Effects of Taste Signaling Protein Abolishment on Gut Inflammation in an Inflammatory Bowel Disease Mouse Model

Summary

Here we present a protocol to investigate the effect of the nullification of gustation-related genes on immune responses in a dextran sulfate sodium (DSS)-induced inflammatory bowel disease (IBD) mouse model.

Abstract

Inflammatory bowel disease (IBD) is one of the immune-related gastrointestinal disorders, including ulcerative colitis and Crohn's disease, that affects the life quality of millions of people worldwide. IBD symptoms include abdominal pain, diarrhea, and rectal bleeding, which may result from the interactions among gut microbiota, food components, intestinal epithelial cells, and immune cells. It is of particular importance to assess how each key gene expressed in intestinal epithelial and immune cells affects inflammation in the colon. G protein-coupled taste receptors, including G protein subunit α-gustducin and other signaling proteins, have been found in the intestines. Here, we use α-gustducin as a representative and describe a dextran sulfate sodium (DSS)-induced IBD model to evaluate the effect of gustatory gene mutations on gut mucosal immunity and inflammation. This method combines gene knockout technology with the chemically induced IBD model, and thus can be applied to assess the outcome of gustatory gene nullification as well as other genes that may exuberate or dampen the DSS-induced immune response in the colon. Mutant mice are administered with DSS for a certain period during which their body weight, stool, and rectal bleeding are monitored and recorded. At different timepoints during administration, some mice are euthanized, then the sizes and weights of their spleens and colons are measured and gut tissues are collected and processed for histological and gene expression analyses. The data show that the α-gustducin knockout results in excessive weight loss, diarrhea, intestinal bleeding, tissue damage, and inflammation vs. wild-type mice. Since the severity of induced inflammation is affected by mouse strains, housing environment, and diet, optimization of DSS concentration and administration duration in a pilot experiment is particularly important. By adjusting these factors, this method can be applied to assess both anti- and pro-inflammatory effects.

Introduction

The two major forms of inflammatory bowel disease (IBD), Crohn's disease (CD), and ulcerative colitis (UC) are characterized by chronic remittent or progressive inflammatory conditions of the intestine with multifactorial etiology^1,2. The development of IBD depends on genetic as well as certain environmental factors such as diet, antibiotic use, and importantly, pathogenic infections. However, the etiology and regulatory molecular mechanisms underlying IBD are still unclear. Hence, numerous chemically induced IBD animal models have been constructed and applied to delineate the pathogenesis and regulatory m....

Protocol

All experiments involving mice were reviewed and approved by the Institutional Animal Care and Use Committees of Zhejiang University. It is advised to wear appropriate personal protective equipment before performing this protocol.

1. Preparation of Mice and DSS

1. Keep the knockout (α-gustducin^-/-) mice and age-, gender-, and body weight-matched wild-type control (α-gustducin^+/+) C57BL/6 mice individually in clean cages.
   NOTE: The knockout mice have been backcrossed with C57BL/6 mice for over 20 generations and have a nearly 100% C57BL/6 genetic background.
2. Dissolve 30 g of dextran s....

Results

A DSS-induced IBD procedure was established by administrating 3% DSS in drinking water to α-gustducin-knockout (KO) and wild-type (WT) mice. Compared to WT mice, the knockout mice exhibited more severe colitis with excessive weight loss, diarrhea, and intestinal bleeding (Figure 1). After a 7 day DSS administration, the differences in tissue integrity were analyzed using H&E staining as the histological method, and more aggravated tissue damage was found .......

Discussion

This method can be employed to quantitively determine the effect of mutations of specific gustatory genes on inflammation in a DSS-induced IBD mouse model. To take full advantage, optimal induction of IBD is a key step. The development of colitis is affected by several factors, including mouse strain, housing environment, intestinal microflora, as well as the genes of interest. It is recommended to perform a pilot experiment with a small number of mice to test different dosages and durations of DSS administration. During.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Antibody
CD45	BD Biosciences	550539
CD3	BD Biosciences	555273
B220	BD Biosciences	550286
CD11b	BD Biosciences	550282
Ly6G	BD Biosciences	551459
Reagent
Dextran Sulfate Sodium Salt (DSS)	MP Biomedicals	2160110
Streptavidin-HRP complex	BD Pharmingen	551011
H&E Staining Kit	BBI Life Sciences	E607318
Phosphate Buffered Saline (PBS)	Sangon Biotech	B548117
FastStart Universal SYBR Green Master(ROX)	Roche	4913850001
MMLV Reverse Transcriptase, GPR	Clontech,TaKaRa	639574
TaKaRa MiniBEST Universal RNA Extraction Kit	TaKaRa	9767
BD 10 mL Syringe	BD Biosciences	309604
Instruments and equipment
balance
scissors
forceps
centrifuge
qPCR machine
staining jars
Software
Imag-Pro Plus	Media Cybernetics, Inc.