Análisis de los receptores notch en la activación proinflamatoria del macrófago

Resumen

Macrophage activation by Toll receptors is one of the early and essential events in the development of a defense response against pathogens. Increasing evidence indicates the involvement of Notch signaling in the control of macrophage polarization and inflammation, but the factors and pathways controlling NOTCH signaling during this process have not been completely delineated yet. For these reasons, in this work we have focused on the characterization of the molecular events that take place in NOTCH signaling in TLR activated macrophages, identifying the ligands and proteases implicated in the process, and especially, we have studied the role of NOTCH3 and NOTCH4 in the proinflammatory gene expression of macrophages. Our results reveal that Delta1 and Delta4 are the main ligands inducing Notch signaling in macrophage, whereas Jagged1 is unable to activate this process. Moreover, ADAM10 is the main ADAM protease implicated in NOTCH processing and activation in macrophages activated through TLRs. We also observe that furin, the enzyme that proteolytically cuts Notch receptors and originates mature forms, is induced by Toll receptor signaling in a NOTCH dependent way, affecting ADAM10 processing and NOTCH signaling. We have also shown that NOTCH3 is the only Notch receptor present in the membrane of resting differentiated macrophages and that TLR4 activation induces a rapid and transient increase of NOTCH3 expression level followed by a gradual fall in its expression, coinciding with a dramatic increase in the expression of Notch1, Notch2 and Notch4. Accordingly, NOTCH3 accumulation in the nucleus preceded that of NOTCH1. We also observed a crosstalk between NOTCH3 and NOTCH1 in the course of macrophage activation by TLR4. In macrophages that lack Notch1 expression, the levels of NOTCH3 are more elevated, whereas the contrary occurs when constitutively active Notch1 is expressed. Thus, Notch1 seems to modulate NOTCH3 activation after macrophage activation. On the other hand, we have observed that although NOTCH1, 2 and 3 contribute to the increased NOTCH signaling observed in macrophages after TLR4 activation, there is a significant and specific role for NOTCH3 receptor in the activation of NF-ĸB and in proinflammatory macrophage activation. In fact, BMDM derived from Notch3 knockout mice or peritoneal macrophages transfected with specific siARN of Notch3 failed to activate proinflammatory cytokines. Apparently, NOTCH3 regulates this process by increasing the phosphorylation of p65 by p38 MAP kinase, which in turn produces an increase in the expression of a subset of genes regulated by NF-ĸB such as iNOS, IFN-β and TNF-α. On the other hand, we have demonstrated that NOTCH4, unlike the rest of Notch receptors, inhibits Notch signaling pathway in macrophages activated through TLR4, and does so by inhibiting the induction of the expression of Notch1 and of the enzymes involved in its processing. In this study, we found that macrophages overexpressing Notch4 have a lower production of proinflammatory cytokines. Notch4 decreased STAT1 and NF-ĸB dependent transcription in response to LPS or IFN-γ, inhibiting in this way the expression of IRF-1, IL6 e IL12. This effect was dependent on an increase of STAT3 Tyr phosphorylation and a decrease of STAT1 phosphorylation. These results suggest that Notch4 negatively regulates the inflammatory response to TLR4 or IFN-γ in macrophages. Finally, we identified Tspan33 as a new Notch target gene in macrophages, as its expression is clearly diminished in macrophages lacking Notch1 and Notch2, but it is enhanced after overexpression of a constitutively active intracellular domain of NOTCH1. Tspan33 expression increases in response to TLR signaling, and this overexpression is enhanced by IFN-γ. TSPAN33 favors NOTCH processing at the membrane by modulating ADAM10 and/or Presenilin1 activity, thus increasing NOTCH signaling in activated macrophages, and in this way Tspan33 favors TLR-induced proinflammatory gene expression. In summary, we have desmostrated the role of the different Notch receptors in the proinflammatory activation of macrophage, and we have identified and characterized some proteins involved in the regulation of their activation. Our results allow a better understanding of the regulation/modulation of the inflammatory response