JHN is supported by the Swiss National Foundation (SNSF 310030_146290).

Mice were bred and kept under specific pathogen-free (SPF) conditions in the animal facility of Ulm University (Ulm, Germany). All animal experiments were performed according to the guidelines of the local animal use and care committee and the National Animal Welfare Law.
1. Construction of the pCFP-OVA Plasmid
<ol>
	<li>Amplify the full size OVA gene using the primers Ova_SpeI_fw (3&#39;-GACCAACTAGTATGGAATTTTGTTTTGATGTATT-5&#39;) and Ova_ClaI_rev (3&#39;- GACCAGATCGATTAAGGGGAAACACATCTGCC-5&#39;)37 using plasmid pCI-OVA (ampicillin resistant)42 as a template. Clone the PCR product (i.e. the OVA coding sequence) into a commercially available phage mid-vector using SpeI and ClaI restrictions sites of the primers and vector to yield the specific OVA-plasmid.</li>
	<li>Amplify the CFP-coding gene together with the strong constitutive promoter Phyper using plasmid pAD1-cCFP (chloramphenicol resistant) 43 as a template and primers CFP_SacII_fw (3&#39;-GATCGACCGCGGTTCTTGAAGACGAAAGGGCC-5&#39;) and CFP_SpeI_rev (3&#39;-GATCGAACTAGTACCACCACCACCACCACCTTTGTAGAGTTCATCCATGC-5&#39;)37.</li>
	<li>Clone the CFP-coding gene into the specific OVA-plasmid (ampicillin resistant) using the SpeI and SacI restriction sites of the primers and vector37. This introduces a spacer of 6 glycine residues between the CFP-coding from OVA-coding sequence.</li>
</ol>
2. Construction of E.coli pCFP-OVA
<ol>
	<li>Grow E.coli strain DH10B in 100 ml of Luria Bertani (LB) medium aerobically at 37 &#176;C on a rotary shaker overnight.</li>
	<li>Mix 800 &#181;l of bacterial culture with 200 &#181;l of glycerol to create glycerol stock and store at -80 &#176;C.</li>
	<li>Add 50 &#181;l of E.coli strain DH10B from glycerol stock in 5 ml of LB medium and grow them aerobically at 37 &#176;C on a rotary shaker overnight.</li>
	<li>Transfer the culture in a baffled 1 L Erlenmeyer flask with 250 ml of LB medium at 37 &#176;C in a rotary shake.</li>
	<li>Grow bacteria to an optical density at 600 nm (OD600) of 0.5 and then chill the culture on ice for 30 min. Centrifuge the bacteria at 5,000 x g for 10 min at 4 &#176;C.</li>
	<li>Resuspend bacteria with 100 ml of ice-cold sterile H2O and centrifuge the bacteria at 5,000 x g for 10 min at 4&#176; C. Resuspend bacteria with 100 ml of ice-cold 10% glycerol and centrifuge the bacteria at 5,000 x g for 10 min at 4 &#176;C. Repeat this last step twice.</li>
	<li>After the final wash step, resuspend bacteria in 700 &#181;l of 10% glycerol. Freeze 50 &#181;l aliquots in liquid nitrogen and store at -80 &#176;C.</li>
	<li>Thaw a 50 &#181;l aliquot on ice and transfer into a pre-chilled electroporation cuvette (2 mm electrode distance).</li>
	<li>Add 5 &#181;l of plasmid DNA (100 ng) to the 50 &#181;l E. coli aliquots. Perform electroporation with a single pulse at 25 &#181;F, 200 ohms (&#937;) and 2.5 kV.</li>
	<li>Resuspend bacteria in 1 ml pre-warmed SOC medium (5 g/L yeast extract, 2 g/L tryptone, 10 mM sodium chloride, 2.5 mM potassium chloride, 10 mM magnesium chloride, 10 mM magnesium sulfate) and incubate at 37 &#176;C aerobically for 1 hr.</li>
	<li>Plate bacteria on LB-agar plates supplemented with ampicillin 100 &#181;g/ml (add 100 &#181;l of a 100 mg/ml ampicillin stock to 100 ml of LB-agar) and incubate overnight aerobically at 37 &#176;C.</li>
	<li>Pick single colonies and re-streak them in LB-agar plates supplemented with ampicillin 100 &#181;g/ml. Incubate overnight aerobically at 37 &#176;C.</li>
	<li>Pick single colonies and grow them overnight in 10 ml of LB media supplemented with ampicillin 100 &#181;g/ml for plasmid isolation. Use commercially available plasmid mini prep kit and elute plasmid DNA with 30 &#181;l dH2O (distilled water) according to manufacturer&#39;s protocol. Verify plasmids by restriction analysis and DNA sequencing37,42,43.</li>
	<li>Set up a culture of positive clones of E. coli pCFP-OVA in 100 ml of LB medium supplemented with ampicillin 100 &#181;g/ml. Grow aerobically at 37 &#176;C on a rotary shaker overnight.</li>
	<li>Mix 800 &#181;l of bacterial culture with 200 &#181;l of pure glycerol and store at -80 &#176;C.</li>
	<li>Centrifuge the rest of the overnight culture at 5,000 x g for 10 min at room temperature (RT) and resuspend the pellet in phosphate buffered saline (PBS). Place 10 &#181;l of the suspension onto glass slide and cover it with a coverslip.</li>
	<li>Observe the samples in a standard fluorescence microscopy to confirm the expression fluorescence of CFP-OVA (CFP excitation at 450 nm, emission at 480 nm). Analyze the bacteria starting with magnification 400X.</li>
	<li>Centrifuge 1 ml of bacteria from overnight cultures at 5,000 x g for 10 min, RT. Resuspend the pellet in 40 &#181;l of PBS and boil at 95 &#176;C for 10 min.</li>
	<li>Centrifuge the samples at 30,000 x g for 10 min and use the supernatants for Western blot analysis37. Use the rabbit-raised anti-OVA polyclonal antibody diluted 1:400.</li>
</ol>
3. Generation of OT-II/Red Mice
<ol>
	<li>Cross a male/female OT-II mouse with a female/male mouse expressing red fluorescent protein (both strains commercially available) in order to generate the OT-II/Red mice37.</li>
</ol>
4. Spleen Cell Isolation
<ol>
	<li>Euthanize the OT-II/Red mice by cervical dislocation after anesthesia by inhalation of any halogenated ether (up to 5% for induction) commercially available.</li>
	<li>Use scissors to cut the left superior abdominal part of the mouse. Under this abdominal part there is the spleen (oval shape with a dark-red color). Use scissors and tweezers to remove it.
	<ol>
		<li>Extract the spleen of the OT-II/Red mice and pass it through a 100 &#181;m cell strainer positioned on a 50 ml tube using the plunger of a 1 ml syringe in order to obtain a single-cell suspension. Wash the strainer twice with 10 ml of PBS supplemented with 1% heat inactivated fetal bovine serum (FBS).</li>
	</ol>
	</li>
	<li>Centrifuge the cell suspension at 390 x g for 5 min at RT. Resuspend the pellet with 5 ml of pre-warmed lysis buffer (144 mM NH4Cl, 17 mM Tris, pH= 7.2) to lyse the red blood cells. Incubate 10 min at 37 &#176;C.</li>
	<li>Wash the sample with 25 ml of PBS supplemented with 1% FBS. Centrifuge at 390 x g for 5 min at RT. Resuspend the pellet in 10 ml of PBS supplemented with 1% FBS.</li>
	<li>Count the cells using a Neubauer counting chamber. Mix 10 &#181;l of the cells suspension with 10 &#181;l of a 0.4% Trypan Blue solution. Incubate for 1-2 min.
	<ol>
		<li>Fill the hemocytometer chamber with 10 &#181;l of stained cells. Count cells under the microscope in four 1 x 1 mm2 squares of one chamber and determine the average number of cells per square. Viable cells appear white, dead cells appear blue. To determine the number of cells/ml multiply cell number by factor of dilution. If 2-4 quadrants are being counted, divide by the number of quadrants.</li>
	</ol>
	</li>
</ol>
5. CD4 + CD62 L+ T Cell Enrichment
<ol>
	<li>Enrich spleen cells for CD4+ CD62L+ T cells via magnetic isolation kit using the principles of negative or positive selection. Here, use negative selection to isolate CD4+ T cells and use positive selection to further enrich for the CD62L+ population within the CD4+ T cell pool. 
	NOTE: In the negative selection procedure, the cells, which will be not isolated (here: all CD4 negative cells) are labeled with specific antibodies coupled with magnetic microbeads. During the column washing steps the CD4+ T cells will pass through the column so that they can be easily collected. In the positive selection procedure cells to be isolated are labeled with appropriate antibody coupled with magnetic microbeads. During the column washing steps the labeled cells will stay in the column and they can be collected by removing the column from the magnetic field and rinsing it with the buffer.</li>
	<li>For the negative selection procedure, resuspend 107 total spleen cells in 40 &#181;l of cold PBS supplemented with 0.5% bovine serum albumin (BSA). Add 10 &#181;l of biotin labeled antibody cocktail supplied with the magnetic isolation kit (specific antibodies binding the non-CD4+ T cells) and incubate 15 min on ice.
	<ol>
		<li>Add 30 &#181;l of cold PBS supplemented with 0.5% BSA and 20 &#181;l of anti-biotin microbeads. Incubate 20 min on ice.</li>
		<li>Add to the sample 10 ml of PBS supplemented with 0.5% BSA and centrifuge at 390 x g for 5 min. Repeat this step twice. Resuspend the cells in 1 ml of cold PBS supplemented with 0.5% BSA.</li>
		<li>Place the column specific for the negative selection procedure in the magnetic field and rinse with 3 ml of cold PBS supplemented with 0.5% BSA onto the column. Add the cell suspension onto the column and wash 3 times with 3 ml of cold PBS supplemented with 0.5% BSA.</li>
		<li>Centrifuge cells of the effluent at 390 x g for 5 min. Effluent passing through the column contains unlabeled CD4+ T cells. Resuspend the pellet in 1 ml of PBS supplemented with 0.5% BSA and count the cells.</li>
		<li>Count the cells using a Neubauer counting chamber. Mix 10 &#181;l of the cells suspension with 10 &#181;l of a 0.4% Trypan Blue solution. Incubate for 1-2 min.
		<ol>
			<li>Fill the hemocytometer chamber with 10 &#181;l of stained cells. Count cells under the microscope in four 1 x 1 mm2 squares of one chamber and determine the average number of cells per square. Viable cells appear white, dead cells appear blue. To determine the number of cells/mL multiply cell number by factor of dilution. If 2-4 quadrants are being counted, divide by the number of quadrants.</li>
		</ol>
		</li>
	</ol>
	</li>
	<li>For the positive selection procedure, add 10 &#181;l of CD62L microbeads per 107 pre-enriched CD4+ T cell fraction. Incubate 15 min on ice.
	<ol>
		<li>Add to the sample 10 ml of PBS supplemented with 0.5% BSA and centrifuge at 390 x g for 5 min. Repeat this step twice. Resuspend in 500 &#181;l of cold PBS supplemented with 0.5% BSA.</li>
		<li>Place the specific column for the negative selection procedure in the magnetic field and rinse with 500 &#181;l of cold PBS supplemented with 0.5% BSA onto the column. Add cell suspension onto the column and wash 3 times with 500 &#181;l of cold PBS supplemented with 0.5% BSA.</li>
		<li>Remove the column from the magnetic field and place it on a suitable collection tube. Add 1 ml of PBS supplemented with 0.5% BSA onto the column and flush out CD4+ CD62L+ T cells retained in the column by firmly pushing the plunger into the column. 
		NOTE: The isolated CD4+ CD62L+ T cells are now ready for downstream applications, such as in vivo experiments. According to the manufacturer&#39;s instruction from the CD4+ CD62L+ T cells isolation kit, the microbeads are not toxic. Due to the small size (around 50 nm), they do not activate cells and they will not saturate cell surface epitopes. Therefore the isolated cells can be transferred into immunodeficient mice to induce colitis37,44,45.</li>
		<li>Count the cells using a Neubauer counting chamber. Mix 10 &#181;l of the cells suspension with 10 &#181;l of a 0.4% Trypan Blue solution. Incubate for 1-2 min.
		<ol>
			<li>Fill the hemocytometer chamber with 10 &#181;l of stained cells. Count cells under the microscope in four 1 x 1 mm2 squares of one chamber and determine the average number of cells per square. Viable cells appear white, dead cells appear blue. To determine the number of cells/ml multiply cell number by factor of dilution. If 2 - 4 quadrants are being counted, divide by the number of quadrants.</li>
		</ol>
		</li>
	</ol>
	</li>
</ol>
6. Induction of Antigen-driven Colitis
<ol>
	<li>Inject intraperitoneally 3 x 105 OT-II/Red CD4+ CD62L+ T cells into CX3CR1GFP/+ x RAG-/- mice. For induction of antigen-specific colitis, administer E. coli pCFP-OVA every second day at a dose of 1 x 108 colony-forming unit (CFU) in 100 &#181;l of PBS per animal by gavage or oral instillation behind the incisors.</li>
	<li>Monitor twice weekly the body weight of transplanted mice and their clinical condition (presence of blood in the stool, stool consistency and activity of mice).</li>
</ol>
7. Tissue Samples for Histopathological Examination
<ol>
	<li>Take tissue samples from the large intestine of mice, fix in neutral-buffered formalin (10%) and embed in paraffin as previously described36,46.</li>
	<li>Section the paraffin samples on a microtome, mount on slides, and perform the H&#38;E stain47,48.</li>
	<li>Categorize the histology of the large intestine as published previously47,48: normal (score 0); mild colitis (score 1), moderate colitis (score 2) and severe colitis (score 3).</li>
</ol>
8. Isolation of Colonic Lamina Propria Cells
<ol>
	<li>Euthanize transplanted mice by cervical dislocation after anesthesia by inhalation of any halogenated ether (up to 5% for induction) commercially available.</li>
	<li>Place the animal down with the belly facing up. Use forceps to grab the skin anteriorly to the urethral opening. Use scissors to cut the skin along the ventral midline from the groin to the chest. Pull the skin back on the sides.</li>
	<li>Cut through the transparent peritoneal muscle wall and opening up the body cavity. Identify the colon that goes from the anus to the caecum49. Cut the 2 extremities and free the tissue from any fat.</li>
	<li>Open the colon longitudinally using a dissection scissors. Use a tweezers to shake the colon tissue in a petri dish containing 25 ml of PBS supplemented with 1% FBS to remove debris and mucous. Cut the colon into 5 - 8 mm pieces.</li>
	<li>Place the colon segments in a 50 ml tube containing 20 ml of the Wash Buffer. The Wash buffer contains 500 ml DPBS, 10mM Hepes and 5mM ethylenediaminetetraacetic acid (EDTA).</li>
	<li>Vortex the tube for 30 sec at maximal speed.</li>
	<li>Incubate the tube with the colon segments in a water bath at 37 &#176;C with 200 rpm shaking for 10 min.</li>
	<li>After the incubation, vortex the tube for 30 sec at maximal speed.</li>
	<li>Discard the supernatants and place the tissue samples in a new 50 ml tube containing 20 ml of the Wash buffer. Repeat steps 6-9 three times</li>
	<li>Discard the supernatants and place the tissue samples in a petri dish containing 25 ml of PBS. Use a tweezers to shake gently the tissue samples in the petri dish for few seconds to remove residual epithelial cells. Repeat this step three times.</li>
	<li>Cut colonic tissues into 2 x 2 mm2 pieces and digest them by incubation in 10 ml of Roswell Park Memorial Institute (RPMI) medium with 0.5 mg/ml collagenase type VIII from Clostridium histolyticum and 10 U/ml of DNaseI.</li>
	<li>Vortex the tube for 30 sec at maximal speed.</li>
	<li>Incubate the colon fragments in a water bath at 37 &#176;C with 200 rpm shaking for 35 min and vortex the tube for 30 sec at maximal speed every 5 min.</li>
	<li>At the end of the incubation vortex the tube for 30 sec at maximal speed.</li>
	<li>Collect the digested fragments by passing them through a 70 &#181;m cell strainer positioned on a new 50 ml tube.</li>
	<li>Wash the sample by adding 20 ml of DPBS and centrifuge at 400 g for 5 min. Then resuspend the cells in 1 ml of DPBS.</li>
	<li>Count the cells using a Neubauer counting chamber. Mix 10 &#181;l of the cells suspension with 10 &#181;l of a 0.4% Trypan Blue solution. Incubate for 1-2 min.
	<ol>
		<li>Fill the hemocytometer chamber with 10 &#181;l of stained cells. Count cells under the microscope in four 1 x 1 mm2 squares of one chamber and determine the average number of cells per square. Viable cells appear white, dead cells appear blue. To determine the number of cells/ml multiply cell number by factor of dilution. If 2-4 quadrants are being counted, divide by the number of quadrants.</li>
	</ol>
	</li>
</ol>
9. Extracellular Staining for FCM Analysis
<ol>
	<li>Centrifuge the cLP cells 5 min at 390 x g. Resuspend the cells in 1 ml of FACS A (PBS supplemented with 1% BSA and 0.1% sodium azide) 
	NOTE: Sodium azide is highly toxic. It can cause hypotension, hypothermia, headache convulsions or death. Dilute solutions of sodium azide (0.1 to 1.0%) usually do not present extraordinary dangers to the user, but they are eye and skin irritants. Therefore, use sodium azide only in the chemical hood while wearing a laboratory coat and a double pair of disposable nitrile gloves.</li>
	<li>Incubate the cells for 15 min at RT with the monoclonal antibody (mAb) 2.4G2 directed against the Fc&#947;RIII/II CD16/CD32 (0.5-1 &#181;g mAb/106 cells) diluted in FACS A.</li>
	<li>Add 200 &#181;l of FACS A to the cells and centrifuge 5 min at 390 x g. Repeat this step twice. Incubate the cells with 0.5 ng/106 cells of the relevant mAb for 20 min at 4 &#176;C. Dilute the antibodies in FACS A.</li>
	<li>Add 200 &#181;l of FACS A to the cells and centrifuge 5 min at 390 x g. Repeat this step twice. Resuspend the samples in 100 &#181;l of FACS A.</li>
	<li>Analyze T cells from CX3CR1GFP/+ x RAG-/- mice reconstituted with OT-II/Red CD4 T cells at the flow cytometer37.</li>
	<li>Analyze data using commercially available software.</li>
</ol>
10. Confocal Microscopy Analysis
<ol>
	<li>Analyze the colon of CX3CR1GFP/+ mice reconstituted or not with OT-II/Red CD4+CD62L+ T cell and fed orally every second day with 1 x 108 CFU of E. coli strain DH10B pCFP-OVA.</li>
	<li>Remove the colon from the mice using scissors and tweezers. Open it longitudinally using a dissection scissors.</li>
	<li>Cut a 5-8 mm piece of colon sample using scissors, put onto glass slides and cover with coverslips.</li>
	<li>Analyze the colon samples with a confocal microscope at a magnification of 40/1.30.</li>
	<li>Detect the CX3CR1+ MPs, labeled with green fluorescein protein (GFP), using a filter set with excitation at 488 nm and emission at 509 nm.</li>
	<li>Detect the CD4+ T cell expressing red protein using a filter set with excitation at 554 nm and emission at 586 nm.</li>
	<li>Detect E.coli pCFP-OVA using a filter set with excitation at 450 nm and emission at 480 nm.</li>
	<li>Define an area of interest and generate scatter diagrams as previously described50 to carry out the co-localization analysis. Use 2 different formulas: the Overlap Coefficient according to Manders and the Pearson&#39;s Coefficient.</li>
</ol>

The authors have nothing to disclose.

As with every other model, the antigen-driven colitis model described above may present few issues that the investigator performing the technique must be aware of. When injecting the OT-II/Red+ CD4+ T CD62L+cells in the hosts, the investigator must be very gentle and careful to insert the needle into the peritoneal cavity. Failure to do so may result in the tearing of the intestine of the mouse which could lead to death, or a subcutaneous administration of cells which will not induce any ...

The intestine is the largest surface of the body that is exposed to the external environment. Vast arrays of resident microbes colonize the human intestine to form the intestinal microbiota (or microflora). This is estimated to consist of up to 100 trillion microbial cells and constitutes one of the most densely populated bacterial habitats known in biology1-3. In the GIT bacteria colonize an intestinal niche where they survive and multiply4. In return, the microbiota endows the host with additional functional features not encoded on its genome1. For example the microbiota stimulates the proliferation of epithelial cells, produces vitamins that hosts cannot produce by themselves, regulates metabolism and protects against pathogens4-6. Given this beneficial relationship, some authors have suggested that humans are &#34;super-organisms&#34; or &#34;holobionts&#34; that are a mix of bacterial and human genes7,8. Given the beneficial impact of the microbiota on the (human) host, the intestinal immune system needs to tolerate commensal microbes to enable their existence in the lumen but also kill the pathogens that invade from luminal side9-11. The intestinal immune system has developed mechanisms to distinguish between harmless and potentially harmful luminal microbes; however these mechanisms are not yet well understood12. Maintaining intestinal integrity requires a tightly regulated immune homeostasis to keep the balance between tolerance and immunity13. An imbalance in immune homeostasis contributes to the induction of intestinal diseases such as inflammatory bowel disease (IBD)3,14.
There are two major types of IBD: Crohn&#39;s disease (CD) and ulcerative colitis (UC). Patients with these diseases usually suffer from rectal bleeding, severe diarrhea and abdominal pain15,16. The single cause of IBD is still unknown, but a combination of genetic factors, environmental influences and dysregulated immune responses might be the key event for disease development15.
Animal models for IBD have been used for over 50 years. In the last few decades new IBD model systems have been developed to test the various hypotheses concerning the pathogenesis of IBD17,18. The best-characterized model of chronic colitis is the T-cell transfer model that induces disruption of T-cell homeostasis19,20. This model involves transferring naive T cells from immunocompetent mice into hosts that lack T and B-cells (such as RAG-/- and SCID mice)16,21. The development of disease in this model is monitored for 3-10 weeks by evaluating the presence of diarrhea, reduced physical activity, and loss of body weight. This is so called the wasting syndrome16. Compared to the healthy mice the colonic tissue of transplanted hosts is thicker, shorter and heavier16. Using the T cell transfer model, it is possible to understand how different T cell populations can contribute to the pathogenesis of IBD22. The T cell transfer model does not analyze the interactions between APCs and T cells in the disease process in an antigen-specific manner. It has been shown that an interaction between myeloid cells and lymphoid cells could be responsible for the development of intestinal inflammation23. Although many aspects of IBD have been clarified, the initial events that lead to the disease development still need to be clearly understood.
It has been shown that in the absence of microbiota transfer colitis cannot be established24. Recently, several theories suggest that IBD could be a result of an immune response against commensal bacteria25. Authors have also proposed that commensal bacteria are essential to induce inflammation in the distal intestine26. In germ free (GF) animals the intestinal immune system is generally impaired27,28, but a colonization of these mice with a mixture of specific-pathogen-free bacteria results in the development of the fully-competent intestinal immune system29. Hence, the microbiota seems to be a key element in the pathogenesis of IBD, either as a mechanism that predisposes to or protects against the development of intestinal inflammation30,31. Current theories suggest that IBD is a result of microbial imbalance, called dysbiosis, in genetically predisposed patients32, but it is not clear yet if the dysbiosis is the cause or the consequence of the disease12. Considering the role of microorganisms in the development of IBD, in vitro experiments showed that CD4+ T cells can be activated by APCs pulsed with intestinal bacteria33,34.
Moreover, it has been shown that antigens from different commensal bacterial species, such as E. coli, Bacteroides, Eubacterium and Proteus, are able to activate CD4+ T cells35. This indicates that presentation of bacterial antigens to T cells is of importance for the development of IBD. To reduce the complexity of multiple antigens derived by the microflora in the disease process, an E.coli strain has been created that produces the OVA antigen. Transfer colitis was induced by injecting OVA-specific T cells into RAG-/- animals colonized with OVA-expressing E. coli.
This model is based on recent evidence suggesting that CX3CR1+ MPs, a major cell subset in the colonic lamina propria (cLP)36, are interacting with CD4+ T cells during transfer colitis37. MPs sample the intestinal lumen for particulate antigen, such as bacteria, using their dendrites36, 38,39. Previous studies demonstrated that MPs can also take up soluble antigens, such as OVA, introduced into the intestinal lumen40,41. Given the abundance of CX3CR1+ MPs in the cLP, it is possible that these cells can sample luminal bacteria and interact with CD4 T cells. Confocal imaging of mice transplanted with OVA-specific CD4+ T cells colonized with E. coli CFP-OVA, show that CX3CR1+ MPs are in contact with OT-II CD4+ T cell during the development of antigen-driven colitis. This model enables the study of the antigen presentation process between intestinal APCs and T cells specific only for particular antigen-expressing bacteria in the gut lumen.

<table><tbody><tr><td>LB Broth, Miller (Luria-Bertani)</td><td>Difco</td><td>244620</td><td></td></tr><tr><td>Rotary Shake</td><td>Reiss Laborbedarf e. K.</td><td>Model 3020 GFL</td><td></td></tr><tr><td>2 mm gap couvettes&amp;nbsp;</td><td>Peqlab Biotechnologie GmbH</td><td>71-2020</td><td></td></tr><tr><td>Glycerol</td><td>Sigma-Aldrich</td><td>G5516-100ML</td><td></td></tr><tr><td>Gene Pulser Xcell system&amp;nbsp;</td><td>BioRad Laboratories GmbH</td><td>1652660</td><td></td></tr><tr><td>LB Agar, Miller (Luria-Bertani)</td><td>Difco</td><td>244510</td><td></td></tr><tr><td>Ampicillin</td><td>Sigma-Aldrich</td><td>A9393-5G</td><td></td></tr><tr><td>SOC Medium</td><td>Sigma-Aldrich</td><td>S1797-100ML</td><td></td></tr><tr><td>High Pure Plasmid Isolation Kit</td><td>Roche</td><td>11754777001</td><td></td></tr><tr><td>Agarose</td><td>Carl Roth GmbH &amp;amp; Co</td><td>3810.1</td><td></td></tr><tr><td>EDTA</td><td>Sigma-Aldrich</td><td>E9884-100G</td><td></td></tr><tr><td>Tris-HCl</td><td>Sigma-Aldrich</td><td>T5941</td><td></td></tr><tr><td>Glacial acetic acid</td><td>Sigma-Aldrich</td><td>537020&amp;nbsp;</td><td></td></tr><tr><td>Gel chamber&amp;nbsp;</td><td>PEQLAB Biotechnology GmbH</td><td>40-0708</td><td></td></tr><tr><td>Loading Dye</td><td>Thermo Fisher</td><td>R0611</td><td></td></tr><tr><td>GeneRuler 1 kb DNA Ladder&amp;nbsp;</td><td>Thermo Fisher</td><td>SM0312</td><td></td></tr><tr><td>Ethidium bromide solution</td><td>Carl Roth GmbH &amp;amp; Co. KG</td><td>2218.3</td><td></td></tr><tr><td>Photo-documentation system&amp;nbsp;</td><td>Decon Science Tech GmbH</td><td>DeVision G&amp;nbsp;</td><td></td></tr><tr><td>DNA sequencing&amp;nbsp;</td><td>MWG-Biotech GmbH</td><td></td><td></td></tr><tr><td>Phosphate buffered saline (PBS)</td><td>Biochrom</td><td>L182-50</td><td></td></tr><tr><td>Fluorescent microscope&amp;nbsp;</td><td>Zeiss</td><td>HBO 100</td><td></td></tr><tr><td>Mini-PROTEAN Tetra System</td><td>Bio-Rad Laboratories GmbH</td><td>1658005</td><td></td></tr><tr><td>PageRuler Prestained Protein Ladder&amp;nbsp;</td><td>Fermentas, St. Leon-Rot, Germany</td><td></td><td></td></tr><tr><td>IstanBlue Solution</td><td>Expedeon, Cambridgeshire, United Kingdom</td><td></td><td></td></tr><tr><td>Nitrocellulose membrane&amp;nbsp;</td><td>Macherey-Nagel GmbH &amp;amp; Co. KG</td><td>741280</td><td></td></tr><tr><td>Electro blotter&amp;nbsp;</td><td>Biometra GmbH</td><td>846-015-600</td><td></td></tr><tr><td>Bovine Serum Albumins (BSA)</td><td>Sigma-Aldrich</td><td>A6003-25G</td><td></td></tr><tr><td>Anti-Ovalbumin antibody&amp;nbsp;</td><td>Abcam</td><td>ab181688</td><td></td></tr><tr><td>Anti-rabbit IgG&amp;nbsp; HRP</td><td>Sigma-Aldrich</td><td>A0545&amp;nbsp;</td><td></td></tr><tr><td>Pierce ECL Plus Western Blotting Substrate</td><td>Pierce Biotechnology, Thermo Fischer Scientific Inc</td><td>32132</td><td></td></tr><tr><td>Forene</td><td>Abbott</td><td>2594.00.00</td><td></td></tr><tr><td>FBS</td><td>Invitrogen</td><td>10500-064</td><td></td></tr><tr><td>Falcon Cell Strainers</td><td>Fischer Scientific&amp;nbsp;</td><td>08-771-2</td><td></td></tr><tr><td>Ammonium chloride</td><td>Sigma-Aldrich</td><td>254134-5G</td><td></td></tr><tr><td>Tris Base</td><td>Sigma-Aldrich</td><td>10708976001</td><td></td></tr><tr><td>CD4&lt;sup&gt;+&lt;/sup&gt; CD62 L&lt;sup&gt;+&lt;/sup&gt; T isolation kit&amp;nbsp;</td><td>Miltenyi Biotec</td><td>130-093-227&amp;nbsp;</td><td></td></tr><tr><td>MACS LS Columns&amp;nbsp;</td><td>Miltenyi Biotec</td><td>130-042-401</td><td></td></tr><tr><td>MACS MS Columns&amp;nbsp;</td><td>Miltenyi Biotec</td><td>130-042-201</td><td></td></tr><tr><td>MidiMACS Separator</td><td>Miltenyi Biotec</td><td>130-042-302</td><td></td></tr><tr><td>MiniMACS Separator</td><td>Miltenyi Biotec</td><td>130-042-102</td><td></td></tr><tr><td>MACS MultiStand</td><td>Miltenyi Biotec</td><td>130-042-303</td><td></td></tr><tr><td>Feeding Needle 20G</td><td>SouthPointe Surgical Supply, Inc</td><td>FN-7903</td><td></td></tr><tr><td>Formalin solution, neutral buffered, 10%</td><td>Sigma-Aldrich</td><td>HT501128</td><td></td></tr><tr><td>Paraffin</td><td>Sigma-Aldrich</td><td>1496904</td><td></td></tr><tr><td>Hematoxylin</td><td>Sigma-Aldrich</td><td>H9627</td><td></td></tr><tr><td>Eosin Y</td><td>Sigma-Aldrich</td><td>230251&amp;nbsp;</td><td></td></tr><tr><td>Dithiothreitol</td><td>Sigma-Aldrich</td><td>D9779&amp;nbsp;</td><td></td></tr><tr><td>Collagenase type VIII</td><td>Sigma-Aldrich</td><td>C-2139</td><td></td></tr><tr><td>Roswell Park Memorial Institute (RPMI) medium</td><td>AppliChem</td><td>A2044, 9050</td><td></td></tr><tr><td>Sodium azide</td><td>Sigma-Aldrich</td><td>438456</td><td></td></tr><tr><td>Mouse BD Fc Block</td><td>BD Pharmingen</td><td>553141</td><td></td></tr><tr><td>FITC-conjugated mAb binding V&amp;szlig; 5.1, 5.2&amp;nbsp;</td><td>BD Pharmingen</td><td>553189</td><td></td></tr><tr><td>APC-conjugated mAb binding CD4 GK1.5&amp;nbsp;</td><td>eBioscience</td><td>17-0041-83</td><td></td></tr><tr><td>FACS Calibur&amp;nbsp;</td><td>BD Biosciences</td><td></td><td></td></tr><tr><td>FCS Express V3 software</td><td>DeNovo</td><td></td><td></td></tr><tr><td>Meta scanning confocal microscope&amp;nbsp;</td><td>Zeiss</td><td>LSM 710&amp;nbsp;</td><td></td></tr><tr><td>Zeiss Workstation</td><td>Zeiss</td><td>LSM 7</td><td></td></tr><tr><td>Zeiss ZEM software&amp;nbsp;</td><td>Zeiss</td><td>v4.2.0.121</td><td></td></tr><tr><td>Maxisorp immuno plates&amp;nbsp;</td><td>NUNC, Roskilde</td><td>442404</td><td></td></tr><tr><td>Streptavidin conjugated alkaline phosphatase</td><td>Jackson Immuno Research</td><td>016-050-084</td><td></td></tr><tr><td>Alkaline phosphatase substrate 4-Nitrophenyl phosphate disodium salt hexahydrate</td><td>Sigma-Aldrich</td><td>71768-5G</td><td></td></tr><tr><td>mAb R4-6A2</td><td>BD Biosciences</td><td>551216</td><td></td></tr><tr><td>mAb XMG1.2&amp;nbsp;</td><td>BD Biosciences</td><td>554410</td><td></td></tr><tr><td>TECAN microplate-ELISA reader</td><td>Tecan</td><td></td><td></td></tr><tr><td>EasyWin software</td><td>Tecan</td><td></td><td></td></tr><tr><td>DNase I recombinant</td><td></td><td>4536282001</td><td></td></tr></tbody></table>

development of an antigen-driven colitis model to study presentation of antigens by antigen presenting cells to t cells

Inflammatory bowel disease (IBD) is a chronic inflammation which affects the gastrointestinal tract (GIT). One of the best ways to study the immunological mechanisms involved during the disease is the T cell transfer model of colitis. In this model, immunodeficient mice (RAG-/- recipients) are reconstituted with naive CD4+ T cells from healthy wild type hosts.
This model allows examination of the earliest immunological events leading to disease and chronic inflammation, when the gut inflammation perpetuates but does not depend on a defined antigen. To study the potential role of antigen presenting cells (APCs) in the disease process, it is helpful to have an antigen-driven disease model, in which a defined commensal-derived antigen leads to colitis. An antigen driven-colitis model has hence been developed. In this model OT-II CD4+ T cells, that can recognize only specific epitopes in the OVA protein, are transferred into RAG-/- hosts challenged with CFP-OVA-expressing E. coli. This model allows the examination of interactions between APCs and T cells in the lamina propria.

To establish an antigen-driven colitis model an E. coli strain has been constructed that contains a plasmid in which the gene for the CFP is fused to the coding sequence for the chicken ovalbumin protein and the fusion construct is expressed under control of the strong constitutive promoter Phyper (Figure 1A). Fluorescent microscopy shows that the recombinant E. coli pCFP-OVA, but not the parental E. coli DH10B, expr...

Watch this Scientific Journal Video about Development of an Antigen-driven Colitis Model to Study Presentation of Antigens by Antigen Presenting Cells to T Cells at JoVE.com

Development of an Antigen-driven Colitis Model to Study Presentation of Antigens by Antigen Presenting Cells to T Cells

In this antigen-driven colitis model, OT-II CD4+ T cells expressing a red fluorescent protein were adoptively transferred into RAG-/- mice that express a green fluorescent protein in mononuclear phagocytes (MPs). The hosts were challenged with Escherichia coli (E.coli) expressing the ovalbumin protein (OVA) fused to a cyan fluorescent protein (CFP).

Inflammatory bowel disease (IBD) is a chronic inflammation which affects the gastrointestinal tract (GIT). One of the best ways to study the immunological ...

development-an-antigen-driven-colitis-model-to-study-presentation

Research

JoVE Journal

Immunology and Infection

9.0K Views.  University College Cork. In this antigen-driven colitis model, OT-II CD4+ T cells expressing a red fluorescent protein were adoptively transferred into RAG-/- mice that express a green fluorescent protein in mononuclear phagocytes (MPs). The hosts were challenged with Escherichia coli (E.coli) expressing the ovalbumin protein (OVA) fused to a cyan fluorescent protein (CFP).

Video: Development of an Antigen-driven Colitis Model to Study Presentation of Antigens by Antigen Presenting Cells to T Cells

Modeling Colitis-Associated Cancer with Azoxymethane (AOM) and Dextran Sulfate Sodium (DSS)

Individuals with inflammatory bowel disease (IBD), such as Crohn's disease (CD) or ulcerative colitis (UC) are at increased risk of developing colorectal cancer (CRC) over healthy individuals. This risk is proportional to the duration and extent of disease, with a cumulative incidence as high as 30% in individuals with longstanding UC with widespread colonic involvement.1 Colonic dysplasia in IBD and colitis associated cancer (CAC) are believed to develop as a result of repeated cycles of epithelial cell injury and repair while these cells are bathed in a chronic inflammatory cytokine milieu.2 While spontaneous and colitis-associated cancers share the quality of being adenocarcinomas, the sequence of underlying molecular events is believed to be different.3 This distinction argues the need for specific animal models of CAC.
Several mouse models currently exist for the study of CAC. Dextran sulfate sodium (DSS), an agent with direct toxic effects on the colonic epithelium, can be administered in drinking water to mice in multiple cycles to create a chronic inflammatory state. With sufficient duration, some of these mice will develop tumors.4 Tumor development is hastened in this model if administered in a pro-carcinogenic setting. These include mice with genetic mutations in tumorigenesis pathways (APC, p53, Msh2), as well as mice pre-treated with genotoxic agents (azoxymethane [AOM], 1,2-dimethylhydrazine [DMH]).5 
The combination of DSS with AOM as a model for colitis associated cancer has gained popularity for its reproducibility, potency, low price, and ease of use. Though they have a shared mechanism, AOM has been found to be more potent and stable in solution than DMH. While tumor development in other models generally requires several months, mice injected with AOM and subsequently treated with DSS develop adequate tumors in as little as 7-10 weeks.6, 7 Finally, AOM and DSS can be administered to mice of any genetic background (knock out, transgenic, etc.) without cross-breeding to a specific tumorigenic strain. Here, we demonstrate a protocol for inflammation-driven colonic tumorigenesis in mice utilizing a single injection of AOM followed by three seven-day cycles of DSS over a 10 week period. This model induces tumors with histological and molecular changes closely resembling those occurring in human CAC and provides a highly valuable model for the study of oncogenesis and chemoprevention in this disease.8

Modeling Colitis-Associated Cancer with Azoxymethane AOM and Dextran Sulfate Sodium DSS

We demonstrate a protocol in which administration of the genotoxic agent azoxymethane (AOM) followed by three cycles of the pro-inflammatory agent dextran sulfate sodium (DSS) rapidly and consistently generates colon tumors in mice with morphologic and molecular similarities to those seen in human colitis-associated cancer.

Individuals with inflammatory bowel disease (IBD), such as Crohn's disease (CD) or ulcerative colitis (UC) are at increased risk of developing colorectal ...

Medicine

Isolation and Characterization of Dendritic Cells and Macrophages from the Mouse Intestine

Within the intestine reside unique populations of innate and adaptive immune cells that are involved in promoting tolerance towards commensal flora and food antigens while concomitantly remaining poised to mount inflammatory responses toward invasive pathogens1,2. Antigen presenting cells, particularly DCs and macrophages, play critical roles in maintaining intestinal immune homeostasis via their ability to sense and appropriately respond to the microbiota3-14. Efficient isolation of intestinal DCs and macrophages is a critical step in characterizing the phenotype and function of these cells. While many effective methods of isolating intestinal immune cells, including DCs and macrophages, have been described6,10,15-24, many rely upon long digestions times that may negatively influence cell surface antigen expression, cell viability, and/or cell yield. Here, we detail a methodology for the rapid isolation of large numbers of viable, intestinal DCs and macrophages. Phenotypic characterization of intestinal DCs and macrophages is carried out by directly staining isolated intestinal cells with specific fluorescence-labeled monoclonal antibodies for multi-color flow cytometric analysis. Furthermore, highly pure DC and macrophage populations are isolated for functional studies utilizing CD11c and CD11b magnetic-activated cell sorting beads followed by cell sorting.

Here, we detail a methodology for the rapid isolation of mouse intestinal dendritic cells (DCs) and macrophages. Phenotypic characterization of intestinal DCs and macrophages is performed using multi-color flow cytometric analysis while magnetic bead enrichment followed by cell sorting is used to yield highly pure populations for functional studies.

Within the intestine reside unique populations of innate and adaptive immune cells that are involved in promoting tolerance towards commensal flora and ...

The Citrobacter rodentium Mouse Model: Studying Pathogen and Host Contributions to Infectious Colitis

This protocol outlines the steps required to produce a robust model of infectious disease and colitis, as well as the methods used to characterize Citrobacter rodentium infection in mice. C. rodentium is a gram negative, murine specific bacterial pathogen that is closely related to the clinically important human pathogens enteropathogenic E. coli and enterohemorrhagic E. coli. Upon infection with C. rodentium, immunocompetent mice suffer from modest and transient weight loss and diarrhea. Histologically, intestinal crypt elongation, immune cell infiltration, and goblet cell depletion are observed. Clearance of infection is achieved after 3 to 4 weeks. Measurement of intestinal epithelial barrier integrity, bacterial load, and histological damage at different time points after infection, allow the characterization of mouse strains susceptible to infection.
The virulence mechanisms by which bacterial pathogens colonize the intestinal tract of their hosts, as well as specific host responses that defend against such infections are poorly understood. Therefore the C. rodentium model of enteric bacterial infection serves as a valuable tool to aid in our understanding of these processes. Enteric bacteria have also been linked to Inflammatory Bowel Diseases (IBDs). It has been hypothesized that the maladaptive chronic inflammatory responses seen in IBD patients develop in genetically susceptible individuals following abnormal exposure of the intestinal mucosal immune system to enteric bacteria. Therefore, the study of models of infectious colitis offers significant potential for defining potentially pathogenic host responses to enteric bacteria. C. rodentium induced colitis is one such rare model that allows for the analysis of host responses to enteric bacteria, furthering our understanding of potential mechanisms of IBD pathogenesis; essential in the development of novel preventative and therapeutic treatments.

The Citrobacter rodentium Mouse Model: Studying Pathogen and Host Contributions to Infectious Colitis

Citrobacter rodentium infection provides a valuable model to study enteric bacterial infections as well as host immune responses and colitis in mice. This protocol outlines the measurement of barrier integrity, pathogen load and histological damage allowing for the thorough characterization of pathogen and host contributions to murine infectious colitis.

This protocol  outlines the steps required to produce a robust model of infectious disease and  colitis, as well as the methods used to characterize ...

Visualization of Estrogen Receptors in Colons of Mice with TNBS-Induced Crohn's Disease using Immunofluorescence

Crohn&#39;s disease is the most diagnosed type of inflammatory bowel disease. Chronic inflammation developing in the intestine leads to peristalsis disorder and damage of intestinal mucosa and seems to be associated with an increased risk of colon neoplastic transformation. Accumulating evidence indicates that estrogens and estrogen receptors affect not only hormone-sensitive tissues, but also other tissues not directly related to estrogens, such as the lungs or colon. Here, we describe the protocol for the successful immunofluorescence staining of estrogen receptors in colon obtained from a murine model of TNBS-induced Crohn&#39;s disease. A detailed protocol for the induction of Crohn&#39;s disease in mice and intestine preparation is provided as well as a step-by-step immunohistochemical procedure using formalin-fixed paraffin-embedded intestine sections. The described methods are not only useful for estrogen receptor detection and estrogen signaling investigation in vivo but can also be applied to for other proteins which may be involved in the development of colitis.

The protocol presents a complete validated TNBS-induced murine model of Crohn&#39;s disease and methods for visualization of estrogen receptors by immunohistochemistry using immunofluorescence of formalin-fixed colon sections embedded in paraffin.

Crohn&#39;s disease is the most diagnosed type of inflammatory bowel disease. Chronic inflammation developing in the intestine leads to peristalsis ...

Biochemistry

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 ...

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 ...

Visualization of IL-22-expressing Lymphocytes Using Reporter Mice

Reporter mice have been widely used to observe the localization of expression of targeted genes. This protocol focuses on a strategy to establish a new transgenic reporter mouse model. We chose to visualize interleukin (IL) 22 gene expression because this cytokine has important activities in the intestine, where it contributes to repair tissues damaged by inflammation. Reporter systems offer considerable advantages over other methods of identifying products in vivo. In the case of IL-22, other studies had first isolated cells from tissues and then re-stimulated the cells in vitro. IL-22, which is normally secreted, was trapped inside cells using a drug, and intracellular staining was used to visualize it. This method identifies cells capable of producing IL-22, but it does not determine whether they were doing so in vivo. The reporter design includes inserting a gene for a fluorescent protein (tdTomato) into the IL-22 gene in such a way that the fluorescent protein cannot be secreted and therefore remains trapped inside the producing cells in vivo. Fluorescent producers can then be visualized in tissue sections or by ex vivo analysis through flow cytometry. The actual construction process for the reporter included recombineering a bacterial artificial chromosome that contained the IL-22 gene. This engineered chromosome was then introduced into the mouse genome. Homeostatic IL-22 reporter expression was observed in different mouse tissues, including the spleen, thymus, lymph nodes, Peyer&#39;s patch, and intestine, by flow cytometry analysis. Colitis was induced by T-cell (CD4+CD45RBhigh) transfer, and reporter expression was visualized. Positive T cells were first present in the mesenteric lymph nodes, and then they accumulated inside the lamina propria of the distal small intestine and colon tissues. The strategy using BACs gave good-fidelity reporter expression compared to IL-22 expression, and it is simpler than knock-in procedures.

We describe here a transgenic reporter mouse model to visualize the IL-22-producing cells inside different mouse tissues. This method can be used to track the location of other cytokines or secretary proteins in the mouse.

Reporter mice have been widely used to observe the localization of expression of targeted genes. This protocol focuses on a strategy to establish a new ...

Murine Distal Colostomy, A Novel Model of Diversion Colitis in C57BL/6 Mice

Diversion colitis (DC) is a frequent clinical condition occurring in patients with bowel segments excluded from the fecal stream as a result of a diverting enterostomy. The etiology of this disease remains ill-defined but appears to differ from that of classical inflammatory bowel diseases such as Crohn's disease and ulcerative colitis. Research aimed to decipher the pathophysiological mechanisms leading to the development of this disease has been severely hampered by the lack of an appropriate murine model. This protocol generates a murine model of DC that facilitates the study of the immune system's role and its interaction with the microbiome in the development of DC. In this model using C57BL/6 animals, distal parts of the colon are excluded from the fecal stream by creating a distal colostomy, triggering the development of mild to moderate inflammation in the excluded bowel segments and reproducing the hallmark lesions of human DC with a moderate systemic inflammatory response. In contrast to the rat model, a large number of genetically-modified murine models on the C57BL/6 background are available. The combination of these animals with our model allows the potential roles of individual cytokines, chemokines, or receptors of bioactive molecules (e.g., interleukin (IL)-17; IL-10, chemokine CXCL13, chemokine receptors CXCR5 and CCR7, and the sphingosine-1-phosphate receptor 4) to be assessed in the pathogenesis of DC. The availability of congenic mouse strains on the C57BL/6 background largely facilitates transfer experiments to establish the roles of distinct cell types involved in the etiology of DC. Finally, the model offers the opportunity to assess the influences of local interventions (e.g., modification of the local microbiome or local anti-inflammatory therapy) on mucosal immunity in affected and non-affected bowel segments and the on systemic immune homeostasis.

Murine distal colostomy provides a murine model for human diversion colitis, a predominantly lymphocytic colitis in colon segment excluded from the fecal stream.

Diversion colitis (DC) is a frequent clinical condition occurring in patients with bowel segments excluded from the fecal stream as a result of a ...

Investigating Target Gene Function in a CD40 Agonistic Antibody-induced Colitis Model using CRISPR/Cas9-based Technologies

The immune system functions to defend humans against foreign invaders such as bacteria and viruses. However, disorders of the immune system may lead to autoimmunity, inflammatory disease, and cancer. The inflammatory bowel diseases (IBD)-Crohn's disease (CD) and ulcerative colitis (UC)-are chronic diseases marked by relapsing intestinal inflammation. Although IBD is most prevalent in Western countries (1 in 1,000), incident rates are increasing around the world. Through association studies, researchers have linked hundreds of genes to the pathology of IBD. However, the elaborate pathology behind IBD and the high number of potential genes pose significant challenges in finding the best therapeutic targets. Additionally, the tools needed to functionally characterize each genetic association introduce many rate-limiting factors such as the generation of genetically modified mice for each gene. To investigate the therapeutic potential of target genes, a model system has been developed using clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated endonuclease (Cas9)-based technologies and a cluster of differentiation 40 (CD40) agonistic antibody. The present study shows that CRISPR/Cas9-mediated editing in the immune system can be used to investigate the impact of genes in vivo. Limited to the hematopoietic compartment, this approach reliably edits the resulting reconstituted immune system. CRISPR/Cas9-edited mice are generated faster and are far less expensive than traditional genetically modified mice. Furthermore, CRISPR/Cas9 editing of mice has significant scientific advantages compared to generating and breeding genetically modified mice such as the ability to evaluate targets that are embryonic lethal. Using CD40 as a model target in the CD40 agonistic antibody-induced colitis model, this study demonstrates the feasibility of this approach.

Here, we describe the methodology to knock out a gene of interest in the immune system using clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated endonuclease (Cas9)-based technologies and the evaluation of these mice in a cluster of differentiation 40 (CD40) agonistic antibody-induced colitis model.

The immune system functions to defend humans against foreign invaders such as bacteria and viruses. However, disorders of the immune system may lead to ...

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 ...

Development of an Antigen-driven Colitis Model to Study Presentation of Antigens by Antigen Presenting Cells to T Cells

Summary

Explore More Videos

Modeling Colitis-Associated Cancer with Azoxymethane (AOM) and Dextran Sulfate Sodium (DSS)

Isolation and Characterization of Dendritic Cells and Macrophages from the Mouse Intestine

The Citrobacter rodentium Mouse Model: Studying Pathogen and Host Contributions to Infectious Colitis

Visualization of Estrogen Receptors in Colons of Mice with TNBS-Induced Crohn's Disease using Immunofluorescence

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

Induction of Murine Intestinal Inflammation by Adoptive Transfer of Effector CD4⁺CD45RB^high T Cells into Immunodeficient Mice

Visualization of IL-22-expressing Lymphocytes Using Reporter Mice

Murine Distal Colostomy, A Novel Model of Diversion Colitis in C57BL/6 Mice

Investigating Target Gene Function in a CD40 Agonistic Antibody-induced Colitis Model using CRISPR/Cas9-based Technologies

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