Journal: eLife
Article Title: Inhibition of ErbB kinase signalling promotes resolution of neutrophilic inflammation
doi: 10.7554/eLife.50990
Figure Lengend Snippet: Tail fin transection was performed as indicated by the red line ( A , upper image). Zebrafish larvae ( mpx :GFP) were pre-treated at two dpf with DMSO, tyrphostin AG825 [Tyr, 10 µM] ( B , minimum n = 28 larvae per condition), or CP-724714 [10 µM] ( C , minimum n = 42 larvae per condition) for 16 hr followed by injury. egfra and erbb2 crispants were generated and injured at two dpf ( D , minimum n = 36 larvae per condition). The number of neutrophils at the site of injury was determined at 4 and 8 hpi by counting GFP-positive neutrophils. To enumerate neutrophils across the whole body, uninjured inhibitor treated larvae (three dpf) ( E , minimum n = 23 larvae per condition) or crispants (two dpf) ( F , minimum n = 28 larvae per condition) were imaged by fluorescent microscopy (A, lower image). Apoptosis was measured at the site of injury after 8 hr by TSA and TUNEL double staining ( G ) (white arrow indicates TUNEL positive neutrophil, scale bar 10 μM) of mpx: GFP tyrphostin AG825 [Tyr, 10 µM] or CP-724714 [10 µM] treated larvae at three dpf ( H , minimum n = 35 larvae per condition). Uninjured inhibitor treated larvae were assessed for neutrophil apoptosis in the CHT at three dpf ( I , minimum n = 27 larvae per group). Apoptosis at the tail fin injury site of egfra erbb2 crispants at two dpf was also measured at eight hpi ( J , minimum n = 26 larvae per group). All data collated from at least three independent experiments, displayed as mean ± SEM. Each icon shows one data point from one individual larvae. Statistically significant differences were calculated by two-way ANOVA with Sidak post-test ( B–D ) or one-way ANOVA with Dunnett’s post-test(E), Students’ t-test ( F ), Kruskal-Wallis test with Dunn’s post-test ( H–I ) or Mann-Whitney U test ( J ), and indicated as *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001.
Article Snippet: C57BL/6 mice (female, 9–10 weeks old) were nebulised with LPS (3 mg per group of 8 mice) ( Pseudomonas aeruginosa , Sigma-Aldrich) and immediately injected intraperitoneally (i.p.) with either Tyrphostin AG825 (Tocris Bioscience) at 20 mg/Kg in 10% DMSO v/v in vegetable oil (eight mice, treatment group) or an equivalent volume of 10% DMSO v/v in vegetable oil (eight mice, control group) ( ; ).
Techniques: Generated, Microscopy, TUNEL Assay, Double Staining, MANN-WHITNEY
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![Neutrophils were incubated with DMSO or 20 µM or 40 µM erbstatin (Erb, A and B ) or tyrphostin AG825 (Tyr, C ) for 6 hr. Apoptosis was assessed by Annexin V/TO-PRO-3 staining by flow cytometry and the percentage apoptosis calculated as Annexin V single plus Annexin V/TO-PRO-3 dual positive events. ( A ) Representative quadrant plot of Erbstatin-treated neutrophils showing distribution of Annexin V and TO-PRO-3 positive events. ( D–E ) Neutrophils were incubated with DMSO or Erbstatin [40 µM] in the presence or absence of the pan caspase inhibitor, Q-VD-OPh [1 µM] for 6 hr ( D ) or 20 hr ( E ). Apoptosis was assessed by light microscopy. The data are expressed as mean percentage apoptosis ± SEM from 3 ( B ), 4 (C and E), or 5 ( D ) independent experiments. Statistical differences were calculated by one-way ANOVA (with Dunnett’s post test ( B–C ) and two-way ANOVA with Sidak ( D–E ) post-tests) and indicated as *p≤0.05, **p≤0.01, ***p≤0.001, ****p≤0.0001.](https://pub-med-central-images-cdn.bioz.com/pub_med_central_ids_ending_with_9918/pmc06839918/pmc06839918__elife-50990-fig2-figsupp1.jpg)
![Neutrophils were incubated with DMSO, Erbstatin [Erb, 40 µM] ( A ) or tyrphostin AG825 [Tyr, 50 µM] ( B ) in the presence of DMSO or N 6 -MB-cAMP [500 µM and 1 mM] for 20 hr. Neutrophils isolated from COPD patients were incubated with DMSO or tyrphostin AG825 [50 µM] in the presence of DMSO or N 6 -MB-cAMP [500 µM] for 20 hr ( C ). Neutrophils isolated from COPD patients and age-matched healthy control subjects (HC) were incubated with DMSO, erbstatin ( D ) [20, 40 µM] or tyrphostin AG825 ( E ) [25, 50 µM] in the presence or absence of GMCSF [50 u/mL] for 20 hr. Apoptosis was assessed by light microscopy. The data are expressed as mean percentage apoptosis ± SEM from 4 to 6 independent experiments. ( F ) Neutrophils were incubated with DMSO or tyrphostin AG825 [Tyr, 50 µM] for 60 min before the addition of GMCSF [50 u/mL] for 15 or 30 mins. ( G ) Neutrophils were incubated with DMSO, tyrphostin AG825 [50 µM] for 60 min before the addition of GMCSF [50 u/mL] for a further 7 hr. Cells were lysed, subjected to SDS-PAGE electrophoresis and membranes probed for p-AKT, Mcl-1 or loading controls, AKT and P38. Images are representative of 3 independent experiments. Charts show densitometric values of 3 individual immunoblots and are expressed as a ratio of target (p-AKT or Mcl-1) over loading control (AKT or P38, respectively). Statistically significant differences were calculated by one-way ANOVA with Sidak post-test ( A–C, F–G ) or two-way ANOVA with Sidak post-test ( D–E ) and indicated as *p<0.05, **p<0.01, ***p<0.001.](https://pub-med-central-images-cdn.bioz.com/pub_med_central_ids_ending_with_9918/pmc06839918/pmc06839918__elife-50990-fig3.jpg)
![Tail fin transection was performed as indicated by the red line ( A , upper image). Zebrafish larvae ( mpx :GFP) were pre-treated at two dpf with DMSO, tyrphostin AG825 [Tyr, 10 µM] ( B , minimum n = 28 larvae per condition), or CP-724714 [10 µM] ( C , minimum n = 42 larvae per condition) for 16 hr followed by injury. egfra and erbb2 crispants were generated and injured at two dpf ( D , minimum n = 36 larvae per condition). The number of neutrophils at the site of injury was determined at 4 and 8 hpi by counting GFP-positive neutrophils. To enumerate neutrophils across the whole body, uninjured inhibitor treated larvae (three dpf) ( E , minimum n = 23 larvae per condition) or crispants (two dpf) ( F , minimum n = 28 larvae per condition) were imaged by fluorescent microscopy (A, lower image). Apoptosis was measured at the site of injury after 8 hr by TSA and TUNEL double staining ( G ) (white arrow indicates TUNEL positive neutrophil, scale bar 10 μM) of mpx: GFP tyrphostin AG825 [Tyr, 10 µM] or CP-724714 [10 µM] treated larvae at three dpf ( H , minimum n = 35 larvae per condition). Uninjured inhibitor treated larvae were assessed for neutrophil apoptosis in the CHT at three dpf ( I , minimum n = 27 larvae per group). Apoptosis at the tail fin injury site of egfra erbb2 crispants at two dpf was also measured at eight hpi ( J , minimum n = 26 larvae per group). All data collated from at least three independent experiments, displayed as mean ± SEM. Each icon shows one data point from one individual larvae. Statistically significant differences were calculated by two-way ANOVA with Sidak post-test ( B–D ) or one-way ANOVA with Dunnett’s post-test(E), Students’ t-test ( F ), Kruskal-Wallis test with Dunn’s post-test ( H–I ) or Mann-Whitney U test ( J ), and indicated as *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001.](https://pub-med-central-images-cdn.bioz.com/pub_med_central_ids_ending_with_9918/pmc06839918/pmc06839918__elife-50990-fig6.jpg)