Renal anemia: from relative insufficiency of EPO to imbalance of erythropoiesis and eryptosis.

Chronic kidney disease has emerged as a major health issue both in China and worldwide. Renal anemia frequently occurs in patients with chronic kidney disease, and its severity and incidence rate increase as the disease progresses. Over the last 30 years, the administration of exogenous EPO and EPO stimulants has been employed to alleviate renal anemia, suggesting that a relative deficiency in EPO may be a primary cause. However, this approach has overshadowed other contributing factors, particularly eryptosis, which results from the reduced lifespan of red blood cells. Numerous studies reveal that there are nephrogenic and extrarenal EPO secretion indicating that an absolute deficiency of EPO is not always present in patients. Therefore, this paper speculates that renal anemia may arise when EPO-driven erythropoiesis fails to adequately compensate for aggravating eryptosis. Other factors including iron metabolism disorder, uremic toxin accumulation, inflammatory state, oxidative stress, and secondary hyperparathyroidism affect EPO reactivity bone marrow hematopoiesis and eryptosis, leading to an imbalance between red blood cell production and destruction, and cause anemia ultimately. More further studies on the pathogenesis and treatment of renal anemia would be expected to provide evidence to support our opinion.

Exposure to Waterpipe Smoke Disrupts Erythrocyte Homeostasis of BALB/c Mice.

The prevalence of waterpipe tobacco smoking (WPS) is increasing worldwide and is relatively high among youth and young adults. It has been shown, both experimentally and clinically, that WPS exposure adversely affects the cardiovascular and hematological systems through the generation of oxidative stress and inflammation. Our study aimed to evaluate the impact of WPS exposure on erythrocytes, a major component of the hematological system, of BALB/c mice. Here, we assessed the effect of nose-only WPS exposure for four consecutive weeks on erythrocyte inflammation, oxidative stress, and eryptosis. The duration of the session was 30 min/day, 5 days/week. Control mice were exposed to air. Our results showed that the levels of C-reactive protein, lipid peroxidation (LPO), superoxide dismutase, and total nitric oxide (NO) were significantly increased in the plasma of WPS-exposed mice. The number of erythrocytes and the hematocrit were significantly decreased in WPS-exposed mice compared with the control group. Moreover, there was an increase in the erythrocyte fragility in mice exposed to WPS compared with those exposed to air. The levels of lactate dehydrogenase, LPO, reduced glutathione, catalase, and NO were significantly increased in the red blood cells (RBCs) of WPS-exposed mice. In addition, erythrocytes of the WPS-exposed group showed a significant increase in ATPase activity, Ca2+, annexin V binding, and calpain activity. Taken together, our findings suggest that WPS exposure elevated inflammation and oxidative stress in the plasma and induced hemolysis in vivo. It also caused alterations of RBCs oxidative stress and eryptosis in vitro. Our data confirm the detrimental impact of WPS on erythrocyte physiology.

Inhibition of malaria and babesiosis parasites by putative red blood cell targeting small molecules.

Background: Chemotherapies for malaria and babesiosis frequently succumb to the emergence of pathogen-related drug-resistance. Host-targeted therapies are thought to be less susceptible to resistance but are seldom considered for treatment of these diseases. Methods: Our overall objective was to systematically assess small molecules for host cell-targeting activity to restrict proliferation of intracellular parasites. We carried out a literature survey to identify small molecules annotated for host factors implicated in Plasmodium falciparum infection. Alongside P. falciparum, we implemented in vitro parasite susceptibility assays also in the zoonotic parasite Plasmodium knowlesi and the veterinary parasite Babesia divergens. We additionally carried out assays to test directly for action on RBCs apart from the parasites. To distinguish specific host-targeting antiparasitic activity from erythrotoxicity, we measured phosphatidylserine exposure and hemolysis stimulated by small molecules in uninfected RBCs. Results: We identified diverse RBC target-annotated inhibitors with Plasmodium-specific, Babesia-specific, and broad-spectrum antiparasitic activity. The anticancer MEK-targeting drug trametinib is shown here to act with submicromolar activity to block proliferation of Plasmodium spp. in RBCs. Some inhibitors exhibit antimalarial activity with transient exposure to RBCs prior to infection with parasites, providing evidence for host-targeting activity distinct from direct inhibition of the parasite. Conclusions: We report here characterization of small molecules for antiproliferative and host cell-targeting activity for malaria and babesiosis parasites. This resource is relevant for assessment of physiological RBC-parasite interactions and may inform drug development and repurposing efforts.

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.

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.

Indicaxanthin prevents eryptosis induced by cigarette smoke extract by interfering with active Fas-mediated signaling.

A physiological mechanism of programmed cell death called eryptosis occurs in aged or damaged red blood cells (RBCs). Dysregulated eryptosis contributes to abnormal microcirculation and prothrombotic risk. Cigarette smoke extract (CSE) induces a p38 MAPK-initiated, Fas-mediated eryptosis, activating the death-inducing signaling complex (DISC). Indicaxanthin (Ind) from cactus pear fruits, is a bioavailable dietary phytochemical in humans and it is able to incorporate into RBCs enhancing their defense against numerous stimuli. This in vitro work shows that Ind, at concentrations that mimic plasma concentrations after a fruit meal, protects erythrocytes from CSE-induced eryptosis. CSE from commercial cigarettes was prepared in aqueous solution using an impinger air sampler and nicotine content was determined. RBCs were treated with CSE for 3 h in the absence or presence of increasing concentrations of Ind (from 1 to 5 µM). Cytofluorimetric measurements indicated that Ind reduced CSE-induced phosphatidylserine externalization and ceramide formation in a concentration-dependent manner. Confocal microscopy visualization and coimmunoprecipitation experiments revealed that Ind prevented both CSE-triggered Fas aggregation and FasL/FADD/caspase 8 recruitment in the membrane, indicating inhibition of DISC assembly. Ind inhibited the phosphorylation of p38 MAPK, caspase-8/caspase-3 cleavage, and caspase-3 activity induced by CSE. Finally, Ind reduced CSE-induced ATP depletion and restored aminophospholipid translocase activity impaired by CSE treatment. In conclusion, Ind concentrations comparable to nutritionally relevant plasma concentrations, can prevent Fas-mediated RBC death signaling induced by CSE, which suggests that dietary intake of cactus pear fruits may limit the deleterious effects of cigarette smoking.

Bufalin reprograms erythrocyte lifespan through p38 MAPK and Rac1 GTPase.

Ample evidence indicates that bufalin (BFN), a cardiotonic steroid in Bufo toad toxin, possesses a potent anticancer activity mainly by stimulating apoptosis in cancer cells. Human red blood cells (RBCs) undergo eryptosis which contributes to a plethora of pathological conditions. No reports, however, have examined the potential toxicity of BFN to RBCs. This study aims to characterize the biochemical mechanisms governing the influence of BFN on the physiology and lifespan of RBCs. Isolated RBCs from healthy volunteers were exposed to anticancer concentrations of commercially available BFN from the skin of Bufo gargarizans (10-200 µM) for 24 h at 37 °C. Photometric assays were used to estimate hemolysis and hemolytic markers, and flow cytometry was used to detect eryptotic markers. Phosphatidylserine externalization was captured by fluorescein isothiocyante-labeled annexin V, cellular dimensions by light scatter patterns, and intracellular Ca2+ and reactive oxygen species (ROS) by fluorogenic dyes Fluo4/AM and 2',7'-dichlorodihydrofluorescein diacetate (H2DCFDA), respectively. BFN caused Ca2+-independent hemolysis and release of LDH, AST, CK, and K+, and increased annexin V-bound cells, cytosolic Ca2+, cell shrinkage, and ROS levels. BFN also disrupted Na+ and Mg2+ trafficking, and was sensitive to PEG 8000, sucrose, SB203580, and NSC 23766. In whole blood, BFN depleted hemoglobin stores, increased fragmented RBCs, and was selectively toxic to reticulocytes, lymphocytes, and platelets. In conclusion, BFN elicits premature RBC death, subject to regulation by p38 MAPK and Rac1 GTPase, and is detrimental to other peripheral blood cells. Altogether, these novel findings prompt cautious consideration of the toxin in anticancer therapy.

Marine Polysaccharides Carrageenans Enhance Eryptosis and Alter Lipid Order of Cell Membranes in Erythrocytes.

Aim In the current study, hemocompatibility of three major commercially available types of carrageenans (ι, κ and λ) was investigated focusing on eryptosis. MATERIALS AND METHODS: Carrageenans of ι-, κ- and λ-types were incubated with washed erythrocytes (hematocrit 0.4%) at 0-1-5-10 g/L for either 24 h or 48 h. Incubation was followed by flow cytometry-based quantitative analysis of eryptosis parameters, including cell volume, cell membrane scrambling and reactive oxygen species (ROS) production, lipid peroxidation markers and confocal microscopy-based evaluation of intracellular Ca2+ levels, assessment of lipid order in cell membranes and the glutathione antioxidant system. Confocal microscopy was used to assess carrageenan cellular internalization using rhodamine B isothiocyanate-conjugated carrageenans. RESULTS: All three types of carrageenans were found to trigger eryptosis. Pro-eryptotic properties were type-dependent and λ-carrageenan had the strongest impact inducing phosphatidylserine membrane asymmetry, changes in cell volume, Ca2+ signaling and oxidative stress characterized by ROS overproduction, activation of lipid peroxidation and severe glutathione system depletion. Eryptosis induction by carrageenans does not require their uptake by erythrocytes. Changes in physicochemical properties of cell membrane were also type-dependent. No carrageenan-induced generation of superoxide and hydroxyl radicals was observed in cell-free milieu. CONCLUSIONS: Our findings suggest that ι-, κ- and λ-types trigger eryptosis in a type-dependent manner and indicate that carrageenans can be further investigated as potential eryptosis-regulating therapeutic agents.

Plasma levels of BCMA-positive extracellular vesicles correlate to response and side effects in myeloma patients treated with belantamab-mafodotin.

In myeloma patients, high levels of soluble BCMA (sBCMA) can limit the efficacy of BCMA-directed therapies. Belantamab-mafodotin is a BCMA antibody-drug conjugate and shows good overall response rates in heavily pretreated patients but progression-free survival data are poor. As the drug induces apoptosis, we hypothesized that sBCMA includes extracellular vesicles (EV) and thus evaluated numbers of BCMA-EV before and during belantamab therapy in 10 myeloma patients. BCMA-EV were significantly higher in patients prior to Belantamab (median: 3227/µl; p = .013) than in other myeloma patients before therapy (n = 10; 1082/µl) or healthy volunteers (n = 10; 980/µl). During therapy, BCMA-EV showed a significant increase to a maximum of 8292/µl (p = .028). Maximal changes in BCMA-EV (Δmax = BCMA-EV at C1/maximal BCMA-EV) showed a strong inverse, logarithmic correlation (r = -.950; p < .001) with FLC ratio changes (Δmax = FLC ratio at C1/minimal FLC ratio) and BCMA-EV peaks often preceded FLC progression. Correlating increase of LDH and BCMA-EV levels, together with clinical symptoms, point to a mafodotin-induced eryptosis. In summary, BCMA-EV are a part of sBCMA, peak levels precede progression, and their measurement might be helpful in identifying resistance mechanisms and side effects of BCMA targeted therapies.

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.

Eriocitrin Disrupts Erythrocyte Membrane Asymmetry through Oxidative Stress and Calcium Signaling and the Activation of Casein Kinase 1α and Rac1 GTPase.

BACKGROUND: Hemolysis and eryptosis result in the premature elimination of circulating erythrocytes and thus contribute to chemotherapy-related anemia, which is extremely prevalent in cancer patients. Eriocitrin (ERN), a flavanone glycoside in citrus fruits, has shown great promise as an anticancer agent, but the potential toxicity of ERN to human erythrocytes remains unstudied. METHODS: Erythrocytes were exposed to anticancer concentrations of ERN (10-100 µM) for 24 h at 37 °C, and hemolysis and associated markers were quantified using colorimetric assays. Eryptosis was assessed by flow cytometric analysis to detect phosphatidylserine (PS) exposure by annexin-V-FITC, intracellular Ca2+ using Fluo4/AM, and oxidative stress with 2-,7-dichlorodihydrofluorescin diacetate (H2DCFDA). ERN was also tested against specific signaling inhibitors and anti-hemolytic agents. RESULTS: ERN caused significant, concentration-dependent hemolysis at 20-100 µM. ERN also significantly increased the percentage of eryptotic cells characterized by Ca2+ elevation and oxidative stress. Furthermore, the hemolytic activity of ERN was significantly ameliorated in the presence of D4476, NSC23766, isosmotic urea and sucrose, and polyethylene glycol 8000 (PEG). In whole blood, ERN significantly elevated MCV and ESR, with no appreciable effects on other peripheral blood cells. CONCLUSIONS: ERN promotes premature erythrocyte death through hemolysis and eryptosis characterized by PS externalization, Ca2+ accumulation, membrane blebbing, loss of cellular volume, and oxidative stress. These toxic effects, mediated through casein kinase 1α and Rac1 GTPase, can be ameliorated by urea, sucrose, and PEG. Altogether, these novel findings are relevant to the further development of ERN as an anticancer therapeutic.

Stimulation of Hemolysis and Eryptosis by ß-Caryophyllene Oxide.

BACKGROUND: Eryptosis stimulated by anticancer drugs can lead to anemia in patients. ß-caryophyllene oxide (CPO) is an anticancer sesquiterpene present in various plants; however, its effect on the structure and function of human red blood cells (RBCs) remains unexplored. The aim of this study was to investigate the hemolytic and eryptotic activities and underlying molecular mechanisms of CPO in human RBCs. METHODS: Cells were treated with 10-100 µM of CPO for 24 h at 37 °C, and hemolysis, LDH, AST, and AChE activities were photometrically assayed. Flow cytometry was employed to determine changes in cell volume from FSC, phosphatidylserine (PS) externalization by annexin-V-FITC, intracellular calcium by Fluo4/AM, and oxidative stress by 2',7'-dichlorodihydrofluorescein diacetate (H2DCFDA). Cells were also cotreated with CPO and specific signaling inhibitors and antihemolytic agents. Furthermore, whole blood was exposed to CPO to assess its toxicity to other peripheral blood cells. RESULTS: CPO induced concentration-responsive hemolysis with LDH and AST leakage, in addition to PS exposure, cell shrinkage, Ca2+ accumulation, oxidative stress, and reduced AChE activity. The toxicity of CPO was ameliorated by D4476, staurosporin, and necrosulfonamide. ATP and PEG 8000 protected the cells from hemolysis, while urea and isotonic sucrose had opposite effects. CONCLUSIONS: CPO stimulates hemolysis and eryptosis through energy depletion, Ca2+ buildup, oxidative stress, and the signaling mediators casein kinase 1α, protein kinase C, and mixed lineage kinase domain-like pseudokinase. Development of CPO as an anticancer therapeutic must be approached with prudence to mitigate adverse effects on RBCs using eryptosis inhibitors, Ca2+ channel blockers, and antioxidants.

Tamoxifen induces eryptosis through calcium accumulation and oxidative stress.

Chemotherapy-related anemia is a major obstacle in anticancer therapy. Tamoxifen (TAM) is an antiestrogen prescribed for breast cancer patients with hemolytic potential and apoptotic properties in nucleated cells. However, the eryptotic activity of TAM has hitherto escaped the efforts of investigators. RBCs from apparently healthy volunteers were treated with 1-50 µM of TAM for 24 h at 37 °C. Hemoglobin leakage and LDH, AST, and AChE activities were photometrically determined while K+, Na+, and Mg2+ were detected by ion-selective electrode. Flow cytometry was used to identify eryptotic cells by annexin-V-FITC, intracellular Ca2+ by Fluo4/AM, sell size and morphology by FSC and SSC signals, respectively, and oxidative stress by H2DCFDA. Whole blood was also exposed to 30 µM of TAM for 24 h at 37 °C to examine the toxicity of TAM to WBCs and platelets. TAM caused Ca2+-independent, dose-responsive hemolysis accompanied by K+, LDH, and AST leakage without improving the mechanical stability of RBCs in hypotonic environments. TAM treatment also increased the proportion of cells positive for annexin-V-FITC, Fluo4, and DCF, along with diminished FSC and SSC signals and AChE activity. Notably, TAM toxicity was aggravated by sucrose but abrogated by vitamin C, PEG 8000, and urea. Moreover, TAM exhibited distinct cytotoxic profiles against leukocytes and platelets. TAM-induced eryptosis is characterized by breakdown of membrane asymmetry, inhibition of AChE activity, Ca2+ accumulation, cell shrinkage, and oxidative stress. Vitamin C, PEG 8000, and urea may hold promise to subvert the undesirable toxic effects of TAM on RBCs.

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.

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.

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.

Desipramine induces eryptosis in human erythrocytes, an effect blunted by nitric oxide donor sodium nitroprusside and N-acetyl-L-cysteine but enhanced by Calcium depletion.

Background: Desipramine a representative of tricyclic antidepressants (TCAs) promotes recovery of depressed patients by inhibition of reuptake of neurotransmitters serotonin (SER) and norepinephrine (NE) in the presynaptic membrane by directly blocking their respective transporters SERT and NET.Aims: To study the effect of desipramine on programmed erythrocyte death (eryptosis) and explore the underlying mechanisms.Methods: Phosphatidylserine (PS) exposure on the cell surface as marker of cell death was estimated from annexin-V-binding, cell volume from forward scatter in flow cytometry. Hemolysis was determined photometrically, and intracellular glutathione [GSH]i from high performance liquid chromatography.Results: Desipramine dose-dependently significantly enhanced the percentage of annexin-V-binding cells and didn´t impact glutathione (GSH) synthesis. Desipramine-induced eryptosis was significantly reversed by pre-treatment of erythrocytes with either nitric oxide (NO) donor sodium nitroprusside (SNP) or N-acetyl-L-cysteine (NAC). The highest inhibitory effect was obtained by using both inhibitors together. Calcium (Ca2+) depletion aggravated desipramine-induced eryptosis. Changing the order of treatment, i.e. desipramine first followed by inhibitors, could not influence the inhibitory effect of SNP or NAC.Conclusion: Antidepressants-caused intoxication can be treated by SNP and NAC, respectively. B) Patients with chronic hypocalcemia should not be treated with tricyclic anti-depressants or their dose should be noticeably reduced.

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.

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.

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.