ml, clone 3E12A2), anti-TLR2 mAb (100 μg/ml, R&D system), or a mixture of anti-CLEC5A mAb and anti-TLR2 mAb for 30 min at room temperature before incubation with SARS-CoV-2.

Techniques: Infection, Real-time Polymerase Chain Reaction, Microscopy, Software, Derivative Assay, Staining

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

Article Title: MAFB Determines Human Macrophage Anti-Inflammatory Polarization: Relevance for the Pathogenic Mechanisms Operating in Multicentric Carpotarsal Osteolysis.

doi: 10.4049/jimmunol.1601667

Figure Lengend Snippet: FIGURE 3. MAFB controls the global anti-inflammatory transcriptional signature of M-MØ. (A) Experimental design. (B) Number of genes whose expression is higher (UP) or lower (DOWN) in siMAFB M-MØ compared with siControl M-MØ (adjusted p , 0.05). (C) Validation of microarray results by quantitative RT-PCR in independent samples of siMAFB and siControl M-MØ (n = 4, *p , 0.05, **p , 0.005, ***p , 0.0005). Data are indicated as mean 6 SD of mRNA levels in siMAFB M-MØ relative to siControl M-MØ. (D) CLEC5A Western analysis in M-MØ treated as indicated. Shown is one experiment of three performed on independent samples. (E) GSEA analysis on the t statistic–ranked list of genes obtained from the siMAFB–M-MØ versus siControl–M-MØ limma analysis, using the proinflammatory (top) and anti-inflammatory (bottom) gene sets previously defined (49). Red and blue arrows indicate the location in the ranked list of those genes whose expression is higher or lower, respectively, in siMAFB M-MØ. (F) Venn diagram analysis of the genes with significantly lower expression in siMAFB M-MØ compared with the GM-MØ– and M-MØ–specific gene signatures (genes that are differ- entially expressed between GM-MØ and M-MØ by .8-fold, GSE68061). (G and H) ELISA of CCL2 and IL-10 protein levels in culture media from M-MØ treated as indicated (n = 4 for CCL2, n = 7 for IL-10, *p , 0.05).

Article Snippet: Protein detection was carried out using Abs against MAFB (sc-10022; Santa Cruz) and CLEC5A (MAB2384; R&D Systems).

Techniques: Expressing, Biomarker Discovery, Microarray, Quantitative RT-PCR, Western Blot, Enzyme-linked Immunosorbent Assay

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

Article Title: CLEC5A Mediates Macrophage Function and COPD Pathologies 1

doi: 10.4049/jimmunol.1500978

Figure Lengend Snippet: Immunofluorescent analysis of CLEC5A in lungs of mice exposed to (A) FA or (B) CS for 6 months. Arrows indicate alveolar macrophage (representative of 4 mice). CLEC5A surface expression was measured on (C) peripheral blood monocytes and (D and E) alveolar macrophage (F4/80hiCD11blo) from FA- and CS- exposed mice. Dashed line represents isotype control, solid line represents CLEC5A (n=5 mice). (F) Average number of CLEC5A+ cells and median fluorescence intensity of CLEC5A on alveolar macrophage. (G) CLEC5A expression on lung macrophage of never smokers (NS) and current smoker (S) (n=4 subjects/group). (H) Average number of CLEC5A+ cells and median fluorescence intensity of CLEC5A on alveolar macrophage.

Article Snippet: CLEC5A was assessed with α-human CLEC5A antibody (clone 283834, R&D Systems) and α-CD45 (eBioscience) using a FACSCalibur and FlowJo software.

Techniques: Expressing, Fluorescence

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

Article Title: CLEC5A Mediates Macrophage Function and COPD Pathologies 1

doi: 10.4049/jimmunol.1500978

Figure Lengend Snippet: BAL macrophage (5×104) were stimulated with (A) control IgG, α-CLEC5A activating antibody (10 ug/ml), LPS (10 ng/ml), or both for 24hrs. Similarly, BAL macrophage (5×104) were stimulated with (B) control IgG, α-CLEC5A activating antibody (10 ug/ml), GM-CSF (20 ng/ml), or both for 24hrs. TNFα was measured by ELISA. Data are mean +/− sem of 3 experiments performed in duplicate. * p<0.05 compared to FA mice.

Article Snippet: CLEC5A was assessed with α-human CLEC5A antibody (clone 283834, R&D Systems) and α-CD45 (eBioscience) using a FACSCalibur and FlowJo software.

Techniques: Enzyme-linked Immunosorbent Assay

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

Article Title: CLEC5A Mediates Macrophage Function and COPD Pathologies 1

doi: 10.4049/jimmunol.1500978

Figure Lengend Snippet: (A and B) WT and Clec5a−/− mice were exposed to CS for up to 6 months to evaluate lung remodeling as assessed by MLI (Data are mean +/− SEM, n=7–8). (C) Pulmonary inflammation was assessed by enumerating the total cells in BAL. (D) Cytokine levels in BAL of WT and Clec5a−/− mice (n=5–8). (E) mRNA expression levels in BAL macrophage of WT and Clec5a−/− mice (n=5–6). * p<0.05 compared to FA mice. # p<0.05 compared to wild-type mice.

Article Snippet: CLEC5A was assessed with α-human CLEC5A antibody (clone 283834, R&D Systems) and α-CD45 (eBioscience) using a FACSCalibur and FlowJo software.

Techniques: Expressing

Journal: Journal of Immunology Research

Article Title: Elevated Expression of C-Type Lectin Domain Family 5-Member A (CLEC5A) and Its Relation to Inflammatory Parameters and Disease Course in Adult-Onset Still's Disease

doi: 10.1155/2020/9473497

Figure Lengend Snippet: The percentages and MFI of CLEC5A-expressing monocytes and granulocytes in patients with adult-onset Still's disease (AOSD). Representative examples of flow cytometric histograms of CLEC5A-expressing monocytes or granulocytes obtained from peripheral blood of one active AOSD patient ((a) A) or ((b) A) and one healthy subject ((a) B) or ((b) B). Comparisons of the percentages and mean fluorescence intensity (MFI) of CLEC5A-expressing monocytes ((c) and (d)) or granulocytes ((e) and (f)) between AOSD patients and healthy control subjects. The data are presented as box-plot diagrams, in which the box encompasses the 25th percentile (lower bar) to the 75th percentile (upper bar). The horizontal line within the box indicates the median value for each group. The p values were determined by using the nonparametric Mann-Whitney U test.

Article Snippet: The blots were blocked with 5% milk in PBS with 0.1% Tween-20 (PBST) (Bionovas, Inc., Washington, DC, USA) for 30 min at room temperature, and subsequently incubated with specific anti-CLEC5A antibody (Aviva Systems Biology, San Diego, CA, USA), anti-NLRP3 antibody (Cell Signaling Technology, Beverly, MA, USA), anti-caspase-1 antibody (Abcam, Cambridge, MA, USA), anti-IL-1 β antibody (Novus Biologicals, LLC, Littleton, CO, USA), anti-IL-18 antibody (Medical & Biology Laboratories Co, Ltd., Naka-ku, Nagoya, Japan), and anti- α -tubulin (1: 5000, Santa Cruz Biotechnology, Dallas, Texas, USA) at 4°C overnight.

Techniques: Expressing, Fluorescence, Control, MANN-WHITNEY

Journal: Journal of Immunology Research

Article Title: Elevated Expression of C-Type Lectin Domain Family 5-Member A (CLEC5A) and Its Relation to Inflammatory Parameters and Disease Course in Adult-Onset Still's Disease

doi: 10.1155/2020/9473497

Figure Lengend Snippet: The mRNA expression levels of CLEC5A and DAP12 and the level of inflammasome-related cytokines IL-1 β and IL-18 in patients with AOSD. The comparisons of relative mRNA expression levels of CLEC5A and DAP12 on PBMCs ((a) and (b), respectively) between 17 AOSD patients and 9 healthy subjects. The horizontal line indicates the median value for each group. The correlation between CLEC5A expression and DAP12 expression in all subjects (c) or AOSD patients (d). Correlation coefficients ( γ ) and p value were obtained by the nonparametric Spearman's rank correlation test. The comparisons in plasma levels of proinflammatory cytokines including IL-1 β (e) and IL-18 (f) from active AOSD patients and healthy controls (HC). The data are presented as box-plot diagrams, in which the box encompasses the 25th percentile (lower bar) to the 75th percentile (upper bar). The horizontal line within the box indicates the median value for each group. The p values were determined by using the nonparametric Mann-Whitney U test. CLEC5A: C-type lectin domain family 5-member A; DAP12: DNAX activation protein 12; AOSD: adult-onset Still's disease; PBMCs: peripheral blood mononuclear cells.

Article Snippet: The blots were blocked with 5% milk in PBS with 0.1% Tween-20 (PBST) (Bionovas, Inc., Washington, DC, USA) for 30 min at room temperature, and subsequently incubated with specific anti-CLEC5A antibody (Aviva Systems Biology, San Diego, CA, USA), anti-NLRP3 antibody (Cell Signaling Technology, Beverly, MA, USA), anti-caspase-1 antibody (Abcam, Cambridge, MA, USA), anti-IL-1 β antibody (Novus Biologicals, LLC, Littleton, CO, USA), anti-IL-18 antibody (Medical & Biology Laboratories Co, Ltd., Naka-ku, Nagoya, Japan), and anti- α -tubulin (1: 5000, Santa Cruz Biotechnology, Dallas, Texas, USA) at 4°C overnight.

Techniques: Expressing, Clinical Proteomics, MANN-WHITNEY, Activation Assay

Journal: Journal of Immunology Research

Article Title: Elevated Expression of C-Type Lectin Domain Family 5-Member A (CLEC5A) and Its Relation to Inflammatory Parameters and Disease Course in Adult-Onset Still's Disease

doi: 10.1155/2020/9473497

Figure Lengend Snippet: The correlations between CLEC5A expression and inflammatory parameters as well as proinflammatory cytokines in 34 patients with adult-onset Still's disease (AOSD).

Article Snippet: The blots were blocked with 5% milk in PBS with 0.1% Tween-20 (PBST) (Bionovas, Inc., Washington, DC, USA) for 30 min at room temperature, and subsequently incubated with specific anti-CLEC5A antibody (Aviva Systems Biology, San Diego, CA, USA), anti-NLRP3 antibody (Cell Signaling Technology, Beverly, MA, USA), anti-caspase-1 antibody (Abcam, Cambridge, MA, USA), anti-IL-1 β antibody (Novus Biologicals, LLC, Littleton, CO, USA), anti-IL-18 antibody (Medical & Biology Laboratories Co, Ltd., Naka-ku, Nagoya, Japan), and anti- α -tubulin (1: 5000, Santa Cruz Biotechnology, Dallas, Texas, USA) at 4°C overnight.

Techniques: Expressing, Activity Assay

Journal: Journal of Immunology Research

Article Title: Elevated Expression of C-Type Lectin Domain Family 5-Member A (CLEC5A) and Its Relation to Inflammatory Parameters and Disease Course in Adult-Onset Still's Disease

doi: 10.1155/2020/9473497

Figure Lengend Snippet: The difference in the effects of CLEC5A knockdown on NLRP3-inflammasome expression in THP-1 cells between AOSD patients and healthy controls. (a) CLEC5A expression in THP-1 cells was knocked down by CLEC5A-specific siRNA (Dharmafect) and a nonspecific siRNA (negative control) for 48 hours. (b) Cells were stimulated with 10% plasma from AOSD patients ( n = 6) and HC ( n = 4) for 6 h at 37°C, and cell lysates were harvested for the determination of (c) NLRP3, (d) caspase-1, (e) IL-1 β , and (f) IL-18 by Western blotting. α -Tubulin is used as loading control. AOSD: adult-onset Still's disease; HC: healthy controls; IL: interleukin. Bars and error bars indicate mean and standard deviation, respectively. The p values were determined by Bonferroni's posttest.

Article Snippet: The blots were blocked with 5% milk in PBS with 0.1% Tween-20 (PBST) (Bionovas, Inc., Washington, DC, USA) for 30 min at room temperature, and subsequently incubated with specific anti-CLEC5A antibody (Aviva Systems Biology, San Diego, CA, USA), anti-NLRP3 antibody (Cell Signaling Technology, Beverly, MA, USA), anti-caspase-1 antibody (Abcam, Cambridge, MA, USA), anti-IL-1 β antibody (Novus Biologicals, LLC, Littleton, CO, USA), anti-IL-18 antibody (Medical & Biology Laboratories Co, Ltd., Naka-ku, Nagoya, Japan), and anti- α -tubulin (1: 5000, Santa Cruz Biotechnology, Dallas, Texas, USA) at 4°C overnight.

Techniques: Knockdown, Expressing, Negative Control, Clinical Proteomics, Western Blot, Control, Standard Deviation

Journal: Journal of Immunology Research

Article Title: Elevated Expression of C-Type Lectin Domain Family 5-Member A (CLEC5A) and Its Relation to Inflammatory Parameters and Disease Course in Adult-Onset Still's Disease

doi: 10.1155/2020/9473497

Figure Lengend Snippet: The differences in CLEC5A levels and plasma cytokine levels in AOSD patients with different patterns of disease course. The differences in the percentages and MFI of CLEC5A-expressing granulocytes ((a) and (b), respectively) or monocytes ((c) and (d), respectively), and plasma levels of IL-1 β (e) and IL-18 (f) in AOSD patients with different patterns of disease course. Data are presented as box-plot diagrams, with the box encompassing the 25th percentile (lower bar) to the 75th percentile (upper bar). The horizontal line within the box indicates median value, respectively, for each group. The p values were determined by the Mann-Whitney U test.

Article Snippet: The blots were blocked with 5% milk in PBS with 0.1% Tween-20 (PBST) (Bionovas, Inc., Washington, DC, USA) for 30 min at room temperature, and subsequently incubated with specific anti-CLEC5A antibody (Aviva Systems Biology, San Diego, CA, USA), anti-NLRP3 antibody (Cell Signaling Technology, Beverly, MA, USA), anti-caspase-1 antibody (Abcam, Cambridge, MA, USA), anti-IL-1 β antibody (Novus Biologicals, LLC, Littleton, CO, USA), anti-IL-18 antibody (Medical & Biology Laboratories Co, Ltd., Naka-ku, Nagoya, Japan), and anti- α -tubulin (1: 5000, Santa Cruz Biotechnology, Dallas, Texas, USA) at 4°C overnight.

Techniques: Clinical Proteomics, Expressing, MANN-WHITNEY

Journal: Journal of Immunology Research

Article Title: Elevated Expression of C-Type Lectin Domain Family 5-Member A (CLEC5A) and Its Relation to Inflammatory Parameters and Disease Course in Adult-Onset Still's Disease

doi: 10.1155/2020/9473497

Figure Lengend Snippet: The change of CLEC5A levels on immune cells from AOSD patients after therapy. The changes of (a) the frequencies or (b) the mean fluorescence index (MFI) of CLEC5A-expressing monocytes and granulocytes in AOSD patients after 6-month therapy. Data are presented as box-plot diagrams, with the box encompassing the 25th percentile (lower bar) to the 75th percentile (upper bar). The horizontal line within the box indicates median value, respectively, for each group. The p values were determined by the Wilcoxon signed rank test.

Article Snippet: The blots were blocked with 5% milk in PBS with 0.1% Tween-20 (PBST) (Bionovas, Inc., Washington, DC, USA) for 30 min at room temperature, and subsequently incubated with specific anti-CLEC5A antibody (Aviva Systems Biology, San Diego, CA, USA), anti-NLRP3 antibody (Cell Signaling Technology, Beverly, MA, USA), anti-caspase-1 antibody (Abcam, Cambridge, MA, USA), anti-IL-1 β antibody (Novus Biologicals, LLC, Littleton, CO, USA), anti-IL-18 antibody (Medical & Biology Laboratories Co, Ltd., Naka-ku, Nagoya, Japan), and anti- α -tubulin (1: 5000, Santa Cruz Biotechnology, Dallas, Texas, USA) at 4°C overnight.

Techniques: Fluorescence, Expressing