Name: development of an antigen-driven colitis model to study presentation of antigens by antigen presenting cells to t cells
Uploaded: 2016-09-18T15:00:00.000+00:00
Duration: 6 min 57 s
Description: 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

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 disease.
Typically, mice will gain weight during the first week after cell transfer. The investigator should weigh the mice at the same time at each weight time point and use the same weighing balance throughout the whole procedure. Sometimes the experimental mice, especially when their weight at the beginning of the experiment is below 18 g, do not develop any signs of the wasting syndrome. However, these animals might still develop significant intestinal inflammation under macroscopic evaluation.
When working with genetically modified bacteria, contaminations can occur. To avoid these issues it is highly recommended to check the E.coli expressing CFP-OVA using the fluorescent microscope to analyze if the bacteria are fluorescent and are rod-shaped (the typical E.coli shape).
The proposed antigen driven-colitis relies on the observation that CX3CR1+ MPs sample commensal luminal bacteria in the steady state and during inflammatory conditions36,37. The induction of T cells responses in the antigen-driven colitis model has been demonstrated by the highly activated phenotype of OT-II CD4+ T cells from the colons of CX3CR1GFP/+ x RAG-/- mice challenged with the antigen, as compared to the T cells isolated from the CX3CR1GFP/+ x RAG-/- mice that were not challenged with antigen37. This is consistent with previous studies in which, after adoptive transfer of OT-II T cells in RAG-/- mice, colitis was only induced when the hosts were challenged with OVA-expressing E. coli 51.
Confocal imaging suggests that CX3CR1+ phagocytes are located close to the intestinal epithelium so that they can sample E. coli pCFP-OVAand interact with OT-II/Red+ CD4+ T cells. After CX3CR1+ phagocytes have sampled E. coli pCFP-OVA the luminal antigen is delivered to the CD103+ dendritic cells (DCs) by CX3CR1+ phagocytes. CD103+ DCs are able to migrate to the mesenteric lymph node (MLN) to prime CD4 T cells37,52. Primed T cells assemble in the cLP where CX3CR1+ phagocytes show the OVA antigen to effector T cells to induce inflammation. The delivery and presentation of the antigen by CX3CR1+ phagocytes could be a key event in the development of colitis.
The ability to induce colitis with a defined bacterium expressing a specific antigen provides the opportunity to study the necessary conditions for the development of colitis24. This model shows that a specific response to an antigenic peptide (OVA expressed by E. coli) is sufficient to induce intestinal inflammation in RAG-/- hosts, suggesting that T cells may react to specific antigen from the intestinal microflora. A single antigen can induce intestinal inflammation in the animal models but it cannot be assumed that single antigens are the cause of human IBD. Moreover, in the transfer colitis model there are naive CD4+ T cells that have unknown specificity and therefore may be reactive to multiple, as of yet unidentified antigens, from the microbiota. Major analysis and studies are needed to better clarify the role of a specific antigen in the pathogenesis of mucosal inflammation.
In recent years the usage of different animal model of colitis has led to an expansion in the knowledge of the pathogenesis of IBD53. However, due to the complexity and to the pathogenesis of the disease, there is not a definitive cure54. Using the described model, it is possible to address several aspects of IBD that are not well clarified yet, such as (i) interactions between monocytes and dendritic cells, (ii) migration of intestinal DCs to the MLN, (iii) antigen presentation, (iv) homing of effector T cells from MLN to the lamina propria and (v) activation of effector T cells in the lamina propria by CX3CR1+ phagocytes. However, further studies are needed to confirm if the CX3CR1+ phagocyte/CD4+ T cell interaction plays a key role in the IBD pathogenesis. Mice cannot be truly representative of humans and this must be kept in mind when mouse models are used to study human IBD2.

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, expresses CFP (Figure 1B). CFP-OVA- producing E. coli is able to activate OT-II cells in vitro and induce IFN-&#947; production in contrast to an E. coli strain expressing CFP only (Figure 2). Figure 3A shows ex vivo 3D confocal imaging of the colonic tissue of CX3CR1GFP/+ mice fed with E. coli pCFP-OVA. E. coli pCFP-OVA can be detected within colonic crypts located close to intestinal epithelial cells and co-localizes with CX3CR1+ phagocytes. To determine the relative proportion of E. coli pCFP-OVA internalized by defined CX3CR1+ phagocytes, the blue and green florescence intensity were determined by scatter blots. The analysis revealed that CX3CR1+ cells sampled 11.9 &#177; 1.5% of E. coli pCFP-OVA detected in the colon (Figure 3B). In OT-II/Red animals, CD4+ T cells are characterized by Red expression and V&#946; 5.1 expression shown by flow cytometry (Figure 4). Figure 5A shows a general overview of the antigen driven colitis model. OT-II/Red+ CD4+ T CD62L+ cells were adoptively transferred into CX3CR1GFP/+ x RAG-/- recipients that were then gavaged every second day with E. coli pCFP-OVA. When challenged with E. coli pCFP-OVA, transferred CX3CR1GFP/+ x RAG-/- mice lose body weight and develop clinical signs of colitis (Figure 5B). Figure 6 shows ex vivo 3D confocal imaging of colonic tissue 7, 14 and 21 days after reconstitution of heterozygous CX3CR1GFP/+ x RAG-/- recipients with OT-II/Red+ cells challenged with E. coli pCFP-OVA. Seven days after cell transfer only a few CX3CR1+ phagocytes have sampled E. coli pCFP-OVA and OT-II/Red+ cells were not detected. Fourteen days after cell transfer, CX3CR1+ phagocytes that sampled E. coli pCFP-OVA were located close to OT-II/Red+ cells. Twenty-one days after cell transfer a high number of CX3CR1+ cells that have sampled E. coli pCFP-OVA and OT-II/Red+ cells, dispersed throughout the cLP in close proximity to CX3CR1+ phagocytes.
<img alt="Figure 1" src="/files/ftp_upload/54421/54421fig1.jpg" /> 
Figure 1: CFP-OVA Producing E. coli. (A) Map of pCFP-OVA with relevant restriction sites, the gene encoding OVA and the bright blue fluorescent protein CFP. (B) Bright field and fluorescent microscopic images of wild type E. coli DH10B, E. coli DH10B pCFP and E. coli DH10B pCFP-OVA. Scale bar: 10 &#181;m. <a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/54421/54421fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" src="/files/ftp_upload/54421/54421fig2.jpg" /> 
Figure 2: OT-II Cells Stimulated with CFP-OVA Produce IFN-&#947;. After isolation from spleen, OT-II cells were stimulated with the indicated numbers of E. coli DH10B pCFP-OVA. Bacterial cells were inactivated following 2 hr incubation with 5 &#181;g/ml gentamicin. After 72 hr of culture, supernatants were collected and IFN-&#947; concentrations determined by ELISA. Experiments were carried out in triplicates and data presented as mean &#177; SEM. <a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/54421/54421fig2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" src="/files/ftp_upload/54421/54421fig3.jpg" /> 
Figure 3: CX3CR1+ Phagocytes Sample E. coli pCFP-OVA. (A) cLP tissue of CX3CR1GFP/+ mice gavaged with 1 x 108 E. coli pCFP-OVA for 7 days and analyzed by ex vivo confocal microscopy. Magnification 40X/1.30. Scale bar = 30 &#181;m. (B) The percentage of internalized E. coli pCFP-OVA quantitated and data presented as mean &#177; SEM. In the Mann-Whitney test p &#60; 0.05 was considered statistically significant. <a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/54421/54421fig3large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 4" src="/files/ftp_upload/54421/54421fig4.jpg" /> 
Figure 4: Characterization of OT-II/Red+CD4+ T Cells. (A) OT-II transgenic animals were crossed with animals expressing the red fluorescent protein to obtain OT-II/Red+ cells. OT-II/Red+ cells were isolated from spleens of OT-II/Red transgenic animals, stained for CD4 and V&#223;5.1 and analyzed by flow cytometry. <a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/54421/54421fig4large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 5" src="/files/ftp_upload/54421/54421fig5.jpg" /> 
Figure 5: OVA-driven Transfer Colitis. (A) Schematic representation of the antigen driven colitis model. OT-II/Red+ CD4+ T CD62L+cells were adoptively transferred into CX3CR1GFP/+ x RAG -/- mice and hosts were gavaged every second day with E. coli pCFP-OVA. (B) Body weight of the indicated groups was measured twice a week. Mean &#177; SEM body weight loss (%) is presented. In the Mann-Whitney test p &#60;0.05 was considered statistically significant. <a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/54421/54421fig5large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 6" src="/files/ftp_upload/54421/54421fig6.jpg" /> 
Figure 6: CX3CR1+ MPs Communicate with OT-II Cells During OVA-driven Transfer Colitis. (A) Colonic tissue samples were examined by ex vivo 3D confocal imaging at day 7, 14 and 21 after transfer of OT-II/Red+ CD4+ T CD62L+cells in CX3CR1GFP/+ x RAG-/- recipients. Magnification 40X/1.30. Scale bar = 30 &#181;m.

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

Universidad San Sebastian

Research

JoVE Journal

Immunology and Infection

8.9K 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

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 ...

Medicine

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

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

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

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

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

Imaging CD4 T Cell Interstitial Migration in the Inflamed Dermis

The ability of CD4 T cells to carry out effector functions is dependent upon the rapid and efficient migration of these cells in inflamed peripheral tissues through an as-yet undefined mechanism. The application of multiphoton microscopy to the study of the immune system provides a tool to measure the dynamics of immune responses within intact tissues. Here we present a protocol for non-invasive intravital multiphoton imaging of CD4 T cells in the inflamed mouse ear dermis. Use of a custom imaging platform and a venous catheter allows for the visualization of CD4 T cell dynamics in the dermal interstitium, with the ability to interrogate these cells in real-time via the addition of blocking antibodies to key molecular components involved in motility. This system provides advantages over both in vitro models and surgically invasive imaging procedures. Understanding the pathways used by CD4 T cells for motility may ultimately provide insight into the basic function of CD4 T cells as well as the pathogenesis of both autoimmune diseases and pathology from chronic infections.

The mechanisms that govern the interstitial motility of CD4 effector T cells at sites of inflammation are relatively unknown. We present a non-invasive approach to visualize and manipulate in vitro-primed CD4 T cells in the inflamed ear dermis, allowing for study of the dynamic behavior of these cells in situ.

The ability of CD4 T cells to carry out effector functions is dependent upon the rapid and efficient migration of these cells in inflamed peripheral ...

An Efficient and High Yield Method for Isolation of Mouse Dendritic Cell Subsets

Dendritic cells (DCs) are professional antigen-presenting cells primarily responsible for acquiring, processing and presenting antigens on antigen presenting molecules to initiate T-cell-mediated immunity. Dendritic cells can be separated into several phenotypically and functionally heterogeneous subsets. Three important subsets of splenic dendritic cells are plasmacytoid, CD8&#945;Pos and CD8&#945;Neg cells. The plasmacytoid DCs are natural producers of type I interferon and are important for anti-viral T cell immunity. The CD8&#945;Neg DC subset is specialized for MHC class II antigen presentation and is centrally involved in priming CD4 T cells. The CD8&#945;Pos DCs are primarily responsible for cross-presentation of exogenous antigens and CD8 T cell priming. The CD8&#945;Pos DCs have been demonstrated to be most efficient at the presentation of glycolipid antigens by CD1d molecules to a specialized T cell population known as invariant natural killer T (iNKT) cells. Administration of Flt-3 ligand increases the frequency of migration of dendritic cell progenitors from bone marrow, ultimately resulting in expansion of dendritic cells in peripheral lymphoid organs in murine models. We have adapted this model to purify large numbers of functional dendritic cells for use in cell transfer experiments to compare in vivo proficiency of different DC subsets.

Distinct dendritic cell subsets exist as rare populations in lymphoid organs, and therefore are challenging to isolate in sufficient numbers and purity for immunological experiments. Here we describe a high efficiency, high yield method for isolation of all of the currently known major subsets of mouse splenic dendritic cells.

Dendritic cells (DCs) are professional antigen-presenting cells primarily responsible for acquiring, processing and presenting antigens on antigen ...

Purification of the Membrane Compartment for Endoplasmic Reticulum-associated Degradation of Exogenous Antigens in Cross-presentation

Dendritic cells (DCs) are highly capable of processing and presenting internalized exogenous antigens upon major histocompatibility class (MHC) I molecules also known as cross-presentation (CP). CP plays an important role not only in the stimulation of na&#239;ve CD8+ T cells and memory CD8+ T cells for infectious and tumor immunity but also in the inactivation of self-acting na&#239;ve T cells by T cell anergy or T cell deletion. Although the critical molecular mechanism of CP remains to be elucidated, accumulating evidence indicates that exogenous antigens are processed through endoplasmic reticulum-associated degradation (ERAD) after export from non-classical endocytic compartments. Until recently, characterizations of these endocytic compartments were limited because there were no specific molecular markers other than exogenous antigens. The method described here is a new vesicle isolation protocol, which allows for the purification of these endocytic compartments. Using this purified microsome, we reconstituted the ERAD-like transport, ubiquitination, and processing of the exogenous antigen in vitro, suggesting that the ubiquitin-proteasome system processed the exogenous antigen after export from this cellular compartment. This protocol can be further applied to other cell types to clarify the molecular mechanism of CP.

The method described here is a new vesicle isolation protocol, which allows for the purification of the cellular compartments where exogenous antigens are processed by endoplasmic reticulum-associated degradation in cross-presentation.

Dendritic cells (DCs) are highly capable of processing and presenting internalized exogenous antigens upon major histocompatibility class (MHC) I ...

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 ...

Analysis of Granulocyte-Macrophage Colony-Stimulating Factor-Producing T Helper Cells in a Mouse Model of Contact Hypersensitivity

Parallel to traditional Th1/Th2/Th17/Treg lineages, granulocyte-macrophage colony-stimulating factor-producing T helper (Th-GM) cells have been identified as a distinct subset of T helper cells (GM-CSF+ IFN-&#947;- IL-17A- IL-22- effector CD4+ T cells) in human and mice. Contact hypersensitivity (CHS) is considered an excellent animal model for allergic contact dermatitis (ACD) in human, manifesting an intact T cell-mediated immune response. To provide a standardized and comprehensive assay to analyze the Th-GM cell subset in the T cell-dependent immune response in vivo, a murine CHS model was induced by sensitization/challenge with a reactive, low-molecular-weight, organic hapten, 2,4-dinitrofluorobenzene (DNFB). The Th-GM subset in effector CD4+ T cells generated upon immunization with the hapten was analyzed by flow cytometry. We found that Th-GM was mainly expanded in lesions and draining lymph nodes in the DNFB-induced CHS mouse model. This method can be applied to further study the biology of Th-GM cells and pharmacological research of therapeutic strategies centered on GM-CSF in various conditions, such as ACD.

Here, we present a simple and standardized method of analyzing the granulocyte-macrophage colony-stimulating factor-producing T helper subset in vivo.

Parallel to traditional Th1/Th2/Th17/Treg lineages, granulocyte-macrophage colony-stimulating factor-producing T helper (Th-GM) cells have been identified ...

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

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