Maternal micronutrients can modify colonic mucosal microbiota maturation in murine offspring

Abstract

Epidemiologic data suggest that early nutritional exposures may inflict persistent changes in the developing mammalian “super-organism” (i.e., the host and its residing microbiota). Such persistent modifications could predispose young adults to inflammatory bowel diseases (IBD). We recently observed that the dietary supplementation of four micronutrients to dams augmented colitis susceptibility in murine offspring in association with mucosal microbiota composition changes. In this study the effects of the four micronutrients on the microbiota of dams and female mice was examined. Additionally, age dependent microbiota composition shifts during pediatric development were delineated from the previous offspring data sets. Maternal and adult female microbiota did not separate secondary to the nutritional intervention. Significant microbiota composition changes occurred from postnatal day 30 (P30) to P90 at the level of 1 phylum and 15 genera. Most of these changes were absent or opposite in the maternally supplemented offspring. Nutritionally induced alterations in mucosal microbiota maturation may be contributors to colitis susceptibility in mammals.

Introduction

Microbiota are an inherent part of mammalian “superorganisms,”¹ and intimately interact with the host.² The commensal microbiota is also an important component in the pathogenesis of inflammatory bowel diseases (IBD) along with mucosal enterocytes and the associated immune system.^3,4 Monozygotic twin discordance rates and other epidemiologic data suggest that nutritional factors may play an important role in the development of these disorders.^5-7 The incidence of IBD peaks in young adults, suggesting that critical insults may influence the development of its key physiologic components (microbiota, gut mucosa, immune system) from fertilization through adolescence and induce IBD. Prenatal nutritional influences may produce persistent modifications in immune, and mucosal-enterocyte maturation. These modifications may in turn change the postnatal intercalating networks between the microbiota and the mucosa associated immune system, thereby increasing host susceptibility toward chronic intestinal inflammation. Therefore, understanding the details of mammalian maturation and its environmental vulnerability within the key biologic components of IBD can provide insights into its pathogenesis.⁸ We have recently shown that maternal methyl-donor (MD: betaine, choline, folate, vitamin B12) supplementation can induce persistent microbiota changes in murine offspring in association with mucosal DNA methylation and gene expression alteration, and a significant increase in colitis susceptibility.⁹ In this study, we examined the effects of MD supplementation on the maternal microbiota and interrogated the previous offspring data set in the context of physiologic changes from infancy to young adulthood. The impact of maternal MD supplementation on the normally occurring pediatric microbiota maturation was also studied.

Materials and Methods

Animals and Tissue Collection

Three C57BL/6J female mice, 10 weeks of age (10w) (The Jackson Laboratory, Bar Harbor, ME, USA), were randomly allocated to receive control (NIH-31), and three 10w females to receive a methyl-donor (MD) supplemented diet (NIH-31 supplemented to 5 mg/Kg folic acid /2.5x control/; 0.5 mg/Kg vitamin B12 /~8x control/; 5 g/Kg betaine /not determined in control/; and 5.76 g/Kg choline /~3x control/ (TD#01308, Harlan-Teklad, Madison, WI, USA))¹⁰ for 2 weeks. At 12 weeks of age the females were mated with age matched C57BL/6J males, then were kept on the same diet throughout pregnancy and lactation. Each dam and its offspring were caged separately during lactation. Following lactation (on the day of weaning), the dams were killed by CO₂ asphyxiation between 1 and 3 p.m. without previous food restriction. Colonic mucosa was collected as described before after washing with ice-cold normal saline.⁸

An independent experiment was conducted with six (3–3 in separate cages) 10w C57BL/6J female mice, which were placed on NIH-31 diet for 10 d, then 1 animal was switched between the cages to eliminate cage effects and 3 mice received MD supplemented diet and 3 received NIH-31 diet for an additional 10 d. Fresh fecal samples (collected from each mouse in a sterile, litter free container following spontaneous defecation) were collected at day 10 and day 20 of the experiment. At day 20 following fecal collection, the animals were sacrificed and colonic mucosa was collected as above. The protocol was approved by the Institutional Animal Care Committee for Baylor College of Medicine.

DNA Extraction for Microbial Studies

Mucosal scrapings and fecal samples were centrifuged at 14,000 rpm for 30 sec and re-suspended in 500μl RLT buffer (Qiagen, Valencia, CA) (with β- mercaptoethanol). Sterile 5mm steel beads (Qiagen, Valencia, CA) and 500μl sterile 0.1mm glass beads (Scientific Industries, Inc., NY, USA) were added for complete bacterial lyses in a Qiagen TissueLyser (Qiagen, Valencia, CA), run at 30Hz for 5min. Samples were centrifuged briefly and 100μl of 100% ethanol was added to a 100μl aliquot of the sample supernatant. This mixture was added to a DNA spin column, and DNA recovery protocols were followed as instructed in the QIAamp DNA Mini Kit (Qiagen, Valencia, CA) starting at step 5 of the Tissue Protocol. DNA was eluted and diluted to a final concentration of 20ng/μl.

Massively Parallel bTEFAP and Bacterial Diversity Data Analysis

Massively parallel bacterial tag-encoded FLX-Titanium amplicon pyrosequencing (bTEFAP) utilizes Titanium reagents and procedures, and a one-step PCR, mixture of Hot Start and HotStar high fidelity taq polymerases, utilizing primers 28F to 519R (amplifying the V1-V3 [numbered in relation to E. coli rRNA] region of the 16S rRNA gene, for the dam samples) or 357R to 926F region (amplifying the V3-V5 regions, for the independent female experiments). The bTEFAP procedures were performed at the Research and Testing Laboratory, (Lubbock, TX, USA) and the Alkek Center for Metagenomics and Microbiome Research Center (Houston, TX, USA), respectively. The previously obtained offspring microbiota data⁹ was re-analyzed from the perspective of age dependent changes. Clostridia analyses were based on clusters delineated in Krogius-Kurikka et al.¹¹ Raw 454 sequence data was processed using a combination of software tools. Multiplexed reads were assigned to their originating samples, quality trimmed (average > = 25, no barcode mismatches allowed), and filtered (minimum length = 200, no homopolymers > 10bp, maximum number of ambiguous bases per read = 1) using Mothur.¹² Trimmed reads were then normalized across all samples using an in-house script designed to randomly choose a user-specified number of reads from each sample. In the microbiota analysis of the dam colonic mucosa after normalization of sequence reads 3200 reads per sample were chosen. In the dietary switch experiment 15,000 reads per sample were chosen. These normalized read sets were then subjected to OTU based analysis utilizing CloVR.¹³ CloVR is an application that integrates multiple state-of-the-art analysis tools into a single program. Chimeric sequences were detected and removed via UCHIME,¹⁴ phylogenetic distance metrics were generated by QIIME (OTU defined as 97% identity),¹⁵ and statistics were generated by Metastats.¹⁶ Sequences with identity scores (to known or well characterized 16S rRNA sequences) greater than 97% identity (< 3% divergence) were resolved at the species level, between 95% and 97% at the genus level, and below 85% to the phylum. P values are the results of uncorrected, unpaired, two-tailed T tests, analysis of variance (ANOVA), or Mann-Whitney U test between the experimental groups.

Results

Methyl-donor Supplementation Did Not Separate Colonic Mucosal and Fecal Microbiota in Female Mice

The effect of MD supplementation on maternal colonic mucosal microbiota composition was investigated. Neither weighted nor unweighted principal component analysis (PCA) of the data revealed MD induced microbiota separation in the dams (Fig. 1). We used weighted PCA to limit for outliers and increase the robustness of the microbiota comparisons for the purposes of the figure. The negative result on microbiota separation is consistent with our findings that MD supplementation after 30 d of life did not affect dextran sulfate sodium (DSS) induced colitis susceptibility⁹ since the severity of DSS colitis is significantly influenced by intestinal microbiota composition.¹⁷ These observations argue that MD supplementation does not induce maternal microbiota separation and suggest that it is unlikely that the inherited maternal microbiota altered the offspring colitis phenotypes. Rather, the reprogramming of immunologic development upon maternal MD supplementation is likely to occur in the offspring. The reprogrammed immune system then could lead to altered gut microbiota development.

Figure 1. Weighted principal component analysis of maternal colonic mucosal microbiome on control (blue circles) and MD supplemented (red circles) diets at the time of weaning. No separation can be delineated between the two groups. The axis numbers represent relative position within two-dimensional space.

There are significant differences between luminal/fecal and mucosal microbiota.¹⁸ Although the mucosal microbiota may be more relevant in modulating colitis susceptibility,¹⁹ there is limited definite data to support this notion. Therefore, we examined the effects of MD supplementation on fecal microbiota composition in female mice. Similar to our findings with the dam mucosal microbiota, a distinct separation upon MD supplementation could not be observed in feces (p = 0.39 using Bray-Curtis dissimilarity as the “distance” metric; Fig. S1).

Microbiota Composition Changes From Infancy to Young Adulthood in Mice

In depth analysis of normal intestinal mucosal microbiota changes during pediatric development in mice has not been performed. Therefore, we examined bacterial taxa differences between infancy (P30) and young adulthood (P90)²⁰ on control diet. Phyla, genera, and species abundance values were compared with heat-maps (dual hierarchical clustering) ANOVA, and non-parametric U test.

The relative percent abundance of Cyanobacteria decreased significantly from P30 to P90 (~0.13% to ~0.003%, respectively; p = 0.005) at the phylum level. Firmicutes (~85–91% on average) and Bacteroidetes (~8–14% on average), the two most abundant phyla remained stable, which pertained to Firmicutes/Bacteroidetes ratios as well. This result opposes findings in human fecal samples where an increase in Firmicutes/Bacteroidetes ratios was observed from infancy to adulthood.²¹

Dual hierarchical clustering at the genera level revealed a significant separation between the P30 mucosal and the P90 mucosal microbiome in the majority (80%) of the studied samples (Fig. S2). The abundance of 15 colonic mucosa associated genera changed significantly during maturation from P30 to P90 (Table 1). Five of these changes affected more than 1% of the microbiota composition between the two age groups.

Table 1. Genera (in alphabetical order) in the colonic mucosal samples of mice on control diet, which significantly changed in abundance by ANOVA between P30 and P90 (n = 5 in each group). Changes that affected more than 1% of the microbiome composition are in bold.

Genus	P30 (%)	P90 (%)	ANOVA p value	U-test p value
Alistipes	4.65E-02	0.614176	0.013283	0.0079
Anaeroplasma	0.169782	4.59E-02	0.022692	0.0317
Aphanizomenon	3.37E-02	0	0.037244	na
Butyrivibrio	0.182158	0.403409	0.018769	0.0159
Clostridium	27.04	41.65	0.037	ns
Desulfonispora	0	0.073129	0.046371	na
Lactobacillus	46.4501	19.88615	0.006079	0.0317
Oribacterium	0	3.04E-02	0.046653	na
Rikenella	3.64225	0.226022	0.004008	0.0079
Roseburia	3.106611	6.698188	0.01973	ns
Ruminococcus	0.447055	1.472997	0.030784	0.0079
Sanguibacter	4.72E-02	0	0.002918	na
Streptomyces	6.02E-02	6.11E-03	0.005594	0.0159
Synechococcus	3.55E-02	0	0.00021	na
Tannerella	9.66E-03	0.56055	0.026981	0.0079

Species level comparison between the infant and young adult colonic mucosal microbiota showed several significant alterations that affected more than 0.1% of the composition (Table 2). In the meantime, the number of different species detected (diversity, 117–153 species/sample) did not vary notably between the two age groups (data not shown). Our results underscore that prominent colonic mucosal microbiota maturation occurs post-suckling in mice both at the genus and species levels.

Table 2. Species (in alphabetical order) with significant abundance changes affecting more than 0.1% of the microbiome composition between P30 and P90 in control offspring. Changes that affected more than 1% of the microbiome composition are in bold (n = 5 in each group).

Species	P30 (%)	P90 (%)	T test p value	U-test p value
Alistipes shahii	0.003221	0.509409	0.010634	0.0079
Anaeroplasma abactoclasticum	0.169782	0.045891	0.022692	0.0317
Butyrivibrio fibrisolvens	0.072357	0.184297	0.040553	ns
Clostridium cocleatum	0.160392	0.003922	0.025454	0.0079
Clostridium disporicum	0.00498	0.402437	0.02022	0.0159
Clostridium fimetarium	0.066349	0.46116	0.003428	0.0079
Clostridium lactatifermentans	0.119912	0.38473	0.006274	0.0159
Clostridium nexile	0.991654	2.972706	0.030372	0.0079
Clostridium orbiscindens	1.321404	1.916829	0.043277	ns
Clostridium saccharolyticum	0.508086	1.253163	0.02129	ns
Clostridium symbiosum	0.617129	1.050736	0.048492	0.0317
Eubacterium xylanophilum	0.097667	1.076145	0.002492	0.0079
Lactobacillus johnsonii	46.44254	19.84106	0.006047	0.0317
Rikenella microfusus	3.64225	0.226022	0.004008	0.0079
Roseburia intestinalis	2.861264	6.212425	0.020636	ns
Ruminococcus bromii	0.21542	0.723837	0.001033	0.0079
Tannerella forsythensis	0.009662	0.56055	0.026981	0.0079

Maternal MD Supplementation Disrupts Pediatric Microbiota Maturation

We wished to examine how maternal methyl-donor supplementation influenced pediatric microbiota maturation in offspring compared with controls. The same taxa comparisons were performed as with the control diet samples.

As opposed to controls, the percent of phylum abundance of Tenericutes (~0.12 to ~0.023% from P30 to P90, p = 0.024) and Spirochetes (0 to ~0.15% from P30 to P90, p = 0.0023) changed significantly.

Dual hierarchical clustering at the genera level revealed a significant separation between the P30 mucosal and the P90 mucosal microbiota in the majority (80%) of the samples studied (Fig. S3). The abundance of 12 colonic mucosa associated genera changed significantly from P30 to P90 in the maternally MD supplemented offspring (Table 3). Four of these changes affected more than 1% of the microbiota composition between the two age groups.

Table 3. Genera (in alphabetical order) with significant abundance changes P30 and P90 in maternally MD supplemented offspring. Changes that affected more than 1% of the microbiome composition are in bold (n = 5 in each group).

Genus	P30 (%)	P90 (%)	ANOVA p value	U-test p value
Alicyclobacillus	0	0.047652	0.022759	na
Anaeroplasma	0.112505	2.33E-02	0.033207	ns
Anaerovibrio	0	2.56E-02	0.015342	na
Bacteroides	7.416936	4.459703	0.043795	ns
Butyrivibrio	0.510524	0.247474	0.027185	ns
Lactobacillus	2.513577	15.24864	0.028771	0.0317
Prochlorococcus	0	1.83E-02	0.043762	na
Sanguibacter	6.44E-02	3.53E-03	0.013397	0.0159
Staphylococcus	3.875108	0.204078	0.01691	ns
Streptomyces	7.91E-02	0	0.032693	na
Treponema	0	0.151721	0.002296	na
Turicibacter	0.195556	17.02062	0.013304	0.0079

Only four (Butyrivibrio, Lactobacillus, Sanguibacter, Streptomyces) genera with a significant maturation change were shared by the control and MD supplemented offspring. Furthermore, in case of Butyrivibrio, and Lactobacillus, the changes in the MD offspring opposed that of controls. The abundance of Sanguibacter and Streptomyces decreased in both control and MD offspring, but these genera contributed with less than 0.1% to the microbiota composition in all groups studied. The developmental impact of Clostridia, more specifically Clostridium clusters IV and XIVa on intestinal inflammatory responses has been recently observed.²² Therefore, we compared overall Clostridia and Clostridium IV, XVIa cluster changes in our experimental groups (Fig. 2). While Clostridia increased significantly from P30 to P90 in the control groups (numeric data in Table 1), there was an opposite trend (p = 0.08) for this in the MD supplemented offspring. Reverse trends were likewise present in the age dependent abundance shifts within the Clostridium IV and XVIa clusters (Fig. 2).

Figure 2. Age dependent Clostridia abundance shifts. Maternal MD supplementation prominently modified the physiologic abundance changes in the offspring colonic mucosa. Age dependent changes between P30 and P90 were significant (p < 0.05) in controls for the Clostridium genus and the Clostridium IV cluster. There was a close to significant decline (p = 0.08) in Clostridia (upper panel) within the MD supplemented group between P30 and P90. There was an overall opposing trend between the two (control: increase; MD supplemented: decrease) groups for Clostridium in general, and for the two specific clusters observed (*:p < 0.05, ***:p < 0.001 for abundance comparison between control and MD supplemented offspring at P30; n = 5 per group).

As with the evaluations of the genera, the putative species level comparisons revealed that the P30 to P90 colonic mucosal microbiota changes differed significantly between control and MD supplemented offspring (Table 4). Only two species (Anaeroplasma abactoclasticum, and Lactobacillus johnsonii) with a significant maturation change were shared by the control and MD supplemented offspring. Among these species, only the abundance of Anaeroplasma abactoclasticum decreased similarly in both groups. On the contrary, the abundance of Lactobacillus johnsonii decreased in controls, but increased in MD supplemented offspring between P30 and P90 (Fig. 3).

Table 4. Species (in alphabetical order) with significant abundance changes affecting more than 0.1% of the microbiome composition between P30 and P90 in maternally MD supplemented offspring. Changes that affected more than 1% of the microbiome composition are in bold (n = 5 in each group).

Species	P30 (%)	P90 (%)	T test p value	U-test p value
Anaeroplasma abactoclasticum	0.112504	0.023298	0.033207	ns
Butyrivibrio hungatei	0.305563	0.103453	0.001088	0.0079
Clostridium celerecrescens	2.260847	0.624948	0.003757	0.0079
Clostridium clostridioforme	1.862933	0.539839	0.008125	0.0079
Clostridium herbivorans	3.0931	1.008053	0.007184	0.0079
Clostridium xylanovorans	0.414296	1.117788	0.047631	ns
Eubacterium biforme	0.006791	16.86542	0.013054	0.0079
Eubacterium cellulosolvens	0.101246	0.010584	0.012172	0.0317
Eubacterium hallii	1.244567	0.32501	0.011346	0.0079
Lactobacillus johnsonii	2.328881	14.23389	0.032528	0.0317
Roseburia cecicola	0.068143	0.249224	0.014587	0.0159
Staphylococcus saprophyticus	3.501773	0.029451	0.017324	ns
Syntrophococcus sucromutans	1.4262	0.114639	0.022863	0.0079

Figure 3. The three most abundant species affected by age dependent maturation in the experimental cohorts. In all three cases, MD supplementation remarkably modified the developmental abundance changes in the colonic mucosa (error bars: standard deviation of the mean; n = 5 in each group).

These findings highlight prenatal and suckling MD supplementation induces remarkably modified colonic mucosal microbiota maturation compared with controls in mice despite of the two offspring groups receiving the same diets after weaning.

Discussion

Age dependent intestinal microbiota composition changes in humans have mostly been assessed in fecal samples with frequently less in depth methodology as utilized herein.^21,23 However, the mucosa-associated microbiota may be more relevant for intestinal inflammatory processes than the fecal flora.¹⁹ Additionally, previous aging related studies mainly focused on microbial population shifts during infancy,^24,25 or senescence²⁶ since the general consensus is that by about 1–2 y of age the intestinal (fecal) microbiota attains a largely similar composition as in adulthood.^24,27 Yet, pediatric developmental changes in the main pathologic components involving intestinal inflammation (microbiota, mucosa and immune system) may be relevant in regards to the developmental origins of IBD, for these disorders usually present in young adulthood.⁸ The importance of microbial composition changes during infancy (but not pediatric development) has been observed in mammalian models in respect to mucosal immune responses²⁸ and colitis susceptibility.²² This phenomenon may be designated as microbe induced imprinting/programming of the immune system and underscores the importance of the intercalating relationship between the key components of IBD pathogenesis.³ In light of this interplay, it is also conceivable that the developmental programming of colonic mucosal and immunologic maturation can also imprint/program age dependent microbiota shifts in the intestinal mucosa. Epigenetic molecular processes are central participants in mammalian developmental programming and differentiation.²⁹ DNA methylation, utilizing the mammalian one carbon pool, is the most stable, and potentially the most relevant epigenetic mechanism in this respect.^29,30 Early developmental nutritional changes, such as maternal MD supplementation (feeding into the mammalian one-carbon pool) has been observed to modify the establishment of DNA methylation at select genomic loci in mice.³¹ Similarly behaving loci have been recently revealed by us in humans highlighting the potential importance of nutritional imprinting/programming in the development of common human diseases.³² Colonic mucosal DNA methylation modifications specifically have been shown to associate with microbiota alterations in murine models of human IBD.^8,33 However, one must first understand physiologic changes during maturation to reveal the impact and possible consequences of modifications in such developmental processes.

In this work, we first determined that considerable microbiota maturation occurs under physiologic conditions in the colonic mucosa of mice even during the post-suckling period. This maturation significantly involved 15 bacterial genera and 17 different species. Similar patterns have been observed in fecal samples from BALB/c mice during the same developmental period (i.e., post-weaning increase in Clostridia and decrease in Lactobacilli).²² Importantly, the physiologic mucosal bacterial maturation determined herein parallel both DNA methylation and gene expression changes and associate with an increased susceptibility to colitis in the same mouse strain (C57BL/6J).⁸ The decreased abundance of Lactobacillus johnsonii for instance may contribute to increased colitis susceptibility since oral L. johnsonii administration has been shown to decrease the severity of experimentally induced large intestinal inflammation in mice.³⁴ Similar reciprocal interactions between the host and the microbiota have already been recognized (see discussion²⁴).

Following the observation of the normal (regular diet, wild type mice) colonic mucosal microbiota shifts during post-suckling (post-weaning) development we turned to examine how maternal MD supplementation might affect this process. This dietary intervention has been observed to induce prolonged augmentation in offspring colitis susceptibility in association with persistent colonic mucosal DNA methylation changes. Longstanding effects on the mucosal microbiota were also observed, but the impact of maternal MD supplementation on the developmental patterning of the microbiota during the post-suckling period was not addressed.⁹ We found that the pediatric maturation of the colonic microbiota remarkably differed in the maternally MD supplemented offspring including highly abundant genera and species (Fig. 2, 3). The differences in maternal MD supplementation induced offspring microbiota shifts appeared to be independent from the maternal microbiota (Fig. 1). The maturation divergence involved Clostridium clusters IV and XIVa, which have been shown to affect intestinal immunologic responses if their abundance was modified at a critical developmental stage in mice.²² Consequently, our findings implicate that the developmental dietary intervention induced alteration of normal colonic-mucosa-associated-microbiota-shifts may have contributed to the observed increase in colitis susceptibility in the MD supplemented offspring. The significant dietary impact on the intestinal microbiota and associated disease predilection in pediatric subpopulations has been recently emphasized.³⁵ However, the potential effects of such dietary differences on prenatal/early postnatal development relevant for modulating age dependent microbiota shifts has not been addressed. Our study underscores that even relatively modest (2.5–8 fold increase in four water soluble vitamins/micronutrients) and transient nutritional exposures during critical developmental periods can majorly influence the successive maturation of mammalian organs/components relevant not only for IBD, but gastrointestinal diseases in general.^2,36,37 The findings are consistent with the paradigm of the interactive, environmentally responsive roles of the microbiota and the host in mammalian developmental immunologic programming.³⁸

This work includes the first in-depth assessment of mammalian colonic mucosal microbiota shifts in the post-weaning pediatric developmental period and how those may be influenced by nutritional/metabolic imprinting. Our observations can support the unraveling of similar pathways in humans, which may be relevant for common gastrointestinal diseases, such as IBD.

Schaible TD, Harris RA, Dowd SE, Smith CW, Kellermayer R. Maternal methyl-donor supplementation induces prolonged murine offspring colitis susceptibility in association with mucosal epigenetic and microbiomic changes. Hum Mol Genet. 2011;20:1687–96. doi: 10.1093/hmg/ddr044.