detection anti human il 17 antibodies  (R&D Systems)

Journal: Clinical and Translational Medicine

Article Title: Reciprocal crosstalk between Th17 and mesothelial cells promotes metastasis‐associated adhesion of ovarian cancer cells

doi: 10.1002/ctm2.1604

Figure Lengend Snippet: IL‐17A and TNF synergistically induce genes involved in mesenchymal transition of mesothelial cells. (A) Synergistically induced genes of mesothelial cells treated as in Figure (n = 5 patients). Jitter plot of the calculated additive and the actually observed induction (FC) by TNF+IL‐17A treatment. The plot shows genes with a ≥2‐fold ratio (= 1 log2 unit) of observed/expected median fold change. (B) Molecular Signature (MSigDB) hallmark pathway analysis. ⁵⁴ The bubble graph presents the 10 most significantly enriched pathways of synergistic candidate genes defined in (A) and the relative overlap with all genes of the respective pathway.(C) RT‐qPCR of EMT marker gene expression after treatment of mesothelial cells for 48 h with rhIL‐17A, rhTNF or both cytokines. Expression values were normalized to the untreated control (cntr). (D) qPCR analysis of selected EMT‐transcription factor genes performed as in panel B. Bar plots show the mean±SD of biological replicates (n = 5). *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001 were determined by by one‐way ANOVA followed by Dunnett's multiple comparison test; ns: not significant.

Article Snippet: After deparaffinization, rehydration and antigen retrieval at pH 6, the following antibodies were used for double staining: anti‐hCD4 (Agilent, IR649), anti‐hRORγt (LS‐B4659‐100, Biozol, Eiching, Germany), and anti‐hIL‐17A (MAB3171, Biotechne, Minneapolis, USA).

Techniques: Quantitative RT-PCR, Marker, Expressing, Comparison

Journal: Clinical and Translational Medicine

Article Title: Reciprocal crosstalk between Th17 and mesothelial cells promotes metastasis‐associated adhesion of ovarian cancer cells

doi: 10.1002/ctm2.1604

Figure Lengend Snippet: Clinical associations of Th17 cells with OC. (A) Kaplan‐Meier curves showing the association of IL17A gene expression with overall survival of OC. The analysis was performed using the KM‐Plotter database ⁵⁵ with auto‐selected cut‐offs for n = 373 patients (RNA‐Seq data for OC in the pan‐cancer dataset). HR: hazard ratio. (B) SOMAscan signals for IL‐17A and TNF in OC ascites (n = 70), matched plasma samples from OC‐patients (OC‐plasma; n = 20) and plasma from healthy donors (HD‐plasma; n = 10). Boxplots show the mean, minimum, maximum and quantiles; data points represent different patients. ****p<0.0001 determined by Kruskal‐Wallis test. (C) Quantification by flow cytometry of IL‐17A + /TNF + double‐positive cells in CD14‐depleted OC ascites cells (n = 9) among CD4 + , CD8 + , γδ, and MAIT cells. (D) Analysis as in panel C for matched OC PBMCs (n = 9). (E) Representative immunostaining of an omental micrometastasis for CD4 (green) and IL‐17A (brown). (F) Representative pictures showing immunostaining of CD4 (green) and IL‐17A (brown) in tumor‐free omentum, tumor‐distant omentum and tumor tissue in omental micrometastases. Arrows indicate CD4 + /IL‐17A + double‐positive cells. (G) Quantification of CD4 + /IL‐17A + double‐positive cells in tumor‐free omentum (n = 5), tumor‐distant omentum (n = 8) and tumor tissue (n = 8). Bar plots in panels C, D and G indicate the mean±SD of biological replicates. ***p<0.001, ****p<0.0001 determined by one‐way ANOVA followed by Dunnett's multiple comparison test. ns: not significant.

Article Snippet: After deparaffinization, rehydration and antigen retrieval at pH 6, the following antibodies were used for double staining: anti‐hCD4 (Agilent, IR649), anti‐hRORγt (LS‐B4659‐100, Biozol, Eiching, Germany), and anti‐hIL‐17A (MAB3171, Biotechne, Minneapolis, USA).

Techniques: Expressing, RNA Sequencing Assay, Flow Cytometry, Immunostaining, Comparison

Journal: Clinical and Translational Medicine

Article Title: Reciprocal crosstalk between Th17 and mesothelial cells promotes metastasis‐associated adhesion of ovarian cancer cells

doi: 10.1002/ctm2.1604

Figure Lengend Snippet: IL‐17A and TNF alter the transcriptome of mesothelial cells towards an inflammatory and mesenchymal phenotype. (A) Mesothelial cells from 5 patients were treated for 48 h with rhIL‐17A, rhTNF or both cytokines prior to bulk RNA‐Seq analysis. (B) Principal component analysis of RNA‐Seq data from mesothelial cells treated as described in (A). Colors: treatment conditions; shapes: patients (n = 5); colored circles: manually added highlights of groups separated by conditions (pc1). (C) Heatmap of differential gene expression (FC relative to untreated cells; n = 5). (D) Venn diagram showing the numbers of differentially regulated genes for different treatment conditions. (E) Molecular Signature (MSigDB) hallmark pathway analysis. ⁵⁴ The bubble graph displays the 10 most significantly enriched pathways for each treatment and the relative overlap with all genes of the respective pathway.

Article Snippet: After deparaffinization, rehydration and antigen retrieval at pH 6, the following antibodies were used for double staining: anti‐hCD4 (Agilent, IR649), anti‐hRORγt (LS‐B4659‐100, Biozol, Eiching, Germany), and anti‐hIL‐17A (MAB3171, Biotechne, Minneapolis, USA).

Techniques: RNA Sequencing Assay, Expressing

Journal: Clinical and Translational Medicine

Article Title: Reciprocal crosstalk between Th17 and mesothelial cells promotes metastasis‐associated adhesion of ovarian cancer cells

doi: 10.1002/ctm2.1604

Figure Lengend Snippet: IL‐17A‐ and TNF‐induced transcriptional changes can be identified in a subset of mesothelial cells from OC patients. (A) Mesothelial cells were enriched from tumor‐free omentum by MACS depletion of EpCAM + and CD45 + cells prior to targeted scRNA‐Seq. (B) Two‐dimensional embedding of scRNA‐Seq data by t‐SNE. Cells were clustered via Louvain and annotated to cell type using the marker genes listed in the Methods and Materials section. The bar chart shows distribution of cell types in the enriched cell population (see also Figure ). (C) Two‐dimensional embedding (UMAP) and Louvain clustering (filtered as described in Methods and Materials) of mesothelial cells identified in panel B. (D) Number IL‐17A/TNF‐induced genes expressed in different clusters. Across all clusters, expression of 67 genes was detectable out of 85 genes included in the scRNA‐Seq analysis. Embedding was performed as in panel C. (E) Visualization of fibroblast (DCN, FBLN1, MMP2) and mesothelial (ITLN1) marker gene expression in the Louvain clusters identified in panel C. (F) Heatmap of IL‐17A+TNF induced genes expressed in Louvain clusters from panel C (filtered for genes expressed in at least 20% of the cells in at least one cluster).

Article Snippet: After deparaffinization, rehydration and antigen retrieval at pH 6, the following antibodies were used for double staining: anti‐hCD4 (Agilent, IR649), anti‐hRORγt (LS‐B4659‐100, Biozol, Eiching, Germany), and anti‐hIL‐17A (MAB3171, Biotechne, Minneapolis, USA).

Techniques: Marker, Expressing

Journal: Clinical and Translational Medicine

Article Title: Reciprocal crosstalk between Th17 and mesothelial cells promotes metastasis‐associated adhesion of ovarian cancer cells

doi: 10.1002/ctm2.1604

Figure Lengend Snippet: IL‐17A and TNF perturb mesothelial monolayer integrity and promote adhesion of tumor cells. (A) Phalloidin staining of mesothelial cells treated with rhIL‐17A, rhTNF or both cytokines for 96 h. Medium and treatment were renewed after 48 h. Representative pictures are shown. (B, C) Quantification of ellipticity (B; Imaris software) and sphericity (C; ImageJ software) of experiments shown in panel A. Four random images were analyzed for each treatment condition and n = 4 patients. (D) ZO‐1 (green) and Hoechst (blue) staining of mesothelial cells treated as in panel A. The pictures are representative of three experiments with cells from different patients. (E) Quantification of ZO‐1 area at cell‐cell contacts with ImageJ software and the Labkit plugin. Three random images for each treatment condition were analyzed and n = 3 patients. (F) Schematic overview of the experimental design of the tumor cell adhesion assay. Mesothelial cells were seeded on 96‐well plates coated with collagen I, stimulated with rhIL‐17, rhTNF or both cytokines, and cultured for 4–5 days till formation of a dense monolayer. Subsequently, wells were washed and CellTracker Green‐stained tumor cells were added for 6 h. (G) Pictures of adherent OC‐tumor cells treated as described in panel F. Representative images are shown. (H) Quantification of adhesion assays as in panel G. Attached tumor cells were quantified by measuring the relative area covered by fluorescently labeled tumor cells using ImageJ software (n = 3 patients). The bar plots show the mean ±SD for biological replicates.*p<0.05, **p<0.01, ****p<0.0001 were determined in panels B, E and H by one‐way ANOVA followed by Dunnett's multiple comparison test, in panel C by Kruskal‐Wallis test followed by Dunn's multiple comparison test: ns: not significant.

Article Snippet: After deparaffinization, rehydration and antigen retrieval at pH 6, the following antibodies were used for double staining: anti‐hCD4 (Agilent, IR649), anti‐hRORγt (LS‐B4659‐100, Biozol, Eiching, Germany), and anti‐hIL‐17A (MAB3171, Biotechne, Minneapolis, USA).

Techniques: Staining, Software, Cell Adhesion Assay, Cell Culture, Labeling, Comparison

Journal: Clinical and Translational Medicine

Article Title: Reciprocal crosstalk between Th17 and mesothelial cells promotes metastasis‐associated adhesion of ovarian cancer cells

doi: 10.1002/ctm2.1604

Figure Lengend Snippet: IL‐17A and TNF synergistically direct the secretome of mesothelial cells towards a Th17‐promoting environment. (A) Affinity proteomics analysis of the mesothelial cell secretome after 48 h treatment with rhIL‐17A, rhTNF or both cytokines. (B) Heatmap showing the top 50 significantly upregulated proteins after combined treatment (p <0.05 by paired t test; synergism defined as FC of combined treatment > added individual FC values x 1.5). FC values were protein‐wise normalized. (C) Bubble graph of synergistically induced proteins. Relative expression normalized to combined IL‐17A/TNF treatment is shown. (D) Schematic overview of the experimental design. CM from mesothelial cells treated with rhIL‐17A and rhTNF and from untreated cells were collected after 24 h. CD4 + cells from PBMCs were treated with CM for 8 days. Induction of Th17 was measured by flow cytometry. (E) Representative staining of CD4 + cells after treatment with CM from untreated compared to IL‐17A/TNF‐treated mesothelial cells. (F) Increase (FC) of IL‐17A + /IFNγ − cell frequency after treatment as in panel E. Bars show the mean ±SD of n = 11 biological replicates. ***p<0.001 was determined by two‐tailed unpaired t‐test.

Article Snippet: After deparaffinization, rehydration and antigen retrieval at pH 6, the following antibodies were used for double staining: anti‐hCD4 (Agilent, IR649), anti‐hRORγt (LS‐B4659‐100, Biozol, Eiching, Germany), and anti‐hIL‐17A (MAB3171, Biotechne, Minneapolis, USA).

Techniques: Expressing, Flow Cytometry, Staining, Two Tailed Test

Journal: Frontiers in Immunology

Article Title: Dual targeting of mTOR/IL-17A and autophagy by fisetin alleviates psoriasis-like skin inflammation

doi: 10.3389/fimmu.2022.1075804

Figure Lengend Snippet: Fisetin treatment modulates several targets and signaling pathways in IL-17A/TNFα-stimulated primary keratinocytes. (A) Venn diagram showing the genes that were differentially regulated with >2-fold increase (shown as upward arrow) or decrease (downward arrow) from each comparison across the different groups of treatment and activation with cytokines in keratinocytes. (B) Metascape analysis at systems level of transcriptome profiles of 3 groups in circus plot: Activated vs Control (green), Activated + Fisetin vs Activated (red) and Activated + Rapamycin vs Activated (blue) in vitro . The plot shows how 1.5-fold changes differentially expressed genes are overlapped: Each arc represents as individual gene. Dark orange color represents the genes shared by multiple groups which forms purple link. On the contrary, the light orange arc represents unique gene to that list from the group. Light blue color represents different genes but shared ontology terms. There is a trend to see more common functional overlap that means subset of genes of the same biological process. (C) Differential enrichment analysis illustrates the transcriptomic enrichment analysis of genes involved in different important ontology terms (GO/KEGG) in cytokine-activated keratinocytes compared with control represented by circles. Enrichment study of GO/KEGG and hall mark gene sets. The overlap between the input gene lists increases with the thickness of pink linkages. The detailed enrichment study is shown in Figure S1 online. (D) All input gene lists were also merged into one list and resulted in a PPI (Protein-Protein Interaction) network. Network nodes are displayed as pies. Color code for pie sector represents a gene list and is consistent with the colors used for Activated vs Control (green), Activated + Fisetin vs Activated (red) and Activated + Rapamycin vs Activated (blue). To identify neighborhoods where proteins are densely connected, the MCODE algorithm was used to this network with biological meaning, which shows that a cytokine mediated signaling, cellular response to cytokine stimulus and autophagosome organization are related to transcriptome expression in the treatment group differentially. (E) The heatmap shows differential expression of mTOR pathway components and autophagy related transcriptome in each replicate of 3 groups. Overlaps at the gene level, where purple curves link identical genes in three groups, including the shared term level, where blue curves link genes that belong to the same enriched ontology term are shown in a circular 3 plot (C) .

Article Snippet: The coating antibody was anti-human IL- 17A (clone 41809.111; R&D Systems) and the detection antibody was biotinylated polyclonal goat anti-human IL-17A (R&D Systems).

Techniques: Protein-Protein interactions, Comparison, Activation Assay, Control, In Vitro, Functional Assay, Expressing, Quantitative Proteomics

Journal: Frontiers in Immunology

Article Title: Dual targeting of mTOR/IL-17A and autophagy by fisetin alleviates psoriasis-like skin inflammation

doi: 10.3389/fimmu.2022.1075804

Figure Lengend Snippet: Fisetin suppresses mTOR kinase activity and reduces phosphorylated mTOR pathway components in cytokine-activated human epidermal keratinocytes. (A) ELISA analysis of mTOR kinase activity in control, fisetin-treated, or cytokine [rhIL-6/IL-22 (15ng/ml each)]-treated primary keratinocytes lysates. (B) Western blot analysis of differential protein expression levels of p-Akt Ser473 , p-AMPK (Thr 172 )?, p-mTOR Ser2448 , and p-raptor (Ser 792 ) in fisetin (15µM) treated, rapamycin (100nM) or (IL-17A+TNFα)-activated HEKa compared with untreated control cells. (C) Graphs of the semi-quantified intensity of bands for p-Akt, p-AMPK, p-mTOR, and p-raptor. Bar represents means ± SD of three separate experiments each performed in triplicate. *P < 0.05 , **P < 0.01 , ***P < 0.001 , and ****P < 0.0001 vs. control for IL-6/IL-22-treated only or vs. IL-22 or IL-17A+TNFα for fisetin-treated cells. ns, not significant.

Article Snippet: The coating antibody was anti-human IL- 17A (clone 41809.111; R&D Systems) and the detection antibody was biotinylated polyclonal goat anti-human IL-17A (R&D Systems).

Techniques: Activity Assay, Enzyme-linked Immunosorbent Assay, Control, Western Blot, Expressing

Journal: Frontiers in Immunology

Article Title: Dual targeting of mTOR/IL-17A and autophagy by fisetin alleviates psoriasis-like skin inflammation

doi: 10.3389/fimmu.2022.1075804

Figure Lengend Snippet: Fisetin induces the expression of autophagy markers in cytokine-stimulated primary epidermal keratinocytes. (A) Representative photographs of the immunofluorescence staining for actin (red) and LC3A/B (green) and the DAPI staining (blue; for nucleus) in fisetin (15 µM), rapamycin (100 nM) or IL-17A+TNFa)-treated HEKa compared with inactivated control keratinocytes/HEKa. (B) Western blot analysis for differential protein levels of autophagy markers Atg5, Beclin1 and SQSTM1 in fisetin- or rapamycin-treated primary keratinocytes activated with cytokines (IL-17A+TNFα) in vitro . (C) Semi-quantification of the bands for LC3A/B, Atg5, and Beclin1 and SQSTM1. Bar is means ± SD of three separate experiments. *P <0.05, **P < 0.01, and ***P < 0.001 vs. control for activated (IL-17A+TNFα) only and activated with keratinocytes treated with fisetin or rapamycin. When contrasting the treatment groups for fisetin and rapamycin, the # is used. * is used when we compared with the activated group. ns, not significant.

Article Snippet: The coating antibody was anti-human IL- 17A (clone 41809.111; R&D Systems) and the detection antibody was biotinylated polyclonal goat anti-human IL-17A (R&D Systems).

Techniques: Expressing, Immunofluorescence, Staining, Control, Western Blot, In Vitro

Journal: Frontiers in Immunology

Article Title: Dual targeting of mTOR/IL-17A and autophagy by fisetin alleviates psoriasis-like skin inflammation

doi: 10.3389/fimmu.2022.1075804

Figure Lengend Snippet: Fisetin reduces IL-17A production by activated CD4 + T lymphocyte alone and when co-cultured with keratinocytes in vitro. (A) Binding affinity of fisetin to IL-17A, predicted using Autodock vina and interaction of fisetin in binding pocket of IL-17A shown in stick and mesh model. (B) This interaction involves hydrophilic and hydrophobic interactions with different residues shown in 2D layout. ELISA assay for the levels of IL-17A secretion by anti-CD3/anti-CD28 activated purified human CD4+ T lymphocytes under different conditions: (C) anti-CD3/anti-CD28 activated CD4+ T lymphocytes cultured alone and then primed with IL-1β and IL-23 to enhance IL-17A production in the presence or absence of fisetin. (D) anti-CD3/anti-CD28 activated CD4+ T lymphocytes co-cultured with NHEKs in the presence or absence of fisetin (10 µM) or Vit-D3 (0.1 µM). Cells were cultured for 48 h in the indicated conditions and secreted IL-17A in the condition culture media was assayed by ELISA. Bar represents Means ± SE resulted from at least three separate experiments and paired t test was used to compare values between treatments. **P <0.01 and ***P <0.001 (–). indicates non-activated or treated cells. ns, not significant.

Article Snippet: The coating antibody was anti-human IL- 17A (clone 41809.111; R&D Systems) and the detection antibody was biotinylated polyclonal goat anti-human IL-17A (R&D Systems).

Techniques: Cell Culture, In Vitro, Binding Assay, Enzyme-linked Immunosorbent Assay, Purification

Journal: Frontiers in Immunology

Article Title: Dual targeting of mTOR/IL-17A and autophagy by fisetin alleviates psoriasis-like skin inflammation

doi: 10.3389/fimmu.2022.1075804

Figure Lengend Snippet: Fisetin normalizes and enhances the expression of differentiation marker loricrin, and reduces the expression of IL-17 and the infiltration of F4/80+ and pan-CD45+ immune cells in IMQ mice skin lesions. (A) Immunofluorescence staining of loricrin (green) and macrophage cell activation marker F4/80 (red) in vehicle-, IMQ-, and fisetin+IMQ or and rapamycin+IMQ-treated mouse skin. (B) Quantification of the fluorescent intensity of loricrin and F4/80+ cells per 20× field view. (C) Immunofluorescence staining of IL-17 (green) and pan CD45+ T lymphocytes (red) in vehicle-, IMQ, fisetin+IMQ-treated, and rapamycin+IMQ-treated mouse skin. DNA in nuclei was counterstained with DAPI (blue). The merged image shows colocalization of IL-17 with some CD45+ cells, which delineates IL-17 + CD45 + T lymphocytes especially in IMQ-treated mice. (D) Quantification of the fluorescent intensity of IL-17 (green) and pan CD45+ T lymphocytes (red) per 20× field view. Shown in bar graph are mean± SD, and paired t test was used to compare values between different treatments, average reading from each group (n=6). *P <0.05 and **P <0.01 . (-) indicates control group and ns indicates non-significant.

Article Snippet: The coating antibody was anti-human IL- 17A (clone 41809.111; R&D Systems) and the detection antibody was biotinylated polyclonal goat anti-human IL-17A (R&D Systems).

Techniques: Expressing, Marker, Immunofluorescence, Staining, Activation Assay, Control

Journal: Frontiers in Immunology

Article Title: Dual targeting of mTOR/IL-17A and autophagy by fisetin alleviates psoriasis-like skin inflammation

doi: 10.3389/fimmu.2022.1075804

Figure Lengend Snippet: List of immune mediators that are significantly modulated upon treatment of IMQ-induced mice skin lesions with fisetin or rapamycin. Each group of mice had 6 mice (n=6) and control group was n=5.

Article Snippet: The coating antibody was anti-human IL- 17A (clone 41809.111; R&D Systems) and the detection antibody was biotinylated polyclonal goat anti-human IL-17A (R&D Systems).

Techniques: Control