Prenatal methyl-donor supplementation augments colitis in young adult mice

Abstract

Inflammatory bowel diseases (IBD) have become highly prevalent in developed countries. Environmentally triggered exaggerated immune responses against the intestinal microbiome are thought to mediate the disorders. The potential dietary origins of the disease group have been implicated. However, the effects of environmental influences on prenatal developmental programming in respect to orchestrating postnatal microbiome composition and predilection towards mammalian colitis have not been examined. We tested how transient prenatal exposure to methyl donor micronutrient (MD) supplemented diets may impact predilection towards IBD in a murine dextran sulfate sodium (DSS) colitis model. Prenatal MD supplementation was sufficient to modulate colonic mucosal Ppara expression (3.2 fold increase; p=0.022) and worsen DSS colitis in young adulthood. The prenatal dietary exposure shifted the postnatal colonic mucosal and cecal content microbiomes. Transfer of the gut microbiome from prenatally MD supplemented young adult animals into germ free mice resulted in increased colitis susceptibility in the recipients compared to controls. Therefore, the prenatal dietary intervention induced the postnatal nurturing of a colitogenic microbiome. Our results show that prenatal nutritional programming can modulate the mammalian host to harbor a colitogenic microbiome. These findings may be relevant for the nutritional developmental origins of IBD.

Figure 1. Prenatal MD supplementation is sufficient to worsen young adult male colitis.

Dams were provided either a control (C) or methyl-donor (MD) diet before, during and following (lactation) pregnancy. Each litter was crossed fostered. Groups were designated as following: MD-MD if they had received MD diet in utero and lactation; C-C for control diet in utero and lactation; MD-C for MD in utero and control in lactation and the last group were C-MD that received control in utero and MD in lactation. Offspring were transferred to control diet at weaning (P21) and tested for susceptibility to colitis by 3% DSS exposure for 5 days in drinking water at P90. (A) The MD-MD and MD-C male mice showed significantly (*p<0.05) increased weight loss compared to the C-C. (B) Colonic lengths were significantly shorter (*p<0.05) in the MD-MD and MD-C compared to the negative control C-C. N=8-12 per group. (C) Histological severity of inflammation was consistent with the MD-MD group suffering a more severe colitis than control. There was a trend approaching significance for microscopically increased colitis in the MD-C group as well. N=5.

Figure 2. Increased expression of Ppara in prenatally MD supplemented young adult mice.

Ppara expression by real-time RT-PCR measurements from colonic mucosal scrapings showed increased expression in the MD-C animals compared to C-C at P90 (U test, p=0.022; n=6-7).

Figure 3. Fecal (F) and colonic mucosal (M) microbiome separation in prenatally MD supplemented (MD-C) young adult mice.

Cecal content (feces) and colonic mucosal scrapings from non-DSS exposed C-C and MD-C animals were processed for microbiome analysis at P90. Principal coordinates analysis (PCoA) of weighted UniFrac measures showed separation of fecal and mucosal microbiomes in the prenatally MD supplemented (MD-C) mice compared to the control cross fostered group (C–C). N=5 per group.

Figure 4. Genera level separation of colonic mucosal and fecal microbiomes upon prenatal MD supplementation.

Combined representation of the 10-10 most prominently (by T test p value) differing genera in the mucosal and fecal (cecal content) samples, respectively, secondary to prenatal MD supplementation. Anaerosptipes and Akkermansia overlapped between the mucosal and fecal comparisons. Hence 18 genera are depicted. Sixteen (90%) out of the 18 genera changed similarly in abundance between mucosa (M) and feces (F) upon prenatal MD supplementation at P90. Prenatally MD supplemented (MD-C) and control cross fostered groups (C–C) are compared. N=5.

Figure 5. Significantly augmented colitis in germ free recipients of MD-C feces.

5-5 germ free male mice were gavaged with pooled cecal content (feces) from either prenatally MD supplemented animals (MD-C), or controls (C–C). After 5 days the animals were tested for colitis susceptibility by 5% DSS exposure in drinking water for 5 days. At day 8, the mice were euthanatized. Colonic length was measured and colonic tissue was examined histologically. (A) Weight loss upon 5% DSS challenge was significantly greater in the recipients of MD-C feces at days 6, 7, and 8 (*p<0.05). (B) The increased severity of colitis was also reflected by significant colonic length shortening in the recipients of MD-C feces (p=0.0099) compared to control. (C) A greater degree of tissue damage and inflammation (p=0.0028) was observed in the MD-C fecal recipients compared to the recipients of control stool. N=5.