Knocking 'em Dead: Pore-Forming Proteins in Immune Defense.

Immune cells use a variety of membrane-disrupting proteins [complement, perforin, perforin-2, granulysin, gasdermins, mixed lineage kinase domain-like pseudokinase (MLKL)] to induce different kinds of death of microbes and host cells, some of which cause inflammation. After activation by proteolytic cleavage or phosphorylation, these proteins oligomerize, bind to membrane lipids, and disrupt membrane integrity. These membrane disruptors play a critical role in both innate and adaptive immunity. Here we review our current knowledge of the functions, specificity, activation, and regulation of membrane-disrupting immune proteins and what is known about the mechanisms behind membrane damage, the structure of the pores they form, how the cells expressing these lethal proteins are protected, and how cells targeted for destruction can sometimes escape death by repairing membrane damage.

Influenza Virus Z-RNAs Induce ZBP1-Mediated Necroptosis.

Influenza A virus (IAV) is a lytic RNA virus that triggers receptor-interacting serine/threonine-protein kinase 3 (RIPK3)-mediated pathways of apoptosis and mixed lineage kinase domain-like pseudokinase (MLKL)-dependent necroptosis in infected cells. ZBP1 initiates RIPK3-driven cell death by sensing IAV RNA and activating RIPK3. Here, we show that replicating IAV generates Z-RNAs, which activate ZBP1 in the nucleus of infected cells. ZBP1 then initiates RIPK3-mediated MLKL activation in the nucleus, resulting in nuclear envelope disruption, leakage of DNA into the cytosol, and eventual necroptosis. Cell death induced by nuclear MLKL was a potent activator of neutrophils, a cell type known to drive inflammatory pathology in virulent IAV disease. Consequently, MLKL-deficient mice manifest reduced nuclear disruption of lung epithelia, decreased neutrophil recruitment into infected lungs, and increased survival following a lethal dose of IAV. These results implicate Z-RNA as a new pathogen-associated molecular pattern and describe a ZBP1-initiated nucleus-to-plasma membrane "inside-out" death pathway with potentially pathogenic consequences in severe cases of influenza.

PARP5A and RNF146 phase separation restrains RIPK1-dependent necroptosis.

Phase separation is a vital mechanism that mediates the formation of biomolecular condensates and their functions. Necroptosis is a lytic form of programmed cell death mediated by RIPK1, RIPK3, and MLKL downstream of TNFR1 and has been implicated in mediating many human diseases. However, whether necroptosis is regulated by phase separation is not yet known. Here, we show that upon the induction of necroptosis and recruitment by the adaptor protein TAX1BP1, PARP5A and its binding partner RNF146 form liquid-like condensates by multivalent interactions to perform poly ADP-ribosylation (PARylation) and PARylation-dependent ubiquitination (PARdU) of activated RIPK1 in mouse embryonic fibroblasts. We show that PARdU predominantly occurs on the K376 residue of mouse RIPK1, which promotes proteasomal degradation of kinase-activated RIPK1 to restrain necroptosis. Our data demonstrate that PARdU on K376 of mouse RIPK1 provides an alternative cell death checkpoint mediated by phase separation-dependent control of necroptosis by PARP5A and RNF146.

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.

Genetic Regulation of RIPK1 and Necroptosis.

The receptor-interacting protein kinase 1 (RIPK1) is recognized as a master upstream regulator that controls cell survival and inflammatory signaling as well as multiple cell death pathways, including apoptosis and necroptosis. The activation of RIPK1 kinase is extensively modulated by ubiquitination and phosphorylation, which are mediated by multiple factors that also control the activation of the NF-κB pathway. We discuss current findings regarding the genetic modulation of RIPK1 that controls its activation and interaction with downstream mediators, such as caspase-8 and RIPK3, to promote apoptosis and necroptosis. We also address genetic autoinflammatory human conditions that involve abnormal activation of RIPK1. Leveraging these new genetic and mechanistic insights, we postulate how an improved understanding of RIPK1 biology may support the development of therapeutics that target RIPK1 for the treatment of human inflammatory and neurodegenerative diseases.

Caspase-8-Dependent Inflammatory Responses Are Controlled by Its Adaptor, FADD, and Necroptosis.

Cell death pathways regulate various homeostatic processes. Autoimmune lymphoproliferative syndrome (ALPS) in humans and lymphoproliferative (LPR) disease in mice result from abrogated CD95-induced apoptosis. Because caspase-8 mediates CD95 signaling, we applied genetic approaches to dissect the roles of caspase-8 in cell death and inflammation. Here, we describe oligomerization-deficient Caspase-8F122GL123G/F122GL123G and non-cleavable Caspase-8D387A/D387A mutant mice with defective caspase-8-mediated apoptosis. Although neither mouse developed LPR disease, removal of the necroptosis effector Mlkl from Caspase-8D387A/D387A mice revealed an inflammatory role of caspase-8. Ablation of one allele of Fasl, Fadd, or Ripk1 prevented the pathology of Casp8D387A/D387AMlkl-/- animals. Removing both Fadd alleles from these mice resulted in early lethality prior to post-natal day 15 (P15), which was prevented by co-ablation of either Ripk1 or Caspase-1. Our results suggest an in vivo role of the inflammatory RIPK1-caspase-8-FADD (FADDosome) complex and reveal a FADD-independent inflammatory role of caspase-8 that involves activation of an inflammasome.

TAM Kinases Promote Necroptosis by Regulating Oligomerization of MLKL.

Necroptosis, a cell death pathway mediated by the RIPK1-RIPK3-MLKL signaling cascade downstream of tumor necrosis factor α (TNF-α), has been implicated in many inflammatory diseases. Members of the TAM (Tyro3, Axl, and Mer) family of receptor tyrosine kinases are known for their anti-apoptotic, oncogenic, and anti-inflammatory roles. Here, we identify an unexpected role of TAM kinases as promoters of necroptosis, a pro-inflammatory necrotic cell death. Pharmacologic or genetic targeting of TAM kinases results in a potent inhibition of necroptotic death in various cellular models. We identify phosphorylation of MLKL Tyr376 as a direct point of input from TAM kinases into the necroptosis signaling. The oligomerization of MLKL, but not its membranal translocation or phosphorylation by RIPK3, is controlled by TAM kinases. Importantly, both knockout and inhibition of TAM kinases protect mice from systemic inflammatory response syndrome. In conclusion, this study discovers that immunosuppressant TAM kinases are promoters of pro-inflammatory necroptosis, shedding light on the biological complexity of the regulation of inflammation.

Identification of necroptosis-associated miRNA signature for predicting prognosis and immune landscape in stomach adenocarcinoma.

PURPOSE: This study aims to identify the potential necroptosis related genes (NRGs)-associated miRNAs signature and explore the impact on the prognosis of stomach adenocarcinoma (STAD). METHODS: Employing rigorous methodologies, we utilized univariate Cox, Lasso and multivariate Cox regression analyses to develop a prognostic signature. Kaplan-Meier (K-M) and ROC curves were applied to assess the prognostic value of signature in a training group and an independent test group. Furthermore, we conducted Gene Set Enrichment Analysis (GSEA) for enrichment of tumor-related pathways. The risk score was calculated for each patient based on the expression of miRNAs which were enrolled in the signature. Patients were stratified into high- and low-risk groups. The immune cell infiltration and immunotherapy were compared between the two groups. Finally, the diagnostic potential of the miRNA was explored by RT-qPCR. RESULTS: We constructed a prognostic model based on 6 NRGs-associated miRNAs. K-M plots underscored superior survival outcomes in the low-risk group. GSEA results revealed the enrichment of several tumor-related pathways in the high-risk group. Notably, CD8+ T cells, Tregs and activated memory CD4+ T cells exhibited negative correlations with the risk score. Additionally, a few immune checkpoint genes, such as CTLA4, PD1 and PD-L1, were significantly upregulated in the low-risk group. Furthermore, the serum expression levels of all these 6 miRNAs were significantly elevated in STAD patients. CONCLUSIONS: Our study identified a robust risk score derived from a signature of 6 NRGs-associated miRNAs, demonstrating high efficacy for prognosis of STAD. These results not only contributed to our understanding of STAD pathogenesis, but also held promise for potential clinical applications, particularly in the realm of personalized immunotherapy for STAD patients.

Caspase-8 is the molecular switch for apoptosis, necroptosis and pyroptosis.

Caspase-8 is the initiator caspase of extrinsic apoptosis1,2 and inhibits necroptosis mediated by RIPK3 and MLKL. Accordingly, caspase-8 deficiency in mice causes embryonic lethality3, which can be rescued by deletion of either Ripk3 or Mlkl4-6. Here we show that the expression of enzymatically inactive CASP8(C362S) causes embryonic lethality in mice by inducing necroptosis and pyroptosis. Similar to Casp8-/- mice3,7, Casp8C362S/C362S mouse embryos died after endothelial cell necroptosis leading to cardiovascular defects. MLKL deficiency rescued the cardiovascular phenotype but unexpectedly caused perinatal lethality in Casp8C362S/C362S mice, indicating that CASP8(C362S) causes necroptosis-independent death at later stages of embryonic development. Specific loss of the catalytic activity of caspase-8 in intestinal epithelial cells induced intestinal inflammation similar to intestinal epithelial cell-specific Casp8 knockout mice8. Inhibition of necroptosis by additional deletion of Mlkl severely aggravated intestinal inflammation and caused premature lethality in Mlkl knockout mice with specific loss of caspase-8 catalytic activity in intestinal epithelial cells. Expression of CASP8(C362S) triggered the formation of ASC specks, activation of caspase-1 and secretion of IL-1ß. Both embryonic lethality and premature death were completely rescued in Casp8C362S/C362SMlkl-/-Asc-/- or Casp8C362S/C362SMlkl-/-Casp1-/- mice, indicating that the activation of the inflammasome promotes CASP8(C362S)-mediated tissue pathology when necroptosis is blocked. Therefore, caspase-8 represents the molecular switch that controls apoptosis, necroptosis and pyroptosis, and prevents tissue damage during embryonic development and adulthood.

Necroptosis pathway emerged as potential diagnosis markers in spinal cord injury.

The present research focused on identifying necroptosis-related differentially expressed genes (NRDEGs) in spinal cord injury (SCI) to highlight potential therapeutic and prognostic target genes in clinical SCI. Three SCI-related datasets were downloaded, including GSE151371, GSE5296 and GSE47681. MSigDB and KEGG datasets were searched for necroptosis-related genes (NRGs). Differentially expressed genes (DEGs) and NRGs were intersected to obtain NRDEGs. The MCC algorithm was employed to select the first 10 genes as hub genes. A protein-protein interaction (PPI) network related to NRDEGs was developed utilizing STRING. Several databases were searched to predict interactions between hub genes and miRNAs, transcription factors, potential drugs, and small molecules. Immunoassays were performed to identify DEGs using CIBERSORTx. Additionally, qRT-PCR was carried out to verify NRDEGs in an animal model of SCI. Combined analysis of all datasets identified 15 co-expressed DEGs and NRGs. GO and KEGG pathway analyses highlighted DEGs mostly belonged to pathways associated with necroptosis and apoptosis. Hub gene expression analysis showed high accuracy in SCI diagnosis was associated with the expression of CHMP7 and FADD. A total of two hub genes, i.e. CHMP7, FADD, were considered potential targets for SCI therapy.

Identification of necroptosis-related genes in ankylosing spondylitis by bioinformatics and experimental validation.

The pathogenesis of ankylosing spondylitis (AS) remains unclear, and while recent studies have implicated necroptosis in various autoimmune diseases, an investigation of its relationship with AS has not been reported. In this study, we utilized the Gene Expression Omnibus database to compare gene expressions between AS patients and healthy controls, identifying 18 differentially expressed necroptosis-related genes (DENRGs), with 8 upregulated and 10 downregulated. Through the application of three machine learning algorithms-least absolute shrinkage and selection operation, support vector machine-recursive feature elimination and random forest-two hub genes, FASLG and TARDBP, were pinpointed. These genes demonstrated high specificity and sensitivity for AS diagnosis, as evidenced by receiver operating characteristic curve analysis. These findings were further supported by external datasets and cellular experiments, which confirmed the downregulation of FASLG and upregulation of TARDBP in AS patients. Immune cell infiltration analysis suggested that CD4+ T cells, CD8+ T cells, NK cells and neutrophils may be associated with the development of AS. Notably, in the group with high FASLG expression, there was a significant infiltration of CD8+ T cells, memory-activated CD4+ T cells and resting NK cells, with relatively less infiltration of memory-resting CD4+ T cells and neutrophils. Conversely, in the group with high TARDBP expression, there was enhanced infiltration of naïve CD4+ T cells and M0 macrophages, with a reduced presence of memory-resting CD4+ T cells. In summary, FASLG and TARDBP may contribute to AS pathogenesis by regulating the immune microenvironment and immune-related signalling pathways. These findings offer new insights into the molecular mechanisms of AS and suggest potential new targets for therapeutic strategies.

Necroptosis inhibitors: mechanisms of action and therapeutic potential.

Necroptosis is a type of programmed cell death that is morphologically similar to necrosis. This type of cell death is involved in various pathophysiological disorders, including inflammatory, neurodegenerative, infectious, and malignant diseases. Receptor-interacting protein kinase 1 (RIPK1), RIPK3, and mixed lineage kinase domain-like protein (MLKL) pseudokinase constitute the core components of the necroptosis signaling pathway and are considered the most promising targets for therapeutic intervention. The discovery and characterization of necroptosis inhibitors not only accelerate our understanding of the necroptosis signaling pathway but also provide important drug candidates for the treatment of necroptosis-related diseases. Here, we will review recent research progress on necroptosis inhibitors, mechanisms of action and their potential applications for disease treatment.

Analysis and experimental validation of necroptosis-related molecular classification, immune signature and feature genes in Alzheimer's disease.

Necroptosis, a programmed cell death pathway, has been demonstrated to be activated in Alzheimer's disease (AD). However, the precise role of necroptosis and its correlation with immune cell infiltration in AD remains unclear. In this study, we conducted non-negative matrix factorization clustering analysis to identify three subtypes of AD based on necroptosis-relevant genes. Notably, these subtypes exhibited varying necroptosis scores, clinical characteristics and immune infiltration signatures. Cluster B, characterized by high necroptosis scores, showed higher immune cell infiltration and was associated with a more severe pathology, potentially representing a high-risk subgroup. To identify potential biomarkers for AD within cluster B, we employed two machine learning algorithms: the least absolute shrinkage and selection operator regression and Random Forest. Subsequently, we identified eight feature genes (CARTPT, KLHL35, NRN1, NT5DC3, PCYOX1L, RHOQ, SLC6A12, and SLC38A2) that were utilized to develop a diagnosis model with remarkable predictive capacity for AD. Moreover, we conducted validation using bulk RNA-seq, single-nucleus RNA-seq, and in vivo experiments to confirm the expression of these feature genes. In summary, our study identified a novel necroptosis-related subtype of AD and eight diagnostic biomarkers, explored the roles of necroptosis in AD progression and shed new light for the clinical diagnosis and treatment of this disease.

Utilizing a novel model of PANoptosis-related genes for enhanced prognosis and immune status prediction in kidney renal clear cell carcinoma.

Kidney renal clear cell carcinoma (KIRC) is the most common histopathologic type of renal cell carcinoma. PANoptosis, a cell death pathway that involves an interplay between pyroptosis, apoptosis and necroptosis, is associated with cancer immunity and development. However, the prognostic significance of PANoptosis in KIRC remains unclear. RNA-sequencing expression and mutational profiles from 532 KIRC samples and 72 normal samples with sufficient clinical data were retrieved from the Cancer Genome Atlas (TCGA) database. A prognostic model was constructed using differentially expressed genes (DEGs) related to PANoptosis in the TCGA cohort and was validated in a Gene Expression Omnibus (GEO) cohorts. Incorporating various clinical features, the risk model remained an independent prognostic factor in multivariate analysis, and it demonstrated superior performance compared to unsupervised clustering of the 21 PANoptosis-related genes alone. Further mutational analysis showed fewer VHL and more BAP1 alterations in the high-risk group, with alterations in both genes also associated with patient prognosis. The high-risk group was characterized by an unfavorable immune microenvironment, marked by reduced levels of CD4 + T cells and natural killer cells, but increased M2 macrophages and regulatory T cells. Finally, the risk model was predictive of response to immune checkpoint blockade, as well as sensitivity to sunitinib and paclitaxel. The PANoptosis-related risk model developed in this study enables accurate prognostic prediction in KIRC patients. Its associations with the tumor immune microenvironment and drug efficacy may offer potential therapeutic targets and inform clinical decisions.

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.

Leveraging single-cell sequencing analysis and bulk-RNA sequencing analysis to forecast necroptosis in cutaneous melanoma prognosis.

Cutaneous melanoma, a malignancy of melanocytes, presents a significant challenge due to its aggressive nature and rising global incidence. Despite advancements in treatment, the variability in patient responses underscores the need for further research into novel therapeutic targets, including the role of programmed cell death pathways such as necroptosis. The melanoma datasets used for analysis, GSE215120, GSE19234, GSE22153 and GSE65904, were downloaded from the GEO database. The melanoma data from TCGA were downloaded from the UCSC website. Using single-cell sequencing, we assess the heterogeneity of necroptosis in cutaneous melanoma, identifying distinct cell clusters and necroptosis-related gene expression patterns. A combination of 101 machine learning algorithms was employed to construct a necroptosis-related signature (NRS) based on key genes associated with necroptosis. The prognostic value of NRS was evaluated in four cohorts (one TCGA and three GEO cohorts), and the tumour microenvironment (TME) was analysed to understand the relationship between necroptosis, tumour mutation burden (TMB) and immune infiltration. Finally, we focused on the role of key target TSPAN10 in the prognosis, pathogenesis, immunotherapy relevance and drug sensitivity of cutaneous melanoma. Our study revealed significant heterogeneity in necroptosis among melanoma cells, with a higher prevalence in epithelial cells, myeloid cells and fibroblasts. The NRS, developed through rigorous machine learning techniques, demonstrated robust prognostic capabilities, distinguishing high-risk patients with poorer outcomes in all cohorts. Analysis of the TME showed that high NRS scores correlated with lower TMB and reduced immune cell infiltration, indicating a potential mechanism through which necroptosis influences melanoma progression. Finally, TSPAN10 has been identified as a key target for cutaneous melanoma and is highly associated with poor prognosis. The findings highlight the complex role of necroptosis in cutaneous melanoma and introduce the NRS as a novel prognostic tool with potential to guide therapeutic decisions.

HIST3H2A promotes the progression of prostate cancer through inhibiting cell necroptosis.

In recent years, there has been an increase in the incidence and mortality rates of prostate cancer (PCa). However, the specific molecular mechanisms underlying its occurrence and development remain unclear, necessitating the identification of new therapeutic targets. Through bioinformatics analysis, we discovered a previously unstudied differential gene called HIST3H2A in prostate cancer. Our study revealed that HIST3H2A is highly expressed in PCa tissues, as confirmed by analysis of both the GEO and UALCAN databases. Further analysis using the KEGG database demonstrated that HIST3H2A regulates the pathway of programmed necroptosis in cells. Additionally, we observed significant up-regulation of HIST3H2A in PCa tissues and cell lines. HIST3H2A was found to regulate cell proliferation, migration, invasion, and the epithelial-mesenchymal transition (EMT) process in tumors. Notably, HIST3H2A's role in regulating programmed necroptosis in prostate cancer cells differs from its role in apoptosis. In vitro and in vivo experiments collectively support the key role of HIST3H2A in promoting the development of prostate cancer, highlighting its potential as a therapeutic target for patients with PCa.

The study of the mechanism of non-coding RNA regulation of programmed cell death in diabetic cardiomyopathy.

Diabetic cardiomyopathy (DCM) represents a distinct myocardial disorder elicited by diabetes mellitus, characterized by aberrations in myocardial function and structural integrity. This pathological condition predominantly manifests in individuals with diabetes who do not have concurrent coronary artery disease or hypertension. An escalating body of scientific evidence substantiates the pivotal role of programmed cell death (PCD)-encompassing apoptosis, autophagy, pyroptosis, ferroptosis, and necroptosis-in the pathogenic progression of DCM, thereby emerging as a prospective therapeutic target. Additionally, numerous non-coding RNAs (ncRNAs) have been empirically verified to modulate the biological processes underlying programmed cell death, consequently influencing the evolution of DCM. This review systematically encapsulates prevalent types of PCD manifest in DCM as well as nascent discoveries regarding the regulatory influence of ncRNAs on programmed cell death in the pathogenesis of DCM, with the aim of furnishing novel insights for the furtherance of research in PCD-associated disorders relevant to DCM.

Protection of Quiescence and Longevity of IgG Memory B Cells by Mitochondrial Autophagy.

The development of long-lived immune memory cells against pathogens is critical for the success of vaccines to establish protection against future infections. However, the mechanisms governing the long-term survival of immune memory cells remain to be elucidated. In this article, we show that the maintenance mitochondrial homeostasis by autophagy is critical for restricting metabolic functions to protect IgG memory B cell survival. Knockout of mitochondrial autophagy genes, Nix and Bnip3, leads to mitochondrial accumulation and increases in oxidative phosphorylation and fatty acid synthesis, resulting in the loss of IgG+ memory B cells in mice. Inhibiting fatty acid synthesis or silencing necroptosis gene Ripk3 rescued Nix-/-Bnip3-/- IgG memory B cells, indicating that mitochondrial autophagy is important for limiting metabolic functions to prevent cell death. Our results suggest a critical role for mitochondrial autophagy in the maintenance of immunological memory by protecting the metabolic quiescence and longevity of memory B cells.

Necroptosis pathways in tumorigenesis.

Necroptosis is a caspase-independent form of programmed cell death executed by the receptor interacting protein kinase 1 (RIPK1)-RIPK3-mixed lineage kinase domain-like protein (MLKL) signaling cascade, deregulation of which can cause various human diseases including cancer. Escape from programmed cell death is a hallmark of cancer, leading to uncontrolled growth and drug resistance. Therefore, it is crucial to further understand whether necroptosis plays a key role in therapeutic resistance. In this review, we summarize the recent findings of the link between necroptosis and cancer, and discuss that targeting necroptosis is a new strategy to overcome apoptosis resistance in tumor therapy.