Mouse Paneth cell antimicrobial function is independent of Nod2

Abstract

Objective Although polymorphisms of the NOD2 gene predispose to the development of ileal Crohn's disease, the precise mechanisms of this increased susceptibility remain unclear. Previous work has shown that transcript expression of the Paneth cell (PC) antimicrobial peptides (AMPs) α-defensin 4 and α-defensin-related sequence 10 are selectively decreased in Nod2−/− mice. However, the specific mouse background used in this previous study is unclear. In light of recent evidence suggesting that mouse strain strongly influences PC antimicrobial activity, we sought to characterise PC AMP function in commercially available Nod2−/− mice on a C57BL/6 (B6) background. Specifically, we hypothesised that Nod2−/− B6 mice would display reduced AMP expression and activity. Design Wild-type (WT) and Nod2−/− B6 ileal AMP expression was assessed via real-time PCR, acid urea polyacrylamide gel electrophoresis and mass spectrometry. PCs were enumerated using flow cytometry. Functionally, α-defensin bactericidal activity was evaluated using a gel-overlay antimicrobial assay. Faecal microbial composition was determined using 454-sequencing of the bacterial 16S gene in cohoused WT and Nod2−/− littermates. Results WT and Nod2−/− B6 mice displayed similar PC AMP expression patterns, equivalent α-defensin profiles, and identical antimicrobial activity against commensal and pathogenic bacterial strains. Furthermore, minimal differences in gut microbial composition were detected between the two cohoused, littermate mouse groups. Conclusions Our data reveal that Nod2 does not directly regulate PC antimicrobial activity in B6 mice. Moreover, we demonstrate that previously reported Nod2-dependent influences on gut microbial composition may be overcome by environmental factors, such as cohousing with WT littermates.

Host Defense Peptide Resistance Contributes to Colonization and Maximal Intestinal Pathology by Crohn's Disease-Associated Adherent-Invasive Escherichia coli

“…Early studies suggested that mutant NOD2 alleles could produce defects in the production of some ␣-defensins from humans (17) and mice (18) and offered a mechanism to account for an altered microbial composition and inflammatory response in CD. However, the precise role of NOD2 in CD remains uncertain based on human (19,20) and better-controlled mouse studies (21) that show antimicrobial functions of Paneth cells are independent of NOD2 status. Other antimicrobial peptide classes, such as ␤-defensin 2 and ␤-defensin 3, are increased in inflamed Crohn's disease lesions (22,23), while the antimicrobial monokine MIG (monokine induced by gamma interferon) (24) and LL-37 are elevated in the mucosa of ulcerative colitis (UC) patients (25,26), suggesting that bacteria living at the mucosal surface with an active role in inflammatory bowel disease (IBD) pathogenesis would require mechanisms to resist antimicrobial peptide defenses in the inflamed gut.…”

Nature vs. Nurture: The Gut Microbiome and Genetics in the Development of Gastrointestinal Disease

“…A potential explanation for this finding is the regulation of β-defensin 2 by NOD2, the expression of which is inducible by the presence of commensal bacteria [69,81]. Still, other animal studies have suggested these microbial changes seen in NOD2-deficient mice are dependent on housing conditions, as similar changes were noted in wild type mice when cohoused with the NOD2-deficient mice [82,83]. Clearly, there is plentiful evidence to suggest a relationship between the gut microbiome and NOD2; however, more research will be needed to further determine the extent of this relationship and its importance.…”

Pathogenesis of Ulcerative Colitis and Crohn’s Disease: Similarities, Differences and a Lot of Things We Do Not Know Yet

“…However, this observation has been questioned by others, claiming that the reduced alpha-defensin expression is simply the consequence of inflammation and independent of NOD2 status [58]. Furthermore, in a recent study on C57BL/6 mice, NOD2 deficient animals presented with equivalent defensin levels and identical antimicrobial activity against commensal and pathogenic bacterial strains as their wild type littermates, and their gut microbial composition was also similar [59]. Nevertheless, other animal experiments have suggested distinct mechanisms connecting NOD2 deficiency to CD.…”