Anti-CD40-Induced Colitis in RAG2KO Mice

Inflammatory bowel disease (IBD)

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].

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:

Typical results

The following are examples of data from studies with anti-CD40-induced colitis in RAG2KO mice run at Hooke.

Clinical readouts

Body weights, stool scores, and disease activity indices (mean + SEM)

Green circles=No colitis, Blue squares=Vehicle, Red triangles=Anti-IL-12p40
Body weights over time relative to Day -2

Stool scores over time

Disease activity index (DAI) over time

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).

Colon weight and colon weight:length ratio (mean ± SEM)

Green open circles=No colitis, Blue open squares=Vehicle, Red open triangles=Anti-IL-12p40
Colon weight on Day 5 after anti-CD40 injection

Colon weight:length ratio on Day 5 after anti-CD40 injection

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).

Histological analysis - H&E (mean ± SEM)

Green open circles=No colitis, Blue open squares=Vehicle, Red open triangles=Anti-IL-12p40

Colon histology scores
Illustrative images of H&E staining in Swiss-rolled colons

Colon histology (H&E; mean ± SEM)

For a higher resolution image, click here or 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).

Histological analysis - Alcian blue (mean ± SEM)

Green open circles=No colitis, Blue open squares=Vehicle, Red open triangles=Anti-IL-12p40
Goblet cell area relative to mucosal tissue area

Illustrative images of Alcian blue staining in Swiss-rolled colons

Colon histology (Alcian blue)

For a higher resolution image, click here or 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 concentrations in colon culture supernatants (mean ± SEM)

Green open circles=No colitis, Blue open squares=Vehicle, Red open triangles=Anti-IL-12p40
Concentrations of IL-1β, IFNγ, and TNFα in colon culture supernatants from mice euthanized on Day 5 after anti-CD40 injection

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).

Cytokine concentrations in serum (Day 1; mean ± SEM)

Green open circles=No colitis, Blue open squares=Vehicle, Red open triangles=Anti-IL-12p40
Concentrations of IL-1β, IFNγ, and TNFα in serum from Day 1 after anti-CD40 injection

References

[1] Danese S et al., Gut 53(7):1035-1043 (2004)
[2] Uhlig HH et al., Immunity 25(2):309-318 (2006)