Sex differences in response to viral infections are well documented . In many cases, these differences are modulated by the effects of gonadal hormones, including testosterone and estradiol, on immune responses to infection . This communication confirms an important role for testicular and ovarian secretions in the response to viral infection, and also shows that sex chromosome complement impacts the pathogenicity of CVB3 but not IAV infections in mice.
Host immunity can play a significant role in either recovery or pathogenesis from both picornavirus and influenza virus infections. For picornaviruses, host T cell immunity is particularly detrimental and in CVB3 myocarditis , Theiler's virus encephamyelitis , and encephalomyocarditis virus-induced diabetes , virus infection induces autoimmunity to tissue antigens by antigenic mimicry or epitope spreading. Autoimmune effectors rather than direct virus cytopathic effects are the dominant mechanism of morbidity and mortality in response to these viruses. T regulatory cell activity abrogates autoimmune T cell responses, morbidity and mortality following picornavirus infection . In the present study, activation of T regulatory cells was increased in Gdx XYF and XYM mice compared to Gdx XXM and XXF mice, resulting in preferential protection of these mice from CVB3. Previous studies have linked activation of FoxP3+ T regulatory cells with resistance to CVB3 induced myocarditis, which is consistent with the current finding [39, 45]. Foxp3, the T regulatory cell transcriptional factor, is encoded on the X chromosome. It is therefore highly probable that the increased T regulatory cell response in Gdx XY mice will explain the increased resistance of these animals to CVB3 induced disease, although this will need to be confirmed by depletion of these cells prior to CVB3 infection. The major question that remains is why the XY sex chromosome complement would give rise to enhanced Foxp3 expression when this gene is present on the X chromosome. It is unlikely that the XY sex chromosome complement is directly affecting expression of the Foxp3 gene, but may establish conditions that are favorable to T regulatory cell activation.
The dominance of CVB3-induced autoimmune pathogenesis is also shown by the disassociation between myocarditis and cardiac virus titers. T cell depletion reduces cardiac injury but has no effect on virus titers in the heart. If cardiac injury resulted from direct virus infection and replication, then T cell depleted male mice would not be protected when virus load in the myocardium remained high. The inability of T cells to promote CVB3 clearance was initially shown in 1974 using both athymic (nude) and thymectomized, irradiated, bone marrow reconstituted animals . Since then, various studies have demonstrated that T cell independent antibodies are rapidly induced by picornavirus infections, including CVB3 and foot-and-mouth disease virus, which, along with macrophages, are the primary mediators of virus clearance [40, 46, 47].
Protection against IAV infection represents a delicate balance between immune responses protecting versus causing pathology in the host. Immediately following infection, proinflammatory responses, including production of cytokines (for example, TNFα, IL-6, and IL-1β) and chemokines (for example, chemokine (C-C motif) ligand 2 (CCL2) and CCL3), are initiated by macrophages, dendritic cells, and epithelial cells in the respiratory tract, which activate humoral and cell-mediated immune responses to promote virus clearance and protection of the host from subsequent infection. There is, however, growing evidence that these early proinflammatory events can lead to severe disease and even death through a process termed 'immunopathology' . Immune memory and long-term protective immunity against IAV is mediated by pre-existing antibodies as well as memory B and T cells . Young adult female mice produce significantly higher proinflammatory cytokine and chemokine responses and experience greater morbidity and mortality during IAV infection than males, which appears to involve the effects of infection on circulating levels of estradiol . Influenza A virus infection of female mice disrupts reproductive function resulting in persistently low levels of estradiol and progesterone, heightened proinflammatory responses, and reduced rates of survival . Consequently, the outcome of IAV infection is severe for both gonadally-intact and Gdx female mice and exogenous administration of estradiol to Gdx females significantly reduces the induction of proinflammatory responses and increases rates of survival . Taken together, our previous data combined with data from the current study illustrate that gonadal secretions, but not sex chromosome complement, play a role in modulating responses to IAV infection.
Gonadectomy of female mice substantially increased pathogenicity in females infected with CVB3, but had little effect in IAV infected females. In males, Gdx had the opposite effect, with Gdx protecting CVB3 infected males, but increasing pathogenesis in IAV infected Gdx males. A key difference between IAV and CVB3 infections is in the crucial role of adaptive immunity to clear influenza virus [50, 51], whereas T lymphocytes and virus neutralizing antibodies are of limited value for elimination of picornaviruses [40, 47]. This difference in the requirement for adaptive immune responses for recovery from IAV and CVB3 likely contributes to why Gdx is protective against CVB3 and promotes pathogenicity of IAV infection.
The response of FCG mice to infection with CVB3 and the development of CVB3-induced myocarditis in XX animals is similar to the responses of FCG mice in experimental models of EAE and lupus, in which the XX sex chromosome complement produces significantly more severe disease than the XY complement . In EAE and lupus models, XX mice had lower levels of Th2 cytokines than XY mice. Consequently, Th2-mediated responses promote resistance against EAE , whereas Th1 and Th17 cell activation is crucial to EAE severity . In the current study, XY mice developed less severe CVB3-induced myocarditis and had more T regulatory cells in their hearts than XX mice. T regulatory cells inhibit proinflammatory responses through direct cell-cell contact with effector T cells, interaction with antigen presenting cells resulting increased indoleamine-2,3-dioxygenase (IDO) production, and secretion of soluble factors including IL-10 and transforming growth factor (TGF)β [54–56]. The effect of sex chromosome complement on T regulatory cells was not addressed in previous experiments of EAE and lupus in FCG mice ; T regulatory cells, however, are associated with resistance against EAE . An inherent deficiency in T regulatory cell activation in individuals either having two X chromosomes or lacking a Y chromosome is consistent with autoimmune diseases that typically show a strong female bias [23, 58].
Three genetic differences between XX and XY mice could contribute to a sex chromosome effect. The Y chromosome may encode genes that normally have an XY-specific effect. Secondly, the double dose of X genes in XX mice, relative to XY mice, could cause constitutively higher expression of some X genes . The process of X inactivation greatly reduces the number of X genes that show such sex differences in expression. There are, however, a small number genes that escape X inactivation resulting in meaningful differences in gene expression . Thirdly, X genes that receive a parental imprint might be expressed higher in one sex than the other, because only females receive a paternal X imprint. Further genetic studies are needed to resolve the chromosome of origin of the sex chromosome effects reported here, and the genes, especially those that code for immune-related proteins, that are responsible.
Sex differences in circulating levels of gonadal hormones contribute significantly to sex differences in response to viral infections ; there is, however, little evidence that gonadal secretions have lasting effects that are maintained for several weeks after removal of the gonads. These effects, often called 'organizational' effects of gonadal hormones , may account for the differences between Gdx males and females in response to CVB3 and IAV infection. Thus, the immune system, like the brain and genitalia, may reflect lifelong sex differences established by sex differences in patterns of gonadal secretions early during development . Alternatively, the differences in gonadal males and females seen in response to CVB3 may be caused by direct effects of Sry on non-gonadal tissues.