Role of TAM in immune functions and inflammatory responses of
macrophages
Beyond ERα-positive mammary cancer, TAM is used at high dosages for
off-target indications, such as infections. Thus, we asked whether the
immune activity of TAM could alter cell uptake ability, a key step in
microbe elimination by macrophages. Peritoneal macrophages collected
from RosaTdTomato mice were treated with fluorescent polymeric
nanoparticles, named polymersomes, that were taken up by cells in a
time-dependent manner. TAM induced a faster accumulation of
nanoparticles and a higher number of cells involved in the phagocytosis
process (Figure 6). As expected, similar results are obtained when cells
are treated with 4HT.
Next, we evaluated whether TAM regulates the immune responses of
macrophages induced by microbial signals, such as LPS. This bacterial
endotoxin increased the mRNA levels coding inflammatory proteins, such
as TNFα, IL6 and IL1β, with a more potent effect observed shortly after
stimulation (Figure 7A). Notably, TAM increased both the short and
long-term LPS effects on TNFα mRNA. Interestingly, the long-term
treatment with TAM alone induced a 5-fold increase in this cytokine
mRNA. On the other hand, the induction of IL1β by LPS is significantly
reduced by TAM at all time points analyzed, with a reduced expression of
this cytokine when TAM was assayed alone. On the other hand, induction
of IL6 by LPS is affected by TAM only shortly after treatment. To
substantiate the biological relevance of these results, we analyzed the
amount of these inflammatory cytokines that is secreted by macrophages
in response to the above-mentioned signals. As expected, TNFα and IL6
proteins are modulated by TAM and LPS in accordance with the effects on
the mRNA (Figure 7B). Strikingly, IL1β levels are 8-fold higher when TAM
is combined with LPS at both time points analyzed, in contrast to what
observed for IL1β mRNA. Superimposable immunomodulatory effects and
increased IL1β protein levels were obtained with 4HT (see Supplementary
Figure 3). After its biosynthesis, IL1β is secreted by macrophages
through the activity of the NLRP3 inflammasome complex and the
transformation of pro-caspase1 into the active caspase-1 enzyme,
allowing the active form of IL1β to be produced and secreted by cells.
Gene expression analyses did not show any effect of TAM on NLRP3
inflammasome components (Nlrp3, Pycard and Caspase-1, see Supplementary
Figure 4). Interestingly, the results of Figure 7C showed the appearance
of active caspase-1 in macrophages treated with TAM, as well as 4HT, at
all the time points tested. These results help explaining the increase
in IL1β protein levels following TAM+LPS treatment, despite the
reduction in mRNA levels, triggered by a dual mechanism of TAM-induced
caspase-1 activation and LPS-increased cytokine expression. This
inflammatory burst induced by TAM+LPS treatment did not result in cell
toxicity, while TAM was able to attenuate the proliferative effects of
LPS on macrophages (See Supplementary Figure 5). Since the PI3K/Akt
pathway has been reported to regulate inflammasome activation, we asked
whether its induction by TAM may also be involved in caspase-1
activation. However, the presence of LY294,002 did not modify the effect
of TAM and 4HT (see Figure 7C), suggesting that PI3K/Akt activation is
not required for TAM and 4HT-induced caspase-1 activation.
Taken together, these results demonstrate that pharmacological
concentrations of TAM are able to alter the immune functions of
macrophages and their phenotypic activation induced by inflammatory
conditions, by promoting a TAM-specific macrophage phenotype that may
counteract pathogen infections.