Intestinal Usp22 loss elevates acute colitis burden resulting in bone fragility

In our previous DSS experiments we could observe severe inflammatory reactions in Usp22^-/- animals even several weeks after the end of the treatment. Moreover, our results indicated a potential interplay between ablated Usp22 levels, increased inflammation and therefore elevated intestinal tumorigenesis. Since there were still inflammatory reactions long time after terminating DSS treatment, we assumed that these symptoms are even stronger when the colitis is highly acute. Thus, we aimed to observe the consequences of Usp22 during the acute phase of intestinal inflammation. For this purpose, we induced colitis in small mouse cohorts (Table 18) and sacrificed these animals two days after terminating the DSS treatment. To determine the disease activity index, stool consistency, Guaiac score and body weight were recorded daily. We could observe diarrhea and the presence of occult blood more frequently after the loss of Usp22 than in wild type animals (Figure 38A, B). Moreover, these parameters were also worsened in APC mutant compared to APC^+/+ mice. While the weight loss was not significantly changed among the groups (data not shown), clear differences were detected in the DAI (Figure 38C, D). The disease activity index increased in a similar rate in all groups, however, the burden was highest in APC^1638N/+, Usp22^-/- animals.

Table 18: Sizes of the experimental mouse cohorts in which the effects of Usp22 ablation was observed during acute colitis.

APC^+/+ APC^1638N/+

Usp22^+/+ Usp22^-/- Usp22^+/+ Usp22

-/-Acute colitis 6 4 3 3

123

Figure 38: Acute colitis is correlated with increased DAI in Usp22^-/- mice. During DSS administration (A) stool consistency and (B) bleeding intensity were recorded to determine the disease activity index. Stool was softer and bloodier in Usp22^-/- mice. (C) The daily calculated disease activity index rose similarly in all mice (D) but the average values of the entire time period was highest in APC^1638N/+, Usp22^-/- animals. Mean ± SEM, Student's t-test, n-numbers indicated in Table 18.

124

After sacrificing animals, blood was extracted and inner organs were isolated. A striking difference was observed while comparing APC wild type and mutant mice to each other. When extracting blood we noticed that it was paler in APC^1638N/+ mice. Thus, we determined the ratio between hematocrit and serum. In APC wild types we could measure between 55-60%

hematocrit in a total blood sample while the ratio was approximately 48% in APC^1638N/+, Usp22^+/+ and 38% in APC^1638N/+, Usp22^-/- mice (Figure 39A). Accordingly, kidneys (Figure 39B) and livers (Figure 39C) were extremely pale in these animals.

Figure 39: The anemic effect of an APC mutation is slightly aggravated by Usp22 loss.

The relative amount of hematocrit was lower in blood samples of APC^1638N/+ mice, especially after Usp22loss. (B) Livers and (C) kidneys were pale in APC mutated animals while (D, E) their spleen was enlarged and heavier. Mean ± SEM, Student's t-test, n-numbers indicated in Table 18.

125

In accordance with Qadri and colleagues (Qadri et al., 2012) spleens were enlarged in mice carrying a heterozygous mutation in the APC gene (Figure 39D, E). Interestingly, spleens of APC^1638N/+ animals were, although not significant, even bigger after the loss of Usp22.

As expected, the acute inflammatory reaction had an effect on the length of the intestinal organs. While the colon length was similar for the majority of animals, APC^1638N/+, Usp22^-/- mice were characterized by shorter colons (Figure 40A, B). In the small intestine only mild effects were observed. On average, SIs of APC^1638N/+, Usp22^-/- were slightly shorter; however, this difference was not significant (Figure 40C).

Figure 40: Acute colitis results in decreased colon lengths in APC^1638N/+, Usp22^-/- animals.

(A, B) Colons and (C) small intestines were measured after sacrificing animals. Colons were slightly shorter in APC^1638N/+, Usp22^-/- mice while the small intestines were largely unaffected.

Mean ± SEM, Student's t-test, n-numbers indicated in Table 18.

126

Colons were stained with H&E to assess the overall organ morphology and the inflammation burden. As expected, acute colitis led to inflammation-associated epithelial damage in all animals (Figure 41A).

Figure 41: Intestinal loss of Usp22 increases burden during acute colitis. (A) Two days after terminating the DSS administration, mice were dissected and colons were stained with H&E. While in all animals epithelial integrity was disturbed, in Usp22^-/- mice more colon segments were characterized by a complete absence of crypt structures. Scale bar: 100 µm.

(B) The average H-score was elevated upon the loss of Usp22 and accordingly (C) more colon segments were affected by severe inflammation-induced epithelial damage in these mice.

Mean ± SEM, Student's t-test, n-numbers indicated in Table 18.

127

In Usp22^+/+ mice several organ regions were detected in which the integrity of the epithelium was disturbed. However, in the majority of colon regions only mild signs of inflammation were observed. In contrast, Usp22^-/- animals were characterized by severe epithelial damage and in large segments of the organ no crypt structures were detected at all. In the minority of tissue, normal crypts could be found. Accordingly, the average H-score (Figure 41B) and the percentage of severely damaged tissue was elevated upon the loss of Usp22 (Figure 41C).

These findings were independent from the APC status of experimental animals.

Figure 42: Acute colitis results in increased bone fragility in APC^+/+, Usp22^-/- mice. (A, B) To determine bone fragility, femora were placed into a Zwick device which applied pressure onto the bone leading to fracture. The loss of Usp22 did not show any effect in APC^1638N/+ mice with regards to (C) yield load, (D) Fmax, (E) failure load and (F) stiffness. However, these parameters revealed that bones of APC^+/+, Usp22^-/- mice are significantly more fragile than femora of Usp22 wild types. Mean ± SEM, Student's t-test, n-numbers indicated in Table 18.

Generally, it was observed in IBD patients that intestinal inflammation correlated with decreased bone mineral density and increased fracture risk (Ali et al., 2009; Bernstein et al., 2000). To determine whether the loss of Usp22 and acute colitis have an impact on bones, we observed the bone fragility. Using a Zwick device (Figure 42A, B), the biomechanical properties

128

of femora were measured in cooperation with Dr. med. Dominik Saul (Department of Trauma, Orthopedics and Reconstructive Surgery, UMG). With these analyses the applied strength during bone deformation (Figure 42C; yield load) and bone fracture (Figure 42D; Fmax) was determined. Moreover, the maximum forces applied before breaking the femora (Figure 42E;

failure load) and the bone stiffness (Figure 42F) were quantified. In APC^1638N/+ mice, no significant difference was detected between Usp22^+/+ and Usp22^-/- animals. Interestingly, the bone stiffness was significantly decreased in APC^+/+, Usp22^-/- mice compared to their Usp22 wild type littermates. Interestingly, inflammatory reactions were sufficient to show an effect on bone fragility in APC^+/+ but not APC^1638N/+ animals with a homozygous Usp22 deletion. In summary, these data reveal that effects of acute colitis are aggravated in Usp22^-/- mice.

129