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. 2014 Aug;49(8):1242-51.
doi: 10.1016/j.jpedsurg.2014.01.060.

Intestinal dysbiosis and bacterial enteroinvasion in a murine model of Hirschsprung's disease

Joseph F Pierre  1 , Amanda J Barlow-Anacker  1 , Christopher S Erickson  1 , Aaron F Heneghan  1 , Glen E Leverson  1 , Scot E Dowd  2 , Miles L Epstein  3 , Kenneth A Kudsk  4 , Ankush Gosain  5
Affiliations

Intestinal dysbiosis and bacterial enteroinvasion in a murine model of Hirschsprung's disease

Joseph F Pierre et al. J Pediatr Surg. 2014 Aug.

Abstract

Background/purpose: Hirschsprung's disease (HSCR), characterized by the absence of ganglia in the distal colon, results in functional obstruction. Despite surgical resection of the aganglionic segment, around 40% of patients suffer recurrent life threatening Hirschsprung's-associated enterocolitis (HAEC). The aim of this study was to investigate whether gut microbiota and intestinal immunity changes contribute to the HAEC risk in an HSCR model.

Methods: Mice with neural crest conditional deletion of Endothelin receptor B (EdnrB) and their littermate controls were used (EdnrB-null and EdnrB-het). Bacterial DNA was prepared from cecal contents of P16-18 and P21-24 animals and pyrosequencing employed for microbiome analysis. Ileal tissue was isolated and secretory phospholipase A2 (sPLA2) expression and activity determined. Enteroinvasion of Escherichia coli into ileal explants was measured using an ex vivo organ culture system.

Results: EdnrB-het and EdnrB-nulls displayed similar flora, sPLA2 expression and activity at P16-18. However, by P21-24, EdnrB-hets demonstrated increased Lactobacillus and decreased Bacteroides and Clostridium, while EdnrB-nulls exhibited reciprocal changes. EdnrB-nulls also showed reduced sPLA2 expression and luminal activity at this stage. Functionally, EdnrB-nulls were more susceptible to enteroinvasion with E. coli ex vivo and released less sPLA2 than EdnrB-hets.

Conclusions: Initially, EdnrB-het and EdnrB-nulls contain similar cecal flora but then undergo reciprocal changes. EdnrB-nulls display dysbiosis, demonstrate impaired mucosal defense, decreased luminal sPLA2 and increased enteroinvasion of E. coli just prior to robust colonic inflammation and death. These findings suggest a role for the intestinal microbiome in the development of HAEC.

Keywords: Dysbiosis; Hirschsprung’s disease; Hirschsprung’s-associated enterocolitis; Microbiome; Mucosal immunity; Secretory phospholipase A2.

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Figures

Figure 1
Figure 1. Survival of EdnrB-null animals and histologic evidence of enterocolitis
(A) EdnrB-null animals were followed until moribund and then sacrificed. Survival in days is plotted and the mean (28.2 days) and median (29 days) survival are indicated. (B) H&E staining of P21 and P26 EdnrB-het and -null colon samples (20x magnification). Black arrows indicate neutrophil infiltration in the P26 EdnrB-null samples. Insert shows higher magnification of neutrophils in the P26 EdnrB-null colon. Scale bar = 100μm.
Figure 2
Figure 2. Hierarchal clustering and ANOVA analysis of EdnrB-het and –null Phyla and Genera
(A) Ward’s minimum variance cluster analysis was employed to compare the microbiota composition of EdnrB-het and –null animals at the phylum level. First order branching (semi-partial R-squared=0.6119) separated a group containing 45% EdnrB-null Late, 30% EdnrB-null Early and 25% EdnrB-het Early (upper group), from a group containing 46% EdnrB-het Late, 21% EdnrB-null Late, 18% EdnrB-het Early and 14% EdnrB-null Early (lower group). (B) The top three phyla found at the early time point were analyzed between groups. The EdnrB-hets demonstrated increased Firmicutes and decreased Bacteroidetes over time (*p<0.05). EdnrB-nulls displayed fewer Firmicutes than EdnrB-hets at both the early and late time points (*p<0.05). Additionally, EdnrB-nulls had increased representation of Bacteroidetes and Proteobacteria, as compared to EdnrB-hets at the later time point (*p<0.05). (C) Ward’s minimum variance cluster analysis was employed to compare the microbiota composition of EdnrB-het and –null animals at the genus level. First order branching (semi-partial R-squared=0.4886) separated a group containing 45% EdnrB-null Late, 27% EdnrB-null Early and 27% EdnrB-het Early (upper group), from a group containing 79% EdnrB-het Late, 14% EdnrB-het Early and 7% EdnrB-null Early (lower group). (D) The top three genera found at the early time point were analyzed between groups. EdnrB-het animals demonstrate increased Lactobacillus over time, while EdnrB-null animals demonstrate a decrease (*p<0.05). Additionally, At the late time point, EdnrB-null animals demonstrate decreased Lactobacillus and increased Bacteroides and Clostridium as compared to EdnrB-het animals (*p<0.05).
Figure 3
Figure 3. Principal coordinate analysis of Unifrac distances for sequences obtained from EdnrB-het and EdnrB-null animals at early and late time points
2D representation of the first two principal coordinates. Axis percentages indicate the amount of variability explained by each coordinate. 95% confidence interval ellipses of EdnrB-het early (blue) and EdnrB-null early (yellow) animals demonstrate similarity of the flora as determined by overlapping confidence intervals. 95% confidence interval ellipses of EdnrB-het late (green) and EdnrB-null late (red) animals demonstrates distinct flora as determined by non-overlapping confidence intervals. This separation appears to be based primarily on the first principal coordinate (x-axis).
Figure 4
Figure 4. Microbial sequence diversity
Three methods were used to assess alpha diversity of the microbiome: (A) Rarefaction (Operational Taxonomic Unit, OTUs, at a level of sequence similarity >97%). (B) Chao1 Index. (C) Shannon-Weaver Index. By two-way ANOVA with Bonferonni post-hoc analysis, EdnrB-het late animals differ from the other groups (* p=0.004).
Figure 5
Figure 5. Small intestinal tissue sPLA2 expression and wash fluid sPLA2 activity
(A) Representative immunohistochemical images of Paneth cell sPLA2 (red) with DAPI cell nucleus stain (blue) in ileal segments. sPLA2 was expressed at the early time point (P16) in both animal groups, but was not visible in EdnrB-nulls at the late time point (P22) (B) PAS staining demonstrating that Paneth cells (black arrows) appear to be present in normal numbers in EdnrB-null animals at the later time point. (C) At P16–P18, EdnrB-het and EdnrB-null animals showed similar luminal levels of sPLA2 activity. However, by P21–24, EdnrB-null mice had significantly decreased luminal levels of sPLA2 activity compared to EdnrB-hets (* p<0.01). Scale bars = 100μm.
Figure 6
Figure 6. Recovered bacteria from tissue segments and sPLA2 activity in secretions after ex vivo intestinal segment culture (EVISC)
(A) EdnrB-null ileal segments had significantly more enteroinvasive E. coli recovered compared to EdnrB-het, demonstrating increased enteroinvasive susceptibility (* p<0.05). (B). sPLA2 activity from secretions of ileal segments during enteroinvasion challenge was significantly decreased in EdnrB-null animals compared to EdnrB-hets (* p<0.05).
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