Diet and/or exercise interventions led to a significant decrease in the incidence of diabetes over a 6-year period among those with IGT.

Necrosis can be induced by stimulating death receptors with tumor necrosis factor (TNF) or other agonists; however, the underlying mechanism differentiating necrosis from apoptosis is largely unknown. We identified the protein kinase receptor-interacting protein 3 (RIP3) as a molecular switch between TNF-induced apoptosis and necrosis in NIH 3T3 cells and found that RIP3 was required for necrosis in other cells. RIP3 did not affect RIP1-mediated apoptosis but was required for RIP1-mediated necrosis and the enhancement of necrosis by the caspase inhibitor zVAD. By activating key enzymes of metabolic pathways, RIP3 regulates TNF-induced reactive oxygen species production, which partially accounts for RIP3's ability to promote necrosis. Our data suggest that modulation of energy metabolism in response to death stimuli has an important role in the choice between apoptosis and necrosis.

Nerves are a common feature of the microenvironment, but their role in tumor growth and progression remains unclear. We found that the formation of autonomic nerve fibers in the prostate gland regulates prostate cancer development and dissemination in mouse models. The early phases of tumor development were prevented by chemical or surgical sympathectomy and by genetic deletion of stromal β2- and β3-adrenergic receptors. Tumors were also infiltrated by parasympathetic cholinergic fibers that promoted cancer dissemination. Cholinergic-induced tumor invasion and metastasis were inhibited by pharmacological blockade or genetic disruption of the stromal type 1 muscarinic receptor, leading to improved survival of the mice. A retrospective blinded analysis of prostate adenocarcinoma specimens from 43 patients revealed that the densities of sympathetic and parasympathetic nerve fibers in tumor and surrounding normal tissue, respectively, were associated with poor clinical outcomes. These findings may lead to novel therapeutic approaches for prostate cancer.

Necroptosis has emerged as an important pathway of programmed cell death in embryonic development, tissue homeostasis, immunity and inflammation1–8. RIPK1 is implicated in inflammatory and cell death signalling9–13 and its kinase activity is believed to drive RIPK3-mediated necroptosis14,15. Here we show that kinase-independent scaffolding RIPK1 functions regulate homeostasis and prevent inflammation in barrier tissues by inhibiting epithelial cell apoptosis and necroptosis. Intestinal epithelial cell (IEC)-specific RIPK1 knockout caused IEC apoptosis, villus atrophy, loss of goblet and Paneth cells and premature death in mice. This pathology developed independently of the microbiota and of MyD88 signalling but was partly rescued by TNFR1 (also known as TNFRSF1A) deficiency. Epithelial FADD ablation inhibited IEC apoptosis and prevented the premature death of mice with IEC-specific RIPK1 knockout. However, mice lacking both RIPK1 and FADD in IECs displayed RIPK3-dependent IEC necroptosis, Paneth cell loss and focal erosive inflammatory lesions in the colon. Moreover, a RIPK1 kinase inactive knock-in delayed but did not prevent inflammation caused by FADD deficiency in IECs or keratinocytes, showing that RIPK3-dependent necroptosis of FADD-deficient epithelial cells only partly requires RIPK1 kinase activity. Epidermis-specific RIPK1 knockout triggered keratinocyte apoptosis and necroptosis and caused severe skin inflammation that was prevented by RIPK3 but not FADD deficiency. These findings revealed that RIPK1 inhibits RIPK3-mediated necroptosis in keratinocytes in vivo and identified necroptosis as a more potent trigger of inflammation compared with apoptosis. Therefore, RIPK1 is a master regulator of epithelial cell survival, homeostasis and inflammation in the intestine and the skin.

Receptor-interacting protein kinase 1 (RIPK1) regulates cell death and inflammation through kinase-dependent and -independent functions. RIPK1 kinase activity induces caspase-8-dependent apoptosis and RIPK3 and mixed lineage kinase like (MLKL)-dependent necroptosis. In addition, RIPK1 inhibits apoptosis and necroptosis through kinase-independent functions, which are important for late embryonic development and the prevention of inflammation in epithelial barriers. The mechanism by which RIPK1 counteracts RIPK3-MLKL-mediated necroptosis has remained unknown. Here we show that RIPK1 prevents skin inflammation by inhibiting activation of RIPK3-MLKL-dependent necroptosis mediated by Z-DNA binding protein 1 (ZBP1, also known as DAI or DLM1). ZBP1 deficiency inhibited keratinocyte necroptosis and skin inflammation in mice with epidermis-specific RIPK1 knockout. Moreover, mutation of the conserved RIP homotypic interaction motif (RHIM) of endogenous mouse RIPK1 (RIPK1) caused perinatal lethality that was prevented by RIPK3, MLKL or ZBP1 deficiency. Furthermore, mice expressing only RIPK1 in keratinocytes developed skin inflammation that was abrogated by MLKL or ZBP1 deficiency. Mechanistically, ZBP1 interacted strongly with phosphorylated RIPK3 in cells expressing RIPK1, suggesting that the RIPK1 RHIM prevents ZBP1 from binding and activating RIPK3. Collectively, these results show that RIPK1 prevents perinatal death as well as skin inflammation in adult mice by inhibiting ZBP1-induced necroptosis. Furthermore, these findings identify ZBP1 as a critical mediator of inflammation beyond its previously known role in antiviral defence and suggest that ZBP1 might be implicated in the pathogenesis of necroptosis-associated inflammatory diseases.

Z-DNA and Z-RNA are left-handed double helix nucleic acid structures with poorly understood biological function 1 – 3 . Z-DNA binding protein 1 (ZBP1, also known as DAI or DLM-1) is a nucleic acid sensor containing two Zα domains that bind Z-DNA 4 , 5 and Z-RNA 6 – 8 . ZBP1 mediates host-defence against certain viruses 6 , 7 , 9 – 14 by sensing viral nucleic acids 6 , 7 , 10 . RIPK1 deficiency or mutation of its RIP homotypic interaction motif (RHIM) triggers ZBP1-dependent necroptosis and inflammation in mice 15 , 16 , however, the mechanisms inducing ZBP1 activation in the absence of viral infection remain elusive. Here we show that Zα-dependent sensing of endogenous ligands induces ZBP1-mediated perinatal lethality in mice expressing RIPK1 with mutated RHIM ( Ripk1 mR/mR ) and skin inflammation in mice with epidermis-specific RIPK1 deficiency (RIPK1 E-KO ), as well as colitis in mice with intestinal epithelial-specific FADD deficiency (FADD IEC-KO ). Consistently, functional Zα domains were required for ZBP1-induced necroptosis in fibroblasts that express RIPK1 with mutated RHIM or were treated with caspase inhibitors. Moreover, inhibition of nuclear export triggered Zα-dependent activation of RIPK3 in the nucleus resulting in cell death, suggesting that ZBP1 may recognise Z-form nucleic acids (Z-NA) in the nucleus. We found that ZBP1 constitutively bound cellular double stranded RNA (dsRNA) in a Zα-dependent manner. Furthermore, endogenous retroelement (ERE)-derived complementary reads were detected in epidermal RNA, suggesting that ERE-derived dsRNA may act as Zα domain ligand triggering ZBP1 activation. Collectively, our results provide evidence that sensing of endogenous Z-NA by ZBP1 triggers RIPK3-dependent necroptosis and inflammation, which could underlie the development of chronic inflammatory conditions particularly in patients with mutations in the RIPK1 and CASPASE-8 genes 17 – 20 .

The programmed death-1 (PD-1) costimulatory receptor inhibits T and B cell responses and plays a crucial role in peripheral tolerance. PD-1 has recently been shown to inhibit T cell responses during chronic viral infections such as HIV. In this study, we examined the role of PD-1 in infection with Mycobacterium tuberculosis, a common co-infection with HIV. PD-1-deficient mice showed dramatically reduced survival compared with wild-type mice. The lungs of the PD-1 −/− mice showed uncontrolled bacterial proliferation and focal necrotic areas with predominantly neutrophilic infiltrates, but a lower number of infiltrating T and B cells. Proinflammatory cytokines, such as TNF-α, IL-1, and especially IL-6 and IL-17 were significantly increased in the lung and sera of infected PD-1 −/− mice, consistent with an aberrant inflammation. Microarray analysis of the lungs infected with M. tuberculosis showed dramatic differences between PD-1 −/− and control mice. Using high-stringency analysis criteria (changes of twofold or greater), 367 transcripts of genes were differentially expressed between infected PD-1 −/− and wild-type mice, resulting in profoundly altered inflammatory responses with implications for both innate and adaptive immunity. Overall, our studies show that the PD-1 pathway is required to control excessive inflammatory responses after M. tuberculosis infection in the lungs.co-inhibitory | costimulatory | infection

Necrotic death of macrophages has long been known to be present in atherosclerotic lesions but has not been studied. We examined the role of receptor interacting protein (RIP) 3, a mediator of necrotic cell death, in atherosclerosis and found that RIP3(-/-);Ldlr(-/-) mice were no different from RIP3(+/+);Ldlr(-/-) mice in early atherosclerosis but had significant reduction in advanced atherosclerotic lesions. Similar results were observed in Apoe(-/-) background mice. Bone marrow transplantation revealed that loss of RIP3 expression from bone-marrow-derived cells is responsible for the reduced disease progression. While no difference was found in apoptosis between RIP3(-/-);Ldlr(-/-) and RIP3(+/+);Ldlr(-/-) mice, electron microscopy revealed a significant reduction of macrophage primary necrosis in the advanced lesions of RIP3(-/-) mice. In vitro cellular studies showed that RIP3 deletion had no effect on oxidized low-density lipoprotein (LDL)-induced macrophage apoptosis, but prevented macrophage primary necrosis occurring in response to oxidized LDL under caspase inhibition or RIP3 overexpression conditions. RIP3-dependent necrosis is not postapoptotic, and the increased primary necrosis in advanced atherosclerotic lesions most likely resulted from the increase of RIP3 expression. Our data demonstrate that primary necrosis of macrophages is proatherogenic during advanced atherosclerosis development.