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: Investigative ophthalmology & visual science

Article Title: S100A8/A9 Promotes Dendritic Cell-Mediated Th17 Cell Response in Sjögren's Dry Eye Disease by Regulating the Acod1/STAT3 Pathway.

doi: 10.1167/iovs.66.1.35

Figure Lengend Snippet: FIGURE 3. S100A8/A9 promotes DC-driven Th17 cell responses in vitro. (A, B) CD4+T cells isolated from NOD mice were cultured in the presence or absence of recombinant S100A8/A9 protein (4 μg/mL) under Th17-polarizing conditions for 3 days (indicated by T cells only). Shown are representative flow cytometric plots (left panel) and percentages of CD4+IL-17+T cells (right panel) (n = 3) (A) and ELISA analysis for the secretion of IL-17 in the culture supernatants (n = 4) (B). (C–G) BMDCs were stimulated with or without recombinant S100A8/A9 protein for 24 hours and then co-cultured with CD4+T cells sorted from NOD mice under Th17-polarizing conditions for 3 days (indicated by T cells co-cultured with DCs). Shown are representative flow cytometric plots (left panel) and percentages of CD4+IL-17+T cells (right panel) in the co-cultured systems (n = 3) (C); ELISA quantification of IL-17 levels in the supernatants of T cells co-cultured with S100A8/A9-treated and vehicle-treated DCs (n = 6) (D); and qRT-PCR analysis of the expression of Th17-related genes in the co-cultured cells (n = 5), including Il17a (E), Il17f (F), and RORγ t (G). (H) Flow cytometry analysis of T-cell proliferation in two groups based on CFSE signals. Data in A to G were analyzed using two-tailed Student’s t-test. Data are reported as mean ± SD. *P < 0.05; **P < 0.01; ns, no significance.

Article Snippet: Supernatant IL-17 levels were measured using a Mouse IL17 DuoSet ELISA Kit (DY421; R&D Systems) according to the manufacturer’s protocol.

Techniques: In Vitro, Isolation, Cell Culture, Recombinant, Enzyme-linked Immunosorbent Assay, Quantitative RT-PCR, Expressing, Flow Cytometry, Two Tailed Test

Journal: Investigative ophthalmology & visual science

Article Title: S100A8/A9 Promotes Dendritic Cell-Mediated Th17 Cell Response in Sjögren's Dry Eye Disease by Regulating the Acod1/STAT3 Pathway.

doi: 10.1167/iovs.66.1.35

Figure Lengend Snippet: FIGURE 4. S100A8/A9 is responsible for the maturation and function of BMDCs. (A) Flow cytometric analysis and statistics of MHC II, CD80, and CD40 expression on BMDCs stimulated with or without recombinant S100A8/A9 protein (n = 3). (B–D) qRT-PCR analysis of gene expression, including proinflammatory cytokines (B, D) and chemokines (C) in vehicle-treated or S100A8/A9-treated BMDCs (n = 3). (E) DCs were treated with vehicle or recombinant S100A8/A9 protein for 24 hours and then co-cultured with CD4+T cells in the presence or absence of anti-IL-23p19. Shown is the ELISA analysis of IL-17 levels in the supernatants of the coculture system (n = 4). Two-tailed Student’s t-test (A–C), Mann–Whitney test (D), and one-way ANOVA test (E) were used to assess statistical significance. Data are reported as mean ± SD. *P < 0.05; **P < 0.01; ns, no significance.

Article Snippet: Supernatant IL-17 levels were measured using a Mouse IL17 DuoSet ELISA Kit (DY421; R&D Systems) according to the manufacturer’s protocol.

Techniques: Expressing, Recombinant, Quantitative RT-PCR, Gene Expression, Cell Culture, Enzyme-linked Immunosorbent Assay, Two Tailed Test, MANN-WHITNEY

Journal: Investigative ophthalmology & visual science

Article Title: S100A8/A9 Promotes Dendritic Cell-Mediated Th17 Cell Response in Sjögren's Dry Eye Disease by Regulating the Acod1/STAT3 Pathway.

doi: 10.1167/iovs.66.1.35

Figure Lengend Snippet: FIGURE 7. The activation of STAT3 signaling contributes to the promoting effect of S100A8/A9 on DC-driven Th17 cell responses by induc- ing the transcription of Il23a. (A, B) DCs were pretreated with Stattic (a STAT3 inhibitor) or DMSO for 1 hour followed by stimulation with S100A8/A9 and then co-cultured with CD4+T cells under Th17-polarizing conditions. Shown are ELISA quantification of IL-17 in the supernatant of co-cultured cells (n = 4) (A) and flow cytometric analysis of the percentages of CD4+IL-17+T cells in the co-cultured cells (n = 4) (B). (C–E) qRT-PCR analysis of relative mRNA expression of Il23a (C), Il6 (D) and Il1b (E) in S100A8/A9-stimulated DCs pretreated with or without Stattic (n = 6). (F) Representative flow cytometry plots of CD11c+MHC II+ cells in vehicle-treated, S100A8/A9-treated, and S100A8/A9 plus Stattic-treated DCs. (G) Representative images of immunofluorescence staining for p-STAT3 in S100A8/A9-treated or vehicle- treated DCs. Scale bar: 5 μm. (H) STAT3 occupancy on the Il23a locus in BMDCs through analysis of the published ChIP-seq databases (GSE36099 and GSE27161). (I) Schematic diagram of predicted STAT3 binding sites in the Il23a promoter region. (J) ChIP-qPCR analysis of the enrichment of p-STAT3 in the Il23a promoter region in DC2.4 treated with S100A8/A9 or vehicle (n = 3). One-way ANOVA test (A, B, D), Kruskal–Wallis test (C, E), and two-tailed Student’s t-test (J) were used. Data are reported as mean ± SD. *P < 0.05; **P < 0.01; ns, no significance.

Article Snippet: Supernatant IL-17 levels were measured using a Mouse IL17 DuoSet ELISA Kit (DY421; R&D Systems) according to the manufacturer’s protocol.

Techniques: Activation Assay, Cell Culture, Enzyme-linked Immunosorbent Assay, Quantitative RT-PCR, Expressing, Flow Cytometry, Immunofluorescence, Staining, ChIP-sequencing, Binding Assay, ChIP-qPCR, Two Tailed Test