RIPK1 and RIPK3 Kinases Promote Cell-Death-Independent Inflammation by Toll-like Receptor 4

Abstract

Summary Macrophages are a crucial component of the innate immune system in sensing pathogens and promoting local and systemic inflammation. RIPK1 and RIPK3 are homologous kinases, previously linked to activation of necroptotic death. In this study we have described roles for these kinases as master regulators of pro-inflammatory gene expression induced by lipopolysaccharide, independent of their well-documented cell death functions. In primary macrophages, this regulation was elicited in the absence of caspase-8 activity, required the adaptor molecule TRIF, and proceeded in a cell autonomous manner. RIPK1 and RIPK3 kinases promoted sustained activation of Erk, cFos and NFκB, which were required for inflammatory changes. Utilizing genetic and pharmacologic tools, we showed that RIPK1 and RIPK3 account for acute inflammatory responses induced by lipopolysaccharide in vivo; notably, this regulation did not require exogenous manipulation of caspases. These findings identified a new pharmacologically accessible pathway that may be relevant to inflammatory pathologies.

Complex Pathologic Roles of RIPK1 and RIPK3: Moving Beyond Necroptosis

“…Upon activation, RIPK1 and RIPK3 become phosphorylated on multiple sites either by autophosphorylation or cross-phosphorylation and this is also required for necroptosis [32]. Of note, multiple phospho-specific antibodies against RIPK1 (Ser14/15, Ser166) and RIPK3 (Ser232 (mouse)/Ser227 (human)) have been developed and provide new useful tools to analyze initiation of necroptosis [33-37]. …”

“…For example, work by our and other labs suggested that RIPK1 kinase activity in L929 and other cell lines is associated with new synthesis and release of TNFα [70-73]. We examined this regulation in macrophages stimulated with LPS and caspase inhibitors and showed that it is mediated by kinase activities of RIPK1 and RIPK3, but necroptosis and MLKL are dispensable [37]. Rather, the responses were mediated by recruitment of inflammatory effectors, in this case Erk1/2, to necrosome-like RIPK1/RIPK3 aggregates (Fig.…”

“…One potential clue to understanding this discrepancy comes from the observation that deficiency in MLKL, which has been previously shown to mediate necroptosis exclusively, led to the increased systemic inflammation, and this was not seen in RIPK3-deficient mice [113]. This may reflect our recent observation that caspase-8 inhibition in macrophages promoted inflammatory gene expression, which was blocked in Ripk3 -/- , but not Mlkl -/- cells [37]. Further, this raises the possibility that the combination of intact pro-inflammatory signaling by RIPK1/RIPK3 and deficient apoptosis and necroptosis provides an optimal environment for the development of autoimmune disease.…”

“…Overall, this work provided a clear demonstration of tumor promoting activity of RIPK1/RIPK3-mediated inflammation through myeloid-dependent immune suppression. Notably, a number of tumor promoting cytokines and chemokines beyond CXCL1, including CXCL2, GM-CSF and IL6, are induced by necrosomes [37]. This may indicate that tumors that are known to be promoted by these inflammatory mediators may be generally targeted by RIPK1 inhibitors.…”

The Implications of Several Forms of Programmed Necrosis for Cancer Therapy

“…RIP3 induces the expression of cytokines that can activate natural killer T cells for Cancer Development anti-tumor activity. In contrast to RIP1 and RIP3, MLKL seems to be dispensable for cytokine synthesis [42,43]. Necroptosis may also be induced by autophagy.…”