Journal: Cell reports

Article Title: TNFR2 Signaling Enhances ILC2 Survival, Function, and Induction of Airway Hyperreactivity

doi: 10.1016/j.celrep.2019.11.102

Figure Lengend Snippet: KEY RESOURCES TABLE

Article Snippet: Recombinant mouse TNF-a , BioLegend , Cat # 575204.

Techniques: Recombinant, Enzyme-linked Immunosorbent Assay, Staining, Software

Journal: bioRxiv

Article Title: MAFB surrogates the glucocorticoid receptor ability to induce tolerogenesis in dendritic cells

doi: 10.1101/2021.07.27.453975

Figure Lengend Snippet: (A) Schematic representation of the experimental approach, comparing dendritic cell (DC) with tolerogenic dendritic cell (tolDC) differentiation. (B) DC and tolDC were cocultured with CD8+ cells for 5 days. The final CFSE signal of CD8+ cells is shown (left panel). CD8+ with only CD3/CD28 T-activator beads (C+) or alone (C-) are also shown. In the right panel, the average proliferation of the quadruplicate is shown (mean ± standard error of the mean (SEM)). (C) IL-10, TNFα, IL-12p70 and IL1-β production of DC and tolDC, after 5 days of differentiation and 24 h of LPS (10 ng/μL) and IFNg (20 ng/μL) stimuli. P-values of paired t-tests are shown. (D) Box-plots of CD80, CD83, CD86 and HLA-DR surface expression (Median Fluorescence Intensity) in DCs and tolDCs in steady-state or stimulated with LPS (10 ng/μL) and IFNg (20 ng/μL) (ns p > 0.05, ** p < 0.01, *** p ≤ 0.001). (E) Gene expression heatmap of differentially expressed genes comparing tolDCs with DCs and also displaying the gene expression values of the precursor cell type (MO) (logFC > 0.5, FDR < 0.05). Scaled fluorescence values of expression arrays are shown, ranging from -2 (lower gene expression, green) to +2 (higher gene expression, orange). (F) Gene ontology (GO) over-representation of GO Biological Process categories. Fold change of tolDC induced genes over background and -log10(FDR) of Fisher’s exact tests are shown. (G) Discriminant regulon expression analysis (DoRothEA) of tolDC compared with DC. Only transcription factors with FDR < 0.05 are shown. NES and logFC of transcription factor expression are depicted. (H) T-distributed stochastic neighbor embedding (t-SNE) plot of the aggregated and batch-corrected gene expression data from our study (MO, DC and tolDC) and two additional public datasets (GSE40484 (moMAC, moDC, cDC2, CM (Classical MOs) and NCM (Non-Classical MOs) and GSE99056 (M-MAC (M2 Macrophages) and GM-MAC (M1 Macrophages)). The 4 different groups obtained using k-means clustering are represented with grey ellipses of multivariate t-distributions.

Article Snippet: Enzyme-linked immunosorbent assays (ELISA) were performed, following the manufacturer’s instructions: Human IL-10, Human IL-12p70, and Human TNFα from BioLegend, and Human IL-1β from ThermoFisher.

Techniques: Expressing, Fluorescence, Gene Expression

Journal: bioRxiv

Article Title: MAFB surrogates the glucocorticoid receptor ability to induce tolerogenesis in dendritic cells

doi: 10.1101/2021.07.27.453975

Figure Lengend Snippet: (A) Volcano plot comparing tolDCs treated with control siRNA (siCTL) and MAFB siRNA (siMAFB). Dashed lines indicate significance thresholds (FDR < 0.05, absolute logFC > 0.5). tolDC-induced and tolDC-repressed genes are shown in blue and orange, respectively. (B) Gene set enrichment analysis (GSEA) of tolDCs (siCTL) vs. tolDCs (siMAFB), using tolDC-induced and tolDC-repressed gene sets. The running enrichment score is represented and the normalized enrichment score (NES) is shown above (FDR < 0.01). (C) DNA methylation heatmap of previously obtained differentially methylated CpGs (C1-CpGs and C2-CpGs) in tolDCs (siCTL) and tolDCs (siMAFB). Scaled β-values are shown (lower DNA methylation levels in blue and higher methylation levels in red). On the right side, violin plots of Cluster 1 (C1) and Cluster 2 (C2) depict β-values (ns p > 0.05, *** p ≤ 0.001). (D) Methylated CpG set enrichment analysis (mCSEA) of tolDCs (siCTL) vs. tolDCs (siMAFB), using MAFB-only CpGs, GR/MAFB CpGs and GR-only CpGs as CpG-sets (depending on the overlap of CpGs with GR or MAFB peaks). The running enrichment score is represented and the normalized enrichment score (NES) and FDR are shown above. (E) Box-plots of median fluorescence intensity (MFI) of CD14, CD16, CD163 and CD1a flow cytometry data from DCs (siCTL), tolDCs (siCTL) and tolDCs (siMAFB) (n = 7) (ns p > 0.05, * p < 0.05, ** p ≤ 0.01). (F) Box-plots of supernatant concentration from DCs (siCTL), tolDCs (siCTL) and tolDCs (siMAFB) (n = 7) of IL-10 in steady-state and stimulated conditions (LPS 10 ng/μL and IFNγ 20 ng/μL) and IL-12p70 and TNFα under stimulated conditions (pg/mL). TNFα and IL-12p70 in steady state were not detected. (ns p > 0.05, * p < 0.05, ** p ≤ 0.01) (G) DC (siCTL), tolDC (siCTL) and tolDC (siMAFB) were cocultured with CD8+ cells for 5 days (n = 4). The final CFSE signal of CD8+ cells is shown (left panel). CD8+ with only CD3/CD28 T-activator beads (C+) or alone (C-) are also shown. On the right panel, the average proliferation of the quadruplicate is shown (mean ± standard error of the mean (SEM)) (** p ≤ 0.01, *** p ≤ 0.001).

Article Snippet: Enzyme-linked immunosorbent assays (ELISA) were performed, following the manufacturer’s instructions: Human IL-10, Human IL-12p70, and Human TNFα from BioLegend, and Human IL-1β from ThermoFisher.

Techniques: Control, DNA Methylation Assay, Methylation, Fluorescence, Flow Cytometry, Concentration Assay

Journal: bioRxiv

Article Title: Staphylococci planktonic and biofilm environments differentially affect macrophage immune activation and osteoclastogenic differentiation

doi: 10.1101/2021.07.26.453923

Figure Lengend Snippet: RAW264.7 cells were cultivated in CM 1:1 diluted in fresh growth media (DMEM high glucose + 10% FCS + 1% Pen/Strep) and immune function was investigated. A) Surface marker profile of macrophages. Cells were stimulated with CM for 20 hours and surface protein levels of relevant TLRs, MHC complex II and co-stimulatory proteins were measured by FACS analysis. Data are presented as median expression levels measured by fluorescence intensity. n=5 experiments, p-values are calculated by Mann-Whitney U test. B) Gene expression analysis of relevant cytokines. Cells were stimulated with CM for 4 hours and mRNA levels of pro- (IL-1β, IL-6, TNF-α and IFN-β) and anti-inflammatory (IL-10) cytokines were quantified by RT-qPCR. Data are presented as relative gene expression of gene of interest related to the reference gene HPRT-1. n=5 experiments, p-values are calculated by Mann-Whitney U test. C) Protein amounts of relevant cytokines in the supernatant. Cells were stimulated with CM for 20 hours and protein concentration of pro- (IL-1α, IL-6, TNF-α and IFN-β) and anti-inflammatory (IL-10) cytokines were quantified in the supernatant by multi cytokine bead array (CBA; LegendPlex). Data are presented as absolute concentration (pg/ml). n=5 experiments, p-values are calculated by Mann-Whitney U test. D) Activation of NF-κB and IRF-3 signaling in macrophages. Cells were stimulated with CM for 4 hours and presence of phospho-NFκB p65 and phospho-IRF3 as activated forms of the transcription factors was visualized by Western Blot. HSP90 was used as loading control. PC: positive control (1 µg Pam3CSK4 + 100 nM CpG), n=4 experiments. E) Free bacterial DNA content in CM. DNA was extracted from 1 ml CM and amplified by qPCR using SA specific primers for gyrase B (product length: 147 bp) and SE specific primers for gyrase A (product length: 194 bp). PCR products were visualized by agarose gel electrophoresis. Numbers below bands show the mean Ct value of n=3 CM approaches. F) Cell stress/ ROS production of macrophages. Cells were stimulated with CM for 2 hours and cell stress was measured by FACS analysis using an oxidative stress reagent. Data are presented as median ROS production measured by fluorescence intensity. n=4 experiments. G) Phagocytic activity of macrophages. Cells were stimulated with CM for 24 hours and phagocytic uptake of fluorescence coupled beads was investigated by FACS analysis. Data are presented as median bead uptake measured by fluorescence intensity. n=5 experiments (one experiment was excluded from statistics as overall intensities were much higher but showed the same trend). A-C+F+G) A positive control (PC) was included in each respective experiment and data are shown in Suppl. Fig. 4.

Article Snippet: Cells then were transferred into suitable well plate formats, treated with CM 1:1 diluted in fresh cell growth media, TLR-ligands (Pam3CSK4: 25 ng/ml and CpG ODN 1668: 25 ng/ml, both InvivoGen, USA), sodium L- or D-lactate (5, 10, 15 and 20 mM, both Sigma-Aldrich, Germany) or recombinant murine cytokines (IL-10: 25 ng/ml, Peprotech, USA; TNF-α: 25 ng/ml, eBioscience, Germany or IFN-β: 0.02, 0.2 and 2 ng/ml, BioLegend, USA) ± recombinant mouse RANKL (50 ng/ml, R&D systems, UK) and analyzed for immune function, signal transduction, metabolic activity and differentiation (OC / MGC formation) after respective time points.

Techniques: Marker, Expressing, Fluorescence, MANN-WHITNEY, Gene Expression, Quantitative RT-PCR, Protein Concentration, Concentration Assay, Activation Assay, Western Blot, Control, Positive Control, Amplification, Agarose Gel Electrophoresis, Activity Assay

Journal: bioRxiv

Article Title: Staphylococci planktonic and biofilm environments differentially affect macrophage immune activation and osteoclastogenic differentiation

doi: 10.1101/2021.07.26.453923

Figure Lengend Snippet: RAW264.7 cells were cultivated in CM 1:1 diluted in fresh growth media (DMEM high glucose + 10% FCS + 1% Pen/Strep) and CM + RANKL-induced multinucleated giant cell (MGC) formation was investigated. In experiments with a cultivation time above 2 days 50 µM β-mercapto ethanol was added to the media to reduce cell proliferation and avoid overgrowing of the cells (here: MGC formation after 5 days). A) Cells were stimulated with CM and RANKL (50 ng/ml) for 5 days. At day 5 cells were fixed, stained for TRAP and nuclei and total numbers of formed MGCs per well were counted. MGCs were defined as TRAP-negative multinucleated giant cells with at least 3 nuclei. Data are presented as MGC numbers per well. n=5 experiments in duplicates (mean of duplicates was included in statistics), p-values are calculated by Mann-Whitney U test. B) Visualization of formed MGCs after 5 days of differentiation. TRAP activity is shown by violet color development and nuclei are stained in blue. Upper picture is showing spontaneous MGC formation in medium (negative control), lower picture is showing formed MGCs stimulated with SE planktonic CM + RANKL. C) Activation of Stat6 and Stat3 signaling in macrophages. Cells were stimulated with CM and RANKL (50 ng/ml) for 2 days and presence of phospho-Stat6 and phospho-Stat3 as activated forms of the transcription factors was visualized by Western Blot. IL-4 and IL-10 (both: 25 ng/ml for 1 hour) were used as positive controls for respective Stat-pathway activation. Β-Actin was used as loading control. n=4 experiments. D) Gene expression analysis of relevant cytokines. Cells were stimulated with CM and RANKL (50 ng/ml) for 2 days and mRNA levels of pro-(IL-1β, IL-6 and TNF-α) and anti-inflammatory (IL-10 and IL-4) cytokines were quantified by RT-qPCR. Data are presented as relative gene expression of gene of interest related to the reference gene HPRT-1. n=5 experiments, p-values are calculated by Mann-Whitney U test. E) Protein amounts of relevant cytokines in the supernatant. Cells were stimulated with CM and RANKL (50 ng/ml) for 2 days and protein concentration of pro-(IL-1α, IL-6, TNF-α and IFN-β) and anti-inflammatory (IL-10) cytokines were quantified in the supernatant by multi cytokine bead array (CBA; LegendPlex). Data are presented as absolute concentration (pg/ml). n=5 experiments, p-values are calculated by Mann-Whitney U test. F) Gene expression analysis of iNOS. Cells were stimulated with CM and RANKL (50 ng/ml) for 2 days and mRNA levels of iNOS were quantified by RT-qPCR. Data are presented as relative gene expression of gene of interest related to the reference gene HPRT-1. n=5 experiments, p-values are calculated by Mann-Whitney U test. G) NO release by macrophages. Cells were stimulated with CM and RANKL (50 ng/ml) for 2 days and NO content in the supernatant was quantified by Griess reaction. Data are presented as concentration (µM) calculated by OD at 540 nm. n=5 experiments, p-values are calculated by Mann-Whitney U test.

Article Snippet: Cells then were transferred into suitable well plate formats, treated with CM 1:1 diluted in fresh cell growth media, TLR-ligands (Pam3CSK4: 25 ng/ml and CpG ODN 1668: 25 ng/ml, both InvivoGen, USA), sodium L- or D-lactate (5, 10, 15 and 20 mM, both Sigma-Aldrich, Germany) or recombinant murine cytokines (IL-10: 25 ng/ml, Peprotech, USA; TNF-α: 25 ng/ml, eBioscience, Germany or IFN-β: 0.02, 0.2 and 2 ng/ml, BioLegend, USA) ± recombinant mouse RANKL (50 ng/ml, R&D systems, UK) and analyzed for immune function, signal transduction, metabolic activity and differentiation (OC / MGC formation) after respective time points.

Techniques: Staining, MANN-WHITNEY, Activity Assay, Negative Control, Activation Assay, Western Blot, Control, Gene Expression, Quantitative RT-PCR, Protein Concentration, Concentration Assay

Journal: bioRxiv

Article Title: Staphylococci planktonic and biofilm environments differentially affect macrophage immune activation and osteoclastogenic differentiation

doi: 10.1101/2021.07.26.453923

Figure Lengend Snippet: RAW264.7 cells were cultivated in growth media (DMEM high glucose + 10% FCS + 1% Pen/Strep) and effect of TLR-ligands (TLR-2: Pam3CSK4, P3 1 µg/ml, TLR-9: CpG 100 nM) or cytokines (IL-10 or TNF-α, both 25 ng/ml) ± RANKL stimulation on macrophage differentiation and late immune response was investigated. In experiments with a cultivation time above 2 days 50 µM β-mercapto ethanol was added to the media to reduce cell proliferation and avoid overgrowing of the cells (here: OC/MGC formation after 5 days). A+B) Cells were stimulated with different stimuli ± RANKL (50 ng/ml) for 5 days. At day 5 cells were fixed, stained for TRAP and nuclei and total numbers of formed osteoclasts (A) or MGCs (B) per well were counted. Osteoclasts were defined as TRAP-positive multinucleated giant cells with at least 3 nuclei. MGCs were defined as TRAP-negative multinucleated giant cells with at least 3 nuclei. Data are presented as OC / MGC numbers per well. n=3 experiments in duplicates, mean + SD are shown. C) Morphological appearance of formed OC and MGC in TRAP/nuclei stain after stimulation with CpG + RL for 5 days. D-F) Gene expression analysis of NFATc1 as osteoclastogenesis marker (D) and pro- (TNF-α, E) and anti- (IL-10, F) inflammatory cytokines. Cells were stimulated with different stimuli and RANKL (50 ng/ml) for 2 days and mRNA levels of NFATc1, TNF-α and IL-10 were quantified by RT-qPCR. Data are presented as relative gene expression of gene of interest related to the reference gene HPRT-1. n=3 experiments, mean + SD are shown. G) Activation of Stat3 signaling in macrophages. Cells were stimulated with different stimuli and RANKL (RL; 50 ng/ml) for 2 days and presence of phospho-Stat3 as activated form of the transcription factor was visualized by Western Blot. IL-10 stimulation (25 ng/ml for 1 hour) was used as positive control for Stat3-pathway activation. Β-Actin was used as loading control. n=3 experiments.

Article Snippet: Cells then were transferred into suitable well plate formats, treated with CM 1:1 diluted in fresh cell growth media, TLR-ligands (Pam3CSK4: 25 ng/ml and CpG ODN 1668: 25 ng/ml, both InvivoGen, USA), sodium L- or D-lactate (5, 10, 15 and 20 mM, both Sigma-Aldrich, Germany) or recombinant murine cytokines (IL-10: 25 ng/ml, Peprotech, USA; TNF-α: 25 ng/ml, eBioscience, Germany or IFN-β: 0.02, 0.2 and 2 ng/ml, BioLegend, USA) ± recombinant mouse RANKL (50 ng/ml, R&D systems, UK) and analyzed for immune function, signal transduction, metabolic activity and differentiation (OC / MGC formation) after respective time points.

Techniques: Staining, Gene Expression, Marker, Quantitative RT-PCR, Activation Assay, Western Blot, Positive Control, Control

Journal: Frontiers in Immunology

Article Title: Proinflammatory Differentiation of Macrophages Through Microparticles That Form Immune Complexes Leads to T- and B-Cell Activation in Systemic Autoimmune Diseases

doi: 10.3389/fimmu.2019.02058

Figure Lengend Snippet: MDM differentiated with MP-IC, and mainly with MP, induce the activation of autologous B cells from patients with RA. (A) From left to right: representative light microscopy pictures of MDM unstim alone; MDM unstim co-cultured with B cells; MDM differentiated in the presence of RMP or RMP-IC from patients with RA and co-cultured with B cells. (B) Representative histograms of CD80 expression on B cells from HC (top) and patients with RA (below) cultured alone (light blue) and with anti-BCR plus CD40L (yellow) or co-cultured with MDM differentiated without (Unstim, black) or with RMP (gray) and RMP-IC (green). Blue histograms represent the FMO control. (C) The frequency of CD80, CD86, CD69, and CD95 in B cells from patients with RA ( n = 7) and HC ( n = 6) co-cultured with MDM differentiated without (Unstim) or with RMP and RMP-IC. (D) The frequency of dead B cells (positive for LIVE-DEAD probe) from patients with RA ( n = 7) and HC ( n = 6) cultured alone (Unstim, in complete medium) and with anti-BCR plus CD40L (positive control) or co-cultured with MDM differentiated without (Unstim) or with RMP and RMP-IC. (E) BAFF and APRIL (Top panel) levels in supernatants of MDM from patients with RA ( n = 5) and HC ( n = 5) differentiated without (Unstim) or with RMP and RMP-IC. IgG and IgM (below panel) levels in supernatants from co-cultures of MDM differentiated with or without RMP and RMP-IC with autologous B cells from HC ( n = 5) and RA ( n = 5) patients. Comparisons among the groups were performed using ANOVA II and the Bonferroni post-hoc test.

Article Snippet: Recombinant human (rh) CD40 Ligand (CD40L), rhIFN-γ, and rhIL-4 were purchased from R&D Systems; rhIL-2 from Biolegend (San Diego, CA, USA) and the affinity-purified F(ab') 2 fragment anti-human IgM (anti-BCR) and F(ab') 2 anti-IgG fragment Alexa Fluor 488 conjugated from Jackson ImmunoResearch (New Baltimore, PA, USA).

Techniques: Activation Assay, Light Microscopy, Cell Culture, Expressing, Control, Positive Control

Journal: Frontiers in Immunology

Article Title: Proinflammatory Differentiation of Macrophages Through Microparticles That Form Immune Complexes Leads to T- and B-Cell Activation in Systemic Autoimmune Diseases

doi: 10.3389/fimmu.2019.02058

Figure Lengend Snippet: MDM differentiated with MP and MP-IC induce the activation and plasmablast differentiation of autologous LB from patients with SLE. (A) The frequency of CD80, CD86, CD69, and CD95 in B cells from patients with SLE ( n = 7) and HC ( n = 6) co-cultured with MDM differentiated without (Unstim) or with LMP and LMP-IC. (B) The frequency of dead B cells (positive for LIVE-DEAD probe) from patients with SLE ( n = 7) and HC ( n = 6) cultured alone (Unstim, in complete medium) and with anti-BCR plus CD40L (positive control) or co-cultured with MDM differentiated without (Unstim) or with LMP and LMP-IC. (C) BAFF and APRIL levels in the supernatants of MDM from patients with SLE ( n = 5) and HC ( n = 5) differentiated without (Unstim) or with LMP and LMP-IC. (D) Representative gating strategy to determine the frequency of plasmablasts after the co-culture of B cells with MDM differentiated without (Unstim) or with LMP and LMP-IC. (E) The frequency of plasmablasts from B cells cultured alone (Unstim, in complete medium), with anti-BCR plus CD40L (positive control), or co-cultured with autologous MDM from patients with SLE ( n = 7) and HC ( n = 6) differentiated without (Unstim) or with LMP and LMP-IC. (F) IgG and IgM levels in supernatants from the co-cultures of MDM differentiated with or without LMP and LMP-IC with autologous B cells from HC ( n = 5) and patients with SLE ( n = 5). Comparisons among the groups were performed using ANOVA II and the Bonferroni post-hoc test.

Article Snippet: Recombinant human (rh) CD40 Ligand (CD40L), rhIFN-γ, and rhIL-4 were purchased from R&D Systems; rhIL-2 from Biolegend (San Diego, CA, USA) and the affinity-purified F(ab') 2 fragment anti-human IgM (anti-BCR) and F(ab') 2 anti-IgG fragment Alexa Fluor 488 conjugated from Jackson ImmunoResearch (New Baltimore, PA, USA).

Techniques: Activation Assay, Cell Culture, Positive Control, Co-Culture Assay

Journal: International Journal of Molecular Sciences

Article Title: Anti-Inflammatory Mechanism of Neural Stem Cell Transplantation in Spinal Cord Injury

doi: 10.3390/ijms17091380

Figure Lengend Snippet: The effects of NSCs on the regulation of macrophage activation in vitro. ( A , B ) Mouse bone marrow-derived macrophages (BMDMs) were cultured in DMEM supplemented with 5% newborn calf serum and 15% L929 cell conditioned media for seven days in vitro. ( A ) A phase of the macrophage; ( B ) BMDMs expressed the macrophage marker F4/80 (green), and the nuclei were stained with DAPI (Blue); ( C ) BMDMs were cultured alone or co-cultured with NSCs by using transwells for 12 h, then incubated with 10 ng/mL IFN-γ for 12 h. Next, the mRNA levels of iNOS, TNF-α, IL-1β, IL-6 and IL-10 were detected by quantitative real-time PCR ( n = 6). Results are displayed as the mean ± SD. * p < 0.05; ( D ) BMDMs were treated with or without NSCs for 24 h and then were induced by 10 ng/mL IFN-γ for 24 h. The BMDMs’ supernatants were collected, and the production of TNF-α and IL-1β was examined by ELISA ( n = 6). Data are represented as the mean ± standard error. * p < 0.05. Scale bar = 20 μm.

Article Snippet: The mouse TNF-α ELISA kit (430901) and mouse IL-1β ELISA kit (432601) were from Biolegend (San Diego, CA, USA).

Techniques: Activation Assay, In Vitro, Derivative Assay, Cell Culture, Marker, Staining, Incubation, Real-time Polymerase Chain Reaction, Enzyme-linked Immunosorbent Assay