Journal: medRxiv

Article Title: Monocytes are the main source of STING-mediated IFN-α production

doi: 10.1101/2022.03.11.22272208

Figure Lengend Snippet: Impact of aging on monocyte counts (a) and IFN-α response (b) after cGAMP stimulation for males and females. Correlation coefficients “ R” and probabilities of correlation were calculated using Pearson test. (a) Correlation between the absolute number of monocytes/mm 3 (log 10 ) and age (years). (b) Correlation between the cGAMP-stimulated release of IFN-α (log 10 ) and the absolute number of monocytes/mm 3 (log 10 ). (c) PBMC from 5 donors were depleted of CD14 + cells (> 98% depletion) using magnetic particles or mock-depleted and stimulated with cGAMP (20 µg/ml) or R848 (1 µg/ml) for 24 hours. IFN-α production was measured by ELISA. Paired Student’s t -test was used for statistical analysis. (d, e) Total PBMCs (5 × 10 5 cells/well) or monocyte-enriched cells (% CD14 + cells > 77% at 3 × 10 5 cells/well) were stimulated with cGAMP or left untreated (NS). The production of IFN-α (d) or IP-10/CXCL-10 (e) was then assessed in 24 hour-supernatants. Median values in pg/ml for [IFN-α] or [IP-10] concentrations normalized to 3 × 10 5 cells/well are shown in parentheses. (f, g) Intracellular analysis of IFN-α synthesis in 18 hour-stimulated monocytes, gated as CD11c pos HLA-DR pos cells, from male or female donors. Brefeldin A was added for the last 3 hours of culture. Frequencies of IFN-α pos monocytes are shown from individual female or male donors stimulated or not with cGAMP. Statistical differences between groups were assessed using the Kruskal-Wallis’s test corrected for multiple comparisons.

Article Snippet: Positive fraction containing CD14 cells was resuspended in R10 medium and 3.10 5 cells were seeded in 96-well plates and stimulated with 20 µg/ml of cGAMP (Invivogen France).

Techniques: Enzyme-linked Immunosorbent Assay

Journal: PLoS ONE

Article Title: Robust and Highly-Efficient Differentiation of Functional Monocytic Cells from Human Pluripotent Stem Cells under Serum- and Feeder Cell-Free Conditions

doi: 10.1371/journal.pone.0059243

Figure Lengend Snippet: The protocol is composed of 5 steps. CD14-positive cells that are sorted between step-4 are differentiated into dendritic cells by step 5-1 or into macrophages by step 5-2. FL-3: Flt-3 ligand, TPO: Thrombopoietin.

Article Snippet: The CD14 + monocytic lineage-directed cell fraction in supernatant was positively sorted by autoMACS pro (Miltenyi Biotec) with CD14 MicroBeads (Miltenyi Biotec) on days 15–28.

Techniques:

Journal: PLoS ONE

Article Title: Robust and Highly-Efficient Differentiation of Functional Monocytic Cells from Human Pluripotent Stem Cells under Serum- and Feeder Cell-Free Conditions

doi: 10.1371/journal.pone.0059243

Figure Lengend Snippet: (A) Flow cytometric analysis of monocytic lineage cells derived sequentially from pluripotent stem cells. An analysis of adherent cells on day 6 and supernatant cells on day 13 and 18 is shown. (B) May-Giemsa staining of CD14 + monocyte-like cells derived from KhES1 on day 16 (left) and primary human monocytes (right). (C) Esterase staining for CD14 + monocyte-like cells derived from KhES1 on day 16. (D) The percentage of CD14 + cells within the total floating cells derived from KhES1/iPS-201B7 was evaluated from day 13 to day 28. (E) May-Giemsa staining (left) and phase contrast image (right) of mature DCs derived from pluripotent stem cells. (F) Flow cytometric analysis of immature/mature DCs derived from pluripotent stem cells. (G) Phase contrast image and flow cytometric analysis of macrophages derived from pluripotent stem cells.(H) RT-PCR analysis of monocytic lineage cells derived from KhES1/iPS-201B7 clones for expression of monocytic lineage marker genes ( PU.1, c-MAF, TLR4, CCL17 and CCL18 ). Peripheral blood monocytes and peripheral blood monocyte-derived mature DCs were used as positive controls.(A–C, E–G) The data from KhES1-derived cells are shown as representative.

Article Snippet: The CD14 + monocytic lineage-directed cell fraction in supernatant was positively sorted by autoMACS pro (Miltenyi Biotec) with CD14 MicroBeads (Miltenyi Biotec) on days 15–28.

Techniques: Derivative Assay, Staining, Reverse Transcription Polymerase Chain Reaction, Clone Assay, Expressing, Marker

Journal: The EMBO Journal

Article Title: Secretion of mitochondrial DNA via exosomes promotes inflammation in Behçet's syndrome

doi: 10.15252/embj.2022112573

Figure Lengend Snippet: A–C mtDNA levels in the CS of HC‐derived leukocytes following PAMP/DAMP stimulation, with or without DNase I treatment. CD14 + monocytes (A), neutrophils (B), and lymphocytes (C) isolated from the peripheral blood of HCs were PAMP/DAMP stimulated utilizing: AL (ATP and LPS), NIG (nigericin and LPS), LPS, PMA, fMLP, CpG (ODN2006), cGAMP, or IM (ionomycin). The COXIII levels in purified DNA in CS were measured using qPCR after removing cell debris and DNase I treatment or mock digestion. D mtDNA levels in the CS of THP‐1 cells induced to undergo primary necrosis (1 st Nec), secondary necrosis (2 nd Nec), apoptosis (Apo), or pyroptosis (Pyro). After removal of cell debris, the supernatant was untreated (left, middle) or treated with Triton‐X100 (right), then undigested (left) or digested (middle, right) with DNase I. mtDNA levels were measured in purified DNA. E, F mtDNA levels in 16K‐ (E) and 100K‐EV (F) fractions in the CS of THP‐1 cells and CD14 + monocytes induced to undergo 1 st Nec, 2 nd Nec, Apo, or Pyro. 16K‐EVs and 100K‐EVs were separated using centrifugation at 16,000 g and 100,000 g , respectively. mtDNA levels in these EVs were measured by qPCR. G, H Secretion of mtDNA in 100K‐EVs by pyroptotic stimulation. THP1 cells were stimulated with LPS alone or LPS + ATP (G). HC–derived CD14 + monocytes were intracellularly stimulated with LPS enclosed in liposomes utilizing Avalanche‐Omni (H). mtDNA levels in 100K‐EVs were measured after 100K‐EVs were isolated from CS. I Deposition of dsDNA in 100K‐EVs. 100K‐EVs isolated from the CS of THP‐1 cells after LPS/ATP stimulation were fixed and stained with an anti‐dsDNA antibody, and then visualized with a secondary antibody conjugated to 10‐nm gold particles. Images were obtained using transmission immunoelectron microscopy. Black dots (white arrows) indicate the dsDNA deposition. Scale bar, 100 nm. Data information: Statistical analyses were performed using an ANOVA with Tukey's post‐hoc test (D–G) (mean ± SD; ** P < 0.01; NS, not statistically significant) and a Student's t ‐test (H) (mean ± SD; ** P < 0.01). For panel A‐C (mean ± SD). The data are representative of three independent experiments. Source data are available online for this figure.

Article Snippet: Human CD14 microbeads , Miltenyi Biotec , 130‐050‐201 , .

Techniques: Derivative Assay, Isolation, Purification, Centrifugation, Liposomes, Staining, Transmission Assay, Immuno-Electron Microscopy

Journal: The EMBO Journal

Article Title: Secretion of mitochondrial DNA via exosomes promotes inflammation in Behçet's syndrome

doi: 10.15252/embj.2022112573

Figure Lengend Snippet: A Leakage of mtDNA from the mitochondria into the cytosol. THP‐1 cells under normal, apoptotic, or pyroptotic conditions were stained with the PicoGreen (green) and MitoTracker‐Deep Red (red) dyes and visualized using confocal microscopy. Scale bar, 5 μm (left). Quantification of the number of PicoGreen‐positive dots in the cytosol (right). B Leaked cytosolic mtDNA levels following cell death induction. After removing the nuclei, the mitochondrial and cytosolic fractions from THP‐1 cells were isolated under normal, apoptotic, or pyroptotic conditions. DNA was purified from the cytosolic fraction, and the mtDNA levels were measured. C, D Cytosolic mtDNA levels in Casp1‐KO or Gsdmd‐KO cells undergoing pyroptosis. mtDNA and mitochondria in WT, Casp1‐KO, and Gsdmd‐KO‐THP‐1 cells induced to undergo pyroptosis were stained with the PicoGreen (green) and MitoTracker‐Deep Red (red) dyes and visualized using confocal microscopy. Scale bar, 5 μm (left). Quantification of the number of PicoGreen‐positive dots in the cytosol (right) (C). mtDNA level in the cytosolic fraction was measured by qPCR (D). E Mitochondria localization of NT‐gasdermin‐D. Ten minutes after THP1 cells were induced pyroptosis, mitochondria fraction was isolated and Western blotting was performed using anti‐gasdermin‐D antibody. F mtDNA level in 100K‐EVs. 100K‐EVs were isolated from CS of WT, Casp1‐KO, Gsdmd‐KO THP1 cells induced to undergo pyroptosis, and mtDNA level in 100K‐EVs was measured. G mtDNA levels in 100K‐EVs from pyroptotic cells with inhibited caspase‐1 or mROS. CD14 + monocytes were pre‐treated with Mito‐TEMPO or Ac‐YVAD‐cmk and then stimulated with LPS and ATP. The levels of mtDNA in 100K‐EVs in CS were measured. H mtDNA levels in 100K‐EVs from caspase‐1‐restored Casp1‐KO‐THP‐1 cells. The levels of mtDNA were measured in 100K‐EVs secreted by pyroptotic WT, Casp1‐KO, and FL‐Casp1‐Casp1‐KO‐THP‐1 cells. I mtDNA levels in 100K‐EVs from cells overexpressing aCasp1 or NT‐Gsdmd. aCasp1‐p10/p20 or NT‐Gsdmd were introduced into WT THP‐1 cells, and the extracellular mtDNA levels within 100K‐Evs were measured. Data information: Statistical analyses were performed using a Steel–Dwass test (A, C) (median; 25 th and 75 th percentiles; minimum and maximum value of a population; ** P < 0.01), and an ANOVA with Tukey's post‐hoc test (B, D, F–I) (mean ± SD; * P < 0.05, ** P < 0.01; NS, not statistically significant). The data are representative of two (E) and three (A–D, F–I) independent experiments. Source data are available online for this figure.

Article Snippet: Human CD14 microbeads , Miltenyi Biotec , 130‐050‐201 , .

Techniques: Staining, Confocal Microscopy, Isolation, Purification, Western Blot

Journal: The EMBO Journal

Article Title: Secretion of mitochondrial DNA via exosomes promotes inflammation in Behçet's syndrome

doi: 10.15252/embj.2022112573

Figure Lengend Snippet: A–C Increased inflammasome activation and pyroptosis in BS monocytes. HC ( n = 4)‐ and BS ( n = 6) ‐derived CD14 + monocytes were stimulated with LPS or ATP. The bioactivity of IL‐1β (Α) and the levels of LDH (B) in CS were measured. The levels of active caspase‐1 (p10) and mature IL‐1β (p17) in CS of HC ( n = 6)‐ and BS ( n = 6)‐CD14 + monocytes were evaluated through western blotting using anti‐Casp1 (p10) and anti–IL‐1β (p17) antibodies (left), respectively. The concentrations of caspase‐1 (p10) and IL‐1β (p17) in gel bands were determined using the ImageJ software, and statistical analyses were performed (right) (C). D Secretion levels of mtDNA via 100K‐EVs in BS monocytes. Neutrophils and CD14 + monocytes isolated from the peripheral blood of HC or BS patients were stimulated with nigericin or LPS. After DNA was purified from 100K‐EVs in the CS, the mtDNA levels were measured using qPCR. E Caspase‐1‐dependent over‐secretion of mtDNA in BS monocytes. HC and BS monocytes were pre‐treated with or without Ac‐YVAD‐cmk, and the cells were stimulated with LPS or ATP. The levels of mtDNA in 100K‐EVs in the CS were measured using qPCR. F Deposition of dsDNA and 8‐OHdG in MVBs of BS monocytes. After inducing pyroptosis via LPS and ATP, transmission immunoelectron microscopy was performed using an anti‐dsDNA (upper) antibody and an anti‐8‐OHdG (lower) antibody and a secondary antibody conjugated to 10‐nm gold particles. Black dots (white arrows) indicate dsDNA‐ (left) or 8‐OHdG‐ (right) positive depositions. Scale bar, 1 μm. G–I Increased secretion of mtDNA in 100K‐EVs by an NLRP3‐mutant derivative. Full‐length NLRP3 (WT‐NLRP3) and an NLRP3 mutant (V200M‐NLRP3) were restored into NLRP3‐KO (FL‐NLRP3‐NLRP3‐KO and V200M‐NLRP3‐NLRP3‐KO) THP‐1 cells (G). The cells were subsequently stimulated with LPS and ATP. The levels of mtDNA in 100K‐EVs (H) and IL‐1β in the CS (I) were measured. Data information: Statistical analyses were performed using an ANOVA with Tukey's post‐hoc test (A, C–E, H, I) (mean ± SD; * P < 0.05, ** P < 0.01; NS, not statistically significant) and a Mann–Whitney U test (B) (median; 25 th and 75 th percentile; minimum and maximum value of a population; ** P < 0.01). The data are representative of two (A–F) and three (G–I) independent experiments. Source data are available online for this figure.

Article Snippet: Human CD14 microbeads , Miltenyi Biotec , 130‐050‐201 , .

Techniques: Activation Assay, Derivative Assay, Western Blot, Software, Isolation, Purification, Transmission Assay, Immuno-Electron Microscopy, Mutagenesis, MANN-WHITNEY

Journal: The EMBO Journal

Article Title: Secretion of mitochondrial DNA via exosomes promotes inflammation in Behçet's syndrome

doi: 10.15252/embj.2022112573

Figure Lengend Snippet: mROS production in CD14 + monocytes. HC and BS monocytes were stained with MitoPB (green) and MitoSox (red), and mROS production was visualized using confocal microscopy. Scale bar, 5 μm. Mitochondrial membrane potential in CD14 + monocytes. HC‐ and BS‐derived CD14 + monocytes were stimulated with LPS, and the reduction in the MMP was monitored by FACS using MitoTracker Green (X‐axis) and MitoTracker‐Deep Red (Y‐axis) (left). The increase in the proportion of MMP‐reduced cells (gated population) following LPS stimulation was quantified (right). Effect of caspase‐1 on the increasing MMP of BS monocytes. BS‐derived CD14 + monocytes were stimulated with LPS in the presence or absence of Ac‐YVAD‐cmk, and the reduction in the MMP was monitored using FACS. Data information: Statistical analyses were performed using a Mann–Whitney U test (B, C) (median; 25 th and 75 th percentiles; minimum and maximum value excluding outliers; * P < 0.05). The data are representative of two (A) and three (B, C) independent experiments. Source data are available online for this figure.

Article Snippet: Human CD14 microbeads , Miltenyi Biotec , 130‐050‐201 , .

Techniques: Staining, Confocal Microscopy, Membrane, Derivative Assay, MANN-WHITNEY

Journal: The EMBO Journal

Article Title: Secretion of mitochondrial DNA via exosomes promotes inflammation in Behçet's syndrome

doi: 10.15252/embj.2022112573

Figure Lengend Snippet:

Article Snippet: Human CD14 microbeads , Miltenyi Biotec , 130‐050‐201 , .

Techniques: Western Blot, Purification, Produced, Affinity Purification, Flow Cytometry, Confocal Microscopy, Electron Microscopy, Amplification, Recombinant, Blocking Assay, Adjuvant, Enzyme-linked Immunosorbent Assay, LDH Cytotoxicity Assay, Isolation, DNA Extraction, dsDNA Assay, Real-time Polymerase Chain Reaction, Plasmid Preparation, Cloning, Mutagenesis, Sequencing, Transfection, DNA Ligation, Fractionation

Journal: Cardiovascular Diabetology

Article Title: Adiponectin levels and expression of adiponectin receptors in isolated monocytes from overweight patients with coronary artery disease

doi: 10.1186/1475-2840-10-14

Figure Lengend Snippet: Flow cytometric analysis of adiponectin receptors in isolated monocytes . (A) Dot plot presentation of isolated mononuclear cells. Cells subpopulations were distinguished by granularity (side scatter) versus staining with CD14-PE monoclonal antibody. Monocytes (CD14-PE + ) were gated (R). (B) Analysis of Adipo-R2 log fluorescence of the gated monocytes. (C) Dot plot of the Adipo-R2 expression versus CD14-PE. The upper right quadrant includes the cells showing positive expression for both adipoR2 and CD14 antibodies (green colour).

Article Snippet: The monocyte fraction was simultaneously stained with anti-CD14-PE monoclonal antibody (BD Biosciences, San Jose, CA, USA).

Techniques: Isolation, Staining, Fluorescence, Expressing