Supplemental Data from "17-β estradiol and 17α-ethinyl estradiol exhibit immunologic and epigenetic regulatory effects in NZB/WF1 female mice"

17α-ethinyl estradiol (EE), a synthetic analog of natural estrogen 17- estradiol (E2), is extensively used in hormonal contraceptives and estrogen replacement therapy, and has also been found in sewage effluents. Given that E2 is a well-known immunomodulator, surprisingly there has been only limited information on the cellular and molecular immunologic consequences of exposure to EE. To address this fundamental gap, we directly compared the effects of EE with E2 on splenic leukocytes of NZB/WF1 mice during the pre-autoimmune period. We found that EE and E2 have common, as well as distinctive, immunologic effects, with EE exposure resulting in more profound effects. Both EE and E2 increased numbers of splenic neutrophils, enhanced neutrophil serine proteases and myeloperoxidase expression, promoted the production of nitric oxide (NO) and monocyte chemoattractant protein (MCP-1), and altered adaptive immune T cell subsets. However, activation of splenic leukocytes through TCR or TLR4 revealed not only common (IL-10), but also hormone-specific alterations of cytokines (IFNgamma, IL-1beta, TNFalpha, IL-2). Further, in EE-exposed mice, TLR9 stimulation suppressed IFNgamma, in contrast to increased IFNgamma from E2-exposed mice. EE and E2 regulated common and hormone-specific expression of immune-related genes. Further, EE exposure resulted in more marked alterations in miRNA expression levels than E2. Only EE was able to reduce global DNA methylation significantly in splenic leukocytes. Together, our novel data revealed that EE and E2 exposure confers more similar effects in innate immune system related cell development and responses, but has more differential regulatory effects in adaptive immune related cell development and responses.