CD4+CD45RBhigh-Induced Colitis in SCID Mice

Inflammatory bowel disease (IBD)

Inflammatory bowel disease (IBD) is an inflammation of all or part of the human gastrointestinal (GI) tract. The most common forms of IBD are ulcerative colitis, which affects the colon, and Crohn's disease, which can affect any part of the GI tract (but most often colon and terminal ileum).

CD4+CD45RBhigh-induced colitis in SCID mice

We consider this the closest rodent model to IBD in human patients, and see excellent disease development in this model at Hooke. Pathological findings and weight loss resemble findings in people, and most IBD therapies efficacious in people also reduce colitis in this model.

The main difference between this model and human disease is the location of lesions - in mice pathological findings are restricted to the colon, while in people they can be present in any part of the GI system, although they are usually present in the colon.

Colitis in this model is believed to develop as a result of an imbalance between different CD4 cell populations [1, 2].

Disease is induced by injecting purified CD4+CD45RBhigh cells into severe combined immunodeficient (SCID) mice (which lack T and B cells). Only CD4+CD45RBlow cell populations contain regulatory T cells; CD4+CD45RBhigh populations do not.

Colitis does not develop when CD4+ cells (containing both CD4+CD45RBlow and CD4+CD45RBhigh cells) are injected, or when separately purified CD4+CD45RBlow cells (containing Tregs) and CD4+CD45RBhigh cells are co-injected into mice. Therefore it appears that regulatory T cells prevent colitis development.

Colitis also does not develop when germ-free SCID mice are used [3]. This implies that the CD4+CD45RBhigh cells generate an immune response against commensal bacteria in the colon; if Tregs are not present to down-regulate the immune response, disease develops.

Colitis appears to be mediated primarily by Th1 cells. Proinflammatory cytokines play an important role in colitis development; blocking IFNγ, IL-12/IL-23 or TNF reduces or blocks disease development [4, 5]. The role of Th17 cells is unclear; similar to findings in human patients, anti-IL-17A antibodies do not inhibit disease in this model.

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Disease induction

Spleens from male BALB/c mice are collected to obtain CD4+ and CD4+CD45RBhigh cells. We prepare a spleen cell suspension, then isolate CD4+ cells using Dynal® Mouse CD4 Negative Isolation kits. Some CD4+ cells are put aside for use in control groups, while the remainder are sorted with a MoFlo cell sorter to isolate CD4+CD45RBhigh cells (which are also CD25-). This purification removes almost all regulatory T cells (most of which have CD4+CD45RBlow phenotype).

Colitis is then induced by injecting the purified CD4+CD45RBhigh cells intravenously into recipient SCID mice. After 3 to 5 weeks, colitis develops as the cells are activated in the colon.

We inject a group of SCID mice with CD4+ cells to establish a negative control group for colitis development.

Colitis induction in SCID mice
Colitis induction in SCID mice

Clinical results

Clinically, the presence of colitis is indicated by weight loss and stool softening. The time to disease onset varies from lab to lab; at Hooke clinical disease usually starts developing 2 to 3 weeks after the cell transfer. Colitis becomes severe 4 to 6 weeks after the cell transfer.


Typical results from studies at Hooke are shown below.

Our standard in vivo readouts are body weight and stool score, both taken three times per week. Disease activity index (DAI) is then calculated from body weight and stool scores.

Body weight
Body weight
Disease activity index (DAI)
Disease activity index (DAI)

At the end of the study mice are euthanized and colons collected, cleaned and analyzed:

Colon weight
Colon weight
Colon weight:length ratio
Colon weight:length ratio
Myeloperoxidase (MPO)
Myeloperoxidase (MPO)

Histology is the main readout in these studies. Histological analysis is performed on Swiss rolled colons; scoring is performed blind.

Typical results of histological analysis
Typical results of histological analysis

Analyses offered

Sometimes our customers order analysis of all groups in advance, but we generally encourage customers to wait for initial results before deciding how much analysis to order.

We usually quote a price for your base study, together with prices for additional analysis that you can order later. This gives you flexibility to analyze only a sample of animals, choose particular groups for analysis, or even skip analysis completely if the results aren't promising. Our scientists will review your initial results with you, and can recommend the best analyses to maximize return from your study.

We generally ask for 5 days notice to schedule tissue collection or analysis (but we do our best to accommodate last-minute orders).

Histological and MPO analysis can be requested within 30 days after termination of a study.

The following are the most commonly ordered analyses in colitis studies.

MPO analysis

Myeloperoxidase (MPO) analysis is performed on colon tissue homogenate; each sample is run in triplicates.

Histological analysis of colons

One H&E section (section of Swiss roll preparation of colon) is prepared and analyzed for each mouse.

Flow cytometry prep - lamina propria cells

The proximal 2/3 of colons (otherwise used for histology) will be digested with collagenase, and lamina propria cells will be isolated on Percoll gradient. A separate cell suspension will be prepared from each animal and cells counted.

Cells will be placed in culture with phorbol myristate acetate (PMA, 50 ng/mL), ionomycin (0.5 µg/mL) and brefeldin (1 µg/mL) and incubated for 4 to 5 hours, then washed.

Flow cytometric analysis of cells

Cells are stained and analyzed by flow cytometry. The customer selects up to 4 antibodies for each analysis. Multiple analyses can be performed on each animal.

We normally have a wide range of antibodies in stock and routinely perform 9 color analysis. We perform both surface and intracellular staining.

Below, mice received either CD4+ cells (negative control for colitis) or CD4+CD45RBhigh cells (colitis-induced). At the end of the study, colon lamina propria cells were isolated and stained with CD4 APC, CD25 PE, and FoxP3 Alexa Fluor® 488 antibodies (top 4 panels), or CD4 APC, IL-17A PE, and IFNγ FITC (bottom 2 panels).

Expression of CD4, CD25, FoxP3, IL-17A, and IFNγ

Cytokine analysis of tissue homogenate

Tissue homogenate is prepared at 50 mg/mL in 1 M Tris, 0.1% Triton-X buffer in the presence of protease inhibitor cocktail. Homogenate is analyzed using either CBA or ELISA.

Typical results from a Hooke study are shown below.

Cytokine concentrations in colon tissue homogenate

Cytokine concentrations in colon tissue homogenate


[1] Powrie F et al, Int Immunol 5:1461 (1993)
[2] Powrie F et al, J Exp Med 183:2669 (1996)
[3] Stepankova R et al, Inflamm Bowel Dis 13:1202 (2007)
[4] Powrie F et al, Immunity 1:553 (1994)
[5] Liu Z et al, Eur. J Immunol 31:1550 (2001)