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

Summary

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).

Abstract

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.

Introduction

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 biology^1-3. 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 vita....

Protocol

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

1. Amplify the full size OVA gene using the primers Ova_SpeI_fw (3'-GACCAACTAGTATGGAATTTTGTTTTGATGTATT-5') and Ova_ClaI_rev (3'- GACCAGATCGATTAAGGGGAAACACATCTGCC-5')³⁷ using plasmid pCI-OVA (ampicillin resistant)⁴² as a template. Clone the PCR product (i.e. the OVA codi....

Results

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 P_hyper (Figure 1A). Fluorescent microscopy shows that the recombinant E. coli pCFP-OVA, but not the parental E. coli DH10B, expr.......

Discussion

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 .......

Materials

List of materials used in this article

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