Immunomodulatory effects of 4HT in macrophages.
Since E2 induces a pro-resolving and anti-inflammatory phenotype in
macrophages through ERα-mediated effects on gene expression (Pepe et
al., 2017, 2018), we asked whether E2 transcriptional activity could be
mimicked or antagonized by 4HT. Primary cultures of peritoneal
macrophages from female mice, chosen as experimental model to more
faithfully mimic the reactivity and function of resident macrophages,
were treated with increasing 4HT concentrations and assayed for the mRNA
levels of Vegfα , a prominent E2-responsive gene in macrophages
(Pepe et al., 2017). Only 10 µM 4HT elicited Vegfα expression,
while dose-dependent positive effects were observed with E2, as expected
(Figure 1A). We excluded that high drug concentrations could be toxic to
the cells, as cell viability was not affected (see Supplementary Figure
1). These results suggest that 4HT may behave as a mild agonist, with
estrogen-like effects emerging only at the highest concentrations. We
therefore tested the combined activity of 4HT and E2, expecting an
increase of hormonal effects in case of 4HT agonist activity.
Unexpectedly, the positive effects triggered by E2 were significantly
reduced by 4HT (Figure 1B), when this drug was tested in a 10:1 or
higher 4HT/E2 ratio and in combinations with 0.001 and 0.1 μM E2
(0.001 μM E2 + 0.1 or 1 μM 4HT; 0.1
μM E2 + 1 μM 4HT). When equimolar to 0.1 μM E2, the inhibitory activity
of 4HT was lost, in accordance with the higher ERα binding affinity of
E2 compared with 4HT (Rich et al., 2002). On the other hand, the
positive effects observed with 10 μM 4HT were still present when assayed
with E2 and, notably, were additive with 10 μM E2,
reaching a 14-fold induction as compared with the 6-fold inductions of
each individual treatment (Figure 1B). Altogether, these results
demonstrate that 4HT acts as an ERα-antagonist in macrophages when used
in the nanomolar concentrations range, while higher amounts act by
different mechanisms to induce unanticipated immunomodulatory macrophage
responses, which can be added on the effects of high estrogenic levels.