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. 2012 May;26(5):1960-9.
doi: 10.1096/fj.10-177980. Epub 2012 Jan 20.

Probiotics stimulate enterocyte migration and microbial diversity in the neonatal mouse intestine

Geoffrey A Preidis  1 , Delphine M Saulnier, Sarah E Blutt, Toni-Ann Mistretta, Kevin P Riehle, Angela M Major, Susan F Venable, Milton J Finegold, Joseph F Petrosino, Margaret E Conner, James Versalovic
Affiliations

Probiotics stimulate enterocyte migration and microbial diversity in the neonatal mouse intestine

Geoffrey A Preidis et al. FASEB J. 2012 May.

Abstract

Beneficial microbes and probiotics show promise for the treatment of pediatric gastrointestinal diseases. However, basic mechanisms of probiosis are not well understood, and most investigations have been performed in germ-free or microbiome-depleted animals. We sought to functionally characterize probiotic-host interactions in the context of normal early development. Outbred CD1 neonatal mice were orally gavaged with one of two strains of human-derived Lactobacillus reuteri or an equal volume of vehicle. Transcriptome analysis was performed on enterocyte RNA isolated by laser-capture microdissection. Enterocyte migration and proliferation were assessed by labeling cells with 5-bromo-2'-deoxyuridine, and fecal microbial community composition was determined by 16S metagenomic sequencing. Probiotic ingestion altered gene expression in multiple canonical pathways involving cell motility. L. reuteri strain DSM 17938 dramatically increased enterocyte migration (3-fold), proliferation (34%), and crypt height (29%) compared to vehicle-treated mice, whereas strain ATCC PTA 6475 increased cell migration (2-fold) without affecting crypt proliferative activity. In addition, both probiotic strains increased the phylogenetic diversity and evenness between taxa of the fecal microbiome 24 h after a single probiotic gavage. These experiments identify two targets of probiosis in early development, the intestinal epithelium and the gut microbiome, and suggest novel mechanisms for probiotic strain-specific effects.

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Figures

Figure 1.
Figure 1.
Human-derived L. reuteri did not persist in the neonatal mouse small intestine. FISH using a Cy3-conjugated probe (red) to label L. reuteri (arrows); DAPI (blue) labeled host cell nuclei. Similar results were observed with both probiotic strains; L. reuteri 17938 shown in the ileum of 8-d-old mice, ×200 view.
Figure 2.
Figure 2.
L. reuteri altered enterocyte RNA pathways involving cell motility. A, B) IPA of differentially expressed enterocyte genes showing the 5 most significantly altered canonical pathways and the highest-scoring protein interaction network for each probiotic strain vs. PBS (n=3 mice/group), 24 h after gavage with strain 17938 (A) or strain 6475 (B). C) Immunohistochemistry reveals diminished production of stabilizing cytoskeletal protein β-actin (product of ACTB gene in above networks) in enterocytes. Compared to PBS controls (left panel), decreased labeling at the basement membrane was found in mice treated with L. reuteri strains 17938 (center panel) or 6475 (right panel). However, smooth muscle cell β-actin (arrowheads) was labeled in all treatment groups. Red represents up-regulated genes; green represents down-regulated genes; gray represents genes not differentially expressed; white represents genes with no array data; solid lines/arrows represent direct interactions; dashed lines/arrows represent indirect interactions.
Figure 3.
Figure 3.
Probiotics increased enterocyte migration and proliferation in the ileum in a strain-specific manner. A) Immunohistochemistry for BrdU at different times after injection. Graphs show mean ± sd position of foremost and least-progressed BrdU-labeled enterocytes. B) Crypt cell proliferation, determined 4 h post-BrdU. C) Crypt height at the same time points. Mice were gavaged daily with probiotics or vehicle. n = 7 mice/group for migration and crypt height analyses; n = 15 mice/group for proliferation analysis; ×200 view; hpi, hours postinjection; dpi, days postinjection. *P < 0.05, **P < 0.01, ***P < 0.001 vs. PBS controls; #P < 0.05, ##P < 0.01, ###P < 0.001 between strains.
Figure 4.
Figure 4.
Probiotics increased phylogenetic diversity of the distal intestinal microbiome, as revealed by 16S rRNA V3-V5 sequences obtained by pyrosequencing. Microbial DNA was derived from pooled stool samples from 8-d-old pups, both before and 4 h, 24 h, and 48 h after a single gastric gavage. A) Shannon's diversity index. B) Species richness or α diversity, defined as the total number of unique OTUs per sample. C) Pielou's index of species evenness. Each data point represents the mean of 2 sequencing reactions/pooled sample. Values are means ± sd; n = 5 pools of 20 mice/treatment group, sampled at multiple time points. *P < 0.05 vs. PBS controls.
Figure 5.
Figure 5.
Probiotics remodeled the distal intestinal microbiome. OTUs revealed by 16S rRNA V3-V5 sequences obtained by pyrosequencing. DNA was derived from stool pooled from 20 mice/group of 8-d-old pups before or 24 h after a single gastric gavage.
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