N-ethyl-N-nitrosourea-induced mutation in ubiquitin-specific peptidase 18 causes hyperactivation of IFN-αß signaling and suppresses STAT4-induced IFN-γ production, resulting in increased susceptibility to Salmonella typhimurium - PubMed (original) (raw)

N-ethyl-N-nitrosourea-induced mutation in ubiquitin-specific peptidase 18 causes hyperactivation of IFN-αß signaling and suppresses STAT4-induced IFN-γ production, resulting in increased susceptibility to Salmonella typhimurium

Etienne Richer et al. J Immunol. 2010.

Abstract

To deepen our knowledge of the natural host response to pathogens, our team undertook an in vivo screen of mutagenized 129S1 mice with Salmonella Typhimurium. One mutation affecting Salmonella susceptibility was mapped to a region of 1.3 Mb on chromosome 6 that contains 15 protein-coding genes. A missense mutation was identified in the Usp18 (ubiquitin-specific peptidase 18) gene. This mutation results in an increased inflammatory response (IL-6, type 1 IFN) to Salmonella and LPS challenge while paradoxically reducing IFN-gamma production during bacterial infection. Increased STAT1 phosphorylation correlated with impaired STAT4 phosphorylation, resulting in overwhelming IL-6 secretion but reduced IFN-gamma production during infection. The reduced IFN-gamma levels, along with the increased inflammation, rationalize the S. Typhimurium susceptibility in terms of increased bacterial load in target organs and cytokine-induced septic shock and death.

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Figures

Figure 1

Figure 1. ENU recessive screen breeding strategy and mapping of the Ity9 mutant pedigree to chromosome 6

(a) Schematic representation of the breeding scheme used to identify the mutant family by screening the G3 mice and to confirm the heritability of the phenotype using the N2 mice. Females and males are respectively represented by circles and by square while gray filling denote the presence of heterozygous mutant allele and white filling homozygous mutant alleles. (b) Fine mapping of the Ity9 locus to a 1.3Mb region on chromosome 6 (build 37.1). White boxes represent 129S1 alleles and black boxes represent heterozygous or DBA2/J alleles. Recombinants in this region were selected for progeny testing to characterize their susceptibility to Salmonella infection by survival analysis. Families demonstrating an early death phenotype (prior to day 8) were classified as affected.

Figure 2

Figure 2. Identification of a mutation in Usp18 underlying the Ity9 phenotype

(a) A guanine to thymine transversion causing a leucine to a phenylalanine missense mutation at the position 361 of USP18 was identified in the mutant pedigree. (b) The alignment of USP18 orthologs showing the conservation of the mutated amino acid in a broad range of eukaryotes. Identical amino acids are highlighted in yellow while conserved amino acids are highlighted in blue (c) Alignment of the segment of the Usp18 sequence of inbred and wild derived mice compared to the Usp18Ity9/Usp18Ity9 mutated strain. The G-C transversion in Mus spretus causes a leucine to valine amino acid change. Identical nucleotides are highlighted in yellow while conserved nucleotides are highlighted in blue. The sequence alignments were done with AlignX (InVitrogen).

Figure 3

Figure 3. Usp18Ity9 allele confers Salmonella Typhimurium Susceptibility

(a) Survival curves of N2 animals issued from affected F1 daughter produced in the original screen littermates infected with 1000 CFUs IV according to their Usp18 genotype. Usp18Ity9/Usp18Ity9 are represented by closed triangle (n=18) while Usp18Ity9/Usp18+ (n=42) and Usp18+/Usp18+ (n=17) are represented by closed and open boxes, respectively. Results are representative of 5 independent experiments. (b) Survival curves of F1 mice issued from a cross between _Usp18_Ity9/Usp18+ and Usp18−/Usp18− mice. Usp18Ity9/Usp18− are represented by closed triangles (n=22) while Usp18+/Usp18− are represented by closed boxes. Usp18Ity9/Usp18Ity9 and compound heterozygous _Usp18_Ity9/Usp18− mice are as susceptible to infection, validating the candidacy of Usp18 as the gene underlying Ity9. Results are representative of 2 independent experiments. (c) Survival curves of Usp18−/Usp18− FVB congenic mice (circles) and FVB mice (squares) confirming the susceptibility of USP18 deficient mice to Salmonella infection. Log-Rank (Mantel-Cox) _P_=0.004.

Figure 4

Figure 4. Usp18Ity9/Usp18Ity9 mice have an increased bacterial load as a consequence of impaired IFN-γ production

Spleen (a) and liver (b) CFUs 3 and 6 days post-infection with 1000 CFUs of Salmonella Typhimurium IV. Usp18Ity9/Usp18+ (open circles) and Usp18Ity9/Usp18Ity9 (closed circles). One tailed Mann-Whitney test was performed for the spleen (a) *_P_=0.0186 **_P_=0.0017 and liver (b) *_P_=0.0216 and **_P_=0.0082. (c) Spleen and liver CFUs 6 days post-infection with 1000 CFUs of Salmonella Typhimurium IV. Usp18+/Usp18− (open circles) and Usp18Ity9/Usp18− (closed circles). Two tailed T-Test _P_=0.0002 (spleen) and _P_=0.0004 (liver). Of note two Usp18Ity9/Usp18− mice were found dead at day 6 post-infection, excluding them from the CFU analysis. (d) Spleen and liver CFUs 3 days post-infection with 1000 CFUs of Salmonella Typhimurium IV. Usp18−/Usp18− (closed circles) and FVB (open circles). Mann Whitney _P_=0.0286 (liver). (e) IFN-γ production from explanted splenocytes from Salmonella Typhimurium infected mice incubated for 4h ex vivo in Usp18Ity9/Usp18+ (light grey) and Usp18Ity9/Usp18Ity9 (dark grey) littermates. 2 way ANOVA _P_=0.0211 N=6/group. (f) Serum IFN-γ concentration of uninfected mice or mice infected with Salmonella Typhimurium at day 1 and day 6 post-infection in Usp18Ity9/Usp18+ (light grey) and Usp18Ity9/Usp18Ity9 (dark grey) littermates. Two-tailed Mann Whitney D6 _P_=0.0087, N=6/group. (g) 24h Kinetic of IFN-γ production from explanted splenocytes stimulated with IFN-β or IL-12. 2 way ANOVA (interaction) _P_=0.0003 N=3/group, independent 2 way ANOVA according to the genotype for each time point * 2h _P_=0.0149, *** 8h _P_=0.0007, ** 24h _P_=0.0074. These results are representative of results obtained in 3 (a,b,e,f), 2 (c,g) or 1 (d) independent experiments.

Figure 5

Figure 5. Usp18Ity9/Usp18Ity9 mice are more susceptible to LPS challenge

Survival of Usp18Ity9/Usp18Ity9 (closed triangles) and control littermates Usp18Ity9/Usp18+ (closed boxes) challenged with 10 µg (a), 100 µg (b) or 500 µg of LPS IV. Survival curves comparison for the 500 µg challenge _P_=0.0002 (***). (d) Serum IL-6 concentration in Usp18Ity9/Usp18+ (light grey) and Usp18Ity9/Usp18Ity9 (dark grey) mice infected with Salmonella Typhimurium at day 0, day 1 and day 6 post-infection. 2 way ANOVA P<0.0001 N=3/group. IL-6 (e) and TNF (f) production of explanted splenocytes from Usp18Ity9/Usp18+ (light grey) and Usp18Ity9/Usp18Ity9 (dark grey) littermates stimulated overnight with an array of PAMPs. 2 way ANOVA (e) _P_=0.0023 (f) _P_=0.0084 N=3/group. 2 tailed T-Test were also performed for each treatment indendently (e) _P_=0.05 (*) P=0.002 (**). These results are representative of results obtained in 3 (a–d) or 2 (e,f) independent experiments.

Figure 6

Figure 6. Critical role of Stat4 in the host response to Salmonella Typhimurium infection in vivo

(a,b) Western blot of protein extracts from (a) BMDM stimulated with 100 U/mL of IFN-β for 0.5, 1, 2 and 4 h probed with anti-phosphoSTAT1 (pSTAT1), anti-STAT1 and anti-GAPDH. Explanted splenocytes from mice uninfected (D0) or infected (D6) with a 1000 CFUs of Salmonella Typhimurium IV for 6 days and either left unstimulated or stimulated with 100U/mL of Ifn-β probed with (b) anti-pSTAT1, anti-STAT1, anti-β-ACTIN and (c) anti-phosphoSTAT4 (pSTAT4), STAT4 and anti-β-ACTIN. These results are representative of results obtained with 6 independent mice. (d) Survival curves of Balb/cJ (empty circles), Stat4tm1Gru/Stat4tm1Gru (empty squares), Stat4tm1Gru/Stat4wt (filled squares) and 129S1 (filled circles) mice infected with 1000 CFUs IV of Salmonella. Log-Rank (Mantel-Cox) for Balb/cJ and Stat4tm1Gru/Stat4tm1Gru _P_=0.0006, Stat4tm1Gru/Stat4tm1Gru and Stat4tm1Gru/Stat4wt _P_=0.0004, Stat4tm1Gru/Stat4wt and 129S1 _P_=0.0004. MSTs are 5.2±0.4 days for BALB/cJ, 6.6±0.7 days for Stat4tm1Gru/ Stat4tm1Gru and 8.2±1.2 days for Stat4tm1Gru/Stat4wt. (e) Bacterial burden in the liver and spleen of Stat4tm1Gru/Stat4tm1Gru (filled circles) and Stat4tm1Gru/Stat4wt (empty circles) mice infected with 1000 CFUs IV of Salmonella 4 days post-infection. Mann-Whitney Two-tailed *** _P_=0.0002 and * _P_=0.0262. (f) Serum IFN-γ concentration of Stat4tm1Gru/Stat4tm1Gru (filled circles) and Stat4tm1Gru/Stat4wt (empty circles) mice infected with Salmonella Typhimurium at day 4 post-infection. *** Unpaired t test Two=−tailed with Welch's correction P<0.0001.

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