Phd in EXPERIMENTAL MEDICINE

Thesis title: Ruolo dell’estrogeno nella regolazione della risposta immunitaria umana: focus sui recettori degli estrogeni

Estrogens, in particular 17β-estradiol (E2), play a critical role in regulating immune function and contribute to the sex-related differences observed in immune-mediated responses. Their biological effects are mediated by two distinct forms of intracellular receptors, ERα and ERβ, which act as ligand-dependent nuclear transcription factors, thereby inducing “genomic” effects. Although most human immune cells express both receptors, their subcellular localization and their specific contribution to the phenotypic and functional modulation of immune responses remain poorly defined. Moreover, although the immunosuppressive activity of estrogens during pregnancy is well known, the mechanisms underlying this effect have not yet been fully elucidated. The aims of this research project were to analyze the expression and distribution of ERα and ERβ in human immune system cells—specifically monocytes, dendritic cells (DC), T lymphocytes, and invariant Natural Killer T cells (iNKT)—and to evaluate the effects of E2, used at physiological concentrations typical of pregnancy, on the innate immune response mediated by human monocytes-differentiated DCs and on the adaptive immune response mediated by human T and iNKT cells. To this end, the following were analyzed: i) receptor expression under basal conditions and after E2 stimulation; ii) the effects of the hormone on DC differentiation, maturation, cytokine secretion, and antigen-presenting function; iii) the antigen-specific response by T lymphocytes and iNKT in terms of proliferation and cytokine production. The results show that, in T lymphocytes and DCs from healthy donors, E2, at concentrations typical of pregnancy, induces a partial localization of ERα in lipid rafts—specialized regions of the plasma membrane rich in cholesterol and sphingolipids. Furthermore, it was observed that E2 does not interfere with monocyte differentiation into DC, suggesting that activation of ERα/ERβ does not alter the cytokine-dependent signaling pathways involved in this process. In differentiated DCs, E2 does not induce DC maturation per sè but is able to inhibit the LPS-induced upregulation of class II major histocompatibility complex (MHC) molecules and the consequent antigen presentation and proliferation of CD4⁺ T lymphocytes in a mixed lymphocyte reaction (MLR). Finally, E2 was found to act directly on T lymphocytes, reducing the production of pro-inflammatory cytokines (IFN-γ, TNF-α, and IL-17) and inhibiting the expansion of α-galactosylceramide (α-GalCer)-specific iNKT cells. In conclusion, the data obtained during this doctoral project confirms the immunosuppressive role of estrogen observed during pregnancy— essential for maintaining maternal-fetal tolerance—and shows how this effect translates into a significant modulation of both innate and adaptive human immune responses. Moreover, the identification of ERα in lipid rafts of T lymphocytes and DC opens new perspectives on the possible involvement of these membrane microdomains in the activation of non-genomic signaling pathways induced by E2, potentially responsible for the rapid and fine regulation of immune responses. The inhibition, by estrogens at high concentrations, of the antigen-specific response of iNKT cells opens a new scenario regarding the possible effects of these hormones in regulating the activity of this lymphocyte population, which plays a crucial role in both infectious processes and the development of autoimmune diseases. Overall, E2, at high concentrations reached during pregnancy, can selectively modulate the function of immune system cells, representing an important protective mechanism against autoimmune diseases. The data obtained suggests the potential use of phytoestrogens or selective estrogen receptor agonists for the development of new therapeutic strategies in immune-mediated diseases.