Trans-epithelial immune cell transfer during suckling modulates delayed-type hypersensitivity in recipients as a function of gender

Abstract

Introduction: Breast feeding has long term effects on the developing immune system which outlive passive immunization of the neonate. We have investigated the transfer of milk immune cells and examined the result of transfer once the recipients were adult.

Methods: Non-transgenic mouse pups were foster-nursed by green fluorescent protein (GFP) transgenic dams for 3 weeks and the fate of GFP+ cells was followed by FACS analysis, immunohistochemistry and RT-PCR for GFP and appropriate immune cell markers. Pups suckled by non-transgenic dams served as controls.

Results: Despite a preponderance of B cells and macrophages in the stomach contents of the pups, most cells undergoing trans-epithelial migration derived from the 3-4% of milk cells positive for T lymphocyte markers. These cells homed to the spleen and thymus, with maximal accumulation at 3-4 weeks. By sensitizing dams with an antigen which elicits a T cell-mediated delayed-type-hypersensitivity (DTH) response, we determined that nursing by a sensitized dam (compared to a non-sensitized dam) amplified a subsequent DTH response in females and yet suppressed one in males.

Discussion: These results suggest that clinical evaluation weighing the pros and cons of nursing male versus female children by mothers with genetically-linked hypersensitivity diseases, such as celiac disease and eczema, or those in regions of the world with endemic DTH-eliciting diseases, such as tuberculosis, may be warranted.

Figure 1. Viability and type of immune cells present in whey clot at week 2.

A) Propidium Iodide staining and analysis by flow cytometry of cells isolated from the whey clot from the stomach of a pup at 2 weeks. B) percentage of milk CD4+, CD8+, γδ TCR+ (GD), Foxp3+, B220+, CD3+, and F4/80+ cells found within the clot, as determined by flow cytometric analysis.

Figure 2. GFP+ cells cross the epithelium of the small intestine at 1 and 2 weeks of nursing.

A, RT-PCR for GFP at 1, 2, 3, and 4 week time points of flushed small intestine of pups foster-nursed by GFPtg dams. The negative control used was small intestine from a non-GFP nursed B6 mouse and the positive control used was small intestine from a GFPtg mouse. B–E, sections of small intestine at weeks 1–4, respectively. Staining of GFP+ cells was enhanced with an anti-GFP antibody.

Figure 3. GFP+ cells crossing the epithelium of the small intestine are not macrophages, B cells, mammary epithelial cells or stem cells.

Small intestine of a B6 pup nursed by a GFPtg dam stained with anti-GFP antibody and tomato lectin (A), anti-F4/80 antibody (C), anti-B220 antibody (E). Small intestine of a non-GFPtg nursed B6 control mouse stained with anti-GFP antibody and tomato lectin (B), anti-F4/80 antibody (D), or anti-B220 antibody (F). For panels A and B, anti-GFP staining is pseudo-colored green and tomato lectin is pseudo-colored red. For F4/80 and B220 staining, anti-GFP staining is in green and F4/80 and B220 is in red. Tomato lectin staining is at 200× magnification and F4/80 and B220 staining is at 1000× magnification. G, RT-PCR for β-casein at weekly time points of small intestine of pups foster-nursed by GFPtg dams. Lactating mammary gland was used as the positive control. H, similar RT-PCR for Oct-4 at weekly time points. Testis was used as the positive control.

Figure 4. Cells crossing the epithelium of the small intestine are CD4+ and CD8+ T cells.

A, small intestine of B6 pup nursed by a GFPtg dam and B, non-GFPtg nursed B6 control stained with anti-GFP antibody (green) and anti-CD4 antibody (red); C, small intestine of B6 pup nursed by a GFPtg dam and D, non-GFPtg nursed B6 control stained with anti-GFP antibody (green) and anti-CD8 antibody (red); E, Small intestine of B6 pup nursed by a GFPtg dam and F, non-GFPtg nursed B6 control stained with anti-GFP antibody (green) and anti-γδ TCR antibody (red); G, Quantification of cell types traversing the small intestine at weeks 1 and 2 (combined), as determined by immunofluorescent staining for GFP and either CD4 or CD8.

Figure 5. CD4+ cells migrate to the spleen and CD8+ cells migrate to the thymus.

RT-PCR for GFP at weekly time points and FACS analysis of cells positive for GFP and an additional marker (CD4 or CD8 or γδ TCR (GD) or Foxp3). A, spleen; B, thymus from pups nursed by GFPtg dams. The negative controls used were spleens and thymi from non-GFPtg-nursed B6 mice, and the positive controls used were spleens and thymi from GFPtg mice. *, p<0.05 compared to non-GFPtg nursed controls.

Figure 6. Effect of Maternal Sensitization on DTH response to Candida in the pups.

B6 pups were foster-nursed by GFPtg dams that were either sensitized or not to Candida. Foster-nursed pups were sensitized with an intradermal injection of fixed Candida albicans into both flanks and then challenged 7 days later with Candida albicans protein antigen in the footpad. Footpad swelling was measured 24 hours later. Statistical analysis by both Mann Whitney (first number) and t test (second number) are shown. M, male; F, female; NS, non-sensitized; S, sensitized.