Anti-CD40-Induced Colitis in RAG2KO 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).
The CD40-CD40 ligand (CD154) costimulatory system is implicated in the pathogenesis of IBD due to its role in cell-mediated immunity and to overexpression of CD40 and CD40L in intestinal tissue from patients with Crohn's disease and ulcerative colitis [1].
Anti-CD40-induced colitis in RAG2KO mice
Stimulation of CD40 by an agonist anti-CD40 antibody in T and B cell-deficient mice results in an activation of an innate immune response in the gut that includes upregulation of IL-23 mRNA and IL-12 mRNA by myeloid cells, elevated proinflammatory serum cytokines, and inflammation of the colon [2]. According to the literature, this model can be induced in germ-free mice, suggesting that development of colitis after anti-CD40 injection is not dependent on an immune response against colonic commensal bacteria [2].
This model is short, with significant elevation in serum cytokines on Day 1 after anti-CD40 injection and colon pathology by Day 5. The model is well-suited to studying innate immune responses in IBD. Inhibition of TNF, IFNγ, or IL-12/IL-23 p40 reduces or blocks disease development [2].
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Disease induction
RAG2KO mice are administered anti-CD40 antibodies on Day 0 (disease induction).
By Day 1, significantly elevated proinflammatory cytokines in serum are detectible vs. naïve mice.
By Day 2 or 3, mice reach a peak in disease activity index (DAI), calculated as a sum of stool scores and body weight loss scores.
By Day 5, colon weight to length ratio is significantly increased over naïve mice, and significant colon pathology is observable.
Naïve RAG2KO mice injected with PBS in lieu of anti-CD40 antibodies are used as a negative control for colitis.
Readouts
Our standard in vivo readouts are body weights and stool scores. Cytokine concentration in serum can also be measured during the study.
At the end of the study the following measurements and analyses are typically performed:
- Colon weight and length
- Colon histology
- Colon culture supernatant cytokine concentration
- Flow cytometric analysis of lamina propria and/or lymphatic organs
Some customers order analysis of all groups in advance, but we generally encourage customers to wait for initial results before deciding which analyses to order.
We usually quote a price for your base study, together with prices for additional analysis that may be ordered 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 day's notice to schedule tissue collection or analysis (but we do our best to accommodate last-minute orders).
Typical results
The following are examples of data from studies with anti-CD40-induced colitis in RAG2KO mice run at Hooke.
Clinical readouts
Clinical readouts (mean ± SD)
| Treatment | # mice |
Relative end body weight (% of Day -2) |
p value | End stool score |
p value | End DAI score |
p value |
|---|---|---|---|---|---|---|---|
| No colitis | 5 | 103.1 ± 4.1 | - | 0.2 ± 0.4 | - | 0.4 ± 0.5 | - |
| Vehicle | 10 | 86.0 ± 9.8 | <0.001^ | 2.4 ± 1.2 | 0.002^ | 5.1 ± 2.4 | 0.001^ |
| Anti-IL-12p40 | 10 | 100.1 ± 3.2 | <0.001* | 0.5 ± 0.5 | 0.002* | 0.8 ± 0.8 | <0.001* |
^ Compared to No colitis.
* Compared to Vehicle.
Relative end body weights (%) compared using a one-way ANOVA followed by Tukey's multiple comparisons test.
End stool and DAI scores compared using Kruskal-Wallis tests followed by Dunn's multiple comparisons tests.
Colon measurements
Typical colon measurements from mice euthanized on Day 5 after anti-CD40 injection are shown below. Groups were compared using one-way ANOVAs followed by Tukey's multiple comparisons tests (***p<0.001).
Histological analysis of colons
Histological analysis is performed on Swiss-rolled colons. One H&E section is prepared and analyzed for each mouse. Scoring is based on mucosal inflammation, epithelial hyperplasia, goblet cell loss, and lamina propria inflammation. Below are typical results from mice euthanized on Day 5 after anti-CD40 injection. Groups were compared a Kruskal-Wallis test followed by Dunn's multiple comparisons test (**p<0.01, ***p<0.001).
For a higher resolution image, click on the image above. Scale bars are 1 mm.
Goblet cell loss can also be quantified computationally in sections stained with Alcian blue. Below are typical results from mice euthanized on Day 5 after anti-CD40 injection. Groups were compared using a one-way ANOVA followed by Tukey's multiple comparisons test (***p<0.001).
For a higher resolution image, click on the image above. Scale bars are 1 mm.
Cytokine analysis of colon culture supernatants
Supernatants from colon cultures are analyzed using a multiplex platform. Below are results from mice euthanized on Day 5 after anti-CD40 injection. For each cytokine, groups were compared using a one-way ANOVA followed by Tukey's multiple comparisons test (**p<0.01, ***p<0.001).
Cytokine analysis of serum
Serum is analyzed using a multiplex platform. Below are results from mice on Day 1 after anti-CD40 injection. For each cytokine, groups were compared using a one-way ANOVA followed by Tukey's multiple comparisons test (***p<0.001).
References
[1] Danese S et al., Gut 53(7):1035-1043 (2004)[2] Uhlig HH et al., Immunity 25(2):309-318 (2006)
Please contact Hooke at or with questions or for a quotation.
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