il-17a neutralizing antibody clone 17f3  (Bio X Cell)

Journal: JCI Insight

Article Title: Targeting IL-17A/glucocorticoid synergy to CSF3 expression in neutrophilic airway diseases

doi: 10.1172/jci.insight.132836

Figure Lengend Snippet: (A) RNA-seq analysis of human ASMCs (hASMCs) untreated (UNT) and treated for 4 hours with IL-17A (100 ng/mL), DEX (1 μM), or both. Shown are representative DEX-sensitive and DEX-resistant genes. Z scores were calculated as log(FPKM) values. (B) Real-time PCR analysis of mRNA expression of indicated cytokines in hASMCs treated as indicated in A. (C) WT human CSF3 promoter luciferase plasmids were transfected into A549 cells. After 24 hours, the cells were treated as indicated in A for 6 hours, followed by luciferase assay. The luciferase activity is expressed as fold induction relative to untreated control. (D) mRNA degradation assay. hASMCs were pretreated with DEX for 4 hours, followed by treatment with actinomycin D (5 μg/mL) in the absence and presence of IL-17A (100 ng/mL). mRNA decay rate was measured as a ratio (percentage) of remaining mRNA at indicated time points to the amount of mRNA at time point 0. Data represent mean ± SEM (n = 3 biological replicates). *P < 0.05 (2-tailed Student’s t test). (E) Schematic representation of the WT and mutated human CSF3 luciferase reporter constructs. (F) NR3C1 binding sequence logo. (G) WT or mutant human CSF3 luciferase constructs were transfected into A549 cells, followed by luciferase assay as indicated in C. (H) ChIP analysis of DEX-induced binding of GR to the human CSF3 promoter. Nucleic extracts from untreated and treated hASMCs were immunoprecipitated with IgG or anti-GR, followed by DNA purification and RT-PCR quantitation with primers spanning the putative NR3C1 binding site of the CSF3 promoter. TS, transcription start site; GR, glucocorticoid receptor. For panels B, C, G, and H, data represent mean ± SD (n = 3 technical replicates). All data are representative of 3 independent experiments.

Article Snippet: Anti–IL-17A neutralizing antibody (clone 17F3), anti-Ly6G antibody (clone 1A8), and isotype control IgG2a (clone 2A3) were obtained from BioXCell.

Techniques: RNA Sequencing, Real-time Polymerase Chain Reaction, Expressing, Luciferase, Transfection, Activity Assay, Control, Degradation Assay, Construct, Binding Assay, Sequencing, Mutagenesis, Immunoprecipitation, DNA Purification, Reverse Transcription Polymerase Chain Reaction, Quantitation Assay

Journal: JCI Insight

Article Title: Targeting IL-17A/glucocorticoid synergy to CSF3 expression in neutrophilic airway diseases

doi: 10.1172/jci.insight.132836

Figure Lengend Snippet: (A) Eight-week-old WT C57BL/6 female mice (n = 5 per group) were subjected to the HDM-CFA acute severe asthma model. Isotype antibody (Control), DEX, anti-CSF3, and anti–IL-17A neutralizing antibodies or combined DEX and antibodies were administered to the mice (as described in Methods). Twenty-four hours after challenging, total cell and neutrophil counts in the BAL were quantified. (B) mRNA expression of lung tissues was quantified by real-time PCR. AU, fold induction relative to unchallenged control mice. Data represent mean ± SEM. One-way ANOVA was performed, followed by Tukey’s multiple-comparisons test. The multiplicity-adjusted P values were calculated for the indicated comparisons. The above data are representative of 3 independent experiments.

Article Snippet: Anti–IL-17A neutralizing antibody (clone 17F3), anti-Ly6G antibody (clone 1A8), and isotype control IgG2a (clone 2A3) were obtained from BioXCell.

Techniques: Control, Expressing, Real-time Polymerase Chain Reaction

Journal: JCI Insight

Article Title: Targeting IL-17A/glucocorticoid synergy to CSF3 expression in neutrophilic airway diseases

doi: 10.1172/jci.insight.132836

Figure Lengend Snippet: (A) Chemical structure of C3G. (B) Human and mouse ASMCs (hASMCs and mASMCs) were treated for 24 hours with IL-17A in the presence of DMSO (vehicle control) or the indicated concentrations of C3G. The concentrations of CXCL1 (GROα in humans and KC in mice) in the culture medium were measured by ELISA. Results are presented as a percentage of the amount of CXCL1 in the culture medium of DMSO-treated cells, which was set at 100%. Data are representative of 3 independent experiments. Error bars represent the SEM of technical replicates. (C and D) Eight-week-old WT C57BL/6 female mice (n = 5 per group) were subjected to the HDM-CFA acute asthma model. PBS (Control), DEX, C3G, or a combination of DEX and C3G were administered to the mice (as described in Methods). Twenty-four hours after challenging, total and neutrophil counts in the BAL were quantified (C). Representative BAL cells were prepared by cytospin and lung tissues were stained with H&E (D). All scale bars (red): 100 μm. (E) mRNA expression of lung tissues was quantified by real-time PCR. (F and G) OVA323–339-specific Th17 cells were adoptively transferred into 8-week-old female C57BL/6 mice (n = 5 mice per treatment group), which were treated as described in Methods. Twenty-four hours after the last challenge, total cell and neutrophil counts in the BAL were quantified. AU, fold induction relative to unchallenged control mice. Data represent mean ± SEM. One-way ANOVA was performed, followed by Tukey’s multiple-comparisons test. The multiplicity-adjusted P values were calculated for the indicated comparisons. The above data are representative of 3 independent experiments.

Article Snippet: Anti–IL-17A neutralizing antibody (clone 17F3), anti-Ly6G antibody (clone 1A8), and isotype control IgG2a (clone 2A3) were obtained from BioXCell.

Techniques: Control, Enzyme-linked Immunosorbent Assay, Staining, Expressing, Real-time Polymerase Chain Reaction

Journal: JCI Insight

Article Title: Targeting IL-17A/glucocorticoid synergy to CSF3 expression in neutrophilic airway diseases

doi: 10.1172/jci.insight.132836

Figure Lengend Snippet: (A) Ten-week-old female SMA-specific (Acta2-specific) Il17rc-deficient mice (SMA-Il17rcfl/fl) and control mice (SMA-Il17rcfl/+) were subjected to the HDM-CFA type-17 acute severe asthma mode (n = 5 mice per group). Twenty-four hours after challenging, total and neutrophil counts in the BAL were quantified. Data represent mean ± SEM. Statistical analysis was performed using 2-tailed Student’s t test. (B) Representative BAL cells were prepared by cytospin and lung tissues were stained with H&E. All scale bars (red): 100 μm. (C) Lung cells were stained with anti-CD45, anti-CD11b, and anti-Ly6G antibodies, followed by TUNEL assay using biotinylated dNTP and streptavidin-FITC. The apoptotic neutrophils were gated as CD45+CD11b+Ly6G+FITC+ cells. Representative dot blots from 2 independent experiments are shown. (D) BM-derived neutrophils were cultured in the presence of the conditioned media collected from mouse ASMCs treated for 24 hours as indicated: IL-17A (100 ng/mL), DEX (1 μM), C3G (100 μM), anti-CSF3 antibody (0.5 μg/mL). Flow cytometry gating live neutrophils as annexin V negative and propidium iodide negative. (E) BM-derived neutrophils were loaded into the upper chamber in Transwells, and the conditioned media collected from mouse ASMCs treated for 24 hours as indicated were added to the bottom chamber. After 6 hours, the number of migrating neutrophils was counted. For D and E, data represent mean ± SD (n = 3 technical replicates). The above data are representative of 3 independent experiments.

Article Snippet: Anti–IL-17A neutralizing antibody (clone 17F3), anti-Ly6G antibody (clone 1A8), and isotype control IgG2a (clone 2A3) were obtained from BioXCell.

Techniques: Control, Staining, TUNEL Assay, Derivative Assay, Cell Culture, Flow Cytometry

Journal: JCI Insight

Article Title: Targeting IL-17A/glucocorticoid synergy to CSF3 expression in neutrophilic airway diseases

doi: 10.1172/jci.insight.132836

Figure Lengend Snippet: In IL-17A–mediated neutrophilic airway diseases, IL-17A is upregulated and induces neutrophil-migrating chemokines (CXCL1 and CXCL2) and survival cytokine (CSF3) to elicit neutrophilic inflammation in the lung. DEX treatment can partially reduce expression of CXCL1 and CXCL2 but greatly induce CSF3 expression in synergy with IL-17A and/or other cytokines (e.g., TNF). The dramatically increased neutrophil-survival potential counteracts the reduced neutrophil-migrating potential caused by DEX treatment, which is often not beneficial in neutrophilic inflammation and sometimes may worsen the disease pathology. In the presence of IL-17A blockers (anti–IL-17A Ab or C3G), IL-17A–induced neutrophil-migrating CXCL1 and CXCL2 were further reduced and the synergistic effect between IL-17A and DEX on CSF3 was disrupted so that the DEX-mediated neutrophil-promoting effect was minimized. Neu, neutrophil; red arrow, increase; +, promoting; –, inhibiting.

Article Snippet: Anti–IL-17A neutralizing antibody (clone 17F3), anti-Ly6G antibody (clone 1A8), and isotype control IgG2a (clone 2A3) were obtained from BioXCell.

Techniques: Expressing