Although

we found that Lgals3−/− TREG cells produce higher amounts of IL-10 than WT TREG cells that could influence susceptibility to L. major infection, we cannot rule out the possibility that this endogenous lectin could also influence IL-10 production by other immune cells, including macrophages or B cells. This effect Torin 1 in vitro is important given recent studies showing the role of IL-10-producing B cells in controlling susceptibility to L. major infection [37]. Moreover, we previously found that macrophages from Lgals3−/– mice produce higher amounts of IL-10 in comparison with WT mice [7], suggesting that IL-10 may serve as a general effector target of the immunoregulatory activity of galectin-3. These results raise the question of whether galectin-3 could play a pivotal role in controlling IL-10 gene transcription and ultimately limiting TREG cell functionality. Our findings add to the recently documented role of galectin-3 in modulating the severity of L. major infection by facilitating neutrophil recruitment to sites of infection [38]. Thus, distinct galectin-3-regulated mechanisms may dictate susceptibility to L. major infection. Notch receptors and their ligands are important factors that contribute to the generation, expansion,

and function of TREG cells [22]. Notch-3 expression is a hallmark of TREG cells and Notch-3-mediated signaling positively regulates the expansion of TREG cells [39]. We found that Notch-1 and Notch-3 receptors Z-VAD-FMK are differentially expressed on TREG cells from WT versus Lgals3−/− mice. Surprisingly, in our model,

Notch-3 expression was found to be downregulated in TREG cells from infected Lgals3−/− mice. Despite this fact, we detected high levels of Tyrosine-protein kinase BLK Hes-1 transcripts in Lgals3−/− mice, suggesting a more pronounced activation of this pathway. In fact, Anastasi et al. [39] showed that transgenic mice overexpressing the active intracellular domain of Notch-3 display increased accumulation of TREG cells in lymphoid organs and increased expression of IL-10. Activation of Notch signaling directly affects TREG-cell function by regulating Foxp3 expression through RBP-J- and Hes1-dependent mechanisms [40, 41]. In addition, recent reports show that Notch signaling regulates IL-10 production by Th1 cells through a STAT4-dependent mechanism that converts pro-inflammatory Th1 cells into T cells with regulatory activity [42]. These observations led us to propose that increased IL-10 production in Lgals3−/− mice during infection was, at least in part, associated with higher activation of Notch signaling in these cells. This hypothesis has been confirmed by the fact that in vitro differentiated TREG cells from Lgals3−/− mice produced more IL-10 and were more resistant to inhibition of the Notch pathway.