the effect of vitamin d deficiency on VDR and ZO-1 in the colon tissue during a citrobacter rodentium infection of wild type and CYP27B1KO mice

Open Access
Mcgill, Kelly J
Area of Honors:
Interdisciplinary in Biochemistry and Molecular Biology and Veterinary and Biomedical Sciences
Bachelor of Science
Document Type:
Thesis Supervisors:
  • Margherita Teresa-Anna Cantorna, Thesis Supervisor
  • Dr. Sarah Ellen Ades, Honors Advisor
  • Scott Brian Selleck, Faculty Reader
  • Robert John Vansaun, Honors Advisor
  • vitamin D
  • citrobacter rodentium infection
  • inflammation
  • tight junction proteins
  • immune challenge
Though traditionally and historically associated with bone metabolism and calcium absorption, recent developments in the field of immunology have demonstrated an important role for vitamin D with regard to immune function. The Cantorna laboratory has studied the interactions of vitamin D with the immune system in face of an immune challenge. They used a bacterial enteric infection and mice that were incapable of responding to vitamin D as an indirect way of assessing the potential role of vitamin D with regard to an immune challenge. The experiment determined that the vitamin D receptor knock out (VDR KO) mice had decreased Citrobacter rodentium fecal loads and a quicker recovery compared to wild-type (WT) mice. The VDR KO mice also had a stronger immune response; they had more interleukin-22 (IL-22)-producing innate lymphoid cells (ILCs) and increased expression of antibacterial peptides compared to the WT mice. The inability of the mice to respond to vitamin D without the vitamin D receptor (VDR) suggested that the down regulation of vitamin D may benefit the animal by enabling a stronger response to the immune challenge. Contradictorily, a different study examined vitamin D deficiency’s influence on infection-induced changes in intestinal epithelial barrier by challenging WT mice that were ether vitamin D sufficient or vitamin D-deficient with an enteric infection. The researchers determined that the vitamin D deficient mice infected with C. rodentium had increased epithelial barrier dysfunction. They concluded that vitamin D deficiency increased susceptibility by allowing for greater intestinal injuries. The aim of the experiment was to determine if the VDR and ZO-1 were down regulated in the colon tissue during an enteric infection in order to better understand how the mice responded to the immune challenge. The expected outcome was that the VDR and ZO-1 expression in the colon would be decreased compared to baseline levels in order to allow a stronger immune response in the face of an enteric infection. C. rodentium was used to infect three different mice groups in the experiment: vitamin D sufficient wild type C75BL/6; vitamin D deficient wild type C57BL/6 mice; and vitamin D deficient cytochrome 27B1 (CYPKO) mice. The distal colon tissue was harvested at the peak day of infection (day 14) and carried out RNA extraction using TRIzol RNA protocol, a reverse transcription reaction, and quantitative PCR to measure the VDR RNA and ZO-1 RNA levels. The three groups were compared with a two-way ANOVA statistical analysis with a p<0.05 as significant. The VDR expression at peak infection or day 14 was down regulated significantly compared to baseline day 0 expression levels in all three groups with a p value <0.001. There was no statistical difference (P>0.05) in down regulation between the vitamin D deficient or vitamin D sufficient mice or between the WT or cyp27B1 KO mice. In addition the VDR colonic expression, the normalized ZO-1 colonic expression level changed significantly between the WT D- and CYPKO D- mice on day 14 with a p-value <0.05. The vitamin D status of the mouse did not have statistical significance between day 0 and day 14. The type of mouse, WT or CYPKO, had a p-value less than 0.05 when the day 0 and day 14 normalized ZO-1 colonic expression. This down regulation may have occurred in order to promote the production of antimicrobial peptides or a stronger Th1 cell response. A kinetic study of this down regulation will take place to determine if it occurs earlier on in the infection, gradually throughout or closer to peak day of infection.