Creating and maintaining the gastrointestinal ecosystem: what we know and need to know from gnotobiology

Abstract

Studying the cross talk between nonpathogenic organisms and their mammalian hosts represents an experimental challenge because these interactions are typically subtle and the microbial societies that associate with mammalian hosts are very complex and dynamic. A large, functionally stable, climax community of microbes is maintained in the murine and human gastrointestinal tracts. This open ecosystem exhibits not only regional differences in the composition of its microbiota but also regional differences in the differentiation programs of its epithelial cells and in the spatial distribution of its component immune cells. A key experimental strategy for determining whether "nonpathogenic" microorganisms actively create their own regional habitats in this ecosystem is to define cellular function in germ-free animals and then evaluate the effects of adding single or several microbial species. This review focuses on how gnotobiotics-the study of germ-free animals-has been and needs to be used to examine how the gastrointestinal ecosystem is created and maintained. Areas discussed include the generation of simplified ecosystems by using genetically manipulatable microbes and hosts to determine whether components of the microbiota actively regulate epithelial differentiation to create niches for themselves and for other organisms; the ways in which gnotobiology can help reveal collaborative interactions among the microbiota, epithelium, and mucosal immune system; and the ways in which gnotobiology is and will be useful for identifying host and microbial factors that define the continuum between nonpathogenic and pathogenic. A series of tests of microbial contributions to several pathologic states, using germ-free and ex-germ-free mice, are proposed.

FIG. 1

Epithelial renewal in the small intestine. (A) Whole-mount preparation of the small intestine from an adult chimeric mouse raised with a conventional microbiota. The mouse was produced by using materials from two inbred strains: embryonic stem cells from one strain (129/Sv) were introduced in blastocysts from another strain (C57BL/6; abbreviated B6). The small intestine of the resulting adult chimera is composed of patches of 129/Sv crypt-villus units and patches of B6 crypt-villus units. All B6 epithelial cells in this mouse contained a locus known as ROSA26, which directs the production of an E. coli β-galactosidase marker (63, 210). This marker allows B6 cells to be identified with a simple histochemical stain (5-bromo-4-chloro-3-indolyl-β-d-galactopyranoside [X-Gal]). Positively stained B6 cells appear blue. None of the 129/Sv epithelial cells contain the ROSA26 locus, and their lack of β-galactosidase makes them appear white. The whole-mount is seen from above. Villi appear as tongue-like projections. The base of each villus is surrounded by crypts, which cannot be seen. Each crypt is monoclonal: it contains epithelial cells that are either all B6 or all 129/Sv but not a mixture of both. (B) Villi located near the border of patches of monoclonal B6-ROSA26 crypts and patches of monoclonal 129/Sv crypts appear striped (arrows). These villi are composed of vertical coherent columns of wholly 129/Sv (white) epithelial cells emanating from monoclonal 129/Sv crypts and adjacent columns of B6 (blue) epithelial cells emanating from monoclonal B6 crypts. The borders of these cellular columns are very distinct, illustrating the highly organized migration of epithelial cells from the base to the tip of the villus. (C) Section from the whole-mount preparation. The section has been stained with X-Gal plus nuclear fast red. The villus on the right is supplied by a B6-ROSA26 crypt that contains an entirely β-galactosidase-positive population of blue cells and by a 129/Sv crypt that contains only β-galactosidase-negative cells. One of the crypts is boxed. (D) Estimates of small-intestine epithelial cell dynamics in the crypt-villus units of mice and humans. Data from reference .

FIG. 2

Epithelial cell renewal in the colon. (A) Whole-mount preparation of colon from the B6-ROSA26–129/Sv chimeric mouse in Fig. 1. The colon does not have villi. Cells emerge from colonic crypts and are incorporated into surface epithelial cuffs that surround the orifice of each crypt. These cuffs represent homologs of small-intestine villi. (B) Section from the colon that has been stained with X-Gal and nuclear fast red. Like small-intestine crypts, colonic crypts are monoclonal. Surface epithelial cuffs associated with adjacent 129/Sv and B6-ROSA26 crypts are indicated by arrows. (C) Estimated values for colonic crypt cell populations. Data from reference . These estimates are less accurate than the corresponding estimates for small-intestine crypts.