Name: effects of taste signaling protein abolishment on gut inflammation in an inflammatory bowel disease mouse model
Uploaded: 2018-11-09T15:30:00
Description: Watch this Scientific Journal Video about Effects of Taste Signaling Protein Abolishment on Gut Inflammation in an Inflammatory Bowel Disease Mouse Model at JoVE.com

This work is supported by grants from the National Natural Sciences Foundation of China (81671016, 31471008, and 31661143030) and National Institutes of Health (DC010012, DC015819) and by the Siyuan Foundation.

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
<ol>
	<li>Keep the knockout (&#945;-gustducin-/-) mice and age-, gender-, and body weight-matched wild-type control (&#945;-gustducin+/+) C57BL/6 mice individually in clean cages. 
	NOTE: The knockout mice h...

<ol>
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A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+transient+receptor+potential+channel+expressed+in+taste+receptor+cells.">A transient receptor potential channel expressed in taste receptor cells.</a> Nature Neuroscience. 5 (11), 1169-1176 (2002).</li><li>Shigemura, N., Ninomiya, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Recent+Advances+in+Molecular+Mechanisms+of+Taste+Signaling+and+Modifying.">Recent Advances in Molecular Mechanisms of Taste Signaling and Modifying.</a> International Review of Cell and Molecular Biology. 323, 71-106 (2016).</li><li>Bezen&#231;on, C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Murine+intestinal+cells+expressing+Trpm5+are+mostly+brush+cells+and+express+markers+of+neuronal+and+inflammatory+cells.">Murine intestinal cells expressing Trpm5 are mostly brush cells and express markers of neuronal and inflammatory cells.</a> Journal of Comparative Neurology. 509 (5), 514-525 (2008).</li><li>Lu, P., Zhang, C. -. 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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...

The two major forms of inflammatory bowel disease (IBD), Crohn&#39;s disease (CD), and ulcerative colitis (UC) are characterized by chronic remittent or progressive inflammatory conditions of the intestine with multifactorial etiology1,2. 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 mechanisms and evaluate the effectiveness of human therapeutics3.
Taste receptors are G protein-coupled receptors (GPCRs) and are classified as two major types: type I (T1Rs), and type II (T2Rs) that detect sweet, umami, and bitter compounds. Taste signaling cascades are initiated by tastant binding to T1Rs or T2Rs, activating the heterotrimeric G proteins consisting of &#945;-gustducin and a G&#946;&#947; dimer and leading to release of the G&#946;&#947; subunits. The G&#946;&#947; moiety in turn stimulates the downstream effector enzyme phospholipase C-&#946;2 (PLC-&#946;2). Activated PLC-&#946;2 then hydrolyzes phosphatidylinositol-4,5-bisphosphate into two intracellular secondary messengers [inositol-1,4,5-trisphosphate (IP3) and diacylglycerol], and IP3 binds to and open its channel-receptor IP3R3, releasing calcium ions from the endoplasmic reticulum. This eventually leads to the opening of transient receptor potential ion channel Trpm5 and release of the neurotransmitter ATP onto the gustatory nerves4,5,6,7. Yet, the signaling pathways of salty and sour tastes are different and independent from sweet, umami, and bitter tastes8. In addition, the components of taste GPCRs and downstream proteins exist in various extra-oral tissues. Recent studies indicated that &#945;-gustducin, the principal component of taste signaling, is found to be expressed in the intestinal mucosa. Further studies are needed to understand the functions of these taste signaling components in extra-oral tissues9,10.
The method described here is used to characterize functions of the gustatory signaling proteins expressed in extra-oral tissues. We combine a transgenic mouse line developed for delineating signaling cascades in taste buds with the chemically induced colitis model. Largely due to its procedural simplicity and pathological similarities to human ulcerative colitis, the dextran sulfate sodium (DSS)-induced IBD model has been most widely used among the various chemically induced colitis models11. In this study, we used &#945;-gustducin-deficient mice as a representative mouse line to reveal novel functions of &#945;-gustducin in gut mucosal immunity and inflammation by 1) analyzing morphological changes in the tissue and 2) assaying differences in the expression of cytokines related to inflammation in the colon. This method can be used to quantitatively and qualitatively determine the contributions of gustatory signaling proteins (and other proteins expressed in the gut) to tissue damage and intestinal inflammation, when genetically modified mouse lines for the genes of interest are available. Advantages of this method are enabling users to obtain integrated data resulting from actions of both the chemical DSS and deficiency of the gene of interest. This method can be further improved to increase its sensitivity and reveal subtle intestinal changes at the cellular and molecular levels.

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	<tbody>
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			<td height="20" style="height:20px;width:269px;">Antibody</td>
			<td style="width:139px;"></td>
			<td style="width:87px;"></td>
			<td style="width:64px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">CD45</td>
			<td>BD Biosciences</td>
			<td>550539</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">CD3</td>
			<td>BD Biosciences</td>
			<td>555273</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">B220</td>
			<td>BD Biosciences</td>
			<td>550286</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">CD11b</td>
			<td>BD Biosciences</td>
			<td>550282</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Ly6G</td>
			<td>BD Biosciences</td>
			<td>551459</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Reagent</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Dextran Sulfate Sodium Salt (DSS)</td>
			<td>MP Biomedicals</td>
			<td>2160110</td>
			<td></td>
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			<td height="20" style="height:20px;">Streptavidin-HRP complex</td>
			<td>BD Pharmingen</td>
			<td>551011</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">H&#38;E Staining Kit</td>
			<td>BBI Life Sciences</td>
			<td>E607318</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Phosphate Buffered Saline (PBS)</td>
			<td>Sangon Biotech</td>
			<td>B548117</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">FastStart Universal SYBR Green Master(ROX)</td>
			<td>Roche</td>
			<td>4913850001</td>
			<td></td>
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		<tr height="21">
			<td height="21" style="height:21px;">MMLV Reverse Transcriptase, GPR</td>
			<td>Clontech,TaKaRa</td>
			<td>639574</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">TaKaRa MiniBEST Universal RNA Extraction Kit&#160;</td>
			<td>TaKaRa</td>
			<td>9767</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">BD 10 ml Syringe</td>
			<td>BD Biosciences</td>
			<td>309604</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Instruments and equipment</td>
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			<td height="20" style="height:20px;">balance</td>
			<td></td>
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			<td height="20" style="height:20px;">scissors&#160;</td>
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			<td height="20" style="height:20px;">forceps</td>
			<td></td>
			<td></td>
			<td></td>
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		<tr height="20">
			<td height="20" style="height:20px;">centrifuge</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">qPCR machine</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">staining jars</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Software</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Imag-Pro Plus&#160;</td>
			<td colspan="2">Media Cybernetics, Inc.&#160;</td>
			<td></td>
		</tr>
	</tbody>
</table>

effects of taste signaling protein abolishment on gut inflammation in an inflammatory bowel disease mouse model

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 &#945;-gustducin and other signaling proteins, have been found in the intestines. Here, we use &#945;-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 &#945;-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.

A DSS-induced IBD procedure was established by administrating 3% DSS in drinking water to &#945;-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&#38;E staining as the histological method, and more aggravated tissue damage was f...

Watch this Scientific Journal Video about Effects of Taste Signaling Protein Abolishment on Gut Inflammation in an Inflammatory Bowel Disease Mouse Model at JoVE.com

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

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.

Inflammatory bowel disease (IBD) is one of the immune-related gastrointestinal disorders, including ulcerative colitis and Crohn's disease, that affects ...

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