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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K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dextran+Sulfate+Sodium+(DSS)-Induced+Colitis+in+Mice.">Dextran Sulfate Sodium (DSS)-Induced Colitis in Mice.</a> Current Protocols in Immunology. 104 (1), 11-14 (2014).</li><li>Chassaing, B., Darfeuille-Michaud, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+Commensal+Microbiota+and+Enteropathogens+in+the+Pathogenesis+of+Inflammatory+Bowel+Diseases.">The Commensal Microbiota and Enteropathogens in the Pathogenesis of Inflammatory Bowel Diseases.</a> Gastroenterology. 140 (6), 1720-1728 (2011).</li><li>Chandrashekar, J., Hoon, M. A., Ryba, N. J. P., Zuker, C. 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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. -. H., Lifshitz, L. M., ZhuGe, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Extraoral+bitter+taste+receptors+in+health+and+disease.">Extraoral bitter taste receptors in health and disease.</a> The Journal of General Physiology. 149 (2), 181-197 (2017).</li><li>Wirtz, S., Neufert, C., Weigmann, B., Neurath, M. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chemically+induced+mouse+models+of+intestinal+inflammation.">Chemically induced mouse models of intestinal inflammation.</a> Nature Protocols. 2, 541-546 (2007).</li><li>Chassaing, B., Aitken, J. D., Malleshappa, M., Vijay-Kumar, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dextran+sulfate+sodium+(DSS)-induced+colitis+in+mice.">Dextran sulfate sodium (DSS)-induced colitis in mice.</a> Current Protocols in Immunology. 104 (25), (2014).</li><li>Feng, P., 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=Aggravated+gut+inflammation+in+mice+lacking+the+taste+signaling+protein+&#945;-gustducin.">Aggravated gut inflammation in mice lacking the taste signaling protein &#945;-gustducin.</a> Brain, Behavior, and Immunity. 71, 23-27 (2018).</li><li>Feng, P., 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=Immune+cells+of+the+human+peripheral+taste+system:+Dominant+dendritic+cells+and+CD4+T+cells.">Immune cells of the human peripheral taste system: Dominant dendritic cells and CD4 T cells.</a> Brain, Behavior, and Immunity. 23 (6), 760-766 (2009).</li><li>Livak, K. J., Schmittgen, T. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Analysis+of+relative+gene+expression+data+using+real-time+quantitative+PCR+and+the+2(-Delta+Delta+C(T))+Method.">Analysis of relative gene expression data using real-time quantitative PCR and the 2(-Delta Delta C(T)) Method.</a> Methods. 25 (4), 402-408 (2001).</li><li>Kim, J. J., Shajib, M. S., Manocha, M. M., Khan, W. I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Investigating+Intestinal+Inflammation+in+DSS-induced+Model+of+IBD.">Investigating Intestinal Inflammation in DSS-induced Model of IBD.</a> Journal of Visualized Experiments. (60), 3678 (2012).</li><li>Axelsson, L. -. G., Landstr&#246;m, E., Goldschmidt, T. J., Gr&#246;nberg, A., Bylund-Fellenius, A. -. 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S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Murine+Colitis+Modeling+using+Dextran+Sulfate+Sodium+(DSS).">Murine Colitis Modeling using Dextran Sulfate Sodium (DSS).</a> Journal of Visualized Experiments. (35), 1652 (2010).</li><li>Howitt, M. R., 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=Tuft+cells,+taste-chemosensory+cells,+orchestrate+parasite+type+2+immunity+in+the+gut.">Tuft cells, taste-chemosensory cells, orchestrate parasite type 2 immunity in the gut.</a> Science. 351 (6279), 1329-1333 (2016).</li></ol>

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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			<td height="20" style="height:20px;width:269px;">Antibody</td>
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			<td style="width:64px;"></td>
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			<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>
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			<td height="21" style="height:21px;">Ly6G</td>
			<td>BD Biosciences</td>
			<td>551459</td>
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			<td height="20" style="height:20px;">Reagent</td>
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			<td></td>
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			<td height="21" style="height:21px;">Dextran Sulfate Sodium Salt (DSS)</td>
			<td>MP Biomedicals</td>
			<td>2160110</td>
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			<td height="20" style="height:20px;">Streptavidin-HRP complex</td>
			<td>BD Pharmingen</td>
			<td>551011</td>
			<td></td>
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			<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>
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			<td height="21" style="height:21px;">FastStart Universal SYBR Green Master(ROX)</td>
			<td>Roche</td>
			<td>4913850001</td>
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			<td height="21" style="height:21px;">MMLV Reverse Transcriptase, GPR</td>
			<td>Clontech,TaKaRa</td>
			<td>639574</td>
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			<td height="21" style="height:21px;">TaKaRa MiniBEST Universal RNA Extraction Kit&#160;</td>
			<td>TaKaRa</td>
			<td>9767</td>
			<td></td>
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			<td height="21" style="height:21px;">BD 10 ml Syringe</td>
			<td>BD Biosciences</td>
			<td>309604</td>
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			<td height="20" style="height:20px;">Instruments and equipment</td>
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			<td height="20" style="height:20px;">balance</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>
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		<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>
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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 ...

This method can help answer key questions in the inflammatory biodisease field about ulcerative colitis and Crohn's disease. The main advantage of this technique is that it can be used to evaluate first anti and proinfammatory effects of signaling proteins. The implication of this technique extends the survey of gas in new advances and inflammation.Though this method can provide insights into genetic effect on gut inflammation. It can also be applied to other factors such as diets and microbiome. Generally, individuals new to this semester will struggle because processing disease scar tissue is tricky.Visual demonstration of this method is critical as dissection and the tissue preparation steps are difficult to learn because mystalia and the cell tissue structures may be altered. To induce dextran sulfate sodium, or DDS colitis, replace the regular drinking water with 3%DSS solution, add libidium for seven days. Measuring each mouses'body weight, DSS solution consumption, and stool production daily.At the end of the seven day treatment, place the mouse in the supine position and use small scissors to make a three centimeter long midline incision in the abdomen. Harvest the spleen and measure its size. Then use forceps to lift the colon to allow it to be separated from the surrounding mesentery.And extract the whole colon until the cecum and rectum are visible. Transect the tissue at the colonosecal margin. And deep within the pelvis to free the proximal and distal colon, respectively.Measure and record the length of the isolated colon. And use a 10 milliliter syringe equipped with a gavage needle to flush the colon with 10 milliliters of ice cold PBS to remove the feces and blood until the illuate runs clear. For histological identification, divide the tissue samples into equally sized proximal, middle, and distal sections.And fix the tissues in 4%paraformaldehyde overnight at 4 degrees Celsius. For hematoxylin and eosin staining of the isolated colon tissue, the next morning, submerge the tissue pieces in 30%sucrose and PBS overnight in individual 15 milliliter tubes for cryo protection of the samples. The next day, embedd the tissues in optimal cutting temperature or OCT compound.And cool the tissues in minus 20 degrees Celsius until the OCT hardens. Place the OCT blocks in the cryostat and set the thickness dial to 12 micrometers. Then slice and collect 12 micrometer thick frozen sections onto glass microscope slides.When all the tissues have been sectioned, warm the slides at 65 degrees Celsius on a hot plate for 20 minutes. And wash the heated sections briefly in distilled water before staining with hematoxylin staining solution for five minutes. Remove the excess hematoxylin solution and run in tap water for five minutes.And differentiate the sections with 0.5%hydrochloric acid in ethanol for 30 seconds, followed by a one minute rinse under running tap water. Next, wash the samples for one minute in PBS. Followed by another five minute wash under running tap water.At the end of the wash, submerge the tissues in an ascending ethanol series for 10 seconds per immersion. Followed by counterstaining in eosin for two minutes. For dehydration of the samples, submerge the slides in 95%ethanol and two changes of 100%ethanol for five minutes per immersion.Followed by clearing with two, five minute changes in xylene. Then score the tissue damage to the proximal, middle, and distal colons of each experimental group. For immunohistochemical analysis, after warming the sections for 20 minutes on the hot plate, wash the slides three times in PBS for 10 minutes per wash.And eliminate the endogenous peroxidase with a 10 minute 3%hydrogen peroxide incubation. At the end of the incubation, wash the samples three times in PBS as demonstrated and block any nonspecific binding with blocking buffer for at least one hour at room temperature. Then, replace the blocking buffer with the primary antibody cocktail of interest.For an overnight incubation at 4 degrees Celsius. The next morning, aspirate the excess primary antibody solution and wash the slides three times in PBS. After the last wash, incubate the slides with the appropriate biotinylated secondary antibody cocktail.Followed by labeling with streptavidin horseradish peroxidase complex at room temperature. To visualize the immunoreactive cells, label the samples with DAB until a light or dark brown color develops. And counterstain the sections with hematoxylin and 0.3%diluted ammonia.When the slides have dried, use a light microscope to obtain bright field images of the tissue sections under a 10x magnification. And use an image processing program to identify and quantify the population of the marked immune cells in both the epithelium and the lamina propria. For gene expression analysis in DSS treated colons, extract RNA from minus 80 degrees Celsius thawed colonic tissue samples according to standard RNA extraction protocols.And mix one microgram with the total RNA with 2.5 microliters of 20 millimolar oligo deoxythymidine 12 through 18 primers and one microliter of moloney murine leukemia virus reverse transcriptase. After a 60 minute incubation at 42 degrees Celsius, mix one microliter of the cDNA with 0.5%microliters of the appropriate forward and reverse quantitative PCR primers for the cytokines of interest and 2x fluorescent green dye. Then run the quantitative PCR under the appropriate parameters and calculate the relative gene expression according to standard protocols.Compared to wildtype mice, alpha gustducin knockout mice exhibit a more severe colitis with excessive weight loss, diarrhea and intestinal bleeding. After seven days of DSS administration, histological analysis of the colon sections reveal a more aggravated tissue damage within the proximal, middle, and distal colons of the knockout mice, compared to their wildtype counterparts. This excessive immune activation leads to the induction of colitis with a robust infiltration of various inflammatory cells in the knockout animal colon as observed by immunohistochemical analysis.Further, quantitative PCR analysis demonstrates higher levels of TNF alpha and inferon gamma expression but lower expression of IL-5, 10, and 13, in knockout compared to wildtype mouse colons with no difference in TGF beta one expression observed. While attempting this procedure, it's important to remember to optimize textorous southvater sodium dosage and demonstration duration. Forwarding this procedure, other method like obtaining culture can be performed in order to understand additional questions like the effect of inflammation in intestinal tissue origination.After it's development, this technique paves a way for researchers in the field of gut inflammation to explore the contributions of gene mutations to the severity of biodisease erodings and other vertebrates. Don't forget that working with disease gut tissue and its contents can be extremely hazardous and the repercussions such as wearing things that are appropriate, personal protection permanent should always be taken while performing this procedure.

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Trichuris muris is a natural pathogen of mice and is biologically and antigenically similar to species of Trichuris that 
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examine factors that control CD4+ T helper (Th) cell activation as well as changes in the intestinal epithelium. The immune response that occurs in 
resistant inbred strains, such as C57BL/6 and BALB/c, is characterized by Th2 polarized cytokines (IL-4, IL-5 and IL-13) and expulsion of worms while Th1-associated 
cytokines (IL-12, IL-18, IFN-&gamma;) promote chronic infections in genetically susceptible AKR/J mice2-6. Th2 cytokines promote physiological changes in 
the intestinal microenvironment including rapid turnover of IECs, goblet cell differentiation, recruitment and changes in epithelial permeability and smooth muscle 
contraction, all of which have been implicated in worm expulsion7-15. Here we detail a protocol for propagating Trichuris muris eggs which can 
be used in subsequent experiments. We also provide a sample experimental harvest with suggestions for post-infection analysis. Overall, this protocol will provide 
researchers with the basic tools to perform a Trichuris muris mouse infection model which can be used to address questions pertaining to Th proclivity in 
the gastrointestinal tract as well as immune effector functions of IECs.

Trichuris muris Infection: A Model of Type 2 Immunity and Inflammation in the Gut

Trichuris muris infection is an intestinal model of Th2 immunity where resistant mice generate a protective Th2 response and susceptible mice generate a pathological Th1 response.

Trichuris muris is a natural pathogen of mice and is biologically and antigenically similar to species of Trichuris that 
infect humans and livestock1. ...

Differentiating Functional Roles of Gene Expression from Immune and Non-immune Cells in Mouse Colitis by Bone Marrow Transplantation

To understand the role of a gene in the development of colitis, we compared the responses of wild-type mice and gene-of-interest deficient knockout mice to colitis. If the gene-of-interest is expressed in both bone marrow derived cells and non-bone marrow derived cells of the host; however, it is possible to differentiate the role of a gene of interest in bone marrow derived cells and non- bone marrow derived cells by bone marrow transplantation technique. To change the bone marrow derived cell genotype of mice, the original bone marrow of recipient mice were destroyed by irradiation and then replaced by new donor bone marrow of different genotype. When wild-type mice donor bone marrow was transplanted to knockout mice, we could generate knockout mice with wild-type gene expression in bone marrow derived cells. Alternatively, when knockout mice donor bone marrow was transplanted to wild-type recipient mice, wild-type mice without gene-of-interest expressing from bone marrow derived cells were produced. However, bone marrow transplantation may not be 100% complete. Therefore, we utilized cluster of differentiation (CD) molecules (CD45.1 and CD45.2) as markers of donor and recipient cells to track the proportion of donor bone marrow derived cells in recipient mice and success of bone marrow transplantation. Wild-type mice with CD45.1 genotype and knockout mice with CD45.2 genotype were used. After irradiation of recipient mice, the donor bone marrow cells of different genotypes were infused into the recipient mice. When the new bone marrow regenerated to take over its immunity, the mice were challenged by chemical agent (dextran sodium sulfate, DSS 5%) to induce colitis. Here we also showed the method to induce colitis in mice and evaluate the role of the gene of interest expressed from bone-marrow derived cells. If the gene-of-interest from the bone derived cells plays an important role in the development of the disease (such as colitis), the phenotype of the recipient mice with bone marrow transplantation can be significantly altered. At the end of colitis experiments, the bone marrow derived cells in blood and bone marrow were labeled with antibodies against CD45.1 and CD45.2 and their quantitative ratio of existence could be used to evaluate the success of bone marrow transplantation by flow cytometry. Successful bone marrow transplantation should show a vast majority of donor genotype (in term of CD molecule marker) over recipient genotype in both the bone marrow and blood of recipient mice.

Bone marrow transplantation provides a way to change the genotype of the bone marrow derived cells. If the gene of interest is expressed in both bone marrow derived cells and non-bone marrow derived cells, bone marrow transplantation can change the bone marrow derived cells to a different genotype without changing the non-bone marrow derived cell genotype.

To understand the role of a gene in the development of colitis, we compared the responses of wild-type mice and gene-of-interest deficient knockout mice ...

A Mouse Model for Pathogen-induced Chronic Inflammation at Local and Systemic Sites

Chronic inflammation is a major driver of pathological tissue damage and a unifying characteristic of many chronic diseases in humans including neoplastic, autoimmune, and chronic inflammatory diseases. Emerging evidence implicates pathogen-induced chronic inflammation in the development and progression of chronic diseases with a wide variety of clinical manifestations. Due to the complex and multifactorial etiology of chronic disease, designing experiments for proof of causality and the establishment of mechanistic links is nearly impossible in humans. An advantage of using animal models is that both genetic and environmental factors that may influence the course of a particular disease can be controlled. Thus, designing relevant animal models of infection represents a key step in identifying host and pathogen specific mechanisms that contribute to chronic inflammation.
Here we describe a mouse model of pathogen-induced chronic inflammation at local and systemic sites following infection with the oral pathogen Porphyromonas gingivalis, a bacterium closely associated with human periodontal disease. Oral infection of specific-pathogen free mice induces a local inflammatory response resulting in destruction of tooth supporting alveolar bone, a hallmark of periodontal disease. In an established mouse model of atherosclerosis, infection with P. gingivalis accelerates inflammatory plaque deposition within the aortic sinus and innominate artery, accompanied by activation of the vascular endothelium, an increased immune cell infiltrate, and elevated expression of inflammatory mediators within lesions. We detail methodologies for the assessment of inflammation at local and systemic sites. The use of transgenic mice and defined bacterial mutants makes this model particularly suitable for identifying both host and microbial factors involved in the initiation, progression, and outcome of disease. Additionally, the model can be used to screen for novel therapeutic strategies, including vaccination and pharmacological intervention.

Animal models have proven to be invaluable tools in defining host and pathogen specific mechanisms that contribute to the development of chronic inflammation. Here we describe a mouse model of oral infection with the human pathogen Porphyromonas gingivalis and detail methodologies to assess the progression of inflammation at local and systemic sites.

Chronic inflammation is a major driver of pathological tissue damage and a unifying characteristic of many chronic diseases in humans including ...

Co-immunoprecipitation of the Mouse Mx1 Protein with the Influenza A Virus Nucleoprotein

Studying the interaction between proteins is key in understanding their function(s). A very powerful method that is frequently used to study interactions of proteins with other macromolecules in a complex sample is called co-immunoprecipitation. The described co-immunoprecipitation protocol allows to demonstrate and further investigate the interaction between the antiviral myxovirus resistance protein 1 (Mx1) and one of its viral targets, the influenza A virus nucleoprotein (NP). The protocol starts with transfected mammalian cells, but it is also possible to use influenza A virus infected cells as starting material. After cell lysis, the viral NP protein is pulled-down with a specific antibody and the resulting immune-complexes are precipitated with protein G beads. The successful pull-down of NP and the co-immunoprecipitation of the antiviral Mx1 protein are subsequently revealed by western blotting. A prerequisite for successful co-immunoprecipitation of Mx1 with NP is the presence of N-ethylmaleimide (NEM) in the cell lysis buffer. NEM alkylates free thiol groups. Presumably this reaction stabilizes the weak and/or transient NP&#8211;Mx1 interaction by preserving a specific conformation of Mx1, its viral target or an unknown third component. An important limitation of co-immunoprecipitation experiments is the inadvertent pull-down of contaminating proteins, caused by nonspecific binding of proteins to the protein G beads or antibodies. Therefore, it is very important to include control settings to exclude false positive results. The described co-immunoprecipitation protocol can be used to study the interaction of Mx proteins from different vertebrate species with viral proteins, any pair of proteins, or of a protein with other macromolecules. The beneficial role of NEM to stabilize weak and/or transient interactions needs to be tested for each interaction pair individually.

This co-immunoprecipitation protocol allows to study the interaction between the influenza A virus nucleoprotein and the antiviral Mx1 protein in human cells. The protocol emphasizes the importance of N-ethylmaleimide for successful co-immunoprecipitation of Mx1 and influenza A virus nucleoprotein.

Studying the interaction between proteins is key in understanding their function(s). A very powerful method that is frequently used to study interactions ...

An Optimized Method for Isolating and Expanding Invariant Natural Killer T Cells from Mouse Spleen

The ability to rapidly secrete cytokines upon stimulation is a functional characteristic of the invariant natural killer T (iNKT) cell lineage. iNKT cells are therefore characterized as an innate T cell population capable of activating and steering adaptive immune responses. The development of improved techniques for the culture and expansion of murine iNKT cells facilitates the study of iNKT cell biology in in vitro and in vivo model systems. Here we describe an optimized procedure for the isolation and expansion of murine splenic iNKT cells.
Spleens from C57Bl/6 mice are removed, dissected and strained and the resulting cellular suspension is layered over density gradient media. Following centrifugation, splenic mononuclear cells (MNCs) are collected and CD5-positive (CD5+) lymphocytes are enriched for using magnetic beads. iNKT cells within the CD5+ fraction are subsequently stained with &#945;GalCer-loaded CD1d tetramer and purified by fluorescence activated cell sorting (FACS). FACS sorted iNKT cells are then initially cultured in vitro using a combination of recombinant murine cytokines and plate-bound T cell receptor (TCR) stimuli before being expanded in the presence of murine recombinant IL-7. Using this technique, approximately 108 iNKT cells can be generated within 18-20 days of culture, after which they can be used for functional assays in vitro, or for in vivo transfer experiments in mice.

Here we present an adapted protocol that can be used to generate a large number of murine invariant natural killer T cells from mouse spleen. The protocol outlines an approach by which splenic iNKT cells can be enriched for, isolated and expanded in vitro using a limited number of animals and reagents.

The ability to rapidly secrete cytokines upon stimulation is a functional characteristic of the invariant natural killer T (iNKT) cell lineage. iNKT cells ...

Establishment of a High-throughput Setup for Screening Small Molecules That Modulate c-di-GMP Signaling in Pseudomonas aeruginosa

Bacterial resistance to traditional antibiotics has driven research attempts to identify new drug targets in recently discovered regulatory pathways. Regulatory systems that utilize intracellular cyclic di-GMP (c-di-GMP) as a second messenger are one such class of target. c-di-GMP is a signaling molecule found in almost all bacteria that acts to regulate an extensive range of processes including antibiotic resistance, biofilm formation and virulence. The understanding of how c-di-GMP signaling controls aspects of antibiotic resistant biofilm development has suggested approaches whereby alteration of the cellular concentrations of the nucleotide or disruption of these signaling pathways may lead to reduced biofilm formation or increased susceptibility of the biofilms to antibiotics. We describe a simple high-throughput bioreporter protocol, based on green fluorescent protein (GFP), whose expression is under the control of the c-di-GMP responsive promoter cdrA, to rapidly screen for small molecules with the potential to modulate c-di-GMP cellular levels in Pseudomonas aeruginosa (P. aeruginosa). This simple protocol can screen upwards of 3,500 compounds within 48 hours and has the ability to be adapted to multiple microorganisms.

Establishment of a High-throughput Setup for Screening Small Molecules That Modulate c-di-GMP Signaling in Pseudomonas aeruginosa

This article describes the high-throughput assay that has been successfully established to screen large libraries of small molecules for their potential ability to manipulate cellular levels of cyclic di-GMP in Pseudomonas aeruginosa, providing a new powerful tool for antibacterial drug discovery and compound testing.

Bacterial resistance to traditional antibiotics has driven research attempts to identify new drug targets in recently discovered regulatory pathways. ...

Visualizing the Effects of Sputum on Biofilm Development Using a Chambered Coverglass Model

Biofilms consist of groups of bacteria encased in a self-secreted matrix. They play an important role in industrial contamination as well as in the development and persistence of many health related infections. One of the most well described and studied biofilms in human disease occurs in chronic pulmonary infection of cystic fibrosis patients. When studying biofilms in the context of the host, many factors can impact biofilm formation and development. In order to identify how host factors may affect biofilm formation and development, we used a static chambered coverglass method to grow biofilms in the presence of host-derived factors in the form of sputum supernatants. Bacteria are seeded into chambers and exposed to sputum filtrates. Following 48 hr of growth, biofilms are stained with a commercial biofilm viability kit prior to confocal microscopy and analysis. Following image acquisition, biofilm properties can be assessed using different software platforms. This method allows us to visualize key properties of biofilm growth in presence of different substances including antibiotics.

This protocol describes the visualization of biofilm development following exposure to host-factors using a slide chamber model. This model allows for direct visualization of biofilm development as well as analysis of biofilm parameters using computer software programs.

Biofilms consist of groups of bacteria encased in a self-secreted matrix. They play an important role in industrial contamination as well as in the ...

Precision-cut Mouse Lung Slices to Visualize Live Pulmonary Dendritic Cells

Inhalation of allergens and pathogens elicits multiple changes in a variety of immune cell types in the lung. Flow cytometry is a powerful technique for quantitative analysis of cell surface proteins on immune cells, but it provides no information on the localization and migration patterns of these cells within the lung. Similarly,&#160;chemotaxis assays can be performed to study the potential of cells to respond to chemotactic factors in vitro, but these assays do not reproduce the complex environment of the intact lung. In contrast to these aforementioned techniques, the location of individual cell types within the lung can be readily visualized by generating Precision-cut Lung Slices (PCLS), staining them with commercially available, fluorescently tagged antibodies, and visualizing the sections by confocal microscopy. PCLS can be used for both live and fixed lung tissue, and the slices can encompass areas as large as a cross section of an entire lobe. We have used this protocol to successfully visualize the location of a wide variety of cell types in the lung, including distinct types of dendritic cells, macrophages, neutrophils, T cells and B cells, as well as structural cells such as lymphatic, endothelial, and epithelial cells. The ability to visualize cellular interactions, such as those between dendritic cells and T cells, in live, three-dimensional lung tissue, can reveal how cells move within the lung and interact with one another at steady state and during inflammation. Thus, when used in combination with other procedures, such as flow cytometry and quantitative PCR, PCLS can contribute to a comprehensive understanding of cellular events that underlie allergic and inflammatory diseases of the lung.

We describe a method for generating Precision-cut Lung Slices (PCLS) and immunostaining them to visualize the localization of various immune cell types in the lung. Our protocol can be extended to visualize the location and function of many different cell types under a variety of conditions.

Inhalation of allergens and pathogens elicits multiple changes in a variety of immune cell types in the lung. Flow cytometry is a powerful technique for ...

An IL-8 Transiently Transgenized Mouse Model for the In Vivo Long-term Monitoring of Inflammatory Responses

Airway inflammation is often associated with bacterial infections and represents a major determinant of lung disease. The in vivo determination of the pro-inflammatory capabilities of various factors is challenging and requires terminal procedures, such as bronchoalveolar lavage and the removal of lungs for in situ analysis, precluding longitudinal visualization in the same mouse. Here, lung inflammation is induced through the intratracheal instillation of Pseudomonas aeruginosa culture supernatant (SN) in transiently transgenized mice expressing the luciferase reporter gene under the control of a heterologous IL-8 bovine promoter. Luciferase expression in the lung is monitored by in vivo bioluminescent image (BLI) analysis over a 2.5- to 48-h timeframe following the instillation. The procedure can be repeated multiple times within 2 - 3 months, thus permitting the evaluation of the inflammatory response in the same mice without the need to terminate the animals. This approach permits the monitoring of pro- and anti-inflammatory factors acting in the lung in real time and appears suitable for functional and pharmacological studies.

An IL-8 Transiently Transgenized Mouse Model for the In Vivo Long-term Monitoring of Inflammatory Responses

The method described here allows for the visualization of IL-8 promoter-dependent inflammation activation in the lungs of mice through non-invasive bioluminescence imaging (BLI). The same animal can be subjected to BLI multiple times for up to two months from the time of delivery of the luciferase reporter construct.

Airway inflammation is often associated with bacterial infections and represents a major determinant of lung disease. The in vivo determination of the ...

Dissecting Multi-protein Signaling Complexes by Bimolecular Complementation Affinity Purification (BiCAP)

The assembly of protein complexes is a central mechanism underlying the regulation of many cell signaling pathways. A major focus of biomedical research is deciphering how these dynamic protein complexes act to integrate signals from multiple sources in order to direct a specific biological response, and how this becomes deregulated in many disease settings. Despite the importance of this key biochemical mechanism, there is a lack of experimental techniques that can facilitate the specific and sensitive deconvolution of these multi-molecular signaling complexes.
Here this shortcoming is addressed through the combination of a protein complementation assay with a conformation-specific nanobody, which we have termed Bimolecular Complementation Affinity Purification (BiCAP). This novel technique facilitates the specific isolation and downstream proteomic characterization of any pair of interacting proteins, to the exclusion of un-complexed individual proteins and complexes formed with competing binding partners. 
The BiCAP technique is adaptable to a wide array of downstream experimental assays, and the high degree of specificity afforded by this technique allows more nuanced investigations into the mechanics of protein complex assembly than is currently possible using standard affinity purification techniques.

Dissecting Multi-protein Signaling Complexes by Bimolecular Complementation Affinity Purification BiCAP

This manuscript describes the protocol for Bimolecular Complementation Affinity Purification (BiCAP). This novel method facilitates the specific isolation and downstream proteomic characterization of any two interacting proteins, while excluding un-complexed individual proteins as well as complexes formed with competing binding partners.

The assembly of protein complexes is a central mechanism underlying the regulation of many cell signaling pathways. A major focus of biomedical research ...