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notably, this regulation did not require exogenous manipulation of caspases. These ?ndings identi?ed a new pharmacologically accessible pathway that may be relevant to in?am- matory pathologies. INTRODUCTION RIPK1 and RIPK3 are homologous Ser/Thr kinases, which attracted interest as central inducers of regulated necrotic cell death, termed '

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necroptosis'

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(Christofferson and Yuan, 2010;

Degterev et al., 2005;

Linkermann and Green, 2014;

Pasparakis and Vandenabeele, 2015;

Silke et al., 2015). A range of triggers, including extracellular factors linked to innate immune regula- tion, such as ligands for tumor necrosis factor receptor (TNFR), interferon-alpha receptor (IFNaR), and Toll-like receptor (TLR) families, as well as viral infection and genotoxic stressors, have been associated with RIPK1 and RIPK3 activation. These stimuli have been shown to induce necroptosis in vitro under conditions in which the apical apoptotic cysteine protease, caspase-8, is in- hibited pharmacologically or genetically (Degterev et al., 2005;

He et al., 2011;

Kaiser et al., 2013;

Tenev et al., 2011;

Thapa et al., 2013;

Upton et al., 2010, 2012). Necroptosis is best under- stood in the context of TNF-a signaling, and the kinase activity of RIPK3 has been shown to play a central role in its activation (Cho et al., 2009;

He et al., 2009;

Zhang et al., 2009). RIPK3 functions within an insoluble amyloid-like RIPK1 and RIPK3 '

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necrosome'

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complex, formation of which requires catalytic activities of both kinases (Cho et al., 2009), and serves as a signaling plat- form for the activation of a downstream pseudo-kinase MLKL, a critical mediator of cell lysis and necroptotic death (Wang et al., 2014). In contrast to the well-established roles for kinase activities of RIPK1 and RIPK3 in directing cell death, emerging evidence suggests additional roles for RIPK1 and RIPK3 in the direct regu- lation of pro-in?ammatory signaling (Christofferson et al., 2012;

Lukens et al., 2013;

McNamara et al., 2013;

Wong et al., 2014). In macrophages, activation of innate immune receptors TLR3 and TLR4, in the presence of a pan-caspase inhibitor zVAD.fmk (zVAD), engages the RIPK1, RIPK3, and MLKL pathway in a manner that is dependent on the RHIM-containing adapter pro- tein TRIF. (He et al., 2011;

Kaiser et al., 2013;

Schworer et al., 2014). Upregulation of in?ammatory gene expression by TLR4 is an important component of both physiologic innate immune

46 Immunity 45, 46C59, July 19,

2016 ?

2016 Elsevier Inc. responses to gram-negative bacterial cell membrane compo- nent, lipopolysaccharide (LPS), and a contributor to various in- ?ammatory pathologies (Kawai and Akira, 2010). Given the chal- lenge of separating pro-in?ammatory from pro-necroptotic consequences of RIPK1 and RIPK3 activation, we reasoned that the former regulation might have been overlooked in the pre- vious works. Here, we report roles for kinase activities of RIPK1 and RIPK3 in LPS-induced expression of in?ammatory cytokines that manifest independently from the pro-death functions of these molecules. We have demonstrated that activation of RIPK1 in macrophages by LPS in the presence of zVAD robustly increased expression of a broad range of in?ammatory mole- cules. RIPK3 also contributed to the RIPK1 kinase-dependent in?ammation, but in a context dependent manner. We have shown that this regulation is independent of necroptosis and oc- curs in Mlkl?/? macrophages protected from cell death. We have further established that prolonged activation of Erk1/2 MAPK, which might occur through direct engagement by RIPK1 and RIPK3 '