Journal: PLoS ONE

Article Title: Distinct Contributions of TNF Receptor 1 and 2 to TNF-Induced Glomerular Inflammation in Mice

doi: 10.1371/journal.pone.0068167

Figure Lengend Snippet: A: Compartment-specific expression of TNFR1 and TNFR2 mRNA in glomeruli and tubulointerstitial tissue isolated from normal mouse kidney as analyzed by quantitative real-time PCR. Values were normalized to rRNA expression used as reference gene. ∗ p<0.05. B: Immunohistochemistry on frozen sections for TNFR1 and TNFR2 protein demonstrates a prominent glomerular expression of both receptors (black color product) in normal mouse kidney. Original magnification x200.

Article Snippet: Protein expression of renal TNFR1 and TNFR2 was detected on 4 µm cryostat sections of OCT-embedded tissue stained with polyclonal goat anti-mouse TNFR1 and polyclonal goat anti-mouse TNFR2 antibodies (both from R&D Systems, Wiesbaden, Germany).

Techniques: Expressing, Isolation, Real-time Polymerase Chain Reaction, Immunohistochemistry

Journal: PLoS ONE

Article Title: Distinct Contributions of TNF Receptor 1 and 2 to TNF-Induced Glomerular Inflammation in Mice

doi: 10.1371/journal.pone.0068167

Figure Lengend Snippet: A: Expression of TNFR1 mRNA in unstimulated, TNF-stimulated and IFN-γ-stimulated glomerular endothelial cells. Cells were stimulated with indicated concentrations of TNF and/or IFN-γ for 5 hours. B: Expression of TNFR2 mRNA in unstimulated, TNF-stimulated and IFN-γ-stimulated glomerular endothelial cells. Cells were stimulated as in A. C: Expression of TNFR1 mRNA in unstimulated, TNF-stimulated and IFN-γ-stimulated mesangial cells. Cells were stimulated with indicated concentrations of TNF and/or IFN-γ for 24 hours. D: Expression of TNFR2 mRNA in unstimulated, TNF-stimulated and IFN-γ-stimulated glomerular endothelial cells. Cells were stimulated as in C. mRNA expression levels were analyzed by quantitative real-time PCR. Values were normalized to GAPDH expression used as reference gene. ∗ p<0.05, ∗∗ <0.01 ( A-D ). E: TNFR1 surface expression in unstimulated, TNF-stimulated and IFN-γ-stimulated glomerular endothelial cells in vitro analyzed by flow cytometry. Cells were stimulated with indicated concentrations of TNF and/or IFN-γ for 5 hours. F: TNFR2 surface expression in unstimulated, TNF-stimulated and IFN-γ-stimulated glomerular endothelial cells. Cells were stimulated as in E. G: Flow cytometric analysis of TNFR1 surface expression in unstimulated, TNF-stimulated and IFN-γ-stimulated mesangial cells in vitro. Cells were treated with indicated concentrations of TNF and IFN-γ for 24 hours. H: TNFR2 surface expression in unstimulated, TNF-stimulated and IFN-γ-stimulated mesangial cells. Cells were stimulated as in G. The percentage of TNFR-positive cells was determined in comparison to the respective isotype controls. ∗ p<0.05 versus medium control ( E-H ). Data are mean and SD of 3 to 6 independent experiments per group.

Article Snippet: Protein expression of renal TNFR1 and TNFR2 was detected on 4 µm cryostat sections of OCT-embedded tissue stained with polyclonal goat anti-mouse TNFR1 and polyclonal goat anti-mouse TNFR2 antibodies (both from R&D Systems, Wiesbaden, Germany).

Techniques: Expressing, Real-time Polymerase Chain Reaction, In Vitro, Flow Cytometry, Comparison

Journal: PLoS ONE

Article Title: Distinct Contributions of TNF Receptor 1 and 2 to TNF-Induced Glomerular Inflammation in Mice

doi: 10.1371/journal.pone.0068167

Figure Lengend Snippet: Expression profiling by microarrays was performed on isolated glomeruli from wildtype, Tnfr1,2 −/−, Tnfr1 −/− and Tnfr2 −/− mice after stimulation with 50 ng/ml TNF for 12 hours ex vivo. A: Significance analysis using SAM detected differential regulation of 290 unique genes in Tnfr1,2 −/− glomeruli with at least 1.5-fold difference compared to wildtype (black data points). 219 genes were down-regulated (upper left), and 71 genes were up-regulated (lower right). B: In Tnfr1 −/− mice 219 differentially regulated genes were identified, with lower expression of 159 genes and increased expression of 60 genes. C: In Tnfr2 -deficient glomeruli only 5 genes were differentially expressed, all of them with decreased expression compared to wildtype. In all three genotypes, expression of the majority of genes with fluorescence signals above background level did not significantly differ from wildtype (grey data points). Data represent the mean log2 value of fluorescence signals from three independent experiments per genotype.

Article Snippet: Protein expression of renal TNFR1 and TNFR2 was detected on 4 µm cryostat sections of OCT-embedded tissue stained with polyclonal goat anti-mouse TNFR1 and polyclonal goat anti-mouse TNFR2 antibodies (both from R&D Systems, Wiesbaden, Germany).

Techniques: Expressing, Isolation, Ex Vivo, Fluorescence

Journal: PLoS ONE

Article Title: Distinct Contributions of TNF Receptor 1 and 2 to TNF-Induced Glomerular Inflammation in Mice

doi: 10.1371/journal.pone.0068167

Figure Lengend Snippet: Expression profiling by microarrays was performed on isolated glomeruli from wildtype, Tnfr1 −/− and Tnfr2 −/− mice 12 after stimulation with 50 ng/ml TNF for 12 hours ex vivo. A negativ “fold change” (black to green) indicates decreased expression and a postive “fold change” (black to red) indicates increased expression in a glomerular sample compared with the average of all analyzed samples. A : Compared to wildtype 219 significantly regulated genes were found in Tnfr1 −/− glomeruli. The number of genes identified in Tnfr1 −/− glomeruli was lower than in Tnfr1,2 −/− glomeruli, but all genes differentially expressed in Tnfr1 −/− glomeruli were regulated similarly in Tnfr1,2 −/− glomeruli, although for some genes pair-wise comparison between Tnfr1,2 −/− to wildtype did not reach significance or was below the pre-defined 1.5-fold cut-off (see also ). B : 5 differentially expressed genes in Tnfr2 −/− glomeruli are listed according to fold-change values compared to wildtype. These genes are not differentially expressed in Tnfr1 −/− glomeruli, but 4 of the 5 genes are similarly regulated in Tnfr1,2−/− mice. ( Tnfr1 −/−) and S4 ( Tnfr2 −/−) list all differentially expressed genes with respective probe set IDs, gene bank accession numbers, gene names and fold-changes according to fold-change values compared to wildtype.

Article Snippet: Protein expression of renal TNFR1 and TNFR2 was detected on 4 µm cryostat sections of OCT-embedded tissue stained with polyclonal goat anti-mouse TNFR1 and polyclonal goat anti-mouse TNFR2 antibodies (both from R&D Systems, Wiesbaden, Germany).

Techniques: Expressing, Isolation, Ex Vivo, Comparison

Journal: PLoS ONE

Article Title: Distinct Contributions of TNF Receptor 1 and 2 to TNF-Induced Glomerular Inflammation in Mice

doi: 10.1371/journal.pone.0068167

Figure Lengend Snippet: Expression of proinflammatory chemokines and adhesion molecules in glomeruli isolated from control mice (black bars), Tnfr1 −/− (dark grey bars), Tnfr2 −/− (bright grey bars), and Tnfr1,2 −/− mice (white bars) was anlayzed after challenge with indicated concentrations of TNF in vitro for 12 or 24 hours. The mRNA levels of CCL2/MCP-1 ( A ), CCL5/RANTES ( B ), ICAM-1 ( C ), VCAM-1 ( D ), E-selectin ( E ), and P-selectin ( F ) were determined by quantitative real-time PCR, and values were normalized to 18S rRNA as reference gene. Data shown are means and SE of n = 3 to 4 per group; ∗ p<0.05 versus wildtype.

Article Snippet: Protein expression of renal TNFR1 and TNFR2 was detected on 4 µm cryostat sections of OCT-embedded tissue stained with polyclonal goat anti-mouse TNFR1 and polyclonal goat anti-mouse TNFR2 antibodies (both from R&D Systems, Wiesbaden, Germany).

Techniques: Expressing, Isolation, In Vitro, Real-time Polymerase Chain Reaction

Journal: PLoS ONE

Article Title: Distinct Contributions of TNF Receptor 1 and 2 to TNF-Induced Glomerular Inflammation in Mice

doi: 10.1371/journal.pone.0068167

Figure Lengend Snippet: Rab6B expression was analyzed in glomeruli isolated from control mice (black bars), Tnfr1 −/− (dark grey bars), Tnfr2 −/− (bright grey bars), and Tnfr1,2 −/− mice (white bars) after challenge with indicated concentrations of TNF in vitro for 12 or 24 hours. The mRNA levels of Rab6B were determined by quantitative real-time PCR, and values were normalized to 18S rRNA as reference gene. Data shown are means and SE of n = 3 per group; ∗ p<0.05, ∗∗ p<0.01 versus wildtype.

Article Snippet: Protein expression of renal TNFR1 and TNFR2 was detected on 4 µm cryostat sections of OCT-embedded tissue stained with polyclonal goat anti-mouse TNFR1 and polyclonal goat anti-mouse TNFR2 antibodies (both from R&D Systems, Wiesbaden, Germany).

Techniques: Expressing, Isolation, In Vitro, Real-time Polymerase Chain Reaction

Journal: PLoS ONE

Article Title: Distinct Contributions of TNF Receptor 1 and 2 to TNF-Induced Glomerular Inflammation in Mice

doi: 10.1371/journal.pone.0068167

Figure Lengend Snippet: pMCs were isolated from control mice (black bars), Tnfr1 −/− (dark grey bars), Tnfr2 −/− (bright grey bars), and Tnfr1,2 −/− mice (white bars) after stimulation with indicated concentrations of TNF in vitro for 12 hours. The mRNA levels of CCL2/MCP-1 ( A ), CCL5/RANTES ( B ), ICAM-1 ( C ), VCAM-1 ( D ), E-selectin ( E ), and P-selectin ( F ) were determined by quantitative real-time PCR, and values were normalized to 18S rRNA as reference gene. Data shown are means and SE of two independently performed experiments.

Article Snippet: Protein expression of renal TNFR1 and TNFR2 was detected on 4 µm cryostat sections of OCT-embedded tissue stained with polyclonal goat anti-mouse TNFR1 and polyclonal goat anti-mouse TNFR2 antibodies (both from R&D Systems, Wiesbaden, Germany).

Techniques: Isolation, In Vitro, Real-time Polymerase Chain Reaction

Journal: PLoS ONE

Article Title: Distinct Contributions of TNF Receptor 1 and 2 to TNF-Induced Glomerular Inflammation in Mice

doi: 10.1371/journal.pone.0068167

Figure Lengend Snippet: Concentrations of CCL2/MCP-1, CCL5/RANTES, and CXCL10/IP-10 were measured by ELISA in culture supernatants of wildtype, Tnfr1 −/−, Tnfr2 −/−, and Tnfr1,2 −/− glomeruli 12 hours after stimulation with 50 ng/ml of TNF. Data shown are means and SD of n = 3 per group; ∗ p<0.05 versus wildtype.

Article Snippet: Protein expression of renal TNFR1 and TNFR2 was detected on 4 µm cryostat sections of OCT-embedded tissue stained with polyclonal goat anti-mouse TNFR1 and polyclonal goat anti-mouse TNFR2 antibodies (both from R&D Systems, Wiesbaden, Germany).

Techniques: Enzyme-linked Immunosorbent Assay

Journal: PLoS ONE

Article Title: Distinct Contributions of TNF Receptor 1 and 2 to TNF-Induced Glomerular Inflammation in Mice

doi: 10.1371/journal.pone.0068167

Figure Lengend Snippet: Compartment-specific flow cytometry was performed on glomerular tissue prepared from wildtype, Tnfr1 −/−, Tnfr2 −/−, and Tnfr1,2 −/− mice 8 hours after intraperitoneal injection of 5 µg TNF. Leukocyte subpopulations were identified as follows: A: CD45 + leukocytes, B: CD45 + Ly6G + F4/80 − neutrophils, C: CD45 + F4/80 + Ly6G − mononuclear phagocytes, and D: CD45 + CD3ε + T cells. Leukocyte numbers are expressed as percentage of all glomerular cells. ∗ p<0.05, ∗∗ p<0.01 versus wildtype.

Article Snippet: Protein expression of renal TNFR1 and TNFR2 was detected on 4 µm cryostat sections of OCT-embedded tissue stained with polyclonal goat anti-mouse TNFR1 and polyclonal goat anti-mouse TNFR2 antibodies (both from R&D Systems, Wiesbaden, Germany).

Techniques: Flow Cytometry, Injection

Journal: bioRxiv

Article Title: Characterizing the polarization continuum of macrophage subtypes M1, M2a and M2c

doi: 10.1101/2022.06.13.495868

Figure Lengend Snippet: A and B) Flow cytometry analysis of dexamethasone treatment for 48 hours on M1 induced macrophages to determine plasticity of inflammatory macrophages. Percentage positive of population shown. Bar shows mean with each point representing individual samples (N=6, ns p>0.05), with a minimum of 10,000 events per sample, significance tested using a two-tailed t-test. A) Flow cytometry analysis included subtype specific markers in addition to markers of the inflammatory response as including: CD14, CD169, CD163, CD16, CD86, CD80, CD209 and CD206. B) Analysis included markers synonymous with COVID-19 infection and inflammation: CD119, CD210, CD192, ACE2, CD120a and CD121a. C) Heatmap visualization of proteomic comparison showing significantly up or downregulated proteins when comparing dexamethasone treated samples against M1 as a baseline.

Article Snippet: Primary antibodies used: CD14 VioBlue (BD Pharminogen, Cat: 558121), CD16 FITC (Miltenyi Biotec, Cat: 130-113-392), CD80 FITC (BioLegend, Cat: 305205), CD86 PE (BioLegend, Cat: 305405), CD119 FITC (Miltenyi Biotec, Cat: 130-099-929), CD120a APC (BioLegend, Cat: 369905), CD121a (BD Biosciences, Cat: 551388), CD163 PE (Miltenyi Biotec, Cat: 130-097628), CD169 APC (Miltenyi Biotec, Cat: 130-098-643), CD192 PE (Miltenyi Biotec, Cat: 130-118-477), CD206 APC (BD Pharminogen, Cat: 550889), CD209 (BioRad, Cat: MCA2318T), CD210 PE (Miltenyi Biotec, Cat: 130-121-426) and ACE2 (R&D Systems, Cat: AF-933-SP).

Techniques: Flow Cytometry, Two Tailed Test, Infection, Comparison

Journal: bioRxiv

Article Title: Characterizing the polarization continuum of macrophage subtypes M1, M2a and M2c

doi: 10.1101/2022.06.13.495868

Figure Lengend Snippet: A) Analysis of the presence of antibodies towards the Spike protein from COVID-19 infection in convalescent plasma samples (CP) and controls using an enzyme linked immunosorbent assay (ELISA). Graph shows the mean and standard deviation for the data (control N=8, CP N=7, **** p<0.0001), significance tested using a two-tailed unpaired t-test. B) Yield of CD14 + cells isolated as a percentage from PBMNCs is significantly reduced for convalescent plasma cones when compared to control samples. Scatter plot with the mean and standard deviation of the sample group ((N=6, * (p<0.05), significance tested using a two-tailed unpaired t-test. C) Representative histograms of flow cytometry analysis of the CD14 + isolated cells immediately after isolation using key macrophage markers, IgG control in grey, control in blue and CP samples in red. Histograms are representative of N=6 with a minimum of 10,000 events per sample. D) Surface markers used to investigate macrophage specific markers immediately after isolation for control and CP samples, including: CD14, CD169, CD163, CD16, CD86, CD80, CD209, CD206, CD119, CD210, CD192, ACE2, CD120a and CD121a. E) Median fluorescence intensity of flow cytometry markers tested in C). All flow cytometry experiments show the mean and standard deviation for the data (N=9, ns p≥0.05), with a minimum of 10,000 events per sample, significance tested using a two-tailed Mann-Whitney test.

Article Snippet: Primary antibodies used: CD14 VioBlue (BD Pharminogen, Cat: 558121), CD16 FITC (Miltenyi Biotec, Cat: 130-113-392), CD80 FITC (BioLegend, Cat: 305205), CD86 PE (BioLegend, Cat: 305405), CD119 FITC (Miltenyi Biotec, Cat: 130-099-929), CD120a APC (BioLegend, Cat: 369905), CD121a (BD Biosciences, Cat: 551388), CD163 PE (Miltenyi Biotec, Cat: 130-097628), CD169 APC (Miltenyi Biotec, Cat: 130-098-643), CD192 PE (Miltenyi Biotec, Cat: 130-118-477), CD206 APC (BD Pharminogen, Cat: 550889), CD209 (BioRad, Cat: MCA2318T), CD210 PE (Miltenyi Biotec, Cat: 130-121-426) and ACE2 (R&D Systems, Cat: AF-933-SP).

Techniques: Infection, Enzyme-linked Immunosorbent Assay, Standard Deviation, Two Tailed Test, Isolation, Flow Cytometry, Fluorescence, MANN-WHITNEY