Erythronecroptosis: an overview of necroptosis or programmed necrosis in red blood cells.

Necroptosis is considered a programmed necrosis that requires receptor-interacting protein kinase 1 (RIPK1), receptor-interacting protein kinase 3 (RIPK3), and pore-forming mixed lineage kinase domain-like protein (MLKL) to trigger a regulated cell membrane lysis. Membrane rupture in necroptosis has been shown to fuel innate immune response due to release of damage-associated molecular patterns (DAMPs). Recently published studies indicate that mature erythrocytes can undergo necroptosis as well. In this review, we provide an outline of multiple cell death modes occurring in erythrocytes, discuss possible immunological aspects of diverse erythrocyte cell deaths, summarize available evidence related to the ability of erythrocytes to undergo necroptosis, outline key involved molecular mechanisms, and discuss the potential implication of erythrocyte necroptosis in the physiology and pathophysiology. Furthermore, we aim to highlight the interplay between necroptosis and eryptosis signaling in erythrocytes, emphasizing specific characteristics of these pathways distinct from their counterparts in nucleated cells. Thus, our review provides a comprehensive summary of the current knowledge of necroptosis in erythrocytes. To reflect critical differences between necroptosis of nucleated cells and necroptosis of erythrocytes, we suggest a term erythronecroptosis for necroptosis of enucleated cells.

Ginsenoside Rh2 Regulates the Calcium/ROS/CK1α/MLKL Pathway to Promote Premature Eryptosis and Hemolysis in Red Blood Cells.

Ginsenoside Rh2 (GRh2) exhibits significant potential as an anticancer agent; however, progress in developing chemotherapeutic drugs is impeded by their toxicity toward off-target tissues. Specifically, anemia caused by chemotherapy is a debilitating side effect and can be caused by red blood cell (RBC) hemolysis and eryptosis. Cells were exposed to GRh2 in the antitumor range and hemolytic and eryptotic markers were examined under different experimental conditions using photometric and cytofluorimetric methods. GRh2 caused Ca2+-independent, concentration-responsive hemolysis in addition to disrupted ion trafficking with K+ and Cl- leakage. Significant increases in cells positive for annexin-V-fluorescein isothiocyanate, Fluo4, and 2,7-dichlorofluorescein were noted upon GRh2 treatment coupled with a decrease in forward scatter and acetylcholinesterase activity. Importantly, the cytotoxic effects of GRh2 were mitigated by ascorbic acid and by blocking casein kinase 1α (CK1α) and mixed lineage kinase domain-like (MLKL) signaling. In contrast, Ca2+ omission, inhibition of KCl efflux, and isosmotic sucrose aggravated GRh2-induced RBC death. In whole blood, GRh2 selectively targeted reticulocytes and lymphocytes. Altogether, this study identified novel mechanisms underlying GRh2-induced RBC death involving Ca2+ buildup, loss of membrane phospholipid asymmetry and cellular volume, anticholinesterase activity, and oxidative stress. These findings shed light on the hematologic toxicity of GRh2 which is crucial for optimizing its utilization in cancer treatment.

Peritoneal Inflammation in PD-Related Peritonitis Induces Systemic Eryptosis: In Vitro and In Vivo Assessments.

Erythrocytes (RBCs) have a highly specialized and organized membrane structure and undergo programmed cell death, known as eryptosis. Our preliminary data show a significant increase in the eryptosis during peritoneal dialysis (PD)-associated peritonitis. The objectives of the present study were assessment of the incrementation of eryptosis in PD patients with peritonitis, evaluation of the relationship between systemic eryptosis in peritonitis and specific peritonitis biomarkers in PD effluent (PDE), and confirmation of the induction of eryptosis by peritonitis in a vitro setting. We enrolled 22 PD patients with peritonitis and 17 healthy subjects (control group, CTR). For the in vivo study, eryptosis was measured in freshly isolated RBCs. For the in vitro study, healthy RBCs were exposed to the plasma of 22 PD patients with peritonitis and the plasma of the CTR group for 2, 4, and 24 h. Eryptosis was evaluated by flow cytometric analyses in vivo and in vitro. PDE samples were collected for biomarkers analysis.The percentage of eryptotic RBCs was significantly higher in PD patients with peritonitis than in CTR (PD patients with peritonitis: 7.7; IQR 4.3-14.2, versus CTR: 0.8; IQR 0.7-1.3; p < 0.001). We confirmed these in vivo results by in vitro experiments: healthy RBCs incubated with plasma from PD patients with peritonitis demonstrated a significant increase in eryptosis compared to healthy RBCs exposed to plasma from the control group at all times. Furthermore, significant positive correlations were observed between eryptosis level and all analyzed peritoneal biomarkers of peritonitis. We investigated a potential connection between systemic eryptosis and peritoneal biomarkers of peritonitis. Up-regulation of inflammatory markers could explain the increased rate of systemic eryptosis during PD-related peritonitis.

Synergistic immunotherapy targeting cancer-associated anemia: prospects of a combination strategy.

Cancer-associated anemia promotes tumor progression, leads to poor quality of life in patients with cancer, and even obstructs the efficacy of immune checkpoint inhibitors therapy. However, the precise mechanism for cancer-associated anemia remains unknown and the feasible strategy to target cancer-associated anemia synergizing immunotherapy needs to be clarified. Here, we review the possible mechanisms of cancer-induced anemia regarding decreased erythropoiesis and increased erythrocyte destruction, and cancer treatment-induced anemia. Moreover, we summarize the current paradigm for cancer-associated anemia treatment. Finally, we propose some prospective paradigms to slow down cancer-associated anemia and synergistic the efficacy of immunotherapy. Video Abstract.

Extracorporeal photopheresis and the cellular mechanisms: Effects of 8-methoxypsoralen and UVA treatment on red blood cells, platelets and reactive oxygen species.

BACKGROUND AND OBJECTIVES: Extracorporeal photopheresis (ECP) is a widespread cellular therapy for graft-versus-host disease, autoimmune diseases and Sézary disease. One of the main effects of ECP is the apoptosis of leukocytes, but the therapeutic mechanisms are not completely known. The aim of this study was to investigate the effects on red blood cells, platelets and the induction of reactive oxygen species. MATERIALS AND METHODS: We used human cells from healthy blood donors to simulate in vitro the composition in an apheresis bag. Cells were treated with 8-methoxypsoralen (8-MOP) and UVA. Red blood cell stability, platelet activity and induction of reactive oxygen species were analysed. RESULTS: After 8-MOP and UVA treatment, the red blood cells showed high cell integrity with low levels of eryptosis and no increase of free haemoglobin or red blood cell distribution width (RDW). Red blood cell immune-associated antigens CD59 and CD147 were hardly affected by the treatment. Platelet glycoproteins CD41, CD62P and CD63 indicated strong platelet activation after 8-MOP and UVA treatment. Reactive oxygen species were slightly but not significantly induced by the treatment. CONCLUSION: The effect of the ECP therapy is probably not exclusively mediated by leukocytes. Platelet activation is another striking effect caused by the treatment of the apheresis product with 8-MOP/UVA. However, since we could hardly identify any evidence for eryptosis or haemolysis, it is unlikely that red blood cell eryptosis is part of the therapeutic mechanism. Further research on this topic seems to be promising.

Myricetin-induced suicidal erythrocyte death.

BACKGROUND: Myricetin, a type of flavonol commonly found in fruits and herbs, has demonstrated anticancer properties by triggering the process of apoptosis or programmed cell death in tumor cells. Despite the absence of mitochondria and nuclei, erythrocytes can undergo programmed cell death, also known as eryptosis.This process is characterized by cell shrinkage, externalization of phosphatidylserine (PS) on the cell membrane, and the formation of membrane blebs. The signaling of eryptosis involves Ca2+ influx, the formation of reactive oxygen species (ROS), and the accumulation of cell surface ceramide. The present study explored the effects of myricetin on eryptosis. METHODS AND RESULTS: Human erythrocytes were exposed to various concentrations of myricetin (2-8 µM) for 24 h. Flow cytometry was used to assess the markers of eryptosis, including PS exposure, cellular volume, cytosolic Ca2+ concentration, and ceramide accumulation. In addition, the levels of intracellular ROS were measured using the 2',7'-dichlorofluorescin diacetate (DCFDA) assay. The myricetin-treated (8 µM) erythrocytes significantly increased Annexin-positive cells, Fluo-3 fluorescence intensity, DCF fluorescence intensity, and the accumulation of ceramide. The impact of myricetin on the binding of annexin-V was significantly reduced, but not completely eliminated, by the nominal removal of extracellular Ca2+. CONCLUSION: Myricetin triggers eryptosis, which is accompanied and, at least in part, caused by Ca2+ influx, oxidative stress and increase of ceramide abundance.

Metabolic exhaustion and casein kinase 1α drive deguelin-induced premature red blood cell death.

1. Deguelin (DGN), a retinoid isolated from many plants, exhibits a potent anticancer activity against a wide spectrum of tumour cells. There is a dearth of evidence, however, regarding the toxicity of DGN to red blood cells (RBCs). This is relevant given the prevalent chemotherapy-associated anaemia observed in cancer patients.2. RBCs were exposed to 1-100 µM of DGN for 24 h at 37 °C. Haemolysis and related markers were photometrically measured while flow cytometry was employed to detect phosphatidylserine exposure through Annexin-V-FITC binding and light scatter properties. Additionally, cytosolic Ca2+ and reactive oxygen species were quantified using Fluo4/AM and H2DCFDA, respectively. DGN was also tested against specific signalling inhibitors in addition to vitamin C and ATP.3. DGN caused a significant increase in Annexin-V-positive cells which was accompanied by cell shrinkage without Ca2+ elevation or oxidative stress. DGN also elicited dose-responsive haemolysis which was ameliorated by preventing KCl efflux and in the presence of sucrose, D4476, and ATP. In whole blood, DGN significantly reduced the reticulocyte count and increased platelet distribution width and large cell count.4. DGN triggers premature RBC eryptosis and haemolysis through casein kinase 1α and ATP depletion, and exhibits a specific toxicity towards reticulocytes and platelets.

Redox Status of Erythrocytes as an Important Factor in Eryptosis and Erythronecroptosis.

Overall, reactive oxygen species (ROS) signalling significantly contributes to initiation and mo-dulation of multiple regulated cell death (RCD) pathways. Lately, more information has become available about RCD modalities of erythrocytes, including the role of ROS. ROS accumulation has therefore been increasingly recognized as a critical factor involved in eryptosis (apoptosis of erythrocytes) and erythro-necroptosis (necroptosis of erythrocytes). Eryptosis is a Ca2+-dependent apoptosis-like RCD of erythrocytes that occurs in response to oxidative stress, hyperosmolarity, ATP depletion, and a wide range of xenobiotics. Moreover, eryptosis seems to be involved in the pathogenesis of multiple human diseases and pathological processes. Several studies have reported that erythrocytes can also undergo necroptosis, a lytic RIPK1/RIPK3/MLKL-mediated RCD. As an example, erythronecroptosis can occur in response to CD59-specific pore-forming toxins. We have systematically summarized available studies regarding the involvement of ROS and oxidative stress in these two distinct RCDs of erythrocytes. We have focused specifically on cellular signalling pathways involved in ROS-mediated cell death decisions in erythrocytes. Furthermore, we have summarized dysregulation of related erythrocytic antioxidant defence systems. The general concept of the ROS role in eryptotic and necroptotic cell death pathways in erythrocytes seems to be established. However, further studies are required to uncover the complex role of ROS in the crosstalk and interplay between the survival and RCDs of erythrocytes.

The Cytotoxic Effect of Septic Plasma on Healthy RBCs: Is Eryptosis a New Mechanism for Sepsis?

Sepsis is a life-threatening multiple-organ dysfunction induced by infection and is one of the leading causes of mortality and critical illness worldwide. The pathogenesis of sepsis involves the alteration of several biochemical pathways such as immune response, coagulation, dysfunction of endothelium and tissue damage through cellular death and/or apoptosis. Recently, in vitro and in vivo studies reported changes in the morphology and in the shape of human red blood cells (RBCs) causing erythrocyte death (eryptosis) during sepsis. Characteristics of eryptosis include cell shrinkage, membrane blebbing, and surface exposure to phosphatidylserine (PS), which attract macrophages. The aim of this study was to evaluate the in vitro induction of eryptosis on healthy RBCs exposed to septic plasma at different time points. Furthermore, we preliminary investigated the in vivo levels of eryptosis in septic patients and its relationship with Endotoxin Activity Assay (EAA), mortality and other biological markers of inflammation and oxidative stress. We enrolled 16 septic patients and 16 healthy subjects (no systemic inflammation in the last 3 months) as a control group. At diagnosis, we measured Interleukin-6 (IL-6) and Myeloperoxidase (MPO). For in vitro study, healthy RBCs were exposed to the plasma of septic patients and CTR for 15 min, 1, 2, 4 and 24 h. Morphological markers of death and eryptosis were evaluated by flow cytometric analyses. The cytotoxic effect of septic plasma on RBCs was studied in vitro at 15 min, 1, 2, 4 and 24 h. Healthy RBCs incubated with plasma from septic patients went through significant morphological changes and eryptosis compared to those exposed to plasma from the control group at all time points (all, p < 0.001). IL-6 and MPO levels were significantly higher in septic patients than in controls (both, p < 0.001). The percentage of AnnexinV-binding RBCs was significantly higher in septic patients with EAA level ≥0.60 (positive EAA: 32.4%, IQR 27.6-36.2) compared to septic patients with EAA level <0.60 (negative EAA: 14.7%, IQR 5.7-30.7) (p = 0.04). Significant correlations were observed between eryptosis and EAA levels (Spearman rho2 = 0.50, p < 0.05), IL-6 (Spearman rho2 = 0.61, p < 0.05) and MPO (Spearman rho2 = 0.70, p < 0.05). In conclusion, we observed a quick and great cytotoxic effect of septic plasma on healthy RBCs and a strong correlation with other biomarkers of severity of sepsis. Based on these results, we confirmed the pathological role of eryptosis in sepsis and we hypothesized its use as a biomarker of sepsis, potentially helping physicians to face important treatment decisions.

Molecular Mechanisms and Pathophysiological Significance of Eryptosis.

Despite lacking the central apoptotic machinery, senescent or damaged RBCs can undergo an unusual apoptosis-like cell death, termed eryptosis. This premature death can be caused by, or a symptom of, a wide range of diseases. However, various adverse conditions, xenobiotics, and endogenous mediators have also been recognized as triggers and inhibitors of eryptosis. Eukaryotic RBCs are unique among their cell membrane distribution of phospholipids. The change in the RBC membrane composition of the outer leaflet occurs in a variety of diseases, including sickle cell disease, renal diseases, leukemia, Parkinson's disease, and diabetes. Eryptotic erythrocytes exhibit various morphological alterations such as shrinkage, swelling, and increased granulation. Biochemical changes include cytosolic Ca2+ increase, oxidative stress, stimulation of caspases, metabolic exhaustion, and ceramide accumulation. Eryptosis is an effective mechanism for the elimination of dysfunctional erythrocytes due to senescence, infection, or injury to prevent hemolysis. Nevertheless, excessive eryptosis is associated with multiple pathologies, most notably anemia, abnormal microcirculation, and prothrombotic risk; all of which contribute to the pathogenesis of several diseases. In this review, we provide an overview of the molecular mechanisms, physiological and pathophysiological relevance of eryptosis, as well as the potential role of natural and synthetic compounds in modulating RBC survival and death.

A Mixture of Dietary Plant Sterols at Nutritional Relevant Serum Concentration Inhibits Extrinsic Pathway of Eryptosis Induced by Cigarette Smoke Extract.

Cell death program of red blood cells (RBCs), called eryptosis, is characterized by activation of caspases and scrambling of membrane phospholipids with externalization of phosphatidylserine (PS). Excessive eryptosis confers a procoagulant phenotype and is implicated in impairment of microcirculation and increased prothrombotic risk. It has recently been reported that cigarette smokers have high levels of circulating eryptotic erythrocytes, and a possible contribution of eryptosis to the vaso-occlusive complications associated to cigarette smoke has been postulated. In this study, we demonstrate how a mixture of plant sterols (MPtS) consisting of ß-sitosterol, campesterol and stigmasterol, at serum concentration reached after ingestion of a drink enriched with plant sterols, inhibits eryptosis induced by cigarette smoke extract (CSE). Isolated RBCs were exposed for 4 h to CSE (10-20% v/v). When RBCs were co-treated with CSE in the presence of 22 µM MPtS, a significant reduction of the measured hallmarks of apoptotic death like assembly of the death-inducing signaling complex (DISC), PS outsourced, ceramide production, cleaved forms of caspase 8/caspase 3, and phosphorylated p38 MAPK, was evident. The new beneficial properties of plant sterols on CSE-induced eryptosis presented in this work open new perspectives to prevent the negative physio-pathological events caused by the eryptotic red blood cells circulating in smokers.

Stimulation of Hemolysis and Eryptosis by α-Mangostin through Rac1 GTPase and Oxidative Injury in Human Red Blood Cells.

BACKGROUND: Chemotherapy-related anemia is prevalent in up to 75% of patients, which may arise due to hemolysis and eryptosis. Alpha-mangostin (α-MG) is a polyphenolic xanthonoid found in the mangosteen tree (Garcinia mangostana) whose antitumor medicinal properties are well-established. Nevertheless, the potential toxic effects of α-MG on red blood cells (RBCs) have, as of yet, not been as well studied. METHODS: RBCs were exposed to 1-40 µM of α-MG for 24 h at 37 °C. Hemolysis and related markers were measured using colorimetric assays, eryptotic cells were identified through Annexin-V-FITC, Ca2+ was detected with Fluo4/AM, and oxidative stress was assessed through H2DCFDA using flow cytometry. The toxicity of α-MG was also examined in the presence of specific signal transduction inhibitors and in whole blood. RESULTS: α-MG at 10-40 µM caused dose-dependent hemolysis with concurrent significant elevation in K+, Mg2+, and LDH leakage, but at 2.5 µM it significantly increased the osmotic resistance of cells. A significant increase was also noted in Annexin-V-binding cells, along with intracellular Ca2+, oxidative stress, and cell shrinkage. Moreover, acetylcholinesterase activity was significantly inhibited by α-MG, whose hemolytic potential was significantly ameliorated by the presence of BAPTA-AM, vitamin C, NSC23766, and isosmotic sucrose but not urea. In whole blood, α-MG significantly depleted intracellular hemoglobin stores and was selectively toxic to platelets and monocytes. CONCLUSIONS: α-MG possesses hemolytic and eryptotic activities mediated through Ca2+ signaling, Rac1 GTPase activity, and oxidative injury. Also, α-MG leads to accelerated cellular aging and specifically targets platelet and monocyte populations in a whole blood milieu.

Rosmarinic Acid Elicits Calcium-Dependent and Sucrose-Sensitive Eryptosis and Hemolysis through p38 MAPK, CK1α, and PKC.

BACKGROUND: Rosmarinic acid (RA) possesses promising anticancer potential, but further development of chemotherapeutic agents is hindered by their toxicity to off-target tissue. In particular, chemotherapy-related anemia is a major obstacle in cancer therapy, which may be aggravated by hemolysis and eryptosis. This work presents a toxicity assessment of RA in human RBCs and explores associated molecular mechanisms. METHODS: RBCs isolated from healthy donors were treated with anticancer concentrations of RA (10-800 µM) for 24 h at 37 °C, and hemolysis and related markers were photometrically measured. Flow cytometry was used to detect canonical markers of eryptosis, including phosphatidylserine (PS) exposure by annexin-V-FITC, intracellular Ca2+ by Fluo4/AM, cell size by FSC, and oxidative stress by H2DCFDA. Ions and pH were assessed by an ion-selective electrode, while B12 was detected by chemiluminescence. RESULTS: RA elicited concentration-dependent hemolysis with AST and LDH release but rescued the cells from hypotonic lysis at sub-hemolytic concentrations. RA also significantly increased annexin-V-positive cells, which was ameliorated by extracellular Ca2+ removal and isosmotic sucrose. Furthermore, a significant increase in Fluo4-positive cells and B12 content and a decrease in FSC and extracellular pH with KCl efflux were noted upon RA treatment. Hemolysis was augmented by blocking KCl efflux and was blunted by ATP, SB203580, staurosporin, D4476, isosmotic urea, and PEG 8000. CONCLUSIONS: RA stimulates Ca2+-dependent and sucrose-sensitive hemolysis and eryptosis characterized by PS exposure, Ca2+ accumulation, loss of ionic regulation, and cell shrinkage. These toxic effects were mediated through energy deprivation, p38 MAPK, protein kinase C, and casein kinase 1α.

Mechanisms of 8-aminoquinoline induced haemolytic toxicity in a G6PDd humanized mouse model.

Primaquine (PQ) and Tafenoquine (TQ) are clinically important 8-aminoquinolines (8-AQ) used for radical cure treatment of P. vivax infection, known to target hepatic hypnozoites. 8-AQs can trigger haemolytic anaemia in individuals with glucose-6-phosphate dehydrogenase deficiency (G6PDd), yet the mechanisms of haemolytic toxicity remain unknown. To address this issue, we used a humanized mouse model known to predict haemolytic toxicity responses in G6PDd human red blood cells (huRBCs). To evaluate the markers of eryptosis, huRBCs were isolated from mice 24-48 h post-treatment and analysed for effects on phosphatidylserine (PS), intracellular reactive oxygen species (ROS) and autofluorescence. Urinalysis was performed to evaluate the occurrence of intravascular and extravascular haemolysis. Spleen and liver tissue harvested at 24 h and 5-7 days post-treatment were stained for the presence of CD169+ macrophages, F4/80+ macrophages, Ter119+ mouse RBCs, glycophorin A+ huRBCs and murine reticulocytes (muRetics). G6PDd-huRBCs from PQ/TQ treated mice showed increased markers for eryptosis as early as 24 h post-treatment. This coincided with an early rise in levels of muRetics. Urinalysis revealed concurrent intravascular and extravascular haemolysis in response to PQ/TQ. Splenic CD169+ macrophages, present in all groups at day 1 post-dosing were eliminated by days 5-7 in PQ/TQ treated mice only, while liver F4/80 macrophages and iron deposits increased. Collectively, our data suggest 8-AQ treated G6PDd-huRBCs have early physiological responses to treatment, including increased markers for eryptosis indicative of oxidative stress, resulting in extramedullary haematopoiesis and loss of splenic CD169+ macrophages, prompting the liver to act as the primary site of clearance.

Blockage of the Na-K-ATPase signaling-mediated oxidant amplification loop elongates red blood cell half-life and ameliorates uremic anemia induced by 5/6th PNx in C57BL/6 mice.

We have previously demonstrated that the Na-K-ATPase signaling-mediated oxidant amplification loop contributes to experimental uremic cardiomyopathy and anemia induced by 5/6th partial nephrectomy (PNx). This process can be ameliorated by systemic administration of the peptide pNaKtide, which was designed to block this oxidant amplification loop. The present study demonstrated that the PNx-induced anemia is characterized by marked decreases in red blood cell (RBC) survival as assessed by biotinylated RBC clearance and eryptosis as assessed by annexin V binding. No significant change in iron homeostasis was observed. Examination of plasma samples demonstrated that PNx induced significant increases in systemic oxidant stress as assessed by protein carbonylation, plasma erythropoietin concentration, and blood urea nitrogen. Systemic administration of pNaKtide, but not NaKtide (pNaKtide without the TAT leader sequence) and a scramble "pNaKtide" (sc-pNaKtide), led to the normalization of hematocrit, RBC survival, and plasma protein carbonylation. Administration of the three peptides had no significant effect on PNx-induced increases in plasma erythropoietin and blood urea nitrogen without notable changes in iron metabolism. These data indicate that blockage of the Na-K-ATPase signaling-mediated oxidant amplification loop ameliorates the anemia of experimental renal failure by increasing RBC survival.NEW & NOTEWORTHY The anemia of CKD is multifactorial, and the current treatment based primarily on stimulating bone marrow production of RBCs with erythropoietin or erythropoietin analogs is unsatisfactory. In a murine model of CKD that is complicated by anemia, blockade of Na-K-ATPase signaling with a specific peptide (pNaKtide) ameliorated the anemia primarily by increasing RBC survival. Should these results be confirmed in patients, this strategy may allow for novel and potentially additive strategies to treat the anemia of CKD.

Increased suicidal erythrocyte death in patients with hepatitis B-related acute-on-chronic liver failure.

Anemia is a common complication of hepatitis B-related acute-on-chronic liver failure (HB-ACLF). Eryptosis, a suicidal erythrocyte death characterized by phosphatidylserine (PS) externalization and red blood cell-derived microparticle (RMP) generation, decreases erythrocyte lifespan. Herein, we investigated whether enhanced eryptosis is involved in the anemia pathophysiology associated with HB-ACLF. PS exposure, cell volume, cytosolic Ca2+, and reactive oxygen species (ROS) production were determined using flow cytometry. RMPs were extracted using a polyethylene glycol (PEG)-based method. We found that hemoglobin (Hb) and hematocrit (Hct) were significantly lower in patients with HB-ACLF than in healthy controls (HC), patients with chronic hepatitis B (CHB), and patients with cirrhosis. The direct antiglobulin test positive rate was 75.9% in patients with HB-ACLF while its intensity was associated with anemia. The ratio of abnormal erythrocytes was higher in patients with HB-ACLF than in HC, CHB, and cirrhosis. The percentage of PS-exposed erythrocytes was higher in patients with HB-ACLF (2.07 ± 0.11%) compared with HC (0.37 ± 0.05%), CHB (0.38 ± 0.03%), and cirrhosis (0.38 ± 0.04%). The cytosolic Ca2+ and ROS abundance were also higher in patients with HB-ACLF compared with HC, patients with CHB, and patients with cirrhosis, and were inversely correlated with the anemia in patients with HB-ACLF. PS exposure of erythrocytes collected from HC was significantly pronounced following incubation in plasma from patients with HB-ACLF compared with incubation in plasma from HC. The protein concentration and RMPs size significantly increased in patients with HB-ACLF compared with HC. Thus, the anemia in patients with HB-ACLF is associated with increased eryptosis, which is partially triggered by increased cytosolic Ca2+ and oxidative stress.NEW & NOTEWORTHY Acute chronic liver failure (ACLF) is a critical syndrome characterized by multiple organ failures and high short-term mortality. A common complication of HB-ACLF is anemia, however, the mechanism of anemia in HB-ACLF remains to be elucidated. We confirm that the accelerated eryptosis is involved in the pathophysiology of anemia associated with HB-ACLF, which progressively aggravates the clinical outcome. Our study illustrates the mechanism regarding the anemia pathogenesis of HB-ACLF, which may be utilized further toward therapeutic ends.

Geraniin inhibits whole blood IFN-γ and IL-6 and promotes IL-1ß and IL-8, and stimulates calcium-dependent and sucrose-sensitive erythrocyte death.

Correlations between circulating cytokine levels and disease states are well established, and pharmacological modulation of the immune response is thus an important aspect of the assessment of investigational new drugs. Moreover, chemotherapy-related anemia is a major obstacle in cancer treatment. Geraniin (GRN), a tannin extracted from Geranium and other plants, possesses promising antitumor potential. However, the effect of GRN on whole blood (WB) cytokine response and RBC physiology remains unexplored. Heparinized blood from consented, healthy adults was challenged with 100 ng/mL of lipopolysaccharide (LPS) with and without pretreatment with 10 µM of GRN for 24 h at 37 °C, and tumor necrosis factor-α (TNF-α), interferon-γ (IFN-γ), interleukin-1ß (IL-1ß), IL-6, IL-8, and IL-10 were assayed by ELISA. Moreover, single-cell RBC suspensions were treated with 5-100 µM of GRN for 24 or 48 h at 37 °C and cytotoxicity and canonical eryptotic markers were examined by flow cytometry. It was revealed that GRN significantly attenuated LPS-induced IFN-γ levels, increased IL-1ß, decreased IL-6 only in absence of LPS, and aggravated LPS-induced IL-8 while together with LPS significantly diminished IL-10. Furthermore, GRN induced dose-responsive, Ca2+-dependent, and sucrose-sensitive hemolysis, along with phosphatidylserine exposure and Ca2+ accumulation with no appreciable cell shrinkage or oxidative damage. GRN was also selectively toxic to platelets, significantly delayed reticulocyte maturation, and significantly disrupted leukocyte proportions. In conclusion, GRN regulates the WB cytokine response and promotes premature hemolysis and eryptosis. This study provides insights into the therapeutic utility of GRN in a highly relevant cellular model system.

Silvernanoparticle-induced hemolysis confounded with direct antiglobulin test-negative autoimmune hemolytic anemias diagnosis.

BACKGROUND: We describe here the first patient with recurrent hemolysis related to disinfectant containing silver nanoparticles (AgNps). METHODS: A 58-year-old chemist repeatedly experienced DAT-negative (Coombs-negative) hemolysis during the last 5 years. He was treated with a number of immunosuppressive drugs including 18 times rituximab. The attempt to treat him with cyclosporine A served only to increase the rate of hemolysis. Only by chance, we revealed that the patient regularly used a hand disinfectant containing AgNps. Serological testing was performed using standard techniques. Eryptosis was measured by binding annexin to exposed phosphatidylserine (PS) of the circulating red blood cells (RBCs). RESULTS: Antiglobulin tests remained negative, and PS exposing RBCs were detected two times during the last hemolytic episodes. Hemolysis completely disappeared following discontinuation of AgNp containing products. CONCLUSION: AgNps are increasingly being used in a large variety of products. Recently, it was reported that they induce in vitro prohemolytic and procoagulant effects via oxidative stress and eryptosis. The clinical findings imply the hemolysis was provoked by the patient's regular use of cleansing products containing AgNps. Our finding might help to explain the etiology of hemolytical disorders that may remain obscure in many cases.

What Should Be Responsible for Eryptosis in Chronic Kidney Disease?

BACKGROUND: Renal anemia is an important complication of chronic kidney disease (CKD). In addition to insufficient secretion of erythropoietin (EPO) and erythropoiesis disorders, the impact of eryptosis on renal anemia demands attention. However, a systemic analysis concerning the pathophysiology of eryptosis has not been expounded. SUMMARY: The complicated conditions in CKD patients, including oxidative stress, osmotic stress, metabolic stress, accumulation of uremic toxins, and iron deficiency, affect the normal skeleton structure of red blood cells (RBCs) and disturbs ionic homeostasis, causing phosphatidylserine to translocate to the outer lobules of the RBC membrane that leads to early elimination and/or shortening of the RBC lifespan. Inadequate synthesis of RBCs cannot compensate for their accelerated destruction, thus exacerbating renal anemia. Meanwhile, EPO treatment alone will not reverse renal anemia. A variety of eryptosis inhibitors have so far been found, but evidence of their effectiveness in the treatment of CKD remains to be established. KEY MESSAGES: In this review, the pathophysiological processes and factors influencing eryptosis in CKD were elucidated. The aim of this review was to underline the importance of eryptosis in renal anemia and determine some promising research directions or possible therapeutic targets to correct anemia in CKD.

Cigarette Smoke Extract Induces p38 MAPK-Initiated, Fas-Mediated Eryptosis.

Eryptosis is a physiological mechanism for the clearance of senescent or damaged erythrocytes by phagocytes. Excessive eryptosis is stimulated under several pathologies and associated with endothelial injury and thrombosis. Cigarette smoke (CS) is an established risk factor for vascular diseases and cigarette smokers have high-levels of eryptotic erythrocytes. This study, for the first time, investigates the mechanism by which CS damages red blood cells (RBCs). CS extract (CSE) from commercial cigarettes was prepared and standardized for nicotine content. Cytofluorimetric analysis demonstrated that treatment of human RBCs with CSE caused dose-dependent, phosphatidylserine externalization and cell shrinkage, hallmarks of apoptotic death. CSE did not affect cellular levels of Ca2+, reactive oxygen species (ROS) or glutathione (GSH). Immununoprecipitation and immunoblotting revealed the assembly of the death-inducing signaling complex (DISC) and oligomerization of Fas receptor as well as cleaved caspase-8 and caspase-3 within 6 h from the treatment. At the same time-interval, CSE elicited neutral sphyngomielinase (nSMase) activity-dependent ceramide formation and phosphorylation of p38 MAPK. Through specific inhibitors' nSMase, caspase-8 or p38 MAPK activities, we demonstrated that p38 MAPK activation is required for caspase-8-mediated eryptosis and that ceramide generation is initiator caspase-dependent. Finally, ex vivo analysis detected phosphorylated p38 MAPK (p-p38) and Fas-associated signaling complex in erythrocytes from cigarette smokers. In conclusion, our study demonstrates that CSE exposure induces in erythrocytes an extrinsic apoptotic pathway involving p38 MAPK-initiated DISC formation followed by activation of caspase-8/caspase-3 via ceramide formation.