FIGURE SUMMARY
Title

Silica crystals activate toll-like receptors and inflammasomes to promote local and systemic immune responses in zebrafish

Authors
Tyrkalska, S.D., Pedoto, A., Martínez-López, A., Ros-Lucas, J.A., Mesa-Del-Castillo, P., Candel, S., Mulero, V.
Source
Full text @ Dev. Comp. Immunol.

Fig. 1. Silica crystals promotes neutrophil recruitment and neutrophilia in zebrafish larvae. SiO2 crystals or PBS were injected in the hindbrain ventricle (HBV, dash line) of 2 dpf Tg(mpx:eGFP). Neutrophil recruitment (A, B), number in the whole body (C, D) and dispersion (E, F) were analyzed by fluorescence microscopy from 1 to 48 hpi (A, B) or 24 hpi (C-F). Each dot represents one individual and the mean ± S.E.M. for each group is also shown. P values were calculated using one-way ANOVA and Tukey multiple range test. ns, not significant, **p ≤ 0.01, ***p ≤ 0.001. Bars: 100 μm (A), 500 μm (C), 250 μm (E).

Fig. 2. Silica crystals promotes macrophage recruitment and monocytosis in zebrafish larvae. SiO2 crystals or PBS were injected in the hindbrain ventricle (HBV, dash line) of 2 dpf Tg(mfap4:mCherry). Macrophage recruitment (A, B) and number in the whole body (C, D) and the head (E) were analyzed by fluorescence microscopy from 1 to 48 hpi (A, B) or 24 hpi (C-E). Each dot represents one individual and the mean ± S.E.M. for each group is also shown. P values were calculated using one-way ANOVA and Tukey multiple range test. ns, not significant, ***p ≤ 0.001. Bars: 100 μm (A), 500 μm (C).

Fig. 3. Silica crystals promotes local and systemic inflammation in zebrafish larvae. SiO2 crystals or PBS were injected in the hindbrain ventricle (HBV, dash line) of 2 dpf Tg(NFkB-RE:eGFP) (A-D) and Tg(il1b:eGFP) (E-H). Nfkb activation (A-D) and Il1b production (E-H) were analyzed by fluorescence microscopy from 1 to 48 hpi (A, B, E, F) or 24 hpi (C, D, G, H). Each dot represents one individual and the mean ± S.E.M. for each group is also shown. P values were calculated using one-way ANOVA and Tukey multiple range test. ns, not significant, *p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001. Bars: 100 μm (A, E), 500 μm (C, G).

Fig. 4. The Cxcl8/Cxcr2 axis is required for the silica crystal-induced systemic inflammation. SiO2 crystals or PBS were injected in the hindbrain ventricle (HBV) of 2 dpf Tg(lyz:DsRED2) (A), Tg(mfap4:mCherry) (B) and wild type (C-M) larvae in the presence of either DMSO or the Cxcr2 inhibitor SB225002 (SB). Neutrophil (A) and macrophage (B) recruitment and number were analyzed at 6, 12 and 24 hpi by fluorescence microscopy, while the transcript levels of the indicated genes (C-M) were analyzed at 12 hpi by RT-qPCR in larval head and tail. Each dot represents one individual and the mean ± S.E.M. for each group is also shown. One representative RT-qPCR experiment from three biological replicates is shown. P values were calculated using one-way ANOVA and Tukey multiple range test. ns, not significant, *p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001.

Fig. 5. Silica crystals signal through the canonical inflammasome in zebrafish. SiO2 crystals or PBS were injected in the hindbrain ventricle (HBV) of 2 dpf Tg(lyz:DsRED2) (A) and wild type (B-M) larvae in the presence of either DMSO or the caspase-1 inhibitor VX-765 (VX). Neutrophil recruitment and number were analyzed at 6, 12 and 24 hpi by fluorescence microscopy (A), while the transcript levels of the indicated genes (B-L) were analyzed at 12 hpi by RT-qPCR in larval head and tail. Caspase-1 activity levels were analyzed at 24 hpi in larval head and tail (M). Each dot represents one individual and the mean ± S.E.M. for each group is also shown. One representative RT-qPCR and caspase-1 activity experiment from three biological replicates is shown. P values were calculated using one-way ANOVA and Tukey multiple range test. ns, not significant, *p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001.

Fig. 6. Silica crystals signal through the Nlrp3/Caspa/Gsdme inflammasome in zebrafish. Tg(lyz:DsRED2) (A, D, E, H), Tg(mfap4:mCherry) (B, F, I) and Tg(NFkB-RE:eGFP) (C, G, J) 1–8 cell stage embryos were microinjected with control, gsdmea/b (A-D), nlrp3 (E-G) or il1b (H-J) crRNA/Cas9 complexes. At 2 dpf, SiO2 crystals or PBS were injected in the larval hindbrain ventricle (HBV) in the presence of either DMSO or the caspase-1 inhibitor VX-765 (VX) (D). Neutrophil (A, D, E, H) and macrophage (B, F, I) recruitment and number, and Nfkb activity (C, G, J) were analyzed at 6, 12 and 24 hpi by fluorescence microscopy. Each dot represents one individual and the mean ± S.E.M. for each group is also shown. P values were calculated using one-way ANOVA and Tukey multiple range test. ns, not significant, *p ≤ p0.05, **p ≤ 0.01, ***p ≤ 0.001.

Fig. 7. Silica crystals signal through Myd88 in zebrafish. SiO2 crystals or PBS were injected in the hindbrain ventricle (HBV) of 2 dpf wild type and Myd88-deficient larvae. The transcript levels of the indicated genes were analyzed at 12 hpi by RT-qPCR in larval head and tail. The data are shown as the mean ± S.E.M. One representative experiment from three biological replicates is shown. P values were calculated using one-way ANOVA and Tukey multiple range test. ns, not significant, *p 0.05, **p ≤ 0.01, ***p ≤ 0.001.

Fig. 8. Silica crystals induce fibrotic markers in zebrafish larvae. SiO2 crystals or PBS were injected in the hindbrain ventricle (HBV) of 2 dpf wild type larvae alone (A-F). The transcript levels of the indicated genes were analyzed from 6 to 120 hpi in larval head and tail (A-F) by RT-qPCR. The data are shown as the mean ± S.E.M. One representative experiment from three biological replicates is shown. P values were calculated using one-way ANOVA and Tukey multiple range test. *p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001.

Acknowledgments
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