The Journal of Immunology N-Ethyl-N-Nitrosourea–Induced Mutation in Ubiquitin-Specific Peptidase 18 Causes Hyperactivation of IFN-ab Signaling and Suppresses STAT4-Induced IFN-g Production, Resulting in Increased Susceptibility to Salmonella Typhimurium Etienne Richer,* ,†,‡ Caitlin Prendergast, ‡ Dong-Er Zhang, x,{ Salman T. Qureshi, †,‡ Silvia M. Vidal,* ,‡,‖ and Danielle Malo* ,†,‡ 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-g production during bacterial infection. Increased STAT1 phosphorylation correlated with impaired STAT4 phosphorylation, resulting in overwhelming IL-6 secretion but reduced IFN-g production during infection. The reduced IFN-g 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. The Journal of Immunology , 2010, 185: 3593–3601. S almonella 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. Accu- mulated data in humans suggest that predisposition to pediatric primary infection with Salmonella involves functional polymor- phisms within genes in the IL-12-dependent, IFN-g–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 crit- ical 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 (SLC11A1 G169D in C57BL/6J and BALB/cJ; TLR4 P712H in C3H/HeJ or PKLR I90N 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 muta- gen 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 *Department of Human Genetics, † Department of Medicine, ‡ Centre for the Study of Host Resistance, and ‖ Department of Microbiology and Immunology, McGill Uni- versity, Montre ´al, Que ´bec, Canada; and x Department of Pathology and { Division of Biological Sciences, Moores University of California San Diego Cancer Center, University of California San Diego, La Jolla, CA 92093 Received for publication March 17, 2010. Accepted for publication July 6, 2010. 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 Sante ´ du Que ´bec fellowship. S.V. and S.Q. hold Canada Research Chairs. D.M. is a McGill Dawson Scholar. Address correspondence and reprint requests to Dr. Danielle Malo, McGill Life Sciences Complex, Bellini Building, 3649 Promenade Sir-William-Osler, Montre ´al, Que ´bec, H3G 0B1, Canada. E-mail address: danielle.malo@mcgill.ca 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 (a and b) receptor; Ity9, immunity to Typhimurium, locus 9; MST, mean survival time; PAMP, pathogen-associated molec- ular pattern; pSTAT1, anti-phosphoSTAT1; pSTAT4, anti-phosphoSTAT4; SNP, single nucleotide polymorphism; S. Typhimurium, Salmonella enterica serovar Typhi- murium; USP18, ubiquitin-specific peptidase 18. 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