Jackson Labs had previously reported that nearby construction partially affected the diabetes incidence in their NOD/LtJ colony in 2006 (http://type1diabetes.jax.org/images/fine-mapping/1976%20cumulative%20inc.jpg
). Therefore, as we were aware of nearby construction upon undertaking a new diabetes incidence study in September 2010, we closely monitored the diabetes onset and incidence in the female NOD mouse experimental control group. Notably, at the HMR-specific pathogen-free animal house facility, diabetes onset in female NOD mice from the NOD/LtJ-HMR colony is typically between 12 to 14 weeks of age, and the incidence of diabetes reaches approximately 70%–80% at 32 weeks of age ().
Figure 1 Nearby construction during the gestation period impacts diabetes onset and cumulative incidence in offspring. Depicted is a typical cumulative incidence of diabetes for eighteen female NOD/LtJ-HMR mice (open squares). Two NOD/LtJ-HMR female mice were (more ...)
Two of the three female NOD mice, that had been placed in breeding pairs, were in gestation during the construction period—September 15 to 18, 2010 (). We monitored the incidence of diabetes in the seven female offspring born in October 2010 from these mothers. We observed a delay in diabetes onset, which now initiated at 22 weeks of age for female NOD mice born from mothers subject to construction stress during gestation (). Moreover, we found a significantly reduced cumulative incidence of diabetes, where only two of the seven female NOD mice developed diabetes within 32 weeks in the NOD/LtJ-HMR mouse colony ().
Notably, the incidence of diabetes in was monitored in female NOD mice inbred for 11 to 13 generations in a relatively small animal house cohort, generally comprised of 3 to 5 breeder pairs. Although breeder replacement is performed on average every 14 weeks to prevent potential genetic selection for diabetes resistance, we could not entirely exclude the possibility of a genetic drift in the NOD/LtJ-HMR mouse colony [17
]. Indeed, as diabetes onset normally occurs between 12 to 14 weeks of age in female NOD mice of the NOD/LtJ-HMR mouse colony, and since we maintained a few breeder pairs beyond that time point, there is a risk that we selected mice carrying a genetic polymorphism conferring resistance to diabetes.
We thus went on to test the hypothesis that the diabetes onset was delayed and that the incidence was reduced in the NOD/LtJ-HMR mouse colony as a consequence of a genetic drift. We undertook a second diabetes incidence study with six female NOD mice of the NOD/LtJ-HMR colony born in December 2010, more than two months after the construction had been completed. In addition, we purchased twenty 7-week-old female NOD mice from the Jackson Labs NOD/ShiLtJ mouse colony and maintained them in the same conditions as the mice from the NOD/LtJ-HMR colony. The onset of diabetes for female NOD mice from both the NOD/LtJ-HMR and NOD/ShiLtJ mouse colonies was between 12–15 weeks of age and the cumulative incidence of diabetes also reached 70–100% in both mouse colonies by 32 weeks of age (). Together, our data demonstrate that the decrease in disease onset and cumulative incidence observed in the female NOD mice born in October 2010 was not due to a genetic drift in our mouse colony and is likely attributable to the effect of nearby construction.
Figure 2 The lower incidence of diabetes is not due to a genetic drift in our colony. The onset of diabetes was monitored in six female NOD mice from the NOD/LtJ-HMR mouse colony born more than two months after the construction had been completed (open circles) (more ...)
Of interest, all of the six female NOD mice of the NOD/LtJ-HMR colony from were born from one of the two original breeders, wherein the mothers had been subject to construction stress during the gestation two months before. We thus opted to directly compare the diabetes incidence from the four female NOD mice born in early October 2010 (i.e., subject to construction stress during their embryonic development) to the six female NOD mice born in December 2010, where all 10 offspring are of the same breeder pair. We again find a statistical difference in the incidence of diabetes as well as a delay in disease onset (). These results demonstrate that the biological effects causing alterations in diabetes susceptibility due to construction stress are rapidly dissipated overtime and are unlikely to cause permanent modifications to the phenotype.
Figure 3 Nearby construction affects diabetes onset and cumulative incidence in NOD mice born from the same breeder pair. The cumulative incidence of diabetes is compared for female NOD mice born from the same breeder pair at different times, namely, those who (more ...)
As mentioned previously, of the seven NOD female offspring born from mothers subject to construction stress during gestation, only two mice progressed to overt diabetes. Therefore, the environmental stress imposed by the construction during embryonic development appears to delay, but not necessarily impede, the autoimmune reaction towards pancreatic islet antigens. As such, we evaluated the subclinical progression of autoimmunity by quantifying the degree of insulitis and islet cell destruction. As expected, all diabetic NOD mice presented with few pancreatic islets and heavy lymphocyte infiltrates, suggesting an active autoimmune process (). In contrast, the 32-week-old nondiabetic NOD/ShiLtJ mice presented with a greater number of pancreatic islets and with fewer lymphocytic infiltrates than the diabetic NOD mice (). Surprisingly, few pancreatic islets were found in the nondiabetic NOD/LtJ-HMR mice which were subject to construction stress during embryonic development (). Moreover, the few remaining pancreatic islets were heavily infiltrated with lymphocytes (). These results suggest that the stress imposed by the construction did not prevent the onset of the autoimmune response and that the mice were slowly progressing towards overt diabetes.
To further define whether an active autoimmune response was ongoing in the five nondiabetic NOD/LtJ-HMR mice which were subject to construction stress during embryonic development, we determined the serum insulin autoantibody (IAA) levels. Serum IAA levels correlate with autoimmune diabetes onset in both humans and NOD mice and the serum IAA levels eventually decline with disease progression [18
]. Expectedly, the 32-week-old nondiabetic NOD/ShiLtJ mice did not show detectable levels of serum IAA levels, while the diabetic NOD mice exhibited a variable range of serum IAA (). In agreement with the histological observations suggestive of an active autoimmune response in the nondiabetic NOD/LtJ-HMR mice, IAA were present in the serum of these mice (). Taken together, these results suggest that the nondiabetic NOD/LtJ-HMR mice were likely progressing towards overt diabetes. The stress imposed by the nearby construction during the embryonic development, therefore, does not preclude the onset of an autoimmune response towards pancreatic islets, although it significantly delays the progression to overt diabetes.
Figure 5 Serum IAA levels as an indication of an ongoing autoimmune response towards pancreatic islets. The serum IAA levels are shown for every mouse group and segregated according to the health status (i.e., diabetic versus nondiabetic). Note that there are (more ...)
Limits of this current study include the low number of mice analyzed in our cohorts and the difficulty in reproducing similar events. However, similar variations in diabetes incidence have previously been documented by the Jackson Laboratory in much larger NOD animal cohorts. Indeed, the Jackson Laboratory has previously reported a modest effect of nearby construction on the incidence of diabetes in their NOD/ShiLtJ mouse colony (http://type1diabetes.jax.org/images/fine-mapping/1976%20cumulative%20inc.jpg
). In contrast, we report a striking and significant delay in disease onset as well as in the cumulative incidence of disease. The main difference between these two studies lies in the stratification of data. Whereas we stratified the mice according to whether the mothers were subject to construction stress during gestation or not, the data reported by the Jackson Laboratory presents a cumulative incidence study of a larger cohort over an entire year and they did not stratify their data relative to construction events. It is thus likely that the reason we observe a more striking effect of nearby construction on both the onset and incidence of diabetes is due to a stricter stratification of affected mice. To that effect, in our hands, the nearby construction only affected mice that were born from mothers subject to construction stress during gestation and not from mice born from the same parents at a later date. The effect is thus not permanent and unlikely to cause permanent changes in a mouse colony. Together with the results of the larger NOD mouse cohort study from the Jackson Laboratory, the current study performed on a limited number of NOD mice supports the view that nearby construction can significantly affect the incidence of diabetes.
Another factor which could have influenced the incidence of diabetes in our study includes variations in the microbiome of the mouse colony over time. The animal house barrier status is known to influence the incidence of diabetes in NOD mice [1
]. More recently, the microbiome was shown to influence diabetes incidence by causing variations in testosterone hormone levels and other metabolomic changes [11
]. We have not directly characterized the microbiome or the hormone levels of our NOD mouse colony before and after construction. However, the routine sentinel health report status did not vary overtime, nor did the feeding, water, bedding, or procedures for the animal handling (as described in the Methods section). In addition, the fertility of NOD mice was not apparently affected by the construction stress (), suggesting that the potential changes in hormone levels were insufficient to affect the fertility. Admittedly, these represent very crude and indirect measures of the microbiome and hormone levels, and thus we cannot fully exclude the possibility that the variations in diabetes incidence in our study were due to modifications in these parameters. Of interest, changes in the microbiome have been reported to significantly alter testosterone levels without affecting NOD mouse fertility [11
In addition to the microbiome and hormone levels, elevated serum IAA levels from the mothers have also been shown to significantly impact diabetes incidence [26
]. At postpartum, the NOD mothers were 10 and 15 weeks of age for the first litters subject to construction stress (). The serum IAA levels typically peak at 8 weeks of age and slowly wane off overtime [18
], suggesting that the mothers were likely to present detectable serum IAA levels. Notably, first litters of NOD mothers in our animal house facility typically present with a normal incidence of diabetes (not shown). Still, the fact that we did not monitor the serum IAA levels from the mothers presents a limitation of our study.
In summary, many parameters can influence diabetes incidence in NOD mice and there are possible alternative explanations as to why the mouse cohorts subject to construction stress during gestation showed a reduced incidence of diabetes. Nevertheless, the fact that litters born from the same mother, where one litter was subject to construction stress and the other was not, exhibited a significant difference in diabetes incidence suggests that construction stress during gestation modulates biological responses. Our results thus add to the observations from the Jackson Laboratory documenting a potential influence of nearby construction on the incidence of diabetes in NOD mice.