TLR2 chemical inhibition impairs macrophage-mediated dissemination of Mma20 bacteria in zebrafish larvae. (A) Schematic of the experimental workflow. Zebrafish larvae were treated with the TLR2 inhibitor C29 at 1 dpf and then immersed with DsRed-labeled Mma20 at 2.5 dpf for 2.5dpi. (B) Representative fluorescence images showing macrophages containing Mma20 bacteria in different body regions in tlr2 wild-type and C29 treated larvae. Bacteria are shown in magenta. Macrophages are shown in cyan. Scale bar: 100 µm for full-body images; 25 µm for zooms. (C) Survival rates for all groups after Mma20 infection showing no significant differences between the wild type and C29 treated larvae. The results are based on 3 independent experiments. Error bars represent mean ± SD. (D-F) Quantification of full body (D), anterior region (E) and posterior region (F) bacterial burden (fluorescent pixel count) shows no significant difference between the wild type and 1 µM or 5 µM C29 treated larvae. (G-I) Quantification of the number of bacteria-containing macrophages in the full body (G), anterior region (H), and posterior region (I). Wild-type larvae had significantly more infected macrophages than 1 µM or 5 µM C29 treated larvae in full body and anterior region. For posterior region, there is no significant difference between wild-type larvae and 1 µM or 5 µM C29 treated larvae. Macrophages containing bacteria were manually counted across the full z-stack for each region. Each z-slice was examined to verify that individual bacteria-containing macrophages were counted once and only once. The data from tlr2+/+ wild-type (n=27) group, 1 µM C29 treated (n=25) group and 5 µM C29 treated group (n=29) are based on two independent experiments. Statistical significant difference was determined by one-way ANOVA, red arrows point to the median, ns, non-significant, *, P < 0.05, **, P < 0.01, ***, P < 0.001.
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