Name: effects of taste signaling protein abolishment on gut inflammation in an inflammatory bowel disease mouse model
Uploaded: 2018-11-09T15:30:00.000+00:00
Duration: 9 min 31 s
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 have been backcrossed with C57BL/6 mice for over 20 generations and have a nearly 100% C57BL/6 genetic background.</li>
	<li>Dissolve 30 g of dextran sulfate sodium (DSS) powder in 1 L of autoclaved water. Mix until the solution becomes clear to ensure that the final working concentration is 3% (w/v). 
	NOTE: The DSS solution can be stored at room temperature for up to 1 week or at 4 &#176;C until use.</li>
</ol>
2. Induction and Evaluation of DSS Colitis in Mice
<ol>
	<li>Weigh and record each mouse&#39;s initial body weight. Place the mice individually into standard plastic cages and label the cages.</li>
	<li>Replace regular drinking water with 3% DSS solution for a total of 7 days to which both groups of mice have access ad libitum.</li>
	<li>Measure the mouse&#39;s body weight, record DSS solution consumption, and collect and examine the stool of each mouse daily during the DSS administration. Observe the severity of diarrhea and rectal bleeding and convert this to the DSS-induced disease index12.</li>
	<li>During the experiment, the percentage of weight loss compared to initial weight and the disease index are calculated to evaluate the symptoms of colitis. 
	NOTE: The disease index is scored by combining observations of diarrhea and rectal bleeding and are defined as follows: 0 (normal stool, no blood), 1 (soft stool, no blood), 2 (soft stool, little blood), 3 (very soft stool, modest bleeding), and 4 (watery stool, significant bleeding)12. The disease index is analyzed daily for each mouse.</li>
	<li>By the end of 7-day DSS treatment, sacrifice the mice by cervical dislocation and proceed with the remaining experiments.</li>
</ol>
3. Preparation of Tissue Samples
<ol>
	<li>Place the mouse in the supine position and clean the skin of the abdomen with 70% ethanol. Make a 3 cm-long midline incision in the abdomen with a pair of small scissors to expose the abdominal cavity.</li>
	<li>Use a pair of forceps to carefully separate the spleen from other tissues, then remove the spleen and measure its size.</li>
	<li>Identify and lift the colon with forceps and separate it from the surrounding mesentery. Pull out the whole colon until the cecum and rectum are visible.</li>
	<li>Isolate the colon by transecting it at the colonocecal margin and deep in the pelvis to free the proximal and distal colon, respectively. Then, measure and record the length of the isolated colon. Be careful not to damage the colonic tissue during the dissecting procedure.</li>
	<li>Flush the colon with 10 mL of ice-cold phosphate-buffered saline (PBS) with a 10 mL syringe equipped with a gavage needle to remove the feces and blood until the eluate is completely clear.</li>
	<li>For histological identification, divide the tissue samples equally into three parts: proximal, middle, and distal. Then, fix the tissue with 4% paraformaldehyde (PFA) overnight at 4 &#176;C.</li>
	<li>For the analysis of cytokine expression, freeze the entire colon rapidly with liquid nitrogen and store it at -80 &#176;C until use.</li>
</ol>
4. Histological Assessment of the Severity of DSS-induced Colitis
<ol>
	<li>Hematoxylin and eosin (H&#38;E) staining
	<ol>
		<li>After fixation, submerge the tissue in a solution of 30% sucrose in 1x PBS overnight in a 15 mL tube to cryoprotect the samples.</li>
		<li>Embed the tissue in optimal cutting temperature (OCT) compound and place it at -20 &#176;C until the OCT hardens.</li>
		<li>Transfer the OCT block containing the tissue to a cryostat, set the thickness dial in the cryostat to 12 &#181;m, and slice and collect 12 &#181;m-thick frozen sections.</li>
		<li>Heat the collected tissue sections from a cryostat at 65 &#176;C for 20 min on a hot plate.</li>
		<li>Wash the sections briefly in distilled water. Stain them with hematoxylin staining solution for 5 min and subsequently rinse with running tap water for 5 min.</li>
		<li>Differentiate the sections with 0.5% hydrochloric acid-ethanol for 30 s and rinse them in running tap water for 1 min. Then, perform the wash in 1x PBS for 1 min and subsequently rinse with running tap water for 5 min.</li>
		<li>Perform washing of the tissue sections in 70%, 75%, 80%, and 95% alcohol, each for 10 s. Counterstain in eosin staining solution for 2 min.</li>
		<li>Perform dehydration through 95% alcohol and 2 changes of absolute alcohol for 5 min each time. Clear in 2 changes of xylene for 5 min.</li>
		<li>Score tissue damage of the proximal, middle, and distal colon of each mouse for both the gene knockout and wild-type groups based on results of the above H&#38;E staining. 
		&#8203;NOTE: The disease index is a combined score of epithelial damage and inflammation in the mucosa, submucosa, muscularis, and serosa regions, which is defined as follows: 0 (no tissue damage and inflammation), 1 (focal tissue damage and inflammation), 2 (patchy tissue damage and inflammation), and 3 (diffuse tissue damage and inflammation)12,13. Three scores per mouse for the proximal, middle, and distal parts of the colon are then summed to obtain a total score for each animal. The average scores for each group are then calculated.</li>
	</ol>
	</li>
	<li>Immunohistochemistry14
	<ol>
		<li>Heat the collected tissue sections from a cryostat at 65 &#176;C for 20 min on a hot plate. Wash the sections in 1x PBS 3 times, for 10 min each. Incubate the tissue sections in 3% hydrogen peroxide for 10 min to eliminate endogenous peroxidase. Wash the sections 3 more times. Block the tissues with blocking buffer (3% BSA, 0.3% non-ionic detergent, 2% goat serum, 0.1% sodium azide in 1x PBS) at room temperature for 1 h or more.</li>
		<li>Replace the blocking buffer with a solution containing the following immune cell type-specific primary antibodies: CD45 for leukocytes, CD3 for T cells, B220 for B cells, CD11b for macrophages, and Ly6G for neutrophils. Incubate at 4 &#176;C overnight.</li>
		<li>Remove the primary antibodies from tissue sections by aspiration. Wash the sections with 1x PBS 3 times, for 10 min each. Incubate the sections with biotinylated secondary antibody followed by incubation with the streptavidin-HRP complex at room temperature.</li>
		<li>Incubate the sections with 3,3&#39;-diaminobenziding (DAB) solution to develop a light- or dark-brown color and visualize the immunoreactive cells.</li>
		<li>Counterstain the sections with hematoxylin and 0.3% (v/v) diluted ammonia. Take bright-field images at 10X magnification under a microscope.</li>
		<li>Use an image processing program to identify and quantify the population of the marked immune cells in both the epithelium and the lamina propria (underneath the epithelium) by setting 2 masks: use the first mask in the color-cube-based feature to set a color detection threshold and measure the colored DAB-reactive areas; use the second mask to determine total areas of the epithelium and laminar propria in the examined section. Express the immunoreactive cell population as a ratio of the staining area of the infiltrated cells to the total area of the examined tissue.</li>
	</ol>
	</li>
</ol>
5. Gene Expression Assessment of DSS-induced Colitis
<ol>
	<li>Retrieve the colon tissues from the DSS-treated gene knockout and wild-type mice from a -80 &#176;C freezer and add 0.6 mL of lysis buffer to 25 mg of tissue, then homogenize in a homogenizer.</li>
	<li>Follow the RNA extraction kit&#39;s protocol to extract the total RNA, and DNase I is used to eliminate any contaminating genomic DNA.</li>
	<li>Run an agarose gel to check the quality of the extracted RNA. If its 28S RNA band is brighter than 18S band, it is usable. Take 1 &#181;L of the RNA sample to determine the RNA concentration on a microspectrophotometer.</li>
	<li>Mix 1 &#181;g of the total RNA with 2.5 &#181;L of 20 mM oligo (dT)12,13,14,15,16,17,18 primers and 1 &#181;L of Moloney murine leukemia virus (MMLV) reverse transcriptase. Incubate at 42 &#176;C for 60 min to prepare the cDNA.</li>
	<li>Set up real-time PCR reactions by mixing 1&#181;L of the cDNA with 0.5 &#181;L of forward and reverse qPCR primers for TNF, IFN-&#947;, IL-5, IL-13, IL-10, TGF-&#946;1, and &#946;-Actin as a control in addition to 2x fluorescent green dye.</li>
	<li>Run the qPCR with the following parameters: 95 &#176;C for 10 min, followed by 45 cycles of 95 &#176;C for 10 s, 50 &#176;C for 25 s, and 72 &#176;C for 20 s.</li>
	<li>Calculate the relative quantification of gene expression by using the 2-&#916;&#916;Ct method15. Comparatively analyze the gene expression levels in the knockout vs. wild-type mice.</li>
</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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The authors declare that they have no competing financial interests.

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 pilot experiment, gross symptoms such as weight loss, diarrhea, intestinal bleeding, and some microscopic changes such as tissue damage, inflammation associated with immune cell infiltration, and expression-level changes of cytokines should be analyzed and compared to control groups that have drinking water without DSS16,17,18,19. Severity of the induced colitis can be monitored by analyzing daily changes in mouse body weight and scores of the collected stool during the DSS-treatment period. After DSS treatment, the tissue damage can be assessed by H&#38;E staining on frozen sections of the colon, whereas infiltration of immune cells and expression levels of cytokines can be identified and quantified using immunohistochemistry and qPCR. DSS dosage, administration duration, and diet can be adjusted to reveal subtle effects of gustatory gene mutations on the colonic immune responses. However, the sensitivity of this method may still limit its application to studies on the minimal effects of some other genes. In this case, some modifications can be adopted; for example, by minimizing the variables from individual-specific intestinal microflora by administrating antibiotics.
The DSS-induced colitis model has been used the most extensively among chemical agent-induced IBD models, largely due to its simplicity, rapidity, reproducibility, controllability, and most importantly its similarities to human ulcerative colitis, which is useful in evaluating the effectiveness of human therapeutics2. One disadvantage researchers must be aware of is that T and B cells are not required for the development of colitis, which is different from development of the disease in humans. However, it may be useful for studying the role of the innate immune system in the development of acute colitis2.
This study has established a DSS-induced IBD model using &#945;-gustducin-deficient mice, which can be used to investigate novel functions of the G protein &#945; subunit in gut mucosal immunity and inflammation. The results show that mice lacking &#945;-gustducin are more susceptible to DSS-induced colitis and are accompanied by more severe symptoms, including tissue damage, excessive inflammatory responses, and altered expression of potent cytokines13. In agreement with recent findings indicating the involvement of taste-like chemosensory pathways in type II immune responses to gut parasites20, &#945;-gustducin and other taste signaling proteins may have novel and important functions in the intestinal immune balance. However, the exact molecular mechanisms underlying these skewed immune responses remain to be uncovered.
Furthermore, this method can be applied to study effects of other gustatory genes that are expressed in the colon (e.g., the transient receptor potential ion channel Trpm5, which is critical to sweet, bitter, and umami tastes and expressed in human colonic cells7). Finally, the same strategy can be used to discover new functions of key proteins and factors in intestinal immunity and help evaluate the effectiveness of novel treatments of certain human diseases.

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.

<table><tbody><tr><td>&lt;strong&gt;Antibody&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>CD45</td><td>BD Biosciences</td><td>550539</td><td></td></tr><tr><td>CD3</td><td>BD Biosciences</td><td>555273</td><td></td></tr><tr><td>B220</td><td>BD Biosciences</td><td>550286</td><td></td></tr><tr><td>CD11b</td><td>BD Biosciences</td><td>550282</td><td></td></tr><tr><td>Ly6G</td><td>BD Biosciences</td><td>551459</td><td></td></tr><tr><td>&lt;strong&gt;Reagent&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>Dextran Sulfate Sodium Salt (DSS)</td><td>MP Biomedicals</td><td>2160110</td><td></td></tr><tr><td>Streptavidin-HRP complex</td><td>BD Pharmingen</td><td>551011</td><td></td></tr><tr><td>H&amp;amp;E Staining Kit</td><td>BBI Life Sciences</td><td>E607318</td><td></td></tr><tr><td>Phosphate Buffered Saline (PBS)</td><td>Sangon Biotech</td><td>B548117</td><td></td></tr><tr><td>FastStart Universal SYBR Green Master(ROX)</td><td>Roche</td><td>4913850001</td><td></td></tr><tr><td>MMLV Reverse Transcriptase, GPR</td><td>Clontech,TaKaRa</td><td>639574</td><td></td></tr><tr><td>TaKaRa MiniBEST Universal RNA Extraction Kit&amp;nbsp;</td><td>TaKaRa</td><td>9767</td><td></td></tr><tr><td>BD 10 mL Syringe</td><td>BD Biosciences</td><td>309604</td><td></td></tr><tr><td>&lt;strong&gt;Instruments and equipment&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>balance</td><td></td><td></td><td></td></tr><tr><td>scissors&amp;nbsp;</td><td></td><td></td><td></td></tr><tr><td>forceps</td><td></td><td></td><td></td></tr><tr><td>centrifuge</td><td></td><td></td><td></td></tr><tr><td>qPCR machine</td><td></td><td></td><td></td></tr><tr><td>staining jars</td><td></td><td></td><td></td></tr><tr><td>&lt;strong&gt;Software&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>Imag-Pro Plus&amp;nbsp;</td><td>Media Cybernetics, Inc.&amp;nbsp;</td><td></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 found in the proximal, middle, and distal colons of the knockout mice than in WT mice (Figure 2). Furthermore, the excessive immune activation led to colitis with infiltration of various inflammatory cells such as macrophages and neutrophils. Immunohistochemical analyses using a number of markers for the immune cells were carried out to determine whether the &#945;-gustducin deficiency affected immune cell infiltration. Comparative analysis showed that the infiltration of leukocytes, neutrophils, and macrophages was significantly increased in the knockout mice compared to WT control mice (Figure 3). Finally, some cytokine expression levels in the colons of DSS-induced colitis mice were determined using qPCR with gene-specific primers. Results showed that compared to WT mice, the knockout mice had higher expression levels of TNF and IFN-&#947; but lower expression levels of IL-5, IL-13, and IL-10; however, no difference was seen in the expression level of TGF-&#946;1 between the knockout and WT mice (Figure 4).
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/58668/58668fig1.jpg" /> 
Figure 1: DSS administration renders more severe colitis in &#945;-gustducin knockouts (KO). (A) Percentage of body weight loss: KO mice displayed significantly more body weight loss starting from day 3. (B) Colitis disease index based on the severity of diarrhea and rectal bleeding: KO mice showed significantly greater disease indices than WT controls. (C) Colon (upper panel) and spleen (lower panels) from representative WT and KO mice 7 days post-DSS administration: KO mice had significantly shorter colons and larger spleens. Error bars represent SEM (*p &#60; 0.05, **p &#60; 0.005, ***p &#60; 0.0005; ANOVA with post hoc t-tests). Scale bar = 1 cm. This figure has been modified from a previous publication13. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/58668/58668fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/58668/58668fig2.jpg" /> 
Figure 2: &#945;-gustducin KO mice show more severe tissue damage following DSS treatment. (A) H&#38;E staining of colon tissues from WT and &#945;-gustducin KO mice not treated with DSS (water) or treated with DSS for 7 days (DSS). (B) Tissue injury scores based on histological staining of colon tissues from WT and KO mice 7 days after DSS administration. DSS treatment induced some tissue damage in WT colons, which was much worse in the KO specimen. Error bars represent SEM (*p &#60; 0.05). Scale bar = 50 &#181;m. This figure has been modified from a previous publication13. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/58668/58668fig2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/58668/58668fig3.jpg" /> 
Figure 3: &#945;-gustducin KO mice display increased infiltration of immune cells in DSS-induced colitis. (A) Massive immune cell infiltration in the colons of DSS-treated KO mice. (B) Quantification of immune cell numbers: the percentage of immunostained areas divided by the total area of measured tissue based on image analyses. Error bars represent SEM. Scale bar = 50 &#181;m. This figure has been modified from a previous publication13. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/58668/58668fig3large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/58668/58668fig4.jpg" /> 
Figure 4: &#945;-gustducin KO mice display different expression levels of immune cytokines in DSS-induced colitis. DSS treatment increased expression of TNF and IFN-&#947; and decreased expression of IL-5, IL-13, and IL-10 in the KO mice colons compared to WT mice. Real-time quantitative RT-PCR was performed using gene-specific primers. &#946;-actin was used as an internal control gene. Error bars represent SEM (*p &#60; 0.05, **p &#60; 0.005). This figure has been modified from a previous publication13. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/58668/58668fig4large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Table 1" src="/files/ftp_upload/58668/58668tbl1.jpg" /> 
Table 1: List of primers used.

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 ...

effects-taste-signaling-protein-abolishment-on-gut-inflammation-an

Nanyang Technological University

Research

JoVE Journal

Immunology and Infection

8.5K Views.  Zhejiang University. Deze methode kan helpen bij het beantwoorden van belangrijke vragen in het inflammatoire bioziekteveld over colitis ulcerosa en de ziekte van Crohn. Het belangrijkste voordeel van deze techniek is dat het kan worden gebruikt om de eerste anti-en proinfammatory effecten van signalering eiwitten te evalueren. De implicatie van deze techniek breidt het onderzoek van gas in nieuwe vooruitgang en ontsteking uit.Hoewel deze methode kan inzicht geven in het genetische effect op darmontsteking...

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

DNBS/TNBS Colitis Models: Providing Insights Into Inflammatory Bowel Disease and Effects of Dietary Fat

Inflammatory Bowel Diseases (IBD), including Crohn&#39;s Disease and Ulcerative Colitis, have long been associated with a genetic basis, and more recently host immune responses to microbial and environmental agents. Dinitrobenzene sulfonic acid (DNBS)-induced colitis allows one to study the pathogenesis of IBD associated environmental triggers such as stress and diet, the effects of potential therapies, and the mechanisms underlying intestinal inflammation and mucosal injury. In this paper, we investigated the effects of dietary n-3 and n-6 fatty acids on the colonic mucosal inflammatory response to DNBS-induced colitis in rats. All rats were fed identical diets with the exception of different types of fatty acids [safflower oil (SO), canola oil (CO), or fish oil (FO)] for three&#160;weeks prior to exposure to intrarectal DNBS. Control rats given intrarectal ethanol continued gaining weight over the 5 day study, whereas, DNBS-treated rats fed lipid diets all lost weight with FO and CO fed rats demonstrating significant weight loss by 48&#160;hr and rats fed SO by 72&#160;hr. Weight gain resumed after 72&#160;hr post DNBS, and by 5 days post DNBS, the FO group had a higher body weight than SO or CO groups. Colonic sections collected 5&#160;days post DNBS-treatment showed focal ulceration, crypt destruction, goblet cell depletion, and mucosal infiltration of both acute and chronic inflammatory cells that differed in severity among diet groups. The SO fed group showed the most severe damage followed by the CO, and FO fed groups that showed the mildest degree of tissue injury. Similarly, colonic myeloperoxidase (MPO) activity, a marker of neutrophil activity was significantly higher in SO followed by CO fed rats, with FO fed rats having significantly lower MPO activity. These results demonstrate the use of DNBS-induced colitis, as outlined in this protocol, to determine the impact of diet in the pathogenesis of IBD.

DNBS/TNBS induced colitis offers an alternative and inexpensive method to study the pathobiology of inflammatory bowel disease. The protocol discussed in this paper outlines the successful application of DNBS to induce colitis in mice and rats and allows one to thoroughly study host-mediated intestinal responses.

Inflammatory Bowel Diseases (IBD), including Crohn&#39;s Disease and Ulcerative Colitis, have long been associated with a genetic basis, and more recently ...

Medicine

Induction of Murine Intestinal Inflammation by Adoptive Transfer of Effector CD4+CD45RBhigh T Cells into Immunodeficient Mice

There are many different animal models available for studying the pathogenesis of human inflammatory bowel diseases (IBD), each with its own advantages and disadvantages. We describe here an experimental colitis model that is initiated by adoptive transfer of syngeneic splenic CD4+CD45RBhigh T cells into T and B cell deficient recipient mice. The CD4+CD45RBhigh T cell population that largely consists of na&#239;ve effector cells is capable of inducing chronic intestinal inflammation, closely resembling key aspects of human IBD. This method can be manipulated to study aspects of disease onset and progression. Additionally it can be used to study the function of innate, adaptive, and regulatory immune cell populations, and the role of environmental exposures, i.e., the microbiota, in intestinal inflammation. In this article we illustrate the methodology for inducing colitis with a step-by-step protocol. This includes a video demonstration of key technical aspects required to successfully develop this murine model of experimental colitis for research purposes.

Induction of Murine Intestinal Inflammation by Adoptive Transfer of Effector CD4+CD45RBhigh T Cells into Immunodeficient Mice

Here, we present a protocol to induce colonic inflammation in mice by adoptive transfer of syngeneic CD4+CD45RBhigh T cells into T and B cell deficient recipients. Clinical and histopathological features mimic human inflammatory bowel diseases. This method allows the study of the initiation of colonic inflammation and progression of disease.

There are many different animal models available for studying the pathogenesis of human inflammatory bowel diseases (IBD), each with its own advantages ...

Investigating Intestinal Inflammation in DSS-induced Model of IBD

Inflammatory bowel disease (IBD) encompasses a range of intestinal pathologies, the most common of which are ulcerative colitis (UC) and Crohn's Disease (CD). Both UC and CD, when present in the colon, generate a similar symptom profile which can include diarrhea, rectal bleeding, abdominal pain, and weight loss.1 Although the pathogenesis of IBD remains unknown, it is described as a multifactorial disease that involves both genetic and environmental components.2
There are numerous and variable animal models of colonic inflammation that resemble several features of IBD. Animal models of colitis range from those arising spontaneously in susceptible strains of certain species to those requiring administration of specific concentrations of colitis-inducing chemicals, such as dextran sulphate sodium (DSS). Chemical-induced models of gut inflammation are the most commonly used and best described models of IBD. Administration of DSS in drinking water produces acute or chronic colitis depending on the administration protocol.3 Animals given DSS exhibit weight loss and signs of loose stool or diarrhea, sometimes with evidence of rectal bleeding.4,5 Here, we describe the methods by which colitis development and the resulting inflammatory response can be characterized following administration of DSS. These methods include histological analysis of hematoxylin/eosin stained colon sections, measurement of pro-inflammatory cytokines, and determination of myeloperoxidase (MPO) activity, which can be used as a surrogate marker of inflammation.6
The extent of the inflammatory response in disease state can be assessed by the presence of clinical symptoms or by alteration in histology in mucosal tissue. Colonic histological damage is assessed by using a scoring system that considers loss of crypt architecture, inflammatory cell infiltration, muscle thickening, goblet cell depletion, and crypt abscess.7 Quantitatively, levels of pro-inflammatory cytokines with acute inflammatory properties, such as interleukin (IL)-1&beta;, IL-6 and tumour necrosis factor (TNF)-&alpha;,can be determined using conventional ELISA methods. In addition, MPO activity can be measured using a colorimetric assay and used as an index of inflammation.8
In experimental colitis, disease severity is often correlated with an increase in MPO activity and higher levels of pro-inflammatory cytokines. Colitis severity and inflammation-associated damage can be assessed by examining stool consistency and bleeding, in addition to assessing the histopathological state of the intestine using hematoxylin/eosin stained colonic tissue sections. Colonic tissue fragments can be used to determine MPO activity and cytokine production. Taken together, these measures can be used to evaluate the intestinal inflammatory response in animal models of experimental colitis.

Experimental models of inflammatory bowel disease have allowed us to examine the complex innate and adaptive immune responses associated with pathogenesis. Using histological scoring, quantification of pro-inflammatory cytokines and myeloperoxidase activity, one can begin to assess these responses seen in inflammatory bowel disease.

Inflammatory bowel disease (IBD) encompasses a range of intestinal pathologies, the most common of which are ulcerative colitis (UC) and Crohn's Disease ...

Biology

Mechanistic Insight into the Development of TNBS-Mediated Intestinal Fibrosis and Evaluating the Inhibitory Effects of Rapamycin

Significant studies have been carried out to understand effective management of intestinal fibrosis. However, the lack of better knowledge of fibrosis has hindered the development of a preventative drug. Primarily, finding a suitable animal model is challenging in understanding the mechanism of Crohn&#39;s-associated intestinal fibrosis pathology. Here, we adopted an effective method where TNBS chemical exposure to mice rectums produces substantially deep ulceration and chronic inflammation, and the mice then chronically develop intestinal fibrosis. Also, we describe a technique where a rapamycin injection shows inhibitory effects on TNBS-mediated fibrosis in the mouse model. To assess the underlying mechanism of fibrosis, we methodically discuss a procedure for purifying Cx3Cr1+ cells from the lamina propria of TNBS-treated and control mice. This detailed protocol will be helpful to researchers who are investigating the mechanism of fibrosis and pave the path to find a better therapeutic invention for Crohn&#39;s-associated intestinal fibrosis.

In this study, we describe a detailed procedure of TNBS-mediated intestinal fibrosis, which exhibits comparable pathophysiology to Crohn&#39;s fibrosis. We also discuss this approach in light of rapamycin facilitated inhibitory effects on intestinal fibrosis.

Significant studies have been carried out to understand effective management of intestinal fibrosis. However, the lack of better knowledge of fibrosis has ...

Performing Colonoscopic-Guided Pinch Biopsies in Mice and Evaluating Subsequent Tissue Changes

Understanding the tissue and cellular changes that occur in the acute injury response as well as during the wound healing process is of paramount importance when studying diseases of the gastrointestinal (GI) tract. The murine colonic pinch biopsy model is a useful tool to define these processes. Additionally, the interplay between gut luminal content (e.g., microbes) and the colon can be studied. However, wound induction and the ability to track wound closure over time in a reliable manner can be challenging. Moreover, tissue preparation and orientation must be carried out in a standardized way to optimally interrogate histologic and molecular changes. Here, we present a detailed method describing biopsy-induced injury and the monitoring of wound closure through repeat colonoscopies. An approach is described that ensures consistent and reproducible measurements of wound size, the ability to collect the wound bed for molecular analyses as well as visualize the wound bed upon sectioning of tissues. The ability to successfully carry out these techniques allows for studies of the acute injury response, wound healing and luminal-host interactions within the colon.

Here, we provide a detailed description of the procedure to induce colonoscopic-guided pinch biopsies in mice and track wound closure in real time. Additionally, methods for the preparation of tissues for histological, immunohistochemical and molecular analyses of the wound bed are provided.

Understanding the tissue and cellular changes that occur in the acute injury response as well as during the wound healing process is of paramount ...

Systematic Scoring Analysis for Intestinal Inflammation in a Murine Dextran Sodium Sulfate-Induced Colitis Model

Murine colitis models are tools that are extensively employed in studies focused on understanding the pathobiology of inflammatory intestinal disorders. However, robust standards for objective and reproducible quantification of disease severity remain to be defined. Most colitis analysis methods rely on limited histological scoring of small segments of intestine, leading to partial or biased analyses. Here, we combine high-resolution image acquisition and longitudinal analysis of the entire colon to quantify intestinal injury and ulceration in the dextran sodium sulfate (DSS) induced model of murine colitis. This protocol allows for the generation of objective and reproducible results without extensive user training. Here, we provide comprehensive details on sample preparation and image analysis using examples of data from DSS induced colitis. This method can be easily adapted to other models of murine colitis that have significant inflammation associated with mucosal injury. We demonstrate that the fraction of inflamed/injured and eroded/ulcerated mucosa relative to the complete length of the colon closely parallels clinical findings such as weight loss amid DSS-induced disease progression. This histological protocol provides a reliable time and cost-effective aid to standardize analyses of disease activity in an unbiased way in DSS colitis experiments.

Systematic scoring of intestinal inflammation using a free computer-assisted system is a powerful tool to quantitatively compare histopathological changes in colitis models characterized by the presence of ulcers and inflammatory changes. Histological colitis score evaluation strengthens clinical observations and facilitates data interpretation.

Murine colitis models are tools that are extensively employed in studies focused on understanding the pathobiology of inflammatory intestinal disorders. ...

Induction of Intestinal Inflammation by Adoptive Transfer of CBir1 TCR Transgenic CD4+ T Cells to Immunodeficient Mice

With the increase of incidence, inflammatory bowel diseases (IBD), which are chronic diseases affecting the gastrointestinal tract, impose a considerable health and financial burden on individuals and society. Therefore, it is critical to investigate the mechanisms underlying the pathogenesis and development of IBD. Here, a gut microbiota antigen-specific T cell transfer colitis model is described. CBir1 flagellin has been recognized as the immunodominant gut bacterial antigen in experimental colitis and patients with Crohn&#39;s disease. CBir1 TCR transgenic na&#970;ve CD4+ T cells, specific to CBir1 flagellin, can induce chronic colitis after adoptive transfer into immune-deficient Rag1-/- mice. The disease severity is assessed by histopathology. The CD4+ T cell phenotypes in colonic lamina propria are also determined. This model closely resembles the development of IBD, which provides an ideal murine model for investigating the mechanisms driving the pathogenesis of IBD and testing the potential drugs for treating IBD.

Induction of Intestinal Inflammation by Adoptive Transfer of CBir1 TCR Transgenic CD4+ T Cells to Immunodeficient Mice

In this protocol, a gut microbiota antigen-specific T cell adoptive transfer colitis model is described. CD4+ T cells are isolated from CBir1 TCR transgenic mice. These are specific for an immunodominant gut microbiota antigen CBir1 flagellin, which is transferred into recipient Rag1-/- mice, leading to intestinal inflammation.

With the increase of incidence, inflammatory bowel diseases (IBD), which are chronic diseases affecting the gastrointestinal tract, impose a considerable ...

Experimental Rat Model of Pouchitis for Studying Ulcerative Colitis Pathogenesis and Treatment

A Rat Model of Pouchitis Following Proctocolectomy and Ileal Pouch-Anal Anastomosis Using Dextran Sulfate Sodium

Ulcerative colitis (UC) is a chronic immune-mediated disease that affects the entire colon and rectum with a relapsing and remitting course, causing lifelong morbidity. When medical treatment is ineffective, especially in cases of massive gastrointestinal bleeding, perforation, toxic megacolon, or carcinogenesis, surgery becomes the last line of defense to cure UC. Total colorectal resection and ileal pouch-anal anastomosis (IPAA) offer the best chance for long-term treatment. Pouchitis is the most common and troublesome postoperative complication. In this investigation, microsurgery is employed to create an ileal pouch model in experimental rats via IPAA surgery. Subsequently, a sustained rat model of pouchitis is established by inducing inflammation of the ileal pouch with dextran sulfate sodium (DSS). The successful establishment of rat pouchitis is validated through analysis of postoperative general status, weight, food and water intake, fecal data, as well as pouch tissue pathology, immunohistochemistry, and inflammatory factor analysis. This experimental animal model of pouchitis provides a foundation for studying the pathogenesis and treatment of the condition.

Author Spotlight: Developing a Rat Model for Pouchitis Research and Treatment

This protocol describes the method for establishing a rat model of pouchitis. The ileal pouch model was created by performing ileal pouch-anal anastomosis (IPAA) surgery using microsurgical techniques. After the surgery, the rat was treated with 4% dextran sulfate sodium (DSS) for 4 days.

Ulcerative colitis (UC) is a chronic immune-mediated disease that affects the entire colon and rectum with a relapsing and remitting course, causing ...

Investigating the Alleviating Effects of Bacillus cereus Administration on Colitis through Gut Microbiota Modulation

Gut microbiota dysbiosis is thought to exert a role in the progression of colitis. However, the precise standards for probiotic administration in alleviating colitis remain undefined. Most analysis methods rely on limited diversity and abundance of gut microorganisms. Therefore, observational studies cannot establish causation. In this study, we applied antibiotic-induced pseudo-germ-free mice to investigate the role of gut microbiota in regulating the probiotic effects of Bacillus&#160;cereus (B. cereus)&#160;on dextran sulfate sodium (DSS)-induced colitis in mice. This process allows for evaluating the bidirectional regulating effect of B. cereus&#160;supplementation on health and provides stable and reproducible results. Here, the detailed protocols for B. cereus cultivation, gavage operation, stool collection, and antibiotic clearance treatment on colitis mice are provided. The optimization methods are also applicable for other chronic inflammatory-associated disorders. The results showed that B. cereus administration decreased body weight loss, colon length shortening, disease activity index, and histopathological scores. However, treatment with antibiotics suppressed the positive effect of B. cereus on colitis. These results indicate that gut microbiota are needed for the alleviating effects of B. cereus on colitis. Therefore, exploring the beneficial effects of probiotics in this research is a promising approach for developing novel treatment strategies for alleviating the symptoms of chronic inflammatory-associated disorders.

Investigating the Alleviating Effects of Bacillus cereus Administration on Colitis through Gut Microbiota Modulation

Here, we present a protocol to establish the antibiotic-induced pseudo-germ-free dextran sulfate sodium-induced colitis mouse model to investigate the role of gut microbiota in regulating the positive effects of Bacillus cereus on colitis.

Gut microbiota dysbiosis is thought to exert a role in the progression of colitis. However, the precise standards for probiotic administration in ...

A Technique to Generate a Murine Model of Dextran Sulfate Sodium-Induced Colitis

Source: Du, Y., et al. <a href="https://app-jove-com.remotexs.ntu.edu.sg/v/58668">Effects of Taste Signaling Protein Abolishment on Gut Inflammation in an Inflammatory Bowel Disease Mouse Model.</a> J. Vis. Exp. (2018)
This video demonstrates a technique to generate a chemical-induced murine model of colitis &#8212; an inflammatory bowel disease. The mouse is allowed to consume a dextran sodium sulfate (DSS) solution for an adequate duration. Upon consumption, DSS damages the mucus and epithelial layer barrier in the colon, allowing gut microbes to enter the underlying tissue &#8212; where they are sensed by the resident immune cells to mediate an exaggerated immune response, resulting in inflammation.

Source: Du, Y., et al. Effects of Taste Signaling Protein Abolishment on Gut Inflammation in an Inflammatory Bowel Disease Mouse Model. J. Vis. Exp. ...

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

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