Exposure of negative-sense viral RNA in the cytoplasm initiates innate immunity to West Nile virus.

For many RNA viruses, immunity is triggered when RIG-I-like receptors (RLRs) detect viral RNA. However, only a minority of infected cells undergo innate immune activation. By examining these "first responder" cells during West Nile virus infection, we found that specific accumulation of anti- genomic negative-sense viral RNA (-vRNA) underlies innate immune activation and that RIG-I preferentially interacts with -vRNA. However, flaviviruses sequester -vRNA into membrane-bound replication compartments away from cytosolic sensors. We found that single-stranded -vRNA accumulates outside of replication compartments in "first responder" cells, rendering it accessible to RLRs. Exposure of this -vRNA occurs at late timepoints of infection, is linked to viral assembly, and depends on the expression of viral structural proteins. These findings reveal that while most infected cells replicate high levels of vRNA, release of -vRNA from replication compartments during assembly occurs at low frequency and is critical for initiation of innate immunity during flavivirus infection.

Apoptotic cells promote circulating tumor cell survival and metastasis.

During tumor progression and especially following cytotoxic therapy, cell death of both tumor and stromal cells is widespread. Despite clinical observations that high levels of apoptotic cells correlate with poorer patient outcomes, the physiological effects of dying cells on tumor progression remain incompletely understood. Here, we report that circulating apoptotic cells robustly enhance tumor cell metastasis to the lungs. Using intravenous metastasis models, we observed that the presence of apoptotic cells, but not cells dying by other mechanisms, supports circulating tumor cell (CTC) survival following arrest in the lung vasculature. Apoptotic cells promote CTC survival by recruiting platelets to the forming metastatic niche. Apoptotic cells externalize the phospholipid phosphatidylserine to the outer leaflet of the plasma membrane, which we found increased the activity of the coagulation initiator Tissue Factor, thereby triggering the formation of platelet clots that protect proximal CTCs. Inhibiting the ability of apoptotic cells to induce coagulation by knocking out Tissue Factor, blocking phosphatidylserine, or administering the anticoagulant heparin abrogated the pro-metastatic effect of apoptotic cells. This work demonstrates a previously unappreciated role for apoptotic cells in facilitating metastasis by establishing CTC-supportive emboli, and suggests points of intervention that may reduce the pro-metastatic effect of apoptotic cells.

LITAF protects against pore-forming protein-induced cell death by promoting membrane repair.

Pore-forming toxins (PFTs) are the largest class of bacterial toxins and contribute to virulence by triggering host cell death. Vertebrates also express endogenous pore-forming proteins that induce cell death as part of host defense. To mitigate damage and promote survival, cells mobilize membrane repair mechanisms to neutralize and counteract pores, but how these pathways are activated is poorly understood. Here, we use a transposon-based gene activation screen to discover pathways that counteract the cytotoxicity of the archetypal PFT Staphylococcus aureus α-toxin. We identify the endolysosomal protein LITAF as a mediator of cellular resistance to PFT-induced cell death that is active against both bacterial toxins and the endogenous pore, gasdermin D, a terminal effector of pyroptosis. Activation of the ubiquitin ligase NEDD4 by potassium efflux mobilizes LITAF to recruit the endosomal sorting complexes required for transport (ESCRT) machinery to repair damaged membrane. Cells lacking LITAF, or carrying naturally occurring disease-associated mutations of LITAF, are highly susceptible to pore-induced death. Notably, LITAF-mediated repair occurs at endosomal membranes, resulting in expulsion of damaged membranes as exosomes, rather than through direct excision of pores from the surface plasma membrane. These results identify LITAF as a key effector that links sensing of cellular damage to repair.

Oligodendrocyte-derived IL-33 functions as a microglial survival factor during neuroinvasive flavivirus infection.

In order to recover from infection, organisms must balance robust immune responses to pathogens with the tolerance of immune-mediated pathology. This balance is particularly critical within the central nervous system, whose complex architecture, essential function, and limited capacity for self-renewal render it susceptible to both pathogen- and immune-mediated pathology. Here, we identify the alarmin IL-33 and its receptor ST2 as critical for host survival to neuroinvasive flavivirus infection. We identify oligodendrocytes as the critical source of IL-33, and microglia as the key cellular responders. Notably, we find that the IL-33/ST2 axis does not impact viral control or adaptive immune responses; rather, it is required to promote the activation and survival of microglia. In the absence of intact IL-33/ST2 signaling in the brain, neuroinvasive flavivirus infection triggered aberrant recruitment of monocyte-derived peripheral immune cells, increased neuronal stress, and neuronal cell death, effects that compromised organismal survival. These findings identify IL-33 as a critical mediator of CNS tolerance to pathogen-initiated immunity and inflammation.

CELLULAR RNA INTERACTS WITH MAVS TO PROMOTE ANTIVIRAL SIGNALING.

Immune signaling needs to be well-regulated to promote clearance of pathogens, while preventing aberrant inflammation. Interferons (IFNs) and antiviral genes are activated by the detection of viral RNA by RIG-I-like receptors (RLRs). Signal transduction downstream of RLRs proceeds through a multi-protein complex organized around the central adaptor protein MAVS. Recent work has shown that protein complex function can be modulated by RNA molecules providing allosteric regulation or acting as molecular guides or scaffolds. Thus, we hypothesized that RNA plays a role in organizing MAVS signaling platforms. Here, we show that MAVS, through its central intrinsically disordered domain, directly interacts with the 3' untranslated regions of cellular mRNAs. Importantly, elimination of RNA by RNase treatment disrupts the MAVS signalosome, including newly identified regulators of RLR signaling, and inhibits phosphorylation of the transcription factor IRF3. This supports the hypothesis that RNA molecules scaffold proteins in the MAVS signalosome to induce IFNs. Together, this work uncovers a function for cellular RNA in promoting signaling through MAVS and highlights a generalizable principle of RNA regulatory control of cytoplasmic immune signaling complexes.

Oligodendrocyte-derived IL-33 functions as a microglial survival factor during neuroinvasive flavivirus infection.

In order to recover from infection, organisms must balance robust immune responses to pathogens with the tolerance of immune-mediated pathology. This balance is particularly critical within the central nervous system, whose complex architecture, essential function, and limited capacity for self-renewal render it susceptible to both pathogen- and immune-mediated pathology. Here, we identify the alarmin IL-33 and its receptor ST2 as critical for host survival to neuroinvasive flavivirus infection. We identify oligodendrocytes as the critical source of IL-33, and microglia as the key cellular responders. Notably, we find that the IL-33/ST2 axis does not impact viral control or adaptive immune responses; rather, it is required to promote the activation and survival of microglia. In the absence of intact IL-33/ST2 signaling in the brain, neuroinvasive flavivirus infection triggered aberrant recruitment of monocyte-derived peripheral immune cells, increased neuronal stress, and neuronal cell death, effects that compromised organismal survival. These findings identify IL-33 as a critical mediator of CNS tolerance to pathogen-initiated immunity and inflammation.

Human ZBP1 induces cell death-independent inflammatory signaling via RIPK3 and RIPK1.

ZBP1 is an interferon-induced cytosolic nucleic acid sensor that facilitates antiviral responses via RIPK3. Although ZBP1-mediated programmed cell death is widely described, whether and how it promotes inflammatory signaling is unclear. Here, we report a ZBP1-induced inflammatory signaling pathway mediated by K63- and M1-linked ubiquitin chains, which depends on RIPK1 and RIPK3 as scaffolds independently of cell death. In human HT29 cells, ZBP1 associated with RIPK1 and RIPK3 as well as ubiquitin ligases cIAP1 and LUBAC. ZBP1-induced K63- and M1-linked ubiquitination of RIPK1 and ZBP1 to promote TAK1- and IKK-mediated inflammatory signaling and cytokine production. Inhibition of caspase activity suppressed ZBP1-induced cell death but enhanced cytokine production in a RIPK1- and RIPK3 kinase activity-dependent manner. Lastly, we provide evidence that ZBP1 signaling contributes to SARS-CoV-2-induced cytokine production. Taken together, we describe a ZBP1-RIPK3-RIPK1-mediated inflammatory signaling pathway relayed by the scaffolding role of RIPKs and regulated by caspases, which may induce inflammation when ZBP1 is activated below the threshold needed to trigger a cell death response.

RIPK1 and RIPK3 regulate TNFα-induced ß-cell death in concert with caspase activity.

OBJECTIVE: Type 1 diabetes (T1D) is characterized by autoimmune-associated ß-cell loss, insulin insufficiency, and hyperglycemia. Although TNFα signaling is associated with ß-cell loss and hyperglycemia in non-obese diabetic mice and human T1D, the molecular mechanisms of ß-cell TNF receptor signaling have not been fully characterized. Based on work in other cell types, we hypothesized that receptor interacting protein kinase 1 (RIPK1) and receptor interacting protein kinase 3 (RIPK3) regulate TNFα-induced ß-cell death in concert with caspase activity. METHODS: We evaluated TNFα-induced cell death, caspase activity, and TNF receptor pathway molecule expression in immortalized NIT-1 and INS-1 ß-cell lines and primary mouse islet cells in vitro. Our studies utilized genetic and small molecule approaches to alter RIPK1 and RIPK3 expression and caspase activity to interrogate mechanisms of TNFα-induced ß-cell death. We used the ß-cell toxin streptozotocin (STZ) to determine the susceptibility of Ripk3+/+ and Ripk3-/- mice to hyperglycemia in vivo. RESULTS: Expression of TNF receptor signaling molecules including RIPK1 and RIPK3 was identified in NIT-1 and INS-1 ß cells and isolated mouse islets at the mRNA and protein levels. TNFα treatment increased NIT-1 and INS-1 cell death and caspase activity after 24-48 h, and BV6, a small molecule inhibitor of inhibitor of apoptosis proteins (IAPs) amplified this TNFα-induced cell death. RIPK1 deficient NIT-1 cells were protected from TNFα- and BV6-induced cell death and caspase activation. Interestingly, small molecule inhibition of caspases with zVAD-fmk (zVAD) did not prevent TNFα-induced cell death in either NIT-1 or INS-1 cells. This caspase-independent cell death was increased by BV6 treatment and decreased in RIPK1 deficient NIT-1 cells. RIPK3 deficient NIT-1 cells and RIPK3 kinase inhibitor treated INS-1 cells were protected from TNFα+zVAD-induced cell death, whereas RIPK3 overexpression increased INS-1 cell death and promoted RIPK3 and MLKL interaction under TNFα+zVAD treatment. In mouse islet cells, BV6 or zVAD treatment promoted TNFα-induced cell death, and TNFα+zVAD-induced cell death was blocked by RIPK3 inhibition and in Ripk3-/- islet cells in vitro. Ripk3-/- mice were also protected from STZ-induced hyperglycemia and glucose intolerance in vivo. CONCLUSIONS: RIPK1 and RIPK3 regulate TNFα-induced ß-cell death in concert with caspase activity in immortalized and primary islet ß cells. TNF receptor signaling molecules such as RIPK1 and RIPK3 may represent novel therapeutic targets to promote ß-cell survival and glucose homeostasis in T1D.

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.

The Antisocial Network: Cross Talk Between Cell Death Programs in Host Defense.

Nearly all animal cells contain proteins evolved to trigger the destruction of the cell in which they reside. The activation of these proteins occurs via sequential programs, and much effort has been expended in delineating the molecular mechanisms underlying the resulting processes of programmed cell death (PCD). These efforts have led to the definition of apoptosis as a form of nonimmunogenic PCD that is required for normal development and tissue homeostasis, and of pyroptosis and necroptosis as forms of PCD initiated by pathogen infection that are associated with inflammation and immune activation. While this paradigm has served the field well, numerous recent studies have highlighted cross talk between these programs, challenging the idea that apoptosis, pyroptosis, and necroptosis are linear pathways with defined immunological outputs. Here, we discuss the emerging idea of cell death as a signaling network, considering connections between cell death pathways both as we observe them now and in their evolutionary origins. We also discuss the engagement and subversion of cell death pathways by pathogens, as well as the key immunological outcomes of these processes.

De novo necroptosis creates an inflammatory environment mediating tumor susceptibility to immune checkpoint inhibitors.

Cancer immunotherapies using monoclonal antibodies to block inhibitory checkpoints are showing durable remissions in many types of cancer patients, although the majority of breast cancer patients acquire little benefit. Human melanoma and lung cancer patient studies suggest that immune checkpoint inhibitors are often potent in patients that already have intratumoral T cell infiltrate; although it remains unknown what types of interventions can result in an intratumoral T cell infiltrate in breast cancer. Using non-T cell-inflamed mammary tumors, we assessed what biological processes and downstream inflammation can overcome the barriers to spontaneous T cell priming. Here we show a specific type of combination therapy, consisting of oncolytic virus and chemotherapy, activates necroptosis and limits tumor growth in autochthonous tumors. Combination therapy activates proinflammatory cytokines; intratumoral influx of myeloid cells and cytotoxic T cell infiltrate in locally treated and distant autochthonous tumors to render them susceptible to immune checkpoint inhibitors.

Identification of MYC as an antinecroptotic protein that stifles RIPK1-RIPK3 complex formation.

The underlying mechanism of necroptosis in relation to cancer is still unclear. Here, MYC, a potent oncogene, is an antinecroptotic factor that directly suppresses the formation of the RIPK1-RIPK3 complex. Gene set enrichment analyses reveal that the MYC pathway is the most prominently down-regulated signaling pathway during necroptosis. Depletion or deletion of MYC promotes the RIPK1-RIPK3 interaction, thereby stabilizing the RIPK1 and RIPK3 proteins and facilitating necroptosis. Interestingly, MYC binds to RIPK3 in the cytoplasm and inhibits the interaction between RIPK1 and RIPK3 in vitro. Furthermore, MYC-nick, a truncated form that is mainly localized in the cytoplasm, prevented TNF-induced necroptosis. Finally, down-regulation of MYC enhances necroptosis in leukemia cells and suppresses tumor growth in a xenograft model upon treatment with birinapant and emricasan. MYC-mediated suppression of necroptosis is a mechanism of necroptosis resistance in cancer, and approaches targeting MYC to induce necroptosis represent an attractive therapeutic strategy for cancer.

Outcomes of RIP Kinase Signaling During Neuroinvasive Viral Infection.

Neuroinvasive viral diseases are a considerable and growing burden on global public health. Despite this, these infections remain poorly understood, and the molecular mechanisms that govern protective versus pathological neuroinflammatory responses to infection are a matter of intense investigation. Recent evidence suggests that necroptosis, an immunogenic form of programmed cell death, may contribute to the pathogenesis of viral encephalitis. However, the receptor-interacting protein (RIP) kinases that coordinate necroptosis, RIPK1 and RIPK3, also appear to have unexpected, cell death-independent functions in the central nervous system (CNS) that promote beneficial neuroinflammation during neuroinvasive infection. Here, we review the emerging evidence in this field, with additional discussion of recent work examining roles for RIPK signaling and necroptosis during noninfectious pathologies of the CNS, as these studies provide important additional insight into the potential for specialized neuroimmune functions for the RIP kinases.

T cells instruct myeloid cells to produce inflammasome-independent IL-1ß and cause autoimmunity.

The cytokine interleukin (IL)-1ß is a key mediator of antimicrobial immunity as well as autoimmune inflammation. Production of IL-1ß requires transcription by innate immune receptor signaling and maturational cleavage by inflammasomes. Whether this mechanism applies to IL-1ß production seen in T cell-driven autoimmune diseases remains unclear. Here, we describe an inflammasome-independent pathway of IL-1ß production that was triggered upon cognate interactions between effector CD4+ T cells and mononuclear phagocytes (MPs). The cytokine TNF produced by activated CD4+ T cells engaged its receptor TNFR on MPs, leading to pro-IL-1ß synthesis. Membrane-bound FasL, expressed by CD4+ T cells, activated death receptor Fas signaling in MPs, resulting in caspase-8-dependent pro-IL-1ß cleavage. The T cell-instructed IL-1ß resulted in systemic inflammation, whereas absence of TNFR or Fas signaling protected mice from CD4+ T cell-driven autoimmunity. The TNFR-Fas-caspase-8-dependent pathway provides a mechanistic explanation for IL-1ß production and its consequences in CD4+ T cell-driven autoimmune pathology.

Universal Principled Review: A Community-Driven Method to Improve Peer Review.

Despite being a staple of our science, the process of pre-publication peer review has few agreed-upon standards defining its goals or ideal execution. As a community of reviewers and authors, we assembled an evaluation format and associated specific standards for the process as we think it should be practiced. We propose that we apply, debate, and ultimately extend these to improve the transparency of our criticism and the speed with which quality data and ideas become public.

RIPK3 Activation Leads to Cytokine Synthesis that Continues after Loss of Cell Membrane Integrity.

Necroptosis is a form of programmed cell death that is defined by activation of the kinase RIPK3 and subsequent cell membrane permeabilization by the effector MLKL. RIPK3 activation can also promote immune responses via production of cytokines and chemokines. How active cytokine production is coordinated with the terminal process of necroptosis is unclear. Here, we report that cytokine production continues within necroptotic cells even after they have lost cell membrane integrity and irreversibly committed to death. This continued cytokine production is dependent on mRNA translation and requires maintenance of endoplasmic reticulum integrity that remains after plasma membrane integrity is lost. The continued translation of cytokines by cellular corpses contributes to necroptotic cell uptake by innate immune cells and priming of adaptive immune responses to antigens associated with necroptotic corpses. These findings imply that cell death and production of inflammatory mediators are coordinated to optimize the immunogenicity of necroptotic cells.

STING is required for host defense against neuropathological West Nile virus infection.

West Nile Virus (WNV), an emerging and re-emerging RNA virus, is the leading source of arboviral encephalitic morbidity and mortality in the United States. WNV infections are acutely controlled by innate immunity in peripheral tissues outside of the central nervous system (CNS) but WNV can evade the actions of interferon (IFN) to facilitate CNS invasion, causing encephalitis, encephalomyelitis, and death. Recent studies indicate that STimulator of INterferon Gene (STING), canonically known for initiating a type I IFN production and innate immune response to cytosolic DNA, is required for host defense against neurotropic RNA viruses. We evaluated the role of STING in host defense to control WNV infection and pathology in a murine model of infection. When challenged with WNV, STING knock out (-/-) mice displayed increased morbidity and mortality compared to wild type (WT) mice. Virologic analysis and assessment of STING activation revealed that STING signaling was not required for control of WNV in the spleen nor was WNV sufficient to mediate canonical STING activation in vitro. However, STING-/- mice exhibited a clear trend of increased viral load and virus dissemination in the CNS. We found that STING-/- mice exhibited increased and prolonged neurological signs compared to WT mice. Pathological examination revealed increased lesions, mononuclear cellular infiltration and neuronal death in the CNS of STING-/- mice, with sustained pathology after viral clearance. We found that STING was required in bone marrow derived macrophages for early control of WNV replication and innate immune activation. In vivo, STING-/- mice developed an aberrant T cell response in both the spleen and brain during WNV infection that linked with increased and sustained CNS pathology compared to WT mice. Our findings demonstrate that STING plays a critical role in immune programming for the control of neurotropic WNV infection and CNS disease.

Intratumoral activation of the necroptotic pathway components RIPK1 and RIPK3 potentiates antitumor immunity.

Although the signaling events that induce different forms of programmed cell death are well defined, the subsequent immune responses to dying cells in the context of cancer remain relatively unexplored. Necroptosis occurs downstream of the receptor-interacting protein kinases RIPK1 and RIPK3, whose activation leads to lytic cell death accompanied by de novo production of proinflammatory mediators. Here, we show that ectopic introduction of necroptotic cells to the tumor microenvironment promotes BATF3+ cDC1- and CD8+ leukocyte-dependent antitumor immunity accompanied by increased tumor antigen loading by tumor-associated antigen-presenting cells. Furthermore, we report the development of constitutively active forms of the necroptosis-inducing enzyme RIPK3 and show that delivery of a gene encoding this enzyme to tumor cells using adeno-associated viruses induces tumor cell necroptosis, which synergizes with immune checkpoint blockade to promote durable tumor clearance. These findings support a role for RIPK1/RIPK3 activation as a beneficial proximal target in the initiation of tumor immunity. Considering that successful tumor immunotherapy regimens will require the rational application of multiple treatment modalities, we propose that maximizing the immunogenicity of dying cells within the tumor microenvironment through specific activation of the necroptotic pathway represents a beneficial treatment approach that may warrant further clinical development.

The Nucleotide Sensor ZBP1 and Kinase RIPK3 Induce the Enzyme IRG1 to Promote an Antiviral Metabolic State in Neurons.

As long-lived post-mitotic cells, neurons employ unique strategies to resist pathogen infection while preserving cellular function. Here, using a murine model of Zika virus (ZIKV) infection, we identified an innate immune pathway that restricts ZIKV replication in neurons and is required for survival upon ZIKV infection of the central nervous system (CNS). We found that neuronal ZIKV infection activated the nucleotide sensor ZBP1 and the kinases RIPK1 and RIPK3, core components of virus-induced necroptotic cell death signaling. However, activation of this pathway in ZIKV-infected neurons did not induce cell death. Rather, RIPK signaling restricted viral replication by altering cellular metabolism via upregulation of the enzyme IRG1 and production of the metabolite itaconate. Itaconate inhibited the activity of succinate dehydrogenase, generating a metabolic state in neurons that suppresses replication of viral genomes. These findings demonstrate an immunometabolic mechanism of viral restriction during neuroinvasive infection.

Comparing the effects of different cell death programs in tumor progression and immunotherapy.

Our conception of programmed cell death has expanded beyond apoptosis to encompass additional forms of cell suicide, including necroptosis and pyroptosis; these cell death modalities are notable for their diverse and emerging roles in engaging the immune system. Concurrently, treatments that activate the immune system to combat cancer have achieved remarkable success in the clinic. These two scientific narratives converge to provide new perspectives on the role of programmed cell death in cancer therapy. This review focuses on our current understanding of the relationship between apoptosis and antitumor immune responses and the emerging evidence that induction of alternate death pathways such as necroptosis could improve therapeutic outcomes.