- Research article
- Open Access
Sexual dimorphism in immune response genes as a function of puberty
© Lamason et al; licensee BioMed Central Ltd. 2006
- Received: 27 July 2005
- Accepted: 22 February 2006
- Published: 22 February 2006
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.
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.
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.
- Female Mouse
- Sexual Dimorphism
- Immune Response Gene
- FasL Expression
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 . 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 . 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.
Gene expression in spleen during puberty
To define puberty-related changes in immune system function, we performed a series of gene expression profiling experiments using 12,000 gene highly redundant oligonucleotide arrays (Affymetrix U74Av2) on spleens of normal pre-pubertal (3- to 4-week-old), pubertal (6- to 9-week-old) and post-pubertal (24- to 28-week-old) female and male C57BL/6 (B6) mice to identify gender- and age-specific expression programs. We used a microarray data analysis approach that was optimized for signal/noise in tissue samples ; unsupervised hierarchical clustering analyses of these samples showed that the biological variables (age, sex) were dominant over technical and inter-individual variables [12, 13]. Genes involved in cell signaling, cell growth, cell differentiation, extracellular matrix synthesis, morphogenesis, vesicle trafficking, oncogenesis and immune responses were up-regulated during puberty in both male and female spleens (e.g., septin family genes, GDNF, R-ras, Ets family transcription factors, Rab family GTPases, alpha catenin, TGF beta, prolactin-like protein, tenascin-X and IKaros) (see Additional file 1). Other genes specifically down-regulated during puberty belonged to the p53 tumor suppressor pathway, chromatin remodeling, cell cycle, DNA repair, replication and transcription categories (e.g., RAD23a, Dnmt1, Ki 67, mBlm, cdc6 and sak) (see Additional file 2). The majority of puberty-driven gene programs in both female and male spleens were involved in erythropoiesis (e.g., erythropoietin receptor, Duffy blood group), consonant with the fact that erythropoiesis is exceptionally active during puberty . These results suggest that puberty, which enables the initiation and development of female and male reproductive capabilities, is a period of intense molecular reorganization in the spleen, probably in response to gender-specific hormones.
Post-pubertal sex differences in immune response genes
Innate immune response genes differentially expressed in post-pubertal B6 male mice.
Serum Amyloid A
Chitinase 3-like 3
Interferon-inducible protein 1-8p
mSLFN4 (schlafen 4)
Alpha-1 acid glycoprotein
Granulocyte maturation Ly-6G.1
Lipocortin 1 (Annexin A1)
Mast cell protease 8
Mast cell function
Peptidoglycan recognition protein
Formyl peptide receptor-1 like receptor
Integrin beta subunit-like cell-surface protein
Mip 1 gamma (CCl 9)
Chemotaxis of monocytes
Leukotriene B4 12-hydroxydehydrogenase
High affinity IgE receptor alpha subunit
Mast cell activation
Plasminogen activator inhibitor 2 (Serpin b2)
NK and mast cell function
Mast cell function
Adaptive immune response genes differentially expressed in post-pubertal B6 female mice.
TCR beta locus
T cell signaling
Ig-kappa light chain V-J kappa region
Immunoglobulin kappa light chain region
Ig kappa chain 7B6 mRNA
RING zinc finger protein
Ig L-chain gene variable region
IgA V-D-J-heavy chain
B-cell receptor gene
B cell signaling
Carboxyl ester lipase
Guanine nucleotide BP, alpha polypeptide
We found no differences in the expression of adaptive immune response genes in pre-pubertal male and female mice (Figure 1A). Even though the basal levels of some innate immune response pathway genes were higher in pre-pubertal male than pre-pubertal female mice, these differences were not statistically significant (Figure 1B). The difference in the expression of these genes cannot be explained by their chromosomal localization, because these genes are predominantly encoded by autosomes (Tables 1 and 2). However the post-pubertal nature of these differences clearly suggests that indeed many of these genes are influenced by sex hormones.
TLR ligand-induced cytokine/chemokine production in post-pubertal mice shows sexual dimorphism.
2.6 ± 0.5
3.8 ± 0.8
7.2 ± 2.3
6.5 ± 1.3
39.7 ± 4.1
39.4 ± 10.2
20.2 ± 3.5
27 ± 2.8
4.1 ± 1.4
4.7 ± 1.1
80.1 ± 17.2
46 ± 11.2
3.7 ± 0.4
17.3 ± 5.8*
159.2 ± 56.5
171.4 ± 54.2
24.5 ± 1.8
24.8 ± 5.2
29.1 ± 4.1
23.8 ± 3.6
848.7 ± 154.4
1132.7 ± 323
1484.5 ± 56.2
1960.5 ± 316.4
12.7 ± 1.0
13.3 ± 1.1
150.8 ± 10.8
151.8 ± 23.9
14.8 ± 2.7
21.2 ± 4.4
937.5 ± 369.8
1202.3 ± 567.4
21.9 ± 0.9*
14.6 ± 2.2
25.3 ± 4.3*
11.9 ± 2.7
214.2 ± 36.1
174.2 ± 48.3
199.1 ± 21.3
210.7 ± 43.8
9.5 ± 1.3
7.4 ± 0.5
118.3 ± 6.0
94.1 ± 16.7
26.5 ± 3
32.2 ± 7.5
675.0 ± 228.5
772.4 ± 323.6
24.2 ± 1.9
20.0 ± 3.0
20.7 ± 2.1
13.8 ± 3.7
201.4 ± 36.7
192.2 ± 45.59
257.6 ± 17.8
298.1 ± 37.8
6.7 ± 0.5
7.5 ± 0.5
92.6 ± 12.2
105.8 ± 18.0
24.6 ± 2.3
31.3 ± 7.1
644.4 ± 236.3
980.7 ± 362.3
5.9 ± 0.8
7.3 ± 0.7
29.4 ± 7.2
28.2 ± 1.5
4831.3 ± 1210.29
5256.1 ± 881.4
1232.3 ± 272.3
1578.7 ± 145.9
12.9 ± 1.7
14.5 ± 1.6
137.1 ± 19.8
136.3 ± 15.0
28.6 ± 4.5
65.6 ± 13.9*
632.1 ± 292.6
679.2 ± 273.1
10.3 ± 6.9
3.2 ± 0.4
12.5 ± 3.5
6.5 ± 0.2
272.9 ± 29.6
262.6 ± 59.4
295.3 ± 30.6
351.8 ± 38.5
5.4 ± 1.1
6.6 ± 1.2
47.8 ± 17.0
80.9 ± 14.8
15.7 ± 1.4
40.2 ± 12.5
195.7 ± 68.3
279.2 ± 94.0
3.5 ± 1.0
3.5 ± 0.7
9.5 ± 2.2
7.7 ± 1.5
169.5 ± 21
279.2 ± 44.7*
51.5 ± 9.8
80.6 ± 10.5*
6.0 ± 0.6
7.2 ± 1.2
58.4 ± 7.1
96.5 ± 18.0
9.6 ± 3.1
43.5 ± 12.1*
403.4 ± 158.7
619.3 ± 218.3
Role of Fas/FasL pathway in generating sexual dimorphism in Ig gene expression
Because Fas/FasL expression was more pronounced in post-pubertal female mice than in males (Figure 4), we hypothesized that these genes may be influenced by female sex hormones. A putative estrogen response element has been previously described in the FasL promoter, and FasL exists mainly in a membrane-bound form on activated CD8+ cells of the T cell lineage . Thus, we measured FasL by flow cytometry and found that its expression was enhanced by a physiological dose of estrogen (10-8M) on these activated CD8+ T cells (see Additional file 4). Previous studies had shown that reverse signaling through FasL leads to increased proliferation of CD8+ but not CD4+ T cells [20, 21]. We therefore considered the possibility that increased estrogen levels during post-pubertal life enhance FasL expression and lead to downstream activation of CD8+ T cells. These activated CD8+ T cells would be expected to secrete growth factors and cytokines that, in turn, would enhance immunoglobulin gene expression in these post-pubertal female mice.
Effect of activated CD8+ T cell culture supernatants on IgG isotype expression
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 . 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 .
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 . 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 . 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) . The reverse signaling pathway through FasL is functional because of the intact extracellular domain that interacts with FasL. In fact, the MRL-Faslprcg mice exhibit sex differences in disease severity . 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 . 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 . 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 . 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 , in part because of testosterone-induced immunosuppression in post-pubertal males . 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.
We have shown that male and female mice differ significantly in post-pubertal life with respect to their immune functions. We have defined one key molecular pathway in this sexual dimorphism, in which we have attributed a novel function to the Fas-FasL pathway, enhancing immunoglobulin gene expression in post-pubertal female mice. These findings have clear implications not only for studies of autoimmunity but also for transplantation and vaccination.
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 . 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-(dT24) 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 .
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.
Dr. Nagaraju is supported by a grant from the Maryland Arthritis Research Centre, Vernon Lynch Memorial Fellowship in Arthritis Research, an Arthritis Investigator Award from National Arthritis Foundation, and NIH_AR050478. Additional support came from grants from the NIH (NHLBI Programs in Genomic Applications [PGA] HOPGENE U01 HL66614-01, and NINDS N01-NS-1-2339). We thank Drs. Deborah McClellan, Paul Plotz for critical review of this manuscript.
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