Necroptosis, a form of regulated necrotic cell death, is mediated by receptor interacting protein 1 (RIPK1), RIPK3, and mixed lineage kinase domain-like protein (MLKL). However, the mechanism by which necroptosis promotes inflammation is still unclear. Here we report that the expression of cytokines is robustly upregulated in a cell-autonomous manner during necroptosis induced by tumor necrosis factor alpha (TNFα). We demonstrate that TNFα-induced necroptosis leads to two waves of cytokine production. The first wave, more transient and weaker than the second, is in response to TNFα alone; whereas the second wave depends upon the necroptotic signaling. We show that necroptosis promotes the transcription of TNFα-target genes in a cell-intrinsic manner. The activation of both NF-κB and p38 by the necroptotic machinery, RIPK1, RIPK3, and MLKL, is involved in mediating the robust induction of cytokine expression in the second wave. In contrast, necroptosis induced by direct oligomerization of MLKL promotes cytokine production at much lower levels than that of necroptosis induced with TNFα. Thus, we conclude that TNFα-induced necroptosis signaling events mediated by RIPK1 and RIPK3 activation, in addition to the MLKL oligomerization, promotes the expression of cytokines involving multiple intracellular signaling mechanisms including NF-κB pathway and p38. These findings reveal that the necroptotic cell death machinery mounts an immune response by promoting cell-autonomous production of cytokines. Our study provides insights into the mechanism by which necroptosis promotes inflammation in human diseases.

Transforming growth factor β-activated kinase1 (TAK1) encoded by the gene MAP3K7 regulates multiple important downstream effectors involved in immune response, cell death, and carcinogenesis. Hepatocyte-specific deletion of TAK1 inTak1ΔHEPmice promotes liver fibrosis and hepatocellular carcinoma (HCC) formation. Here, we report that genetic inactivation of RIPK1 kinase using a kinase dead knockin D138N mutation inTak1ΔHEPmice inhibits the expression of liver tumor biomarkers, liver fibrosis, and HCC formation. Inhibition of RIPK1, however, has no or minimum effect on hepatocyte loss and compensatory proliferation, which are the recognized factors important for liver fibrosis and HCC development. Using single-cell RNA sequencing, we discovered that inhibition of RIPK1 strongly suppresses inflammation induced by hepatocyte-specific loss of TAK1. Activation of RIPK1 promotes the transcription of key proinflammatory cytokines, such as CCL2, and CCR2+macrophage infiltration. Our study demonstrates the role and mechanism of RIPK1 kinase in promoting inflammation, both cell-autonomously and cell-nonautonomously, in the development of liver fibrosis and HCC, independent of cell death, and compensatory proliferation. We suggest the possibility of inhibiting RIPK1 kinase as a therapeutic strategy for reducing liver fibrosis and HCC development by inhibiting inflammation.

Patients with hypertrophic cardiomyopathy, particularly young adults, can die from arrhythmia, but the mechanism underlying abnormal rhythm formation remains unknown. C57Bl6 × CBA/Ca mice carrying a cardiac actin ( ACTC) E99K (Glu99Lys) mutation reproduce many aspects of human hypertrophic cardiomyopathy, including increased myofilament Ca sensitivity and sudden death in a proportion (up to 40%) of young (28-40 day old) animals. We studied the hearts of transgenic (TG; ACTC E99K) mice and their non-TG (NTG) littermates when they were in their vulnerable period (28-40 days old) and when they were adult (8-12 wk old). Ventricular myocytes were isolated from the hearts of TG and NTG mice at these two time points. We also examined the hearts of mice that died suddenly (SCD). SCD animals had approximately four times more collagen compared with age-matched NTG mice, yet myocyte cell size was normal. Young TG mice had double the collagen content of NTG mice. Contraction and Ca transients were greater in cells from young TG mice compared with their NTG littermates but not in cells from adult mice (TG or NTG). Cells from young TG mice had a greater propensity for Ca waves than NTG littermates, and, despite similar sarcoplasmic reticulum Ca content, a proportion of these cells had larger Ca spark mass. We found that the probability of SCD in young TG mice was increased when the mutation was expressed in animals with a CBA/Ca background and almost eliminated in mice bred on a C57Bl6 background. The latter TG mice had normal cellular Ca homeostasis. NEW & NOTEWORTHY Mice with the actin Glu99Lys hypertrophic cardiomyopathy mutation ( ACTC E99K) are prone to sudden cardiac death around 40 days, associated with increased Ca transients, spark mass, and fibrosis. However, adult survivors have normal Ca transients and spark density accompanied by hypertrophy. Penetrance of the sudden cardiac death phenotype depends on the genetic background of the mouse. Listen to this article's corresponding podcast at http://ajpheart.podbean.com/e/calcium-regulation-in-e99k-mouse-heart/ .