Sex Differences in Mouse Popliteal Lymph Nodes

Abstract

Females have more robust immune responses than males, well-illustrated by the degree of inflammation elicited during delayed-type hypersensitivity (DTH) responses. Here, we have investigated underlying sex differences that may contribute to differential footpad DTH responses using wildtype and four core genotypes (FCG) mice and popliteal lymphnode cellularity and gene expression. DTH responses in XX and XY FCG females showed no role for almost all genes expressed on sex chromosomes. After then filtering-out genes differentially expressed between XX and XY females, only one gene was sexually differentially expressed in wildtype mice, glycosylation-dependent cell adhesion molecule 1 (Glycam1), expressed 7-fold higher in females. Glycam1 facilitates leukocyte entry through high endothelial venules. Consistent with greater Glycam1 expression, female nodes contained twice as many cells. While females had more memory T cells in their nodes, males had a higher percentage of T regulatory cells. This sexual dimorphism in wildtype animals manifested pre-pubertally, was enhanced post-pubertally, and was eliminated by castration. The formation of male gonads is determined by the expression of Sry. Sry overexpression, which does not affect testosterone levels, produced an exaggerated male phenotype. We conclude that Sry expression through formation of the male gonad indirectly negatively impacts the potential for local inflammation.

Figure 1

Delayed-type hypersensitivity response to Candida albicans. Data show the change in footpad swelling in mm over 24 hours for mice sensitized with Candida albicans (ovariectomized FCG-XXF n = 9, and ovariectomized FCG-XYF n = 10 (a)), (FCG-XYM n = 20, FCG-XXM n = 26, FCG-XXF n = 18, FCG-XYF n = 24 (b))or DPBS (ovariectomized FCG-XXF n = 11, and ovariectomized FCG-XYF n = 9 (a)), (FCG-XYM n = 10, FCG-XXM n = 22, FCG-XXF n = 6, FCG-XYF n = 16 (b)) and challenged with Candida albicans purified protein. Panel c is all FCG data normalized to FCG-XXF = 1, and wildtype B6 male normalized to wildtype B6 female = 1. Mann-Whitney statistical analysis was performed and significance was designated as follows: *P < 0.05, **P < 0.01, ***P < 0.001.

Figure 2

Total number of cells in the popliteal lymph nodes of wildtype and Sry Hi mice. Results shown are the average total number of cells counted per two popliteal lymph nodes of wildtype males and females at (a) 5 months of age (Male n = 4 Female n = 4), (b) 3 weeks of age (Male n = 4 Female n = 4), and (c) 6 weeks of age (Male n = 4 Female n = 4). Panel d compares wildtype (n = 5) and Sry Hi (n = 3) males at 3 weeks of age. Mann-Whitney statistical analysis was performed and significance was designated as follows: *P < 0.05, **P < 0.03, ***P < 0.001.

Figure 3

T cells in the popliteal lymph nodes of 3 week old, 6 week old, and 5 month old wildtype mice. Shown are the average total numbers of T cells, CD4+ T cells, CD8+ T cells, and % of CD4+ T cells that were T regulatory (CD25+/FOXP3+) per two popliteal lymph nodes of 3- week old (Male n = 4 Female n = 4) (a–d), 6-week old (Male n = 4 Female n = 4) (e–h), and 5-month old (Male n = 4 Female n = 4) (i–l) males and females. Mann-Whitney statistical analysis was performed and significance was designated as follows: *P < 0.05, **P < 0.03, ***P < 0.01.

Figure 4

T cells in the popliteal lymph nodes of wildtype males and Sry overexpressing males at 3 weeks of age. Shown are the average total numbers of (a) T cells, (c) CD4+ T cells (c) CD8+ T cells, and (d) % of CD4+ T cells that were T regulatory (CD25+/FOXP3+) per two popliteal lymph nodes of wild type males (WT n = 5) and Sry overexpressing males (Sry Hi n = 3). Mann-Whitney statistical analysis was performed and significance was designated as follows: *P < 0.05, **P < 0.03, ***P < 0.01.

Figure 5

T cell numbers in the popliteal lymph node of 6-week old wildtype mice with either gonadectomy or sham surgery. Shown are the average total numbers of (a,c) CD4+ T-cells, (b,d) CD8+ T-cells per two popliteal lymph nodes of 6-week old females with sham surgery (F SHAM n = 5), females with ovariectomy (F OVX n = 5), males with sham surgery (M SHAM n = 5), or males with castration (M CAST n = 7) at 3 weeks of age. Mann-Whitney statistical analysis was performed and significance was designated as follows: *P < 0.05, **P < 0.03, ***P < 0.01.

Figure 6

CD62L+ T cells in the popliteal lymph nodes of 3- and 6-week old wildtype mice. Shown are the average total numbers of CD4+/CD62L+ T cells and CD8+/CD62L+ T-cells, per two popliteal lymph nodes of 3-week old (Male n = 4 Female n = 4) (a,b) and 6-week old (Male n = 4 Female n = 4) (c,d) males and females, and CD62L Mean Fluorescence Intensity (MFI) on the same cell types in the popliteal lymph nodes of 3-week old (e,f) and 6 week old (g,h) males and females. Mann-Whitney statistical analysis was performed and significance was designated as follows: *P < 0.05, **P < 0.03, ***P < 0.01.

Figure 7

Central memory T cells in the popliteal lymphnode of wildtype mice. Shown are average numbers of CD4+ (a,c) and CD8+ (b,d) memory T cells per two lymph nodes of 5-month old male and female animals (Male n = 3 in A and B, and n = 5 in c and d, Female n = 3 in a and b, and n = 5 in c and d). Mann-Whitney statistical analysis was performed and significance was designated as follows: *P < 0.05.

Figure 8

T cell numbers in the inguinal lymphnodes and spleen of 5-month old wildtype males and females. Shown are the average total numbers of T cells (a,e), CD4+ T cells (b,f), CD8+ T cells (c,g), and % of CD4+ T cells that were T regulatory (CD25+/FOXP3+) (d,h) per two inguinal lymph nodes (Male n = 4 Female n = 4) (a–d) and spleen (Male n = 4 Female n = 4) (e–h) of 5-month old males and females. Mann-Whitney statistical analysis was performed and significance was designated as follows: *P < 0.05, **P < 0.03, ***P < 0.01.

Figure 9

Representative gating strategies used to analyze cell types by flow cytometry. Shown are representative dot plots with gating for CD4+/CD62L+ and CD8+/CD62L+ T-cells (a), T-regulatory Cells (CD4+/CD25+/FOXP3+) (b), CD4+ Memory T-cells (CD4+/CD44+/CD62L+) and CD8+ Memory T-cells (CD8+/CD44+/CD62L+) (c), and CCR7+/CD4+ Memory T-cells and CCR7+/CD8+ Memory T-cells (d).