Crohn's Disease-Like Ileitis and the Inhibitory Effect of Sucralose on Streptococci.

To the Editor: Recently, we assessed the effects of the artificial sweetener Splenda (ingredients: sucralose and maltodextrin) in Crohn’s disease (CD) ileitis1 after feeding the product to mice prone to CD (SAMP1/YitFc) and (CD-free) control mice (AKR/J). Here, we would like to address criticisms raised by Lynch et al.,2 who emphasize that high-quality animal studies are important to patients. Because we agree, it is critical for us to clarify our experimental design to demystify Lynch’s criticisms on the scientific and translational value of our statistically significant findings, which are in synchrony with findings from other studies assessing sucralose in rats,3 a study assessing maltodextrin in cells and mice, which was cited in Rodriguez-Palacios et al.,1 and an earlier study of Splenda in rats,4 which was also criticized by 1 of the authors in Lynch’s letter (V. L. Grotz, who works for the manufacturer of Splenda). Examples of the effect on the gut microbiota and inflammation in other organs also support our findings,5, 6 whereas the translational implications of our study to humans are illustrated in recent discussions.7, 8 Overall, Lynch’s letter said that the “conclusions presented are not supported by the collective evidence relevant to the tested product. ” However, it is important to stress that (1) the letter presents no original data supporting the criticisms and (2) from a literature search of the first author (PubMed, B. Lynch), it is evident that as for-profit (food industry) consultants,9 their (seemingly biased) publications often conclude the same: that the products tested “are safe based on lack of significant effects on body weight, food consumption, hematology, clinical chemistry, organ weights, and histopathological examination,”10–13 which are nonspecific outcomes of subclinical inflammatory responses in the gut. Our study design was simple. We offered Splenda in water to mice and compared its effect with that of water alone. The experiment was repeated 3 times, with increasing doses each time. Nothing else was different. We also used an array of highly specific methods, not used by Lynch,10–13 to determine the subclinical and mechanistic impact of Splenda in gut health (RNA quantification and fluorescence in situ hybridization to identify bacteria and the malX gene in gut tissues, metagenomics, 16S rRNA microbiome, a novel quantitative parallel lanes plating culture method, and biochemical markers of inflammation). Despite the simplicity and robustness of our study, from Lynch’s letter, at least 2 major aspects called our attention, indicating that the experimental design was not clear to Lynch, raising concerns about the precision of their criticisms. Lynch implied (1) that we ignored the microbiome variability in our mice and (2) that the CCL21 defect (which we discovered and cited2) was an immune deficiency in SAMP that could render our conclusions invalid (we found that Splenda promotes ileum-associated dysbiosis in CD-SAMP mice but not in “healthy” control mice). On the microbiome: Lynch said that “no data were presented to understand normal variability in the SAMP mouse microbiome with normal dietary fluctuations. ” In this context, we cannot see the rationale of the criticism because there is no such as thing as “normal dietary fluctuations” in standard rodent research, nor did fluctuations occur during our study. Our laboratory mice were not fed varying meals. The diet was pelleted, the same, and ad libitum. Readers may notice that we devoted substantial effort to understanding the microbiome’s ecological variability in SAMP at the colony-and-cage level before the Splenda experiments were conducted (refer to Figs. 1 and 2 in our original published study in1), and we also identified for the first time a novel form of “cyclical bias in microbiome research,”14 which we controlled for to ensure high research quality. The Methods section1 also described novel methods we proposed to control for intersubject pre-experimental fecal microbiota heterogeneity.15, 16 Lastly, we sampled all animals in the morning, alternating sampling across groups, to control for circadian variation. Contrary to ignoring microbiome variability, as Lynch suggests, others have identified our methods on rodent research as novel (see the Commentary by Taconic Inc.17). On CCL21: Lynch said that “SAMP mice also produce little or no CCL21, which is important for normal immune response, and this could predispose the intestinal tissue to bacterial infiltration into the ileal lamina propria. ” To date, SAMP is the best and only polygenic inbred mouse model for CD, for which we have already identified numerous polymorphisms.18 Therefore, we do not understand the rationale for mentioning only 1 of the many biological abnormalities of SAMP mice (CCL21) as a problem in our study, which, as in humans, make them prone to CD. Our surprise with this basic criticism is that regardless of the disease mechanisms that make SAMP mice prone to CD-ileitis, our results arose from comparing 2 identical groups of SAMP mice, 1 of which received Splenda; the other did not. In this context, the significant differences detected can only be certainly attributed to the administration of the product. CCL21 has nothing to do with explaining pronounced differences in the Splenda group; that is, because all SAMP mice had the same genetic/immunological (CCL21) background. Additional minor points in the letter included histology and that gut © 2018 Crohn’s & Colitis Foundation. Published by Oxford University Press. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.