Estrogen immunomodulation in systemic autoimmunity: evidences in in vitro models on a human myeloid cell line

Background: Estrogens have an important role in determining immune system development, regulation and response to stimuli. Estrogens also influence pathogenesis and progression of autoimmune diseases, including Systemic Lupus Erythematosus (SLE), where there is a strong sex bias: female to male prevalence ratio is about 9:1. Estrogens, and in particular 17-β estradiol (E2), act as transcription factor through the binding to its specific receptors, ERα and ERβ. Among genes regulated by E2, Interferon α (IFNα) and B Lymphocyte Stimulator (BLyS) seem to be affected, and BLyS seems to be an IFN-inducible gene. Both cytokines are increased in SLE patients, indicating a link between hormones and cytokines induction in autoimmunity; BLyS over-expression can represent also a sign of the so called “IFN signature” characterizing systemic autoimmune diseases. Objective: The aim of the present study was to evaluate the effects of E2 on BLyS mRNA and protein expression in a human myeloid cell line, in order to propose an in vitro model of estrogen systemic immunomodulation, also considering IFNα as a possible mediator of this signaling pathway. Materials and Methods: U937 monocytes and U937-derived macrophages (treated with 50ng/mL Phorbol 12-Myristate 13-Acetate for 72 hours, Sigma-Aldrich) were exposed to E2 (Sigma-Aldrich) 100nM, 10nM, 1nM or 0.1nM. Cell viability was evaluated with Trypan Blue and Burker Chamber cell counting. Total RNA was extracted after 6, 24 and 48 hour administration, and culture medium was collected for protein quantification. Quantitative PCR was performed for BLyS and IFNα genes. GAPDH was used as reference gene. Primer concentration was 100nM and cDNA quantity was 25ng. Data were analyzed with 2-ΔΔCt method and statistical analysis was performed with REST-384© version 2 using Pair Wise Fixed Reallocation Randomisation Test©. ELISA assay, using a commercially available kit “Quantikine® ELISA” for Human BAFF/BLyS/TNFSF13B (R&D System) was performed for BLyS protein detection in culture supernatants. Data were normalized for total protein and differences in protein levels between treated and control cells during times were performed with multivariate analysis for repeated measures. The same protocols were used for treatment with exogenous human recombinant INFα (hr-IFNα, 1000IU/mL, Enzo Life Sciences), investigating BLyS mRNA expression at 6, 10, 24 and 48 hour and BLyS protein release at 6, 24 and 48 hour administration. Results: E2 did not induce any modulation of BLyS gene expression at any time or concentration used, in both monocytes and derived-macrophages. BLyS protein release was increased during time, and the highest E2 doses induced BLyS protein mobilization in monocytes, while a BLyS release was noticed in derived-macrophages starting from 1nM E2. In monocytes, E2 induced a time-dependent IFNα up-regulation especially at doses 10nM and 1nM, while in differentiated macrophages E2 induced a significant IFNα down-regulation at 24 hours at doses 100nM and 10nM. IFNα treatment induced a significant up-regulation of BLyS, both in monocytes and in derived-macrophages at any time of treatment, but the effect was higher and faster in differentiated cells than in steady-state monocytes. Regarding BLyS protein release, IFNα treatment did not induce a significant BLyS increase compared with that observed in untreated cells. Conclusion: Within 48-hour treatment E2 dose-dependently induces IFNα, but not BLyS expression, while exogenous IFNα affects BLyS transcription but not protein release. These findings suggest that estrogens could primarily have a role in the modulation of cytokines belonging to innate immunity, such as IFNα, with different effects depending on target cell phenotype and milieu. Estrogen modulation of adaptive immune response, here exemplified by BLyS, seems to be the result of estrogen-induced IFNα up-regulation, thus confirming the link between innate and adaptive immune activation in systemic autoimmunity.