Although single nucleotide polymorphisms in the NOD2 (nucleotide-binding oligomerization domain-containing 2) gene have been associated with the pathogenesis of Crohn's disease (CD), little is known about the role of nonpolymorphic wild-type (WT) NOD2 genes in the gut. To date, most animal studies addressing the role of WT NOD2 have been conducted using healthy (ileitis/colitis-free) mouse strains, and have attributed a modulating effect to the gut microbiome, partly based on 16S rRNA sequencing. Recently, we determined that the genetic deletion of the NOD2 gene decreased the severity of Th2-mediated chronic inflammation in a mouse strain characterized by suffering CD-like spontaneous progressive ileitis (SAMP1Yit/Fc; SAMP). The aims of this study were to quantify the effect of the NOD2 deletion on SAMP mice the severity of acute dextran sulfate sodium (DSS)-induced colitis, and to determine the impact of the deletion on the gut microbiome in young mice.Methods:
Given that the mouse gut microbiota quickly responds to dietary changes within 24 hours of feeding manipulations, we quantified the extent of compositional and functional divergence of the gut microbiota from 6-week-old SAMP mice in the disease induction phase (i.e., “pre-inflamed”), when the percentage of abnormal mucosa affected by CD-like “cobblestone lesions” is negligible, 7 days after the implementation of a recently proposed “inter-subject pre-experimental fecal homogenization (IsPreFeH) protocol.” Individual fecal samples collected from each individually-caged mouse were then used for (1) DNA 16S rRNA-based microbiome analysis, and (2) RNA-based functional and metabolic metatranscriptome analysis (n = 3-versus-3). Following IsPreFeH, 16S analysis was also conducted on mice before and/or after DSS-induced colitis (3% drinking water, 7d, n = 3–6/group, 2–3 experiments).Results:
Phylum and family-level based compositional analysis on 16S rRNA amplicon libraries and phylogenomic taxonomic inference from functional mRNA metatranscriptome data revealed no significant differences between SAMP-NOD2−/− and WT mice. Transcript functional abundance also showed no differences in major metabolic subsystem levels, including microbial virulence markers to explain natural differences in disease severity between SAMP-NOD2−/− and WT. Agglomeration of 7473 detected functional roles at a higher-level classification level revealed differences (P < 0.05) in the log-fold changes between groups for only 4 (2.07%) of 193 level-2 metabolic categories, which are within an expected error rate for false discovery (type-1 error) due to chance alone. DSS-treated SAMP-NOD2−/− mice lost significantly less body weight than WT mice, had less colonic interleukin-13 gene (Th2-) expression, and had less evidence of colitis based on endoscopy, histology, and tissue myeloperoxidase activity. Pooled statistical analysis of the 16S data, controlling for the experimental replica, indicate that the fecal 16S-microbiome differences between SAMP-NOD2-/- and WT mice were negligible and non-predictable of NOD2 deletion using logistic regression, compared to the microbiome differences attributable to DSS-induced colitis.Conclusions:
These results support the concept that the anti-inflammatory clinical effect of the NOD2 deletion on intestinal disease severity, at least in young mice during acute intestinal inflammation, in a mouse line prone to spontaneous chronic CD-like ileitis, occurs in a manner that cannot be attributable to changes in the gut microbiota, either naturally due to the gene deficiency, or inducibly due to the administration of DSS.