Involvement of Notch signaling pathway in regulating IL-12 expression via c-Rel in activated macrophages

Macrophages play an important role both in innate and adaptive immune responses. Treatment with interferon (IFN) gamma together with lipopolysaccharide (LPS) activates pro-inflammatory macrophages which secrete various pro-inflammatory cytokines including IL-12. IL-12 promotes a Th1 type immune response by directly controlling the differentiation of CD4(+) T helper 1 cells. Activation of Notch signaling pathway was reported in activated macrophages but the involvement of this signaling pathway in IL-12 expression has not been documented. In this study, we investigated the role of Notch signaling in regulating expression of the IL-12/IL-23 subunit, IL-12p40. Using a gamma-secretase inhibitor (GSI) to inhibit Notch signaling, we observed a profound decrease in il12p40 mRNA levels and IL-12p70 secretion upon IFN gamma/LPS stimulation. On the other hand, overexpression of activated form of Notch1 in activated RAW264.7 macrophage-like cell lines significantly increased the level of il12p40 mRNA. GSI treatment did not affect the expression of irf5, a master regulator of il12p40 transcription in macrophages. Detailed analysis of the signaling cascades that were affected by this inhibition showed that c-Rel nuclear translocation was inhibited and Erk1/2 activation was compromised by GSI treatment. Addition of exogenous tumor necrosis factor (TNF) alpha only partially rescued the expression of il12p40 in the presence of GSI. Unexpectedly, inhibition of Notch signaling using a dominant negative (DN) Mastermind-like (MAML) transcription co-activator, did not affect c-Rel nuclear localization upon activation or il12p40 mRNA levels, suggesting that the transcriptional activity of Notch signaling is dispensable for the activation of c-Rel. These results strongly suggest that Notch signaling in activated macrophages is involved in regulating the expression of il12p40 directly via c-Rel and indirectly via TNF alpha production. (C) 2012 Elsevier Ltd. All rights reserved.