Interferon (IFN) stimulation is required to trigger interferon-stimulated gene (ISG) upregulation in Samhd1-deficient zebrafish larvae. (A) Diagram indicating the protein domains. (B) Transcript levels of samhd1 by reverse transcription PCR (RT-PCR) in the head and tail of zebrafish larvae. The average from three independent experiments, each with 25 pooled heads or tails, is shown. (C) Transcript levels of isg15 by reverse transcription quantitative PCR (RT-qPCR) in the head of zebrafish larvae injected with gRNA (samhd1 or STD)/Cas9 complexes with or without 1 pg/egg of pcDNA-IFNphi3. Bars show the mean ± SEM of three independent experiments, each with 25 pooled heads. Data are represented as fold change from control (STD). P-values were calculated using Student’s t-test in (B) and using one-way ANOVA and Tukey’s multiple range test in (C). ns, not significant. *p ≤ 0.05.

Samhd1 deficiency results in the spontaneous activation of nuclear factor kappa B (NF-κB) and hyperresistance to infection with Salmonella enterica serovar Typhimurium (STM). (A) Real-time visualization of NF-κB activation in Samhd1-deficient zebrafish larvae. The nfkb:eGFP reporter zebrafish line injected with gRNA (samhd1 or STD)/Cas9 complexes were analyzed using fluorescence microscopy and quantified. Each dot represents a larva, and the mean ± SEM for each experimental group is also shown. (B) Representative images of whole larvae are shown, and the region of interest (ROI) used to quantify the fluorescence in the nfkb:eGFP reporter line is indicated as a dot line in the images. (C) Zebrafish one-cell embryos were injected with gRNA (samhd1 or STD)/Cas9 complexes, dechorionated, and infected at 2 days post-fertilization via the yolk sac with STM at a multiplicity of infection (MOI) of 10 or phosphate-buffered saline (PBS), with the number of surviving larvae counted daily during the next 5 days. A total number of >100 specimens/treatment. P-values were calculated using Student’s t test in (A) and long-range test in (C). **p ≤ 0.01, ****p ≤ 0.0001.

Samhd1 deficiency leads to an activated inflammatory state. (A–F) Transcript levels of nfkb1(A), il1b(B), isg15(C), tnfα(D), ifng1r(E), and cxcl8a(F) in larvae injected with gRNA (samhd1 or STD)/Cas9 complexes infected or not with Salmonella enterica serovar Typhimurium (STM) for 24 h assayed using reverse transcription quantitative PCR (RT-qPCR). Bars show the mean ± SEM of three independent experiments, each with 25 pooled larvae. Data are represented as fold change from control. P-values were calculated using one-way ANOVA. ns, not significant. *p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001, ****p ≤ 0.0001.

Pharmacological inhibition of nuclear factor kappa B (NF-κB) abolishes the protection of Samhd1-deficient zebrafish larvae to infection with Salmonella enterica serovar Typhimurium (STM). Zebrafish one-cell embryos were injected with gRNA (samhd1 or crSTD)/Cas9 complexes, dechorionated at 24 h, and treated by bath immersion with 10 nM BAY11-7082 (BAY) (A) or with 10 μM baricitinib (B). Drugs were renewed every day for the next 6 days. Controls were incubated with 0.1% dimethyl sulfoxide (DMSO). Larvae were infected at 2 days post-fertilization via the yolk sac with STM at a multiplicity of infection (MOI) of 10, with the number of surviving larvae counted daily during the next 5 days. A total number of >100 specimens/treatment. P-values were calculated using a long-range test. **p ≤ 0.01, ***p ≤ 0.001, ****p ≤ 0.0001.

Genetic inhibition of nuclear factor kappa B (NF-κB) in macrophages abolishes the hyperresistance of Samhd1-deficient zebrafish larvae to infection with Salmonella enterica serovar Typhimurium. (A) The zebrafish line expressing a dominant negative (DN) form of Nfkbiaa, Tg(UAS:dn-nfkbiaa), was outcrossed with the transgenic line Tg(mpeg1:gal4), and one-cell-stage embryos were injected with gRNA (samhd1 or STD)/Casp9 complexes and infected with STM as previously described. The average from two independent experiments with >80 larvae per group is shown. P-values were calculated using a long-range test. ns, not significant. ***p ≤ 0.001. (B) Model showing the restriction of NF-κB activation by Samhd1 in wild-type macrophages and the hyperresistance of Samhd-1deficient macrophages to STM infection through the induction of NF-κB activation.