Receptor-interacting Serine/threonine-protein Kinase 1 Research Articles

Discovery of Potent and Selective Receptor-Interacting Serine/Threonine Protein Kinase 2 (RIPK2) Inhibitors for the Treatment of Inflammatory Bowel Diseases (IBDs).

Receptor-interacting serine/threonine protein kinase 2 (RIPK2) has been demonstrated to be a promising target for treating inflammatory diseases. Herein, we describe the discovery and optimization of a series of RIPK2 inhibitors derived from an FLT3 inhibitor, CHMFL-FLT3-165. Compound 10w was identified to possess an IC50 value of 0.6 nM for RIPK2 and greater than 50,000-fold selectivity over its family homologous kinase RIPK1 (IC50 > 30 μM). It exhibited high kinase selectivity and inhibited RIPK2 to prevent NOD-induced cytokine production following muramyl dipeptide (MDP) stimulation. In an acute colitis model, compound 10w exerted better therapeutic effects than the JAK inhibitor filgotinib and the RIPK2 inhibitor WEHI-345. These robust results of in vitro and in vivo pharmacodynamic experiments demonstrate that RIPK2 as a therapeutic target shows potential abilities for the treatment of inflammatory bowel diseases.

Taurine inhibits necroptosis helps to alleviate inflammatory and injury induced by Klebsiella infection

Klebsiella infection is widely acknowledged to inflict severe inflammatory damage in bovines. Herein, we demonstrate significant death of EpH4-Ev cells incubated with Klebsiella. And compelling evidence shows that Klebsiella infection increases interactions between the Receptor-interacting serine/threonine-protein kinase 1 (RIPK1) and RIPK3, which promotes phosphorylation of RIPK3 and MLKL to induce necroptosis. However, these changes can be partially reversed by taurine and Nec-1s. Moreover, using taurine and Nec-1s to partially inhibit necroptosis significantly reduce TNF-α, IL-1β and IL-6 levels and NAGase activity induced by Klebsiella infection. Taken together, taurine partially inhibits necroptosis induced by Klebsiella infection and hence alleviates inflammatory and injury in EpH4-Ev cells.

MLKL Regulates Rapid Cell Death-independent HMGB1 Release in RSV Infected Airway Epithelial Cells.

Respiratory syncytial virus (RSV)-induced bronchiolitis is a significant contributor to infant morbidity and mortality. Previously, we identified that necroptosis, a pro-inflammatory form of cell death mediated by receptor-interacting serine/threonine-protein kinase 1 (RIPK1) and RIPK3, and mixed lineage kinase domain like protein (MLKL), occurs in RSV-infected human airway epithelial cells (hAECs), mediating the release of the alarmin high mobility group box 1 (HMGB1). Here, we show that RSV infection of hAECs induces the biphasic release of HMGB1 at 6 (“early”) and 24 (“late”) hours post infection (hpi). The early phase of HMGB1 release at 6 hpi is cell death-independent, however, this release is nonetheless attenuated by inhibition of MLKL (primarily associated with necroptosis). The early release of HMGB1 promotes the late phase of HMGB1 release via the activation of RAGE (receptor for advanced glycation endproducts) and occurs with cell death. Treatment of hAECS with exogenous HMGB1 combined with a pan-caspase inhibitor induces hAEC necroptosis, and is attenuated by the RAGE antagonist, FPS-ZM1. Together, these findings demonstrate that RSV infection of hAECs leads to the early release of HMGB1, followed by a paracrine feed-forward amplification loop that further increases HMGB1 levels and promotes cell death. As the inhibition of MLKL or targeting of HMGB1/RAGE pathway attenuates the release of pro-inflammatory HMGB1 and decreases viral load, this suggests that the pharmacological targeting of these pathways may be of benefit for the treatment of severe RSV bronchiolitis.

Reovirus Activated Cell Death Pathways.

Mammalian orthoreoviruses (ReoV) are non-enveloped viruses with segmented double-stranded RNA genomes. In humans, ReoV are generally considered non-pathogenic, although members of this family have been proven to cause mild gastroenteritis in young children and may contribute to the development of inflammatory conditions, including Celiac disease. Because of its low pathogenic potential and its ability to efficiently infect and kill transformed cells, the ReoV strain Type 3 Dearing (T3D) is clinical trials as an oncolytic agent. ReoV manifests its oncolytic effects in large part by infecting tumor cells and activating programmed cell death pathways (PCDs). It was previously believed that apoptosis was the dominant PCD pathway triggered by ReoV infection. However, new studies suggest that ReoV also activates other PCD pathways, such as autophagy, pyroptosis, and necroptosis. Necroptosis is a caspase-independent form of PCD reliant on receptor-interacting serine/threonine-protein kinase 3 (RIPK3) and its substrate, the pseudokinase mixed-lineage kinase domain-like protein (MLKL). As necroptosis is highly inflammatory, ReoV-induced necroptosis may contribute to the oncolytic potential of this virus, not only by promoting necrotic lysis of the infected cell, but also by inflaming the surrounding tumor microenvironment and provoking beneficial anti-tumor immune responses. In this review, we summarize our current understanding of the ReoV replication cycle, the known and potential mechanisms by which ReoV induces PCD, and discuss the consequences of non-apoptotic cell death—particularly necroptosis—to ReoV pathogenesis and oncolysis.

An IgM monoclonal antibody against domain 1 of CD147 induces non-canonical RIPK-independent necroptosis in a cell type specific manner in hepatocellular carcinoma cells

CD147/Basigin/EMMPRIN is overexpressed in several cancerous tissues and it has been shown to induce matrix metalloproteinases (MMPs) whose expression is associated with cancer metastasis. Thus, targeting CD147 with monoclonal antibodies (mAbs) potentially has therapeutic applications in cancer immunotherapy. Here, we report the use of anti-CD147 mAbs targeting domain 1 of CD147, namely M6-1D4 (IgM), M6-1F3 (IgM), M6-2F9 (IgM) and M6-1E9 (IgG2a), against several human cancer cell lines. Strikingly, IgM but not IgG mAbs against CD147, especially clone M6-1D4, induced acute cellular swelling, and this phenomenon appeared to be specifically found with hepatocellular carcinoma (HCC) cells. Furthermore, molecular investigation upon treating HepG2 cells with M6-1D4 showed unfolded protein response (UPR) activation, autophagosome accumulation, and cell cycle arrest, but without classic apoptosis related features. More interestingly, prolonged M6-1D4 treatment (24 h) resulted in irreversible oncosis leading to necroptosis. Furthermore, treatment with a mixed lineage kinase domain-like psuedokinase (MLKL) inhibitor and partial knockout of MLKL resulted in reduced sensitivity to necroptosis in M6-1D4-treated HepG2 cells. Surprisingly however, the observed necroptotic signaling axis appeared to be non-canonical as it was independent of receptor-interacting serine/threonine-protein kinase (RIPK) phosphorylation. In addition, no cytotoxic effect on human dermal fibroblast (HDF) was observed after incubation with M6-1D4. Taken together, this study provides clues to target CD147 in HCC using mAbs, as well as sheds new light on a novel strategy to kill cancerous cells by the induction of necroptosis.

Linear ubiquitination modification of NR6A1 by LUBAC inhibits RIPK3 kinase activity and attenuates apoptosis of vascular smooth muscle cells

Nuclear receptor subfamily 6 group A member 1 (NR6A1) is involved in promoting the apoptotic process of vascular smooth muscle cells (VSMCs) which is a critical process involved in atherosclerosis, but the action mechanism remains to be determined. Therefore, we studied the underlying mechanisms by which NR6A1 accelerated VSMC apoptosis in atherosclerosis. An atherosclerosis model has been established in apolipoprotein E-deficient rats with a high-fat diet for 12 weeks, which was characterized by pathological aortic plaques, increased lipid depositionand collagen content in aortic tissues, and high cholesterol and triglycerides levels in the serum. NR6A1 was experimentally shown to increase at protein level rather than messenger RNA level in atherosclerotic rats. Immunofluorescence exhibited the main location of NR6A1 in the cell nucleus of rat aortic tissues. By performing ectopic expression experiments, NR6A1 was demonstrated to suppress the viability and expedite the apoptosis of VSMCs, corresponding to augmented caspase-3, caspase-8, and caspase-9 activities. It was further unraveled that NR6A1 could activate receptor-interacting serine/threonine-protein kinase 3 (RIPK3) by inducing its phosphorylation. Conversely, RIPK3 inhibitor GSK872 undermined the proapoptotic effect of NR6A1 on VSMCs. The co-immunoprecipitationassay identified that linear ubiquitin chain assembly complex (LUBAC) can be pulled down by NR6A1. Furthermore. LUBAC inhibited the expression of NR6A1 by promoting its linear ubiquitination, thereby dephosphorylating RIPK3 and consequently inhibiting the VSMC apoptosis. Overall, LUBAC-induced linear ubiquitination of NR6A1 can potentially arrest the apoptosis of VSMCs in atherosclerosis by downregulating RIPK3 and attenuating caspase activity. This finding suggests promising athero-protective targets by limiting VSMC apoptosis.

IL-37 Expression in Patients with Abdominal Aortic Aneurysm and Its Role in the Necroptosis of Vascular Smooth Muscle Cells.

Background Our previous studies have shown that interleukin- (IL-) 37 plays a protective role in patients and animal models with coronary artery disease. However, the role of IL-37 in patients with abdominal aortic aneurysm (AAA), another artery disease, is yet to be elucidated. Methods and Results AAA tissues and plasma samples were obtained from patients with or without surgical intervention. Normal renal aortic tissues were collected from kidney transplant donors. Our findings established that in AAA, IL-37 was distributed in endothelial cells, macrophages, and vascular smooth muscle cells (VSMCs) and that it was chiefly concentrated in VSMCs. Furthermore, the expression was found to be downregulated compared with that in normal artery tissues. Immunofluorescence showed that, unlike normal arteries, IL-37 was translocated to the nucleus of VSMCs in AAA. Moreover, in patients with AAA, the expressions of IL-37, IL-6, and tumor necrosis factor- (TNF-) α were increased in the plasma in comparison with the healthy controls. Correlation analysis revealed that IL-37 was positively correlated with IL-6, TNF-α, age, aneurysm diameter, and blood pressure. Furthermore, human aortic vascular smooth muscle cells (HASMCs) were stimulated with angiotensin II (AngII) in vitro to simulate smooth muscle cell (SMC) damage in AAA. A decrease in IL-37 expression and an increase in receptor-interacting serine/threonine-protein kinase 3 (RIPK3) expression were observed in HASMCs stimulated with AngII. On this basis, inhibition of RIPK3 with GSK'872 significantly attenuated necroptosis. Moreover, the necroptosis rates were significantly lowered in HASMCs treated with recombinant IL-37, whereas the rates were enhanced when the cells were depleted of the interleukin. Immunoblotting results showed that both exogenous and endogenous IL-37 could affect the expressions of RIPK3, NLRP3, and IL-1β. Also, the phosphorylation of RIPK3 and p65 was affected. Meanwhile, IL-37 promoted the transition of SMC from proliferative type to contractile type. Conclusions The expression of IL-37 in VSMCs decreases in patients with AAA, whereas IL-37 supplementation suppresses RIPK3-mediated necroptosis and promotes the transition of VSMCs from proliferative to contractile type.

Loss of Endothelial Cell RIPK3 Leads to Increased Vascular Permeability Following Intestinal Ischemia‐Reperfusion Injury

The receptor‐interacting serine/threonine‐protein kinase 3 (RIPK3) is primarily known for promoting necroptosis, but recent findings suggest that RIPK3 has non‐necroptotic roles as well, particularly in endothelial cells (ECs). Our lab has reported both beneficial and detrimental roles for endothelial RIPK3 during vascular development, which has prompted us to study RIPK3 in the context of different postnatal vascular pathologies. Our current study focuses on roles for endothelial RIPK3 following intestinal ischemia‐reperfusion (I/R) injury in mice. First‐order branches of the superior mesenteric artery that feeds the ileum were clamped for 1 hr to induce ischemia, and clamps were removed subsequently to allow 24 hr reperfusion in tamoxifen‐induced Ripk3;Cdh5(PAC)‐CreERT2 mice (RIPK3ECKO) compared to wildtype littermate control mice (WT) (males and females, 12 weeks old). Unclamped small intestinal regions were considered as sham control in the same mouse. An Evans blue permeability assay was utilized in assessing small intestinal and distal organ vascular permeability. Small intestinal vascular permeability directly downstream of I/R injury was significantly increased in both RIPK3ECKO and WT mice (n=6 mice/group), however permeability was also significantly increased in sham small intestinal regions and in livers of RIPK3ECKO unlike in WT controls (n=6 mice/group). No difference in vascular permeability was observed between RIPK3ECKO and WT mice (n=5 mice/group) at baseline without I/R injury in all organs assessed. However, in uninjured RIPK3ECKO mice we do observe an increase in the CD68+ proinflammatory macrophage marker in the liver and submucosal layer of the small intestine compared to uninjured WT mice (n=5 mice/group). When clodronate liposomes were administered to deplete macrophages and determine whether this CD68+ macrophage population was contributing to the increased permeability in RIPK3ECKO mice following I/R injury, we observed a permeability rescue in the small intestine but not in the liver (n=4‐5 mice/group). This finding suggests that there are organ‐specific differences in macrophage contribution to vascular permeability following I/R injury. Our current studies are evaluating circulating and tissue specific cytokines and endothelial cell adhesion molecules to understand why RIPK3ECKO vasculature becomes more permeable in response to I/R injury. Discovering cellular mechanisms for vascular endothelial RIPK3 following I/R injury could provide novel therapeutic targets to promote vessel recovery after I/R injury.

High Receptor-interacting Serine/Threonine-protein Kinase 3 (RIP3) Expression Serves as an Independent Poor Prognostic Factor for Triple-negative Breast Carcinoma.

Receptor-interacting serine/threonine-protein kinase 3 (RIP3) is a key component related to tumor necrosis factor-dependent necroptosis. RIP3 has been known to be a predictive biomarker in many types of carcinomas. We aimed to investigate whether RIP3 expression is correlated with clinicopathological characteristics and the outcomes of patients with breast carcinoma. We performed immunostaining for RIP3 and analyzed the association of RIP3 expression status with the clinicopathological characteristics and survival of 203 patients with invasive ductal carcinoma of the breast. High RIP3 expression was significantly correlated with lymph node metastasis and human epidermal growth factor receptor 2 positivity. In patients with triple-negative breast carcinoma (TNBC), high RIP3 expression was an independent prognostic factor for disease-free survival (DFS). RIP3-high TNBC showed the lowest DFS rate. High RIP3 expression is associated with aggressive clinical behavior of breast carcinoma. Our data suggest that RIP3 serves as an independent prognostic factor in TNBC.

The Impact of RIPK1 Kinase Inhibition on Atherogenesis: A Genetic and a Pharmacological Approach.

RIPK1 (receptor-interacting serine/threonine-protein kinase 1) enzymatic activity drives both apoptosis and necroptosis, a regulated form of necrosis. Because necroptosis is involved in necrotic core development in atherosclerotic plaques, we investigated the effects of a RIPK1S25D/S25D mutation, which prevents activation of RIPK1 kinase, on atherogenesis in ApoE−/− mice. After 16 weeks of western-type diet (WD), atherosclerotic plaques from ApoE−/− RIPK1S25D/S25D mice were significantly larger compared to ApoE−/− RIPK1+/+ mice (167 ± 34 vs. 78 ± 18 × 103 µm2, p = 0.01). Cell numbers (350 ± 34 vs. 154 ± 33 nuclei) and deposition of glycosaminoglycans (Alcian blue: 31 ± 6 vs. 14 ± 4%, p = 0.023) were increased in plaques from ApoE−/− RIPK1S25D/S25D mice while macrophage content (Mac3: 2.3 ± 0.4 vs. 9.8 ± 2.4%, p = 0.012) was decreased. Plaque apoptosis was not different between both groups. In contrast, pharmacological inhibition of RIPK1 kinase with GSK’547 (10 mg/kg BW/day) in ApoE−/− Fbn1C1039G+/− mice, a model of advanced atherosclerosis, did not alter plaque size after 20 weeks WD, but induced apoptosis (TUNEL: 136 ± 20 vs. 62 ± 9 cells/mm2, p = 0.004). In conclusion, inhibition of RIPK1 kinase activity accelerated plaque progression in ApoE−/− RIPK1S25D/S25D mice and induced apoptosis in GSK’547-treated ApoE−/− Fbn1C1039G+/− mice. Thus, without directly comparing the genetic and pharmacological studies, it can be concluded that targeting RIPK1 kinase activity does not limit atherogenesis.

A pancancer analysis of the carcinogenic role of receptor-interacting serine/threonine protein kinase-2 (RIPK2) in human tumours

BackgroundTo explore the expression and carcinogenic mechanism of RIPK2 in human tumours, and to provide the theoretical basis for the further study of RIPK2.MethodsWe used the TCGA, CPTAC, HPA databases to analyse the expression, mutation, and prognosis of RIPK2 in human tumours. Through the Cbioportal, Ualcan, TIMER2.0, and STRING websites, We understand the genetic variation, immune infiltration and enrichment analysis of RIPK2 related genes.ResultsRIPK2 was highly expressed in most tumours (such as BRCA, COAD and LUSC, etc.), and the high expression of RIPK2 was correlated with tumour stage and prognosis. In addition, Amplification was the main type of RIPK2 in tumour mutation state, and the amplification rate was about 8.5%. In addition, RIPK2 was positively associated with tumour-infiltrating immune cells (such as CD8+ T, Tregs, and cancer-associated fibroblasts). According to the KEGG analysis, RIPK2 may play a role in tumour mainly through NOD-like signaling pathway and NF-kappaB signaling pathway. GO enrichment analysis showed that the RIPK2 is mainly related to I-kappaB kinase/NF-kappaB signaling, Ribonucleoprotein granule and Ubiquitin-like protein ligase binding.ConclusionRIPK2 plays an important role in the occurrence, development and prognosis of malignant tumours. Our pancancer study provided a relatively comprehensive description of the carcinogenic effects of RIPK2 in different tumours, and provided useful information for further study of RIPK2.

Inhibition of RIPK1 by ZJU-37 promotes oligodendrocyte progenitor proliferation and remyelination via NF-\u03baB pathway

Receptor interacting serine/threonine protein kinase 1 (RIPK1) activation and necroptosis have been genetically and mechanistically linked with human multiple sclerosis and neurodegenerative diseases for which demyelination is a common key pathology. Demyelination can be healed through remyelination which is mediated by new oligodendrocytes derived from the adult oligodendrocyte progenitor cells (OPCs). Unfortunately, the efficiency of remyelination declines with progressive aging partially due to the depletion of OPCs following chronic or repeated demyelination. However, to our knowledge, so far there is no drug which enhances proliferation of OPCs, and it is unknown whether inhibiting RIPK1 activity directly affect OPCs, the central player of remyelination. Using TNFα induced RIPK1-dependent necroptosis in Jurkat FADD−/− cells as a cell death assay, we screened from 2112 FDA-approved drugs and the drug candidates of new RIPK1 inhibitors selected by ourselves, and identified ZJU-37, a small molecule modified by introducing an amide bond to Nec-1s, is a new RIPK1 kinase inhibitor with higher potency than Nec-1s which has the best reported potency. We unveil in addition to protecting myelin from demyelination and axons from degeneration, ZJU-37 exhibits a new role on promoting proliferation of OPCs and enhancing remyelination by inhibiting RIPK1 kinase activity with higher potency than Nec-1s. Mechanistically, ZJU-37 promotes proliferation of OPCs by enhancing the transcription of platelet derived growth factor receptor alpha via NF-κB pathway. This work identifies ZJU-37 as a new drug candidate which enhances remyelination by promoting proliferation of OPCs, paving the way for a potential drug to enhance myelin repair.

Ketamine inhibits TNF-\u03b1-induced cecal damage by enhancing RIP1 ubiquitination to attenuate lethal SIRS

Systemic inflammatory response syndrome (SIRS) is a sepsis-associated inflammatory state and a self-defense mechanism against specific and nonspecific stimuli. Ketamine influences many key processes that are altered during sepsis. However, the underlying mechanisms remain incompletely understood. In this study, TNF-α-treated mice, as well as HT-29 and L929 cell models, were applied to characterize TNF-α-induced systemic and local cecal tissue inflammatory responses. Behavioral, biochemical, histological, and molecular biological approaches were applied to illustrate the related processes. Mice with TNF-α-induced SIRS showed systemic and local cecal tissue inflammatory responses, as indicated by increased levels of high mobility group box 1 protein (HMGB1), chemokines (C-X-C motif) ligand 10 (CXCL10), interleukin-6 (IL-6), and IL-10, as well as high mortality. Ketamine pretreatment alleviated death rates, symptoms, and the production of inflammatory cytokines induced by TNF-α in mice. Moreover, ketamine also protected the mice from TNF-α-induced cecal damage by suppressing the phosphorylation of receptor-interacting serine/threonine-protein kinase 3 (RIP3) and mixed lineage kinase domain-like protein (MLKL). In addition, our results showed that ketamine efficiently inhibited TNF-α-induced necroptosis in HT-29 and L929 cells. Furthermore, we explored the mechanism using different L929 cell lines. The results displayed that ketamine inhibited TNF-α-induced necroptosis by enhancing RIP1 ubiquitination and reducing the RIP1-RIP3 and RIP3-MLKL interactions, as well as the formation of necrosomes. Thus, our study may provide a new theoretical and experimental basis for treating diseases characterized by SIRS-associated inflammatory factor storms. Moreover, our exploration may provide potential molecular mechanisms and targets for therapeutic intervention and clinical application of ketamine.

Suppression of RIP1 activity via S415 dephosphorylation ameliorates obesity-related hepatic insulin resistance.

Receptor-interacting serine/threonine-protein kinase 1 (RIP1) is a well-documented key regulator of TNFα-mediated inflammation, apoptosis, and necroptosis, which contribute to the development of obesity-related metabolic diseases such as nonalcoholic steatohepatitis. However, the mechanism regarding how RIP1 influences obesity-related insulin resistance remains elusive. Primary hepatocytes with necrostatin 1 treatment or RIP1 expression were exposed to palmitic acid (PA), prior to the examination of cellular insulin signaling. Phosphorylation sites of RIP1 were detected by liquid chromatography with tandem mass spectrometry, and RIP1 variants with mutated phosphorylation sites were overexpressed in hepatocytes to identify the specific residue that influenced the RIP1-mediated insulin resistance. Adenovirus expressing RIP1 (S415A) mutant were administered into diet-induced obese mice to assess the effects on insulin sensitivity. This study uncovered an aberrant increase in RIP1 activity during the development of obesity-induced insulin resistance. Inhibition of RIP1 activity with necrostatin 1 ameliorated PA- or high-fat diet-caused hepatic insulin resistance. With liquid chromatography with tandem mass spectrometry analysis and mutagenesis screening, S415, a novel phosphorylation site of RIP1, was identified to be responsible for RIP1-mediated insulin resistance. Loss-of-function mutation of S415 efficiently blunted RIP1-evoked insulin resistance in PA-treated hepatocytes or diet-induced obese mice. These findings highlight the diabetogenic role of RIP1 S415 and propose RIP1 as a promising therapeutic target for type 2 diabetes.

Correlation Between Plasma Levels of RIP3 and Acute Ischemic Stroke with Large-Artery Atherosclerosis.

Receptor-interacting serine-threonine protein kinase 3 (RIP3) was previously discovered to be an important medium in the occurrence and development of major atherosclerotic cerebral infarction. However, the role of RIP3 in acute ischemic stroke remains unclear. This study aimed to explore the correlation between plasma levels of RIP3 and acute ischemic stroke with large-artery atherosclerosis (LAA). This prospective study enrolled 116 patients with LAA, 40 healthy controls, and 30 acute ischemic stroke patients with small-artery occlusion. The patients with LAA were divided according to the quartile of plasma levels of RIP3. A logistic regression model was used for comparison. The ROC curve was performed to evaluate the predictive value. In patients with LAA, the RIP3 levels in patients with poor outcomes as well as neurological deterioration were significantly higher than those with good outcomes (P < 0.001) and without neurological deterioration (P = 0.014). Patients in the highest levels of plasma RIP3 quartile were more likely to have neurological deterioration (OR, 11.07; 95% CI, 1.990-61.582) and poor outcomes (OR, 35.970; 95% CI, 5.392-239.980) compared with the lowest. The optimal cut-off value for neurological deterioration was 1127.75 pg/mL (specificity, 66.7%; sensitivity, 69.2%) and that for poor prognosis was 1181.82 pg/mL (specificity, 89.7%; sensitivity, 62.1%). Elevated levels of plasma RIP3 were significantly associated with neurological deterioration and poor prognosis in patients with LAA. A significant increase in plasma RIP3 levels can predict neurological deterioration and the poor prognosis of these patients.

Abstract TP259: Neuroprotective Effects Of Ripk2 Deficiency In Experimental Ischemic Stroke

Background: Receptor-interacting serine-threonine protein kinase 2 (Ripk2) is involved in the signaling pathways of members of the NOD family of cytosolic pattern recognition receptors. Ripk2 has been implicated in the caspase-1 pathway of hypoxia and ischemia-induced neuronal cell death, as well as the pathology of neuroinflammatory diseases like multiple sclerosis. Ischemic stroke is characterized by brain injury resulting from initial hypoxia/ischemia-induced cell death and secondary neuroinflammation. Hypothesis: Since Ripk2 is implicated in both pathways, we hypothesized that the genetic deletion of Ripk2 will improve outcomes for animals after experimental stroke. Methods: We utilized Ripk2 knockout ( Ripk2 -/- ) mice (3-4 months old) and subjected them to 45min of transient middle cerebral artery occlusion (tMCAO) along with wild-type (WT) control littermates. Infarct size was measured at 24h by TTC staining. RNA was isolated from the ipsilateral and contralateral cortices for RT-qPCR. Weight grip test, vertical grid test, and open field behavioral tests were conducted before and up to 21 days after tMCAO with novel object recognition and Y-maze tests occurring at day 28. Results: After 24h, Ripk2 -/- mice had significantly smaller infarct sizes than wild-type (WT) mice. Ripk2 -/- mice also had much lower expression of proinflammatory cytokines ( Tnfα, Il1β, and Il6 ) and chemokines ( Ccl2 and Cxcl1 ) in the ipsilateral cortex relative to WT, implicating a diminished proinflammatory response to ischemia/reperfusion injury in Ripk2 -/- animals. Ripk2 -/- mice displayed significantly better neurological deficit scores compared to WT at 24h, 48h, 7d, 14d, and 21d post-tMCAO. Ripk2 -/- mice also performed much better than WT at acute time points in a battery of behavioral tests, such as the weight grip test, vertical grid test, and open field test. Neurological assessment via novel object recognition and Y-maze tests showed no difference between genotypes at 28d. Conclusions: Our findings support the hypothesis that Ripk2 plays a detrimental role in the pathology of ischemic stroke. Understanding Ripk2’s role in stroke pathology may reveal a novel therapeutic target for the treatment of ischemic stroke.

DOP64 Pathogenic RIPK1 Mutations Cause Infantile-onset IBD with Inflammatory and Fistulizing Features

Abstract Background Genetics plays a key role in the pathogenesis of inflammatory bowel disease (IBD). With the expanding use of next-generation sequencing, &amp;gt;70 different monogenic disorders associated with IBD have been identified, and most of them present in the first years of life. Recently, several patients with severe IBD were identified to harbor pathogenic mutations in Receptor-interacting serine/threonine-protein kinase 1 (RIPK1) gene, which regulates necroptosis, a necrotic cell death mechanism. We present the clinical features, genetic analysis and immune work-up of three patients with infantile-onset IBD resulting from novel RIPK1 mutations. Methods Whole exome sequencing was performed in three patients with severe infantile-onset IBD, along with sanger sequencing for conformation. Mass cytometry time of flight was conducted for in-depth immunophenotyping, including cytokine secretion analysis following lipopolysaccharide (LPS) or Phorbol myristate acetate with ionomycin (PMA-I), on one of the patient’s peripheral blood mononuclear cells, and compared to control subjects and patients with Crohn’s disease. Results All patients, born to consanguineous Muslim families, presented with severe colitis and multiple perianal fistulas in the first months of life, without severe or atypical infections. One of the patients had a partial response to high doses of inlfliximab and azathioprine, while another one failed to respond to adaliumab and later to low dose anakinra, an IL-1 receptor antagonist. Genetic studies identified novel and pathogenic genetic variants in the RIPK1 gene in all patients, that were confirmed by Sanger sequencing. Using mass cytometry time of flight unbiased clustering analysis, we identified peripheral immune dysregulation in one of these patients, characterized by an increase in IFNγ CD8+ T cells along with a decrease in monocytes, dendritic cells and B cells. Moreover, RIPK1-deficient patient’s immune cells exhibited decreased IL-6 production in response to LPS across multiple cell types including T cells B cells and innate immune cells. Conclusion Mutations in RIPK1 should be considered in very young patients presenting with colitis and perianal fistulas. Given RIPK1’s role in both immune cells (and specifically in inflammasome activation), and epithelial cells, it is unclear whether immunosuppressive medications (including IL1 blockade) as well as allogeneic hematopoietic stem cell transplantation can suppress or cure the hyper-inflammatory response in these patients. Additional studies in humans are required to better define the role of RIPK1 in regulating intestinal immune responses, and how treatment can be optimized for patients with RIPK1 deficiency.

Stem Cell Factor SOX9 Interacts with a Cell Death Regulator RIPK1 and Results in Escape of Cancer Stem Cell Death.

High-grade ovarian cancer (HGOC) is the most lethal gynecological cancer, with high metastasis and recurrence. Cancer stem cells (CSCs) are responsible for its apoptosis resistance, cancer metastasis, and recurrence. Thus, targeting CSCs would be a promising strategy for overcoming chemotherapy resistance and improving patient prognosis in HGOC. Among upregulated oncogenic proteins in HGOC, we found that transcription factor SOX9 showed a strong correlation with stemness-regulating ALDH1A1 and was localized predominantly in the cytoplasm of HGOC with lymph node metastasis. In order to address the role of unusual cytoplasmic SOX9 and to explore its underlying mechanism in HGOC malignancy, a Y2H assay was used to identify a necroptotic cell death-associated cytoplasmic protein, receptor-interacting serine/threonine protein kinase 1 (RIPK1), as a novel SOX9-interacting partner and further mapped their respective interacting domains. The C-terminal region containing the transactivation domain of SOX9 interacted with the death domain of R1PK1. Consistent with its stemness-promoting function, SOX9 knockdown in vitro resulted in changes in cell morphology, cell cycle, stem cell marker expression, cell invasion, and sphere formation. Furthermore, in vivo knockdown completely inhibited tumor growth in mouse xenograft model. We propose that cytoplasmic SOX9-mediated cell death suppression would contribute to cancer stem cell survival in HGOC.

TNF-α-Mediated RIPK1 Pathway Participates in the Development of Trigeminal Neuropathic Pain in Rats.

Receptor-interacting serine/threonine-protein kinase 1 (RIPK1) participates in the regulation of cellular stress and inflammatory responses, but its function in neuropathic pain remains poorly understood. This study evaluated the role of RIPK1 in neuropathic pain following inferior alveolar nerve injury. We developed a model using malpositioned dental implants in male Sprague Dawley rats. This model resulted in significant mechanical allodynia and upregulated RIPK1 expression in the trigeminal subnucleus caudalis (TSC). The intracisternal administration of Necrosatin-1 (Nec-1), an RIPK1 inhibitor, blocked the mechanical allodynia produced by inferior alveolar nerve injury The intracisternal administration of recombinant rat tumor necrosis factor-α (rrTNF-α) protein in naive rats produced mechanical allodynia and upregulated RIPK1 expression in the TSC. Moreover, an intracisternal pretreatment with Nec-1 inhibited the mechanical allodynia produced by rrTNF-α protein. Nerve injury caused elevated TNF-α concentration in the TSC and a TNF-α block had anti-allodynic effects, thereby attenuating RIPK1 expression in the TSC. Finally, double immunofluorescence analyses revealed the colocalization of TNF receptor and RIPK1 with astrocytes. Hence, we have identified that astroglial RIPK1, activated by the TNF-α pathway, is a central driver of neuropathic pain and that the TNF-α-mediated RIPK1 pathway is a potential therapeutic target for reducing neuropathic pain following nerve injury.

Nanoencapsulation of Cordycepin Induces Switching from Necroptosis to Apoptosis in Human Oral Cancer Cells (HSC-4) Through Inhibition of Receptor-Interacting Serine/Threonine-Protein Kinase 3 (RIPK3) and Autophagy Modulation

Human oral squamous carcinoma is considered the most common oral cancer; it imposes multiple oral and dental consequences as a result of oral cancer treatment. We previously found that the nanoencapsulation of cordycepin (CS) could inhibit oral cancer cells. However, the mechanism of action was not understood. The aim of this study was to investigate the signaling pathway by which CS and encapsulated nanoparticles (NPs) activate the inhibition of cancer cell growth. We demonstrated that human oral cancer (HSC-4) cells underwent necroptosis when incubated with high concentrations of CS, but not when incubated with either low concentrations of CS or encapsulated CS NPs. High concentrations of CS-induced necroptosis of HSC-4 cells, demonstrated by a reduction in apoptotic ( BAX, Caspase-3, Caspase-8, and Caspase-9) and autophagic genes ( LC3, Atg5, and Atg12). However, low concentrations of CS significantly induced the expression of autophagic gene LC3. Interestingly, encapsulated CS NPs induced a significant increase in apoptotic genes ( P53, BAX, Caspase-3, Caspase-8, and Caspase-9), but a significant decrease in autophagic ( P62, Atg5, and Atg12) and necroptotic genes ( receptor-interacting serine/threonine-protein kinase 3 [RIPK3]) . We also found that encapsulated CS NPs enhanced the accumulation of cellular protein and decreased secreted supernatant protein levels. Moreover, encapsulated CS NPs had higher efficacy in terms of reactive oxygen species (ROS) generation-mediated inhibition of autophagy compared to nonencapsulated CS, suggesting that nanoencapsulation of CS can switch the program of HSC-4 cell death from necroptosis to apoptosis. In conclusion, HSC-4 cells have a defense strategy against CS-induced cell apoptosis, but this problem can be solved through the use of encapsulation combined with nanotechnology.