Neonatal Colonisation Expands a Specific Intestinal Antigen-Presenting Cell Subset Prior to CD4 T-Cell Expansion, without Altering T-Cell Repertoire

Interactions between the early-life colonising intestinal microbiota and the developing immune system are critical in determining the nature of immune responses in later life. Studies in neonatal animals in which this interaction can be examined are central to understanding the mechanisms by which the microbiota impacts on immune development and to developing therapies based on manipulation of the microbiome. The inbred piglet model represents a system that is comparable to human neonates and allows for control of the impact of maternal factors. Here we show that colonisation with a defined microbiota produces expansion of mucosal plasma cells and of T-lymphocytes without altering the repertoire of alpha beta T-cells in the intestine. Importantly, this is preceded by microbially-induced expansion of a signal regulatory protein α-positive (SIRPα+) antigen-presenting cell subset, whilst SIRPα−CD11R1+ antigen-presenting cells (APCs) are unaffected by colonisation. The central role of intestinal APCs in the induction and maintenance of mucosal immunity implicates SIRPα+ antigen-presenting cells as orchestrators of early-life mucosal immune development

Repertoire is not altered on expansion of mucosal CD4 T-cells.

<p>(A) Representative spectratype electropherograms of <i>TRβV</i>29 for a germ-free pig, and a colonised pig from the same litter of piglets. In general, samples from the spleen demonstrate a more Gaussian electropherogram distribution than intestinal samples. RFU: relative fluorescence units, x axis: CDR3 length (base pair size). (B) Hierarchical cluster analysis of CDR3 lengths from 20 <i>TRβV</i> groups and 1 subgroup from the proximal jejunum, distal jejunum and spleen from pigs at 21 days of age.</p

Numbers and ages of GF and colonised pigs in each experiment.

<p>Four experiments were performed with between five and seven pigs in each. Pigs were euthanised and samples taken either at 0 (1 experiment), 5 (1 experiment) or 21 (2 experiments) days of age.</p

T-cell repertoire is different between tissues but not between GF and colonised pigs.

<p>Principle component analysis of CDR3 lengths for all V beta families was performed for a maximum of 25 iterations for convergence using an un-rotated factor solution. <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0033707#pone-0033707-t002" target="_blank">Table 2</a> shows the p-values from a fully factorial general linear model of the first five principal components with colonisation status (GF or colonised) and tissue as fixed factors. A p-value of <0.01 was considered significant using a Bonferroni correction for 5 tests.</p

Colonisation expands mucosal CD4⁺ T cells by 21 days of age.

<p>(A) CD4 staining in jejunal mucosa from a GF pig and (B) a colonised pig at 21 days of age. Sections were stained with antibodies to CD4 (green) and capillary endothelium (MIL11; in blue). In both (A) and (B), (i) shows the two-colour image; (ii) shows the original greyscale image for the blue channel (endothelium); (iii) shows the original greyscale image for the green channel (CD4). Scale bar represents 10 µm. (C) Area of CD4+ T cell staining in jejunal sections from piglets at birth (open diamonds) and GF piglets (open squares) and colonised piglets (black squares) at 5 and 21 days of age (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0033707#pone-0033707-t001" target="_blank">Table 1</a> for numbers of piglets in each experiment). *p<0.05.</p

Colonisation expands IgM and IgA-producing mucosal plasma cells.

<p>(A) IgA staining in jejunal mucosa from a GF pig and (B) a colonised pig at 21 days of age. Sections were stained with antibodies to IgA (red) and capillary endothelium (MIL11; in blue). In both (A) and (B), (i) shows the two-colour image; (ii) shows the original greyscale image for the red channel (IgA); (iii) shows the original greyscale image for the blue channel (endothelium). Scale bar represents 10 µm. (C) IgM staining in jejunal mucosa from a GF pig and (D) a colonised pig at 21 days of age. Sections were stained with antibodies to IgM (red). Scale bar represents 10 µm. (E) Area of IgM and (F) IgA staining in jejunal sections from piglets at birth (open diamonds) and GF piglets (open squares) and colonised piglets (black squares) at 5 and 21days of age (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0033707#pone-0033707-t001" target="_blank">Table 1</a> for numbers of piglets in each experiment). * p<0.01.</p

Colonisation selectively increases SIRPα⁺ APC subsets within the first five days of life.

<p>Intestinal APC (MHCII⁺) subsets were defined by their expression of SIRPα, CD11R1 and CD16. (A–D) show SIRPα⁺ subsets (E–G) show SIRPα⁻ subsets from piglets at birth (n = 5; light grey bars) and GF piglets (white bars) and colonised piglets (dark grey bars) at 5 (n = 3 GF and n = 3 colonised) and 21 (n = 4 GF and n = 6 colonised) days of age (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0033707#pone-0033707-t001" target="_blank">Table 1</a> for numbers of piglets in each experiment). Error bars represent standard errors of the mean. After a Bonferroni correction was applied for multiple tests (ANOVA), a p-value of <0.003 was considered to be significant.*p<0.003. The MHCII⁺ SIRPα⁻ CD11R1⁻CD16⁻ subset is not shown as this represents endothelial MHCII, which was unaffected by colonisation or age.</p

Colonisation affects mucosal MHCII⁺ APC subsets.

<p>(A) Area of MHCII⁺ cell staining in jejunal sections from piglets at birth (open diamonds) and GF piglets (open squares) and colonised piglets (black squares) at 5 and 21 days of age (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0033707#pone-0033707-t001" target="_blank">Table 1</a> for numbers of piglets in each experiment). (B) SIRPα staining in jejunal mucosa from a GF pig and (C) a colonised pig at 21 days of age. (B) (i) and (C) (i) show staining with antibodies to CD16 (red), SIRPα (green) and CD11R1 (blue). (B) (ii) and (C) (ii) show the same sections with the same cells in red and blue but with MHCII DR in green. In both (B) and (C), (iii) shows the red channel (SIRPα); (iv) shows the green channel (CD16), (v) shows the blue channel (CD11R1); (vi) shows the infra-red channel (MHC class II). Scale bar represents 10 µm. (D) Partitioning of MHCII between SIRPα⁺ (green shading) and SIRPα⁻ (purple shading) APC subsets in the intestines of pigs at birth and from both treatment groups at 5 days and 21 days of age (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0033707#pone-0033707-t001" target="_blank">Table 1</a> for numbers of piglets in each experiment).</p