Very-early-onset Inflammatory Bowel Disease in an Infant with a Partial RIPK1 Deletion.

The monogenic causes of very-early-onset inflammatory bowel disease (VEO-IBD) have been defined by genetic studies, which were usually related to primary immunodeficiencies. Receptor-interacting serine/threonine-protein kinase-1 (RIPK1) protein is an important signalling molecule in inflammation and cell death pathways. Its deficiency may lead to various clinical features linked to immunodeficiency and/or inflammation, including IBD. Here, we discuss an infant with malnutrition, VEO-IBD, recurrent infections and polyathritis who has a homozygous partial deletion in RIPK1 gene.

ADAR1 mutation causes ZBP1-dependent immunopathology.

The RNA-editing enzyme ADAR1 is essential for the suppression of innate immune activation and pathology caused by aberrant recognition of self-RNA, a role it carries out by disrupting the duplex structure of endogenous double-stranded RNA species1,2. A point mutation in the sequence encoding the Z-DNA-binding domain (ZBD) of ADAR1 is associated with severe autoinflammatory disease3-5. ZBP1 is the only other ZBD-containing mammalian protein6, and its activation can trigger both cell death and transcriptional responses through the kinases RIPK1 and RIPK3, and the protease caspase 8 (refs. 7-9). Here we show that the pathology caused by alteration of the ZBD of ADAR1 is driven by activation of ZBP1. We found that ablation of ZBP1 fully rescued the overt pathology caused by ADAR1 alteration, without fully reversing the underlying inflammatory program caused by this alteration. Whereas loss of RIPK3 partially phenocopied the protective effects of ZBP1 ablation, combined deletion of caspase 8 and RIPK3, or of caspase 8 and MLKL, unexpectedly exacerbated the pathogenic effects of ADAR1 alteration. These findings indicate that ADAR1 is a negative regulator of sterile ZBP1 activation, and that ZBP1-dependent signalling underlies the autoinflammatory pathology caused by alteration of ADAR1.

Bone Marrow-Derived RIPK3 Mediates Kidney Inflammation in Acute Kidney Injury.

BACKGROUND: Receptor-interacting protein kinase 3 (RIPK3), a component of necroptosis pathways, may have an independent role in inflammation. It has been unclear which RIPK3-expressing cells are responsible for the anti-inflammatory effect of overall Ripk3 deficiency and whether Ripk3 deficiency protects against kidney inflammation occurring in the absence of tubular cell death. METHODS: We used chimeric mice with bone marrow from wild-type and Ripk3-knockout mice to explore RIPK3's contribution to kidney inflammation in the presence of folic acid-induced acute kidney injury AKI (FA-AKI) or absence of AKI and kidney cell death (as seen in systemic administration of the cytokine TNF-like weak inducer of apoptosis [TWEAK]). RESULTS: Tubular and interstitial cell RIPK3 expressions were increased in murine AKI. Ripk3 deficiency decreased NF-κB activation and kidney inflammation in FA-AKI but did not prevent kidney failure. In the chimeric mice, RIPK3-expressing bone marrow-derived cells were required for early inflammation in FA-AKI. The NLRP3 inflammasome was not involved in RIPK3's proinflammatory effect. Systemic TWEAK administration induced kidney inflammation in wild-type but not Ripk3-deficient mice. In cell cultures, TWEAK increased RIPK3 expression in bone marrow-derived macrophages and tubular cells. RIPK3 mediated TWEAK-induced NF-κB activation and inflammatory responses in bone marrow-derived macrophages and dendritic cells and in Jurkat T cells; however, in tubular cells, RIPK3 mediated only TWEAK-induced Il-6 expression. Furthermore, conditioned media from TWEAK-exposed wild-type macrophages, but not from Ripk3-deficient macrophages, promoted proinflammatory responses in cultured tubular cells. CONCLUSIONS: RIPK3 mediates kidney inflammation independently from tubular cell death. Specific targeting of bone marrow-derived RIPK3 may limit kidney inflammation without the potential adverse effects of systemic RIPK3 targeting.

Receptor Interacting Protein Kinase Pathways Regulate Innate B Cell Developmental Checkpoints But Not Effector Function in Mice.

Mutations in the scaffolding domain of Receptor Interacting Protein kinases (RIP) underlie the recently described human autoimmune syndrome, CRIA, characterized by lymphadenopathy, splenomegaly, and autoantibody production. While disease mechanisms for CRIA remain undescribed, RIP kinases work together with caspase-8 to regulate cell death, which is critical for normal differentiation of many cell types. Here, we describe a key role for RIP1 in facilitating innate B cell differentiation and subsequent activation. By comparing RIP1, RIP3, and caspase-8 triple deficient and RIP3, caspase-8 double deficient mice, we identified selective contributions of RIP1 to an accumulation of murine splenic Marginal Zone (MZ) B cells and B1-b cells. We used mixed bone-marrow chimeras to determine that innate B cell commitment required B cell-intrinsic RIP1, RIP3, and caspase-8 sufficiency. RIP1 regulated MZ B cell development rather than differentiation and RIP1 mediates its innate immune effects independent of the RIP1 kinase domain. NP-KLH/alum and NP-Ficoll vaccination of mice doubly deficient in both caspase-8 and RIP3 or deficient in all three proteins (RIP3, caspase-8, and RIP1) revealed uniquely delayed T-dependent and T-independent IgG responses, abnormal splenic germinal center architecture, and reduced extrafollicular plasmablast formation compared to WT mice. Thus, RIP kinases and caspase-8 jointly orchestrate B cell fate and delayed effector function through a B cell-intrinsic mechanism.

Dying cells fan the flames of inflammation.

Inflammatory processes that recruit leukocytes to injured or infected tissues are crucial for tissue repair and the elimination of pathogens. However, excessive or chronic inflammation promotes tissue damage and disease, as in arthritis, atherosclerosis, inflammatory bowel disease, and COVID-19. Intracellular constituents released from dying cells are among the stimuli that trigger proinflammatory gene expression programs in innate immune cells. We explore how programmed cell death mechanisms­apoptosis, necroptosis, and pyroptosis­may contribute to inflammatory disease. We discuss inhibition of cell death as a potential therapeutic strategy, focusing on the targets RIPK1 (receptor interacting serine/threonine kinase 1), NLRP3 (NLR family pyrin domain containing 3), and GSDMD (gasdermin D) as important mediators of lytic cell death. We also consider the potential benefits of limiting membrane rupture rather than cell death by targeting NINJ1.

RIPK1 and TRADD Regulate TNF-Induced Signaling and Ripoptosome Formation.

TNF is a proinflammatory cytokine that is critical for the coordination of tissue homeostasis. RIPK1 and TRADD are the main participants in the transduction of TNF signaling. However, data on the cell fate-controlling functions of both molecules are quite controversial. Here, we address the functions of RIPK1 and TRADD in TNF signaling by generating RIPK1- or TRADD-deficient human cell lines. We demonstrate that RIPK1 is relevant for TNF-induced apoptosis and necroptosis in conditions with depleted IAPs. In addition, TRADD is dispensable for necroptosis but required for apoptosis. We reveal a new possible function of TRADD as a negative regulator of NIK stabilization and subsequent ripoptosome formation. Furthermore, we show that RIPK1 and TRADD do not appear to be essential for the activation of MAPK signaling. Moreover, partially repressing NF-κB activation in both RIPK1 and TRADD KO cells does not result in sensitization to TNF alone due to the absence of NIK stabilization. Importantly, we demonstrate that RIPK1 is essential for preventing TRADD from undergoing TNF-induced ubiquitination and degradation. Taken together, our findings provide further insights into the specific functions of RIPK1 and TRADD in the regulation of TNF-dependent signaling, which controls the balance between cell death and survival.

Cyclophilin D Regulates the Nuclear Translocation of AIF, Cardiac Endothelial Cell Necroptosis and Murine Cardiac Transplant Injury.

Ischemia-reperfusion injury (IRI) is an inevitable consequence of organ transplant procedure and associated with acute and chronic organ rejection in transplantation. IRI leads to various forms of programmed cell death, which worsens tissue damage and accelerates transplant rejection. We recently demonstrated that necroptosis participates in murine cardiac microvascular endothelial cell (MVEC) death and murine cardiac transplant rejection. However, MVEC death under a more complex IRI model has not been studied. In this study, we found that simulating IRI conditions in vitro by hypoxia, reoxygenation and treatment with inflammatory cytokines induced necroptosis in MVECs. Interestingly, the apoptosis-inducing factor (AIF) translocated to the nucleus during MVEC necroptosis, which is regulated by the mitochondrial permeability molecule cyclophilin D (CypD). Furthermore, CypD deficiency in donor cardiac grafts inhibited AIF translocation and mitigated graft IRI and rejection (n = 7; p = 0.002). Our studies indicate that CypD and AIF play significant roles in MVEC necroptosis and cardiac transplant rejection following IRI. Targeting CypD and its downstream AIF may be a plausible approach to inhibit IRI-caused cardiac damage and improve transplant survival.

RIPK1 regulates starvation resistance by modulating aspartate catabolism.

RIPK1 is a crucial regulator of cell death and survival. Ripk1 deficiency promotes mouse survival in the prenatal period while inhibits survival in the early postnatal period without a clear mechanism. Metabolism regulation and autophagy are critical to neonatal survival from severe starvation at birth. However, the mechanism by which RIPK1 regulates starvation resistance and survival remains unclear. Here, we address this question by discovering the metabolic regulatory role of RIPK1. First, metabolomics analysis reveals that Ripk1 deficiency specifically increases aspartate levels in both mouse neonates and mammalian cells under starvation conditions. Increased aspartate in Ripk1-/- cells enhances the TCA  flux and ATP production. The energy imbalance causes defective autophagy induction by inhibiting the AMPK/ULK1 pathway. Transcriptional analyses demonstrate that Ripk1-/- deficiency downregulates gene expression in aspartate catabolism by inactivating SP1. To summarize, this study reveals that RIPK1 serves as a metabolic regulator responsible for starvation resistance.

A20 deficiency in myeloid cells protects mice from diet-induced obesity and insulin resistance due to increased fatty acid metabolism.

Obesity-induced inflammation is a major driving force in the development of insulin resistance, type 2 diabetes (T2D), and related metabolic disorders. During obesity, macrophages accumulate in the visceral adipose tissue, creating a low-grade inflammatory environment. Nuclear factor κB (NF-κB) signaling is a central coordinator of inflammatory responses and is tightly regulated by the anti-inflammatory protein A20. Here, we find that myeloid-specific A20-deficient mice are protected from diet-induced obesity and insulin resistance despite an inflammatory environment in their metabolic tissues. Macrophages lacking A20 show impaired mitochondrial respiratory function and metabolize more palmitate both in vitro and in vivo. We hypothesize that A20-deficient macrophages rely more on palmitate oxidation and metabolize the fat present in the diet, resulting in a lean phenotype and protection from metabolic disease. These findings reveal a role for A20 in regulating macrophage immunometabolism.

RIPK1-Associated Inborn Errors of Innate Immunity.

RIPK1 (receptor-interacting serine/threonine-protein kinase 1) is a key molecule for mediating apoptosis, necroptosis, and inflammatory pathways downstream of death receptors (DRs) and pattern recognition receptors (PRRs). RIPK1 functions are regulated by multiple post-translational modifications (PTMs), including ubiquitination, phosphorylation, and the caspase-8-mediated cleavage. Dysregulation of these modifications leads to an immune deficiency or a hyperinflammatory disease in humans. Over the last decades, numerous studies on the RIPK1 function in model organisms have provided insights into the molecular mechanisms of RIPK1 role in the maintenance of immune homeostasis. However, the physiological role of RIPK1 in the regulation of cell survival and cell death signaling in humans remained elusive. Recently, RIPK1 loss-of-function (LoF) mutations and cleavage-deficient mutations have been identified in humans. This review discusses the molecular pathogenesis of RIPK1-deficiency and cleavage-resistant RIPK1 induced autoinflammatory (CRIA) disorders and summarizes the clinical manifestations of respective diseases to help with the identification of new patients.

Interaction of RIPK1 and A20 modulates MAPK signaling in murine acetaminophen toxicity.

Acetaminophen (APAP)-induced liver necrosis is a form of regulated cell death (RCD) in which APAP activates the mitogen-activated protein kinases (MAPKs) and specifically the c-Jun-N-terminal kinase (JNK) pathway, leading to necrotic cell death. Previously, we have shown that receptor interacting protein kinase-1 (RIPK1) knockdown is also protective against APAP RCD upstream of JNK. However, whether the kinase or platform function of RIPK1 is involved in APAP RCD is not known. To answer this question, we used genetic mouse models of targeted hepatocyte RIPK1 knockout (RIPK1HepCKO) or kinase dead knock-in (RIPK1D138N) and adult hepatocyte specific knockout of the cytoprotective protein A20 (A20HepCKO), known to interact with RIPK1, to study its potential involvement in MAPK signaling. We observed no difference in injury between WT and RIPK1D138N mice post APAP. However, RIPK1HepCKO was protective. We found that RIPK1HepCKO mice had attenuated pJNK activation, while A20 was simultaneously upregulated. Conversely, A20HepCKO markedly worsened liver injury from APAP. Mechanistically, we observed a significant upregulation of apoptosis signal-regulating kinase 1 (ASK1) and increased JNK activation in A20HepCKO mice compared with littermate controls. We also demonstrated that A20 coimmunoprecipitated (co-IP) with both RIPK1 and ASK1, and that in the presence of RIPK1, there was less A20-ASK1 association than in its absence. We conclude that the kinase-independent platform function of RIPK1 is involved in APAP toxicity. Adult RIPK1HepCKO mice are protected against APAP by upregulating A20 and attenuating JNK signaling through ASK1, conversely, A20HepCKO worsens injury from APAP.

RIPK1 Promotes Energy Sensing by the mTORC1 Pathway.

The mechanisms of cellular energy sensing and AMPK-mediated mTORC1 inhibition are not fully delineated. Here, we discover that RIPK1 promotes mTORC1 inhibition during energetic stress. RIPK1 is involved in mediating the interaction between AMPK and TSC2 and facilitate TSC2 phosphorylation at Ser1387. RIPK1 loss results in a high basal mTORC1 activity that drives defective lysosomes in cells and mice, leading to accumulation of RIPK3 and CASP8 and sensitization to cell death. RIPK1-deficient cells are unable to cope with energetic stress and are vulnerable to low glucose levels and metformin. Inhibition of mTORC1 rescues the lysosomal defects and vulnerability to energetic stress and prolongs the survival of RIPK1-deficient neonatal mice. Thus, RIPK1 plays an important role in the cellular response to low energy levels and mediates AMPK-mTORC1 signaling. These findings shed light on the regulation of mTORC1 during energetic stress and unveil a point of crosstalk between pro-survival and pro-death pathways.

Flexible Usage and Interconnectivity of Diverse Cell Death Pathways Protect against Intracellular Infection.

Programmed cell death contributes to host defense against pathogens. To investigate the relative importance of pyroptosis, necroptosis, and apoptosis during Salmonella infection, we infected mice and macrophages deficient for diverse combinations of caspases-1, -11, -12, and -8 and receptor interacting serine/threonine kinase 3 (RIPK3). Loss of pyroptosis, caspase-8-driven apoptosis, or necroptosis had minor impact on Salmonella control. However, combined deficiency of these cell death pathways caused loss of bacterial control in mice and their macrophages, demonstrating that host defense can employ varying components of several cell death pathways to limit intracellular infections. This flexible use of distinct cell death pathways involved extensive cross-talk between initiators and effectors of pyroptosis and apoptosis, where initiator caspases-1 and -8 also functioned as executioners when all known effectors of cell death were absent. These findings uncover a highly coordinated and flexible cell death system with in-built fail-safe processes that protect the host from intracellular infections.

Catalytically inactive RIP1 and RIP3 deficiency protect against acute ischemic stroke by inhibiting necroptosis and neuroinflammation.

Necroptosis, which is mediated by RIP1/RIP3/MLKL (receptor-interacting protein kinase 1/receptor-interacting protein kinase 3/mixed lineage kinase domain-like protein) signaling, is a critical process in the development of acute ischemic stroke. However, it is unclear precisely how necroptosis promotes the pathogenesis of acute ischemic stroke. In this experimental study in mice, we investigated how necroptosis loss-of-function mice, RIP1 kinase-dead mice, RIP3-deficiency mice, and MLKL-deficiency mice could be protected against cerebral injury after acute ischemic stroke. Insoluble RIP1, RIP3, and MLKL were all detected in the infarct area of the study mice, indicating activation of necroptosis. Two types of RIP1 kinase-dead mutant mice (Rip1K45A/K45A or Rip1Δ/Δ) were used to show that catalytically-inactive RIP1 can decrease the infarct volume and improve neurological function after MCAO/R (middle cerebral artery occlusion/reperfusion). Both Rip3-/- mice and Mlkl-/- mice were protected against acute ischemic stroke. In addition, necroptosis loss-of-function mice showed less inflammatory responses in the infarct area. Therefore, necroptosis and its accompanying inflammatory response can lead to acute injury following ischemia stroke. Our study provides new insight into the pathogenetic mechanisms of acute ischemic stroke, and suggests potential therapeutic targets for neuroprotection.

RIP1 promotes proliferation through G2/M checkpoint progression and mediates cisplatin-induced apoptosis and necroptosis in human ovarian cancer cells.

Receptor-interacting protein 1 (RIP1, also known as RIPK1) is not only a tumor-promoting factor in several cancers but also mediates either apoptosis or necroptosis in certain circumstances. In this study we investigated what role RIP1 plays in human ovarian cancer cells. We showed that knockout (KO) of RIP1 substantially suppressed cell proliferation, accompanied by the G2/M checkpoint arrest in two human ovarian cancer cell lines SKOV3 and A2780. On the other hand, RIP1 KO remarkably attenuated cisplatin-induced cytotoxicity, which was associated with reduction of the apoptosis markers PARP cleavage and the necroptosis marker phospho-MLKL. We found that RIP1 KO suppressed cisplatin-induced ROS accumulation in both SKOV3 and A2780 cells. ROS scavenger BHA, apoptosis inhibitor Z-VAD or necroptosis inhibitor NSA could effectively suppress cisplatin's cytotoxicity in the control cells, suggesting that ROS-mediated apoptosis and necroptosis were involved in cisplatin-induced cell death. In addition, blocking necroptosis with MLKL siRNA effectively attenuated cisplatin-induced cytotoxicity. In human ovarian cancer A2780 cell line xenograft nude mice, RIP1 KO not only significantly suppressed the tumor growth but also greatly attenuated cisplatin's anticancer activity. Our results demonstrate a dual role of RIP1 in human ovarian cancer: it acts as either a tumor-promoting factor to promote cancer cell proliferation or a tumor-suppressing factor to facilitate anticancer effects of chemotherapeutics such as cisplatin.

An intestinal organoid-based platform that recreates susceptibility to T-cell-mediated tissue injury.

A goal in precision medicine is to use patient-derived material to predict disease course and intervention outcomes. Here, we use mechanistic observations in a preclinical animal model to design an ex vivo platform that recreates genetic susceptibility to T-cell-mediated damage. Intestinal graft-versus-host disease (GVHD) is a life-threatening complication of allogeneic hematopoietic cell transplantation. We found that intestinal GVHD in mice deficient in Atg16L1, an autophagy gene that is polymorphic in humans, is reversed by inhibiting necroptosis. We further show that cocultured allogeneic T cells kill Atg16L1-mutant intestinal organoids from mice, which was associated with an aberrant epithelial interferon signature. Using this information, we demonstrate that pharmacologically inhibiting necroptosis or interferon signaling protects human organoids derived from individuals harboring a common ATG16L1 variant from allogeneic T-cell attack. Our study provides a roadmap for applying findings in animal models to individualized therapy that targets affected tissues.

RIPK3 collaborates with GSDMD to drive tissue injury in lethal polymicrobial sepsis.

Sepsis is a systemic inflammatory disease causing life-threatening multi-organ dysfunction. Accumulating evidences suggest that two forms of programmed necrosis, necroptosis and pyroptosis triggered by the pathogen component lipopolysaccharide (LPS) and inflammatory cytokines, play important roles in the development of bacterial sepsis-induced shock and tissue injury. Sepsis-induced shock and tissue injury required receptor-interacting protein kinase-3 (RIPK3) and mixed lineage kinase domain-like protein (MLKL) phosphorylation, caspase11 activation and gasdermin D (GSDMD) cleavage. However, the synergistic effect of necroptosis and pyroptosis in the pathological progress of sepsis remains elusive. In this study, we found that blockage of both necroptosis and pyroptosis (double deletion of Ripk3/Gsdmd or Mlkl/Gsdmd) resulted in accumulative protection against septic shock, systemic blood clotting and multi-organ injury in mice. Bone marrow transplantation confirmed that necroptosis and pyroptosis in both myeloid and nonmyeloid cells are indispensable in the progression of sepsis-induced multi-organ injury. Both RIPK3 and GSDMD signaling collaborated to amplify necroinflammation and tissue factor release in macrophages and endothelial cells, which led to tissue injury. Furthermore, cell death induced by inflammatory cytokines and high-mobility group box 1 could be prevented by double ablation of Ripk3/Gsdmd or Mlkl/Gsdmd, suggesting that a positive feedback loop interconnecting RIPK3/MLKL and GSDMD machinery and inflammation facilitated sepsis progression. Collectively, our findings demonstrated that RIPK3-mediated necroptosis and GSDMD-mediated pyroptosis collaborated to amply inflammatory signaling and enhance tissue injury in the process of sepsis, which may shed new light on two potential targets of combined therapeutic interventions for this highly lethal disorder.

Inhibitory effects of JQ1 on listeria monocytogenes-induced acute liver injury by blocking BRD4/RIPK1 axis.

Listeria monocytogenes (LM) is a facultative intracellular bacterium that causes septicemia-associated acute hepatic injury. However, the pathogenesis of this process is still unclear, and there is still a lack of effective therapeutic strategy for the treatment of LM-induced liver injury. In this study, we attempted to explore the effects of necroptosis on bacterial-septicemia-associated hepatic disease and to explore the contribution of JQ1, a selective BRD4 inhibitor, to the suppression of necroptosis and inhibition of LM-triggered hepatic injury. The results indicated that hepatic BRD4 was primarily stimulated by LM both in vitro and in vivo, along with significantly up-regulated expression of receptor-interacting protein kinase (RIPK)-1, RIPK3, and p-mixed lineage kinase-like (MLKL), showing the elevated necroptosis. However, JQ1 treatment and RIPK1 knockout were found to significantly alleviate LM-induced acute liver injury. Histological alterations and cell death in hepatic samples in LM-infected mice were also alleviated by JQ1 administration or RIPK1 deletion. However, JQ1-improved hepatic injury by LM was abrogated by RIPK1 over-expression, suggesting that the protective effects of JQ1 took place mainly in an RIPK1-dependent manner. In addition, LM-evoked inflammatory response in liver tissues were also alleviated by JQ1, which was similar to the findings observed in mice lacking RIPK1. The anti-inflammatory effects of JQ1 were diminished by RIPK1 over-expression in LM-infected mice. Finally, both in vivo and in vitro experiments suggested that JQ1 dramatically improved hepatic mitochondrial dysfunction in LM-injected mice, but this effect was abolished by RIPK1 over-expression. In conclusion, these results indicated that suppressing BRD4 by JQ1 could ameliorate LM-associated liver injury by suppressing necroptosis, inflammation, and mitochondrial dysfunction by inhibiting RIPK1.

RIP1 kinase activity is critical for skin inflammation but not for viral propagation.

Receptor interacting protein kinase 1 (RIP1) is a critical effector of inflammatory responses and cell death activation. Cell death pathways regulated by RIP1 include caspase-dependent apoptosis and caspase-independent necroptosis. The kinase activity of RIP1 has been associated with a number of inflammatory, neurodegenerative, and oncogenic diseases. In this study, we use the RIP1 kinase inhibitor GNE684 to demonstrate that RIP1 inhibition can effectively block skin inflammation and immune cell infiltrates in livers of Sharpin mutant (Cpdm; chronic proliferative dermatitis) mice in an interventional setting, after disease onset. On the other hand, genetic inactivation of RIP1 (RIP1 KD) or ablation of RIP3 (RIP3 KO) or MLKL (MLKL KO) did not affect testicular pathology of aging male mice. Likewise, infection with vaccinia virus or with mouse gammaherpesvirus MHV68 resulted in similar viral clearance in wild-type, RIP1 KD, and RIP3 KO mice. In summary, this study highlights the benefits of inhibiting RIP1 in skin inflammation, as opposed to its lack of relevance for testicular longevity and the response to certain viral infections.