Development, characterization and transcriptional profiling of a mouse model of fatal infectious diarrhea and colitis

Thesis (Ph. D.)--Massachusetts Institute of Technology, Biological Engineering Division, 2007.This electronic version was submitted by the student author. The certified thesis is available in the Institute Archives and Special Collections.Includes bibliographical references (p. 195-208).Citrobacter rodentium is a naturally occurring murine bacterial pathogen which is used to model human diarrheagenic E. coli (EPEC and EHEC) infections in mice. C. rodentium causes colonic hyperplasia and a variable degree of colitis and mortality in the majority of inbred and outbred lines of mice. Differences in C. rodentium-induced disease are determined by the genetic background of the host. Here, C. rodentium infection in resistant outbred Swiss Webster (SW) mice was compared with infection in the cognate inbred FVB strain for the first time. In contrast to subclinical infection in SW mice, adult FVB mice developed overt disease with significant weight loss, severe colitis, and over 75% mortality. Fluid therapy intervention completely prevented mortality in FVB mice, and expression of pro-inflammatory and immunomodulatory genes in the colon was similar in both lines of mice, suggesting that mortality in C. rodentiuminfected FVB mice is due to hypovolemia resulting from severe dehydration. To identify host factors responsible for the development of mortality, gene expression in the distal colon of FVB and SW mice was investigated using a whole mouse genome Affymetrix array.(cont.) Transcripts represented by 1,547 probe sets (3.4%) were differentially expressed between FVB and SW mice prior to infection and at 4 and 9 days post-inoculation. Data analysis suggested that intestinal ion disturbances rather than immune-related processes are responsible for susceptibility in C. rodentium-infected FVB mice. Marked impairment in intestinal ion homeostasis predicted by microarray analysis was confirmed by quantitative RT-PCR and serum electrolyte measurements that showed hypochloremia and hyponatremia in susceptible FVB mice. C. rodentium infection was next characterized in additional inbred strains of Swiss origin. SWR and SJL mice developed minimal morbidity and no mortality in response to the pathogen, demonstrating resistance to disease. Furthermore, C3H mice developed severe diarrhea and gene expression changes comparable to those in infected FVB mice, suggesting common pathogenic mechanisms in susceptible strains. In conclusion, C. rodentium infection in FVB mice is a useful model for fatal infectious diarrhea. These studies contribute to our understanding of C. rodentium pathogenesis and identify possible candidates for susceptibility to fatal enteric bacterial infection.by Diana Borenshtein.Ph.D

Development of Fatal Colitis in FVB Mice Infected with Citrobacter rodentium▿

Citrobacter rodentium is the causative agent of transmissible murine colonic hyperplasia. The disease is characterized by severe but temporary epithelial hyperplasia with limited inflammation in the descending colon of adult mice on a variety of genetic backgrounds. The natural history of infection with this murine pathogen has been characterized in outbred Swiss Webster (SW) mice but not in the cognate inbred FVB strain. In contrast to subclinical infection in SW mice, 12-week-old FVB mice developed overt disease with significant weight loss and mortality beginning by 9 days postinoculation (dpi). By 21 dpi, more than 75% of infected FVB mice died or had to be euthanized, whereas no mortality developed in SW mice. Mortality in FVB mice was fully prevented by fluid therapy. Fecal shedding of bacteria was similar in both groups through 9 dpi; however, a slight but significant delay in bacterial clearance was observed in FVB mice by 12 to 18 dpi. SW mice developed hyperplasia with minimal inflammation in the descending colon. FVB mice developed epithelial cell hyperproliferation, severe inflammation with erosions and ulcers, and epithelial atypia by 6 dpi in the descending colon. In the majority of surviving FVB mice, colonic lesions, including epithelial atypia, were reversible, although a small percentage (5 to 7%) exhibited chronic colitis through 7 months postinoculation. The existence of susceptible and resistant lines of mice with similar genetic backgrounds will facilitate the identification of host factors responsible for the outcome of infection and may lead to the development of novel strategies for preventing and treating infectious colitis

Diarrhea as a cause of mortality in a mouse model of infectious colitis

Background: Comparative characterization of genome-wide transcriptional changes during infection can help elucidate the mechanisms underlying host susceptibility. In this study, transcriptional profiling of the mouse colon was carried out in two cognate lines of mice that differ in their response to Citrobacter rodentium infection; susceptible inbred FVB/N and resistant outbred Swiss Webster mice. Gene expression in the distal colon was determined prior to infection, and at 4 and 9 days post-inoculation using a whole mouse genome Affymetrix array. Results: Computational analysis identified 462 probe sets more than 2-fold differentially expressed between uninoculated resistant and susceptible mice. In response to C. rodentium infection, 5,123 probe sets were differentially expressed in one or both lines of mice. Microarray data were validated by quantitative real time RT-PCR for 35 selected genes and were found to have a 94% concordance rate. Transcripts represented by 1,547 probe sets, were differentially expressed between susceptible and resistant mice regardless of infection status, a host effect. Genes associated with transport were over-represented to a greater extent than even immune response-related genes. Electrolyte analysis revealed reduction in serum levels of chloride and sodium in susceptible animals. Conclusion: The results support the hypothesis that mortality in C. rodentium-infected susceptible mice is associated with impaired intestinal ion transport and development of fatal fluid loss and dehydration. 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 intestinal bacterial infections.National Institutes of Health (U.S.)Biogen Idec (Firm)United States. Public Health Service (T32 ES07020)United States. Public Health Service (P01 CA26731)United States. Public Health Service (P30 ES02109