Daniela Siegmund, Martin Ehrenschwender, Harald Wajant

• TNF is not only a major effector molecule of PAMP/DAMP-activated macrophages, but also regulates macrophage function and viability. We recently demonstrated that TNFR2 triggers necroptosis in macrophages with compromised caspase activity by two cooperating mechanisms: induction of endogenous TNF with subsequent stimulation of TNFR1 and depletion of cytosolic TRAF2-cIAP complexes. Here we show that TNFR2 activation in caspase-inhibited macrophages results in the production of endogenous TNF and TNFR1 stimulation followed by upregulation of A20,TNF is not only a major effector molecule of PAMP/DAMP-activated macrophages, but also regulates macrophage function and viability. We recently demonstrated that TNFR2 triggers necroptosis in macrophages with compromised caspase activity by two cooperating mechanisms: induction of endogenous TNF with subsequent stimulation of TNFR1 and depletion of cytosolic TRAF2-cIAP complexes. Here we show that TNFR2 activation in caspase-inhibited macrophages results in the production of endogenous TNF and TNFR1 stimulation followed by upregulation of A20, TRAF1, IL-6, and IL-1β. Surprisingly, TNFR1-mediated induction of IL-6 and IL-1β was clearly evident in response to TNFR2 stimulation but occurred not or only weakly in macrophages selectively and directly stimulated via TNFR1. Moreover, TNFR2-induced TNFR1-mediated gene induction was largely inhibited by necrostatin-1, whereas upregulation of A20 and TRAF1 by direct and exclusive stimulation of TNFR1 remained unaffected by this compound. Thus, treatment with TNFR2/ZVAD enables TNFR1 in macrophages to stimulate gene induction via a pathway requiring RIPK1 kinase activity. TNFR2/ZVAD-induced production of IL-6 and IL-1β was largely blocked in necroptosis-resistant MLKL- and RIPK3-deficient macrophages, whereas induction of A20 and TRAF1 remained unaffected. In sum, our results show that in caspase-inhibited macrophages TNFR2 not only triggers TNF/TNFR1-mediated necroptosis but also TNF/TNFR1-mediated RIPK3/MLKL-dependent and -independent gene induction.…