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.