TRANSCRIPTOMICS APPROACH TO UNDERSTAND THE ROLE OF VITAMIN A IN CITROBACTER RODENTIUM INFECTION IN THE MOUSE SMALL INTESTINE AND COLON
Open Access
- Author:
- Chai, Zhi
- Graduate Program:
- Integrative and Biomedical Physiology
- Degree:
- Doctor of Philosophy
- Document Type:
- Dissertation
- Date of Defense:
- October 01, 2019
- Committee Members:
- A Catharine Ross, Dissertation Advisor/Co-Advisor
A Catharine Ross, Committee Chair/Co-Chair
Connie Jo Rogers, Committee Member
Gregory C Shearer, Committee Member
Margherita Teresa-Anna Cantorna, Outside Member
Donna Hope Korzick, Program Head/Chair - Keywords:
- RNAseq
Transcriptome
Intestine
Vitamin A
Citrobacter rodentium
mouse - Abstract:
- Background: Both vitamin A (VA) deficiency and diarrheal diseases are serious public health issues worldwide, especially for the children living in resource-limited areas. From epidemiological studies it is estimated that over 20% (or 250 million) of preschool aged children are VA deficient. VA deficiency impairs the normal development, vision, immune functions, and is positively associated with the severity of infectious diseases. On the other hand, diarrheal diseases cause ~0.8 million deaths per year in children under the age of five and rank as the second leading cause of infection-related mortality in this demographic group. Recurrent early childhood diarrhea can contribute to impairments in fitness, growth, and cognition. Vitamin A deficiency is associated with impaired intestinal barrier function and increased risk of mortality due to mucosal infection, whereas VA supplementation can effectively lower the diarrhea-related mortality. Citrobacter rodentium (C. rodentium) infection in mice serves as a model for diarrheal diseases in humans. During C. rodentium infection, vitamin A deficient (VAD) mice displayed higher susceptibility (lower survival rate and slower pathogen clearance) and reduced colonic IL-17 mRNA expression compared to their vitamin A sufficient (VAS) counterparts, suggesting that the hosts with different VA status responded distinctively to C. rodentium infection. In other words, an Interaction effect (VA status × C. rodentium infection) existed. This Interaction effect has not previously been studied on a transcriptomics scale, even though a handful of -omics studies have examined the C. rodentium infection effect in the colon. Moreover, none of these global –omics studies have compared the small intestinal or colonic gene expression profiles of the VAD mice to their VAS counterparts (VA effect). Therefore, a comprehensive evaluation of all the three effects (VA, Infection, and the Interaction effect) under the same study design is warranted. Overall hypothesis: RNAseq can be used to examine and compare the VA effect, Infection effect caused by 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: VAD mice were generated by feeding VAD diet to the pregnant C57/BL6 dams and their post-weaning offspring. 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. 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. Following a review of literature (Chapter 1), Chapter 2 covers the overall design and methodology for this dissertation. The mouse model of VA deficiency and C. rodentium infection were validated to assure the model consistency with previous studies. The RNAseq pipeline that were employed consistently among the three result chapters, including the analytical approaches and the statistical model of the differential expression analysis, were also illustrated in Chapter 2. In Chapter 3, the VA effect on intestinal gene expression in SI and colon were investigated separately, allowing for a comparison of these two sections of the gastrointestinal tract on the levels of both DEGs and co-expression network. Hypothesis: In the intestine, VA-deficiency will alter the expression of genes essential in the retinoid metabolic pathway and the mucosal immune responses. Results: 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. In Chapter 4, we analyzed the intestinal transcriptome in infected versus non-infected mice, and compared the Infection effect of C. rodentium in colon during peak infection, versus that in the SI during early infection. Hypothesis: C. rodentium infection will induce genes relevant to the innate and adaptive immune functions, and at the same time, downregulate genes that are involved in ion and water absorption. Results: 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. In Chapter 5, the Interaction effect (VA status × C. rodentium infection) was characterized in both the SI and the colon. Hypothesis: The Infection response in the VAD host will be associated with an insufficient immunological response and an impaired ion/water absorption, compared with the transcriptional response in the intestines of VAS host. Results: 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.