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
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;- GACCAGATCGATTAAGGGGAAACACA...

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.

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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...

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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 ...

The overall goal of this antigen-driven colitis model is to determine whether CX3, CR1 positive mononuclear phagocytes interact with and activate CD4 positive T cells during antigen-specific colitis and whether this mononuclear phagocyte-dependent effector T cell expansion occurs within the intestinal lamina propria. This method can help answer key questions in the inflammatory bowel disease field, such as what types of interaction occur within antigen presenting cells and the effector T cells in the colonic lamina propria. The main advantage of this technique is that it allows the study of immune cells'interaction and the elements that lead to the pathogencity of inflammatory bowel disease.This method uses the T cell transfer colitis model, in which the red fluorescent antigen-specific naive CD4 T cells are transferred to the immunodeficient host, expressing the green fluorescent protein in mononuclear cells. Cell transfer is followed with the oral gavage of the blue fluorescent antigen-expressing bacteria. The immune events taking place in the intestinal lamina propria of the hosts can be monitored at various stages of disease development.Use dissection scissors to open the colon longitudinally and wash the tissue in a petri dish containing 25 milliliters of PBS to remove any debris and mucus. Cut the colon into five to eight millimeter pieces and place the resulting fragments in 20 milliliters of fresh DPBS without calcium and magnesium, supplemented with ten millimolar HEPES and five millimolar EDTA. Vortex the tube with the sample for 30 seconds on maximum speed.Then, incubate at 37 degrees Celsius for ten minutes with gentle shaking to remove the epithelium. At the end of the incubation, Vortex again for 30 seconds. Then, discard the supernatant containing the epithelial cells.Transfer the tissue pieces into a new tube containing 20 milliliters of fresh PBS, HEPES, and EDTA buffer, and repeat the incubation with the vortexing steps before and after. After the third repetition, all the epithelial cells are removed and the supernatant is clear. Wash the tissue fragments gently in PBS to remove any residual epithelial cells.Cut the tissue fragments into two by two millimeter pieces and transfer the pieces for digestion in ten millimeters of RPI medium, supplemented with 0.5 milligrams per milliliter of collagenase type eight and ten units per milliliter of DNAse one. Vortex the samples for 30 seconds. Then, incubate the tissue pieces at 37 degrees Celsius with shaking for 30 to 35 minutes and vortex the tube every five minutes during the incubation.When the tissue is digested, strain the resulting slurry through a 70 micron cell strainer into a 50 milliliter conical tube. Wash the cell strainer once and spin down the single cell suspension by centrifugation. Finally, wash the cells once in PBS and centrifuge.Then, determine the number of viable cells by trypan blue exclusion. Cyan fluorescent protein ovalbumin protein producing e. coli activate interferon gamma production in OT-II cells in vitro in contrast to e.coli that express CFP only. Ex vivo 3D confocal imaging of the colonic tissue of transgenic green fluorescent protein expressing mice, fed with e. coli PCFP ova demonstrates the presence of the fluorescent protein-expressing bacteria within the colonic crypts near the intestinal epithelial cells and co-localized with the host intestinal phagocytes.In OT-II red animals, the CD4 positive T cells are characterized by their co-expression of red fluorescent protein and V beta five point one. When challenged with e. coli PCFP ova, BNT lymphocyte-deficient green fluorescent protein-expressing transgenic mice that have been adoptively transplanted with OT-II red CD4 positive CD62 ligand positive T cells rapidly lose body weight and develop clinical signs of colitis.Seven days after cell transfer, only a few host phagocytes have sampled e. coli PCFP ova and OT-II red positive cells are not detected. 14 days after cell transfer, host phagocytes that have sampled the e.coli PCFP ova are located in close proximity to the adoptively transferred OT-II red positive cells. By 21 days after cell transfer, a high number of host cells have sampled the e. coli PCFP ova and donor OT-II red positive cells, which are found dispersed throughout the colonic lamina propria, are in close contact with the host intestinal phagocytes.When mastered, this technique can be completed in four hours if it is performed properly. Following this procedure, other methods like staining of the intestinal lymphocytes and serum cytokine analysis can be performed to answer additional questions about the activation state of CD4 T cells and to confirm the severity of the colitis. After each development, this technique paves the way for research in the field of and immunology to explore the little events that lead to inflammatory bowel disease in the mouse model.After watching this video, you should have a good understanding of how to isolate colonic lamina propria immune cells for the downstream ex vivo analysis.

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