Mouse Paneth cell antimicrobial function is independent of Nod2 - PubMed (original) (raw)

Mouse Paneth cell antimicrobial function is independent of Nod2

Michael T Shanahan et al. Gut. 2014 Jun.

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.

Keywords: BACTERIAL INTERACTIONS; CROHN'S DISEASE; IBD - GENETICS; INFLAMMATORY BOWEL DISEASE; INTESTINAL EPITHELIUM.

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Figures

Figure 1

Figure 1

Paneth cell (PC) antimicrobial peptide transcript expression is primarily unaffected by Nod2. Ileal transcript levels of (A) lysozyme-P (Lyz); (B) regenerating islet-derived protein 3 gamma (Reg3γ); (C) angiogenin 4 (Ang4); (D) total PC α-defensins (PanCrp); (E) cryptdin-related sequence (CRS)1C and (F) CRS4C are shown for wild-type (WT) and Nod2−/− mice (n=8–10 mice/group). CRS4C was not detected (ND) in any animal. Copy number is normalised to β-actin and expressed as a fold Δ relative to the WT group. Data are shown as means with SEM. *p<0.05; N.S.—not significant (p>0.6), based on Student's t test.

Figure 2

Figure 2

Paneth cell α-defensin expression is independent of Nod2. mRNA expression of α-defensin isoforms (A) Defa4; (B) Defa20; (C) Defa3; and (D) Defa5 in the ileum of wild-type (WT) and Nod2−/− mice (n=8–10 mice/group). Copy number is normalised to β-actin and expressed as a fold Δ relative to the WT group. Data are shown as means with SEM. ND— not detected; NS— not significant (p>0.1), based on Student's t test. (E) acid urea-polyacrylamide gel electrophoresis demonstrates peptide expression patterns of α-defensin isoforms in the ileum of WT and Nod2−/− mice. First lane is recombinant Defa4 control; each additional lane represents an individual mouse. Individual bands (based on calculated mass determined via mass spectrometry): 1-Defa4 (recombinant); 2,7-Defa5; 3,8-Defa24; 4,9-Defa20/Defa21; 5,10-Defa2; and 6,11-Defa22.

Figure 3

Figure 3

Nod2 does not influence Paneth cell (PC) development. (A) H&E staining of ileal tissue shown at 400 × magnification from wild-type (WT) and Nod2−/− mice was used to quantify intestinal crypts. Panel (B) represents average data from 18–20 high power fields/group (3–4 mice/group). Panel (C) shows immunohistochemical staining of ileal tissue for lysozyme (Lyz), allowing for the quantification of PCs per crypt. Panel (D) represents average data from 40–45 crypts/group (5–6 mice/group). (E,F) Flow cytometry was used to measure the percentage of Lyz+ CD45− PCs from the ileal epithelium of WT and Nod2−/− mice. Data are representative of three independent experiments (NS— not significant, p>0.1, based on Student's t test).

Figure 4

Figure 4

Antimicrobial activity of Paneth cell α-defensins is unaffected by Nod2. (A) wild-type (WT) and Nod2−/− mouse-derived ileal protein extracts were resolved by acid urea-polyacrylamide gel electrophoresis. An excised gel strip containing the α-defensins was placed onto bacteria-laden agarose. Bacterial clearance zones are shown for (B) Escherichia coli NC101, (C) Listeria monocytogenes 10403S, and (D) Salmonella enterica serovar Typhimurium. Similarly sized zones of bacterial growth inhibition are seen for all bacterial strains, regardless of Nod2 status.

Figure 5

Figure 5

Local cage environment overrides Nod2-mediated influences on the intestinal microbiota. Principal coordinates analysis (PcoA) using the first two coordinates of a PcoA based on Bray-Curtis dissimilarity of 454 sequences of 16S rRNA from stool samples. Nod2−/− mice (KO) are indicated by circles, while wild-type (WT) mice are shown with squares. The numbers by each symbol indicate the cage of the animal, and each cage is depicted in a unique colour. PC, principal coordinate.

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