Neratinib reduces neutrophilic inflammation at the tail fin injury site of zebrafish larvae in vivo and induces apoptosis of human neutrophils in vitro. Neutrophils in zebrafish larvae can be enumerated by fluorescence microscopy using the transgenic line TgBAC(mpx:EGFP)i114, in which each GFP-expressing (green) cell is counted as one neutrophil (A). Tail fin transection (A, orange dotted line) was performed after 16 h of treatment with neratinib, and neutrophils at the site of injury enumerated 4 and 8 h post-injury. Larvae treated with neratinib had reduced numbers of neutrophils at the tail fin injury site at both time points in comparison to DMSO-treated larvae (B). Minimum n = 30 larvae per condition across three experimental repeats; data analyzed by two-way ANOVA with Sidak’s multiple comparisons. Total neutrophil number across the whole body of larvae was unchanged after 16 h of treatment with 10 μM of neratinib, in comparison to control DMSO-treated larvae (C). N = 20 larvae per condition across three independent experiments; data analyzed by unpaired t-test. In human neutrophils isolated from whole-blood samples from healthy volunteers, treatment with neratinib in vitro results in a dose-dependent increase in the rate of apoptosis (D). N = 5 healthy blood donors; data analyzed by one-way ANOVA with Dunnett’s multiple comparisons, comparing each neratinib concentration with the DMSO control treatment. Each data point represents data from one larva or human blood donor. Bars show mean ± standard deviation. p-values are indicated, where p <0.05.
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