Activation Of RIPK3 Research Articles

Reduced levels of A20 protein prompted RIPK1-dependent apoptosis and blood-brain barrier breakdown during cerebral ischemia reperfusion injury.

Blood-brain barrier (BBB) leakage is an important cause of the exacerbation of pathological features of cerebral ischemia reperfusion injury (CIRI). However, the specific mechanism of BBB leakage is not clear. It was found that the CIRI resulted in RIPK1 activation and subsequent RIPK1-dependent apoptosis (RDA). Inhibition of RIPK1 significantly reduced BBB breakdown and brain damage. The aim of this study is to investigate the mechanism of RIPK1 in the BBB leakage during CIRI. It was discovered by proteomics that autophagy activation resulting from ischemia and reperfusion significantly downregulated the level of A20 protein. A20 is an important protein that regulates RIPK1 and RDA. It was hypothesized that activation of autophagy caused by ischemic reperfusion led to a decrease in A20 protein, which, in turn, caused the activation of RIPK1 and the occurrence of RDA, leading to leakage of the BBB. The findings in this study revealed the role of RIPK1 in the cell death and BBB leakage upon cerebral ischemia reperfusion injury, and these findings provide a novel perspective for the treatment of ischemic reperfusion.

Downregulation of miR-124-3p suppresses the development of the deep retinal blood vessels by enhancing the Stat1/Ripk1 pathway in mouse retinal microglia

The study aimed to investigate the role of microRNA (miR)-124-3p in retinal angiogenesis in a mouse model. An intravitreal injection of miR-124-3p antagomir was used to knockdown the expression of miR-124-3p in the mouse retina at postnatal day (P)3. Immunofluorescent staining of both retinal frozen sections and whole retina were used to observe retinal vascular development in the P6, P9 and P12 mice, as well as the changes in retinal ganglion cells, astrocytes, Müller cells and microglia. Whole retinal RNA extracted from P9 mice was used for transcriptome sequencing. Following gene set enrichment analysis, the enriched genes caused by miR-124-3p inhibition were analyzed by immunofluorescent staining and western blot. Results indicated that deep vascular development was significantly inhibited by the activation of M1 phenotype microglia. Moreover, there were no notable effects on superficial retinal vascular development, the retinal ganglion cells, astrocytes, and Müller cells. The expression of the Stat1/Irf9/Eif2ak2/Ripk1 axis in the miR-124-3p knockdown group was significantly increased. The microglia penetrated deep into the retina and the activation of Ripk1(+) microglia significantly increased, which was accompanied by an increased level of apoptosis to inhibit the deep vascular sprout. Downregulation of miR-124-3p during the early retinal development can suppress the development of the deep retinal blood vessels by enhancing the expression level of the Stat1/Irf9/Eif2ak2/Ripk1 axis and inducing the cell apoptosis of the activation of Ripk1(+) microglia.

Cyclophosphamide enfeebles myocardial isometric contraction force via RIP1/RIP3/MLKL/TRPM7-mediated necroptosis

This study explores the negative impact of cyclophosphamide (CP) on cardiac contractility by specifically examining its effect on the active and passive tension of the cardiac muscle in-vitro and revealing the mechanism through which CP induces myocardial insult in-vivo. In young male Sprague-Dawley rats, cardiac toxicity was induced by a single intraperitoneal injection of CP (150 mg/kg body weight). Axial heart tissue slices were electrically stimulated, and the total isometric contraction force was measured at varying pretension levels. Blood and tissue biochemical assays, and histological/ immuno-histological assessments were conducted to evaluate the underlying molecular mechanisms. Statistical analysis shows that there is a significant difference between the drugged and the control groups in terms of the active tension values. Moreover, the pre-tension stress significantly affects both the active and passive tension values. CP altered heart, body, and heart-to-body weight, desolated cardiac muscle architecture, surged cardiac enzymes (CK-MB, LDH, and cTn l), augmented myocardial oxidative stressors (MDA), and weakened myocardial antioxidant status (SOD and GSH). Mechanistically, cyclophosphamide prompted the necroptotic trajectory evidenced by the activation of RIPK1, RIPK3, MLKL and TRPM7, the inhibition of caspase 8 and BCL2 and the upregulation of the protein/mRNA expression of TNF-α and TNFR1. This study identifies necroptosis as a key factor in cyclophosphamide-evoked myocardial contractility impairment, highlighting its potential as a target for alleviating antitumor-related myocardial damage. This innovative approach to investigating the underlying mechanisms of CP-induced cardiac toxicity offers valuable insights into the potential of developing new therapies to mitigate cyclophosphamide’s negative impact.

RIPK3 promoter hypermethylation in hepatocytes protects from bile acid-induced inflammation and necroptosis

Necroptosis facilitates cell death in a controlled manner and is employed by many cell types following injury. It plays a significant role in various liver diseases, albeit the cell-type-specific regulation of necroptosis in the liver and especially hepatocytes, has not yet been conceptualized. We demonstrate that DNA methylation suppresses RIPK3 expression in human hepatocytes and HepG2 cells. In diseases leading to cholestasis, the RIPK3 expression is induced in mice and humans in a cell-type-specific manner. Overexpression of RIPK3 in HepG2 cells leads to RIPK3 activation by phosphorylation and cell death, further modulated by different bile acids. Additionally, bile acids and RIPK3 activation further facilitate JNK phosphorylation, IL-8 expression, and its release. This suggests that hepatocytes suppress RIPK3 expression to protect themselves from necroptosis and cytokine release induced by bile acid and RIPK3. In chronic liver diseases associated with cholestasis, induction of RIPK3 expression may be an early event signaling danger and repair through releasing IL-8.

Abstract 3737: A novel mechanism of NF-κB activation by genotoxic chemotherapy

Abstract CARP-1, a perinuclear phospho-protein, is a biphasic regulator of cell survival and apoptosis signaling. Genotoxic drugs induce apoptosis in part by elevating CARP-1 levels while also promote cell survival in part by activating NF-κB that involves CARP-1 interaction with NF-κB kinase subunit γ (aka, NEMO). We previously noted interaction of CARP-1 with RIPK1, and since RIPK1 is known to regulate NF-κB signaling, we investigated whether RIPK1-CARP-1 interaction is involved in genotoxic chemotherapy-dependent apoptosis or NF-κB-activated survival signaling. Mutagenesis and co-IP-WB analyses revealed that a 40-amino acid epitope within the amino-terminal catalytic domain of RIPK1 interacted with CARP-1 (611-640) peptide. Overexpression of this CARP-1/CCAR1-binding (CB) epitope peptide or RIPK1 mutant that has in-frame deletion of CB-epitope resulted in moderate but significant reduction in viabilities of cells treated with Adriamycin or Cisplatin relative to the viability loss noted in cells expressing RIPK1 (WT). However, no significant differences in loss of cell viabilities were noted in cells expressing various substitution mutants of RIPK1 (aspartate (D) 138 to asparagine (N), serine (S) 109 and threonine (T) 110 to alanines (A), lysine (K) 87 to arginine (R), K97 to R, K105 to R, or K115 to R) relative to their RIPK1 (WT) expressing counterparts when treated with genotoxic drugs. Interestingly, CB-epitope mutant, but not RIPK1 (WT), failed to translocate to cytoplasm in Adriamycin-treated cells. Genotoxic drugs activated p65/RelA in cells expressing RIPK1 (WT), while expression of RIPK1 CB-epitope mutant resulted in abrogation of p65/RelA activation by Adriamycin or Cisplatin but not by TNFα. Collectively our data demonstrate that CARP-1 interaction with RIPK1 promotes nuclear export of RIPK1 and consequent NF-κB activation in cells exposed to genotoxic drugs. Citation Format: Jaganathan Venkatesh, Magesh Muthu, Arun K. Rishi. A novel mechanism of NF-κB activation by genotoxic chemotherapy. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 3737.

Role of SESTRIN2/AMPK/ULK1 pathway activation and lysosomes dysfunction in NaAsO2-induced liver injury under oxidative stress

Arsenic, a serious environmental poison to human health, is widely distributed in nature. As the main organ of arsenic metabolism, liver is easily damaged. In the present study, we found that arsenic exposure can cause liver injury in vivo and in vitro, to date the underlying mechanism of which is yet unclear. Autophagy is a process that depends on lysosomes to degrade damaged proteins and organelles. Here, we reported that oxidative stress can be induced and then activated the SESTRIN2/AMPK/ULK1 pathway, damaged lysosomes, and finally induced necrosis upon arsenic exposure in rats and primary hepatocytes, which was characterized by lipidation of LC3II, the accumulation of P62 and the activation of RIPK1 and RIPK3. Similarly, lysosomes function and autophagy can be damaged under arsenic exposure, which can be alleviated after NAC treatment and aggravated by Leupeptin treatment in primary hepatocytes. Moreover, we also found that the transcription and protein expressions of necrotic-related indicators RIPK1 and RIPK3 in primary hepatocytes were decreased after P62 siRNA. Taken together, the results revealed that arsenic can induce oxidative stress, activate SESTRIN2/AMPK/ULK1 pathway to damage lysosomes and autophagy, and eventually induce necrosis to damage liver.

Downregulation of G protein–coupled receptor kinase 4 protects against kidney ischemia-reperfusion injury

Ischemia/reperfusion injury of the kidney is associated with high morbidity and mortality, and treatment of this injury remains a challenge. G protein-coupled receptor kinase 4 (GRK4) plays a vital role in essential hypertension and myocardial infarction, but its function in kidney ischemia/reperfusion injury remains undetermined. Among the GRK subtypes (GRK2-6) expressed in kidneys, the increase in GRK4 expression was much more apparent than that of the other four GRKs 24 hours after injury and was found to accumulate in the nuclei of injured mouse and human renal tubule cells. Gain- and loss-of-function experiments revealed that GRK4 overexpression exacerbated acute kidney ischemia/reperfusion injury, whereas kidney tubule-specific knockout of GRK4 decreased injury-induced kidney dysfunction. Necroptosis was the major type of tubule cell death mediated by GRK4, because GRK4 significantly increased receptor interacting kinase (RIPK)1 expression and phosphorylation, subsequently leading to RIPK3 and mixed lineage kinase domain-like protein (MLKL) phosphorylation after kidney ischemia/reperfusion injury, but was reversed by necrostatin-1 pretreatment (an RIPK1 inhibitor). Using co-immunoprecipitation, mass spectrometry, and siRNA screening studies, we identified signal transducer and activator of transcription (STAT)1 as a GRK4 binding protein, which co-localized with GRK4 in the nuclei of renal tubule cells. Additionally, GRK4 phosphorylated STAT1 at serine 727, whose inactive mutation effectively reversed GRK4-mediated RIPK1 activation and tubule cell death. Kidney-targeted GRK4 silencing with nanoparticle delivery considerably ameliorated kidney ischemia/reperfusion injury. Thus, our findings reveal that GRK4 triggers necroptosis and aggravates kidney ischemia/reperfusion injury, and its downregulation may provide a promising therapeutic strategy for kidney protection.

OASL phase condensation induces amyloid-like fibrillation of RIPK3 to promote virus-induced necroptosis

RIPK3–ZBP1–MLKL-mediated necroptosis is a proinflammatory cell death process that is crucial for antiviral host defence. RIPK3 self-oligomerization and autophosphorylation are prerequisites for executing necroptosis, yet the underlying mechanism of virus-induced RIPK3 activation remains elusive. Interferon-inducible 2′-5′ oligoadenylate synthetase-like (OASL) protein is devoid of enzymatic function but displays potent antiviral activity. Here we describe a role of OASL as a virus-induced necroptosis promoter that scaffolds the RIPK3–ZBP1 non-canonical necrosome via liquid-like phase condensation. This liquid-like platform of OASL recruits RIPK3 and ZBP1 via protein–protein interactions to provide spatial segregation for RIPK3 nucleation. This process facilitates the amyloid-like fibril formation and activation of RIPK3 and thereby MLKL phosphorylation for necroptosis. Mice deficient in Oasl1 exhibit severely impaired necroptosis and attenuated inflammation after viral infection, resulting in uncontrolled viral dissemination and lethality. Our study demonstrates an interferon-induced innate response whereby OASL scaffolds RIPK3–ZBP1 assembly via its phase-separated liquid droplets to facilitate necroptosis-mediated antiviral immunity.

SENP1 prevents steatohepatitis by suppressing RIPK1-driven apoptosis and inflammation

Activation of RIPK1-driven cell death and inflammation play important roles in the progression of nonalcoholic steatohepatitis (NASH). However, the mechanism underlying RIPK1 activation in NASH remains unclear. Here we identified SENP1, a SUMO-specific protease, as a key endogenous inhibitor of RIPK1. SENP1 is progressively reduced in proportion to NASH severity in patients. Hepatocyte-specific SENP1-knockout mice develop spontaneous NASH-related phenotypes in a RIPK1 kinase-dependent manner. We demonstrate that SENP1 deficiency sensitizes cells to RIPK1 kinase-dependent apoptosis by promoting RIPK1 activation following TNFα stimulation. Mechanistically, SENP1 deSUMOylates RIPK1 in TNF-R1 signaling complex (TNF-RSC), keeping RIPK1 in check. Loss of SENP1 leads to SUMOylation of RIPK1, which re-orchestrates TNF-RSC and modulates the ubiquitination patterns and activity of RIPK1. Notably, genetic inhibition of RIPK1 effectively reverses disease progression in hepatocyte-specific SENP1-knockout male mice with high-fat-diet-induced nonalcoholic fatty liver. We propose that deSUMOylation of RIPK1 by SENP1 provides a pathophysiologically relevant cell death-restricting checkpoint that modulates RIPK1 activation in the pathogenesis of nonalcoholic steatohepatitis.

RIPK3 Deficiency Caused By DNA Hypermethylation Blocks R-2-Hydroxyglutarate Induced Necroptosis in AML with IDH Mutation

Background Mutations in isocitrate dehydrogenases (IDH) are frequently identified in 10% - 20% of acute myeloid leukemia (AML) cases. IDH mutations induce a neomorphic enzymatic function that catalyzes the conversion of α-ketoglutarate (α-KG) to the R enantiomer of 2-hydroxyglutarate (R-2HG). α-KG is also a co-substrate, together with oxygen and the co-factor iron, for α-KG-dependent dioxygenases. R-2HG is structurally close to α-KG and competitively inhibits the activity of α-KG-dependent dioxygenases. It has been reported that R-2HG inhibits the growth of IDH-wild-type AML cells by inhibiting the activity of fat mass and obesity-associated protein (FTO) and then reducing MYC and CEBPA expression. Recently, R-2HG was found to suppress aerobic glycolysis and proliferation in myeloid leukemia cells. However, why AML cells carrying IDH mutations tolerated high levels of R-2HG is not clear. Results In line with the previous study, we found that R-2HG markedly inhibited the viability of AML cells in a dose- and time-dependent manner. Interestingly, Necrostatin-1(Nec-1), a necroptosis inhibitor, could significantly prevent cell death induced by R-2HG in AML cell lines, but not Ferrostatin-1 (a ferroptosis inhibitor), Z-VAD (an apoptosis inhibitor), 3-MA (an autophagy inhibitor) and VX-765 (a pyroptosis inhibitor), suggesting that necroptosis contributed to the anti-leukemia activity of R-2HG. We evaluated the expression of genes involved in the necroptosis pathway and found the level of RIPK1 was significantly increased in R-2HG treated SKM-1 and THP-1 cells, while the expressions of RIPK3 and MLKL did not change. The knockdown of RIPK1 reduced the necroptosis caused by R-2HG in SKM-1 and THP-1 cells. Besides, a significant decrease in proliferation was observed when RIPK1 was overexpressed in AML cells which was significantly rescued by Nec-1. Among α-KG-dependent dioxygenases, the expression of lysine demethylase 2B (KDM2B) was negatively correlated with that of RIPK1. Moreover, KDM2B deficiency significantly increased RIPK1 expression and triggered necroptosis. ChIP followed by qPCR (ChIP-qPCR) showed that the RIPK1 promoter region was significantly enriched by H3K4me3, a main target of KDM2B. R-2HG treatment or knockdown of KDM2B activated H3K4me3 and increased RIPK1 expression. By comparing the expression of necroptosis-related genes between R-2HG-resistant and sensitive cell lines (GSE87187), we found that RIPK3, another key player of necroptosis, was under-expressed in R-2HG-resistant cell lines. Consistently, RIPK3 was lowly expressed in IDH-mutant primary AML cells compared to that of IDH wild-type primary AML cells. We found that enhanced RIPK3 levels increased the toxicity of R-2HG in R-2HG-resistant cell lines. Methylation-specific PCR (MS-PCR) revealed that methylation occurred in this region of genomic DNA near the TSS of RIPK3 in R-2HG-resistant cell lines. Primary cells from IDH-mutant AML patients were more sensitive to decitabine than those from IDH wild-type AML patients. Conclusion Herein, we reported that R-2HG triggered necroptosis in AML cell lines and IDH wild-type primary AML cells via KDM2B/RIPK1 signal pathway. The loss of RIPK3 expression resulting from RIPK3 promoter hypermethylation contributed to R-2HG tolerance in IDH mutated primary AML cells and R-2HG-resistant AML cell lines. IDH-mutant AML cells were more sensitive to DNMT inhibitor. These results implicate that restoring RIPK3 activity is a promising way to reverse R-2HG-resistant in AML cells with IDH mutation. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Microglial Dysfunction in Neurodegenerative Diseases via RIPK1 and ROS.

Microglial dysfunction is a major contributor to the pathogenesis of multiple neurodegenerative diseases. The neurotoxicity of microglia associated with oxidative stress largely depends on NF-κB pathway activation, which promotes the production and release of microglial proinflammatory cytokines and chemokines. In this review, we discuss the current literature on the essential role of the NF-κB pathway on microglial activation that exacerbates neurodegeneration, with a particular focus on RIPK1 kinase activity-dependent microglial dysfunction. As upregulated RIPK1 kinase activity is associated with reactive oxygen species (ROS) accumulation in neurodegenerative diseases, we also discuss the current knowledge about the mechanistic links between RIPK1 activation and ROS generation. Given RIPK1 kinase activity and oxidative stress are closely regulated with each other in a vicious cycle, future studies are required to be conducted to fully understand how RIPK1 and ROS collude together to disturb microglial homeostasis that drives neurodegenerative pathogenesis.

PP6 negatively modulates LUBAC-mediated M1-ubiquitination of RIPK1 and c-FLIPL to promote TNFα-mediated cell death

Activation of TNFR1 by TNFα induces the formation of a membrane-associated, intracellular complex termed complex I. Complex I orchestrates a complex pattern of modifications on key regulators of TNF signaling that collectively determines the cell fate by activating pro-survival or executing cell death programs. However, the regulatory mechanism of complex I in cell-fate decision is not fully understood. Here we identify protein phosphatase-6 (PP6) as a previously unidentified component of complex I. Loss of PP6 protects cells from TNFα-mediated cell death. The role of PP6 in regulating cell death requires its phosphatase activity and regulatory subunits. Further mechanistic studies show that PP6 modulates LUBAC-mediated M1-ubiquitination of RIPK1 and c-FLIPL to promote RIPK1 activation and c-FLIPL degradation. We also show that melanoma-associated PP6 inactivating mutants offer resistance to cell death due to the loss of sensitivity to TNFα. Thus, our study provides a potential mechanism by which melanoma-related PP6 inactivating mutations promote cancer progression.

422.7: Necroptosis Plays a Role in Acute Kidney Injury (AKI) and the Progression to Renal Fibrosis Following Ischemia Reperfusion Injury (IRI)

Introduction: In kidney transplantation, ischemia reperfusion injury (IRI), a type of acute kidney injury (AKI), is associated with poorer outcomes including delayed graft function, rejection and chronic kidney disease (CKD) with fibrosis. Therapies to reduce AKI and CKD following transplantation have been investigated but none has been translated into clinical practice. Here we examined necroptosis, a pathway of regulated necrosis that has been implicated in kidney IRI. Necroptosis is triggered by recruitment of the intracellular kinases RIPK1 and RIPK3 and activation of the pseudokinase MLKL. Active MLKL causes cell death by plasma membrane rupture, driving “necroinflammation”. RIPK1 and RIPK3 may also have a cell death-independent role in the development of kidney fibrosis. The aims of this study were to (1) investigate the role of necroptosis in a mouse IRI model of AKI, using knockout mice and small molecule inhibitors, and (2) determine whether inhibition of RIPK1 or RIPK3 after AKI interferes with the progression to CKD. Methods: AKI model: 10-12 week old male MLKL KO mice and wild type (WT) littermates underwent right nephrectomy followed by 18, 20, 22 or 24 min left kidney ischemia. Samples were collected at 24 h to assess renal injury, inflammation and necroptosis. In a separate experiment using the 18 min model, WT mice were treated either before or after IR with a RIPK1 inhibitor Nec-1s, a RIPK3 inhibitor GSK872, or vehicle.CKD-after-AKI model: This model was adapted from the AKI 18 min model by (i) omitting right nephrectomy, (ii) delaying the endpoint to 28 days, and (iii) extending the analyses to include renal fibrosis (Masson’s trichrome staining) and relevant gene expression. WT mice were treated with Nec-1s, GSK872 or vehicle daily on days 3-9. Results: In the AKI model, expression of necroptotic (RIPK1, RIPK3 and MLKL), kidney injury (KIM-1, NGAL) and inflammatory (IL-6, IL-1β, IL-33, TNFα) genes was significantly upregulated following moderate (18 min) and severe (20-24 min) ischemia. MLKL KO mice were protected from moderate but not severe IRI, with lower creatinine (µmol/L) (47.13±6.28 vs 80.78±10.45, p=0.007) and less tubular injury (%) (16.79±2.80 vs 26.95±3.68, p=0.048) compared to WT littermates. WT mice receiving Nec-1s before or after IR showed reduced creatinine and tubular injury compared to vehicle controls, whereas GSK872 was not protective.In the CKD-after-AKI model, vehicle-treated WT mice showed significant renal fibrosis at 28 days compared to sham (fibrosis score 28.68±1.25 vs 2.33±0.11, p=0.0025), with increased expression of RIPK1 (p=0.0079), RIPK3 (p=0.0025), and the pro-fibrotic genes TGFβ (p=0.0025), HIF1α (p=0.0025) and Col-I (p=0.0025). Inhibiting RIPK1 or RIPK3 on days 3-9 resulted in reduced fibrosis.Conclusion: Our data support a role for necroptosis in AKI and fibrosis following IR, and identify inhibition of the necroptosis pathway as a potential therapeutic strategy. NHMRC scholarship GNT1168307.

Abstract 944: Loss of Ripk3 signaling promotes T cell lymphoma development in Tet2 deficient mice

Abstract The TET2 gene is commonly mutated in multiple hematological malignancies and is frequently associated with poor prognosis. TET2 mutations are also found to occur in healthy individuals with no apparent hematological disease. A common factor observed between such individuals and those with the disease is the presence of increased inflammation and cytokine signature. A key mediator of inflammation and cytokine-induced necroptosis and metabolic signaling is the receptor-interacting protein kinase 3 (Ripk3). Ripk3 plays important tumor-promoting and suppressive roles in multiple cancers. Compared to bone marrow (BM) cells isolated from wild-type mice, higher levels of Ripk3 activity can be detected in Tet2-/- BM cells. Based on the evidence that Tet2 mutations alone are insufficient and are known to cooperate with other mutations for full-blown malignant transformation, we hypothesize that loss of Ripk3 signaling might promote the transformation of Tet2 mutant HSPCs To test our hypothesis we crossed Tet2 conditional knockout (Tet2fx/fx Mx1-Cre+) mice with Ripk3-/- mice to generate Tet2 and Ripk3 compound knockout (Tet2-/-Ripk3-/-) mice. We performed in vitro colony assays and in vivo engraftment and reconstitution assays on Bone marrow mononuclear cells (BM-MNC) derived from Tet2-/-Ripk3-/- and Tet2-/- mice. Our results indicate that TNF-α stimulation confers a significant hematopoietic advantage to Tet2-/- HSPCs which is abolished upon loss of Ripk3. Moreover, while Tet2-/- BM-MNC has significantly higher reconstitution ability in vivo, the additional loss of Ripk3 led to reduced engraftment and reconstitution that was comparable to WT BM-MNC. Given that TNF-α stimulation promotes Ripk3 activation, our results indicate that the TNF-RIPK3 signaling axis is essential in conferring enhanced hematopoietic fitness of Tet2-/- HSPCs.Tet2-/-Ripk3-/- mice developed aggressive tumors by 12-15 months of age as characterized by profound hepatosplenomegaly and lymphadenopathy, with substantial lymphocytosis, neutrophilia, anemia, and thrombocytopenia. There was extensive infiltration of tumor cells in the liver and spleen in diseased Tet2-/-Ripk3-/- mice. There was a marked expansion of both CD4+PD1+CXCR5+ follicular T helper cells (Tfh) and CD4+PD1+ peripheral T helper cells (Tph), indicating the development of a T cell lymphoma in the Tet2-/-Ripk3-/- mice. Additionally, disease characteristics including the reduced surface expression of CD3 in the tumor cells, increased levels of classical Th cytokines in the serum, as well as the presence of heterogeneous populations of cells within the tumor tissues recapitulate the pathological features of angioimmunoblastic T cell lymphoma (AITL). We are currently investigating Ripk3 signaling in the tumor cells of AITL patients and whether we can treat such aggressive fatal diseases by reactivating Ripk3 signaling. Citation Format: Kanak Joshi, Shuchi Zinzuwadia, Ryan Mack, Lei Zhang, Shanhui Liu, Mark Sellin, Wei Wei, Peter Breslin, Ameet R. Kini, Jiwang Zhang. Loss of Ripk3 signaling promotes T cell lymphoma development in Tet2 deficient mice [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 944.

Aniline exposure activates receptor-interacting serine/threonineprotein kinase 1 and causes necroptosis of AML12 cells.

With the increased use of aniline, potential impacts on human health cannot be ignored. The hepatotoxicity of aniline is largely unknown and the underlying mechanism remains unclear. Therefore, the aim of the present study was to investigate the hepatotoxicity of aniline and elucidate the underlying mechanism. AML12 cells were exposed to different concentrations of aniline (0, 5, 10, or 20mM) to observe changes to reactive oxygen species (ROS) production and the expression patterns of necroptosis-related proteins (RIPK1, RIPK3, and MLKL). The potential mechanism underlying aniline-induced hepatotoxicity was explored by knockout of RIPK1. The results showed that aniline induced cytotoxicity in AML12 cells in a dose-dependent manner in addition to the production of ROS and subsequent necroptosis of AML12 cells. Silencing of RIPK1 reversed upregulation of necroptosis-related proteins in AML12 cells exposed to aniline, demonstrating that aniline-induced ROS production was related to necroptosis of AML12. Moreover, aniline promoted intracellular RIPK1 activation, suggesting that the RIPK1/ROS pathway plays an important role in aniline-induced hepatotoxicity. NAC could quench ROS and inhibit necroptosis. These results provide a scientific basis for future studies of aniline-induced hepatotoxicity for the prevention and treatment of aniline-induced cytotoxicity.

Nuclear RIPK1 promotes chromatin remodeling to mediate inflammatory response.

RIPK1 is a master regulator of multiple cell death pathways, including apoptosis and necroptosis, and inflammation. Importantly, activation of RIPK1 has also been shown to promote the transcriptional induction of proinflammatory cytokines in cells undergoing necroptosis, in animal models of amyotrophic lateral sclerosis (ALS) and Alzheimer's disease (AD), and in human ALS and AD. Rare human genetic carriers of non-cleavable RIPK1 variants (D324V and D324H) exhibit distinct symptoms of recurrent fevers and increased transcription of proinflammatory cytokines. Multiple RIPK1 inhibitors have been advanced into human clinical trials as new therapeutics for human inflammatory and neurodegenerative diseases, such as ALS and AD. However, it is unclear whether and how RIPK1 kinase activity directly mediates inflammation independent of cell death as the nuclear function of RIPK1 has not yet been explored. Here we show that nuclear RIPK1 is physically associated with the BAF complex. Upon RIPK1 activation, the RIPK1/BAF complex is recruited by specific transcription factors to active enhancers and promoters marked by H3K4me1 and H3K27ac. Activated nuclear RIPK1 mediates the phosphorylation of SMARCC2, a key component of the BAF complex, to promote chromatin remodeling and the transcription of specific proinflammatory genes. Increased nuclear RIPK1 activation and RIPK1/BAF-mediated chromatin-remodeling activity were found in cells expressing non-cleavable RIPK1, and increased enrichment of activated RIPK1 on active enhancers and promoters was found in an animal model and human pathological samples of ALS. Our results suggest that RIPK1 kinase serves as a transcriptional coregulator in nucleus that can transmit extracellular stimuli to the BAF complex to modulate chromatin accessibility and directly regulate the transcription of specific genes involved in mediating inflammatory responses.

Correction to: GLP-1 Receptor Agonist Inhibited the Activation of RIPK1 for Alleviation the Neuronal Death and Neuroinfammation in APP/PS1 Mice

Correction to: GLP-1 Receptor Agonist Inhibited the Activation of RIPK1 for Alleviation the Neuronal Death and Neuroinfammation in APP/PS1 Mice

Osmotic stress activates RIPK3/MLKL-mediated necroptosis by increasing cytosolic pH through a plasma membrane Na+/H+ exchanger.

Necroptosis is a form of cell death triggered by stimuli such as the tumor necrosis factor family of cytokines, which induce necrotic cell death through the RIPK1-RIPK3-MLKL pathway. We report here that necroptosis is also activated by extracellular osmotic stresses. Unlike the previously identified inducers of necroptosis, osmotic stress stimulated necroptosis through the direct activation of the kinase activity of RIPK3 by an increase in cytosolic pH mediated by the Na+/H+ exchanger SLC9A1. Knockout, knockdown, or chemical inhibition of SLC9A1 blocked necroptosis induced by osmotic stresses. Moreover, setting intracellular pH at above-physiological values directly activated RIPK3 and necroptosis. The activation of RIPK3 by osmotic stresses did not require its RHIM domain, the protein-interacting domain required for the activation of RIPK3 when cells respond to other previously identified necroptotic stimuli. These results thus delineate a pathway that activates necroptosis in response to osmotic stresses.

Isolates of Salmonella typhimurium circumvent NLRP3 inflammasome recognition in macrophages during the chronic phase of infection

Inflammasome signaling results in cell death and release of cytokines from the IL-1 family, which facilitates control over an infection. However, some pathogens such as Salmonella typhimurium (ST) activate various innate immune signaling pathways, including inflammasomes, yet evade these cell death mechanisms, resulting in a chronic infection. Here we investigated inflammasome signaling induced by acute and chronic isolates of ST obtained from different organs. We show that ST isolated from infected mice during the acute phase displays an increased potential to activate inflammasome signaling, which then undergoes a protracted decline during the chronic phase of infection. This decline in inflammasome signaling was associated with reduced expression of virulence factors, including flagella and the Salmonella pathogenicity island I genes. This reduction in cell death of macrophages induced by chronic isolates had the greatest impact on the NLRP3 inflammasome, which correlated with a reduction in caspase-1 activation. Furthermore, rapid cell death induced by Casp-1/11 by ST in macrophages limited the subsequent activation of cell death cascade proteins Casp-8, RipK1, RipK3, and MLKL to prevent the activation of alternative forms of cell death. We observed that the lack of the ability to induce cell death conferred a competitive fitness advantage to ST only during the acute phase of infection. Finally, we show that the chronic isolates displayed a significant attenuation in their ability to infect mice through the oral route. These results reveal that ST adapts during chronic infection by circumventing inflammasome recognition to promote the survival of both the host and the pathogen.

Human RIPK3 maintains MLKL in an inactive conformation prior to cell death by necroptosis

The ancestral origins of the lytic cell death mode, necroptosis, lie in host defense. However, the dysregulation of necroptosis in inflammatory diseases has led to widespread interest in targeting the pathway therapeutically. This mode of cell death is executed by the terminal effector, the MLKL pseudokinase, which is licensed to kill following phosphorylation by its upstream regulator, RIPK3 kinase. The precise molecular details underlying MLKL activation are still emerging and, intriguingly, appear to mechanistically-diverge between species. Here, we report the structure of the human RIPK3 kinase domain alone and in complex with the MLKL pseudokinase. These structures reveal how human RIPK3 structurally differs from its mouse counterpart, and how human RIPK3 maintains MLKL in an inactive conformation prior to induction of necroptosis. Residues within the RIPK3:MLKL C-lobe interface are crucial to complex assembly and necroptotic signaling in human cells, thereby rationalizing the strict species specificity governing RIPK3 activation of MLKL.