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Diarrhea as a Cause of Mortality in a Mouse Model of Infectious Colitis

ABSTRACT: Examination of host genome-wide changes upon encounters with pathogens provides insight into the pathogenesis of infection and disease. When performed comparatively, it may shed light on the mechanisms underlying host susceptibility. In this study, gene expression in the mouse colon was investigated in two cognate lines of mice that differ in their response to Citrobacter rodentium infection; susceptible inbred FVB/N (FVB) and resistant outbred Swiss Webster (SW) mice. Gene expression in the distal colon was investigated prior to infection and at 4 and 9 days post-inoculation using a whole mouse genome Affymetrix array. Computational analysis identified 462 (1%) probe sets differentially expressed by more than 2-fold between uninoculated SW and FVB mice. In response to C. rodentium infection, 5,123 probe sets (11.4%) were significantly modulated in one or both lines of mice. Microarray data were validated by quantitative real time RT-PCR on 35 selected genes (r = 0.87, p < 0.001) and were found to have a 94% concordance. Transcripts represented by 1,547 probe sets (3.4%), were differentially expressed between FVB and SW mice regardless of infection status (host effect). Genes associated with transport were over-represented to a greater extent than immune response-related genes (25% vs. 11% of enrichment, respectively). Electrolyte analysis revealed hypochloremia and hyponatremia in susceptible animals. The results support the hypothesis that mortality in C. rodentium-infected FVB mice is associated with impaired ion transport and development of fatal hypovolemia. These studies contribute to our understanding of the pathogenesis of C. rodentium and suggest novel strategies for the prevention and treatment of diarrhea associated with bacterial infections of the intestinal tract. Keywords: temporal change (post infection)

ORGANISM(S): Mus musculus

PROVIDER: GSE8025 | GEO | 2007/12/01

SECONDARY ACCESSION(S): PRJNA100831

REPOSITORIES: GEO

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Project description:Examination of host genome-wide changes upon encounters with pathogens provides insight into the pathogenesis of infection and disease. When performed comparatively, it may shed light on the mechanisms underlying host susceptibility. In this study, gene expression in the mouse colon was investigated in two cognate lines of mice that differ in their response to Citrobacter rodentium infection; susceptible inbred FVB/N (FVB) and resistant outbred Swiss Webster (SW) mice. Gene expression in the distal colon was investigated prior to infection and at 4 and 9 days post-inoculation using a whole mouse genome Affymetrix array. Computational analysis identified 462 (1%) probe sets differentially expressed by more than 2-fold between uninoculated SW and FVB mice. In response to C. rodentium infection, 5,123 probe sets (11.4%) were significantly modulated in one or both lines of mice. Microarray data were validated by quantitative real time RT-PCR on 35 selected genes (r = 0.87, p < 0.001) and were found to have a 94% concordance. Transcripts represented by 1,547 probe sets (3.4%), were differentially expressed between FVB and SW mice regardless of infection status (host effect). Genes associated with transport were over-represented to a greater extent than immune response-related genes (25% vs. 11% of enrichment, respectively). Electrolyte analysis revealed hypochloremia and hyponatremia in susceptible animals. The results support the hypothesis that mortality in C. rodentium-infected FVB mice is associated with impaired ion transport and development of fatal hypovolemia. These studies contribute to our understanding of the pathogenesis of C. rodentium and suggest novel strategies for the prevention and treatment of diarrhea associated with bacterial infections of the intestinal tract. Experiment Overall Design: Global gene expression analysis was performed on the distal colon with 2-3 mice per group. Because no difference for any parameters was observed in uninfected mice at 4 or 9 dpi, the animals were pooled into an uninoculated control groups for each line of mouse. The selection of representative samples for microarray analysis was based on known infection status and colonic lesions. The final number of biological replicates for each condition was n = 5 for uninoculated FVB mice (âFpâ group), n = 4 for uninoculated SW mice (âSpâ group), and n = 3 for infected animals from each line at each time point (âFi4â, âSi4â, âFi9â, and âSi9â respectively).

Project description:Overall hypothesis: RNAseq can be used to examine and compare the vitamin A (VA) effect, Infection effect caused by Citrobacter rodentium (C. rodentium), and the Interaction effect (VA status × C. rodentium infection) in mouse small intestine (SI) and colon, on the scale of global transcriptome. Methods: Vitamin A deficient (VAD) mice were generated by feeding VAD diet to the pregnant C57/BL6 dams and their post-weaning offspring. Vitamin A sufficient (VAS) mice were fed with purified diet containing normal amount of VA. At weaning, mice were maintained on their respective diets and assigned to one of the four treatment groups (non-infected VAD, infected VAD, non-infected VAS and infected VAS) until the end of the experiments. For the infected groups, age-matched VAD or VAS mice were orally inoculated with C. rodentium. Mice during early (SI study) or peak (colon study) C. rodentium infection were euthanized in two separate studies. The SI study focused on SI and analyses were performed on samples collected 5 days post-infection. The colon study analyzed samples on post-infection day 10, the peak of C. rodentium infection. Total mRNA extracted from SI and colon were sequenced using Illumina HiSeq 2500 platform. Three to six biological replicates were sequenced in each group. Differentially Expressed Gene (DEG), Gene Ontology (GO) enrichment, and Weighted Gene Co-expression Network Analysis (WGCNA) were performed to characterize expression patterns and co-expression patterns. Results: a) differentially expression genes (DEGs) corresponding to VA effect were present in both the SI and colon. In SI, DEGs altered by VA were mainly involved in the retinoid metabolic pathway and immunity-related pathways. Novel target genes (e.g. Mbl2, Mmp9, Cxcl14, and Nr0b2) and cell types (based on Tuft cell and mast cell markers) under the regulation of VA were suggested by differential expression analysis coupled with the co-expression network analysis. In regard to the colon, VA demonstrated an overall suppressive effect on the cell division pathway. Finally, the comparison of co-expression modules between SI and colon indicated distinct regulatory networks in these two organs. b) In the SI, no Infection effect was detected during early infection, whereas in the colon, during peak of C. rodentium infection: 1) It has been shown that infection upregulated innate and adaptive immune functions, epithelial proliferation, apoptosis, endoplasmic reticulum stress, and reactive oxygen species production, as well as downregulated ion and water absorption. This helped to confirm the findings in previous studies, consolidating the knowledge-base about the host response to C. rodentium, 2) Pathways (e.g. neuron activity, smooth muscle contraction, vascular constriction, and developmental regulation) and additional ion transporters previously not known to be influenced by C. rodentium were suggested as potential mechanisms responsible for the diarrhea pathology and host response. 3) Retinoic acid receptor (RAR) and vitamin D receptor (VDR) signaling pathways appeared to be downregulated by infection, indicating that the malabsorption of micronutrients might be a danger signal for the host to switch from homeostasis to an anti-infection mode. c) In the SI, no Interaction (VA status × C. rodentium infection) effect was detected during early infection. In colon, 1329 genes corresponded to the Interaction effect. During the peak of C. rodentium infection, the sufficient stimulation of cell division, protein catabolism, apoptosis, transcription regulation, MHC class I, interferons, and cytokine signaling might be of key importance for the resistance of VAS mice. In addition, the proper maintenance of the proliferation, differentiation, migration, and activation of T cells and leukocytes during peak infection may also confer the VAS hosts with higher survival and clearance rate. On the other hand, our data suggested that the higher rate of lethal dehydration observed in infected VAD mice, may be attributed to the disrupted of HCO3- metabolism and an improperly high level of Cl- secretion. Conclusions: VA alters the gene expression profile in both SI and colon, whereas the Infection effect and the Interaction effect were only observed in colon during peak of C. rodentium infection, but not in the SI during early infection. Our studies suggest novel target genes and cell types under the regulation of VA in the SI, and indicate that the resistance of the VAS hosts may be attributed to the concordant control over ion/water absorption, immune functions, as well as the balance between epithelial hyperplasia and apoptosis.

Project description:Opioids analgesics are frequently prescribed in the United States and worldwide. However, serious side effects such as addiction, immunosuppression and gastrointestinal symptoms limit their use. It has been recently demonstrated that morphine treatment results in significant disruption in gut barrier function leading to increased translocation of gut commensal bacteria. Further study indicated distinct alterations in the gut microbiome and metabolome following morphine treatment, contributing to the negative consequences associated with opioid use. However, it is unclear how opioids modulate gut homeostasis in the context of a hospital acquired bacterial infection. In the current study, a mouse model of C. rodentium infection was used to investigate the role of morphine in the modulation of gut homeostasis in the context of a hospital acquired bacterial infection. Citrobacter rodentium is a natural mouse pathogen that models intestinal infection by enteropathogenic Escherichia coli (EPEC) and enterohemorrhagic E. coli (EHEC) and causes attaching and effacing lesions and colonic hyperplasia. Morphine treatment resulted in 1) the promotion of C. rodentium systemic dissemination, 2) increase in virulence factors expression with C. rodentium colonization in intestinal contents, 3) altered gut microbiome, 4) damaged integrity of gut epithelial barrier function, 5) inhibition of C. rodentium-induced increase in goblet cells, and 6) dysregulated IL-17A immune response. This is the first study to demonstrate that morphine promotes pathogen dissemination in the context of intestinal C. rodentium infection, indicating morphine modulates virulence factor-mediated adhesion of pathogenic bacteria and induces disruption of mucosal host defense during C. rodentium intestinal infection in mice. This study demonstrates and further validates a positive correlation between opioid drug use/abuse and increased risk of infections, suggesting over-prescription of opioids may increase the risk in the emergence of pathogenic strains and should be used cautiously. Therapeutics directed at maintaining gut homeostasis during opioid use may reduce the comorbidities associated with opioid use for pain management.

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Diarrhea as a Cause of Mortality in a Mouse Model of Infectious Colitis

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