This study tests a novel hypothesis that 1,25(OH)2D, i.e. the active vitamin D metabolite, augments regenerative capacity of the Lgr5+ intestinal stem cells (ISCs). To test this hypothesis, we are proposing two specific aims.
Specific Aim 1 will determine whether 1,25(OH)2D promotes regenerative capacity of the Lgr5+ ISCs by in vivo tracing of the Lgr5+ ISCs and their progenies in the entire process of intestinal epithelium regeneration. We will use our previously developed strategy to deliver a locally high 1,25(OH)2D concentration to the intestines. Regenerative capacity will be determined by abundance, migration, proliferation, survivability, and differentiation of the Lgr5+ ISCs. In this specific aim, one study will be performed in normal healthy animals (Objective 1A). To determine clinical significance, the other study will be performed in animals induced for experimental colitis (Objective 1B). In both objectives, we will use immunohistochemistry (IHC) to investigate the entire regenerative process of the Lgr5+ ISCs. To do this, we will trace the Lgr5+ ISCs using F1 offspring of the cre-inducible Lgr5-EGFP-IRES-creERT2 (Lgr5-GFP) and the cre-reporter B6.Cg-Gt(ROSA)26Sortm9(CAG-tdTomato)Hze/J (Ai9 or Ai) mice. In the Lgr5-GFP mice, GFP expression is under the control of Lgr5 promoter and enhancer and Cre can be induced by tamoxifen injection. Additionally, the Ai mice express tdTomato when Cre is present independent of Lgr5 expression. Consequently, the F1 offspring (Lgr5-GFP-Ai mice), upon tamoxifen administration to activate the Cre, will express both GFP and tdTomato in crypt Lgr5+ ISCs but will only express tdTomato in the progenies of Lgr5+ ISCs outside the crypts. Since normal intestinal epithelia are replaced every 4-5 days, at days 1, 3, and 5 immediately following the 1,25(OH)2D delivery, we will study abundance of the Lgr5+ ISCs by staining for GFP, migration for tdTomato, proliferation for Brdu, survivability for bcl-2 and caspase 3, and differentiation for markers specific for each type of epithelial cells.
Specific Aim 2 will determine whether the effects of 1,25(OH)2D treatment on regenerative capacity of the Lgr5+ ISCs depend on vitamin D receptor (VDR) using in vitro cultured multipotent Lgr5+ ISC lines. Since 1,25(OH)2D can function through both VDR-dependent and independent mechanisms, this study will investigate whether 1,25(OH)2D acts on the Lgr5+ ISCs directly through VDR signaling or indirectly via a VDR-independent mechanism. We will purify the Lgr5+ ISCs from Lgr5-GFP mice and establish multipotent Lgr5+ ISC lines in a multipotent medium. Using the multipotent Lgr5+ ISC lines, we will reach two objectives. Objective 2A will investigate the effects of 1,25(OH)2D treatment on proliferation (Ki-67) and survivability (bcl-2 and caspase 3) of the multipotent Lgr5+ ISCs with or without VDR knockdown. Objective 2B will examine the effects of 1,25(OH)2D treatment on differentiation of the Lgr5+ ISCs in a differentiation medium with or without VDR knockdown. The ultimate goal is to develop a 1,25(OH)2D- based strategy to enhance epithelium repair for the treatment of inflammatory bowel disease.

Public Health Relevance

Inflammatory bowel disease (IBD) is a chronic inflammatory disorder in the gastrointestinal tract and affects approximately 1.6 million Americans. IBD patients receiving current medications, which block immune responses in the intestine, have shown reduced disease severity, delayed disease progression, and improved life quality but most of time do not go into remission, suggesting that novel therapeutic strategies targeting different mechanisms are needed. This study aims to determine whether intestinal epithelial repair can be promoted by in vivo enhancing the regenerative function of intestinal stem cells in IBD patients, which aim to shed light on whether augmentation of intestinal epithelial repair can be a novel therapeutic strategy for IBD.

Agency
National Institute of Health (NIH)
Institute
National Institute of Allergy and Infectious Diseases (NIAID)
Type
Exploratory/Developmental Grants (R21)
Project #
1R21AI142170-01
Application #
9650304
Study Section
Gastrointestinal Mucosal Pathobiology Study Section (GMPB)
Program Officer
Rothermel, Annette L
Project Start
2019-02-05
Project End
2021-01-31
Budget Start
2019-02-05
Budget End
2020-01-31
Support Year
1
Fiscal Year
2019
Total Cost
Indirect Cost
Name
Loma Linda University
Department
Internal Medicine/Medicine
Type
Schools of Medicine
DUNS #
009656273
City
Loma Linda
State
CA
Country
United States
Zip Code
92350