Journal: International Journal of Cardiology

Article Title: Enhanced IL-17 signalling following myocardial ischaemia/reperfusion injury

doi: 10.1016/j.ijcard.2011.08.849

Figure Lengend Snippet: Primers sequences used for qPCR analysis.

Article Snippet: In a different set of experiments rats were treated with 200 μg of anti-IL-17A polyclonal antibody intraperitoneally (R & D Systems, AF-421-NA) or PBS 2 h before undergoing in vivo ischemia/reperfusion injury.

Techniques:

Journal: International Journal of Cardiology

Article Title: Enhanced IL-17 signalling following myocardial ischaemia/reperfusion injury

doi: 10.1016/j.ijcard.2011.08.849

Figure Lengend Snippet: IL-17A, IL-17F, IL-17 receptor and IL-17 target genes are transcriptionally upregulated following in vivo I/R injury. RNA and protein was extracted from the left ventricles of rats undergoing sham operation or 25 min ischaemia and 2 h reperfusion (I/R). (A) IL-17RA, IL-17A and IL-17F expression was measured by qPCR. (B) IL-17A and IL-17RA protein levels were assessed by western blot, GAPDH was used as a loading control. (C) Expression of the IL-17 target genes IL-6, IL-1, iNOS and MMP-9 was measured by qPCR.

Article Snippet: In a different set of experiments rats were treated with 200 μg of anti-IL-17A polyclonal antibody intraperitoneally (R & D Systems, AF-421-NA) or PBS 2 h before undergoing in vivo ischemia/reperfusion injury.

Techniques: In Vivo, Expressing, Western Blot, Control

Journal: International Journal of Cardiology

Article Title: Enhanced IL-17 signalling following myocardial ischaemia/reperfusion injury

doi: 10.1016/j.ijcard.2011.08.849

Figure Lengend Snippet: IL-17 induces Cxcl1, IL-6 and Socs3 expression in cardiac myocytes. Neonatal rat ventricular myocytes were treated with 10 ng/ml IL-17 for the indicated times and the levels of (A) Cxcl1, (B) IL-6, (C) MCP-1 and (D) Socs3 were measured by qPCR. Experiments were repeated in duplicate, statistical analysis was carried out using a one-way ANOVA followed by Dunnett's post test, *p < 0.05, **p < 0.01, ***p < 0.001. (E) I/R injury and IL-17 have additive effects on Cxcl1 expression. NRVMs were subjected to 4 h ischaemia and 6 h reperfusion with or without 10 ng/ml IL-17 which was added at the time of reperfusion. The expression of Cxcl1 was analysed by qPCR. Statistical analysis was carried out using student's t -test. **p < 0.01, **p < 0.001.

Article Snippet: In a different set of experiments rats were treated with 200 μg of anti-IL-17A polyclonal antibody intraperitoneally (R & D Systems, AF-421-NA) or PBS 2 h before undergoing in vivo ischemia/reperfusion injury.

Techniques: Expressing

Journal: International Journal of Cardiology

Article Title: Enhanced IL-17 signalling following myocardial ischaemia/reperfusion injury

doi: 10.1016/j.ijcard.2011.08.849

Figure Lengend Snippet: IL-17 mediated upregulation of Cxcl1 and IL-6 in cardiac myocytes is MAPK dependent. (A) NRVMs were treated with 10 ng/ml IL-17 for the indicated times and cell lysates were analysed by Western blot using the indicated antibodies. (B) NRVMs were pre-treated for 30 min with the indicated inhibitors followed by IL-17 stimulation for 6 h. The expression of Cxcl1 and IL-6 was measured by qPCR. Statistical analysis was carried out using a student's t -test. *p < 0.05, **p < 0.01, **p < 0.001.

Article Snippet: In a different set of experiments rats were treated with 200 μg of anti-IL-17A polyclonal antibody intraperitoneally (R & D Systems, AF-421-NA) or PBS 2 h before undergoing in vivo ischemia/reperfusion injury.

Techniques: Western Blot, Expressing

Journal: International Journal of Cardiology

Article Title: Enhanced IL-17 signalling following myocardial ischaemia/reperfusion injury

doi: 10.1016/j.ijcard.2011.08.849

Figure Lengend Snippet: IL-17 receptor and IL-17F are transcriptionally upregulated in cardiac myocytes. (A) NRVM were subjected to 4 h in vitro ischaemia and up to 24 h reperfusion in normal media after which the levels of IL-17 receptor and IL-17F were assessed by qPCR. (B) H9c2 cells were subjected to in vitro I/R for the indicated times and the levels of IL-17F and Cxcl1 were measured by qPCR. (C) NRVMs were treated with 200 μM H 2 O 2 for 6 h and the expression of the indicated genes was measured by qPCR. (D) NRMVs were transduced with STAT3C adenovirus at MOI = 100. After 48 h, increased expression of STAT3 was confirmed by Western blot and IL-17F expression was measured by qPCR. *p < 0.05, **p < 0.01, ***p < 0.001, student's t -test, n = 3 per group, repeated in duplicate.

Article Snippet: In a different set of experiments rats were treated with 200 μg of anti-IL-17A polyclonal antibody intraperitoneally (R & D Systems, AF-421-NA) or PBS 2 h before undergoing in vivo ischemia/reperfusion injury.

Techniques: In Vitro, Expressing, Transduction, Western Blot

Journal: International Journal of Cardiology

Article Title: Enhanced IL-17 signalling following myocardial ischaemia/reperfusion injury

doi: 10.1016/j.ijcard.2011.08.849

Figure Lengend Snippet: Blocking IL-17 signalling with a specific anti-IL-17 neutralizing antibody reduced creatine phosphokinase and apoptotic cell death in the myocardium following in vivo I/R injury. Plasma concentration of CPK (A) and apoptosis assessed by the TUNEL assay (B) in sham-operated (control) or rats exposed to in vivo I/R injury (I/R) or I/R plus treatment with IL-17 neutralizing antibody (IL-17 Bo Ab). Immunofluorescence data (left panel) (Data are mean of ± 6 rats **P < 0.001). Anti-IL-17 neutralizing antibody also reduced the increase in cleaved/active caspase-3 expression following in vivo I/R injury (C). Western blot analysis was performed on tissue lysates prepared as in Fig. 3 A above and immunoblotted with a specific antibody against the cleaved and active form of caspase-3 and GAPDH.

Article Snippet: In a different set of experiments rats were treated with 200 μg of anti-IL-17A polyclonal antibody intraperitoneally (R & D Systems, AF-421-NA) or PBS 2 h before undergoing in vivo ischemia/reperfusion injury.

Techniques: Blocking Assay, In Vivo, Clinical Proteomics, Concentration Assay, TUNEL Assay, Control, Immunofluorescence, Expressing, Western Blot

Journal: Immunity

Article Title: The Cytokine IL-17A Limits Th17 Pathogenicity via a Negative Feedback Loop Driven by Autocrine Induction of IL-24

doi: 10.1016/j.immuni.2020.06.022

Figure Lengend Snippet: Spontaneous Uveitis in IL-17A-Deficient Mice Develops in the Context of Increased Expression of Other Th17-Lineage Cytokines (A) Incidence of spontaneous uveitis in Il17a −/− R161H mice and their Il17a +/+ or − R161H littermates was measured. Incidence was determined by fundus examination (upper panel; at least 5 mice per group at weaning at 4 weeks and at least 15 mice per group for all other time points), disease score by histology of eyes collected between 11 and 18 weeks of age (lower panel; at least 4 mice per group at each time point), and representative images of the H&E staining of the retina (100× original magnification). (B) IL-17A-sufficient or IL-17A-deficient R161H cells were adoptively transferred into WT B10.RIII recipients. Data are combined from two experiments with at least 14 mice per point. Disease was scored at the indicated time points using fundoscopy. (C and D) Cells were obtained from the eye-draining lymph nodes (LNs) of IL-17A-sufficient or IL-17A-deficient CD90.2 R161H mice and were polarized under Th17 conditions with IRBP 161–180 peptide for 3 days and adoptively transferred into CD90.1 WT B10.RIII recipients. (C) Cytokine profiles of in vitro -polarized CD4 + T cells were analyzed using intracellular staining. Shown are representative fluorescence-activated cell sorting (FACS) plots (top) and compiled data (bottom) from four independent experiments. (D) Five days after adoptive transfer, eyes (6 or more) were pooled, and the cytokine profiles of eye-infiltrating donor (CD90.2) CD4 + T cells were determined using intracellular staining. Representative FCM plots (top) and compiled data (bottom) from three independent experiments. ∗ p < 0.05, Student’s t test. Data are depicted as mean ± SEM.

Article Snippet: Where specified, 10 μg/ml of anti-human IL-17A antibody or isotype control (R&D systems) was added to the cultures.

Techniques: Expressing, Staining, In Vitro, Fluorescence, FACS, Adoptive Transfer Assay

Journal: Immunity

Article Title: The Cytokine IL-17A Limits Th17 Pathogenicity via a Negative Feedback Loop Driven by Autocrine Induction of IL-24

doi: 10.1016/j.immuni.2020.06.022

Figure Lengend Snippet: IL-17A Inhibits Expression of Other Th17-Lineage Cytokines through the IL-17A Receptor Cells were obtained from the spleen of IL-17A-sufficient or IL-17A-deficient B10RIII. CD4 + CD62L + T cells were isolated and polarized under Th17 conditions with anti-CD3/CD28 antibodies for 3 days. (A) Cytokine profiles of polarized CD4 + T cells were analyzed using intracellular staining and shown as representative FCM plots (top) and compiled data (bottom) from three to five independent experiments. (B) Th17-polarized CD4 + cells were stained with IL-17RA or IL-17RC. Gray filled histogram, isotype control; red histogram, WT Th17 cells; blue histogram, Il17a −/− Th17 cells. Shown are representative histograms (top left plots) and compiled mean fluorescence intensity (MFI) data (top right plots) from four or five independent experiments. Their gene expression was also determined using real-time PCR. Data were compiled from three independent experiments (bottom graphs) and were normalized to GAPDH and expressed as relative to WT Th0 cells. (C) Th17-polarized WT CD4 + T cells with or without indicated isotype control, anti-IL-17RA, or anti-IL-17RC antibodies. Shown are representative FCM plots (left) and compiled data (right) from five independent experiments. (D) Th17-polarized CD4 + T cells were cultured with or without recombinant IL-17A. Shown are representative FCM plots (left) and compiled data (right) from four independent experiments. ∗ p < 0.05, Student’s t test (A and B) and one-way ANOVA (C and D). Data are depicted as mean ± SEM. See also and .

Article Snippet: Where specified, 10 μg/ml of anti-human IL-17A antibody or isotype control (R&D systems) was added to the cultures.

Techniques: Expressing, Isolation, Staining, Control, Fluorescence, Gene Expression, Real-time Polymerase Chain Reaction, Cell Culture, Recombinant

Journal: Immunity

Article Title: The Cytokine IL-17A Limits Th17 Pathogenicity via a Negative Feedback Loop Driven by Autocrine Induction of IL-24

doi: 10.1016/j.immuni.2020.06.022

Figure Lengend Snippet: IL-24 Is Expressed by Th17 Cells, and Its Expression Is Dependent on IL-17A (A) IL-24 expression in WT and Il17a −/− Th17 cells as determined using q-PCR (left, n = 3 or 4, relative to resting CD4 + CD62L + T cells) and ELISA (right, n = 4). (B) Kinetics of IL-24 expression over 3 days of polarization in WT Th17 cells were studied (n = 3, relative to resting CD4 + CD62L + T cells). (C) CD4 + CD62L + T cells from WT and Il17a −/− mice were polarized under Th17 conditions with anti-CD3/CD28 antibodies for 3 days with or without an anti-IL-17A antibody. Shown are representative FCM plots (left) and compiled data (right) from five independent experiments. (D) WT or Il17a −/− mice were immunized with IRBP 161–180 peptide. Cytokine profiles of eye-infiltrating CD4+ T cells were determined using intracellular staining. Representative data of two independent experiments with at least five mice per group. (E) Cells were obtained from the eye-draining LNs of IL-17A-sufficient or IL-17A-deficient CD90.2 R161H mice and were polarized under Th17 conditions with IRBP 161–180 peptide for 3 days. Cells were adoptively transferred into CD90.1 WT B10.RIII recipients. Five days after adoptive transfer, eyeballs (six or more) were pooled, and the cytokine profiles of eye-infiltrating CD90.2 + CD4 + T cells were determined using intracellular staining. Representative FCM plots (right) and compiled data (left) from three independent experiments. ∗ p < 0.05, Student’s t test (A and E) or one-way ANOVA (C and D). Data are depicted as mean ± SEM. See also Figure S3 .

Article Snippet: Where specified, 10 μg/ml of anti-human IL-17A antibody or isotype control (R&D systems) was added to the cultures.

Techniques: Expressing, Enzyme-linked Immunosorbent Assay, Staining, Adoptive Transfer Assay

Journal: Immunity

Article Title: The Cytokine IL-17A Limits Th17 Pathogenicity via a Negative Feedback Loop Driven by Autocrine Induction of IL-24

doi: 10.1016/j.immuni.2020.06.022

Figure Lengend Snippet: IL-24 Inhibits the Effector Functions of Th17 Cells (A–E) CD4 + CD62L + T cells from WT, Il17a −/− mice were isolated and polarized under Th17 conditions with anti-CD3/CD28 antibodies for 3 days with or without (A and B) recombinant IL-24 or (C) anti-IL-24 antibody or isotype control in WT. CD4 + CD62L + T cells were also isolated from Il24 −/− mice for Th17 polarization. (D) Il17a −/− Th17 cells were re-stimulated with anti-CD3/CD28 antibodies for 24 h with or without IL-24. The mRNA expression of SOCS1 and SOCS3 was determined using real-time PCR. (E) Il17a −/− Th17 cells were polarized in presence of different combinations of IL-24 and siRNAs; expression of IL-17F and GM-CSF was determined using flow cytometry. (F–H) Cells were obtained from the draining LNs of IL-17A-sufficient or IL-17A-deficient R161H mice and were polarized under Th17 conditions with IRBP 161–180 peptide for 3 days with Il24 siRNA or scrambled control. (F) The expression of IL-17F and GM-CSF was determined by intracellular cytokine staining. Stability of the knockdown was confirmed using RT-PCR (normalized to control siRNA). (G) Cells were polarized in the presence of control siRNA or Il24 siRNA and were transferred to naive WT recipients. (H) After the adoptive transfer, the recipient mice received recombinant IL-24 (intraperitoneal [i.p.] injection) every other day. In (A)–(F), data are combined from at least three independent experiments. In (G) and (H), data are combined from two independent experiments with at least seven mice per group. ∗ p < 0.05; ∗∗ p < 0.01, Student’s t test (A and C), one-way ANOVA (B, E, and F), and two-way ANOVA with Dunnett’s correction for multi-group comparison (G and H). Data are depicted as mean ± SEM. See also .

Article Snippet: Where specified, 10 μg/ml of anti-human IL-17A antibody or isotype control (R&D systems) was added to the cultures.

Techniques: Isolation, Recombinant, Control, Expressing, Real-time Polymerase Chain Reaction, Flow Cytometry, Staining, Knockdown, Reverse Transcription Polymerase Chain Reaction, Adoptive Transfer Assay, Injection, Comparison

Journal: Immunity

Article Title: The Cytokine IL-17A Limits Th17 Pathogenicity via a Negative Feedback Loop Driven by Autocrine Induction of IL-24

doi: 10.1016/j.immuni.2020.06.022

Figure Lengend Snippet: IL-17A Induces NF-κB Signaling in Th17 Cells (A and B) CD4 + CD62L + T cells from WT mice were isolated and polarized under Th17 conditions with anti-CD3/CD28 antibodies for 3 days. Cells were pulsed with IL-17A. (A and B) Phosphorylation of NF-κBp65, Erk1/2, and p38 was determined using flow cytometry. Data are shown as representative histogram and as compiled data from three (A) and four (B) independent experiments. (C and D) NF-κBp65 translocated into the nucleus after IL-17A re-stimulation. Representative of three (C) and two (D) independent experiments. ∗ p < 0.05 and ∗∗∗∗ p < 0.0001, one-way ANOVA (A–C). Data are depicted as mean ± SEM.

Article Snippet: Where specified, 10 μg/ml of anti-human IL-17A antibody or isotype control (R&D systems) was added to the cultures.

Techniques: Isolation, Phospho-proteomics, Flow Cytometry

Journal: Immunity

Article Title: The Cytokine IL-17A Limits Th17 Pathogenicity via a Negative Feedback Loop Driven by Autocrine Induction of IL-24

doi: 10.1016/j.immuni.2020.06.022

Figure Lengend Snippet: IL-17A Induces the Il24 Gene Promoter Activity through NF-κB (A) The Il24 gene has two potential binding sites for NF-κB (predicted by Genomatix). (B) PCR and real-time PCR showing the result of the two Il24 promoter sequences after the DNA was pulled down by anti-p65 antibody. Left panel shows representative data after 30 min; right panel shows average data compiled from three independent experiments. (C and D) CD4 + CD62L + T cells from WT mice were isolated and polarized under Th17 conditions with anti-CD3/CD28 antibodies for 3 days. Cells were pulsed with IL-17A. Cells were transfected with the indicated reporter constructs (C) Il 24 promoter and (D) Il24 promoter with mutation at Nfkb1 and/or Nfkb2. Fold change in Firefly/Renilla (FL/RL) ratio is plotted with respect to control (n = 8). ∗ p < 0.05, ∗∗∗ p < 0.001, and ∗∗∗∗ p < 0.0001, one-way ANOVA (B and D) and Student’s t test (C). Data are depicted as mean ± SEM.

Article Snippet: Where specified, 10 μg/ml of anti-human IL-17A antibody or isotype control (R&D systems) was added to the cultures.

Techniques: Activity Assay, Binding Assay, Real-time Polymerase Chain Reaction, Isolation, Transfection, Construct, Mutagenesis, Control

Journal: Immunity

Article Title: The Cytokine IL-17A Limits Th17 Pathogenicity via a Negative Feedback Loop Driven by Autocrine Induction of IL-24

doi: 10.1016/j.immuni.2020.06.022

Figure Lengend Snippet: IL-17A Induces Human Th17 Cells to Produce IL-24, Which Suppresses Expression of Th17-Lineage Cytokines (A–F) Naive human CD4 + T cells were isolated using naive CD4 + T cell isolation kit (Miltenyi) and were polarized under Th17 conditions for 14 days. (A) Kinetics of IL-24 expression during Th17 polarization were studied. Cells were collected at the indicated time points and were analyzed using real-time PCR (n = 4). (B) Expression of IL-24 by human Th0 and Th17 cells was determined from supernatant collected by ELISA on day 5 (n = 5). (C, E, and F) Cells were harvested on day 14 and subjected to intracellular staining for the indicated cytokines (C, n = 4; E, n = 5; F, n = 6). (D) Supernatant and cells were harvested on day 5 for ELISA (n = 6) and real-time PCR (n = 8), respectively, to detect IL-24 expression. ∗ p < 0.05, ∗∗ p < 0.01, and ∗∗∗ p < 0.001, paired t test (A, C, and D) and one-way ANOVA (E). (F) Not significant. Data are depicted as mean ± SEM.

Article Snippet: Where specified, 10 μg/ml of anti-human IL-17A antibody or isotype control (R&D systems) was added to the cultures.

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

Journal: Immunity

Article Title: The Cytokine IL-17A Limits Th17 Pathogenicity via a Negative Feedback Loop Driven by Autocrine Induction of IL-24

doi: 10.1016/j.immuni.2020.06.022

Figure Lengend Snippet:

Article Snippet: Where specified, 10 μg/ml of anti-human IL-17A antibody or isotype control (R&D systems) was added to the cultures.

Techniques: Virus, Recombinant, Adjuvant, Sample Prep, Software, Control, High Throughput Screening Assay, Sequencing

Journal: Experimental & Molecular Medicine

Article Title: Downregulation of Orai1 expression in the airway alleviates murine allergic rhinitis

doi: 10.3858/emm.2012.44.3.013

Figure Lengend Snippet: Protein expression of IL-33, IL-17E and TSLP. Expression levels of IL-33, IL-17E and TSLP in the nasal mucosa of the mice in each group were determined by Western blotting.

Article Snippet: The expressions of IL-33, IL-17E and TSLP were also analyzed using rat anti-mouse IL-33 monoclonal antibody (R&D systems, Minneapolis, MN), rat anti-mouse IL-17E monoclonal antibody (R&D systems), and rabbit anti-mouse TSLP polyclonal antibody (ProSci Incorporated, Poway, CA), respectively.

Techniques: Expressing, Western Blot