SKAP2 acts downstream of CD11b/CD18 and regulates neutrophil effector function.

Background: The importance of CD11b/CD18 expression in neutrophil effector functions is well known. Beyond KINDLIN3 and TALIN1, which are involved in the induction of the high-affinity binding CD11b/CD18 conformation, the signaling pathways that orchestrate this response remain incompletely understood. Method: We performed an unbiased screening method for protein selection by biotin identification (BioID) and investigated the KINDLIN3 interactome. We used liquid chromatography with tandem mass spectrometry as a powerful analytical tool. Generation of NB4 CD18, KINDLIN3, or SKAP2 knockout neutrophils was achieved using CRISPR-Cas9 technology, and the cells were examined for their effector function using flow cytometry, live cell imaging, microscopy, adhesion, or antibody-dependent cellular cytotoxicity (ADCC). Results: Among the 325 proteins significantly enriched, we identified Src kinase-associated phosphoprotein 2 (SKAP2), a protein involved in actin polymerization and integrin-mediated outside-in signaling. CD18 immunoprecipitation in primary or NB4 neutrophils demonstrated the presence of SKAP2 in the CD11b/CD18 complex at a steady state. Under this condition, adhesion to plastic, ICAM-1, or fibronectin was observed in the absence of SKAP2, which could be abrogated by blocking the actin rearrangements with latrunculin B. Upon stimulation of NB4 SKAP2-deficient neutrophils, adhesion to fibronectin was enhanced whereas CD18 clustering was strongly reduced. This response corresponded with significantly impaired CD11b/CD18-dependent NADPH oxidase activity, phagocytosis, and cytotoxicity against tumor cells. Conclusion: Our results suggest that SKAP2 has a dual role. It may restrict CD11b/CD18-mediated adhesion only under resting conditions, but its major contribution lies in the regulation of dynamic CD11b/CD18-mediated actin rearrangements and clustering as required for cellular effector functions of human neutrophils.

Reduced myeloid commitment and increased uptake by macrophages of stem cell-derived HPS2 neutrophils.

Hermansky-Pudlak syndrome type 2 (HPS2) is a rare autosomal recessive disorder, caused by mutations in the AP3B1 gene, encoding the ß3A subunit of the adapter protein complex 3. This results in mis-sorting of proteins within the cell. A clinical feature of HPS2 is severe neutropenia. Current HPS2 animal models do not recapitulate the human disease. Hence, we used induced pluripotent stem cells (iPSCs) of an HPS2 patient to study granulopoiesis. Development into CD15POS cells was reduced, but HPS2-derived CD15POS cells differentiated into segmented CD11b+CD16hi neutrophils. These HPS2 neutrophils phenocopied their circulating counterparts showing increased CD63 expression, impaired degranulation capacity, and intact NADPH oxidase activity. Most noticeable was the decrease in neutrophil yield during the final days of HPS2 iPSC cultures. Although neutrophil viability was normal, CD15NEG macrophages were readily phagocytosing neutrophils, contributing to the limited neutrophil output in HPS2. In this iPSC model, HPS2 neutrophil development is affected by a slower rate of development and by macrophage-mediated clearance during neutrophil maturation.

Neutrophils in sickle cell disease: Exploring their potential role as a therapeutic target.

Factors influencing the activation of neutrophils in SCD and the potential neutrophil-mediated ameliorating effects of therapies in SCD.

SIGLEC-5/14 Inhibits CD11b/CD18 Integrin Activation and Neutrophil-Mediated Tumor Cell Cytotoxicity.

Since the successful introduction of checkpoint inhibitors targeting the adaptive immune system, monoclonal antibodies inhibiting CD47-SIRPα interaction have shown promise in enhancing anti-tumor treatment efficacy. Apart from SIRPα, neutrophils express a broad repertoire of inhibitory receptors, including several members of the sialic acid-binding receptor (SIGLEC) family. Here, we demonstrate that interaction between tumor cell-expressed sialic acids and SIGLEC-5/14 on neutrophils inhibits antibody-dependent cellular cytotoxicity (ADCC). We observed that conjugate formation and trogocytosis, both essential processes for neutrophil ADCC, were limited by the sialic acid-SIGLEC-5/14 interaction. During neutrophil-tumor cell conjugate formation, we found that inhibition of the interaction between tumor-expressed sialic acids and SIGLEC-5/14 on neutrophils increased the CD11b/CD18 high affinity conformation. By dynamic acoustic force measurement, the binding between tumor cells and neutrophils was assessed. The interaction between SIGLEC-5/14 and the sialic acids was shown to inhibit the CD11b/CD18-regulated binding between neutrophils and antibody-opsonized tumor cells. Moreover, the interaction between sialic acids and SIGLEC-5/14-consequently hindered trogocytosis and tumor cell killing. In summary, our results provide evidence that the sialic acid-SIGLEC-5/14 interaction is an additional target for innate checkpoint blockade in the tumor microenvironment.

Transfusion of female blood in a rat model is associated with red blood cells entrapment in organs.

Transfusion of red blood cells (RBCs) has been associated with adverse outcomes. Mechanisms may be related to donor sex and biological age of RBC. This study hypothesized that receipt of female blood is associated with decreased post-transfusion recovery (PTR) and a concomitant increased organ entrapment in rats, related to young age of donor RBCs. Donor rats underwent bloodletting to stimulate production of new, young RBCs, followed by Percoll fractionation for further enrichment of young RBCs based on their low density. Control donors did not undergo these procedures. Male rats received either a (biotinylated) standard RBC product or a product enriched for young RBCs, derived from either male or female donors. Controls received saline. Organs and blood samples were harvested after 24 hours. This study found no difference in PTR between groups, although only the group receiving young RBCs from females failed to reach a PTR of 75%. Receipt of both standard RBCs and young RBCs from females was associated with increased entrapment of donor RBCs in the lung, liver, and spleen compared to receiving blood from male donors. Soluble ICAM-1 and markers of hemolysis were higher in recipients of female blood compared to control. In conclusion, transfusing RBCs from female donors, but not from male donors, is associated with trapping of donor RBCs in organs, accompanied by endothelial activation and hemolysis.

Circulating Iron in Patients with Sickle Cell Disease Mediates the Release of Neutrophil Extracellular Traps.

Introduction: Neutrophils promote chronic inflammation and release neutrophil extracellular traps (NETs) that can drive inflammatory responses. Inflammation influences progression of sickle cell disease (SCD), and a role for NETs has been suggested in the onset of vaso-occlusive crisis (VOC). We aimed to identify factors in the circulation of these patients that provoke NET release, with a focus on triggers associated with hemolysis. Methods: Paired serum and plasma samples during VOC and steady state of 18 SCD patients (HbSS/HbSß0-thal and HbSC/HbSß+-thal) were collected. Cell-free heme, hemopexin, and labile plasma iron have been measured in the plasma samples of the SCD patients. NETs formation by human neutrophils from healthy donors induced by serum of SCD patients was studied using confocal microscopy and staining for extracellular DNA using Sytox, followed by quantification of surface coverage using ImageJ. Results: Eighteen patients paired samples obtained during VOC and steady state were available (11 HbSS/HbSß0-thal and 7 HbSC/HbSß+-thal). We observed high levels of systemic heme and iron, concomitant with low levels of the heme-scavenger hemopexin in sera of patients with SCD, both during VOC and in steady state. In our in vitro experiments, neutrophils released NETs when exposed to sera from SCD patients. The release of NETs was associated with high levels of circulating iron in these sera. Although hemin triggered NET formation in vitro, addition of hemopexin to scavenge heme did not suppress NET release in SCD sera. By contrast, the iron scavengers deferoxamine and apotransferrin attenuated NET formation in a significant proportion of SCD sera. Discussion: Our results suggest that redox-active iron in the circulation of non-transfusion-dependent SCD patients activates neutrophils to release NETs, and hence, exerts a direct pro-inflammatory effect. Thus, we propose that chelation of iron requires further investigation as a therapeutic strategy in SCD.

Lack of eosinophil extracellular trap formation due to failure of plasma membrane breakdown in the absence of elastase.

Activated eosinophils are described to release eosinophil extracellular traps (EETs), which consist of the cell's DNA covered with granule-derived antimicrobial peptides. Upon stimulation of eosinophils with the known EET-inducers phorbol 12-myristate 13-acetate, monosodium urate crystals, or Candida albicans, we observed that their plasma membrane became compromised, resulting in accessibility of the nuclear DNA for staining with the impermeable DNA dye Sytox Green. However, we did not observe any DNA decondensation or plasma membrane rupture by eosinophils, which sharply contrasts with neutrophil extracellular trap (NET) formation and the subsequent cell death known as NETosis. Neutrophil elastase (NE) activity is thought to be essential for the cleavage of histones and chromatin decondensation during NETosis. We observed that the neutrophils of a patient with a mutation in ELANE, leading to congenital neutropenia and NE deficiency, were unable to undergo NETosis. Taken together, we may suggest that the natural absence of any NE-like proteolytic activity in human eosinophils explains why EET formation is not observed, even when eosinophils become positive for an impermeable DNA dye in response to stimuli that induce NETosis in neutrophils.

Increased retention of functional mitochondria in mature sickle red blood cells is associated with increased sickling tendency, hemolysis and oxidative stress.

Abnormal retention of mitochondria in mature red blood cells (RBC) has been recently reported in sickle cell anemia (SCA) but their functionality and their role in the pathophysiology of SCA remain unknown. The presence of mitochondria within RBC was determined by flow cytometry in 61 SCA patients and ten healthy donors. Patients were classified according to the percentage of mature RBC with mitochondria contained in the whole RBC population: low (0-4%), moderate (>4% and <8%), or high level (>8%). RBC rheological, hematological, senescence and oxidative stress markers were compared between the three groups. RBC senescence and oxidative stress markers were also compared between mature RBC containing mitochondria and those without. The functionality of residual mitochondria in sickle RBC was measured by high-resolution respirometry assay and showed detectable mitochondrial oxygen consumption in sickle mature RBC but not in healthy RBC. Increased levels of mitochondrial reactive oxygen species were observed in mature sickle RBC when incubated with Antimycin A versus without. In addition, mature RBC retaining mitochondria exhibited greater levels of reactive oxygen species compared to RBC without mitochondria, as well as greater Ca2+, lower CD47 and greater phosphatidylserine exposure. Hematocrit and RBC deformability were lower, and the propensity of RBC to sickle under deoxygenation was higher, in the SCA group with a high percentage of mitochondria retention in mature RBC. This study showed the presence of functional mitochondria in mature sickle RBC, which could favor RBC sickling and accelerate RBC senescence, leading to increased cellular fragility and hemolysis.

Efficient complement-mediated clearance of immunosuppressed T cells by macrophages.

Cancer is one of the leading causes of death worldwide. Treatment outcome is largely dictated by the tumor type, disease stage, and treatment success rates, but also by the variation among patients in endogenous anti-tumor responses. Studies indicate that the presence of neutrophils in the tumor microenvironment is associated with a worse patient outcome due to their ability to suppress local anti-tumor T cell activity. Our previous studies investigated the mechanisms by which neutrophils suppress and damage T cells to become smaller in size (small T cells), debilitating their effector activities. Several studies indicate a role for tumor-associated macrophages in scavenging damaged or dead cells. We hypothesized that the observed lack of small T cells in the TME by confocal microscopy is due to immediate uptake by macrophages. In this study, we confirmed that indeed only the smaller, damaged T cells are taken up by macrophages, once serum-opsonized. Damaged T cells opsonized with complement factor C3 fragments were phagocytosed by macrophages, resulting in almost instantaneous and highly efficient uptake of these small T cells. Inhibition of the complement receptors CR1, CR3 and CR4 expressed by macrophages completely blocked phagocytosis. By contrast, actively proliferating T cells (large T cells) were neither impaired in neutrophil-MDSC activity nor opsonized for phagocytosis by macrophages. Rapid removal of damaged T cells suggests a role of complement and macrophages within the tumor microenvironment to clear suppressed T cells in cancer patients.

Cultured CD71+ erythroid cells modulate the host immune response.

OBJECTIVE: The study aimed to determine the impact of Red Blood Cells (RBCs) generated from peripheral blood mononuclear cells (PBMCs) on T cell proliferation and host response following whole blood stimulation. BACKGROUND: Culturing RBCs is a potential solution for donor shortage. The impact of immature cultured RBCs which express CD71+ on host immune response is not known. METHODS/MATERIALS: PBMCs were seeded in an erythroid expansion medium. CD71+ cells were isolated at days 14 and 21 of culture and incubated with either purified T cells or with LPS-stimulated whole blood. Controls were incubated with medium. RESULTS: At day 9, the percentage of cells that expressed CD45 and CD71 reached to the highest level (32.9%, IQR; 26.2-39.05) while the percentage of cells that expressed CD71 and CD235a reached to the highest level on day 17 (70.2%, IQR; 66.1-72.8). Incubation of T cells with days 14 CD71+ cells and day 21 CD71+ cells increased T cell proliferation. In a whole blood stimulation assay, day 21 CD71+ cells, but not day 14 CD71+ cells, inhibited the production of IL-6 and TNFα. CONCLUSION: Cultured erythroid cells can modulate the immune response by promoting T cell proliferation and inhibiting cytokine secretions following whole blood stimulation.

Humanized MISTRG as a preclinical in vivo model to study human neutrophil-mediated immune processes.

Introduction: MISTRG mice have been genetically modified to allow development of a human myeloid compartment from engrafted human CD34+ haemopoietic stem cells, making them particularly suited to study the human innate immune system in vivo. Here, we characterized the human neutrophil population in these mice to establish a model that can be used to study the biology and contribution in immune processes of these cells in vivo. Methods and results: We could isolate human bone marrow neutrophils from humanized MISTRG mice and confirmed that all neutrophil maturation stages from promyelocytes (CD11b-CD16-) to end-stage segmented cells (CD11b+CD16+) were present. We documented that these cells possessed normal functional properties, including degranulation, reactive oxygen species production, adhesion, and antibody-dependent cellular cytotoxicity towards antibody-opsonized tumor cells ex vivo. The acquisition of functional capacities positively correlated with the maturation state of the cell. We found that human neutrophils were retained in the bone marrow of humanized MISTRG mice during steady state. However, the mature segmented CD11b+CD16+ human neutrophils were released from the bone marrow in response to two well-established neutrophil-mobilizing agents (i.e., G-CSF and/or CXCR4 antagonist Plerixafor). Moreover, the neutrophil population in the humanized MISTRG mice actively reacted to thioglycolate-induced peritonitis and could infiltrate implanted human tumors, as shown by flow cytometry and fluorescent microscopy. Discussion: These results show that functional human neutrophils are generated and can be studied in vivo using the humanized MISTRG mice, providing a model to study the various functions of neutrophils in inflammation and in tumors.

The impact of biological age of red blood cell on in vitro endothelial activation markers.

Introduction: Blood donor characteristics influence red blood cell transfusion outcomes. As donor sex affects the distribution of young to old RBCs in the circulation, we hypothesized that the amount of circulating young RBCs in the blood product are associated with immune suppression. Materials and Methods: Blood samples were collected from healthy volunteers and density fractionated into young and old subpopulations. In an activated endothelial cell model, RBC adhesion to endothelium and secretion of endothelial activation markers were assessed. The impact of RBC biological age was also assessed in a T cell proliferation assay and in a whole blood stimulation assay. Results: After Percoll fractionation, young RBCs contained more reticulocytes compared to old RBCs. Young RBCs associated with lower levels of E-selectin, ICAM-1, and vWF from activated endothelial cells compared to old RBCs. RBC subpopulations did not affect T cell proliferation or cytokine responses following whole blood stimulation. Conclusion: Young RBCs contain more reticulocytes which are associated with lower levels of endothelial activation markers compared to old RBCs.

Biotinylated Platelets: A Promising Labeling Technique?

Labeling of platelets (PLTs) is essential for research purposes, in order to measure the recovery and survival of transfused PLTs in vivo. Biotinylation is a promising new alternative to the gold standard of radioactive labeling. This review highlights 4 key publications that provide significant insights into biotin-labeled PLTs (bioPLTs). Stohlawetz et al. established that transfusion of bioPLTs in human recipients is possible. De Bruin et al. developed a standardized, reproducible protocol for biotinylation of PLTs as a promising method to trace and isolate transfused PLTs in vivo, with reduced levels of PLT activation markers. Muret et al. developed a nonwashing biotin labeling method to implement in a blood bank environment. Finally, in a preclinical study, Ravanat et al. showed that different densities of biotin can be used to concurrently monitor multiple populations of human PLTs in the circulation of the same subject. These studies have made major contributions to the development of bioPLTs as a viable option for use in human research, and indicate that bioPLTs can be safely administered, preferably at a low density of biotin.

Aged versus fresh autologous platelet transfusion in a two-hit healthy volunteer model of transfusion-related acute lung injury.

BACKGROUND: Transfusion-related acute lung injury (TRALI) is a severe complication of blood transfusion that is thought of as a two-hit event: first the underlying patient condition (e.g., sepsis), and then the transfusion. Transfusion factors include human leukocyte antigen antibodies or biologic response modifiers (BRMs) accumulating during storage. Preclinical studies show an increased TRALI risk with longer stored platelets, clinical studies are conflicting. We aim to discover whether longer platelet concentrate (PC) storage time increases TRALI risk in a controlled human experiment. STUDY DESIGN AND METHODS: In a randomized controlled trial, 18 healthy male volunteers received a first hit of experimental endotoxemia (2 ng/kg lipopolysaccharide), and a second hit of fresh (2-day old) or aged (7-day old) autologous PC, or physiological saline. After 6 h, changes in TRALI pathways were determined using spirometry, chest X-ray, and bronchoalveolar lavage (BAL). RESULTS: All subjects reacted adequately to lipopolysaccharide infusion and satisfied SIRS criteria (increased pulse [>90/min] and temperature [>38°C]). There were no differences between the saline, fresh, and aged PC groups in BAL-fluid protein (95 ± 33 µg/ml; 83 ± 21 µg/ml and 104 ± 29 µg/ml, respectively) and relative neutrophil count (1.5 ± 0.5%; 1.9 ± 0.8% and 1.3 ± 0.8%, respectively), nor in inflammatory BAL-fluid BRMs (Interleukin-6, CXCL8, TNFα , and myeloperoxidase), clinical respiratory parameters, and spirometry results. All chest X-rays were normal. CONCLUSIONS: In a human endotoxemia model of autologous platelet transfusion, with an adequate first hit and platelet storage lesion, transfusion of 7-day-old PC does not increase pulmonary inflammation compared with 2-day-old PC.

Measurement of post-transfusion red blood cell survival kinetics in sickle cell disease and ß-Thalassemia: A biotin label approach.

BACKGROUND: Red blood cell (RBC) transfusions are an important treatment modality for patients with sickle cell disease (SCD) and ß-thalassemia. A subgroup of these patients relies on a chronic RBC transfusion regimen. Little is known about RBC survival (RCS) of the transfused allogeneic RBCs. In this study, we aimed to study the RCS kinetics of transfused RBCs in SCD and ß-thalassemia and to investigate factors that determine RCS. METHODS AND MATERIALS: We performed a prospective cohort study on fourteen adults with SCD and ß-thalassemia disease receiving a chronic transfusion regimen. RCS and the influence of donor and patient characteristics on RCS were assessed by simultaneous transfusion of two allogeneic RBCs using RBC biotinylation. Phenotyping of well-known RBC markers over time was performed using flow cytometry. RESULTS: RCS of the two transfused RBC units was similar in most patients. Although intra-individual variation was small, inter-individual variation in RCS kinetics was observed. Most patients demonstrated a non-linear trend in RCS that was different from the observed linear RCS kinetics in healthy volunteers. After an initial slight increase in the proportion of biotinylated RBCs during the first 24 h, a rapid decrease within the first 10-12 days was followed by a slower clearance rate. CONCLUSION: These are the first data to demonstrate that patient-related factors largely determine post-transfusion RCS behavior of donor RBC in SCD and ß-thalassemia, while donor factors exert a negligible effect. Further assessment and modeling of RCS kinetics and its determinants in SCD and ß-thalassemia patients may ultimately improve transfusion therapy.

Prophylactic furosemide to prevent transfusion-associated circulatory overload: a randomized controlled study in rats.

Transfusion-associated circulatory overload (TACO) is the leading cause of transfusion related morbidity and mortality. The only treatment is empirical use of furosemide. Our aim was to investigate if furosemide can prevent TACO. A randomized controlled trial was performed using a previously validated two-hit rat model for TACO. Volume incompliance was induced (first hit) in anemic, anesthetized Lewis rats. Rats were randomized to placebo, low-dose (5 mg kg-1) or high-dose (15 mg kg-1) furosemide-administered prior to transfusion (second-hit) and divided over two doses. Primary outcome was change in left-ventricular end-diastolic pressure (∆LVEDP) pre- compared to post-transfusion. Secondary outcomes included changes in preload, afterload, contractility and systemic vascular resistance, as well as pulmonary outcomes. Furosemide treated animals had a significantly lower ∆LVEDP compared to placebo (p = 0.041), a dose-response effect was observed. ∆LVEDP in placebo was median + 8.7 mmHg (IQR 5.9-11), + 3.9 (2.8-5.6) in the low-dose and 1.9 (- 0.6 to 5.6) in the high-dose group. The effect of furosemide became apparent after 15 min. While urine output was significantly higher in furosemide treated animals (p = 0.03), there were no significant changes in preload, afterload, contractility or systemic vascular resistance. Furosemide rapidly and dose-dependently decreases the rise in hydrostatic pulmonary pressure following transfusion, essential for preventing TACO.

Cancer cells resist antibody-mediated destruction by neutrophils through activation of the exocyst complex.

BACKGROUND: Neutrophils kill antibody-opsonized tumor cells using trogocytosis, a unique mechanism of destruction of the target plasma. This previously unknown cytotoxic process of neutrophils is dependent on antibody opsonization, Fcγ receptors and CD11b/CD18 integrins. Here, we demonstrate that tumor cells can escape neutrophil-mediated cytotoxicity by calcium (Ca2+)-dependent and exocyst complex-dependent plasma membrane repair. METHODS: We knocked down EXOC7 or EXOC4, two exocyst components, to evaluate their involvement in tumor cell membrane repair after neutrophil-induced trogocytosis. We used live cell microscopy and flow cytometry for visualization of the host and tumor cell interaction and tumor cell membrane repair. Last, we reported the mRNA levels of exocyst in breast cancer tumors in correlation to the response in trastuzumab-treated patients. RESULTS: We found that tumor cells can evade neutrophil antibody-dependent cellular cytotoxicity (ADCC) by Ca2+-dependent cell membrane repair, a process induced upon neutrophil trogocytosis. Absence of exocyst components EXOC7 or EXOC4 rendered tumor cells vulnerable to neutrophil-mediated ADCC (but not natural killer cell-mediated killing), while neutrophil trogocytosis remained unaltered. Finally, mRNA levels of exocyst components in trastuzumab-treated patients were inversely correlated to complete response to therapy. CONCLUSIONS: Our results support that neutrophil attack towards antibody-opsonized cancer cells by trogocytosis induces an active repair process by the exocyst complex in vitro. Our findings provide insight to the possible contribution of neutrophils in current antibody therapies and the tolerance mechanism of tumor cells and support further studies for potential use of the exocyst components as clinical biomarkers.

The Effect of Sex-Mismatched Red Blood Cell Transfusion on Endothelial Cell Activation in Critically Ill Patients.

Background: Observational studies suggest that sex-mismatched transfusion is associated with increased mortality. Mechanisms driving mortality are not known but may include endothelial activation. The aim of this study is to investigate the effects of sex-mismatched red blood cell (RBC) transfusions on endothelial cell activation markers in critically ill patients. Study Design and Methods: In patients admitted to the intensive care unit who received a single RBC unit, blood samples were drawn before (T0), 1 h after (T1), and 24 h after transfusion (T24) for analysis of soluble syndecan-1, soluble intercellular adhesion molecule-1, soluble thrombomodulin (sTM), von Willebrand factor antigen, interleukin-6 (IL-6), and tumor necrosis factor-alpha (TNFα). Changes in the levels of these factors were compared between sex-matched and sex-mismatched groups. Results: Of 69 included patients, 32 patients were in the sex-matched and 37 patients were in the sex-mismatched group. Compared to baseline, sex-matched transfusion was associated with significant reduction in sTM level (p value = 0.03). Between-group comparison showed that levels of syndecan-1 and sTM were significantly higher in the sex-mismatched group compared to the sex-matched group at T24 (p value = 0.04 and 0.01, respectively). Also, TNFα and IL-6 levels showed a statistically marginal significant increase compared to baseline in the sex-mismatched group at T24 (p value = 0.06 and 0.05, respectively), but not in the sex-matched group. Discussion: Transfusion of a single sex-mismatched RBC unit was associated with higher syndecan-1 and sTM levels compared to transfusion of sex-matched RBC unit. These findings may suggest that sex-mismatched RBC transfusion is associated with endothelial activation.

Storage of red blood cells in alkaline PAGGGM improves metabolism but has no effect on recovery after transfusion.

Additive solutions are used to limit changes that red blood cells (RBCs) undergo during storage. Several studies have shown better preservation of glucose and redox metabolism using the alkaline additive solution PAGGGM (phosphate-adenine-glucose-guanosine-gluconate-mannitol). In this randomized open-label intervention trial in 20 healthy volunteers, the effect of storage, PAGGGM vs SAGM (saline-adenine-glucose-mannitol), on posttransfusion recovery (PTR) and metabolic restoration after transfusion was assessed. Subjects received an autologous biotinylated RBC concentrate stored for 35 days in SAGM or PAGGGM. As a reference for the PTR, a 2-day stored autologous biotinylated RBC concentrate stored in SAGM was simultaneously transfused. RBC phenotype and PTR were assessed after transfusion. Biotinylated RBCs were isolated from the circulation for metabolomics analysis up to 24 hours after transfusion. The PTR was significantly higher in the 2-day stored RBCs than in 35-day stored RBCs 2 and 7 days after transfusion: 96% (90 to 99) vs 72% (66 to 89) and 96% (90 to 99) vs 72% (66 to 89), respectively. PTR of SAGM- and PAGGGM-stored RBCs did not differ significantly. Glucose and redox metabolism were better preserved in PAGGGM-stored RBCs. The differences measured in the blood bag remained present only until 1 day after transfusion. No differences in RBC phenotype were found besides an increased complement C3 deposition on 35-day RBCs stored in PAGGGM. Our data indicate that despite better metabolic preservation, PAGGGM is not a suitable alternative for SAGM because storage in PAGGGM did not result in an increased PTR. Finally, RBCs recovered from circulation after transfusion showed reversal of the metabolic storage lesion in vivo within a day. This study is registered in the Dutch trial register (NTR6492).

Malaria-associated adhesion molecule activation facilitates the destruction of uninfected red blood cells.

Severe malarial anemia (SMA) is the main cause of malaria-associated infant mortality in malaria endemic countries. One major factor that contributes to SMA is the accumulation of uninfected red blood cells (uRBCs) in the spleen. We report the activation of adhesion molecules Lutheran/basal cell adhesion molecule (Lu/BCAM) and CD44 on uRBCs from Plasmodium falciparum in vitro cultures and patients with malaria that mediates adherence to the splenic extracellular matrix (ECM) components laminin-α5 and hyaluronic acid (HA), respectively. This tight ECM-adhesion molecule interaction was associated with elevated intracellular Ca2+ levels, increased shedding of microvesicles, and Lu/BCAM clustering on altered uRBCs. Moreover, we observed that a soluble parasite-derived factor promoted the adhesive phenotype of uRBCs, as the incubation of RBCs with filtered malaria-conditioned medium reproduced the same adhesive effect in malaria culture-derived uRBCs. Eventually, Lu/BCAM and CD44 activation facilitate the adherence to ECM components of the red pulp, resulting in the enhanced splenic retention of uRBCs. Our results suggest a novel adhesion molecule-dependent mechanism that augments malaria-induced anemia.