Sexual dimorphism in immune response genes as a function of puberty

Abstract

Background: Autoimmune diseases are more prevalent in females than in males, whereas males have higher mortality associated with infectious diseases. To increase our understanding of this sexual dimorphism in the immune system, we sought to identify and characterize inherent differences in immune response programs in the spleens of male and female mice before, during and after puberty.

Results: After the onset of puberty, female mice showed a higher expression of adaptive immune response genes, while males had a higher expression of innate immune genes. This result suggested a requirement for sex hormones. Using in vivo and in vitro assays in normal and mutant mouse strains, we found that reverse signaling through FasL was directly influenced by estrogen, with downstream consequences of increased CD8+ T cell-derived B cell help (via cytokines) and enhanced immunoglobulin production.

Conclusion: These results demonstrate that sexual dimorphism in innate and adaptive immune genes is dependent on puberty. This study also revealed that estrogen influences immunoglobulin levels in post-pubertal female mice via the Fas-FasL pathway.

Figure 1

Post-pubertal, sexually dimorphic gene expression patterns in spleen. Shown is a data visualization of gender-specific gene expression patterns in normal mouse spleen. Six C57BL/6 mouse spleens from each sex at the pre-pubertal (3–4 weeks), pubertal (6–9 weeks) and post-pubertal (24–28 weeks) stages (a total of 36 mice) were analyzed using high-density oligonucleotide arrays. A hierarchical clustering algorithm was applied to group genes based on the similarities in gene expression patterns. The X-axis represents time points (weeks) in female (left) and male (right) mice. The Y-axis represents relative intensity ratios. A: Gene cluster up-regulated in post-pubertal female mice, B: Gene cluster up-regulated in post-pubertal male mice. These clusters were used for subsequent identification of sexually dimorphic immune function pathways.

Figure 2

Serum immunoglobulin levels in pre-pubertal, pubertal and post-pubertal female and male C57BL/6 mice. Serum samples from 6–8 C57BL/6 mice at 3–4 weeks, 6–9 weeks and 24–28 weeks were collected and assayed by ELISA. The samples were tested at dilutions of 1:5,000; 1:10,000; 1:50,000 and 1:100,000. The line graph presents the mean absorbance at 405 nm ± SE for males (blue line) and females (pink line).

Figure 3

Acute phase proteins in post-pubertal mice. A: Serum amyloid A and haptoglobin mRNA expression patterns in pre-pubertal, pubertal and post-pubertal mice. The X-axis represents time points (weeks) in female and male mice, and the Y-axis represents relative intensity ratios. B: Serum amyloid A and haptoglobin protein levels in post-pubertal C57BL/6 mice. Serum amyloid A levels in 24- to 28-week-old C57BL/6 mice. Serum samples (1:200) along with known standards were assayed in 96-well ELISA plates. The concentrations of the test samples were determined from the standard curve by multiplying the interpolated values by the dilution factor (n = 10/sex: male vs. female [μg/ml mean ± SE]: 77.48 ± 25.26 vs. 0.38 ± 0.12; p = 0.009). The working assay range was 11.8-190 μg/ml. Normal serum levels of SAA are generally less than 20 μg/ml. Serum haptoglobin levels were assayed using a colorimetric assay. Normal murine haptoglobin levels range from 0–0.1 mg/ml, and these increase in the acute phase to 0.3–2.0 mg/ml. The assay has a sensitivity of 0.05 mg/ml haptoglobin. (n = 10/sex: male vs. female [mg/ml mean ± SE]: 0.578 ± 0.059 vs. 0.254 ± 0.013; p = 0.00004).

Figure 4

Immunoglobulin isotypes, Fas, and FasL genes show similar expression patterns. Nucleating hierarchical clustering with IgG1 and IgG2b revealed Fas and FasL as co-regulated transcripts in post-pubertal female mouse spleens. These data drove the hypothesis that a non-apoptotic role of Fas/FasL might be responsible for downstream differential IgG isotype expression. The X-axis represents time points (weeks) in female and male mice, and the Y-axis represents relative intensity ratios.

Figure 5

Differences in serum immunoglobulin IgG2a and IgG2b isotypes are abolished in post-pubertal B6 lpr (Fas-deficient) and B6 gld (FasL-deficient) mice. Serum samples from 16- to 20-week-old male and female (5–6 mice/sex) B6, B6 lpr and B6 gld mice were assayed by ELISA. The samples were diluted at 1:5,000 (IgG2a); 1:10,000 (IgG3); 1:50,000 (IgG1 and IGg2b). Bar graphs represent mean absorbance at 405 nm ± SE. Significant differences for IgG2a (p = 0.0009) and IgG2b (p = 0.00005) between post-pubertal male (blue bar) and female (checker red bar) mice are shown with asterisks.

Figure 6

Effect of activated CD8⁺ T cell supernatants on Ig expression in vitro antibody synthesis. A representative example of 3 separate experiments is shown. T cells (5 × 10⁵) were stimulated for 18 h with various stimulants either individually or in combination (CD3/CD28, anti-CD3 and anti-CD28; E, estrogen, (10-8 M and 10-9 M), Fas-Fc (μg/ml: 0.75 and 1.25). The culture supernatants from activated CD8⁺ T cells were collected and added (200 μl) to 1.5 × 10⁶ splenocytes (800 μl) and incubated for 10 days. The culture supernatants were assayed for various immunoglobulin isotypes using ELISA. Graphs represent mean absorbance of triplicates at 405 nm ± SD.

Figure 7

Differences in serum IgG2a but not IgG2b levels are abolished in B6 IFN-gamma knockout mice (B6 GKO). Serum samples from 16- to 20-week-old male and female (4–6 mice/sex) B6 and B6 GKO mice were assayed by ELISA. Graphs represent mean absorbance at 405 nm ± SE. Significant differences for B6 IgG2a (p = 0.005), B6 IgG2b (p = 0.003) and B6 GKO IgG2b (0.001) between post-pubertal male (blue bar) and female (checker red bar) mice are shown with asterisks.