Fig 1.
Activation of the type I IFN receptor (IFNAR) leads to the formation of the interferon stimulated gene factor 3 complex ISGF3 (STAT1:STAT2:IRF9 complex) which binds to the IFN stimulated response element (ISRE) promoter sequence leading to the activation of various interferon stimulated genes (ISGs) such as isg15, irf7 and cxcl10.
Both IFNAR and IFNGR activation leads to the formation of STAT1 homodimers that bind to GAS sequence leading to the production of additional ISGs.
Fig 2.
Survival of C57BL/6, Stat2-/- and Stat1-/- mice after oral S. Typhimurium infection.
Percent survival of C57BL/6 (n = 18) black line, Stat1-/- (n = 11) red line, and Stat2-/- (n = 13) blue line mice for 8 days post oral 109 Salmonella Typhimurium infection following streptomycin pretreatment. The results from three independent experiments with at least 3 mice in each group were combined. Stat2-/- infected mice survived significantly longer, p = 0.0026.
Fig 3.
Transcript levels of various type I IFN stimulated genes in ceca of C57BL/6 and Stat2-/- mice 48 hours after S. Typhimurium infection.
C57BL/6 and Stat2-/- mice were orally infected with 109 STM following streptomycin pretreatment. Transcript levels of Irf7, Isg15, Irgm1, Oas1b, Rsad1 and Cxcl10 were determined by qPCR in the cecum of infected mice 48 post infection. Data was normalized to uninfected mice from each group. Each data point represents one analyzed mouse sample. Mean and SE were calculated by averaging results from three independent experiments. *p <0.05 as determined by Students t-test.
Fig 4.
Transcript levels of genes classically associated with S. Typhimurium infection in ceca of C57BL/6 and Stat2-/- mice 48 hours after S. Typhimurium infection.
C57BL/6 and Stat2-/- mice were orally infected with 109 STM following streptomycin pretreatment. Expression levels of Tnfα, Il6 Ifnγ and Mcp1 were determined by qPCR in the cecum of infected mice 48 post infection. Data were normalized to uninfected mice from each group. Each data point represents one analyzed mouse sample. Mean and SE were calculated by averaging results from three independent experiments. *p <0.05 as determined by Students t-test.
Fig 5.
Bacterial burdens in various organs in C57BL/6 and Stat2-/- infected mice following S. Typhimurium infection.
C57BL/6 and Stat2-/- mice were orally infected with 109 CFU of S. Typhimurium following streptomycin pretreatment. Forty-eight hours following infection mice, were euthanized and the colon contents, cecum, mesenteric lymph nodes (MLN), and spleen were collected and plated for bacterial enumeration. Mean and SE were calculated by averaging results from three independent experiments. *p <0.05 as determined by Students t-test.
Fig 6.
Neutrophil influx and luminal oxygenation promotes S. Typhimurium survival.
C57BL/6 and Stat2-/- mice were orally infected with 109 CFU of S. Typhimurium following streptomycin pretreatment. A. Neutrophils were counted in ten fields in H&E stained tissue sections and the number of neutrophils were averaged for each sample. B. The enzymatic activity of MPO was quantified in fecal samples by ELISA. C. Paraffin embedded sections were stained with anti-MPO antibody (green) as well as DAPI (blue) to mark the nucleus. Images were captured using SPOT imaging software at 40x. C57BL/6 and Stat2-/- mice were orally infected with a 1:1 ratio of wild type S. Typhimurium and cyxA mutant mutant following streptomycin pretreatment. Mice were euthanized 4 days after infection and the competitive index (CI, output ratio of WT/cyxA mutant divided by input ratio of WT/cyxA mutant) was calculated in the D. colon contents and E. liver. F. colons of WT and Stat2-/- mice infected with 1:1 ratio of WT S. Typhimurium: cyxA mutant were collected 4 days post infection and paraffin embedded. Tissues were stained for hypoxia using the pimonidazole hypoxia probe (red) and DAPI (blue). Images were captured at 63x using Leica confocal microscope. Mean and SE were calculated by averaging results from three independent experiments. *p <0.05, ** p<0.01 as determined by Students t-test.
Fig 7.
16S rRNA profiling of the microbiome from the S. Typhimurium infected wild-type and Stat2-/- mice following 5 weeks of co-housing.
A. Phylum level microbiota composition was determined in uninfected and infected C57BL/6 and Stat2-/- mice through 16S rRNA analysis in fecal DNA samples as well as B. cecal DNA samples. C. Relative abundance of Bacteroidetes was determined in fecal DNA samples from uninfected and infected C57BL/6 and Stat2-/- mice D. Relative abundance of Proteobacteria was determined in fecal DNA samples from uninfected and infected C57BL/6 and Stat2-/- mice. E. CFU/g of S. Typhimurium was enumerated in the colon contents of C57BL/6 and Stat2-/- mice 48 hours post infection. Mean and SE were calculated by averaging results *p <0.05, as determined by Students t-test.
Fig 8.
Neutrophil analysis in co-housed wild-type and Stat2-/- mice infected with S. Typhimurium.
A. for the cecum of uninfected and S. Typhimurium infected wild-type C57BL/6 and Stat2-/- mice. B. Average neutrophils (Cumulative histopathology score PMN) numbers enumerated in ten fields from cecum of uninfected and S. Typhimurium infected wild-type C57BL/6 and Stat2-/- mice. C. Flow cytometry analysis of cecal cell suspensions. Neutrophils were gated after duplet and dead cell elimination for the following markers; CD45+, CD3-, CD56-, CD19-, Ly6G+ D. Percent of live cells as determined by flow cytometry for C57BL/6 uninfected, C57BL/6 S. Typhimurium infected and Stat2-/- S. Typhimurium infected mice. E. Superoxide anion (O2-) generation in response to fMLP in the presence or absence of IFNα by bone marrow neutrophils isolated from wild-type and Stat2-/- mice. Mean and SE were calculated by averaging results from three independent experiments. *p <0.05, ** p<0.01 as determined by Students t-test.
Table 1.
Primers used for qPCR.