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.