Intestinal Bacteria and the Infant’s Developing Immune System

Facultative bacteria (those which survive in atmospheric oxygen), such as E. coli and Enterococci, are the first to establish. Without competition from anaerobic bacteria, they may expand rather uninhibited and may reach much higher population numbers at this stage in life than later on. Anaerobic bacteria establish successively, starting with, e.g., Bifidobacteria, Clostridia and Bacteroides.

When more and more anaerobic species establish, the milieu becomes more and more hostile to the facultatives. The facultatives start to decline in numbers and the microflora becomes successively more dominated by anaerobic bacteria. In an adult individual, anaerobes outnumber facultatives by 1000 to 1.

Figure 1. Sources of intestinal microbiota in the newborn infant




The colonisation pattern in infancy is dependent on environmental exposure. Bacteria colonising the infant may derive from the maternal vaginal and perineal flora (this source is not available in infants delivered by caesarean section).

Caesarean section delivered infants therefore show delayed acquisition of some typical faecal bacteria such as E. coli and Bacteroides. Instead, other bacteria may expand in the microflora due to absence of competition. Typical examples include Clostridia (which are spore-formers and are therefore ubiquitous in the environment), Klebsiella and Enterobacter (which may be acquired from food).

Reduced exposure to faeces, such as in caesarean section delivery, or in highly hygienic environments may, thus lead to reduced appearance of certain bacteria (“hygiene-sensitive” bacteria), and increased appearance of other bacteria (“hygiene-favoured” bacteria). Still other bacteria are neutral to hygiene. This group is represented by enterococci, which are resistant to most normal hygienic measures and are equally often found in infants independent of delivery mode. We have recently demonstrated that the colonisation pattern of children without siblings resembles that of infants born by caesarean section, although less pronounced (unpublished). This shows that life-style factors may, indeed, influence intestinal colonization.

Previously we have shown that infants in Sweden acquire E. coli later than infants born in Pakistan and they also have a slower turn-over of individual strains in the microflora during a certain period in time^2,3. In the last 30 years, E. coli and Bacteroides may have become even more uncommon in the early infantile microflora than they were previously⁴. Instead, it appears as if typical skin bacteria such as Staphylococci have expanded in the microflora of Western infants, most likely due to decreased competition from more “professional” intestinal bacteria^4,5,6.

Development of immune function
The intestinal microflora is a major stimulus to the developing immune system. Gram-positive and Gram-negative bacteria give rise to different patterns of mediator release when they interact with human monocytes^7,8,9. The ratio of Gram-negative to Gram-positive bacteria in the gut flora
may therefore, theoretically, influence antigen-presenting cells in the gut mucosa and could thereby influence how innocuous antigens are handled by the gut.

Another factor of importance in determining whether immunity or tolerance to an antigen will develop is the processing by intestinal epithelial cells. Ingestion of a dietary protein leads to specific immune tolerance in animals and people. It is known that development of this type of tolerance – oral tolerance – depends on the passage of the antigen across an intact gut epithelium. This passage alters the antigen and converts it into a tolerogen. Thus, serum collected from an animal fed a protein antigen shortly after feeding will contain a tolerogenic form of the antigen which can be transferred into a naïve recipient and induce tolerance in the latter. It has recently been shown that this process involves loading of antigenic peptides onto major histocompatibility complex class II molecules within the intestinal epithelial cell and export of the complex on membraneous vesicles, so called “tolerosomes”¹⁰.

The composition of the microflora may influence the capacity of the small intestinal epithelium to process dietary antigens into tolerogens – it appears that a complex flora with ample anaerobes is beneficial for tolerogenic processing, whereas colonisation by a single bacterial species is not sufficient¹¹.

Tolerance is mediated by regulatory T cells (Treg). A number of different Treg subtypes exist, but the so called “natural” Tregs appear to play a central role. These cells are CD4+ and are characterised by dense expression of surface CD25, by transcription of the FOXP3 gene and by containing intracellular CTLA-4. Absence of this cell subset in humans and mice lead to the IPEX syndrome characterised by elevated serum IgE, organ-specific autoimmunity, eczema and colonic inflammation¹².

Since not only allergies, but also certain autoimmune diseases (notably type 1-diabetes) and inflammatory bowel disease are currently increasing in Western populations, one may speculate that CD25+ Tregs may be functionally immature in a growing number of Western people due to reduced exposure to some microbes at a critical stage in infancy. It can be noted that CD25+ Tregs from germ-free mice are functionally deficient, compared to the same cell subset from animals reared conventionally¹³.

Role of superantigens
We have found that infants delivered by caesarean section have an increased risk of developing food allergy in the first year of life (unpublished). Conversely, early colonisation by S. aureus strains which produce certain toxins leads to expansion of circulating regulatory T cells and protection from food allergy (Karlsson et al., manuscript in preparation). These toxins, namely S. aureus enterotoxin A, B, C or D, or TSST-1 (toxic shock syndrome toxin-1) share the common property that they bind to the T cell receptor of a large proportion of the T cells, regardless of their antigen specificity.

These toxins therefore induce a very strong T cell activation, hence their name “superantigens”. We speculate that strong stimulation of T cells within or beneath the epithelium by superantigen in infants colonised by toxin-producing strains might render the epithelial cells more capable of tolerogenic processing. Indeed, preliminary data from animal experiments indicate that pretreatment of newborn mice with bacterial superantigen enhances their capacity to develop oral tolerance to a dietary antigen given at a later time-point (Hultcrantz et al., manuscript in preparation).

To speculate, the newborn infant is equipped for facing a world rich in microbes. These induce a forceful activation of the infantile immune system, leading to maturation of tolerogenic processes. In today’s overly hygienic societies, infants are largely deprived of microbial immune stimulating signals due to a severe reduction of the amounts and variety of environmental microbes.

This may hamper the maturation of tolerogenic processes that should be carried out by the small intestinal epithelium. Strong T cell stimulants, such as bacterial superantigen may potentially be developed into an immunostimulant that could induce tolerogenic processes and potentially protect from allergy development.

References

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