Gene expression in IL-10-deficient and wild-type mice following Citrobacter rodentium infection
ABSTRACT: To further understand immune mechanims involved in regulating intestinal inflammation, we employed whole genome microarray expression profiling as a discovery platform to identify genes with the potential of regulating inflammation in the absence of IL-10. Whole colon tissue from IL-10-deficient and C57BL/6 (wild-type) mice was collected 2 weeks after Citrobacter rodentium infection and from uninfected controls. Consistent with the histological and cellular analysis, expression levels of many chemokines and cytokines involved in recruiting leukocytes and promoting inflammation were, on average, lower in IL-10 deficient compared to wild-type mice after infection. An exception to this general trend was IL-27, a cytokine with both pro- and anti-inflammatory properties. Two weeks after Citrobacter rodentium challenge, total RNA was extracted and analyzed from whole colon tissue of infected IL-10-deficient and wild-type mice, and compared to uninfected controls. Each sample contained equal amounts of total RNA from 4-5 female mice which were pooled and used in the experiment.
Project description:Transcriptional profiling of intestinal response to Citrobacter rodentium in wild-type and Nlrp12-deficient mice Four-conditions experiment, Nlrp12-deficient mouse infected by Citrobacter rodentium at day 7 versus non-infected Nlrp12-deficient mice with two biologicals replicates , Wild-type mouse infected by Citrobacter rodentium at day 7 versus non-infected Wild-type mice with two biologicals replicates and Nlrp12-deficient mouse infected by Citrobacter rodentium versus Control mouse infected by Citrobacter rodentium at 2 differents times ( day 0 and post infection at day 7 ) with three biologicals replicates
Project description:An experiment to test the equivalence of the Citrobacter rodentium RegA and E.Coli SMS 3-5 homologue 8 Conditions (4 Genotypes by 2 treatments) in duplicate. Reference design without dye swaps.
Project description:Retinoic-acid receptor-related orphan receptor-γt-positive (RORγt+) innate lymphoid cells (ILCs) produce interleukin (IL)-22 and IL-17, which are critical for protective immunity against enteric pathogens. The molecular mechanism underlying the development and survival of RORγt+ ILCs is not thoroughly understood. Here we show that Dedicator of cytokinesis 8 (DOCK8), a scaffolding protein involved in cytoskeletal rearrangement and cell migration, is essential for the protective immunity against Citrobacter rodentium. A comparative RNA sequencing-based analysis reveals an impaired induction of antimicrobial peptides in the colon of DOCK8-deficient mice, which correlates with high susceptibility to infection and a very low number of IL-22-producing RORγt+ ILCs in their GI tract. Furthermore, DOCK8-deficient RORγt+ ILCs are less responsive to IL-7 mediated signaling, more prone to apoptosis and produce less IL-22 due to a defect in IL-23-mediated STAT3 phosphorylation. Our studies reveal an unsuspected role of DOCK8 for the function, generation and survival of RORγt+ ILCs. Control and DOCK8 KO mice were infected with 2X109 CFU of Citrobacter rodentium and day 8 post infection mice were sacrificed and their colons were harvested (n=5) . Total RNA was purified from the infected colons with RNeasy mini kit (Qiagen). RNA sequencing was performed (pooled RNA sample from five mice in each group) at Genomic Core Facility Southwestern Medical Center, University of Texas.
Project description:Identification of the targets of RegA with and without bicarbonate stimulation by comparing RegA knockout to multicopy RegA transgenics. RegA is an AraC like transcription factor identified in a mutational screen for virulence genes in Citrobacter rodentium, an attaching and effacing pathogen that causes transmissible colonic hyperplasia in mice. This experiment compares the RegA null strain with a multicopy plasmid rescue of this null strain in the presence and absence of bicarbonate with the aim of identifying pathogenesis related genes related to the early and late stages of attachment and effacement. Keywords: genetic modification, transcription factor, induction A strain of Citrobacter rodentium with a knockout of RegA was compared to the same strain rescued with a multicopy plasmid containing the wildtype RegA gene. These strains were analyzed with and without bicarbonate in an unconnected two factor design with dye balanced biological replicates.
Project description:Defense against attaching and effacing (A/E) bacteria requires the sequential generation of IL-23 and IL-22 to induce protective mucosal responses. While the critical source of IL-22 has been identified as CD4+ and Nkp46+ innate lymphoid cells (ILCs), the precise source of IL-23 is unclear. Here, we use genetic techniques to deplete specific classical dendritic cell (cDC) subsets and analyze immunity to the A/E pathogen Citrobacter rodentium. We find that Zbtb46+ cDCs, and specifically Notch2-dependent intestinal CD11b+ cDCs, but not Batf3-dependent CD103+ cDCs, are required for IL-23 production and immunity against C. rodentium. Notch2 controls cDC differentiation at a terminal step mediated by lymphotoxin signaling. Importantly, these results provide the first demonstration of a non-redundant function of CD11b+ cDCs in vivo. Analysis of genes differentially expressed between WT, Batf3 KO and Notch2 KO colons following C. rodentium infection. Mice were infected with 2 x 10^9 C. rodentium and colons harvested at either Day 4 or Day 9.
Project description:to investigate global gene expression of C. rodentium strain ICC169 and its isogenic pstCA and phoB mutants when grown in phosphate-rich and phosphate-low medium Genotype: 3 levels (wt, pstCA, phoB) phosphate: 2 levels (low, high)
Project description:The identification of Atg16L1 as a susceptibility gene has implicated antibacterial autophagy in the pathogenesis of Crohn's disease, a major type of inflammatory bowel disease (IBD). However, the role of Atg16L1 during extracellular bacterial infections of the intestine has not been sufficiently examined and compared to the function of other IBD susceptibility genes such as Nod2. We now find that Atg16L1 mutant mice are extraordinarily resistant to intestinal disease induced by the model bacterial pathogen Citrobacter rodentium. We further demonstrate that Atg16L1 deficiency alters the intestinal environment to mediate an enhanced immune response that is dependent on monocytic cells, and that Atg16L1/Nod2 double mutant mice lose this advantage. These results reveal an unappreciated immuno-suppressive function of an IBD gene, and raise the possibility that gene variants that affect the autophagy pathway were evolutionarily maintained to protect against certain life-threatening infections. Twenty samples have been analyzed. All are colonic tissue from mice. Controls are uninfected WT mice, uninfected Atg16L1 mutant mice (Atg16L1HM) (n=3/genotype). Treatment conditions are tissue from WT and Atg16L1 mutant mice 6 days after C. rodentium infection (n=4/genotype) and 15 days after infection (n=3/genotype).
Project description:Infection by attaching and effacing (A/E) pathogens poses a serious threat to public health, as was highlighted by the recent outbreak of E. coli O157:H7 infection in the United States. Here, by using a murine A/E pathogen, Citrobacter rodentium, we demonstrate that C. rodentium infection is lethal to IL-22-/- mice within two weeks. IL-22, in the early phase of infection, is indispensable for preventing the invasion of bacteria through the intestinal epithelium, and thereby preventing systemic spread and mortality. We also show that IL-23 is required for the early induction of IL-22 during C. rodentium infection. Finally, our data suggest that IL-22 exerts its function by boosting the innate immune responses of colonic epithelial cells, especially though the induction of anti-microbial proteins, RegIIIβ and RegIIIγ. Experiment Overall Design: Control or IL-22-treated mouse colon in triplicate.