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. 2019 Jan 29;8:e39123. doi: 10.7554/eLife.39123

Figure 2. Clemastine activity is host-dependent, dose-responsive, and requires macrophages.

(A) Bacterial burden per animal assessed by Mm:tdTomato fluorescence 5 days post infection (dpi) after treatment with 0.5% DMSO vehicle or 5 µM clemastine. Red dots denote the corresponding image shown in Figure 2B. Representative of five experiments. (B) Representative images from experiment in Figure 2A (red dots). Mm:tdTomato infection treated with 0.5% DMSO vehicle or 5 µM clemastine in 0.5% DMSO. White arrows denote regions of bacterial foci. Scale bar is 500 µm. (C) Quantification of Mm:tdTomato fluorescence at 5 dpi in zebrafish larvae treated with increasing concentrations of clemastine. (D) Mm:tdTomato bacterial broth culture grown in the presence of increasing concentrations of clemastine, compared to 0.5% DMSO, 1% DMSO, no vehicle (-), and 200 µg/mL isoniazid. (E) Bacterial burden of wildtype and heterozygous siblings or irf8st95 mutants, which lack macrophages, treated with 0.5% DMSO or 5 µM clemastine. Blue dots indicate animals in Figure 2F. Data are pooled from two biological replicates. (F) Representative infections from irf8st95 mutants and wildtype/heterozygous siblings from each treatment group in Figure 2E at 5 dpi. Wildtype animals (left) were equally brightened (no change in gamma settings) to show contrast of bacteria and brightfield. The irf8st95-/- example animals (right) were not brightened. (G) Number of bacteria per macrophage during treatment with 0.5% DMSO or 5 µM clemastine, 1 dpi. Each dot represents the mean number of intracellular Mm:mCerulean bacteria inside macrophages of one Tg (mfap4:tdTomato)xt11 animal at 24 hpi infected with ~ 50 CFU. Representative of three independent experiments. (A) Two-tailed, unpaired t-test. (C) Ordinary one-way ANOVA with Tukey’s multiple comparison test, ns1 = 0.0864, ns2 = 0.7799, ns3 = 0.2415. Post-test for linear trend, p<0.0001. (E) Kruskal-Wallis ANOVA for unequal variances with Dunn’s multiple comparisons test, ns1 > 0.9999 (A,C,E) Error bars are s.d. (G) Two-tailed unpaired t-test; error bars are s.e.m. p-Values from statistical tests on untransformed data are provided in Supplementary file 2.

Figure 2.

Figure 2—figure supplement 1. Macrophage function during clemastine treatment.

Figure 2—figure supplement 1.

(A) Bacterial burden per animal assessed by Mm:tdTomato fluorescence 5 days post-infection after treatment with 0.5% DMSO or 5 µM diphenhydramine. (B) Example images from quantification in Figure 2G. Bacteria are Mm:mCerulean and macrophages Tg(mfap4:tdTomato)xt12 are false colored in magenta, maximum intensity projection of 5 z stacks of 10 µm. Scale bar is 100 µm. (C) Number of infected macrophages per fish. Each dot represents the total number of infected macrophages in each animal from experiment in Figure 2G. (D) Stills from Video 1. Scale bar is 100 µm. Maximum intensity projection of 4 z-stacks of 15 µm. White arrows mark bacteria that are present in both initial and final timeframes, and yellow arrows mark individual bacteria that disappear during the timelapse. (E) Ratio of green to red fluorescent bacteria from Mm:aprA’::GFP,smyc::mCherry infection (~ 50 CFU) in wildtype zebrafish larvae over the course of a 5-day infection during treatment with 0.5% DMSO or 5 µM clemastine. (A) Unpaired t-test, ns1 = 0.3147, error bars are s.d. (C) Unpaired t-test, ns1 = 0.1907, error bars are s.d. (E) Unpaired t-tests between treatments for each day, ns1 > 0.9999, ns2 = 0.5505, error bars are s.d. p-Values from all transformed and untransformed data are provided in Supplementary file 3.