ERβ and ERα Differentially Regulate NKT and Vγ4+ T-cell Activation and T-regulatory Cell Response in Coxsackievirus B3 Infected Mice

Abstract

OBJECTIVES Coxsackievirus B3 (CVB3) induced myocarditis is sex dependent with males developing more severe disease than females. Previous studies had shown that sex-associated hormones determine the sex bias with testosterone and progesterone promoting myocarditis while estrogen (E2) is protective. There are two major estrogen receptors: estrogen receptor alpha (ERα) and estrogen receptor beta (ERβ). The goal of the current study was to determine the relative role of these receptors to myocarditis susceptibility and the mechanism of their action. METHODS Female C57Bl/6 wild-type mice and C57Bl/6 mice deficient in ERα, or ERβ were infected intraperitoneally with 102 plaque forming units CVB3. After 7 days, hearts were evaluated for virus titers by plaque forming assay and myocardial inflammation. Lymphoid cells either from the spleen or infiltrating the heart were characterized by labeling with antibodies including CD4, CD25, FoxP3, IFNγ, IL-4, CD11b, CD1d, Vγ4, TCRβ, or with CD1d-tetramer and evaluated by flow cytometry. To confirm that signaling through distinct estrogen receptors controlled myocarditis susceptibility and T-regulatory cell response, male C57Bl/6 mice were treated with the ERα-specific agonist, propyl pyrazole triol (PPT), ERβ agonist, diarylpropionitrile (DPN), or 17-β-estradiol (E2) as a non-specific estrogen receptor agonist. RESULTS Myocarditis, cardiac virus titers, and CD4(+) Th1 (IFNγ) bias were increased in infected ERαKO and decreased in infected ERβKO mice compared to C57Bl/6 controls. CD4(+)Th1 bias and myocarditis severity correlated inversely with numbers of CD4(+)CD25(+)FoxP3(+) T regulatory cells which were decreased in ERαKO and increased in ERβKO mice. Increased T-regulatory cells corresponded to a preferential activation of natural killer T (NKT) cells in ERβKO mice. Male C57Bl/6 mice treated with DPN showed increased myocarditis while those treated with PPT and E2 showed decreased myocarditis corresponding to either decreased (DPN) or increased (PPT/E2) T-regulatory cell responses in male C57Bl/6 mice. DPN and PPT treatment had no effect on T-regulatory cell responses in NKT KO or γδKO mice. CONCLUSION These results demonstrate that ERα and ERβ both modulated CVB3 myocarditis susceptibility but in opposite directions and that their predominant effect is mediated through their ability to alter NKT and Vγ4(+) innate T cell responses in the infected host. It is these innate T cells which positively or negatively modulate T-regulatory cell responses.