RIPK3 Expression Research Articles

IKKε and TBK1 prevent RIPK1 dependent and independent inflammation.

TBK1 and IKKε regulate multiple cellular processes including anti-viral type-I interferon responses, metabolism and TNF receptor signaling. However, the relative contributions and potentially redundant functions of IKKε and TBK1 in cell death, inflammation and tissue homeostasis remain poorly understood. Here we show that IKKε compensates for the loss of TBK1 kinase activity to prevent RIPK1-dependent and -independent inflammation in mice. Combined inhibition of IKKε and TBK1 kinase activities caused embryonic lethality that was rescued by heterozygous expression of kinase-inactive RIPK1. Adult mice expressing kinase-inactive versions of IKKε and TBK1 developed systemic inflammation that was induced by both RIPK1-dependent and -independent mechanisms. Combined inhibition of IKKε and TBK1 kinase activities in myeloid cells induced RIPK1-dependent cell death and systemic inflammation mediated by IL-1 family cytokines. Tissue-specific studies showed that IKKε and TBK1 were required to prevent cell death and inflammation in the intestine but were dispensable for liver and skin homeostasis. Together, these findings revealed that IKKε and TBK1 exhibit tissue-specific functions that are important to prevent cell death and inflammation and maintain tissue homeostasis.

Study of Therapeutic Mechanisms of Bupi Yichang Formula against Colon Cancer Based on Network Pharmacology, Machine Learning, and Experimental Verification.

Bupi Yichang formula (BPYCF) has shown the anti-cancer potential; however, its effects on colon cancer and the mechanisms remain unknown. This study intended to explore the effects of BPYC on colon cancer and its underlying mechanisms. BPYCF-related and colon cancer-related targets were acquired from public databases, followed by differentially expressed genes (DEG) identification. Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) analyses were performed using clusterProfiler. A protein-protein interaction (PPI) network was constructed using STRING database. CytoHubba and MCODE to screen the hub targets. A diagnostic model was built using random forest algorithm. Molecular docking was conducted using PyMOL and AutoDock. High-performance liquid chromatograph-mass spectrometry (HPLC-MS) analysis and in vitro validation were performed. Forty-six overlapping targets of BPYCF-related, colon cancer-related targets, and DEGs were obtained. GO and KEGG analyses showed that the targets were mainly enriched in response to lipopolysaccharide, neuronal cell body, protein serine/threonine/tyrosine, as well as C-type lectin receptor, NOD-like receptor, and TNF signaling pathways. Five targets were identified as the pivotal targets, among which, NOS3, CASP8, RIPK3, and TNFRSF10B were stably docked with the core active component, naringenin. Naringenin was also identified from the BPYCF sample through HPLC-MS analysis. In vitro experiments showed that BPYCF inhibited cell viability, reduced NOS3 expression, and elevated CASP8, RIPK3, and TNFRSF10B expression in colon cancer cells. BPYCF might treat colon cancer mainly by regulating NOS3, CASP8, RIPK3, and TN-FRSF10B. This study first revealed the therapeutic effects and mechanisms of BPYCF against colon cancer, paving the path for the development of targeted therapeutic strategies for this cancer in the clinic.

Simvastatin-Loaded Nanoniosome Protects H9c2 Cells from Oxygen-Glucose Deprivation/ Reperfusion Injury by Downregulating Inflammation.

Simvastatin (SIM) has anti-inflammatory and antioxidant properties against cardiac ischemia/reperfusion injury (I/RI). However, it suffers from low bioavailability and a short half-life. Nanoniosomes are novel drug delivery systems that may increase SIM effectiveness. The present research evaluates the impact of SIM-loaded nanoniosomes on the oxygen-glucose deprivation/reperfusion (OGD/R) injury model of H9c2 cells. Cells were seeded based on five groups: (1) control; (2) OGD/R; (3) OGD/R receiving SIM; (4) OGD/R receiving nanoniosomes; and (5) OGD/R receiving SIM loaded nanoniosomes. OGD/R injury of the H9c2 cells was treated with SIM or SIM loaded nanoniosomes. Cell viability, two inflammatory factors, necroptosis factors, along with HMGB1 and Nrf2 gene expressions were assessed. The cells treated with SIM loaded nanoniosomes showed a significant elevation in the cell viability and a reduction in HMGB1, Nrf2, TNF-α, IL-1β, RIPK1, and ROCK1 expression levels compared to the OGD/R and SIM groups. Based on our findings, nanoniosomes could safely serve as a drug delivery system to counterbalance the disadvantages of SIM, resulting in improved aqueous solubility and stability.

Target deubiquitinase OTUB1 as a therapeatic strategy for BLCA via β-catenin/necroptosis signal pathway.

Ubiquitination, a prevalent and highly dynamic reversible post-translational modification, is tightly regulated by the deubiquitinating enzymes (DUBs) superfamily. Among them, OTU Domain-Containing Ubiquitin Aldehyde-Binding Protein 1 (OTUB1) stands out as a critical member of the OTU deubiquitinating family, playing a pivotal role as a tumor regulator across various cancers. However, its specific involvement in BLCA (BLCA) and its clinical significance have remained ambiguous. This study aimed to elucidate the biofunctions of OTUB1 in BLCA and its implications for clinical prognosis. Our investigation revealed heightened OTUB1 expression in BLCA, correlating with unfavorable clinical outcomes. Through in vivo and in vitro experiments, we demonstrated that increased OTUB1 levels promote BLCA tumorigenesis and progression, along with conferring resistance to cisplatin treatment. Notably, we established a comprehensive network involving OTUB1, β-catenin, necroptosis, and BLCA, delineating their regulatory interplay. Mechanistically, we uncovered that OTUB1 exerts its influence by deubiquitinating and stabilizing β-catenin, leading to its nuclear translocation. Subsequently, nuclear β-catenin enhances the transcriptional activity of c-myc and cyclin D1 while suppressing the expression of RIPK3 and MLKL, thereby fostering BLCA progression and cisplatin resistance. Importantly, our clinical data suggest that the OTUB1/β-catenin/RIPK3/MLKL axis holds promise as a potential biomarker for BLCA.

The effect of curcumin on the necroptosis signaling pathway in colon cancer cells

Colon cancer is the one of the most common types of cancer in humans. A sedentary lifestyle, increasing obesity and the consumption of food additives favor the development and occurrence of colon cancer. It is emphasized that curcumin, a yellow compound isolated from the turmeric plant, is important in preventing cancer. Studies have shown that curcumin has an anticancer effect by driving cancer cells into apoptosis, but studies showing its effect on necroptosis are inconclusive. Necroptosis is a form of programmed cell death mediated by RIP proteins and has been shown to play an important role in cancer. This study aims to determine the effect of curcumin on the necroptosis signaling pathway. For this purpose, HT-29 and HCT-116 colon cancer cells were cultured and exposed to different concentrations of curcumin and MTT experiments were performed to determine the effect on cell viability. The expression levels of RIPK1, RIPK3, and MLKL genes, which are markers of necroptosis, were analyzed by real-time PCR. It was found that the expression level of RIPK1, RIPK3, and MLKL genes significantly increased after exposure of HT -29 cells to 50 µM curcumin. Moreover, the expression of RIPK1 and MLKL genes increased in HCT-116 cells after curcumin administration. Consequently, the current data clearly suggest that curcumin is a prominent driver of necroptotic signaling-mediated colon cancer cell death.

Investigation of the modulatory effects of miR-421 on the necroptosis pathway in colon cancer cells

Colon cancer is a significant cause of cancer death and morbidity worldwide. Despite significant advancements in anti-tumor therapies over recent years, enhancing the overall prognosis still presents a substantial challenge. A prevalent characteristic of many solid tumors, including colon cancer, is tumor necrosis. Recent studies have indicated that under specific conditions, cell death through necrosis can be orchestrated rather than occurring randomly. Receptor-interacting protein (RIP) kinases have been identified to be key regulators of this controlled form of necrosis. In addition, the deregulation of necroptosis has been identified to be a key driving factor during malignant progression of human tumors, including colon cancer. Accordingly, to extend our understanding of the modulatory effects of miR-421 on the necroptosis pathway, herein we performed gain-of-function studies of miR-421 in colon cancer cell lines and analyzed differential expression of receptor integrating serine-threonine kinase-1 (RIPK1), a key regulator of necroptosis in cells. Remarkably, overexpression of miR-421 significantly suppressed the expression of RIPK1 in colon cancer cells, indicating that miR-421 is a key regulatory miRNA targeting RIPK1 activity in colon cancer. Collectively, the findings obtained here strongly suggest that miR-421 can be a druggable target for the modulation of RIK1-mediated necroptotic signaling.

RIPK1 polymorphisms and expression levels: impact on genetic susceptibility and clinical outcome of epithelial ovarian cancer

BackgroundThe aim of this study was to explore the associations of RIPK1 polymorphisms, plasma levels and mRNA expression with susceptibility to epithelial ovarian cancer (EOC) and clinical outcome.MethodsThree hundred and nineteen EOC patients included in a 60-month follow-up program and 376 controls were enrolled. Two tag SNPs (rs6907943 and rs9392453) of RIPK1 were genotyped using polymerase chain reaction (PCR)-restriction fragment length polymorphism (RFLP) method. Plasma levels of RIPK1 and RIPK1 mRNA expression in white blood cells were determined by ELISA and qPCR, respectively.ResultsFor rs9392453, significantly increased EOC risk was found to be associated with C allele (P = 0.002, OR = 1.49, 95%CI 1.15–1.92), and with CT/CC genotypes in the dominant genetic model (P = 0.006, OR = 1.54, 95%CI 1.12–2.08). CC haplotype (rs6907943-rs9392453) was associated with increased EOC susceptibility. CC genotype of rs6907943 and CT/CC genotypes of rs9392453 were associated with early onset (age ≤ 50 years) of EOC (OR = 2.5, 95%CI 1.03–5.88, and OR = 1.64, 95%CI 1.04–2.63, respectively). AC genotype of rs6907943 was associated with better overall survival of EOC patients in the over-dominant genetic model (P = 0.035, HR = 0.41, 95%CI 0.18–0.94). Multivariate survival analysis identified the AC genotype of rs6907943 as an independent protective factor for survival of early onset patients (P = 0.044, HR = 0.12, 95%CI 0.02–0.95). Compared to controls, significantly increased plasma levels of RIPK1 and reduced RIPK1 mRNA expression were observed in patients.ConclusionsOur results suggest that tag SNPs of RIPK1, increased plasma levels of RIPK1 protein and reduced RIPK1 mRNA expression in white blood cells, may influence the susceptibility to EOC. SNP rs6907943 may be a useful marker to distinguish EOC patients with high risk of death.

Endogenous hydrogen sulphide deficiency and exogenous hydrogen sulphide supplement regulate skin fibroblasts proliferation via necroptosis.

An excessive proliferation of skin fibroblasts usually results in different skin fibrotic diseases. Hydrogen sulphide (H2 S) is regarded as an important endogenous gasotransmitter with various functions. The study aimed to investigate the roles and mechanisms of H2 S on primary mice skin fibroblasts proliferation. Cell proliferation and collagen synthesis were assessed with the expression of α-smooth muscle actin (α-SMA), proliferating cell nuclear antigen (PCNA), Collagen I and Collagen III. The degree of oxidative stress was evaluated by dihydroethidium (DHE) and MitoSOX staining. Mitochondrial membrane potential (ΔΨm) was detected by JC-1 staining. Necroptosis was evaluated with TDT-mediated dUTP nick end labelling (TUNEL) and expression of receptor-interacting protein kinase 1 (RIPK1), RIPK3 and mixed lineage kinase domain-like protein (MLKL). The present study found that α-SMA, PCNA, Collagen I and Collagen III expression were increased, oxidative stress was promoted, ΔΨm was impaired and positive rate of TUNEL staining, RIPK1 and RIPK3 expression as well as MLKL phosphorylation were all enhanced in skin fibroblasts from cystathionine γ-lyase (CSE) knockout (KO) mice or transforming growth factor-β1 (TGF-β1, 10 ng/mL)-stimulated mice skin fibroblasts, which was restored by exogenous sodium hydrosulphide (NaHS, 50 μmol/L). In conclusion, endogenous H2 S production impairment in CSE-deficient mice accelerated skin fibroblasts proliferation via promoted necroptosis, which was attenuated by exogenous H2 S. Exogenous H2 S supplement alleviated proliferation of skin fibroblasts with TGF-β1 stimulation via necroptosis inhibition. This study provides evidence for H2 S as a candidate agent to prevent and treat skin fibrotic diseases.

MiR-223-3p Prevents Necroptotic Macrophage Death by Targeting Ripk3 in a Negative Feedback Loop and Consequently Ameliorates Advanced Atherosclerosis.

The formation of large necrotic cores results in vulnerable atherosclerotic plaques, which can lead to severe cardiovascular diseases. However, the specific regulatory mechanisms underlying the development of necrotic cores remain unclear. To evaluate how the modes of lesional cell death are reprogrammed during the development of atherosclerosis, the expression levels of key proteins that are involved in the necroptotic, apoptotic, and pyroptotic pathways were compared between different stages of plaques in humans and mice. Luciferase assays and loss-of-function studies were performed to identify the microRNA-mediated regulatory mechanism that protects foamy macrophages from necroptotic cell death. The role of this mechanism in atherosclerosis was determined by using a knockout mouse model with perivascular drug administration and tail vein injection of microRNA inhibitors in Apoe-/- mice. Here, we demonstrate that the necroptotic, rather than the apoptotic or pyroptotic, pathway is more activated in advanced unstable plaques compared with stable plaques in both humans and mice, which closely correlates with necrotic core formation. The upregulated expression of Ripk3 (receptor-interacting protein kinase 3) promotes the C/EBPβ (CCAAT/enhancer binding protein beta)-dependent transcription of the microRNA miR-223-3p, which conversely inhibits Ripk3 expression and forms a negative feedback loop to regulate the necroptosis of foamy macrophages. The knockout of the Mir223 gene in bone marrow cells accelerates atherosclerosis in Apoe-/- mice, but this effect can be rescued by Ripk3 deficiency or treatment with the necroptosis inhibitors necrostatin-1 and GSK-872. Like the Mir223 knockout, treating Apoe-/- mice with miR-223-3p inhibitors increases atherosclerosis. Our study suggests that miR-223-3p expression in macrophages protects against atherosclerotic plaque rupture by limiting the formation of necrotic cores, thus providing a potential microRNA therapeutic candidate for atherosclerosis.

The Dual Functions of Andrographolide in the Epstein–Barr Virus-Positive Head-and-Neck Cancer Cells: The Inhibition of Lytic Reactivation of the Epstein–Barr Virus and the Induction of Cell Death

Andrographolide, a medicinal compound, exhibits several pharmacological activities, including antiviral and anticancer properties. Previously, we reported that andrographolide inhibits Epstein–Barr virus (EBV) lytic reactivation, which is associated with viral transmission and oncogenesis in epithelial cancers, including head-and-neck cancer (HNC) cells. However, the underlying mechanism through which andrographolide inhibits EBV lytic reactivation and affects HNC cells is poorly understood. Therefore, we investigated these mechanisms using EBV-positive HNC cells and the molecular modeling and docking simulation of protein. Based on the results, the expression of EBV lytic genes and viral production were significantly inhibited in andrographolide-treated EBV-positive HNC cells. Concurrently, there was a reduction in transcription factors (TFs), myocyte enhancer factor-2D (MEF2D), specificity protein (SP) 1, and SP3, which was significantly associated with a combination of andrographolide and sodium butyrate (NaB) treatment. Surprisingly, andrographolide treatment also significantly induced the expression of DNA Methyltransferase (DNMT) 1, DNMT3B, and histone deacetylase (HDAC) 5 in EBV-positive cells. Molecular modeling and docking simulation suggested that HDAC5 could directly interact with MEF2D, SP1, and SP3. In our in vitro study, andrographolide exhibited a stronger cytotoxic effect on EBV-positive cells than EBV-negative cells by inducing cell death. Interestingly, the proteome analysis revealed that the expression of RIPK1, RIPK3, and MLKL, the key molecules for necroptosis, was significantly greater in andrographolide-treated cells. Taken together, it seems that andrographolide exhibits concurrent activities in HNC cells; it inhibits EBV lytic reactivation by interrupting the expression of TFs and induces cell death, probably via necroptosis.

Contribution to the study of inflammasome and programmed cell death in paracoccidioidomycosis oral lesions.

Paracoccidioidomycosis is the most prevalent systemic mycosis in Latin America, with a high incidence in Brazil, Colombia and Venezuela, and constitutes a serious public health problem, a frequent cause of morbidity and disability for work. Some mechanisms of cell death are described as important tools in infectious processes. When apoptosis is blocked, RIPK (Receptor-interacting protein kinase) 3 dependent, a caspase-independent form of cell death, can limit the replication and spread of pathogens. Some molecules that mediate necroptosis include RIPK3 and have been extensively studied due to their signalling mechanism and pathological function. RIPK3 activates NLRP1 and NLRP3-mediated inflammasome formation. Caspase-1 has an important role in processing the cytokines ILβ and IL18 to their active form. Such molecules are part of the inflammasome characterization, whose caspase-1-dependent activation promotes the death of pyroptotic cells and the secretion of proinflammatory cytokines. Knowledge about the mechanisms of pathogen-mediated cell death can be useful for understanding of the pathogenesis of infections and inflammatory conditions. The objective of this work was to identify the mechanisms of programmed cell death and inflammasome components in human oral mucosal lesions of paracoccidioidomycosis through immunohistochemical methods and identification of RIPK-3, IL1β, IL18, NLRP-1 and caspase-1. Thirty specimens were included, and a histopathological analysis of the lesions was performed using haematoxylin-eosin staining. Our results on insitu expression of inflammasome elements and programmed cell death showed increased expression of IL-1β, NLRP-1, caspase-1 and RIPK-3. We suggest that inflammasome complex participate in the immunopathogenesis in paracoccidioidomycosis oral lesions in an interplay with RIPK3.

Decreased gene expression of RIPK1 and RIPK3, necroptosis players, in calves with sepsis

Background With the increase in the world population, the need for livestock-based nutrition is also increasing. In addition, the livestock sector becomes more important as it contributes to the economy However, sepsis has high morbidity and mortality rate in newborn calves and can cause severe economic losses. Therefore, new biomarkers to distinguish sepsis from other diseases are urgently needed in veterinary medicine. In the present study, we investigated for the first time the gene expression levels of necroptosis members, including RIPK1 and RIPK3, and one of the NF-kB activators RIPK2, in calves with sepsis.
 Methods and results We examined the mRNA levels of RIPK1, RIPK3, and RIPK2 using qPCR in 10 healthy Holstein calves and 20 Holstein calves with sepsis due to suffering from enteritis infection between 1-20 days of age. The hematologic parameters, including leukocytes, erythrocytes, hemoglobin, hematocrit, and platelets, were evaluated in the calves included in this study. The results showed that calves with sepsis had prominently lower mRNA levels of RIPK1 and RIPK3 than those in healthy calves. Besides, RIPK2 mRNA expression was absent in healthy calves and calves with sepsis. 
 Conclusions In veterinary medicine decreased RIPK1 and RIPK3 mRNA levels might be biomarkers to diagnose sepsis in calves.

Inhibition of Urban Particulate Matter-Induced Airway Inflammation by RIPK3 through the Regulation of Tight Junction Protein Production.

Urban particulate matter (UPM) is a high-hazard cause of various diseases in humans, including in the respiratory tract, skin, heart, and even brain. Unfortunately, there is no established treatment for the damage caused by UPM in the respiratory epithelium. In addition, although RIPK3 is known to induce necroptosis, its intracellular role as a negative regulator in human lungs and bronchial epithelia remains unclear. Here, the endogenous expression of RIPK3 was significantly decreased 6 h after exposure to UPM. In RIPK3-ovexpressed cells, RIPK3 was not moved to the cytoplasm from the nucleus. Interestingly, the overexpression of RIPK3 dramatically decreased TEER and F-actin formation. Its overexpression also decreased the expression of genes for pro-inflammatory cytokines (IL-6 and IL-8) and tight junctions (ZO-1, -2, -3, E-cadherin, and claudin) during UPM-induced airway inflammation. Importantly, overexpression of RIPK3 inhibited the UPM-induced ROS production by inhibiting the activation of iNOS and eNOS and by regulating mitochondrial fission processing. In addition, UPM-induced activation of the iκB and NF-κB signaling pathways was dramatically decreased by RIPK3, and the expression of pro-inflammatory cytokines was decreased by inhibiting the iκB signaling pathway. Our data indicated that RIPK3 is essential for the UPM-induced inflammatory microenvironment to maintain homeostasis. Therefore, we suggest that RIPK3 is a potential therapeutic candidate for UPM-induced pulmonary inflammation.

SeNPs alleviates BDE-209-induced intestinal damage by affecting necroptosis, inflammation, intestinal barrier and intestinal flora in layer chickens

As environmental pollutants, polybrominated diphenyl ethers (PBDEs) can have toxic effects on living organisms and has a bioaccumulative effect. Low doses of selenium nanoparticles (SeNPs) can exert antioxidant, anti-inflammatory and anti-toxin functions on the organism. This experiment evaluated SeNPs' ability to prevent chicken’s intestinal damage from decabromodiphenyl ether (BDE-209) exposure. Sixty layer chickens were separated into four groups at randomly and equally: Control group, SeNPs group (1 mg/kg SeNPs), BDE-209 group (400 mg/kg BDE-209), and BDE-209 +SeNPs group (400 mg/kg BDE-209 and 1 mg/kg SeNPs), for 42 days. The results showed that BDE-209 increased MDA content, decreased the activities of T-SOD, T-AOC, GSH and iNOS, up-regulated the expression of TNF-α, RIPK1, RIPK3 and MLKL, promoted the production of inflammatory factors, reduced the levels of tight junction proteins (Claudin-1, Occludin, ZO-1). SeNPs attenuated intestinal oxidative stress, necroptosis, inflammation and intestinal barrier damage caused by BDE-209. This protective effect is associated with the MAPK/NF-κB signaling pathway. Moreover, SeNPs restores flora alpha and beta diversity, improves intestinal flora composition and its abundance. It shifts the dysbiosis of intestinal flora caused by BDE-209 to normal. Overall, SeNPs can alleviate BDE-209-induced intestinal barrier damage and intestinal flora disorders, which are associated with intestinal oxidative stress, necroptosis and inflammation.

Hepatocarcinogenesis Is Supported by Sublethal Necroptosis Signaling.

NF-κB activation in hepatocytes with low RIPK3 expression can trigger sublethal necrosome activation.

Necroptosis has a crucial role in the development of chronic periodontitis.

Periodontitis is a non-communicable chronic inflammatory disease that affects the entire periodontium and its severe types cause irreparable destruction. The purpose of this study was to determine the type of cell death in chronic periodontitis (CP) with the expression of receptor-interacting protein kinase (RIPK) type1 and RIPK3 genes. This cross-sectional study was carried out from September 2019 to 2020. The samples (38 participants) were divided into two groups: 20 recently diagnosed CP patients and 18 healthy individuals. Participants' data was collected in the periodontology Department, Dental school, Mashhad University of Medical Sciences and sent to the Immunology Lab for assessment of RIPK1 and RIPK3 expressions using quantitative real time-PCR. The study sample consisted of 30 females (78.9%) and 8 males (21.1%) with a mean age of 34±5 years. The expression of the genes of interest in CPs exhibited an opposite pattern. Although, RIPK3 gene expression was significantly greater in CP patients compared to the control group (P=0.024), the expression of RIPK1 decreased (p<0.001). Moreover, no significant correlation was observed between age and gender with these molecules in CPs. The RIPK3 selectively contributes to necroptosis, therefore, it seems that RIPK3-mediated necroptosis is involved in chronic periodontitis. RIPK1 also participates in necroptosis, but mostly in apoptosis. Therefore, necroptosis as an unprogrammed inflammatory cell death induced by pathogenic damages seems to be another mechanism complicated in periodontitis and could be used as a novel target for CP therapy.

#5648 RIPK3 MEDIATES KIDNEY INJURY INDUCED BY A CYTOKINE STORM

Abstract Background and Aims Receptor-interacting protein kinase 3 (RIPK3) is traditionally involved in necroptosis, a regulated necrosis pathway which has been observed in diseases associated to cell death and inflammation. However, RIPK3 has been also associated to inflammatory responses independent of necroptosis. In previous studies we demonstrated that RIPK3 deficiency does not prevent renal injury but protects from inflammation in folic acid-induced AKI (FA-AKI), suggesting a proinflammatory role independently of cell death. In the present work, we aim to explore the role of RIPK3 in kidney injury and inflammation induced by a cytokine storm. Method Lipopolysaccharide (LPS) was used to induce cytokine storm-AKI in mice. For this, female 12- to 14-week-old wild type (WT), RIPK3-KO, MLKL-KO or NRLP3-KO C57BL/6J mice received a single intraperitoneal (i.p) injection of LPS 5 mg/kg or vehicle and were sacrificed 1h, 4h and 24 hours later. To generate chimera mice, WT and Ripk3-KO receptor mice were irradiated to deplete the autologous bone marrow (BM). BM was extracted from the femur and tibia of donor WT or Ripk3-KO mice, and 107 cells were transferred to irradiated receptor mice by intravenous injection. After 1 month, LPS-AKI was induced. Plasma was collected to assess kidney function. Kidneys were collected for RNA, protein studies, and histologic studies. Additionally, a WT group received 1.65 mg/kg of the necroptosis inhibitor Necrostatin-1 (Nec1) i.p. prior to LPS to evaluate the impact of necroptosis pathway. Liver, lungs and heart were also collected to assess the systemic inflammatory response. Additionally, cultured murine immortalized tubular MCT cells, and primary tubular cells, bone marrow dendritic cells (BMDC) and bone marrow derived macrophages (BMDM) isolated from WT and Ripk3-KO mice were studied. Cells were stimulated with 100 ng/ml LPS for 6h and RNA was studied. To analyze the impact of the inflammatory response on tubular cells, supernatants from LPS-stimulated BMDM and BMDC were collected and used to stimulate MCT cells. RNA expression was studied by RT-PCR and protein expression by Western Blotting. Results The kidney expression of RIPK3 mRNA and protein was upregulated in cytokine storm-AKI in mice, while Ripk3 deficiency improved survival and renal function, with less expression of proinflammatory cytokines and inflammatory infiltrate. Necroptosis did not seem to be implicated in cytokine storm-AKI, since neither Nec1 nor genetic MLKL ablation offered protection on renal function. Systemic inflammation in non-kidney organs was also milder with Ripk3 deficiency. In addition, inflammasome-related proteins were upregulated in cytokine storm-AKI, and this was reduced in Ripk3-KO mice. However, Nlrp3-deficient mice developed kidney injury and inflammation after LPS injection. Next, we explored the role of RIPK3 in BM-derived cells in cytokine storm-AKI by generating chimera mice. In this context, WT mice with Ripk3 deficient BM exhibited less inflammation and better renal function. This supports that RIPK3 from BM cells mediates kidney inflammation and injury during cytokine storm-AKI. In cultured cells, LPS induced RIPK3 expression in BMDMs but not in tubular cells, and RIPK3 mediated IL-6 expression in BMDMs but not in tubular cells. Moreover, IL-6 and conditioned media from LPS-exposed WT macrophages promoted proinflammatory responses in cultured tubular cells, that was partially ameliorated for RIPK3-KO LPS-macrophage conditioned medium. Conclusion In conclusion, RIPK3 mediates kidney injury and systemic inflammation induced by a cytokine storm independently of the necroptosis pathway. These results identify RIPK3 as a therapeutic target for renal inflammatory diseases.

Selenium-Enriched Cardamine violifolia Alleviates LPS-Induced Hepatic Damage and Inflammation by Suppressing TLR4/NODs-Necroptosis Signal Axes in Piglets.

Selenium-enriched Cardamine violifolia (SEC), a cruciferous plant, exerts excellent antioxidant and anti-inflammatory capacity, but its effect on hepatic function is unclear. This study investigated the effect and potential mechanism of SEC on hepatic injury induced by lipopolysaccharide (LPS). Twenty-four weaned piglets were randomly allotted to treatment with SEC (0.3 mg/kgSe) and/or LPS (100 μg/kg). After 28 days of the trial, pigs were injected with LPS to induce hepatic injury. These results indicated that SEC supplementation attenuated LPS-induced hepatic morphological injury and reduced aspartate aminotransferase (AST) and alkaline phosphatase (ALP) activities in plasma. SEC also inhibited the expression of pro-inflammatory cytokines such as interleukin 6 (IL-6) and tumor necrosis factor-alpha (TNF-α) after the LPS challenge. In addition, SEC improved hepatic antioxidant capacity via enhancing glutathione peroxidase (GSH-Px) activity and decreasing malondialdehyde (MDA) concentration. Moreover, SEC downregulated the mRNA expression of hepatic myeloid differentiation factor 88 (MyD88) and nucleotide-binding oligomerization domain proteins 1 (NOD1) and its adaptor molecule receptor interacting protein kinase 2 (RIPK2). SEC also alleviated LPS-induced hepatic necroptosis by inhibiting RIPK1, RIPK3, and mixed-lineage kinase domain-like (MLKL) expression. These data suggest that SEC potentially mitigates LPS-induced hepatic injury via inhibiting Toll-like receptor 4 (TLR4)/NOD2 and necroptosis signaling pathways in weaned piglets.

Arsenic trioxide-induced cytotoxicity in A549 cells: The role of necroptosis.

Lung cancer is one of the deadliest cancers globally. Arsenic trioxide (ATO) is still present as a highly effective drug in treating acute promyelocytic leukemia (APL). Chemotherapy resistance is one of the major problems in cancer therapy. Necroptosis, can overcomes resistance to apoptosis, and can promote cancer treatment. This study examines the necroptosis pathway in A549 cancer cells exposed to ATO. We used the MTT test to determine the ATO effects on the viability of A549 cells at three different time intervals. Also, the reactive oxygen species (ROS) and mitochondrial membrane potential (MMP) were performed in three-time intervals. The effect of ATO on apoptosis was evaluated by Annexin V / PI staining and, the RIPK1 and MLKL gene expression were measured by Real-Time PCR. The ATO has dose and time-dependent cytotoxic effects, so at 24, 48, and 72 h, the IC50 doses were 33.81 '11.44 '2.535 µM respectively. A 50 μM ATO is the most appropriate to increase the MMP loss significantly at all three times. At 24 and 48 h after exposure of cells to ATO, the ROS levels increased. The RIPK1 gene expression increased significantly compared to the control group at concentrations of 50 and 100 μM; however, MLKL gene expression decreased. The A549 cells, after 48 h exposure to ATO at 50 and 100 μM, induces apoptosis and necroptosis. Due to the reduced expression of MLKL, it can be concluded that ATO is probably effective in the metastatic stage of cancer cells.

Variation in CD8 T cell IFNγ differentiation to strains of Toxoplasma gondii is characterized by small effect QTLs with contribution from ROP16.

Toxoplasma gondii induces a strong CD8 T cell response characterized by the secretion of IFNγ that promotes host survival during infection. The initiation of CD8 T cell IFNγ responses in vitro differs widely between clonal lineage strains of T. gondii, in which type I strains are low inducers, while types II and III strains are high inducers. We hypothesized this phenotype is due to a polymorphic "Regulator Of CD8 T cell Response" (ROCTR). Therefore, we screened F1 progeny from genetic crosses between the clonal lineage strains to identify ROCTR. Naïve antigen-specific CD8 T cells (T57) isolated from transnuclear mice, which are specific for the endogenous and vacuolar TGD057 antigen, were measured for their ability to become activated, transcribe Ifng and produce IFNγ in response to T. gondii infected macrophages. Genetic mapping returned four non-interacting quantitative trait loci (QTL) with small effect on T. gondii chromosomes (chr) VIIb-VIII, X and XII. These loci encompass multiple gene candidates highlighted by ROP16 (chrVIIb-VIII), GRA35 (chrX), TgNSM (chrX), and a pair of uncharacterized NTPases (chrXII), whose locus we report to be significantly truncated in the type I RH background. Although none of the chromosome X and XII candidates bore evidence for regulating CD8 T cell IFNγ responses, type I variants of ROP16 lowered Ifng transcription early after T cell activation. During our search for ROCTR, we also noted the parasitophorous vacuole membrane (PVM) targeting factor for dense granules (GRAs), GRA43, repressed the response suggesting PVM-associated GRAs are important for CD8 T cell activation. Furthermore, RIPK3 expression in macrophages was an absolute requirement for CD8 T cell IFNγ differentiation implicating the necroptosis pathway in T cell immunity to T. gondii. Collectively, our data suggest that while CD8 T cell IFNγ production to T. gondii strains vary dramatically, it is not controlled by a single polymorphism with strong effect. However, early in the differentiation process, polymorphisms in ROP16 can regulate commitment of responding CD8 T cells to IFNγ production which may have bearing on immunity to T. gondii.