The immune activity of selective estrogen receptor modulators is gene and macrophage subtype-specific yet converges on Il1b downregulation.

Raloxifene belongs to the family of Selective Estrogen Receptor Modulators (SERMs), which are drugs widely prescribed for Estrogen Receptor alpha (ERα)-related pathologies. Recently, SERMs are being tested in repurposing strategies for ERα-independent clinical indications, including a wide range of microbial infections. Macrophages are central in the fight against pathogen invasion. Despite estrogens have been shown to regulate macrophage phenotype, SERMs activity in these cells is still poorly defined. We investigated the activity of Raloxifene in comparison with another widely used SERM, Tamoxifen, on immune gene expression in macrophages obtained from mouse and human tissues, including mouse peritoneal macrophages, bone marrow-derived macrophages, microglia or human blood-derived macrophages, assaying for the involvement of the ERα, PI3K and NRF2 pathways also under inflammatory conditions. Our data demonstrate that Raloxifene acts by a dual mechanism, which entails ERα antagonism and off-target mediators. Moreover, micromolar concentrations of Raloxifene increase the expression of immune metabolic genes, such as Vegfa and Hmox1, through PI3K and NRF2 activation selectively in peritoneal macrophages. Conversely, Il1b mRNA down-regulation by SERMs is consistently observed in all macrophage subtypes and unrelated to the PI3K/NRF2 system. Importantly, the production of the inflammatory cytokine TNFα induced by the bacterial endotoxin, LPS, is potentiated by SERMs and paralleled by the cell subtype-specific increase in IL1ß secretion. This work extends our knowledge on the biological and molecular mechanisms of SERMs immune activity and indicate macrophages as a pharmacological target for the exploitation of the antimicrobial potential of these drugs.

Tamoxifen Twists Again: On and Off-Targets in Macrophages and Infections.

Beyond the wide use of tamoxifen in breast cancer chemotherapy due to its estrogen receptor antagonist activity, this drug is being assayed in repurposing strategies against a number of microbial infections. We conducted a literature search on the evidence related with tamoxifen activity in macrophages, since these immune cells participate as a first line-defense against pathogen invasion. Consistent data indicate the existence of estrogen receptor-independent targets of tamoxifen in macrophages that include lipid mediators and signaling pathways, such as NRF2 and caspase-1, which allow these cells to undergo phenotypic adaptation and potentiate the inflammatory response, without the induction of cell death. Thus, these lines of evidence suggest that the widespread antimicrobial activity of this drug can be ascribed, at least in part, to the potentiation of the host innate immunity. This widens our understanding of the pharmacological activity of tamoxifen with relevant therapeutic implications for infections and other clinical indications that may benefit from the immunomodulatory effects of this drug.

ERα-independent NRF2-mediated immunoregulatory activity of tamoxifen.

Sex differences in immune-mediated diseases are linked to the activity of estrogens on innate immunity cells, including macrophages. Tamoxifen (TAM) is a selective estrogen receptor modulator (SERM) used in estrogen receptor-alpha (ERα)-dependent breast cancers and off-target indications such as infections, although the immune activity of TAM and its active metabolite, 4-OH tamoxifen (4HT), is poorly characterized. Here, we aimed at investigating the endocrine and immune activity of these SERMs in macrophages. Using primary cultures of female mouse macrophages, we analyzed the expression of immune mediators and activation of effector functions in competition experiments with SERMs and 17ß-estradiol (E2) or the bacterial endotoxin LPS. We observed that 4HT and TAM induce estrogen antagonist effects when used at nanomolar concentrations, while pharmacological concentrations that are reached by TAM in clinical settings regulate the expression of VEGFα and other immune activation genes by ERα- and G protein-coupled receptor 1 (GPER1)-independent mechanisms that involve NRF2 through PI3K/Akt-dependent mechanisms. Importantly, we observed that SERMs potentiate cell phagocytosis and modify the effects of LPS on the expression of inflammatory cytokines, such as TNFα and IL1ß, with an overall increase in cell inflammatory phenotype, further sustained by potentiation of IL1ß secretion through caspase-1 activation. Altogether, our data unravel a novel molecular mechanism and immune functions for TAM and 4HT, sustaining their repurposing in infective and other estrogen receptors-unrelated pathologies.

Reciprocal interference between the NRF2 and LPS signaling pathways on the immune-metabolic phenotype of peritoneal macrophages.

The metabolic and immune adaptation to extracellular signals allows macrophages to carry out specialized functions involved in immune protection and tissue homeostasis. Nuclear factor erythroid 2-related factor 2 (NRF2) is a transcription factor that coordinates cell redox and metabolic responses to stressors. However, the individual and concomitant activation of NRF2 and inflammatory pathways have been poorly investigated in isolated macrophages. We here took advantage of reporter mice for the transcriptional activities of NRF2 and nuclear factor-kB (NFκB), a key transcription factor in inflammation, and observe a persisting reciprocal interference in the response of peritoneal macrophages to the respective activators, tert-Butylhydroquinone (tBHQ) and lipopolysaccharide (LPS). When analyzed separately by gene expression studies, these pathways trigger macrophage-specific metabolic and proliferative target genes that are associated with tBHQ-induced pentose phosphate pathway (PPP) with no proliferative response, and with opposite effects observed with LPS. Importantly, the simultaneous administration of tBHQ + LPS alters the effects of each individual pathway in a target gene-specific manner. In fact, this co-treatment potentiates the effects of tBHQ on the antioxidant enzyme, HMOX1, and the antibacterial enzyme, IRG1, respectively; moreover, the combined treatment reduces tBHQ activity on the glycolytic enzymes, TALDO1 and TKT, and decreases LPS effects on the metabolic enzyme IDH1, the proliferation-related proteins KI67 and PPAT, and the inflammatory cytokines IL-1ß, IL-6, and TNFα. Altogether, our results show that the activation of NRF2 redirects the metabolic, immune, and proliferative response of peritoneal macrophages to inflammatory signals, with relevant consequences for the pharmacological treatment of diseases that are associated with unopposed inflammatory responses.