INTRODUCTION
Sexual dimorphism in innate immunity mainly manifests by lower susceptibility and better outcomes against infections in females, who also display better vaccination responses and transplantation outcomes as well as higher incidence of autoimmune diseases compared to males (Fischer et al., 2015; Jaillon et al., 2019; Klein & Flanagan, 2016; Shepherd et al., 2021). Estrogen hormones contribute to the disparity in immunity by regulating immune cells. Estrogens bind to the estrogen receptors (ERs), ERα and ERβ, which are ligand-activated transcription factors that modulate gene transcription by binding to target genes promoter or by interfering with the activity of other transcription factors (McDonnell and Norris, 2002; Kovats, 2015). Estrogens also induce rapid cytoplasmic responses, such as calcium influx and cAMP formation, mediated by estrogen-activated ERs and G protein-coupled estrogen receptor 1 (GPER1), a membrane ER (Revankar et al., 2005). ER-selective antagonists have been developed to counteract the transcriptional effects of estrogens. These antagonists are defined as selective estrogen receptor modulators (SERMs) for their tissue-selective pharmacological activity; one relevant example is tamoxifen (TAM) which triggers ER-antagonist and agonist responses in the mammary tissue and bone, respectively (Y. Maximov et al., 2013). TAM is widely employed in ERα-positive breast cancers as a precursor drug of the active metabolite, 4-hydroxytamoxifen (4HT), that inhibits cancer cells proliferation through ERα-antagonistic mechanism. In fact, ERs binding affinity of 4HT is similar to the endogenous ligand 17β-estradiol (E2), while TAM affinity is 100-fold lower than E2 (Rich et al., 2002;,Clarke et al., 2003). Interestingly, TAM is currently used in repurposing strategies as recent clinical evidence proved its efficacy in ER-independent cancers and infections by intracellular pathogens (Butts et al., 2014; Ma et al., 2015; Sik Jang et al., 2015; Hasegawa et al., 2018; Montoya and Krysan, 2018; Zheng et al., 2018). In these circumstances, higher doses of TAM are used, reaching plasma and tissue drug concentrations in the micromolar range.
Macrophages are key players in innate immunity and carry out effector and protective functions through the acquisition of distinct phenotypes (Mantovani and Locati, 2009). The classic inflammatory phenotype (also referred to as M1) is activated by inflammatory cytokines and pathogen-derived signals, such as the bacterial endotoxin lipopolysaccharide (LPS), and results in the expression of effector functions including production of inflammatory mediators, such as TNFα and IL1-β, and reactive molecular species that are pivotal for pathogens and cancer cells killing. On the other hand, the macrophage alternative phenotype (also referred to as M2) is stimulated by Th2 cytokines blunts inflammation and promotes tissue remodeling. These two phenotypes simplistically represent the extremes of a spectrum of intermediate functions acquired by macrophages under the combined influence of different endogenous molecules, including estrogens or xenobiotics (Pepe et al., 2017). The transcription factor NRF2 has been recently identified as a molecular player involved in macrophage phenotypic conversion. In resting conditions, NRF2 is bound to Keap1 in an inhibitory complex that leads NRF2 to proteasomal degradation, while an oxidative burst induces Keap1 dissociation and NRF2 migration to the nucleus, where it regulates gene expression upon binding to ARE responsive elements in the promoter regions of NRF2 target genes (Itoh et al., 1999). During inflammation or infections, NRF2 activation in macrophages increases bacterial clearance by phagocytosis and modulates the expression of inflammatory mediators, in parallel with the production of antioxidant proteins that buffer the reactive oxygen species (ROS) generated by macrophages for pathogen killing (Harvey et al., 2011; Kobayashi et al., 2016; Wang et al., 2017b; Bewley et al., 2018).
Through ERα-mediated mechanisms, estrogens have been shown to induce cell expansion and phenotypic switch in macrophages, leading to a faster activation and conversion towards an M2-like phenotype (Villa et al., 2015; Pepe et al., 2017, 2018). This immune activity may explain the better performance of females in some physio-pathological conditions, such as vaccination, infections or neurodegenerative pathologies, and may turn detrimental in others, such as tumors or endometriosis, both conditions sustained by the M2 macrophage phenotype (Vegeto et al., 2010, 2020; Pepe et al., 2018; Vázquez-Martínez et al., 2018). The clinical use of ER antagonists may thus offer therapeutic benefit or adverse effects, depending on the specific pathogenic role of macrophages and drug efficacy in these cells. Despite the wide use of TAM in estrogen-dependent and off target indications, its activity in immune cells is still poorly defined.
The present study was envisioned to understand the hormonal and immunomodulatory activity of SERMs in macrophages. We found that pharmacological levels of 4HT and, more importantly, TAM (i) induce ERα and GPER1-independent immunomodulatory effects in macrophages, that modify cell polarization through Nrf2 activation, (ii) increase phagocytosis and (iii) potentiate the ability to respond to LPS. Our results shed new light on the pharmacological potential and immune regulatory activity of TAM and 4HT, sustaining the use of SERMs in repurposing strategies against infections and other ERα-unrelated pathologies.