Virus and infection

The H3 variant of CVB3 was made from an infectious cDNA clone as previously described (Knowlton et al. 1996). Mice were infected by intraperitoneal injection of 0.5 ml of PBS containing 100 or 50 PFU CVB3 and killed seven days after infection.

Serum testosterone

Eight-week-old mice were bled by tail vein, and the samples were spun down for 20 min at 16,000 g to extract serum. Serum testosterone levels were measured using a testosterone enzyme immunoassay (EIA) kit according to the manufacturer’s instructions (Assay Designs, Ann Arbor, MI). Briefly, serum samples were diluted with 1 part steroid displacement reagent for every 99 parts sample. Each sample was diluted 1:10 and 1:20 in PBS and added to the 96-well plate in duplicate, followed by the primary antibody, and incubated at room temperature for 1 hr while shaking. Wells were washed and conjugate was added to each well and incubated at room temperature for 1 hr while shaking. Wells were washed, and the pNpp substrate was added to each well and incubated at 37° for 1 hr without shaking. Stop solution was added, and the plate was read immediately with optical density at 405 nm.

Lymphocyte isolation

Perfused and intact liver, spleen, and thymus were extracted from 8-week-old mice and placed in 50 ml tubes containing RPMI 1640 media with 10% FCS and shipped overnight on ice to Brown University. Thymocytes were minced and washed once in 1% PBS-serum. To obtain splenic lymphocytes, spleens were minced, passed through nylon mesh (Tetko, Kansas City, MO), washed once in 1% PBS-serum, and then cell suspensions were layered on lympholyte-M (Cedarlane Laboratories Ltd., Canada). Hepatic lymphocytes were prepared by mincing and passing through a 70 mm nylon cell strainer (Falcon, Franklin Lakes, NJ). After washing three times in 1% PBS-serum, cell suspensions were layered on a two-step discontinuous Percoll gradient (Pharmacia Fine Chemicals, Piscataway, NJ). Splenocytes and hepatic lymphocytes were collected after centrifugation for 20 min at 900 g.

Antibodies and flow cytometric analysis

TCRβ-FITC (or APC), CD8α-efluor 450 (or Pacific blue), CD4-APC (or perCP), CD44-efluor 780, NK1.1-PE-Cy7, and CD24-FITC were purchased from eBioscience (San Diego, CA). CD1d Tetramer-PE was obtained from the National Institute of Allergy and Infectious Disease MHC Tetramer Core Facility at Emory University (Atlanta, GA). Cells were suspended in buffer composed of PBS containing 1% FCS. Cells were first incubated with 2.4G2 mAb and stained with mAbs specific for cell surface markers for 20 min at room temperature. Depending on the experiment and the tissue, from 2.5 × 10⁵ to 1 × 10⁶ events were collected in the lymphoid gate on a FACSAria. The data were analyzed using FlowJo (Tree Star Inc., Ashland, OR).

Differences in basal invariant natural killer T cell numbers do not influence the mortality of CVB3-infected B6-ChrY consomic mice

Invariant natural killer T (iNKT) cells are an important immune regulator during CVB3 infections, and the treatment of mice with α-galactosylceramide, an agonist for iNKT cells, protects the mice from CVB3-induced disease (Liu and Huber 2011; Wu et al. 2010). Consequently, Y^Nkt, a locus that leads to a profound deficiency in iNKT cells in male B6-ChrY^{Nkt-129F11/Pas} mice (Wesley et al. 2007), is a functionally relevant candidate for Y^Cvb3. Therefore, we analyzed the number of iNKT cells in the liver, spleen, and thymus of all B6-ChrY consomic strains by staining leukocytes for TCRβ⁺ and CD1d-tetramer⁺ cells and assessing the percentage of iNKT cells by flow cytometry (File S1).

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Figure 3

Y^Nkt polymorphism influences the frequency of iNKT cells in B6-ChrY consomic strains. (A) Thymocytes were analyzed by flow cytometry for the percentage of iNKT cells by staining for TCRβ- and CD1d-expressing cells for each B6-ChrY consomic strain and then normalizing to B6 controls. The data are represented as the difference in the percentage iNKT from B6 (B6 is the baseline). The X-axis labels indicate the mouse strain donating ChrY to B6. The hatched line represents the normalized percentage of iNKT cells seen in B6-ChrY^Nkt-129/Pas mice. The significance of observed differences among the strains was determined by one-way ANOVA followed by Bonferroni's multiple comparison test. (B) Representative liver and thymus staining from (left) male B6-ChrY^129F11/Pas inheriting the ChrY^129F11/Pas from mice by natural breeding and (right) male B6-ChrY^Nkt-129/Pas mice carrying the ChrY^129/Pas transmitted from male founder mice derived from 129/Pas embryonic stem cells. n ≥ 4 mice per group. ^**P < 0.01; ^***P < 0.001; ^****P < 0.0001.

As the various B6-ChrY consomic strains were analyzed throughout multiple experiments, the data derived from each consomic line was normalized to the B6 data from each independent experiment to control for interexperimental variability. Interestingly, we observed a continuous distribution of iNKT cells across the ChrY strains. The analysis of thymic iNKT cells produced three significant groupings (Figure 3A). Similar continuous distributions of iNKT cells were also observed for liver and splenic iNKT cell numbers (data not shown).

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Figure 4

Differences in CVB3-induced mortality are not the result of changes in adult serum testosterone levels among the B6-ChrY consomic strains. Serum testosterone was measured in 8-week-old B6-ChrY consomic male mice and compared with wild-type B6 by EIA. X-axis labels indicate the mouse strain donating ChrY to B6. Only B6-ChrY^RF mice exhibited a significant elevation in testosterone compared with B6. Significance of observed differences was determined by one-way ANOVA, followed by a Dunnett’s multiple comparison test. n = 5 mice per strain. ^***P ≤ 0.001.

Our data confirm that natural ChrY polymorphism results in differences in the percentage of basal iNKT cells in mice. However, it is important to note that further investigation is required to determine whether CVB3 infection itself changes the distribution of iNKT cells among the B6-ChrY consomic strains. Nonetheless, as the strain distribution pattern (SDP) for mortality is discordant with the SDP for iNKT cells in naïve B6-ChrY consomic strains, these data suggest that Y^Cvb3 is not Y^Nkt, and that the variations in basal iNKT cell numbers do not contribute to the phenotypic differences in CVB3-induced mortality. Moreover, these data indicate that the Y^Nkt-129/Pas allele transmitted by male founder mice derived from 129F11/Pas embryonic stem cells is unique, as male mice that inherited the wild-type ChrY^129F11/Pas by natural breeding differed markedly in iNKT cell numbers (Figure 3B). A direct comparison of these two ChrYs could in theory lead to the identification of the locus controlling iNKT cell numbers and possibly, as with yaa (Pisitkun et al. 2006; Subramanian et al. 2006), the identification of a unique translocation in ChrY^Nkt-129/Pas giving rise to a dramatic reduction in iNKT cell numbers compared to naturally occurring Y^Nkt alleles.

Sry allele-dependent variations in prenatal testosterone production do not underlie Y^Cvb3

Sry is a particularly intriguing candidate for Y^Cvb3 because Sry expression by the bipotential cells of the primordial gonad initiates testis differentiation by activating male-specific transcription factors, in particular Sox9, to induce Sertoli cell differentiation. This drives testis formation (Kashimada and Koopman 2010), including differentiation of fetal Leydig cells (Brennan et al. 2003; Clark et al. 2000; Gnessi et al. 2000; Yao et al. 2002), which produce the androgens required for masculinization of the male fetus during embryogenesis (Sriraman et al. 2005). Sertoli cells also contribute to the proper differentiation and function of adult Leydig cells (Loveland and Schlatt 1997; Russell et al. 2001; Yan et al. 2000) responsible for pubertal production of testosterone and sexual maturation (Ge and Hardy 2007). There is increasing evidence that testis determination may not be the only function of Sry, as it is also expressed in the brain, kidney, and adrenal glands of adult males (Ely et al. 2010). The existence of functionally significant Sry polymorphisms is well documented in studies using B6-ChrY consomic strains in which Sry alleles give rise to varying degrees of sex reversal, ranging from normal testis development to permanent sex reversal (Albrecht et al. 2003; Biddle and Nishioka 1988; Eicher and Washburn 1986; Eicher et al. 1982; Nagamine et al. 1987, 1999). Therefore, Sry polymorphisms could lead to differences in neonatal and/or adult testosterone levels, thereby impacting susceptibility to CVB3-induced mortality.

To test our hypothesis, we compared the SDP of Sry polymorphisms with the SDP of CVB3-induced mortality. The B6-ChrY consomic lines can be separated into two categories based on the evolutionary origin of the Mus musculus species donating ChrY, including musculus and domesticus. Within the domesticus ChrY consomic lines, two groups of mice exist based on their Sry alleles (Table 1) (Washburn et al. 2001). Domesticus Group A is defined as having Sry alleles with 13 CAG repeats in glutamine repeat cluster 3, which results in sex reversal when inherited by B6 mice heterozygous for the Chr17 TOrleans mutation. Mice within this group have reduced Sry expression in the fetal gonad, which is insufficient for normal development. Domesticus Group B is defined as having Sry alleles with 12 CAG repeats in glutamine repeat cluster 3 and a 10 bp deletion in the 5′ UTR that allows for normal development in B6 mice heterozygous for the TOrleans mutation and normal Sry expression levels in the fetal gonad. However, our analysis of the consomic lines revealed that the SDP for CVB3-induced mortality is discordant with the SDP for Sry alleles (Table 1). These data suggest that the Sry allelic variations having the potential to influence testis development and neonatal testosterone production are not likely to underlie Y^Cvb3.

We thank Meena Subramanian for technical assistance. This work was supported by the National Institutes of Health (NIH) grants NS061014 (C.T.), NS069628 (C.T.), AI45666 (C.T. and S.A.H.), HL108371 (S.A.H.), and AI046709 (L.B.). L.T. was supported by NIH grant 3R01AI046709, a research supplement to promote diversity in health-related research.