Necroptosis stimulates interferon-mediated protective anti-tumor immunity.

Necroptosis is an inflammatory form of cell suicide that critically depends on the kinase activity of Receptor Interacting Protein Kinase 3 (RIPK3). Previous studies showed that immunization with necroptotic cells conferred protection against subsequent tumor challenge. Since RIPK3 can also promote apoptosis and NF-κB-dependent inflammation, it remains difficult to determine the contribution of necroptosis-associated release of damage-associated molecular patterns (DAMPs) in anti-tumor immunity. Here, we describe a system that allows us to selectively induce RIPK3-dependent necroptosis or apoptosis with minimal NF-κB-dependent inflammatory cytokine expression. In a syngeneic tumor challenge model, immunization with necroptotic cells conferred superior protection against subsequent tumor challenge. Surprisingly, this protective effect required CD4+ T cells rather than CD8+ T cells and is dependent on host type I interferon signaling. Our results provide evidence that death-dependent type I interferon production following necroptosis is sufficient to elicit protective anti-tumor immunity.

A second-generation M1-polarized CAR macrophage with antitumor efficacy.

Chimeric antigen receptor (CAR) T cell therapies have successfully treated hematological malignancies. Macrophages have also gained attention as an immunotherapy owing to their immunomodulatory capacity and ability to infiltrate solid tumors and phagocytize tumor cells. The first-generation CD3ζ-based CAR-macrophages could phagocytose tumor cells in an antigen-dependent manner. Here we engineered induced pluripotent stem cell-derived macrophages (iMACs) with toll-like receptor 4 intracellular toll/IL-1R (TIR) domain-containing CARs resulting in a markedly enhanced antitumor effect over first-generation CAR-macrophages. Moreover, the design of a tandem CD3ζ-TIR dual signaling CAR endows iMACs with both target engulfment capacity and antigen-dependent M1 polarization and M2 resistance in a nuclear factor kappa B (NF-κB)-dependent manner, as well as the capacity to modulate the tumor microenvironment. We also outline a mechanism of tumor cell elimination by CAR-induced efferocytosis against tumor cell apoptotic bodies. Taken together, we provide a second-generation CAR-iMAC with an ability for orthogonal phagocytosis and polarization and superior antitumor functions in treating solid tumors relative to first-generation CAR-macrophages.

Necroptosis at a glance.

Necroptosis, or programmed necrosis, is an inflammatory form of cell death with important functions in host defense against pathogens and tissue homeostasis. The four cytosolic receptor-interacting protein kinase homotypic interaction motif (RHIM)-containing adaptor proteins RIPK1, RIPK3, TRIF (also known as TICAM1) and ZBP1 mediate necroptosis induction in response to infection and cytokine or innate immune receptor activation. Activation of the RHIM adaptors leads to phosphorylation, oligomerization and membrane targeting of the necroptosis effector protein mixed lineage kinase domain-like (MLKL). Active MLKL induces lesions on the plasma membrane, leading to the release of pro-inflammatory damage-associated molecular patterns (DAMPs). Thus, activities of the RHIM adaptors and MLKL are tightly regulated by posttranslational modifications to prevent inadvertent release of immunogenic contents. In this Cell Science at a Glance article and the accompanying poster, we provide an overview of the regulatory mechanisms of necroptosis and its biological functions in tissue homeostasis, pathogen infection and other inflammatory diseases.

Tumor-intrinsic and immune modulatory roles of receptor-interacting protein kinases.

Receptor-interacting protein kinase 1 (RIPK1) and RIPK3 are signaling adaptors that critically regulate cell death and inflammation. Tumors have adapted to subvert RIPK-dependent cell death, suggesting that these processes have key roles in tumor regulation. Moreover, RIPK-driven cancer cell death might bolster durable antitumor immunity. By contrast, there are examples in which RIPKs induce inflammation and aid tumor progression. Furthermore, the RIPKs can exert their effects on tumor growth through regulating the activity of immune effectors in the tumor microenvironment, thus highlighting the context-dependent roles of RIPKs. Here, we review recent advances in the regulation of RIPK activity in tumors and immune cells and how these processes coordinate with each other to control tumorigenesis.

A class of viral inducer of degradation of the necroptosis adaptor RIPK3 regulates virus-induced inflammation.

The vaccine strain against smallpox, vaccinia virus (VACV), is highly immunogenic yet causes relatively benign disease. These attributes are believed to be caused by gene loss in VACV. Using a targeted small interfering RNA (siRNA) screen, we identified a viral inhibitor found in cowpox virus (CPXV) and other orthopoxviruses that bound to the host SKP1-Cullin1-F-box (SCF) machinery and the essential necroptosis kinase receptor interacting protein kinase 3 (RIPK3). This "viral inducer of RIPK3 degradation" (vIRD) triggered ubiquitination and proteasome-mediated degradation of RIPK3 and inhibited necroptosis. In contrast to orthopoxviruses, the distantly related leporipoxvirus myxoma virus (MYXV), which infects RIPK3-deficient hosts, lacks a functional vIRD. Introduction of vIRD into VACV, which encodes a truncated and defective vIRD, enhanced viral replication in mice. Deletion of vIRD reduced CPXV-induced inflammation, viral replication, and mortality, which were reversed in RIPK3- and MLKL-deficient mice. Hence, vIRD-RIPK3 drives pathogen-host evolution and regulates virus-induced inflammation and pathogenesis.

Regulatory mechanisms of RIPK1 in cell death and inflammation.

Receptor Interacting Protein Kinase 1 (RIPK1) and RIPK3 are key adaptors that play critical roles in inflammatory and cell death signaling. Work in recent years have shown that their activities are tightly regulated by ubiquitination, phosphorylation and proteolysis. In addition to these post-translational modifications, the expression and activities of these kinases can further be tuned by environmental changes in pH and oxygen content. Proper control of these regulatory processes is crucial for the RIP kinases to execute their functions in immune responses and tissue homeostasis. In this review, we discuss recent advance in our understanding of the molecular mechanisms that regulate the activities of the RIP kinases. We will also discuss how the different regulatory mechanisms contribute to the functions of RIPK1 and RIPK3 in different pathophysiological settings.

Necroptosis in anti-viral inflammation.

The primary function of the immune system is to protect the host from invading pathogens. In response, microbial pathogens have developed various strategies to evade detection and destruction by the immune system. This tug-of-war between the host and the pathogen is a powerful force that shapes organismal evolution. Regulated cell death (RCD) is a host response that limits the reservoir for intracellular pathogens such as viruses. Since pathogen-specific T cell and B cell responses typically take several days and is therefore slow-developing, RCD of infected cells during the first few days of the infection is critical for organismal survival. This innate immune response not only restricts viral replication, but also serves to promote anti-viral inflammation through cell death-associated release of damage-associated molecular patterns (DAMPs). In recent years, necroptosis has been recognized as an important response against many viruses. The central adaptor for necroptosis, RIPK3, also exerts anti-viral effects through cell death-independent activities such as promoting cytokine gene expression. Here, we will discuss recent advances on how viruses counteract this host defense mechanism and the effect of necroptosis on the anti-viral inflammatory reaction.

Leishmania braziliensis Subverts Necroptosis by Modulating RIPK3 Expression.

Leishmania braziliensis infection causes skin ulcers, typically found in localized cutaneous leishmaniasis (LCL). This tissue pathology associates with different modalities of cell necrosis, which are subverted by the parasite as a survival strategy. Herein we examined the participation of necroptosis, a specific form of programmed necrosis, in LCL lesions and found reduced RIPK3 and PGAM5 gene expression compared to normal skin. Assays using infected macrophages demonstrated that the parasite deactivates both RIPK3 and MLKL expression and that these molecules are important to control the intracellular L. braziliensis replication. Thus, LCL-related necroptosis may be targeted to control infection and disease immunopathology.

Loss-of-Function RNAi Screen to Identify Necrosis-Signaling Molecules.

Over the recent years, genome-wide RNA interference (RNAi) library screens have been instrumental in the identification of key regulators of various biological pathways. The prolific use of this technique is attributed to its amenability to a high-throughput format. Here, we present the step-by-step method to conduct a siRNA screen to identify genes involved in necroptosis, a nonapoptotic form of proinflammatory cell death. The method described here uses MTS cell proliferation assay to measure necroptosis, which is compatible with high-throughput format screening on multiwell microtiter plates. This ensures that the screen can be performed in a timely and efficient manner.

Inflammasome, Inflammation, and Tissue Homeostasis.

Organismal fitness demands proper response to neutralize the threat from infection or injury. At the mammalian intestinal epithelium barrier, the inflammasome coordinates an elaborate tissue repair response marked by the induction of antimicrobial peptides, wound-healing cytokines, and reparative proliferation of epithelial stem cells. The inflammasome in myeloid and intestinal epithelial compartments exerts these effects in part through maintenance of a healthy microbiota. Disease-associated mutations and elevated expression of certain inflammasome sensors have been identified. In many cases, inhibition of inflammasome activity has dramatic effects on disease outcome in mouse models of experimental colitis. Here, we discuss recent studies on the role of distinct inflammasome sensors in intestinal homeostasis and how this knowledge may be translated into a therapeutic setting.

Peptidoglycan-Sensing Receptors Trigger the Formation of Functional Amyloids of the Adaptor Protein Imd to Initiate Drosophila NF-κB Signaling.

In the Drosophila immune response, bacterial derived diaminopimelic acid-type peptidoglycan binds the receptors PGRP-LC and PGRP-LE, which through interaction with the adaptor protein Imd leads to activation of the NF-κB homolog Relish and robust antimicrobial peptide gene expression. PGRP-LC, PGRP-LE, and Imd each contain a motif with some resemblance to the RIP Homotypic Interaction Motif (RHIM), a domain found in mammalian RIPK proteins forming functional amyloids during necroptosis. Here we found that despite sequence divergence, these Drosophila cryptic RHIMs formed amyloid fibrils in vitro and in cells. Amyloid formation was required for signaling downstream of Imd, and in contrast to the mammalian RHIMs, was not associated with cell death. Furthermore, amyloid formation constituted a regulatable step and could be inhibited by Pirk, an endogenous feedback regulator of this pathway. Thus, diverse sequence motifs are capable of forming amyloidal signaling platforms, and the formation of these platforms may present a regulatory point in multiple biological processes.

Distinct Kinase-Independent Role of RIPK3 in CD11c+ Mononuclear Phagocytes in Cytokine-Induced Tissue Repair.

Receptor interacting protein kinase 3 (RIPK3) induces necroptosis, a type of regulated necrosis, through its kinase domain and receptor interacting protein (RIP) homotypic interaction motif (RHIM). In addition, RIPK3 has been shown to regulate NLRP3 inflammasome and nuclear factor κB (NF-κB) activation. However, the relative contribution of these signaling pathways to RIPK3-dependent inflammation in distinct immune effectors is unknown. To investigate these questions, we generated RIPK3-GFP reporter mice. We found that colonic CD11c+CD11b+CD14+ mononuclear phagocytes (MNPs) expressed the highest level of RIPK3 in the lamina propria. Consequently, deletion of the RIPK3 RHIM in CD11c+ cells alone was sufficient to impair dextran sodium sulfate (DSS)-induced interleukin (IL)-23 and IL-1ß expression, leading to severe intestinal inflammation. In contrast, mice expressing kinase inactive RIPK3 were not hypersensitive to DSS. Thus, a key physiological function of RIPK3 is to promote reparative cytokine expression through intestinal CD11c+ MNPs in a kinase- and necroptosis-independent manner.

Necroptosis: Mechanisms and Relevance to Disease.

Necroptosis is a form of regulated cell death that critically depends on receptor-interacting serine-threonine kinase 3 (RIPK3) and mixed lineage kinase domain-like (MLKL) and generally manifests with morphological features of necrosis. The molecular mechanisms that underlie distinct instances of necroptosis have just begun to emerge. Nonetheless, it has already been shown that necroptosis contributes to cellular demise in various pathophysiological conditions, including viral infection, acute kidney injury, and cardiac ischemia/reperfusion. Moreover, human tumors appear to obtain an advantage from the downregulation of key components of the molecular machinery for necroptosis. Although such an advantage may stem from an increased resistance to adverse microenvironmental conditions, accumulating evidence indicates that necroptosis-deficient cancer cells are poorly immunogenic and hence escape natural and therapy-elicited immunosurveillance. Here, we discuss the molecular mechanisms and relevance to disease of necroptosis.

Border Security: The Role of RIPK3 in Epithelium Homeostasis.

Receptor interacting protein kinase 3 (RIPK3) is a crucial inducer of necroptosis. Its activity is controlled by interaction with other signal adaptors through the "RIP homotypic interaction motif" (RHIM). Recent studies revealed a critical function for RIPK3 in the maintenance of epithelial tissue integrity. In mice with genetic deficiency of the apoptosis adaptors FADD or caspase 8, RIPK3 promotes necroptotic cell death of epithelial cells, leading to excessive and lethal inflammation. In contrast, when FADD and caspase 8 functions are intact, RIPK3 serves as a protector of intestinal epithelial integrity by promoting injury-induced wound repair. In the latter case, RIPK3 promotes optimal cytokine expression by cells of hematopoietic origin. Specifically, bone marrow derived dendritic cells (BMDCs) have an obligate requirement for RIPK3 for optimal secretion of mature IL-1ß and other inflammatory cytokines in response to toll-like receptor 4 (TLR4) stimulation. RIPK3 promotes cytokine expression through two complementary mechanisms: NF-κB dependent gene transcription and processing of pro-IL-1ß. We propose that RIPK3 functions in different cell compartments to mediate inflammation through distinct mechanisms.

RIPK3 Slams the Brake on Leukemogenesis.

Evasion of cell death is a key hallmark of cancers. In this issue of Cancer Cell, Höckendorf and colleagues identified RIPK3, an essential kinase for necroptosis, as having a key role in inhibiting acute myeloid leukemia development.

RIPK1 and PGAM5 Control Leishmania Replication through Distinct Mechanisms.

Leishmaniasis is an important parasitic disease found in the tropics and subtropics. Cutaneous and visceral leishmaniasis affect an estimated 1.5 million people worldwide. Despite its human health relevance, relatively little is known about the cell death pathways that control Leishmania replication in the host. Necroptosis is a recently identified form of cell death with potent antiviral effects. Receptor interacting protein kinase 1 (RIPK1) is a critical kinase that mediates necroptosis downstream of death receptors and TLRs. Heme, a product of hemoglobin catabolism during certain intracellular pathogen infections, is also a potent inducer of macrophage necroptosis. We found that human visceral leishmaniasis patients exhibit elevated serum levels of heme. Therefore, we examined the impact of heme and necroptosis on Leishmania replication. Indeed, heme potently inhibited Leishmania replication in bone marrow-derived macrophages. Moreover, we found that inhibition of RIPK1 kinase activity also enhanced parasite replication in the absence of heme. We further found that the mitochondrial phosphatase phosphoglycerate mutase family member 5 (PGAM5), a putative downstream effector of RIPK1, was also required for inhibition of Leishmania replication. In mouse infection, both PGAM5 and RIPK1 kinase activity are required for IL-1ß expression in response to Leishmania However, PGAM5, but not RIPK1 kinase activity, was directly responsible for Leishmania-induced IL-1ß secretion and NO production in bone marrow-derived macrophages. Collectively, these results revealed that RIPK1 and PGAM5 function independently to exert optimal control of Leishmania replication in the host.

Necroptosis-independent signaling by the RIP kinases in inflammation.

Recent advances have identified a signaling cascade involving receptor interacting protein kinase 1 (RIPK1), RIPK3 and the pseudokinase mixed lineage kinase domain-like (MLKL) that is crucial for induction of necroptosis, a non-apoptotic form of cell death. RIPK1-RIPK3-MLKL-mediated necroptosis has been attributed to cause many inflammatory diseases through the release of cellular damage-associated molecular patterns (DAMPs). In addition to necroptosis, emerging evidence suggests that these necroptosis signal adaptors can also facilitate inflammation independent of cell death. In particular, the RIP kinases can drive NF-κB and inflammasome activation independent of cell death. In this review, we will discuss recent discoveries that led to this realization and present arguments why cell death-independent signaling by the RIP kinases may have a more important role in inflammation than necroptosis.

Regulation of RIPK3- and RHIM-dependent Necroptosis by the Proteasome.

Receptor-interacting protein kinase 3 (RIPK3) is a serine/threonine kinase with essential function in necroptosis. The activity of RIPK3 is controlled by phosphorylation. Once activated, RIPK3 phosphorylates and activates the downstream effector mixed lineage kinase domain-like (MLKL) to induce necroptosis. In certain situations, RIPK3 has also been shown to promote apoptosis or cytokine expression in a necroptosis and kinase-independent manner. The ubiquitin-proteasome system is the major pathway for selective degradation of cellular proteins and thus has a critical role in many cellular processes such as cell survival and cell death. Clinically, proteasome inhibition has shown promise as an anti-cancer agent. Here we show that the proteasome inhibitors MG132 and bortezomib activate the RIPK3-MLKL necroptotic pathway in mouse fibroblasts as well as human leukemia cells. Unlike necroptosis induced by classical TNF-like cytokines, necroptosis induced by proteasome inhibitors does not require caspase inhibition. However, an intact RIP homotypic interaction motif (RHIM) is essential. Surprisingly, when recruitment of MLKL to RIPK3 is restricted, proteasome inhibitors induced RIPK3-dependent apoptosis. Proteasome inhibition led to accumulation of K48-linked ubiquitinated RIPK3, which was partially reduced when Lys-264 was mutated. Taken together, these results reveal the ubiquitin-proteasome system as a novel regulatory mechanism for RIPK3-dependent necroptosis.