Characterization of tirap mutant zebrafish larvae. (A) Mutant DNA and protein sequence of Tirap. A point mutation (T to A) in the TIR domain of zebrafish tirap results in a stop codon. (B) Proportion of genotypes. The proportion of genotypes after the incross of tirap^+/- heterozygous. (C) Experimental scheme of body length measurement. (D) Development. The body measurement of tirap^+/+ and tirap^-/- zebrafish at 5 days post fertilization (5 dpf). The measurements are based on 3 independent experiments with tirap^+/+ (n=52) and tirap^-/- (n=52). (E, F) qPCR of tirap^+/+ and tirap^-/- zebrafish embryos. Tirap^+/+ and tirap^-/- embryos were injected with purified LPS (100 µg/ml) or sterile water as a control at 27 hour post fertilization (hpf). The expression level of il1b and fosl1a were analyzed at 1 hour post injection (hpi) by qRT-PCR. Data (mean ± SD) are combined with 3 biological replicates (n=15 embryos/group). Statistical significance was determined by two-way ANOVA with Tukey's multiple comparison method as a post-hoc test. (G, H, I) Experimental scheme and representative image of macrophages and neutrophils in zebrafish larvae (tirap^+/+ and tirap^-/-). Scale bar: 50 µm. (J, K) Quantification of neutrophils and macrophages number in tirap mutant and wild-type zebrafish larvae. Number of neutrophils and macrophages in zebrafish larvae under unchallenged conditions. The number of neutrophils for tirap^+/+ (n=33) and tirap^-/- (n=27) and the number of macrophages for tirap^+/+ (n=36) and tirap^-/- (n=28) are based on two independent experiments. Statistical significant difference was determined by unpaired t-test, ns, non-significant.

Transcriptomic profiles of tirap mutant and myd88 mutant zebrafish larvae measured by RNAseq. (A) Overview of the distribution of differentially expressed genes (DEGs) log2 fold change in tirap^-/- versus tirap^+/+ zebrafish larvae. (B) Overview of the distribution of DEGs log2 fold change in myd88^-/- versus myd88^+/+ zebrafish larvae. DEGs were assessed by padj value less than 0.05. Down-regulated gene sets are shown in blue, and up-regulated gene sets are shown in red. (C) KEGG pathway enrichment analysis of tirap^-/- versus tirap^+/+. (D) KEGG pathway enrichment analysis of myd88^-/- versus myd88^+/+. Significantly enriched KEGG pathway terms for DEGs of tirap^-/- versus tirap^+/+ groups were determined by using the hypergeometric test/Fisher's exact test, with a threshold of p value < 0.05, which were adjusted using the Benjamini and Hochbery FDR correction. For myd88^-/- versus myd88^+/+ group, significantly enriched KEGG pathway terms for DEGs were determined by using the hypergeometric test/Fisher's exact test, with a threshold of p value < 0.1.

Schematic diagram of significantly differentially expressed genes (DEGs) in the representative pathways in tirap mutant zebrafish larvae compared to the wild type controls. (A) Glycolysis and gluconeogenesis, TCA cycle and oxidative phosphorylation related pathways. (B) TLR-associated signaling pathways. (C) Calcium regulation-related pathways. (D) Ribosome-associated pathway. The significance was established as padj value less than 0.05. Red arrows represent significantly upregulated DEGs, blue arrows represent significantly downregulated DEGs. The link between TIRAP and PIK3AP1 refers to Luo, et al.59. The link between CREB and DHPR refers to Antony, et al.44. The link between CREB and PP2A refers to Chen, et al.60. Other links refer to KEGG Pathway Database (https://www.genome.jp/kegg/pathway.html).

Schematic diagram of significantly differentially expressed genes (DEGs) in the representative pathways in myd88 mutant zebrafish larvae compared to the wild type controls. (A) Glycolysis and gluconeogenesis, TCA cycle and oxidative phosphorylation related pathways. (B) TLR-associated signaling pathways. (C) Calcium regulation-related pathways. (D) Ribosome-associated pathway. The significance was established as padj value less than 0.05. Red arrows represent significantly upregulated DEGs, blue arrows represent significantly downregulated DEGs. The link between TIRAP and PIK3AP1 refers to Luo, et al.59. The link between CREB and DHPR refers to Antony, et al.44. Other links refer to KEGG Pathway Database (https://www.genome.jp/kegg/pathway.html).

Metabolic profiles of tirap mutant and myd88 mutant zebrafish larvae measured by ¹H NMR and analysis of PLS-DA and heat map. (A) The representative one-dimensional ¹H NMR spectra of tirap wild type and mutant zebrafish larvae measured by NMR spectrometry. Spectra from chemical shift 0.8 to 4.3 were assigned to specific metabolites. (B) PLS-DA analysis of tirap wild type and mutant groups. n = 3, each replicate represents 120 pooled larvae. (C) Heat map analysis of significantly altered metabolites detected in tirap mutants (P < 0.05). n = 3, each replicate represents 120 pooled larvae. (D) The representative one-dimensional ¹H NMR spectra of myd88 wild type and mutant zebrafish larvae measured by NMR spectrometry. Spectra from chemical shift 0.8 to 4.3 were assigned to specific metabolites. (E) PLS-DA analysis of myd88 wild type and mutant groups. n = 3, each replicate represents 120 pooled larvae. (F) Heat map analysis of significantly altered metabolites detected in myd88 mutants (P < 0.05). Thr threonine, Lac lactate, Asp aspartate, Ser Serine, tCr total creatine (creatine + phosphocreatine), Glc Glucose, 2-Pho 2-Phosphoglycerate, Ala alanine, Gln glutamine, Arg arginine, Leu leucine, Val valine, Gly glycine, Tau taurine, His Histidine, Tyr tyrosine, Mal Malate, Met Methionine, Suc Succinate, Pyr Pyruvate, Glu glutamate, Acet Acetate, Lys lysine, Mel Melatonin, Eth Ethanol, Isol Isoleucine, 2h3m 2-Hydroxy-3-methylvalerate, 2-Hyd 2-Hydroxyglutarate.

Quantification of bacterial burden after M. marinum infection in tirap mutant and wild type zebrafish larvae. (A) tirap^+/+ and tirap^-/- zebrafish were infected with mCherry-labeled M. marinum strain Mma20 by caudal vein injection at 28 hours post fertilization (hpf). (B) Representative images were taken after 4 days of infection (dpi), respectively. (C) Bacterial burdens at 4 dpi were quantified using bacterial fluorescence pixels. Data were combined from 3 independent experiments, for tirap^+/+, n=67, for tirap^-/-, n=65. ****, P <0.0001. Scale bar: 500 µm.

The number of recruited neutrophils upon tail wounding in zebrafish larvae. (A) Schematic representation of experiment. The wounded zebrafish larvae were imaged at designated times 1 hour post wounding (hpw), 3 hpw and 6 hpw. (B) Representative images of tirap^+/+ and tirap^-/- at 1 hpw and 6 hpw. Scale bar: 100 µm. (C) Number of neutrophils recruited to tail region upon tail wounding at 1, 3, 6 hpw. Red stars indicate the representative images shown in the panel B. The number of neutrophils at 1 hpw from one independent experiment for tirap^+/+ (n=23) and tirap^-/- (n=20), at 3 hpw for tirap^+/+ (n= 52) and tirap^-/- (n= 53) and at 6 hpw for tirap^+/+ (n=72) and tirap^-/- (n=57) are based on three independent experiments. Statistically significant difference was determined by nonparametric tests: Mann-Whitney and Kolmogorov-Smirnov, ns, non-significant; **, P < 0.01, ***, P < 0.001.

Quantification of neutrophil behaviour upon tail wounding in zebrafish larvae. (A) Schematic representation of experiment. (B) Representative image of neutrophil migration at frame 120 (3 hpw) (C-D) Quantification of neutrophil migratory capability of 3 dpf tirap larvae upon tail wounding. Mean speed (C) and meandering index (D) were quantified using IMARIS Software. The mean speed of neutrophils is measured as total displacement of neutrophils divided by the number of frames. Data consists of 3 tirap^+/+ and 3 tirap^-/- larvae. Statistically significant difference was determined by unpaired t test with Welch's correction, ns, non-significant; ***, P < 0.001.

Relative expression of immune-related genes upon tail wounding in tirap mutant zebrafish larvae. hpw, hour post wounding; ns, non-significant; *, P < 0.05; **, P < 0.01.

Graphical summary of tirap mutant compared to tlr2 and myd88 mutants in affecting leukocyte migration behavior upon tail wounding in zebrafish larvae. hpw, hour post wounding.