Background
The incidence and severity of human diseases vary between the sexes: For example, autoimmune diseases are generally more common in females than in males and are most marked in women of childbearing age [
1‐
3]. Thus, it appears that susceptibility to autoimmunity is expressed at the time of puberty. Puberty is a period of intense molecular, physiological and anatomical reorganization in the body, and the hormonal changes occurring at the time of puberty lay the framework for biological differences that persist throughout life and may contribute to the variable onset and progression of disease in males and females [
4]. Sex-related differences in disease susceptibility have also been observed in several mouse models of infectious and autoimmune diseases and may be related to differences in the expression patterns of immune response genes [
5,
6].
Immune responses are sexually dimorphic, both in type and magnitude. Two general systems of immunity to infectious agents have been selected during evolution: innate (natural) immunity, and acquired (adaptive or specific) immunity. The innate immune system uses proteins encoded in the germline (on macrophages, mast cells, natural killer cells) to recognize conserved products of infectious non-self (i.e., microbial pathogens), but not non-infectious self (i.e., host proteins) [
7,
8]. In contrast to this relatively inflexible system is the almost infinitely adaptable immune system of lymphocytes [
9]. These two systems are known to interact closely with each other: For example, cellular and soluble components of innate immunity help the adaptive immune response to select and respond to appropriate antigens. Even though these two systems are very well studied, there is a paucity of literature on gender differences as a function of age. Understanding the basis of sex differences in immune response genes is important for developing new approaches to prevention, diagnosis and treatment of infectious and autoimmune diseases.
We studied sexual dimorphism in immune response genes in C57Bl/6 (B6) mice because B6 mice do not spontaneously develop autoimmune diseases. However, when autoimmune-susceptible loci are transferred onto a B6 background, the mice readily manifest a disease phenotype, including profound sex differences in disease severity [
10,
11]. We have now investigated the sex differences in immune response genes in the spleens of pre-pubertal, pubertal and post-pubertal male and female B6 mice using global gene expression profiling. Our data indicate that there is a clear sexual dimorphism after puberty in innate and adaptive immune genes. We have also identified one such pathway, reverse signaling through FasL, as a possible source of the sexual dimorphism in immunoglobulin (Ig) levels that is seen between males and females, since this pathway is affected by estrogen levels.
Discussion
We have shown that male and female mice differ significantly with respect to their immune response genes in post-pubertal life. The innate immune response genes are highly up-regulated in post-pubertal male but not female mice. Post-pubertal male mice also produce higher levels of IL-1α and IL-1β in response to the TLR-2 ligand (Table
3). The biological relevance of these findings can be seen in both infectious and autoimmune disease conditions. Although males are more susceptible than females to many parasitic infections, there are some parasites for which males are more resistant than females and differences in innate and adaptive arms of the immune system may explain this sex reversal. For example, the innate immune response plays a critical role in offering males protection against Toxoplasma
gondii infection [
22,
23]. Our data are consistent with the relative deficiency of innate immune response genes in female mice, as evidenced by their enhanced susceptibility to and higher mortality associated with certain parasitic infections (e.g., T.
gondii). Thus, the relative resistance of the males to T.
gondii infection is likely explained by their high levels of innate immunity-related proteins. Furthermore, it is also known that the 5-lipoxygenase pathway and leukotrienes are integral components of innate immune cells such as macrophages, mast cells and eosinophils [
24]. Recent experiments have clearly demonstrated that 5-lipoxygenase-deficient male mice on an MRL
lpr/
lpr background show a marked decrease in survival, further supporting a protective role for innate immune response genes in autoimmune diseases [
25].
In contrast, adaptive immune response genes are highly up-regulated in post-pubertal female mice. These mice also produce significantly higher levels of cytokines and chemokine that influence antibody production than do post-pubertal males (Table
3). These findings are particularly relevant to autoimmune diseases, in which the adaptive immune system attacks normal self tissue. We propose that enhanced susceptibility to autoimmune disease in post-pubertal life is the result of an altered ratio of adaptive and innate immune response genes. This hypothesis is in fact supported by the finding that genetic defects in innate immune response genes (complement C1q and serum amyloid P) in mice result in spontaneous autoimmune disease [
26‐
28]. It is known that females produce higher levels of Igs than do male mice in response to a variety of antigens, and these effects have been attributed to sex steroids [
29‐
31]. Our results confirm these findings and further indicate that even non-immunized female mice show significantly elevated levels of various Ig isotype genes, and that the levels are even more enhanced in post-pubertal life.
Fas and FasL genes showed spatial and temporal expression patterns similar to those of immunoglobulin genes. The preferential expression of Fas and FasL in post-pubertal females suggested a role for this pathway in generating sexual dimorphism in immunoglobulin gene expression. The observed post-pubertal sex differences in Ig levels in B6 mice were abolished in B6 lpr and B6 gld mice, indicating that the post-pubertal levels of specific Ig isotypes are regulated through Fas/FasL pathway.
Genetic defects in both Fas and FasL are known to cause severe lupus like autoimmune disease on the MRL/Mp genetic background. The gender differences in disease severity (mortality, pancreatitis and autoantibodies) in MRL/Mp mice are abolished when Fas (
lpr) mutation is transferred onto this background, suggesting that MRL
lpr mice are gender-neutral [
32,
33]. It is important to note that in a previous study, transferring the C1q deficiency onto the MRL background did not abolish the gender differences [
34]. Thus, the defects in the Fas-FasL signaling pathway alone abolish the gender differences in lupus-like autoimmune disease in MRL mice. Further supporting this observation is the finding that
lpr mice show spontaneous polyclonal B cell activation and lymphadenopathy [
35]. The male
lpr mice showed significant increases in Ig levels, similar to those seen in females (Figure
5). These results are interesting, especially when correlated with the disease-prone MRL
lpr mouse model of lupus, in which male mice die as early as female mice (50% mortality in both male and female mice by 5.5 months of age). This finding suggests that increased IgG levels in males lead to increases in immune complex-mediated disease, similar to those in female mice.
This hypothesis is further supported by another model of autoimmunity: MRL-Fas
lprcg mice have a phenotype similar to that of MRL
lpr mice because of a defect in Fas-mediated apoptotic signaling (a single amino acid mutation in the cytoplasmic death domain) [
36]. The reverse signaling pathway through FasL is functional because of the intact extracellular domain that interacts with FasL. In fact, the MRL-Fas
lprcg mice exhibit sex differences in disease severity [
37]. These observations suggest that reverse signaling through FasL is involved in generating sex differences in IgG isotypes, and consequently in the frequency of severe disease in female mice.
It has been shown that FasL expression in ovaries is closely correlated with estrogen levels, which vary at different phases of the female estrus cycle. This result suggests that estrogen dynamically controls FasL expression on various cells and may enhance Ig levels only once during each cycle [
38]. To directly establish the role of estrogen in this reverse signaling pathway, we carried out
in vitro stimulation of CD8
+ T cells and assessed Ig isotype levels. We have shown here that FasL expression on activated CD8
+ T cells is influenced by estrogen and have further demonstrated that the culture supernatants from estrogen-activated CD8
+ T cells produce growth factors that enhance
in vitro immunoglobulin levels. These data suggest that reverse signaling through FasL in CD8
+ T cells leads to the production of growth factors that enhance the expression of Ig isotypes and that females are expected to have enhanced Ig switching because of their elevated post-pubertal estrogen levels. It is likely that some of the growth factors secreted by the activated CD8
+ T cells also influence B cell growth, maturation and differentiation.
In addition to their effects on CD8
+ T cells, estrogens affect the production of IFN-γ [
39,
40], which is known to enhance IgG2a responses [
41]. These activated CD8
+ T cells would be expected to secrete growth factors and cytokines, which in turn would affect B cell growth and differentiation, leading to the enhanced immunoglobulin isotype expression in post-pubertal female mice. We therefore assessed the effect of IFN-γ on IgG2a levels in B6 IFN-γ knockout mice. These data suggested that increases in post-pubertal Ig isotype levels may be due to differential expression of cytokines (e.g., IFN-γ) produced by CD8
+ T cells activated through Fas-FasL reverse signaling. Recently, it has been shown that IgG2a-chromatin immune complexes, together with TLR 9 are very efficient in activating autoreactive B cells [
42]. Our findings suggest that the increased IgG2a induced by the estrogen-Fas/FasL- IFN-γ pathway in post-pubertal female mice is one of the susceptibility factors enhancing autoimmunity in females. We speculate that differential expression of cytokines such as TGF-β may be involved in generating IgG2b differences in post-pubertal life.
Ig genes are transiently increased at the time of puberty in male mice (Figure
1A). The exact mechanism by which this increase occurs is not known. It is likely that the transiently elevated levels of estrogen at the time of puberty in males [
43,
44] may enhance FasL expression on CD8
+ T cells. Reverse signaling through FasL may also be responsible for this transient increase in Ig gene expression in male pubertal mice. The molecular basis for the large increase in innate immune response genes in males as compared to the adaptive immune response genes in females is not clear. It is possible that male hormones may regulate some of the innate immune response genes directly.
While the pathway analysis presented here has focused on the estrogen-Fas/FasL- IFN-γ pathway, our data also have implications with regard to male-related immunity. It has been observed that males have a higher mortality due to infectious diseases than do females [
45], in part because of testosterone-induced immunosuppression in post-pubertal males [
46]. The exact molecular mechanisms by which testosterone suppresses the acquired immune system are not yet understood. The data presented here suggest that males have an adequate innate immune response (first line of defense) but a relatively diminished adaptive immune response, which is critical for the elimination of the microorganisms. Thus, the documented higher mortality rates in males worldwide may be due in part to this relatively deficient adaptive immune response.
Methods
Mice
Pre-pubertal (3- to 4 week-old), pubertal (6- to 9-week-old) and post-pubertal (16- to 20-week- and 24- to 28-week-old) C57BL/6 (B6) mice (The Jackson Laboratory) were used for gene expression profiling experiments. Johns Hopkins University is an AAALAC-accredited institution, and the mice were housed and cared for in accordance with institutional guidelines.
Gene expression profiling and analysis
Expression profiling using Affymetrix U74Av2 (12,488 probe sets) was done as previously described [
47]. In brief, six spleens from female and male B6 mice in each age group (pre-pubertal, pubertal and post-pubertal) (a total of 36 mice) were used for expression profiling. The spleens were homogenized in guanidinium thiocyanate homogenization buffer (0.1 M Tris HCl, pH 7.5, with 4.0 M guanidinium thiocyanate and 1% β-mercaptoethanol) using a Polytron homogenizer (Brinkmann). Total RNA was extracted by centrifuging the homogenate at 25,000 rpm for 24 h over a CsCl cushion (5.7 M CsCl with 0.01 M EDTA, pH 7.5). Double-stranded cDNA was synthesized from each aliquot using 8 ug of total RNA and the SuperScript Choice system (Invitrogen) and T7-(dT
24) primer (GENESET Corp). Double-stranded cDNA reactions and all the following steps were done in duplicate for each sample. Double-stranded cDNA was purified using Phase Lock Gel (Eppendorf-5 Prime). Biotin-labeled cRNA was then synthesized from the double-stranded cDNA by
in vitro transcription using a BioArray HighYield RNA Transcript Labelling Kit (Affymetrix). The cRNA was then purified using an RNeasy Mini kit (QIAGEN), fragmented, and hybridized to murine genome U74A chips for 16 h. The GeneChips were then washed and stained on the Affymetrix Fluidics Station 400 following Affymetrix protocols. The stained images were read using a Hewlett-Packard G2500A Gene Array Scanner and stored in an Affymetrix Microarray Laboratory Information Management System (LIMS). Quality control measures included >4-fold cRNA amplification (from total RNA/cDNA), scaling factors <2 to reach a whole-chip normalization of 800, and visual observation of hybridization patterns for chip defects. Probe set analysis was done using Microarray Suite version 5.0. The signal intensity values (absolute analyses) of the probe sets were then loaded into GeneSpring (Silicon Genetics, Redwood city, CA) for further analysis. Gene clusters were identified using statistical analysis of expression based on correlation coefficient. Briefly, a gene differentially regulated at a specific age was selected, and then a gene cluster was generated whose expression pattern correlates to the selected gene with the correlation coefficient of 0.97. All data files available through Public Expression Profiling Resource [
48].
Determination of serum polyclonal isotype-specific Ig levels
Serum samples from the various age groups (B6, B6 lpr and B6 gld mice [16–20 weeks old] and B6 GKO mice [16 weeks old]) were collected and stored in aliquots at -80°C before analysis. The levels of serum polyclonal IgG1, IgG2a, IgG2b, IgG3, IgM, IgA, kappa and lambda light chain antibodies were determined using isotype-specific antibodies. Ig levels in these sera were assayed by solid-phase enzyme-linked immunosorbent assay (ELISA) using goat anti-mouse Ig antibody-coated plates and alkaline phosphatase-conjugated isotype-specific anti-Ig antibodies as developing reagents (Southern Biotechnology). Dilutions of sera (IgG1, IgG2b and κ light chain at 1:10,000; 1:50,000 and 1:100,000; IgG2a, IgG3, IgM, IgA and λ light chain at 1:1,000, 1:5,000 and 1:10,000) from experimental mice were prepared, and the results are expressed as OD405 absorbance values.
Determination of serum amyloid A and serum haptoglobin levels
Mouse serum amyloid A levels were determined using a solid-phase ELISA (Phage Range, Tridelta), and serum haptoglobin levels were determined using a colorimetric assay according to the manufacturer's instructions (Phage Range, Tridelta). Statistical significance was calculated using students t-test. A p value less than 0.05 was considered statistically significant.
Flow cytometric analysis
All antibodies and reagents used for surface and intracellular cytofluorimetric analyses were purchased from Pharmingen. FasL expression was assessed on CD8+ T cells after stimulation with combinations of anti-CD3/anti-CD28; FasFc; and estrogen using anti-FasL antibodies. Cell staining was detected by flow cytometry on FACS Calibur (Becton Dickinson) and analyzed using Cell Quest software.
Purification of CD8+ T cells and in vitro antibody synthesis assays
Spleens from post-pubertal (12- to 14-week-old) female mice were disrupted in PBS containing FBS. CD8+ T cells were enriched using a Spin-Sep murine cell enrichment kit (Stem Cell Technologies) according to the manufacturer's protocol. T cells (5 × 105) were stimulated for 18 h with various stimulants, either individually or in combinations (anti-CD3/anti-CD28; FasFc; and estrogen). These activated CD8+ T cells were washed and added to 1.5 × 106 splenocytes in 24 well plates. On day 3 half of the medium was removed and supplemented with fresh medium and further incubated for 7 days. On day 10 the culture supernatants were collected and assayed for Ig isotypes.
Determination of IFN-gamma in culture supernatants
Purified CD8+ T cells were stimulated with plate-bound anti-CD3/anti-CD28 (2.5 μg/ml/10 μg/ml) and FasFc (2.5 and 1.25 μg/ml) in the presence and absence of estrogen (1 × 10-8M) for 24 h, and the supernatants were assayed for IFN-gamma using a commercial ELISA kit (Quantikine IFN-γ, R&D Systems). Statistical significance was calculated using students t-test. A p value less than 0.05 was considered statistically significant.
In vitro TLR stimulation of splenocytes
Total splenocytes were isolated from post-pubertal male and female B6 mice as described above. Cells (2.5 × 106 cells/ml) were stimulated with the following doses of TLR ligands: lipopolysaccharide (LPS, Sigma), 200 ng/ml; lipoteichoic acid (LTA, Sigma), 5 μg/ml; Poly I:C (Sigma), 50 μg/ml; Pam3CSK4 (EMC Microcollections), 1 ng/ml; Imiquimod, 100 ng/ml; T1 CpG DNA (5'-TCGTCGTTTTGTCGTTTTGTCGTT-3'), 400 ng/ml. After two day incubation with these ligands, supernatants were isolated, and cytokine and chemokine analyses were carried out using SearchLight Technology (Pierce Biotechnology). This system uses multiplexed sandwich ELISAs to quantify up to 16 different cytokines/chemokines per well of a 96-well plate. The results were expressed as pg/ml (mean ± SD). Statistical significance was calculated using students t-test. A p value less than 0.05 was considered statistically significant.
Authors' contributions
RL conducted research, analyzed data and wrote the paper. PZ, RR, AD, JCH, JJC, RA conducted research. PC analyzed data and critically evaluated the paper. AR, EPH designed research and critically evaluated the paper. KN oversaw research, designed and conducted experiments, analyzed data, and wrote the paper.