N-Ethyl-N-Nitrosourea–Induced Mutation in Ubiquitin-Specific Peptidase 18 Causes Hyperactivation of IFN-αβ Signaling and Suppresses STAT4-Induced IFN-γ Production, Resulting in Increased Susceptibility to Salmonella Typhimurium

Etienne Richer, Caitlin Prendergast, Dong-Er Zhang, Salman T. Qureshi, Silvia M. Vidal and Danielle Malo
J Immunol September 15, 2010, 185 (6) 3593-3601; DOI: https://doi.org/10.4049/jimmunol.1000890

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-γ production during bacterial infection. Increased STAT1 phosphorylation correlated with impaired STAT4 phosphorylation, resulting in overwhelming IL-6 secretion but reduced IFN-γ production during infection. The reduced IFN-γ 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.

Salmonella enterica species includes a number of closely related serovars capable of causing serious infections in humans and animals. Typhoid fever caused by S. enterica serovar Typhi is a major public health concern in many developing countries, claiming millions of lives annually. This intracellular Gram-negative bacterium follows a fecal–oral infection route and establishes systemic infection in the host. The outcome of the infection will vary from mild to severe, with some infected people remaining healthy carriers, suggesting an important host genetic contribution to the outcome of this disease. In humans, nontyphoid Salmonella infections, such as S. enterica serovar Typhimurium (S. Typhimurium) and S. Enteritidis infections, usually present as self-limiting gastroenteritis, although a certain percentage of these infections may become invasive and result in septicemia. Accumulated data in humans suggest that predisposition to pediatric primary infection with Salmonella involves functional polymorphisms within genes in the IL-12-dependent, IFN-γ–mediated immunity (1–3).

In mice, the study of natural variation in the host response to infection has led to the identification of genes that have a critical impact during infections (4). Oral infection of mice with S. Typhimurium causes a typhoid-like disease in which the bacteria invade the M cells of the intestine, gain access to the mesenteric lymph nodes, and establish a systemic disease with major sites of replication in the spleen and liver (5). Great phenotypic diversity of the host response to S. Typhimurium is observed among different inbred mouse strains owing to the presence of specific mutations that are inherited as monogenic traits (SLC11A1G169D in C57BL/6J and BALB/cJ; TLR4P712H in C3H/HeJ or PKLRI90N in AcB61 mice) (6–8) or complex patterns (9).

Infectious disease models using inbred and recombinant inbred/congenic mice are inherently limited by the finite natural genetic variation present in these strains. To circumvent this problem, our group has used random mutagenesis with the chemical mutagen N-ethyl-N-nitrosourea (ENU) in the approach to functional genomics of infectious disease susceptibility. Induction of novel mutations, most of which are inherited in a recessive manner, is initially screened by experimental challenge of mice from a three-generation breeding scheme with S. Typhimurium (10). Similar strategies using chemically defined microbial structures and viruses have been successful in identifying novel targets in the mouse genome (11–13) and translating them to human primary immune deficiencies (14). This is the first report of the identification of a novel genetic determinant of susceptibility to a prevalent bacterial disease by direct challenge of ENU-mutagenized mice with the infectious agent.

In this paper, we describe the identification of a Salmonella-susceptible ENU mutant named Ity9 (immunity to Typhimurium locus 9) that carries a nonfunctional allele at the gene encoding ubiquitin-specific peptidase 18 (USP18; Usp18Ity9). USP18 has been shown to have dual independent functions as an ISG15 protease (15) and as a negative regulator of type 1 IFN signaling by binding to IFN (α and β) receptor (IFNAR) 2 (16, 17). Consistent with these functions, USP18 was reported to play a role in host defense as a negative regulator of antiviral activities (18–20).

Mice presenting a point mutation in Usp18 exhibit innate susceptibility to lethal S. Typhimurium infection, as measured by decreased survival and increased bacterial load in spleen and liver. Usp18Ity9/Usp18Ity9 mutants are more susceptible to LPS challenge and display an increased inflammatory response during Salmonella infection that is associated with increased type 1 IFN signaling through the activation of STAT1 and results in increased expression of IL-6 and type 1 IFN regulated genes. Contrasting with these enhanced activation effects, the Usp18Ity9/Usp18Ity9 mutant mice have impaired STAT4 phosphorylation and IFN-γ production during Salmonella infection. Our findings suggest that Salmonella susceptibility in Usp18Ity9/Usp18Ity9 mutant mice can be explained by the observed increase in bacterial load in target organs as a consequence of lower IFN-γ production, and by the heightened inflammatory response triggered by STAT1 hyperactivation.

Materials and Methods

Mice

All animal experiments were performed under conditions specified by the Canadian Council on Animal Care, and the animal use protocol was approved by the McGill University Animal Care Committee. The 129S1, DBA/2J, BALB/cJ, and C.129S2-Stat4tm1Gru/J mice were purchased from The Jackson Laboratory (Bar Harbor, ME). The generation of Usp18−/Usp18− mice has been described previously (21). The Usp18− allele was transferred to a FVB/J background by repeated backcrossing.

ENU mutagenesis

G0 129S1 males were mutagenized by the University of Toronto Centre for Modeling Human Disease group led by Dr. Janet Rossant. The 129S1 males received 150 mg/kg ENU i.p. once at 8–12 wk of age. Infertility of the treated mice following the treatment was confirmed, as well as the recovery of fertility 8–12 wk following the injection.

Genotyping

The genome scan was conducted using a panel of 768 single nucleotide polymorphisms (SNPs) (Mutation Mapping and Developmental Analysis Project, Brigham and Women's Hospital, Harvard Medical School, Boston, MA) (22). Additional genotyping was performed by microsatellite analysis using high-resolution agarose gels or by SNP sequencing. Genotyping of the L361F mutation in Usp18 was performed using custom TaqMan SNP genotyping assay (Applied Biosystems, Streetville, Ontario, Canada). DNA samples were purchased from The Jackson Laboratory. Wild-derived species Mus caroli/EiJ originates from Thailand, and Mus spretus/EiJ originates from Puerto Real, Cadix Province, Spain.

In vivo Salmonella infections

Mice were infected i.v. with 1000 CFUs S. Typhimurium isolate Keller, as previously described (23). Briefly, bacteria were grown in tryptic soy broth to an OD of 0.09, cooled to 4°C, and plated overnight on tryptic soy agar. The day after, the infectious dose was adjusted to 5000 CFUs/ml, and 0.2 ml was injected in the caudal vein of 6- to 8-wk-old mice of both sexes. For CFU determination, the mice were euthanized with CO2 at the given day postinfection, and the spleen and liver were aseptically removed, weighed, and homogenized with a Polytron (Kinematica, Bohemia, NY). The resulting homogenate was diluted in saline and plated on tryptic soy agar to determine the organ bacterial load.

LPS and poly I:C challenge

Mice were injected i.p. with the given amount of LPS K235 (no. L2143; Sigma-Aldrich, Oakville, Ontario, Canada) once or poly I:C (no. P0913; Sigma-Aldrich) daily for 4 d diluted in 0.5 ml saline and monitored for 5 d. Mice were monitored twice daily and euthanized when moribund, in accordance with the McGill University Ethics Committee.

Splenocytes

Spleens were aseptically removed from uninfected or S. Typhimurium-infected mice and transferred in PBS. They were briefly ground between the frosted area of two microscope slides. The cells were then dispersed by passages through 21- and 25-gauge needles to obtain a single-cell suspension. This suspension was layered over Lympholite-M (Cedarlane, Burlington, Ontario, Canada) and centrifuged at room temperature, 1460 × g, 20 min. The buffy coat was collected, diluted in RPMI, and pelleted at 485 × g for 5 min before treatment with RBC lysis solution (Invitrogen, Burlington, Ontario, Canada). After a final centrifugation, the cells were resuspended and their concentration adjusted to 2 × 106 cells/ml. The splenocytes were then either left untreated for 4 h to measure IFN-γ production or stimulated overnight with 1 μM CpG DNA (α DNA), 10 μg/ml zymosan (no. TLRL-ZYN; InvivoGen, San Diego, CA), 10 μg/ml LPS055:B5 (no. L6529; Sigma-Aldrich), 10 μg/ml lipoteichoic acid from S. aureus (no. L2515; Sigma-Aldrich), or 5 μg/ml imiquimod (no. TLRL-IMQ; InvivoGen) to measure IL-6 using a sandwich ELISA. For protein extraction, the splenocytes were left untreated for 4 h and then stimulated for 60 or 90 min with 100 U/ml IFN-β (no. I9032; Sigma-Aldrich, St. Louis, MO). For the kinetics of IFN-γ production, explanted splenocytes were left untreated for 16 h and then stimulated with IFN-β (100 U/ml) or IL-12 (10 ng/ml) for periods varying between 4 h and 24 h.

Bone marrow-derived macrophages

The femurs were collected and kept on ice in HBSS. Both ends were cut and the bone marrow extracted by flushing the femurs with 2 ml RPMI using a 26-gauge needle. Single-cell suspension was obtained by passing the media through a 27-gauge needle. RBCs were lysed for 5 min using an RBC lysis solution. The remaining cells were centrifuged 5 min at 485 × g and resuspended in 10 ml complete media (RPMI, 10% FCS, 20% L929 supernatant), left to adhere overnight in adherent Primaria petri (BD Biosciences, Missisauga, Ontario, Canada). The cell suspension was then transferred to a nonadherent tissue flask and maintained in culture for 5 d with the addition of L929 supernatant at days 2 and 4 prior to cell counting and plating.

Protein extracts

Protein extracts were prepared using the CellLytic-M reagent (no. C2978; Sigma-Aldrich) according to the manufacturer’s protocol. Proteins were quantified with the Non-Interfering Protein Assay Kit (EMD Chemicals, Gibbstown, NJ).

Western blots

Western blots were performed with the Novex system (Invitrogen) using 4–12% Bis-Tris gels according to the manufacturer’s protocols. The polyvinylidene difluoride membranes were blotted sequentially (when applicable) with pSTAT4(Tyr693) (Santa Cruz Biotechnology, Santa Cruz, CA), pSTAT1(Tyr701) (Cell Signaling, Danvers, CA), STAT4 (Cell Signaling), STAT1 (Cell Signaling), and actin (Sigma-Aldrich) or GAPDH (Cell Signaling) Abs, the membranes being stripped with Restore buffer (Fisher, Nepean, Ontario, Canada) in between. The blots were revealed using Immobilon (Millipore, Billerica, MA) and Hyper ECL films (GE Healthcare, Missisauga, Ontario, Canada).

Statistical analysis

Statistical analyses were performed using GraphPad Prism 4 (GraphPad Software, San Diego, CA).

Results

A mutation in Usp18 is responsible for the susceptibility of Ity9 mutant mouse to S. Typhimurium infection

The breeding scheme used to identify the Ity9 pedigree (Fig. 1A) involves generation 1 (G1) mice produced by two independently mutagenized generation 0 (G0) males. This strategy was used to increase the total number of mutations that could be screened in each G3 and to maintain screening of the X chromosome (10). The 129S1/SvImJ (129S1) mice were selected for mutagenesis and subsequently bred with C57BL/6J females to generate the G1 progeny. For each G1 pedigree, four G2 brother–sister pairs were mated to produce G3. Using this breeding scheme, the C57BL/6J SLC11A1Asp169 Salmonella-susceptibility allele was introduced into the G2 population. Animals from the G2 generation were genotyped for Slc11a1, and mice carrying the wild-type resistant allele (SLC11A1Gly169) were selected for further G3 breeding to prevent phenotypic variation caused by SLC11A1. Following phenotyping of G3 mice, the G2 male carrying the susceptibility allele was outcrossed to a DBA/2J female (SLC11A1Gly169), and the N2 animals were screened to confirm the heritability of the phenotype. Initial mapping of the mutation to chromosome 6 was established using six affected mice (Supplemental Fig. 1). Fine mapping with a total of 263 N2 progeny (Fig. 1B) delimited the mutation to a 1.3-Mb interval on chromosome 6. This region of the chromosome comprises 13 annotated genes, 2 predicted genes, and 1 noncoding RNA (Ensembl build 37.1). All the coding sequences and exon/intron boundaries in this region were sequenced, and only one guanine to thymine transversion in the gene Usp18 was found (Fig. 2A). This missense mutation causes a leucine to phenylalanine amino acid change at position 361 of USP18, a highly conserved amino acid through the amniotes (Fig. 2B). To confirm that the SNP for this codon is exclusive to our ENU mutant strain, a broad range of classical inbred and wild-derived Mus subspecies were sequenced (Fig. 2C). This region of the protein is involved in the binding of USP18 to the IFNAR2 (17), suggesting that the Usp18Ity9 mutation may interfere with the activation of type 1 IFN signaling.

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 G3 mice and to confirm the heritability of the phenotype using N2 mice. Females and males are respectively represented by ● and by ▪, whereas gray filling denotes the presence of heterozygous mutant alleles and white filling, homozygous mutant alleles. B, Fine mapping of the Ity9 locus to a 1.3-Mb 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 (arrow) causing a leucine to phenylalanine missense mutation at position 361 of USP18 was identified in the mutant pedigree. B, The alignment of USP18 orthologs showing conservation of the mutated amino acid in a broad range of eukaryotes. Identical amino acids are highlighted in yellow, and conserved amino acids are highlighted in blue. C, Alignment of the segment of the Usp18 sequence of inbred and wild derived mice compared with 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, and conserved nucleotides are highlighted in blue. The sequence alignments were done with AlignX (Invitrogen).

To confirm that Usp18 is indeed responsible for the Ity9 phenotype, we carried out allelic complementation assays in Usp18Ity9/Usp18− compound mutants. Usp18Ity9/Usp18+ mice on a DBA/2J background were crossed to Usp18−/Usp18− mice on an FVB/J background to generate F1 progeny. Of note, in this breeding scheme, all resulting F1 animals carried one copy of the wild-type Slc11a1 gene, precluding the impact of Slc11a1 on the analysis of the disease phenotype. Susceptibility to infection was measured by survival analysis in Usp18Ity9/Usp18− and Usp18+/Usp18− heterozygous mice (Fig. 3A, 3B). Usp18Ity9/Usp18− mice present the same degree of susceptibility to Salmonella infection (Fig. 3B) as do Usp18Ity9/Usp18Ity9 mice (Fig. 3A). The median survival time for Usp18Ity9/Usp18Ity9 does not significantly differ from that observed in Usp18Ity9/Usp18− compound heterozygotes (7.0 d versus 7.5 d; p = 0.1415). These results show the lack of allelic complementation between Usp18Ity9 and Usp18− and confirm that Usp18 is indeed the gene underlying Ity9. We did also evaluate survival of infection in Usp18−/Usp18− mice and compared their degree of susceptibility with that in FVB/J controls (Fig. 3C). Usp18−/Usp18− mice are significantly more susceptible to infection than are FVB/J mice. The mean survival time (MST) is 4.3 ± 1.0 in Usp18−/Usp18−- and 6.2 ± 0.5 in Usp18+/Usp18+ FVB mice (p = 0.004). Both groups of mice presented shorter MST compared with Usp18Ity9/Usp18Ity9 and Usp18Ity9/Usp18− because of the presence of a mutant allele at SLC11A1Asp169.

FIGURE 3.
FIGURE 3.

Usp18Ity9 allele confers S. Typhimurium susceptibility. A, Survival curves of N2 animals issued from affected F1 daughter produced in the original screen littermates infected i.v. with 1000 CFUs according to their Usp18 genotype. Usp18Ity9/Usp18Ity9 mice are represented by ▴ (n = 18), whereas Usp18Ity9/Usp18+ (n = 42) and Usp18+/Usp18+ (n = 17) are represented by ▪ and □, respectively. Results are representative of five independent experiments. B, Survival curves of F1 mice issued from a cross between Usp18Ity9/Usp18+ and Usp18−/Usp18− mice. Usp18Ity9/Usp18− mice are represented by ▴ (n = 22), and Usp18+/Usp18− are represented by ▪. Usp18Ity9/Usp18Ity9 and compound heterozygous Usp18Ity9/Usp18− mice are as susceptible to infection, validating the candidacy of Usp18 as the gene underlying Ity9. Results are representative of two independent experiments. C, Survival curves of Usp18−/Usp18− FVB congenic mice (●) and FVB mice (▪), confirming the susceptibility of USP18-deficient mice to Salmonella infection. Log-rank (Mantel–Cox) p = 0.004. Ity9, immunity to Typhimurium, locus 9.

Usp18Ity9/Usp18Ity9 mice present increased bacterial loads in target organs that are associated with decreased IFN-γ production

The higher susceptibility of the Usp18Ity9/Usp18Ity9 mice in terms of survival (Fig. 3A) correlates with a higher bacterial burden in the spleen (Fig. 4A) and liver (Fig. 4B) of infected mice at 3 and 6 d postinfection. At day 6, when susceptible mice first become moribund, homozygous mutant animals have a bacterial load that is 36-fold higher in the spleen and 55-fold higher in the liver compared with their heterozygous littermates. The complementation assay in Usp18Ity9/Usp18− compound mutant confirms that the USP18L361F mutation is responsible for the Ity9 increased bacterial load in the spleen and liver of mice 6 d postinfection (Fig. 4C). Higher bacterial load after Salmonella infection was also observed in the spleen and liver of Usp18−/Usp18− mice with levels that are 28- and 6-fold higher than in FVB/J controls in the liver and spleen, respectively (Fig. 4D). These results indicate that mice homozygous for the Ity9 mutation have markedly impaired control of Salmonella replication at two major sites during systemic infection, likely contributing to the disease phenotype.

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 d postinfection with 1000 CFUs of S. Typhimurium i.v. Usp18Ity9/Usp18+ (○) and Usp18Ity9/Usp18Ity9 (●). A one-tailed Mann–Whitney U test was performed for the spleen (A) and liver (B). A, *p = 0.0186; **p = 0.0017. B,*p = 0.0216; **p = 0.0082. C, Spleen and liver CFUs 6 d postinfection with 1000 CFUs of S. Typhimurium i.v. Usp18+/Usp18− (○) and Usp18Ity9/Usp18− (●). Two-tailed t test. ***p = 0.0002 (spleen); ***p = 0.0004 (liver). Of note, two Usp18Ity9/Usp18− mice were found dead at day 6 postinfection, excluding them from the CFU analysis. D, Spleen and liver CFUs 3 d postinfection with 1000 CFUs of S. Typhimurium i.v. Usp18−/Usp18− (●) and FVB (○). Mann–Whitney. *p = 0.0286 (liver). E, IFN-γ production from explanted splenocytes from S. Typhimurium-infected mice incubated for 4 h ex vivo in Usp18Ity9/Usp18+ (light gray) and Usp18Ity9/ Usp18Ity9 (dark gray) littermates. Two-way ANOVA. *p = 0.0211; n = 6/group. F, Serum IFN-γ concentration of uninfected mice or mice infected with S. Typhimurium at day 1 and day 6 postinfection in Usp18Ity9/Usp18+ (light gray) and Usp18Ity9/ Usp18Ity9 (dark gray) littermates. Two-tailed Mann–Whitney day 6. **p = 0.0087; n = 6/group. G, The 24-h kinetics of IFN-γ production from explanted splenocytes stimulated with IFN-β or IL-12. Two-way ANOVA (interaction). p = 0.0003; n = 3/group; independent two-way ANOVA according to the genotype for each time point: 2 h, *p = 0.0149; 8 h, ***p = 0.0007; 24 h, **p = 0.0074. These results are representative of results obtained in three (A, B, E, F), two (C, G), or one (D) independent experiment.

A constant feature of the Usp18Ity9 mutation is seen with respect to IFN-γ production during infection with S. Typhimurium (Fig. 4E, 4F). In the past we have observed and reported that, in Salmonella-susceptible mice, increased production of IFN-γ parallels increased bacterial load (23, 24). In Usp18Ity9/Usp18Ity9 mice the situation is different; despite an increased bacterial load in spleen and liver, circulating IFN-γ levels are 2.5 times lower in Usp18Ity9/Usp18Ity9 mice 6 d postinfection compared with wild-type littermates (Fig. 4F). In addition, explanted splenocytes from infected mutant mice produced less IFN-γ compared with control littermates (Fig. 4E). To evaluate the regulation of IFN-γ secretion by Usp18Ity9, in vitro stimulation of splenocytes with IL-12 or IFN-β was performed (Fig. 4G). In the Usp18Ity9/Usp18Ity9 splenocytes, peak IFN-γ production was observed 2 h after IL-12 or IFN-β stimulation, and no detectable levels of IFN-γ were present by 8 h; in contrast, among controls the peak IFN-γ production was observed 4–8 h post stimulation and was sustained for up to 24 h. These observations indicate that both the IL-12– and the IFN-β–dependent pathways leading to IFN-γ production are affected by the Ity9 mutation. Ifng transcription correlates well with protein production, as Ifng mRNA was diminished in the mutant splenocytes compared with control mice infected with S. Typhimurium for 6 d (Supplemental Figs. 2A, 3A). Low levels of IFN-γ could explain higher bacterial growth in the spleen and liver of Salmonella-infected Usp18Ity9/Usp18Ity9 mice.

Usp18Ity9/Usp18Ity9 mice present increased responsiveness to pathogen-associated molecular patterns and Salmonella-induced septic shock

To further investigate the pathogenesis of decreased resistance to infection in Usp18Ity9/Usp18Ity9 mice, we looked at the hematologic manifestations of Salmonella infection and the host inflammatory response to pathogen-associated molecular patterns (PAMPs) in vivo and ex vivo. We have shown previously that mice of different genetic backgrounds infected with S. Typhimurium present a progressive anemia and a lymphopenia during infection (8, 23). During the course of Salmonella infection in Usp18Ity9/Usp18Ity9mice, we observed a mild anemia in combination with a moderate lymphopenia and a marked thrombocytopenia. The lymphopenia was not observed in wild-type animals, and the thrombocytopenia was not as severe (Supplemental Table I). The administration of poly I:C, a TLR3 agonist, in Usp18Ity9/Usp18Ity9 mice had also a dramatic impact on circulating lymphocytes and platelets, reflecting an increased activation of the immune system.

Usp18Ity9/Usp18Ity9 mice showed an increased susceptibility to lethal in vivo challenge with LPS, a major PAMP of Salmonella, as shown by decreased survival after LPS administration in vivo (Fig. 5A–C). This observation suggests an exacerbation of the inflammatory response induced by type 1 IFN in response to TLR activation by LPS or Salmonella. In fact, Salmonella-induced expression of IFN-β mRNA in Usp18Ity9/Usp18Ity9 macrophages was increased compared with that in wild-type littermates (Supplemental Figs. 2B, 3D), supporting a role for TLR-dependent IFN-β–induced shock in our model.

FIGURE 5.
FIGURE 5.

Usp18Ity9/Usp18Ity9 mice are more susceptible to LPS challenge. Survival of Usp18Ity9/Usp18Ity9 (▴) and control littermates Usp18Ity9/Usp18+ (▪) challenged with 10 μg (A), 100 μg (B), or 500 μg (C) of LPS IV. Survival curves comparison for the 500-μg challenge. ***p = 0.0002. D, Serum IL-6 concentration in Usp18Ity9/Usp18+ (light gray) and Usp18Ity9/Usp18Ity9 (dark gray) mice infected with S. Typhimurium at day 0, day 1, and day 6 postinfection. Two-way ANOVA. p < 0.0001; n = 3/group. IL-6 (E) and TNF (F) production of explanted splenocytes from Usp18Ity9/Usp18+ (light gray) and Usp18Ity9/Usp18Ity9 (dark gray) littermates stimulated overnight with an array of PAMPs. Two-way ANOVA. E, p = 0.0023; F, p = 0.0084; n = 3/group. Two-tailed t tests were also performed for each treatment independently (E). *p = 0.05; **p = 0.002. These results are representative of those obtained in three (A–D) or two (E, F) independent experiments.

Early during Salmonella infection, different immune cell types, including macrophages, neutrophils, and lymphocytes, induce cytokines (TNF and IL-6, for example) that mediate inflammation and are involved in the pathogenesis of septic shock (5). A cardinal feature of Salmonella infection in Usp18Ity9/Usp18Ity9 mice is the presence of high IL-6 levels in circulation and in infected tissues and cells. This is observed in vivo in the serum of Usp18Ity9/Usp18Ity9 mice following S. Typhimurium infection (Fig. 5D). Compared with control cells, Usp18Ity9/Usp18Ity9 splenocytes also secreted a significantly higher level of IL-6 following in vitro stimulation with LPS and zymosan (Fig. 5E), although no statistically significant differences in the production of TNF were detected (Fig. 5F). High levels of IL-6 production, together with high expression of Ifnb and additional IFN-regulated genes (Cxcl10, Irf7), in splenocytes (Supplemental Figs. 2A, 3B) and Il15, Isg15, and Mx1 in bone marrow-derived macrophages (BMDMs) (Supplemental Figs. 2B, 3E) during Salmonella infection most likely contribute to the development of septic shock in the Usp18Ity9/Usp18Ity9 mice during infection. The heightened response to LPS can synergize with the increased bacterial load to lower survival of the Usp18Ity9/Usp18Ity9 mice during Salmonella infection.

STAT1 induction and STAT4 repression by type 1 IFN in response to Salmonella infection in Usp18Ity9/Usp18Ity9 mice

With the aim of dissecting the signaling modifications caused by the Usp18Ity9 mutation that could explain the opposite impact of the mutation on IFN-γ and IL-6 production, the expression and phosphorylation of STAT1 and STAT4 were evaluated in BMDMs (Fig. 6A) and splenocytes (Fig. 6B, 6C). STAT1 is known to be phosphorylated after type 1 IFN binding to the IFNAR, and its phosphorylation is increased in Usp18−/Usp18− mice (25). In both cell types, basal STAT1 and phosphorylated STAT1 (pSTAT1) levels were higher in Usp18Ity9/Usp18Ity9 mice than in control littermates (Fig. 6). STAT1 was highly expressed in splenocytes during Salmonella infection (Fig. 6B) and in BMDMs 24 h after stimulation with IFN-β (data not shown). Hyperactivation of the JAK/STAT pathway in the Usp18Ity9/Usp18Ity9 mice is consistent with high IL-6 production and higher susceptibility of the mice to cytokine-mediated septic shock.

FIGURE 6.
FIGURE 6.

Critical role of Stat4 in the host response to S. Typhimurium infection in vivo. A and B, Western blot of protein extracts from (A) BMDMs stimulated with 100 U/ml of IFN-β for 0.5, 1, 2, and 4 h probed with pSTAT1, anti-STAT1, and anti-GAPDH. Explanted splenocytes from mice uninfected (D0) or infected (D6) with a 1000 CFUs of S. Typhimurium i.v. for 6 d and either left unstimulated or stimulated with 100 U/ml of IFN-β probed with (B) anti-pSTAT1, anti-STAT1, anti–β-ACTIN, and (C) pSTAT4, STAT4, and anti–β-ACTIN. These results are representative of results obtained with six independent mice. D, Survival curves of BALB/cJ (○), Stat4tm1Gru/ Stat4tm1Gru (□), Stat4tm1Gru/Stat4wt (▪), and 129S1 (●) mice infected with 1000 CFUs i.v. 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 d for BALB/cJ, 6.6 ± 0.7 d for Stat4tm1Gru/Stat4tm1Gru, and 8.2 ± 1.2 d for Stat4tm1Gru/Stat4wt. E, Bacterial burden in the liver and spleen of Stat4tm1Gru/Stat4tm1Gru (●) and Stat4tm1Gru/Stat4wt (○) mice infected with 1000 CFUs i.v. of Salmonella 4 d postinfection. Mann–Whitney two-tailed. *p = 0.0262; ***p = 0.0002. F, Serum IFN-γ concentration of Stat4tm1Gru/Stat4tm1Gru (●) and Stat4tm1Gru/Stat4wt (○) mice infected with S. Typhimurium at day 4 postinfection. Unpaired t test, two-tailed with Welch’s correction. ***p < 0.0001. pSTAT1, anti-phosphoSTAT1; pSTAT4, anti-phosphoSTAT4.

STAT4 is important for IFN-γ induction, and type 1 IFNs have been reported to activate STAT4. During viral infection, a high STAT1 level negatively correlates with type 1 IFN-induced phosphorylation of STAT4 and the resulting production of IFN-γ (26). In our model, basal STAT4 levels were not different in the splenocytes of Usp18Ity9/Usp18Ity9 mice and control littermates. During infection, pSTAT4 levels decreased at D6 in both the mutant and the control mice; however, lower levels of pSTAT4 were consistently observed in Usp18Ity9/Usp18Ity9 mice when the day 0 (D0) splenocytes were stimulated with IFN-β (Fig. 6C). The ratios of pStat4/Stat4 after quantification of the bands and adjustment for loading were significantly lower in Usp18Ity9/Usp18Ity9 compared with littermate controls by a factor of 26%, both prior infection and at day 6 postinfection. These observations are consistent with the competition between STAT1 and STAT4 at the IFNAR level for phosphorylation. Because STAT4 is central to IFN-γ production through both the IL-12 and the IFN-β pathways, lower pSTAT4 levels contribute to the decreased IFN-γ production observed in Usp18Ity9/Usp18Ity9 mice.

To further demonstrate the importance of STAT4 during infection, we did challenge Stat4 knockout mice (http://jaxmice.jax.org/strain/002826.htm) with S. Typhimurium (Fig. 6D). The Stat4 gene is located on chromosome 1 ∼20 Mb proximal to Slc11a1. Stat4tm1Gru mice were transferred onto a BALB/cJ background, a strain that normally carries a mutant Slc11a1 allele; however, during the creation of the congenic strain, the 129S2 wild-type allele of Slc11a1 was transferred with the Stat4 null allele (data not shown). To create appropriate controls for this particular experiment, we crossed C.129S2-Stat4tm1Gru/J to BALB/cJ mice to create F1 progeny that were backcrossed to C.129S2-Stat4tm1Gru/J. All progeny issued from the backcross carried at least one copy of the wild-type allele at Slc11a1 and were either Stat4−/− or Stat4wt/−. We showed that mice deficient for Stat4 are significantly more susceptible to infection than mice carrying one (Stat4wt/−) (p = 0.0004) or two (129S1) (p < 0.0001) wild-type alleles at Stat4. These results confirm the importance of STAT4 during the acute systemic model of S. Typhimurium infection.

Discussion

The past few years have validated the use of chemical mutagenesis in the mouse as a powerful functional genomics tool to produce genetic variants for immunologic/host defense functions and to study diverse physiologic and pathologic conditions (27, 28). This strategy is particularly attractive because it does not require assumptions about the nature of the immunologic defect and approaches more closely the genetic analysis of natural variation in populations and the possibility to translate the research findings into improved understanding of the molecular basis of susceptibility to infectious diseases in humans. In this paper, we present the identification of a novel Salmonella-susceptibility gene using a direct primary recessive screen with an important bacterial human pathogen.

In this paper, we report the molecular basis of Ity9 as being USP18. The evidence leading to this conclusion is the following: The mutation identified in USP18 (L361F) was the sole coding change within the critical genomic interval; L361 is highly conserved in all mouse strains tested and other animal species; and, finally, complementation assays in vivo using Usp18Ity9/Usp18− compound mutants have shown unambiguously that Usp18 is indeed the gene underlying the locus Ity9. USP18, also known as UBP43, belongs to the deubiquinating protease family. USP18 is expressed in the thymus and macrophages of adult mice, as well as in hematopoietic cell lines, and its expression can be modulated by IFN and LPS. The protein has a de-ISGylation activity specific for the ubiquitin-like protein ISG15 and is involved in the negative regulation of type 1 IFN signaling by interacting with IFNRA2 and limiting JAK–STAT1 activation. Characterization of USP18 confirmed a critical role for this protease in the downregulation of the JAK–STAT pathway by preventing the interaction between JAK1 and the IFNAR2 (17). Some genes involved in the JAK–STAT pathway (Tyk2, Jak3, STAT1) have been shown in mouse models of infection and in rare human immunodeficiencies to play a critical role in the host response to bacterial, viral, and parasitic infections (4).

Overall, the data presented in this paper are consistent with the fact that the mutation identified in Usp18 interferes with the type 1 IFN signaling pathway, resulting in increased inflammatory response triggered by STAT1 hyperactivation that is responsible for the susceptibility of Usp18Ity9/Usp18Ity9 mice to LPS-induced septic shock. Hyperactivation of STAT1 also has an impact on type 1 IFN-mediated STAT4 phosphorylation for IFN-γ production in mutant mice, resulting in increased bacterial load in target organs and increased susceptibility to Salmonella infection. We did not observe any functional difference in phagocytic capabilities, bacterial killing, or pyropoptosis of BMDMs from Usp18Ity9/Usp18Ity9 mice (data not shown), excluding a direct effect of the mutation on macrophage function and highlighting the importance of analyzing the impacts of this mutation at the whole-organism level.

Type 1 IFNs are traditionally viewed and have been best described as antiviral cytokines; they have been shown to play a major role in LPS-induced shock, as revealed by the observation that mice deficient in the production of type 1 IFN as a consequence of a disruption of either TYK2, a JAK protein tyrosine kinase family member engaged at the IFNAR1, or the adaptor protein TRIF are resistant to the toxic effect of LPS (29, 30). IFN-β production by bacterial stimulation can occur through at least two recognition pathways that are either TLR dependent or TLR independent (reviewed in Ref. 31). TLR3, -4, -7, and -9 are involved in the production of type 1 IFN through ligand recognition (dsRNA for TLR3, LPS for TLR4, ssRNA for TLR7, and unmethylated CpG DNA for TLR9) and recruitment of the adaptor proteins MyD88 (TLR4, TLR7, TLR9) and/or TRIF (TLR4, TLR3). In the case of Salmonella recognition, TLR4 stimulation is the most likely candidate pathway leading to the induction of IFN-β. Secreted IFN-β then signals through the IFNAR and stimulates the ISGF3-mediated production of several IFN-inducible genes, a pathway that is hyperactivated in Usp18Ity9/Usp18Ity9 mice. As a consequence, high IL-6 levels detected in the circulation and in the infected tissues of Usp18Ity9/Usp18Ity9 mice most likely contribute to the development of septic shock in these mice (32), a cellular response that may be amplified by increased bacterial load in spleen and liver.

IFN-γ, also known as type 2 IFN, is a key cytokine modulating host defense during Salmonella infections. The injection of IFN-γ (33) or Abs against IFN-γ (34) prior to infection affects the outcome of the infection by, respectively, enhancing or reducing resistance of the mice to bacterial challenge in terms of bacterial replication. During Salmonella infection, NK cells are important IFN-γ producers, and their depletion results in increased bacterial load in spleen and liver (35, 36) and impaired survival to infection (37). IFN-γ is also a key cytokine in human Salmonella infections, as defects in the IL-12/IFN-γ pathway increase susceptibility toward Salmonella and mycobacterial intracellular pathogens (1, 2). The IFN-γ production from cultured splenocytes stimulated with heat-killed Salmonella was later shown to be at least partly dependent on IFN-β–induced STAT4 phosphorylation (38). In fact, the ratio of STAT4 and STAT1 is crucial for IFN-γ production by splenic NK cells during viral (lymphocytic choriomeningitis virus) infection (26). STAT4 was shown to be highly expressed in resting NK cells, which allows them to produce IFN-γ early during infection. Later during the course of lymphocytic choriomeningitis virus infection, STAT1 expression is increased in NK cells and STAT4 phosphorylation is inhibited (26). In our model, an analogous increase in STAT1 expression is seen in splenocytes during the course of infection, which may account for the reduced levels of STAT4 phosphorylation and subsequent IFN-γ deficiency leading to the observed increase in bacterial burden. More importantly, we have shown for the first time using Stat4-deficient mice that STAT4 is an important player in the host response to infection with S. Typhimurium.

Usp18Ity9/Usp18Ity9 mice share several phenotypic similarities with Usp18-deficient mice. Usp18−/Usp18− and Usp18Ity9/Usp18Ity9 mice are more sensitive to LPS-induced shock in vivo, as measured by percentage of mortality (current study and Ref. 39). In the current model of infection, Usp18Ity9/Usp18Ity9 and Usp18−/Usp18− mice showed increased susceptibility to infection with S. Typhimurium in terms of decreased survival and increased bacterial load in spleen and liver. These observations are different from a previous report in which Usp18−/Usp18− mice were shown to exhibit lower bacterial load in spleen and liver compared with wild-type mice after Salmonella infection, although the survival curves between the two groups of mice were not different (39).

The fact that USP18 has been associated in different studies with either decreased or increased susceptibility to Salmonella infection reflects the importance of the genetic background of the mice, as well as the route and dose of infection, in the development of clinical disease. In previous studies, the Usp18− allele was transferred to a mixed C57BL6/129S6 background, where the mutation in the gene Slc11a1 (Asp169), a major determinant of Salmonella susceptibility, segregated. It was shown previously by us (8, 40, 41) and others (42) that the allele present at Slc11a1 (Gly169 versus Asp169) is of most importance during the evaluation of Salmonella susceptibility in mutant mice. In addition, the model of infection was using the i.p. route (i.v. in the current study) with a small inoculum (40 CFUs versus 1000 CFUs). During mild infection, it is conceivable that enhanced type 1 IFN signaling in the absence of USP18 could be transiently beneficial with respect to bacterial growth. In addition, the impact of the route of infection during Salmonella infection has been previously shown to affect disease outcome by mobilizing specific inflammatory responses (43, 44).

Collectively, the current results indicate that in our model, mutations within Usp18 cause susceptibility to Salmonella infection. Susceptibility of Usp18Ity9/Usp18Ity9 mice to infection was also observed using an oral infection model (E. Richer and D. Malo, unpublished observations), confirming the detrimental role of USP18 dysfunction during Salmonella infection. The comparable profiles of survival and Salmonella replication in Usp18Ity9/Usp18Ity9, Usp18−/Usp18−, and compound heterozygous (Usp18Ity9/Usp18−) mice suggest strongly that the Usp18F361 allele at Ity9 is a null allele, identifying a new residue essential for the function of the protein.

In conclusion, these studies establish a critical role of USP18 in host resistance to infection with Salmonella and illustrate the complex interplay between type 1 and type 2 IFN in antimicrobial defense mechanisms, providing better insight into the mechanisms involved in type 1 IFN signaling during Salmonella infection. In humans, only a few genetic factors known to influence the predisposition to pediatric primary infection with Salmonella have been identified and involve polymorphisms within the IL-12/-23/IFN-γ–mediated axis (reviewed in Ref. 3). The patients were defective either in IFN-γ production (as observed in our model) or in responding to IFN-γ, owing to mutations in the genes encoding for IFNGR1, IFNGR2, IL12B, IL12RB1, or STAT1 (2, 45–47). In this paper, we validate the use of ENU mutagenesis to identify novel important genes in host susceptibility to Salmonella infection. These discoveries will be of utmost importance in elucidating novel disease mechanisms, particularly the involvement of type 1 IFN signaling and USP18, in human studies.

Acknowledgments

We thank Janet Rossant for providing the G0 mice and Nadia Prud’homme, Danica Albert, Line Larivière, Isabelle Angers, and Rosalie Wilkinson for technical assistance.

Disclosures The authors have no financial conflicts of interest.

Footnotes

  • This work was supported by National Institutes of Health Grant HL091549 (to D.E.Z.), the New Emerging Team program of the Canadian Institutes of Health Research (to S.M.V., S.T.Q., and D.M.), and the Research Institute of the McGill University Health Centre (to S.M.V., S.T.Q., and D.M.). E.R. received a Fonds de la Recherche en Santé du Québec fellowship. S.V. and S.Q. hold Canada Research Chairs. D.M. is a McGill Dawson Scholar.

  • The online version of this article contains supplemental material.

  • Abbreviations used in this paper:

    BMDM
    bone marrow-derived macrophage
    ENU
    N-ethyl-N-nitrosourea
    IFNAR
    IFN (α and β) receptor
    Ity9
    immunity to Typhimurium, locus 9
    MST
    mean survival time
    PAMP
    pathogen-associated molecular pattern
    pSTAT1
    anti-phosphoSTAT1
    pSTAT4
    anti-phosphoSTAT4
    SNP
    single nucleotide polymorphism
    S. Typhimurium
    Salmonella enterica serovar Typhimurium
    USP18
    ubiquitin-specific peptidase 18.

  • Received March 17, 2010.
  • Accepted July 6, 2010.

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