(d) Serum IL-6 concentration inUsp18Ity9/Usp18+(light grey) andUsp18Ity9/Usp18Ity9(dark grey) mice infected withSalmonellaTyphimurium at day 0, day 1 and day 6 post-infection. with the increased inflammation rationalize theSalmonellaTyphimurium susceptibility in terms of increased bacterial load in target organs and cytokine-induced septic shock and death. == Introduction == Salmonella entericaspecies includes a number of closely related serovars capable of causing serious infections in humans and animals. Typhoid fever caused bySalmonella entericaserovar 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, non typhoidSalmonellasuch asSalmonella entericaserovar Typhimurium (SalmonellaTyphimurium) andSalmonellaEnteritidis infections usually present as self limiting gastroenteritis, although a certain Olodaterol percentage of these infections may become invasive and result in septicemia. Accumulated data in humans Olodaterol suggest that predisposition to pediatric primary infection withSalmonellainvolves functional polymorphisms within genes in the IL-12-dependent, IFN–mediated immunity (13). Olodaterol 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 withSalmonellaTyphimurium causes a typhoid-like disease where 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 toSalmonellaTyphimurium is observed among different inbred mouse strains due Rabbit Polyclonal to GSK3beta to the presence of specific mutations that are inherited as monogenic traits (Slc11a1G169Din C57BL/6J and BALB/cJ; Tlr4P712Hin C3H/HeJ or PklrI90Nin AcB61 mice) (68) 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, functional genomics of infectious disease susceptibility has been approached by our group using random mutagenesis with the chemical mutagen, N-ethyl-N-nitrosourea (ENU). Induction of novel mutations, most of which are inherited in a recessive manner, are initially screened by experimental challenge of mice from a three generation breeding scheme withSalmonellaTyphimurium (10). Similar strategies using chemically defined microbial structures and viruses have been successful in identifying novel targets in the mouse genome (1113) 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 the ENU-mutagenized mice with the infectious agent. In the current report, we describe Olodaterol the identification of aSalmonella-susceptible ENU-mutant namedIty9(Immunity to Typhimurium locus 9) that carries a non functional allele at the gene encoding USP18 (Usp18Ity9). USP18 has been shown to have dual independent functions as an ISG15 protease (15) and as a negative regulator of the type 1 IFN signaling by binding to the IFN (alpha and beta) receptor 2 (IFNAR2) (16,17). Consistent with these functions, USP18 was reported to play a role in host defense as a negative regulator of antiviral activities (1820). Mice presenting a point mutation inUsp18exhibit innate susceptibility to lethalSalmonellaTyphimurium infection as measured by decreased survival and increased bacterial load in spleen and liver.Usp18Ity9/Usp18Ity9mutants are more susceptible to LPS challenge and display an increased inflammatory response duringSalmonellainfection 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, theUsp18Ity9/Usp18Ity9mutant mice have impaired STAT4 phosphorylation and IFN- production duringSalmonellainfection. Our findings suggest thatSalmonellasusceptibility inUsp18Ity9/Usp18Ity9mutant mice can be explained by the observed increased in bacterial load in target organs as a consequence of lower IFN- 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. 129S1, DBA/2J, BALB/cJ and C.129S2-Stat4tm1Gru/J were purchased from The Jackson Laboratory. The generation ofUsp18/Usp18mice has been described previously (21). TheUsp18allele was transferred to a FVB/J background by repeated backcrossing. == ENU mutagenesis == G0 129S1 males were mutagenized by the University of Toronto CMHD group led by Dr. Janet Rossant. The 129S1 males received 150 mg/kg of ENU intraperitonally once at 812.