Figure 3

Inhibition of ripk3 increases mortality in zebrafish larvae with pneumococcal meningitis and attenuates neutrophil recruitment during early stages of pneumococcal meningitis

(A) Gene expression analysis of ripk3 during early stages of pneumococcal meningitis. RT-qPCR was performed on pools of 20 zebrafish larvae injected with 2,000 CFUs of S. pneumoniae PLY+, S. pneumoniae PLY−, or PBS and collected at 3, 6, and 9 hpi. The data represent six biological replicates; F(1, 5) = 54.72, p = 0.0007, PLY+ versus control (CTRL) versus PLY− versus CTRL; determined by two-way ANOVA on log2-transformed values. Multiple comparison analyses of each individual time point showed no statistical differences between both groups: adjusted p = 0.9975, PLY+ versus CTRL versus PLY− versus CTRL at 3 hpi; adjusted p = 0.0676, at 6 hpi; adjusted p = 0.0829, at 9 hpi; determined by Sidak’s post hoc test on log2-transformed values.

(B) Survival curves of 2 dpf zebrafish larvae injected with 200 CFUs of S. pneumoniae D39V into the hindbrain ventricle and control non-infected (ni) larvae treated with 100 μM GSK′872 or vehicle (DMSO). The data represent the mean ± SEM of three biological replicates with 20 larvae per group (n = 60 in total/group); ^∗p = 0.0237; determined by log rank test.

(C) Graph showing differences in number of neutrophils recruited to the hindbrain ventricle of zebrafish larvae injected with 2,000 CFUs of S. pneumoniae D39V upon treatment with 100 μM GSK′872 versus treatment with DMSO. The data represent the mean ± SEM of two biological replicates with 4–5 larvae per group (n = 9–10 in total/group); ^∗∗∗∗p < 0.0001, GSK′872 (100 μM) versus vehicle (DMSO); determined by two-way ANOVA.

(D) Representative confocal imaging of a single Z-slice of zebrafish larvae injected with 2,000 CFUs of red-fluorescently labeled S. pneumoniae into the hindbrain ventricle treated with 100 μM GSK′872 versus treatment with DMSO. Neutrophils are labeled by a GFP fusion to the mpx gene. Scale bar: 100 μm.

(E) Graph of number of neutrophils recruited to the hindbrain ventricle of zebrafish larvae injected with 2,000 CFUs of S. pneumoniae PLY+ or S. pneumoniae PLY− and treatment with 100 μM GSK′872 versus treatment with DMSO. The data represent the mean ± SEM of two biological replicates with 5 larvae per group (n = 10 in total/group); ^∗∗∗p = 0.0006, S. pneumoniae PLY+ (GSK′872, 100 μM) versus S. pneumoniae PLY+ (DMSO); p = 0.6441, S. pneumoniae PLY− (GSK′872, 100 μM) versus S. pneumoniae PLY− (DMSO); determined by two-way ANOVA.