K45A mutation of RIPK1 results in poor necroptosis and cytokine signaling in macrophages, which impacts inflammatory responses in vivo

Abstract

Receptor interacting protein kinase 1 (RIPK1) participates in several cell signaling complexes that promote cell activation and cell death. Stimulation of RIPK1 in the absence of caspase signaling induces regulated necrosis (necroptosis), which promotes an inflammatory response. Understanding of the mechanisms through which RIPK1 promotes inflammation has been unclear. Herein we have evaluated the impact of a K45A mutation of RIPK1 on necroptosis of macrophages and the activation of inflammatory response. We show that K45A mutation of RIPK1 results in attenuated necroptosis of macrophages in response to stimulation with LPS, TNFα and IFNβ in the absence of caspase signaling. Impairment in necroptosis correlated with poor phosphorylation of RIPK1, RIPK3 and reduced trimerization of MLKL. Furthermore, K45A mutation of RIPK1 resulted in poor STAT1 phosphorylation (at S727) and expression of RANTES and MIP-1α following TNF-R engagement in the absence of caspase activation. Our results further indicate that in the absence of stimulation by pathogen-associated molecular patterns (PAMPs), cellular inhibitors of apoptotic proteins (cIAPs) prevent the K45-dependent auto-phosphorylation of RIPK1, leading to resistance against necroptosis. Finally, RIPK1K45A mice displayed attenuated inflammatory response in vivo as they were significantly resistant against endotoxin shock, but highly susceptible against a challenge with Salmonella typhimurium. This correlated with reduced expression of IL-1β and ROS, and poor processing of caspase 8 by RIPK1K45A macrophages. Overall, these results indicate that K45 mediated kinase activity of RIPK1 is not only important for necroptosis but it also has a key role in promoting cytokine signaling and host response to inflammatory stimuli. Apoptosis is considered as the predominant, programmed pathway of cell death; however, recently several pathways of regulated cell death have been shown to operate in cells that are considered highly inflammatory.1,2 Although apoptosis has a major role during fetal development, 3 regulated necrotic cell death does not appear to influence fetal development. 4 Interestingly, receptor interacting protein kinase 1 (RIPK1) deficiency cause embryonic lethality, suggesting a key role of RIPK1 in host survival. 5 Besides the kinase activity, RIPK1 appears to have a scaffolding function, which may influence immune homeostasis. 6,7 Depending on the interacting partners and posttranslational modifications, RIPK1 has a multifaceted role in cell signaling and cell survival.8 Following TNF-R1 signaling, RIPK1 transitions between pro-survival and pro-cell death signaling complexes. 9,10 Necroptosis is a form of regulated necrosis of cells that operates in the absence of caspase activity 9 and is initiated by the engagement of various TLRs or cytokine receptors.11 The first cardinal signaling step in necrosome signaling is the phosphorylation of RIPK1, which leads to RIPK1-RIPK3 interaction and phosphorylation of RIPK3.12 Although necroptotic signaling has been shown to be...

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“…enterica serovar Typhimurium SL1344 (abbreviated as S. typhimurium ) by intravenous injection into the tail vein, as previously described (Shutinoski et al 2016). Mice were sacrificed at 5 dpi, and spleens were collected.…”

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“…Furthermore, RIPK1 can also be activated to induce necroptosis via auto-phosphorylation by its kinase function itself [30]. Upon blockage of the RIPK1 kinase activity via an Asp138Asn mutation in a mouse model (Ripk1D138N/D138N mice), fibroblasts are protected against necroptosis but induction of similar levels of apoptosis like in wildtype was observed.…”

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“…Several pathways of regulated necrosis contribute to the pathogenesis of SIRS and sepsis. This has been demonstrated for necroptosis [112,118,[142][143][144][145][146][147] and pyroptosis [148][149][150][151].…”