Rearing environment affects development of the immune system in neonates

Abstract

Early-life exposure to appropriate microbial flora drives expansion and development of an efficient immune system. Aberrant development results in increased likelihood of allergic disease or increased susceptibility to infection. Thus, factors affecting microbial colonization may also affect the direction of immune responses in later life. There is a need for a manipulable animal model of environmental influences on the development of microbiota and the immune system during early life. We assessed the effects of rearing under low- (farm, sow) and high-hygiene (isolator, milk formula) conditions on intestinal microbiota and immune development in neonatal piglets, because they can be removed from the mother in the first 24 h for rearing under controlled conditions and, due to placental structure, neither antibody nor antigen is transferred in utero. Microbiota in both groups was similar between 2 and 5 days. However, by 12-28 days, piglets reared on the mother had more diverse flora than siblings reared in isolators. Dendritic cells accumulated in the intestinal mucosa in both groups, but more rapidly in isolator piglets. Importantly, the minority of 2-5-day-old farm piglets whose microbiota resembled that of an older (12-28-day-old) pig also accumulated dendritic cells earlier than the other farm-reared piglets. Consistent with dendritic cell control of T cell function, the effects on T cells occurred at later time-points, and mucosal T cells from high-hygiene, isolator pigs made less interleukin (IL)-4 while systemic T cells made more IL-2. Neonatal piglets may be a valuable model for studies of the effects of interaction between microbiota and immune development on allergy.

Fig. 1

A dendrogram of the mean values of the Dice coefficients for pairwise comparison of the denaturing gradient gel electrophoresis (DGGE) profiles of the ileal flora in piglets of different ages reared under low- and high-hygiene conditions. Outlying piglets (i.e. those that are either of a different age or reared under different conditions to the majority of animals in the indicated group) are listed in red. Dashed lines indicate the borders between the five separate (numbered) clusters.

Fig. 2

The variability with age of the microbial flora in the ileum of low- and high-hygiene piglets. Black bars: low-hygiene (farm-reared) piglets; open bars: high-hygiene (isolator-reared) piglets. Error bars represent standard error of the mean.

Fig. 3

Changes in professional and non-professional antigen-presenting cells (APCs) with age in piglets reared under low- and high-hygiene conditions. (a) Images show major histocompatibility complex class II (MHCII) (red) expression by CD16⁺ (green) and endothelial (MIL11⁺; blue) cells in the small intestinal mucosa of low- and high-hygiene pigs at all time-points. Scale bar 10 µm. (b) Changes in CD16⁺MHCII⁺[dendritic cell (DC)] staining with age. (c) Changes in CD16⁺MHCII⁺ staining with age for individual farm-reared piglets. Each point represents a piglet; for comparison, horizontal bars represent the means for each time-point. The outlying piglets from the microbial flora analysis (Fig. 1) are indicated in red. (d) Changes in MIL11⁺MHCII⁺ (capillary endothelial cell) staining with age. Solid line: low-hygiene (farm-reared) piglets; dashed line: high-hygiene (isolator-reared) piglets. *P < 0·05; **P < 0·01. Error bars represent standard error of the mean.

Fig. 4

Cytokine production by whole-cell preparations from the small intestine of piglets reared under low- and high-hygiene conditions. (a) Interleukin (IL)-4 production and (b) IL-2 production in the supernatants from whole-cell preparations from the small intestine 41 h after concanavalin A (ConA) stimulation. Solid line: low-hygiene (farm-reared) piglets; dashed line: high-hygiene (isolator-reared) piglets. **P < 0·01 comparing the effect of rearing environment at each time-point.

Fig. 5

Cytokine production by whole-cell preparations from the small intestine of piglets reared under low- and high-hygiene conditions. (a) Interleukin (IL)-2 production and (b) IL-4 production in the supernatants from whole-cell preparations from the mesenteric lymph node and spleen 41 h after concanavalin A (ConA) stimulation. Solid line: low-hygiene (farm-reared) piglets; dashed line: high-hygiene (isolator-reared) piglets. *P < 0·05 comparing the effect of rearing environment at each time-point.