FIGURE SUMMARY
Title

Inhibition of ErbB kinase signalling promotes resolution of neutrophilic inflammation

Authors
Rahman, A., Henry, K.M., Herman, K.D., Thompson, A.A.R., Isles, H.M., Tulotta, C., Sammut, D., Rougeot, J.J., Khoshaein, N., Reese, A.E., Higgins, K., Tabor, C., Sabroe, I., Zuercher, W.J., Savage, C.O., Meijer, A.H., Whyte, M.K., Dockrell, D.H., Renshaw, S.A., Prince, L.R.
Source
Full text @ Elife

Schematics showing PKIS screen design.

(A) Tail fin transected three dpf Tg(mpx:GFP)i114 zebrafish larvae that had generated an inflammatory response at six hpi were incubated with individual PKIS compounds [25 µM] for a further 6 hr. Larvae were imaged and manually scored between 0–3 on the basis of green fluorescence at the injury site. (B) PKIS compounds were incubated with primary human neutrophils 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 (as indicated by red box).

Pharmacological inhibition and genetic knockdown of <italic>egfra</italic> and <italic>erbb2</italic> by CRISPR/Cas9 reduces neutrophil number at the site of injury in a zebrafish model of inflammation.

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.

Acknowledgments
This image is the copyrighted work of the attributed author or publisher, and ZFIN has permission only to display this image to its users. Additional permissions should be obtained from the applicable author or publisher of the image. Full text @ Elife