Aged Erythrocytes Research Articles

Super-resolution microscopy unveils the nanoscale organization and self-limiting clustering of CD47 in human erythrocytes.

The transmembrane protein CD47, an innate immune checkpoint protein, plays a pivotal role in preventing healthy erythrocytes from immune clearance. Our study utilized stochastic optical-reconstruction microscopy (STORM) and single-molecule analysis to investigate the distribution of CD47 on the human erythrocyte membrane. Contrary to previous findings in mouse erythrocytes, we discovered that CD47 exists in randomly distributed monomers rather than in clusters across the human erythrocyte membrane. Using 2nd antibody-induced crosslinking, we found that CD47 aggregates into stable clusters within minutes. By comparing these STORM results with those of the fully mobile protein CD59 and the cytoskeleton-bound membrane protein glycophorin C under similar conditions, as well as devising two-color STORM co-labeling and co-clustering experiments, we further quantitatively revealed an intermediate, self-limiting clustering behavior of CD47, elucidating its fractional (∼14%) attachment to the cytoskeleton. Moreover, we report reductions in both the amount of CD47 and its clustering capability in aged erythrocytes, providing new insight into erythrocyte senescence. Together, the combination of STORM and 2nd antibody-based crosslinking unveils the unique self-limiting clustering behavior of CD47 due to its fractional cytoskeleton attachment.

Differential regulation by CD47 and thrombospondin-1 of extramedullary erythropoiesis in mouse spleen

Extramedullary erythropoiesis is not expected in healthy adult mice, but erythropoietic gene expression was elevated in lineage-depleted spleen cells from Cd47−/− mice. Expression of several genes associated with early stages of erythropoiesis was elevated in mice lacking CD47 or its signaling ligand thrombospondin-1, consistent with previous evidence that this signaling pathway inhibits expression of multipotent stem cell transcription factors in spleen. In contrast, cells expressing markers of committed erythroid progenitors were more abundant in Cd47−/− spleens but significantly depleted in Thbs1−/− spleens. Single-cell transcriptome and flow cytometry analyses indicated that loss of CD47 is associated with accumulation and increased proliferation in spleen of Ter119−CD34+ progenitors and Ter119+CD34− committed erythroid progenitors with elevated mRNA expression of Kit, Ermap, and Tfrc. Induction of committed erythroid precursors is consistent with the known function of CD47 to limit the phagocytic removal of aged erythrocytes. Conversely, loss of thrombospondin-1 delays the turnover of aged red blood cells, which may account for the suppression of committed erythroid precursors in Thbs1−/− spleens relative to basal levels in wild-type mice. In addition to defining a role for CD47 to limit extramedullary erythropoiesis, these studies reveal a thrombospondin-1-dependent basal level of extramedullary erythropoiesis in adult mouse spleen.

Differential regulation by CD47 and thrombospondin-1 of extramedullary erythropoiesis in mouse spleen.

Extramedullary erythropoiesis is not expected in healthy adult mice, but erythropoietic gene expression was elevated in lineage-depleted spleen cells from Cd47-/- mice. Expression of several genes associated with early stages of erythropoiesis was elevated in mice lacking CD47 or its signaling ligand thrombospondin-1, consistent with previous evidence that this signaling pathway inhibits expression of multipotent stem cell transcription factors in spleen. In contrast, cells expressing markers of committed erythroid progenitors were more abundant in Cd47-/- spleens but significantly depleted in Thbs1-/- spleens. Single-cell transcriptome and flow cytometry analyses indicated that loss of CD47 is associated with accumulation and increased proliferation in spleen of Ter119-CD34+ progenitors and Ter119+CD34- committed erythroid progenitors with elevated mRNA expression of Kit, Ermap, and Tfrc. Induction of committed erythroid precursors is consistent with the known function of CD47 to limit the phagocytic removal of aged erythrocytes. Conversely, loss of thrombospondin-1 delays the turnover of aged red blood cells, which may account for the suppression of committed erythroid precursors in Thbs1-/- spleens relative to basal levels in wild-type mice. In addition to defining a role for CD47 to limit extramedullary erythropoiesis, these studies reveal a thrombospondin-1-dependent basal level of extramedullary erythropoiesis in adult mouse spleen.

Toxicokinetic relationship between the adducts in globin and their cleavage products in the urine: Implications for human biomonitoring

Globin adducts of various chemicals, persisting in organism over the whole lifetime of erythrocytes, have been used as biomarkers of cumulative exposures to parent compounds. After removal of aged erythrocytes from the bloodstream, cleavage products of these adducts are excreted with urine as alternative, non-invasively accessible biomarkers. In our biomonitoring studies on workers exposed to ethylene oxide, its adduct with globin, N-(2-hydroxyethyl)valine, and the related urinary cleavage product N-(2-hydroxyethyl)-L-valyl-L-leucine have been determined. To describe a toxicokinetic relationship between the above types of biomarkers, a general compartmental model for simulation of formation and removal of globin adducts has been constructed in the form of code in R statistical computing environment. The essential input variables include lifetime of erythrocytes, extent of adduct formation following a single defined exposure, and parameters of exposure scenario, while other possible variables are optional. It was shown that both biomarkers reflect the past exposures differently as the adduct level in globin is a mean value of adduct levels across all compartments (subpopulations of erythrocytes of the same age) while excretion of cleavage products reflects the adduct level in the oldest compartment. Application of the model to various scenarios of continuous exposure demonstrated its usefulness for human biomonitoring data interpretation.

Selective elimination of younger erythrocytes in blood circulation and associated molecular changes in benzo (a) pyrene induced mouse model of lung cancer.

Anemia is a common feature in cancer patients. The present research was conducted to explore the mechanisms of induction of anemia in a mouse model of lung cancer. The lung cancer was induced by treating orally with BaP (50 mg/kg body weight, twice a week for four weeks). The erythrocyte kinetics were studied using a double in vivo biotinylation (DIB) technique. ROS production and apoptosis analysis were done by staining with the CMH2DCFDA stain and anti-mouse Annexin V antibody, followed by flow cytometry. The expression of antioxidant, apoptotic, anti-apoptotic and inflammatory genes was analyzed by quantitative PCR (RT-qPCR). BaP-induced tumour reduced body weight and induced persistent haemolytic anaemia. The kinetics data suggest that, though reticulocyte production was enhanced, the proportion of young erythrocytes did not increase in the same proportion. The young aged erythrocytes were selectively eliminated from blood circulation, but intermediate and old aged erythrocytes persisted for a longer duration. The tumour progression leads to a significant increase in ROS production and apoptosis in the erythrocytes. The molecular data suggests that the expression levels of antioxidants (SOD1, catalase, and GPX1) and erythropoietin (Epo) were significantly increased. The anti-inflammatory genes Interleukin-6 (IL-6), Interleukin-10 (IL-10) were significantly decreased.Apoptotic genes Bax, and caspase 3 were significantly decreased while Bcl 2 was significantly increased in the blood of tumour-bearing mice. The overall data suggest that erythrocyte turnover is severely modulated with the progression of tumor. The apoptosis, ROS levels, antioxidant, anti-apoptotic, and Epo gene expressions were increased, but proapoptotic and anti-inflammatory gene expression were suppressed.

Heme oxygenase-1 increases intracellular iron storage and suppresses inflammatory response of macrophages by inhibiting M1 polarization.

Heme oxygenase-1 (HO-1) catalyzes the first and rate-limiting enzymatic step of heme degradation, producing carbon monoxide, biliverdin, and free iron. Most iron is derived from aged erythrocytes by the decomposition of heme, which happened mainly in macrophages. However, the role of HO-1 on iron metabolism and function of macrophage is unclear. The present study investigated the effect of HO-1 on iron metabolism in macrophages, and explored the role of HO-1 on inflammatory response, polarization, and migration of macrophages. HO-1 inducer Hemin or HO-1 inhibitor zinc protoporphyrin was intravenously injected to C57BL/6J mice every 4 d for 28 d. We found that HO-1 was mainly located in the cytoplasm of splenic macrophages of mice. Activation of HO-1 by Hemin significantly increased iron deposition in the spleen, up-regulated the gene expression of ferritin and ferroportin, and down-regulated gene expression of divalent metal transporter 1 and hepcidin. Induced HO-1 by Hemin treatment increased intracellular iron levels of macrophages, slowed down the absorption of extracellular iron, and accelerated the excretion of intracellular iron. In addition, activation of HO-1 significantly decreased the expression of pro-inflammatory cytokines including interleukin (IL)-6, IL-1β, and inducible nitric oxide synthase, but increased the expression of anti-inflammatory cytokines such as IL-10. Furthermore, activation of HO-1 inhibited macrophages to M1-type polarization, and increased the migration rate of macrophages. This study demonstrated that HO-1 was able to regulate iron metabolism, exert anti-inflammatory effects, and inhibit macrophages polarization to M1 type.

Non-alcoholic fatty liver disease as a risk factor for anemia of chronic inflammation (experimental research)

The aim of the study. In recent years, non-alcoholic fatty liver disease (NAFLD) has been considered a hepatic manifestation of the metabolic syndrome. The main consequence of NAFLD is chronic hepatic inflammation, which leads to dyslipidemia, inflammation, increased oxidative stress, and endothelial dysfunction. Immune activation in response to interaction with agents of a metabolic nature induces the release of pro-inflammatory cytokines in the liver, which subsequently cause iron сhomeostasis disorder. This leads to a frequent association of NAFLD with anemia of various etiology. In this regard, we considered it important to assess the severity of the systemic inflammatory response in NAFLD in the experiment in order to -diagnose anemia of chronic inflammation.Materials and methods. The study was carried out on 26 male Wistar rats, which were divided into control and experimental groups. In animals of the experimental group, NAFLD was modeled according to the generally accepted method. In order to assess metabolic disorders, we determined the main biochemical parameters, a complete blood count with the calculation of erythrocyte indices, the concentration of the main pro-inflammatory cytokines – interleukin (IL) 1, IL-6. Results. In laboratory rats with NAFLD, a statistically significant increase of intrahepatic enzymes in blood serum was found. The state of the erythrocyte lineage of hematopoiesis in the experimental group progressively worsened and caused the development of anemic syndrome. Synchronously, a statistically significant increase in serum levels of IL-1, IL-6 was recorded, which confirms the correlation of NAFLD with anemia of chronic inflammation.Conclusions. A high concentration of IL-1, IL-6 cytokines in NAFLD inhibits iron absorption in the duodenum, leads to the activation of macrophages, blocking the release of iron processed from aging erythrocytes into plasma. Further study of the mechanisms of anemia in NAFLD provides important therapeutic targets in the treatment of both NAFLD and its comorbidities.

Casein kinase 1α mediates eryptosis: a review.

Eryptosis is a coordinated non-lytic cell death of erythrocytes characterized by cell shrinkage, cell membrane scrambling, Ca2+ influx, ceramide accumulation, oxidative stress, activation of calpain and caspases. Physiologically, it aims at removing damaged or aged erythrocytes from circulation. A plethora of diseases are associated with enhanced eryptosis, including metabolic diseases, cardiovascular pathology, renal and hepatic diseases, hematological disorders, systemic autoimmune pathology, and cancer. This makes eryptosis and eryptosis-regulating signaling pathways a target for therapeutic interventions. This review highlights the eryptotic signaling machinery containing several protein kinases and its small molecular inhibitors with a special emphasis on casein kinase 1α (CK1α), a serine/threonine protein kinase with a broad spectrum of activity. In this review article, we provide a critical analysis of the regulatory role of CK1α in eryptosis, highlight triggers of CK1α-mediated suicidal death of red blood cells, cover the knowledge gaps in understanding CK1α-driven eryptosis and discover the opportunity of CK1α-targeted pharmacological modulation of eryptosis. Moreover, we discuss the directions of future research focusing on uncovering crosstalks between CK1α and other eryptosis-regulating kinases and pathways.

Sexual Dimorphism of Adult Human Spleen in Haryana State of North India

introduction: Spleen is an important lymphoid organ of human body in the upper left part of the abdomen. Spleen performs various functions as it produces immune response against blood-borne antigens and acts as the graveyard of defective and aged erythrocytes. It exhibits sexual dimorphism. The present study is conducted to evaluate sexual dimorphism in morphometry of spleen (spleen weight, length, width, thickness and surface area) in the Haryana region. Region-wise assessment of spleen size gives a broad perspective of spleen sizes and also helps in understanding the ranges in morphometry of spleen with respect to gender. The study proves helpful in context of providing spleen size for spleen transplantation. materials and methods: After taking permission from Institute Ethics Committee, 30 male and 30 female adult human spleens in the age group of 16-70 years belonging to the Haryana region of North India were included in the study. The study was conducted in the Anatomy department along with collaboration from the Forensic department at Pt. B.D. Sharma PGIMS, Rohtak. The data thus obtained was recorded and analysed using suitable software. results: Statistical significance in dimorphism of splenic weight, length and splenic total surface area in males and females. The results of the present study would be useful for anthropologists, and forensic medicine experts to identify a spleen.

Sickle cell disease as an accelerated aging syndrome.

Sickle cell disease (SCD) is characterized by vaso-occlusion, hemolysis, and systemic manifestations that form the hallmark of the disease. Apart from morbidity, SCD is also associated with increased mortality and decreased quality of life. Aging is a natural phenomenon that is associated with changes at cellular, tissue, and organ levels, in addition to the loss of physical fitness, increased susceptibility to diseases, and a higher likelihood of mortality. Some of the cellular mechanisms involved in normal (or physiological) aging include abnormalities of sphingolipids (ceramides) and reduced length of the telomere. These changes have also been documented in SCD. Cellular, organs, and physical manifestations of SCD resemble an accelerated aging syndrome. Sickle erythrocytes also acquire morphological features similar to that of aged normal erythrocytes and are thus picked up early by the macrophages for destruction. Brain, kidney, heart, innate and adaptive immune system, and musculoskeletal system of patients with SCD exhibit morphological and functional changes that are ordinarily seen in the elderly in the general population. Stroke, silent cerebral infarcts, cardiomegaly, heart failure, pulmonary hypertension, nephropathy with proteinuria, osteopenia, osteoporosis, osteonecrosis, gout, and infections are exceedingly common in SCD. In this review, we have attempted to draw parallels between SCD and accelerated aging syndromes.

The Multiple Facets of Iron Recycling.

The production of around 2.5 million red blood cells (RBCs) per second in erythropoiesis is one of the most intense activities in the body. It continuously consumes large amounts of iron, approximately 80% of which is recycled from aged erythrocytes. Therefore, similar to the “making”, the “breaking” of red blood cells is also very rapid and represents one of the key processes in mammalian physiology. Under steady-state conditions, this important task is accomplished by specialized macrophages, mostly liver Kupffer cells (KCs) and splenic red pulp macrophages (RPMs). It relies to a large extent on the engulfment of red blood cells via so-called erythrophagocytosis. Surprisingly, we still understand little about the mechanistic details of the removal and processing of red blood cells by these specialized macrophages. We have only started to uncover the signaling pathways that imprint their identity, control their functions and enable their plasticity. Recent findings also identify other myeloid cell types capable of red blood cell removal and establish reciprocal cross-talk between the intensity of erythrophagocytosis and other cellular activities. Here, we aimed to review the multiple and emerging facets of iron recycling to illustrate how this exciting field of study is currently expanding.

Aging erythrocyte membranes as biomimetic nanometer carriers of liver-targeting chromium poisoning treatment

Chromium poisoning has become one of the most common heavy metal poisoning occupational diseases with high morbidity and mortality. However, most antidotes detoxify the whole body and are highly toxic. To achieve hepato-targeted chromium poisoning detoxification, a novel hepato-targeted strategy was developed using aging erythrocyte membranes (AEMs) as biomimetic material coated with a dimercaptosuccinic acid (DMSA) nanostructured lipid carrier to construct a biomimetic nano-drug delivery system. The particle size, potential, drug loading, encapsulation rate, in vitro release, and stability of the nanoparticles (NPs) were characterized. Confocal microscopy and flow cytometry showed that the prepared NPs could be phagocytized by RAW264.7 macrophage cells. The efficacy of AEM-DMSA-NPs for targeted liver detoxification was evaluated by in vitro MTT analysis and an in vivo model of chromium poisoning. The results showed that the NPs could safely and efficiently achieve targeted liver chromium poisoning detoxification. All the results indicated that the biomimetic nano-drug delivery system mediated by aging erythrocyte membranes and containing DMSA nanoparticles could be used as a novel therapeutic drug delivery system potentially targeting liver detoxification.

Hemolysis in the spleen drives erythrocyte turnover

AbstractRed pulp macrophages (RPMs) of the spleen mediate turnover of billions of senescent erythrocytes per day. However, the molecular mechanisms involved in sequestration of senescent erythrocytes, their recognition, and their subsequent degradation by RPMs remain unclear. In this study, we provide evidence that the splenic environment is of substantial importance in facilitating erythrocyte turnover through induction of hemolysis. Upon isolating human spleen RPMs, we noted a substantial lack of macrophages that were in the process of phagocytosing intact erythrocytes. Detailed characterization of erythrocyte and macrophage subpopulations from human spleen tissue led to the identification of erythrocytes that are devoid of hemoglobin, so-called erythrocyte ghosts. By using in vivo imaging and transfusion experiments, we further confirmed that senescent erythrocytes that are retained in the spleen are subject to hemolysis. In addition, we showed that erythrocyte adhesion molecules, which are specifically activated on aged erythrocytes, cause senescent erythrocytes to interact with extracellular matrix proteins that are exposed within the splenic architecture. Such adhesion molecule–driven retention of senescent erythrocytes under low shear conditions was found to result in steady shrinkage of the cell and ultimately resulted in hemolysis. In contrast to intact senescent erythrocytes, the remnant erythrocyte ghost shells were prone to recognition and breakdown by RPMs. These data identify hemolysis as a key event in the turnover of senescent erythrocytes, which alters our current understanding of how erythrocyte degradation is regulated.

Water Loss in Aging Erythrocytes Provides a Clue to a General Mechanism of Cellular Senescence

Experimental evidence for age-dependent loss of intracellular water content as a widespread concomitant of cellular senescence is reviewed. Quantitative models are presented, indicating that an age-dependent increase in macromolecular crowding resulting from water loss may be responsible for three observed phenomena: a general age-dependent loss of intracellular protein solubility, a delayed and rapid appearance of high molecular weight aggregates, and an age-dependent transfer of intracellular protein from dilute to concentrated or condensed phases.

Old cells die hard - sympatholysis and the ageing erythrocyte.

Old cells die hard - sympatholysis and the ageing erythrocyte.

Congenital Hemolytic Anemias: Is There a Role for the Immune System?

Congenital hemolytic anemias (CHAs) are a heterogeneous group of rare hereditary conditions including defects of erythrocyte membrane proteins, red cell enzymes, and disorders due to defective erythropoiesis. They are characterized by variable degree of anemia, chronic extravascular hemolysis, reduced erythrocyte life span, splenomegaly, jaundice, biliary lithiasis, and iron overload. Although few data are reported on the role of the immune system in CHAs, several immune-mediated mechanisms may be involved in the pathogenesis of these rare diseases. We reported in ~60% of patients with hereditary spherocytosis (HS), the presence of naturally-occurring autoantibodies (NAbs) directed against different membrane proteins (α- and β-spectrin, band 3, and dematin). Positive HS subjects showed a more hemolytic pattern and NAbs were more evident in aged erythrocytes. The latter is in line with the function of NAbs in the opsonization of damaged/senescent erythrocytes and their consequent removal in the spleen. Splenectomy, usually performed to reduce erythrocyte catheresis and improve Hb levels, has different efficacy in various CHAs. Median Hb increase is 3 g/dL in HS, 1.6–1.8 g/dL in pyruvate kinase deficiency (PKD), and 1 g/dL in congenital dyserythropoietic anemias (CDA) type II. Consistently with clinical severity, splenectomy is performed in 20% of HS, 45% of CDAII, and in 60% of PKD patients. Importantly, sepsis and thrombotic events have been registered, particularly in PKD with a frequency of ~7% for both. Furthermore, we analyzed the role of pro-inflammatory cytokines and found that interleukin 10 and interferon γ, and to a lesser extent interleukin 6, were increased in all CHAs compared with controls. Moreover, CDAII and enzymatic defects showed increased tumor necrosis factor-α and reduced interleukin 17. Finally, we reported that iron overload occurred in 31% of patients with membrane defects, in ~60% of CDAII cases, and in up to 82% of PKD patients (defined by MRI liver iron concentration >4 mg Fe/gdw). Hepcidin was slightly increased in CHAs compared with controls and positively correlated with ferritin and with the inflammatory cytokines interleukin 6 and interferon γ. Overall the results suggest the existence of a vicious circle between chronic hemolysis, inflammatory response, bone marrow dyserythropoiesis, and iron overload.

Elastic hysteresis loop acts as cell deformability in erythrocyte aging

The decrease in cellular deformability shows strong correlation with erythrocyte aging. Cell deformation can be divided into passive deformation and active deformation; however, the active deformation has been ignored in previous studies. In this work, Young's moduli of age-related erythrocytes were tested by atomic force microscopy. Furthermore, the deformation and passive and active deformation values were calculated by respective areas. Our results showed that erythrocytes in the densest fraction had the highest values of the Young's modulus, deformation, and active deformation, but the lowest values of passive deformation. Moreover, values of the deformation and active deformation both increased gradually with erythrocyte aging. The present data indicate that the elastic hysteresis loop between the approach and the retract curve could be regarded as erythrocyte deformability, and cellular deformability could be characterized by energy states. In addition, active deformation might be a crucial mechanical factor for clearing aged erythrocytes. This could provide an important information on erythrocyte biomechanics in the removal of aged cell.

Impaired functional capacity of polarised neonatal macrophages

Neonatal sepsis is accompanied by impaired apoptotic depletion of monocytes and macrophages (MΦ), aberrant cytokine production, impaired cell metabolism, and sustained inflammation. Macrophage-colony stimulating factor (M-CSF) triggers the differentiation from monocytes into MΦ (MΦ-0). Interleukin-10 (IL10) and Interferon-gamma (IFNy) further differentiate MΦ subpopulations, the anti-inflammatory MΦ-IL10 and the pro-inflammatory MΦ-IFNy subtype. We previously have shown significant differences between adult (PBMΦ) and cord blood (CBMΦ) in the metabolism of all subtypes. To test the hypothesis whether the competence to differentiate monocytes into MΦ-0 and to polarise into MΦ-IFNy and MΦ-IL10 was diminished in CBMΦ as compared to PBMΦ, we polarised monocytes by cultivation with M-CSF for 72 h, followed by stimulation with IFNy or IL10, for 48 h. After flow cytometry based immunotyping, we tested four functions: Phagocytosis of GFP-E. coli, uptake of erythrocytes, T-cell proliferation, induction of regulatory T-cells as well as phosphorylation analysis of AKT and STAT1/STAT3. Phosphorylation of STAT-1 and STAT-3, obligatory to differentiate into MΦ-IFNγ, MΦ-0 and MΦ-IL10, was found to be aberrant in CBMΦ. Whereas infected MΦ-0 showed identical phagocytic indices and intracellular degradation, TLR4-expression, NFkB up-regulation, IL10-, IL6-, and TNFα production of CBMΦ-0 were reduced. In addition, the capacity to bind aged erythrocytes and the consecutive IL10 production was lower in CBMΦ-IL10. Polarised PBMΦ-IFNy and PBMΦ-IL10 expressed higher levels of co-stimulatory receptors (CD80, CD86), had a higher capacity to stimulate T-cells and induced higher amounts of regulatory T-cells (all p < 0.05 vs. corresponding CBMΦ). Hypoxia-inducible-factor-1α (HIF-1α) was stronger expressed in CBMΦ-IFNy and upregulated in infected CBMΦ-0, whereas heme-oxygenase 1 (HO-1) expression was similar to adult PBMΦ. Neonatal MΦ-0, MΦ-IFNy and MΦ-IL10 polarisation is impaired with respect to phenotype and functions tested which may contribute to sustained inflammation in neonatal sepsis.

Circulating bilirubin level is determined by both erythrocyte amounts and the proportion of aged erythrocytes in ageing and cardiovascular diseases

BackgroundBilirubin has been involved in the process of ageing and the pathology of ageing-related diseases. Circulating bilirubin is mainly derived from the clearance of disintegrated erythrocytes in the blood. However, the change of serum bilirubin level and its regulation during ageing and in ageing-related diseases remain to be elucidated. MethodsA retrospective study was conducted by analyzing the blood cell test results and liver function results of 14,049 healthy research subjects at the Physical Examination Center and 2052 patients with various types of cardiovascular diseases (CVD) at the Department of Cardiology in Renmin Hospital of Wuhan University. Spearman correlation analysis and linear-regression analysis were used for correlation studies. Differences between male and female were investigated. ResultsWhereas the erythrocyte counts continuously decreased along with age, the proportion of aged erythrocytes was significantly increased in both male and female. The level of total circulating bilirubin was positively correlated with age and erythrocyte counts. The increase of bilirubin was associated with the increased morphological deviation of erythrocytes during ageing. Compared with health controls, the level of circulating bilirubin in CVD patients was significantly decreased consistent with the decline of erythrocyte counts and hemoglobin. ConclusionsAgeing may be accompanied by an increased ageing rate of erythrocytes, which contributes to the ageing-related decline of erythrocyte counts. Both erythrocyte counts and the proportion of aged erythrocytes coordinately might determine the circulating level of bilirubin during ageing. In CVD, the decline of circulating bilirubin may be largely attributed to concurrent anemia.

Hemolysis in the Spleen Drives Erythrocyte Turnover

Erythrocytes circulate for an average of 120 days before they are removed from the circulation. Various processes and factors have been identified that may contribute to degradation of senescent erythrocytes, but this complex process is still not completely understood. Accumulation of removal signals such as phosphatidylserine exposure, changes in CD47 expression and oxidation of proteins and lipids that render them susceptible to complement deposition, may contribute to recognition and degradation by red pulp macrophages (RPM) of the spleen. However, many questions remain on the exact mechanisms that determine the fate of aged erythrocytes. This is well exemplified in a mouse study in which physiologically aged erythrocytes were found to undergo phagocytosis by RPM in vivo but not in vitro. This finding suggested that the splenic architecture may play an important role in facilitating erythrocyte turnover. Loss of membrane deformability may lead to the initial trapping of aged or damaged erythrocytes in the spleen, an event that precedes their degradation by macrophages. Loss of deformability can explain why certain genetic diseases that affect erythrocyte membrane deformability, such as is the case in sickle cell disease and spherocytosis, result in trapping in the spleen, giving rise to anaemia. Next to loss of deformability, activation of adhesion molecules, such as Lu/BCAM and CD44, specifically on aged erythrocytes has been proposed to contribute to retention of erythrocytes within the spleen, leading to their turnover. In this study we provide evidence that the splenic environment is of key importance in facilitating erythrocyte turnover through induction of hemolysis. Upon isolating human spleen RPM we noted that only a small proportion of the macrophages were in the process of phagocytosing intact erythrocytes. Based on a range of variables, including the number of erythrocytes that are cleared daily, the number of RPM present in the spleen, the degradation rate of erythrocytes as well as differential contribution of spleen and liver to erythrocyte turnover, conservative estimates approximate that at least a 30-fold fewer erythrophagocytic events are observed in RPM than anticipated. Detailed characterization of erythrocyte and macrophage subpopulations from human spleen tissue led to the identification of a large population of erythrocytes that are devoid of hemoglobin, so-called erythrocyte ghosts. By in vivo imaging of the spleen and transfusion experiments we further confirmed that senescent erythrocytes that are retained in the spleen are subject to hemolysis, thereby forming erythrocyte ghosts. Of note, we found that the levels of haptoglobin and hemopexin, two plasma proteins that are involved in scavenging of haemoglobin and heme, respectively, correlate well with the rate of hemolysis that was observed in the spleen. Additionally, we show that the erythrocyte adhesion molecules which are specifically activated on aged erythrocytes, Lu/BCAM and CD44, cause senescent erythrocytes to interact with the extracellular matrix of the spleen. This adhesion molecule-driven retention of senescent erythrocytes, under low shear conditions, was found to result in steady shrinkage of the erythrocytes and ultimately resulted in hemolysis and ghost formation. In contrast to intact senescent erythrocytes, the remnant erythrocyte ghosts were found to be immediately recognized and rapidly degraded (1-3 hours) by RPM, thereby explaining the lack of phagocytosis of intact erythrocytes in the spleen. Together, these data identify hemolysis and ghost formation as key events in the turnover of senescent erythrocytes, which alters our current understanding of how erythrocyte degradation is regulated. Disclosures No relevant conflicts of interest to declare.