Stroke constitutes the second highest cause of morbidity and mortality worldwide while also impacting the world economy, triggering substantial financial burden in national health systems. High levels of blood glucose, homocysteine, and cholesterol are causative factors for atherothrombosis. These molecules induce erythrocyte dysfunction, which can culminate in atherosclerosis, thrombosis, thrombus stabilization, and post-stroke hypoxia. Glucose, toxic lipids, and homocysteine result in erythrocyte oxidative stress. This leads to phosphatidylserine exposure, promoting phagocytosis. Phagocytosis by endothelial cells, intraplaque macrophages, and vascular smooth muscle cells contribute to the expansion of the atherosclerotic plaque. In addition, oxidative stress-induced erythrocytes and endothelial cell arginase upregulation limit the pool for nitric oxide synthesis, leading to endothelial activation. Increased arginase activity may also lead to the formation of polyamines, which limit the deformability of red blood cells, hence facilitating erythrophagocytosis. Erythrocytes can also participate in the activation of platelets through the release of ADP and ATP and the activation of death receptors and pro-thrombin. Damaged erythrocytes can also associate with neutrophil extracellular traps and subsequently activate T lymphocytes. In addition, reduced levels of CD47 protein in the surface of red blood cells can also lead to erythrophagocytosis and a reduced association with fibrinogen. In the ischemic tissue, impaired erythrocyte 2,3 biphosphoglycerate, because of obesity or aging, can also favor hypoxic brain inflammation, while the release of damage molecules can lead to further erythrocyte dysfunction and death.

Erythrocytes (red blood cells) interact with both the immune and the metabolic systems. However, emerging data indicate that they can also function as mediators in immunometabolic interactions. High-fat diet results in increased erythrocyte cholesterol, externalized phosphatidylserine, bound MCP1, myeloperoxidase and the generation of reactive oxygen species. As a result, a pro-inflammatory effect of the erythrocyte ensues subsequently triggering macrophage inflammation, chemotaxis, erythrophagocytosis and endothelial activation. Furthermore, as a consequence of metabolic syndrome or obesity, important immunoregulatory molecules of erythrocytes, such as CD47, Glycophorin A and microvesicles are affected. Finally, inflammation-induced lipid remodelling of erythrocytes possibly partakes in a positive feedback loop with inflammation. These studies strongly indicate that erythrocytes contribute to the immunometabolic cross-talk, mainly by linking the systemic metabolic status with innate immunity and inflammation. Further exploration of the implicated mechanisms could lead to potent therapeutic targets for metaflammation-related diseases.

Metabolic dysfunction-associated steatohepatitis (MASH) is a major cause of a worldwide clinical and financial burden. Despite the tremendous efforts for untangling the molecular mechanisms, there is still a need for defining specific therapeutic targets. In this study, the author will focus on the role of erythrocyte death and hepatic erythrophagocytosis in MASH. Evidence indicates that erythrolysis prior to erythrophagocytosis protects against the development of MASH, while phagocytosis of intact erythrocytes culminates in hepatic inflammation. Furthermore, understanding the balance between erythrolysis and intact erythrocyte engulfment could lead to the development of new strategies for the treatment of MASH.

BACKGROUND: Erythrophagocytosis constitutes a major pathogenic mechanism of metabolic dysfunction associated fatty liver disease (MAFLD). Our previous research established a quantitative thin-layer chromatography (TLC) technique for sphingomyelin, revealing reduced levels in the red blood cells (erythrocytes) of patients with metabolic dysfunction associated fatty liver disease (MAFLD). This reduction was accompanied by erythrocyte sphingosine accumulation, a driver of pro-inflammatory erythrophagocytosis, though sphingosine 1-phosphate release remained stable. To better understand erythrocyte sphingosine metabolism, we adapted our quantitative TLC method to analyze sphingosine within the erythrocyte-conditioned media (ECM) of MAFLD patients. Methodology: Separation was performed on 10X10cm Silica gel 60 F254 plates using a mobile phase of chloroform, methanol, acetic acid, and water (60:50:1:4 v/v/v/v). The dynamic range, linearity, and range of linearity were assessed by analysing sphingosine levels from 0.1 to 100μg/spot. We validated the system’s precision and sensitivity by performing triplicate analyses of sphingosine standards (1.25, 2.5, and 5μg). The limits of detection and quantification were derived from the calibration curve’s slope and standard deviation (3.3 XSD/slope for LOD; 10 XSD/slope for LOQ). Accuracy was assessed via recovery tests at 100%, 200%, and 300% of a 2.5μg load. We confirmed specificity by evaluating the retention factors against other lipid species. This protocol was applied to Folch-extracted lipids from the ECM (5 × 10 ⁷ cells/ml) of four MAFLD patients and four healthy controls, spiked with 5μg of sphingosine. Findings: The calibration model, based on combined Green and Blue color intensities, followed the linear equation y = -11.171x + 353.25(R ² = 0.94). Interday precision values were 0.21%, 1.65%, and 0.44%, while recovery rates (accuracy) ranged from 94.5% to 98.7%. The measured LOD and LOQ were 0.75μg and 1.21μg, respectively. The sensitivity was calculated at 90ng. Statistical analysis showed no significant variance in sphingosine concentrations in erythrocyte-conditioned media between the MAFLD group and the control group. Summary: The described thin layer chromatography is accurate, precise, sensitive, with good limits of detection and quantification, and most importantly is low-cost and time-efficient. Using this method, we show that while erythrocytes of MAFLD patients exhibit sphingosine accumulation, the utilisation of exogenous sphingosine from their erythrocytes is not affected. This suggests that the metabolic shift may be driven by increased sphingosine supply from the plasma.

Background: Hepatic erythrophagocytosis is augmented in NASH and amplifies inflammation and fibrosis. Although various pro-phagocytic signals have been identified on erythrocytes of NASH patients, the role of bound thrombospondin-1 (TSP-1), which acts as an “eat-me” signal, arginase-1, which regulates the levels of nitric oxide in erythrocytes, and phosphatidylethano-lamine (PE) which can amplify erythrophagocytosis and hepatic inflammation have not been explored. Hence, we sought to investigate the levels of arginase-1 and TSP-1 in erythrocyte lysate and PE in erythrocyte membranes of NASH patients. Methods: Twenty-four patients and 14 healthy controls participated in our study. The levels of TSP-1 and arginase were quantified by ELISA in erythrocyte lysates, and the levels of PE in erythrocyte membranes by thin layer chro-matography. Results: Erythrocytes of NAFLD patients exhibit lower levels of arginase-1 and TSP-1 (p<0.01). Erythrocyte-bound TSP-1 levels correlated with the levels of erythrocyte surface CD47. Phosphatidylethanolamine was increased in erythrocytes of NASH patients and was accompanied by increased release, indicating exposure. Conclusion: Our results imply reduced TSP-1 binding by erythrocytes which could allow free TSP-1 molecules to act on macrophages, enhancing erythrophagocytosis. Increased PE which could amplify inflammation after efferocytosis, while downregulation of arginase-1 could lead to defective efferocytosis.