Receptor Interacting Protein Kinase1 (RIPK1) is a key regulator of necroptosis and plays an important role in various inflammatory signaling and cell death processes. GSK963 has been reported to inhibit RIPK1 with limited anti-necroptotic effect. We designed and prepared a series of phenylisoxazolidine analogs based on GSK963 to improve their anti-necroptotic activity. After several turns of structure activity relationship (SAR) studies, compound 22, encoded with KWML-22, was found to have good anti-necroptotic activity (EC50 = 30.0 nM) and enzymatic activity (IC50 = 6.9 nM) against RIPK1. In a TNF-α-induced inflammation in vivo model, a dose of 10 mg/kg protected mice from hypothermia and death. Our results suggest that 22 is a promising lead compound for the study of inflammatory diseases.

The specific role of efferocytosis-related long noncoding RNAs (ERLncRNAs) in Clear Cell Renal Cell Carcinoma (ccRCC) has not been thoroughly examined. This study aims to identify and validate a signature of ERLncRNAs for prognostic prediction and characterization of the immune landscape in individuals with ccRCC.

Analysis of ccRCC samples was conducted by utilizing clinical and RNA sequencing information obtained from The Cancer Genome Atlas (TCGA). Pearson correlation analysis was utilized to identify lncRNAs associated with efferocytosis, which was then used to create a new prognostic model through univariate Cox regression, Least Absolute Shrinkage and Selection Operator (LASSO) regression, and stepwise multivariate Cox analysis. In order to investigate the biological significance, we performed a functional enrichment analysis to assess how well the model predicts outcomes. Differences in the immune landscape were observed through a comparison of immune cell infiltration, tumor mutational burden (TMB), and tumor microenvironment (TME) characteristics. Following this, drug sensitivity analysis was conducted.

This led to the identification of a unique signature consisting of seven ERLncRNAs (LINC01615, RUNX3-AS1, FOXD2-AS1, AC002070.1, LINC02747, LINC00944, and AC092296.1). Model performance was measured by Kaplan-Meier curves and receiver operating characteristic (ROC) curves. The nomogram and C-index provided additional validation of the strong correlation between the risk signature and clinical decision-making.

On the whole, our innovative signature exhibits potential for prognostic prediction and assessment of immunotherapeutic response in patients with ccRCC.

Necroptosis is a programmed form of cell death. Receptor-interacting serine/threonine protein kinase l (RIPK1) is a crucial protein kinase that regulates the necroptosis pathway. Increased expression of death receptor family ligands such as tumor necrosis factor (TNF) increases the susceptibility of cells to apoptosis and necroptosis. RIPK1, RIPK3, and mixed-lineage kinase-like domain (MLKL) proteins mediate necrosis. RIPK1-mediated necroptosis further promotes cell death and inflammation in the pathogenesis of liver injury, skin diseases, and neurodegenerative diseases. The N-terminal kinase domain of RIPK1 is significant in the induction of cell death and can be used as a vital drug target for inhibitors. In this paper, we outline the pathways of necroptosis and the role RIPK1 plays in them and suggest that targeting RIPK1 in therapy may help to inhibit multiple cell death pathways.

Necroptosis initiated by the host sensor Z-NA binding protein 1 (ZBP1) is essential for host defense against a growing number of viruses, including herpes simplex virus 1 (HSV-1). Studies with HSV-1 and other necroptogenic stimuli in murine settings have suggested that ZBP1 triggers necroptosis by directly complexing with the kinase RIPK3. Whether this is also the case in human cells, or whether additional co-factors are needed for ZBP1-mediated necroptosis, is unclear. Here, we show that ZBP1-induced necroptosis in human cells requires RIPK1. We have found that RIPK1 is essential for forming a stable and functional ZBP1-RIPK3 complex in human cells, but is dispensable for the formation of the equivalent murine complex. The receptor-interacting protein (RIP) homology interaction motif (RHIM) in RIPK3 is responsible for this difference between the 2 species, because replacing the RHIM in human RIPK3 with the RHIM from murine RIPK3 is sufficient to overcome the requirement for RIPK1 in human cells. These observations describe a critical mechanistic difference between mice and humans in how ZBP1 engages in necroptosis, with important implications for treating human diseases.

Receptor-interacting serine/threonine kinase 1 (RIPK1) regulates inflammatory signaling and induces apoptosis and necroptosis. Pharmacological inhibition of RIPK1 kinase activity has demonstrated efficacy in animal models of neurodegenerative, autoimmune and inflammatory diseases. SIR9900 is a potent and selective novel small molecule RIPK1 inhibitor. This first-in-human, phase I, randomized, double-blind, placebo-controlled study evaluated the safety, pharmacokinetics, and pharmacodynamics of single (3-200 mg) and multiple (3-60 mg daily for 10 days) ascending oral doses of SIR9900 in healthy adult (18-64 years, n = 80) and elderly participants (≥ 65 years, multiple doses 30 mg, n = 8). The study included a food effect component. Overall, SIR9900 was safe and well tolerated with no concerning dose-dependent trends in safety observed. SIR9900 was rapidly absorbed with a plasma maximum concentration time (Tmax) of 3.0-4.0 h and plasma half-life (t1/2) of 31.92-37.75 h following single doses. Similar Tmax and t1/2 results were observed following multiple doses. Systemic exposure to SIR9900 increased in a dose-proportional manner and was similar between adult and elderly participants. No appreciable food effect was observed. The cerebrospinal fluid to unbound plasma ratio was 1.15. A robust pharmacodynamic effect was demonstrated with approximately 90% peripheral target engagement at 3 h post-dose, and sustained RIPK1 inhibition over the 10-day treatment period. The promising safety, pharmacokinetic, and pharmacodynamic profile of SIR9900 with central nervous system penetrating potential in healthy adult and elderly participants supports its further clinical development in patients with inflammatory and degenerative diseases, particularly in the central nervous system.

Sepsis is a life-threatening syndrome characterized by organ dysfunction caused by a dysregulated host response to infection. Sepsis-associated acute liver injury (SA-ALI) is a frequent and serious complication of sepsis that considerably impacts both short-term and long-term survival outcomes. In intensive care units (ICUs), the mortality rate of patients with SA-ALI remains high, mostly due to the absence of effective early diagnostic markers and suitable therapeutic strategies. Recent studies have demonstrated the importance of non-coding RNAs (ncRNAs) in the development and progression of SA-ALI. This review focuses on the critical roles of ncRNAs, including microRNAs (miRNAs), long non-coding RNAs (lncRNAs), and circular RNAs (circRNAs), in regulating "cytokine storms", oxidative stress, mitochondrial dysfunction, and programmed cell death in SA-ALI, and summarizes the current state and limitations of existing studies on lncRNAs and circRNAs in SA-ALI. By integrating advancements in high-throughput sequencing technologies, this review provides novel insights into the dual potential of ncRNAs as diagnostic biomarkers and therapeutic targets, offers new ideas for SA-ALI diagnosis and treatment research and highlights potential challenges in clinical translation.

Sepsis is a life-threatening condition resulting from pathogen infection and characterized by organ dysfunction. Programmed cell death (PCD) during sepsis has been associated with the development of multiple organ dysfunction syndrome (MODS), impacting various physiological systems including respiratory, cardiovascular, renal, neurological, hematological, hepatic, and intestinal systems. It is well-established that pathogen infections lead to immune dysregulation, which subsequently contributes to MODS in sepsis. However, recent evidence suggests that sepsis-related opportunistic pathogens can directly induce organ failure by promoting PCD in parenchymal cells of each affected organ. This study provides an overview of PCD in damaged organ and the induction of PCD in host parenchymal cells by opportunistic pathogens, proposing innovative strategies for preventing organ failure in sepsis.

Sepsis is a complicated clinical disease caused by a defective host response to infection, leading to elevated morbidity and fatality globally. Sepsis patients have a significant risk of life-threatening organ damage, including hearts, brains, lungs, kidneys, and livers. Nevertheless, the molecular pathways driving organ injury in sepsis are not well known. Ferroptosis, a non-apoptotic cell death, occurs due to iron metabolism disturbance and lipid peroxide buildup. Multiple studies indicate that ferroptosis has a significant role in decreasing inflammation and lipid peroxidation during sepsis. Ferroptosis inhibitors and medications, aimed at the most studied ferroptosis process, including Xc-system, Nrf2/GPX4 axis, and NCOA4-FTH1-mediated ferritinophagy, alleviating sepsis effectively. However, few clinical trials demonstrated ferroptosis-targeted drugs's effectiveness in sepsis. Our study examines ferroptosis-targeted medicinal agents and their potential benefits for treating sepsis-associated organ impairment. This review indicates that ferroptosis suppression by pharmaceutical means may be a useful therapy for sepsis-associated organ injury.

It has been established that N-acetyltransferase (murine NAT1 (mNAT1) and human NAT2 (hNAT2)) mediates insulin sensitivity in type 2 diabetes. Here we show that mNAT1 deficiency leads to a decrease in cellular spermidine-a natural polyamine exhibiting health-protective and anti-ageing effects-but understanding of its mechanism is limited. We identify that mNAT1 and hNAT2 modulate a type of post-translational modification involving acetylated spermidine, which we name acetylhypusination, on receptor-interacting serine/threonine-protein kinase 1 (RIPK1)-a key regulator of inflammation and cell death. Spermidine supplementation decreases RIPK1-mediated cell death and diabetic phenotypes induced by NAT1 deficiency in vivo. Furthermore, insulin resistance and diabetic kidney disease mediated by vascular pathology in NAT1-deficient mice can be blocked by inhibiting RIPK1. Finally, we demonstrate a decrease in spermidine and activation of RIPK1 in the vascular tissues of human patients with diabetes. Our study suggests a role for vascular pathology in diabetes onset and progression and identifies the inhibition of RIPK1 kinase as a potential therapeutic approach for the treatment of type 2 diabetes.

Tumor necrosis factor (TNF)-induced receptor-interacting serine/threonine protein kinase 1 (RIPK1)-mediated cell death, including apoptosis and necroptosis, is increasingly recognized as a major driver of inflammatory diseases. Cell death checkpoints normally suppress RIPK1 kinase to safeguard the organism from its detrimental consequences. However, the mechanisms licensing RIPK1 kinase activity when a protective checkpoint is disabled remain unclear. Here, we identified S-palmitoylation as a licensing modification for RIPK1 kinase. TNF induces RIPK1 palmitoylation, mediated by DHHC5 and dependent on K63-linked ubiquitination of RIPK1, which enhances RIPK1 kinase activity by promoting the homo-interaction of its kinase domain and promotes cell death upon cell death checkpoint blockade. Furthermore, DHHC5 is amplified by fatty acid in the livers of mice with metabolic dysfunction-associated steatohepatitis, contributing to increased RIPK1 cytotoxicity observed in this condition. Our findings reveal that ubiquitination-dependent palmitoylation licenses RIPK1 kinase activity to induce downstream cell death signaling and suggest RIPK1 palmitoylation as a feasible target for inflammatory diseases.

The hypersecretion of cytokines triggers life-threatening systemic inflammatory response syndrome (SIRS), leading to multiple organ dysfunction syndrome (MODS) and mortality. Although both coagulopathy and necroptosis have been identified as important factors in the pathogenesis of SIRS, the specific cell types that undergo necroptosis and the interrelationships between coagulopathy and necroptosis remain unclear. In this study, we utilized visualization analysis via intravital microscopy to demonstrate that both anticoagulant heparin and nonanticoagulant heparin (NAH) pretreatment protect mice against TNF-α-induced mortality in SIRS. Moreover, the deletion of Mlkl or Ripk3 resulted in decreased coagulation and reduced mortality in TNF-α-induced SIRS. These findings suggest that necroptosis plays a key role upstream of coagulation in SIRS-related mortality. Furthermore, using a genetic lineage tracing mouse model (Tie2-Cre;Rosa26-tdT), we tracked endothelial cells (ECs) and verified that EC necroptosis is responsible for the vascular damage observed in TNF-α-treated mice. Importantly, Mlkl deletion in vascular ECs in mice had a similar protective effect against lethal SIRS by blocking EC necroptosis to protect the integrity of the endothelium. Collectively, our findings demonstrated that RIPK3-MLKL-dependent necroptosis disrupted vascular integrity, resulting in coagulopathy and multiorgan failure, eventually leading to mortality in SIRS patients. These results highlight the importance of targeting vascular EC necroptosis for the development of effective treatments for SIRS patients.

Tumor necrosis factor (TNF) induces systemic inflammatory response syndrome (SIRS), and severe SIRS can serve as a model for studying animal death caused by organ failure. Through strategic cecectomy, we demonstrate that necroptosis in the cecum initiates the death process in TNF-treated mice, but it is not the direct cause of death. Instead, we show that it is the cardiac dysfunction downstream of cecum damage that ultimately leads to the death of TNF-treated mice. By in vivo and ex vivo physiological analyses, we reveal that TNF and the damage-associated molecular patterns (DAMPs) released from necroptotic cecal cells jointly target cardiac endothelial cells, triggering caspase-8 activation and subsequent cardiac endothelial damage. Cardiac endothelial damage is a primary cause of the deterioration of diastolic function in the heart of TNF-treated mice. Our research provides insights into the pathophysiological process of TNF-induced lethality.

Caspase-8, a renowned cysteine-aspartic protease within its enzyme family, initially garnered attention for its regulatory role in extrinsic apoptosis. With advancing research, a growing body of evidence has substantiated its involvement in other cell death processes, such as pyroptosis and necroptosis, as well as its modulatory effects on inflammasomes and proinflammatory cytokines. PANoptosis, an emerging concept of cell death, encompasses pyroptosis, apoptosis, and necroptosis, providing insight into the often overlapping cellular mortality observed during disease progression. The activation or deficiency of caspase-8 enzymatic activity is closely linked to PANoptosis, positioning caspase-8 as a key regulator of cell survival or death across various physiological and pathological processes. Aberrant expression of caspase-8 is closely associated with the development and progression of a range of inflammatory diseases, including immune system disorders, neurodegenerative diseases (NDDs), sepsis, and cancer. This paper delves into the regulatory role and impact of caspase-8 in these conditions, aiming to elucidate potential therapeutic strategies for the future intervention.

Tumor necrosis factor alpha (TNFα), as a key pro-inflammatory cytokine, plays a central role in joint diseases. In recent years, numerous models of TNFα-induced cartilage inflammation have been developed. However, due to the significant differences between these models and the lack of consensus in their construction, it becomes difficult to compare the results of different studies. Therefore, we summarized and compared these models based on important parameters for model construction, such as cell source, cytokine concentration, stimulation time, mechanical stimulation, and more. We attempted to analyze the advantages and disadvantages of each model and provide a compilation of the analytical methods used in previous studies. Currently, TNFα chondrocyte inflammation models can be categorized into four main types: monolayer-based, construct-based, explant-based TNFα chondrocyte inflammation models, and miscellaneous TNFα chondrocyte inflammation models. The most commonly used models were the monolayer-based TNFα chondrocyte inflammation models (42.86% of cases), with 10 ng/mL TNFα being the most frequently used concentration. The most frequently used chondrocyte cell passage is passage 1 (50%). Human tissues were most frequently used in experiments (51.43%). Only five articles included models with mechanical stimulations. We observed variations in design conditions between different models. This systematic review provides the essential experimental characteristics of the available chondrocyte inflammation models with TNFα, and it provides a platform for better comparison between existing and new studies in this field. It is essential to perform further experiments to standardize each model and to find the most appropriate experimental parameters.

The interplay between the immune system, coagulation, and endothelium is critical in regulating the host response to infection. However, in sepsis and other critical illnesses, a dysregulated immune response can lead to excessive alterations in these mechanisms, resulting in coagulopathy, endothelial dysfunction, and multi-organ dysfunction. This review aims to provide a comprehensive analysis of the pathophysiological mechanisms that govern the complex interplay between immune dysfunction, endothelial dysfunction, and coagulation in sepsis. It emphasises clinical significance, evaluation methods, and potential therapeutic interventions. Understanding these mechanisms is essential for developing effective treatments that can modulate the immune response, mitigate thrombosis, restore endothelial function, and ultimately improve patient survival.

Necroptosis initiated by the host sensor Z-NA Binding Protein-1 (ZBP1) is essential for host defense against a growing number of viruses, including Herpes Simplex Virus-1 (HSV-1). Studies with HSV-1 and other necroptogenic stimuli in murine settings have suggested that ZBP1 triggers necroptosis by directly complexing with the kinase RIPK3. Whether this is also the case in human cells, or whether additional co-factors are needed for ZBP1-mediated necroptosis, is unclear. Here, we show that ZBP1-induced necroptosis in human cells requires RIPK1. We have found that RIPK1 is essential for forming a stable and functional ZBP1-RIPK3 complex in human cells, but is dispensable for the formation of the equivalent murine complex. The RIP Homology Interaction Motif (RHIM) in RIPK3 is responsible for this difference between the two species, because replacing the RHIM in human RIPK3 with the RHIM from murine RIPK3 is sufficient to overcome the requirement for RIPK1 in human cells. These observations describe a critical mechanistic difference between mice and humans in how ZBP1 engages in necroptosis, with important implications for treating human diseases.

The purpose of this study was to investigate early stage dynamic changes in relevant indicators in neurocritical patients to identify biomarkers that can predict a poor prognosis at an early stage (1-4 days after admission). This study retrospectively collected clinical data, inflammatory indicators, and nutritional indicators from 77 patients at the neurology intensive care unit. The 3-month modified Rankin scale score was used as the outcome indicator. A linear mixed model was used to analyze changes in inflammatory indicators and nutritional indicators in neurocritical patients over time from 1-4 days after admission. Logistic regression was used to determine the independent risk factors for a poor prognosis in neurocritical patients and to construct a predictive model. The predictive efficacy of the model was verified using leave-one-out cross-validation and decision curve analysis methods. The analysis results showed that 1-4 days after admission, the inflammatory indicators of white blood cell and absolute monocyte counts and the nutritional indicators of body cell mass(BCM), fat-free mass, body cell mass/phase angle (BCM/PA), intracellular water, extracellular water, and skeletal muscle index increased overall, while the nutritional indicators of albumin and visceral fat area decreased overall. The logistic multivariate regression model showed that the Charlson comorbidity index (CCI) (odds ratio (OR) = 2.526, 95% CI [1.202, 5.308]), hemoglobin (Hb)(on admission)-Hb(min) (OR = 1.049, 95% CI [1.015, 1.083), BCM(on admission) (OR = 0.794, 95% CI [0.662, 0.952]), and the change in BCM/PA 1-4 days after admission (OR = 1.157, 95% CI [1.070, 1.252]) were independent risk factors for a poor prognosis in neurocritical patients. The predictive analysis showed that the predictive power of Model 1 with BCM/PA (area under the curve (AUC) = 0.95, 95% CI (0.90, 0.99)) was 93%, 65%, 141%, and 133% higher than that of Model 2 without BCM/PA, the CCI, the APACHE II score, and the NRS2002 score (all P < 0.05), respectively. The CCI, Hb(on admission)-Hb(min), BCM(on admission), and an increase in BCM/PA 1-4 days after admission were independently associated with a poor prognosis in neurocritical patients. Of these variables, BCM/PA may be a valid indicator for early stage prediction of a poor prognosis in neurocritical patients.

Sepsis is a life-threatening disease due to a dysregulated host response to infection, with an unknown regulatory mechanism for prognostic necroptosis-related genes (NRGs). Using GEO datasets GSE65682 and GSE134347, we identified six NRG biomarkers ( ATRX , TSC1 , CD40 , BACH2 , BCL2 , and LEF1 ) with survival and diagnostic significance through Kaplan-Meier (KM) and receiver operating characteristic (ROC) analyses. Afterward, the ingenuity pathway analysis (IPA) highlighted enrichment in hepatic fibrosis pathways and BEX2 protein. Moreover, we examined their regulatory targets and functional links with necroptotic signaling molecules via miRDB, TargetScan, Network analyst, and GeneMANIA. The molecular regulatory network displayed that hsa-miR-5195-3p and hsa-miR-145-5p regulated ATRX, BACH2, and CD40, while YY1 showed strong connectivity, concurrently controlling LEF1, ATRX, BCL2, BACH2, and CD40. CD40 exhibited similar expression patterns to RIPK3 and MLKL, and LEF1 was functionally associated with MLKL. Additionally, DrugBank analysis identified paclitaxel, docetaxel, and rasagiline as potential BCL2-targeting sepsis treatments. Finally, real-time quantitative PCR confirmed ATRX, TSC1, and LEF1 downregulation in sepsis samples, contrasting CD40's increased expression in CTL samples. In conclusion, ATRX , TSC1 , CD40 , BACH2 , BCL2 , and LEF1 may be critical regulatory targets of necroptosis in sepsis, providing a basis for further necroptosis-related studies in sepsis.

Necrosome activation following TLR- or cytokine receptor-signaling results in cell death by necroptosis which is characterized by the rupture of cell membranes and the consequent release of intracellular contents to the extracellular milieu. While necroptosis exacerbates various inflammatory diseases, the mechanisms through which the inflammatory responses are regulated are not clear. We show that the necrosome activation of macrophages results in an upregulation of various pathways, including the mitogen-activated protein kinase (MAPK) cascade, which results in an elevation of the inflammatory response and consequent expression of several cytokines and chemokines. Programming for this upregulation of inflammatory response occurs during the early phase of necrosome activation and proceeds independently of cell death but depends on the activation of the receptor-interacting protein kinase-1 (RipK1). Interestingly, necrosome activation also results in an upregulation of IFNβ, which in turn exerts an inhibitory effect on the maintenance of inflammatory response through the repression of MAPK-signaling and an upregulation of Zfp36. Activation of the interferon-induced gene factor-3 (ISGF3) results in the expression of ZFP36 (TTP), which induces the post-transcriptional degradation of mRNAs of various inflammatory cytokines and chemokines through the recognition of AU-rich elements in their 3'UTR. Furthermore, ZFP-36 inhibits IFNβ-, but not TNFα- induced necroptosis. Overall, these results reveal the molecular mechanism through which IFNβ, a pro-inflammatory cytokine, induces the expression of ZFP-36, which in turn inhibits necroptosis and halts the maintenance of the inflammatory response.

CD28 and 4-1BB costimulatory endodomains included in chimeric antigen receptor (CAR) molecules play a critical role in promoting sustained antitumor activity of CAR-T cells. However, the molecular events associated with the ectopic and constitutive display of either CD28 or 4-1BB in CAR-T cells have been only partially explored. In the current study, we demonstrated that 4-1BB incorporated within the CAR leads to cell cluster formation and cell death in the forms of both apoptosis and necroptosis in the absence of CAR tonic signaling. Mechanistic studies illustrate that 4-1BB sequesters A20 to the cell membrane in a TRAF-dependent manner causing A20 functional deficiency that in turn leads to NF-κB hyperactivity, cell aggregation via ICAM-1 overexpression, and cell death including necroptosis via RIPK1/RIPK3/MLKL pathway. Genetic modulations obtained by either overexpressing A20 or releasing A20 from 4-1BB by deleting the TRAF-binding motifs of 4-1BB rescue cell cluster formation and cell death and enhance the antitumor ability of 4-1BB-costimulated CAR-T cells.

Disulfidptosis is a prevalent apoptotic mechanism, intrinsically linked to cancer prognosis. However, the specific involvement of disulfidptosis-related long non-coding RNA (DRLncRNAs) in Kidney renal clear cell carcinoma (KIRC) remains incompletely understood. This study aims to elucidate the potential prognostic significance of disulfidptosis-related LncRNAs in KIRC.

Expression profiles and clinical data of KIRC patients were retrieved from the TCGA database to discern differentially expressed DRLncRNAs correlated with overall survival. Cox univariate analysis, Lasso Regression, and Cox multivariate analysis were used to construct a clinical prediction model.

Six signatures, namely FAM83C.AS1, AC136475.2, AC121338.2, AC026401.3, AC254562.3, and AC000050.2, were established to evaluate overall survival (OS) in the context of Kidney renal clear cell carcinoma (KIRC) in this study. Survival analysis and ROC curves demonstrated the strong predictive performance of the associated signature. The nomogram exhibited accurate prognostic predictions for overall patient survival, offering substantial clinical utility. Gene set enrichment analysis revealed that risk signals were enriched in various immune-related pathways. Furthermore, the risk features exhibited significant correlations with immune cells, immune function, immune cell infiltration, and immune checkpoints.

This study has unveiled, for the first time, six disulfdptosis-related LncRNA signatures, laying a solid foundation for enhanced and precise prognostic predictions in KIRC.

Receptor-interacting serine/threonine-protein kinase 1 (RIPK1) functions as a critical stress sentinel that coordinates cell survival, inflammation, and immunogenic cell death (ICD). Although the catalytic function of RIPK1 is required to trigger cell death, its non-catalytic scaffold function mediates strong pro-survival signaling. Accordingly, cancer cells can hijack RIPK1 to block necroptosis and evade immune detection. We generated a small-molecule proteolysis-targeting chimera (PROTAC) that selectively degraded human and murine RIPK1. PROTAC-mediated depletion of RIPK1 deregulated TNFR1 and TLR3/4 signaling hubs, accentuating the output of NF-κB, MAPK, and IFN signaling. Additionally, RIPK1 degradation simultaneously promoted RIPK3 activation and necroptosis induction. We further demonstrated that RIPK1 degradation enhanced the immunostimulatory effects of radio- and immunotherapy by sensitizing cancer cells to treatment-induced TNF and interferons. This promoted ICD, antitumor immunity, and durable treatment responses. Consequently, targeting RIPK1 by PROTACs emerges as a promising approach to overcome radio- or immunotherapy resistance and enhance anticancer therapies.

Receptor-interacting protein kinase 1 (RIPK1) plays a crucial role in controlling inflammation and cell death. Its function is tightly controlled through post-translational modifications, enabling its dynamic switch between promoting cell survival and triggering cell death. Phosphorylation of RIPK1 at various sites serves as a critical mechanism for regulating its activity, exerting either activating or inhibitory effects. Perturbations in RIPK1 phosphorylation status have profound implications for the development of severe inflammatory diseases in humans. This review explores the intricate regulation of RIPK1 phosphorylation and dephosphorylation and highlights the potential of targeting RIPK1 phosphorylation as a promising therapeutic strategy for mitigating human diseases.

Activation of receptor-interacting protein kinase 1 (RIPK1), a broadly expressed serine/threonine protein kinase, by pro-inflammatory cytokines and pathogens can result in apoptosis, necroptosis, or inflammation. RIPK1 inhibition has been shown to reduce inflammation and cell damage in preclinical studies and may have therapeutic potential for degenerative and inflammatory diseases. SIR2446 is a potent and selective novel small molecule RIPK1 kinase inhibitor. This phase I, randomized, double-blind, placebo-controlled study in Australia (ACTRN12621001621808) evaluated the safety (primary objective), pharmacokinetics, and pharmacodynamics of single (3-600 mg) and multiple (5-400 mg for 10 days) ascending oral doses of SIR2446M (SIR2446 magnesium salt form) in healthy adults from Nov 24, 2021, until May 01, 2023. All treatment-emergent adverse events (TEAEs) were mild/moderate. The most reported TEAEs were vascular access site pain, headache, and rash morbilliform. SIR2446M plasma half-lives ranged from 11 to 19 h and there were no major deviations from dose proportionality for maximum concentration and area under the curve across doses. Renal excretion of unchanged SIR2446 was minimal. No marked accumulation was observed (mean accumulation ratio, 1.2-1.6) after multiple daily doses. A high-fat meal mildly reduced the exposure but was not considered clinically significant. SIR2446M had a rapid and sustained inhibitory effect on the activity of RIPK1, with an overall 90% target engagement at repeated doses ranging from 30 to 400 mg in peripheral blood mononuclear cells ex vivo stimulated to undergo necroptosis. The favorable safety, pharmacokinetic, and pharmacodynamic profile of SIR2446M in healthy participants supports its further clinical development in patients with degenerative and inflammatory diseases.

Necroptosis is a lytic form of regulated cell death reported to contribute to inflammatory diseases of the gut, skin and lung, as well as ischemic-reperfusion injuries of the kidney, heart and brain. However, precise identification of the cells and tissues that undergo necroptotic cell death in vivo has proven challenging in the absence of robust protocols for immunohistochemical detection. Here, we provide automated immunohistochemistry protocols to detect core necroptosis regulators - Caspase-8, RIPK1, RIPK3 and MLKL - in formalin-fixed mouse and human tissues. We observed surprising heterogeneity in protein expression within tissues, whereby short-lived immune barrier cells were replete with necroptotic effectors, whereas long-lived cells lacked RIPK3 or MLKL expression. Local changes in the expression of necroptotic effectors occurred in response to insults such as inflammation, dysbiosis or immune challenge, consistent with necroptosis being dysregulated in disease contexts. These methods will facilitate the precise localisation and evaluation of necroptotic signaling in vivo.

Necroptosis is a caspase-independent modality of cell death implicated in many inflammatory pathologies. The execution of this pathway requires the formation of a cytosolic platform that comprises RIPK1 and RIPK3 which, in turn, mediates the phosphorylation of the pseudokinase MLKL (S345 in mouse). The activation of this executioner is followed by its oligomerisation and accumulation at the plasma-membrane where it leads to cell death via plasma-membrane destabilisation and consequent permeabilisation. While the biochemical and cellular characterisation of these events have been amply investigated, the study of necroptosis involvement in vivo in animal models is currently limited to the use of Mlkl-/- or Ripk3-/- mice. Yet, even in many of the models in which the involvement of necroptosis in disease aetiology has been genetically demonstrated, the fundamental in vivo characterisation regarding the question as to which tissue(s) and specific cell type(s) therein is/are affected by the pathogenic necroptotic death are missing. Here, we describe and validate an immunohistochemistry and immunofluorescence-based method to reliably detect the phosphorylation of mouse MLKL at serine 345 (pMLKL-S345). We first validate the method using tissues derived from mice in which Caspase-8 (Casp8) or FADD are specifically deleted from keratinocytes, or intestinal epithelial cells, respectively. We next demonstrate the presence of necroptotic activation in the lungs of SARS-CoV-infected mice and in the skin and spleen of mice bearing a Sharpin inactivating mutation. Finally, we exclude necroptosis occurrence in the intestines of mice subjected to TNF-induced septic shock. Importantly, by directly comparing the staining of pMLKL-345 with that of cleaved Caspase-3 staining in some of these models, we identify spatio-temporal and functional differences between necroptosis and apoptosis supporting a role of RIPK3 in inflammation independently of MLKL versus the role of RIPK3 in activation of necroptosis.

Sepsis is defined as life-threatening organ dysfunction caused by a dysregulated host response to infection. Studies have indicated that immune dysfunction plays a central role in the pathogenesis of sepsis. Dendritic cells (DCs) play a crucial role in the emergence of immune dysfunction in sepsis. The major manifestations of DCs in the septic state are abnormal functions and depletion in numbers, which are linked to higher mortality and vulnerability to secondary infections in sepsis. Apoptosis is the most widely studied pathway of number reduction in DCs. In the past few years, there has been a surge in studies focusing on regulated cell death (RCD). This emerging field encompasses various forms of cell death, such as necroptosis, pyroptosis, ferroptosis, and autophagy-dependent cell death (ADCD). Regulation of DC's RCD can serve as a possible therapeutic focus for the treatment of sepsis. Throughout time, numerous tactics have been devised and effectively implemented to improve abnormal immune response during sepsis progression, including modifying the functions of DCs and inhibiting DC cell death. In this review, we provide an overview of the functional impairment and RCD of DCs in septic states. Also, we highlight recent advances in targeting DCs to regulate host immune response following septic challenge.

Caspase-8 (Casp8) serves as an initiator of apoptosis or a suppressor of necroptosis in context-dependent manner. Members of the p90 RSK family can phosphorylate caspase-8 at threonine-265 (T265), which can inactivate caspase-8 for bypassing caspase-8-mediated blockade of necroptosis and can also decrease caspase-8 level by promoting its degradation. Mutating T265 in caspase-8 to alanine (A) in mice blocked TNF-induced necroptotic cecum damage but resulted in unexpectedly massive injury in the small intestine. Here, we show RSK1, RSK2, and RSK3 redundantly function in caspase-8 phosphorylation, and the duodenum is the most severely affected part of the small intestine when T265 phosphorylation of caspase-8 was prevented. Eliminating caspase-8 phosphorylation resulted in a duodenum-specific increase in basal caspase-8 protein level, which shall be responsible for the increased sensitivity to TNF-induced damage. Apoptosis of intestinal epithelial cells (IECs) was predominant in the duodenum of TNF-treated Rsk1-/-Rsk2-/-Rsk3-/- and Casp8T265A/T265A mice, though necroptosis was also observed. The heightened duodenal injury amplified systemic inflammatory responses, as evidenced by the contribution of hematopoietic cells to the sensitization of TNF-induced animal death. Further analysis revealed that hematopoietic and non-hematopoietic cells contributed differentially to cytokine production in response to the increased cell death. Collectively, RSKs emerges as a previously overlooked regulator that, via tissue/organ-constrained inactivating caspase-8 and/or downregulating caspase-8 protein level, controls the sensitivity to TNF-induced organ injury and animal death.

Sepsis results from systemic, dysregulated inflammatory responses to infection, culminating in multiple organ failure. Here, we demonstrate the utility of CD5L for treating experimental sepsis caused by cecal ligation and puncture (CLP). We show that CD5L's important features include its ability to enhance neutrophil recruitment and activation by increasing circulating levels of CXCL1, and to promote neutrophil phagocytosis. CD5L-deficient mice exhibit impaired neutrophil recruitment and compromised bacterial control, rendering them susceptible to attenuated CLP. CD5L-/- peritoneal cells from mice subjected to medium-grade CLP exhibit a heightened pro-inflammatory transcriptional profile, reflecting a loss of control of the immune response to the infection. Intravenous administration of recombinant CD5L (rCD5L) in immunocompetent C57BL/6 wild-type (WT) mice significantly ameliorates measures of disease in the setting of high-grade CLP-induced sepsis. Furthermore, rCD5L lowers endotoxin and damage-associated molecular pattern (DAMP) levels, and protects WT mice from LPS-induced endotoxic shock. These findings warrant the investigation of rCD5L as a possible treatment for sepsis in humans.

This study aimed to evaluate the role of receptor-interacting protein kinase-3 (RIPK3) in the diagnosis, estimation of disease severity, and prognosis of premature infants with necrotising enterocolitis (NEC).

RIPK3, lactic acid (LA), and C-reactive protein (CRP) levels were measured in the peripheral blood of 108 premature infants between 2019 and 2023, including 24 with stage II NEC, 18 with stage III NEC and 66 controls. Diagnostic values of the indicators for NEC were evaluated via receiver operating characteristic (ROC) curve analysis.

Plasma RIPK3 and LA levels upon NEC suspicion in neonates with stage III NEC were 32.37 ± 16.20 ng/mL. The ROC curve for the combination of RIPK3, LA, CRP for NEC diagnosis were 0.925. The time to full enteral feeding (FEFt) after recovery from NEC was different between two expression groups of plasma RIPK3 (RIPK3 < 20.06 ng/mL and RIPK3 ≥ 20.06 ng/mL).

Plasma RIPK3 can be used as a promising marker for the diagnosis and estimation of disease severity of premature infants with NEC and for the guidance on proper feeding strategies after recovery from NEC.