Angiotensin-converting enzyme 2 (ACE2) and accessory proteases (TMPRSS2 and CTSL) are needed for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) cellular entry, and their expression may shed light on viral tropism and impact across the body. We assessed the cell-type-specific expression of ACE2, TMPRSS2 and CTSL across 107 single-cell RNA-sequencing studies from different tissues. ACE2, TMPRSS2 and CTSL are coexpressed in specific subsets of respiratory epithelial cells in the nasal passages, airways and alveoli, and in cells from other organs associated with coronavirus disease 2019 (COVID-19) transmission or pathology. We performed a meta-analysis of 31 lung single-cell RNA-sequencing studies with 1,320,896 cells from 377 nasal, airway and lung parenchyma samples from 228 individuals. This revealed cell-type-specific associations of age, sex and smoking with expression levels of ACE2, TMPRSS2 and CTSL. Expression of entry factors increased with age and in males, including in airway secretory cells and alveolar type 2 cells. Expression programs shared by ACE2 + TMPRSS2 + cells in nasal, lung and gut tissues included genes that may mediate viral entry, key immune functions and epithelial-macrophage cross-talk, such as genes involved in the interleukin-6, interleukin-1, tumor necrosis factor and complement pathways. Cell-type-specific expression patterns may contribute to the pathogenesis of COVID-19, and our work highlights putative molecular pathways for therapeutic intervention.

Phosphatidylserine (PS), IntroductionThe well-preserved asymmetric phospholipid distribution in the red blood cell (RBC) is lost in a subpopulation of sickle cells. 1,2 Exposure of phosphatidylserine (PS) on RBCs has been shown to accelerate thrombin-forming processes 3 and is therefore thought to contribute to an imbalance in hemostasis in the sickle cell patient. We have previously reported a correlation between the extent of PS exposure in sickle cell patients and their risk for stroke, as assessed by transcranial Doppler rates. 4 Similar PS-exposing populations have been reported in a few other diseases, such as ␤-thalassemia and chronic kidney failure, 5-7 but are found most consistently and abundantly in sickle cell anemia.The mechanisms by which PS-exposing cells are formed and persist in the circulation of sickle cell patients are not known. The absence of PS-exposing subpopulations in hereditary spherocytosis, hereditary elliptocytosis, or ␣-thalassemia, 6,8 indicates that PS exposure cannot simply be attributed to anemia or a higher rate of erythropoiesis. It is unclear how the primary defect in sickle cell anemia, a point mutation in hemoglobin leading to polymerization of RBCs under deoxygenating conditions, could result in the formation of PS-exposing cells. Phospholipid asymmetry is maintained by the adenosine 5Ј-triphosphate (ATP)-dependent aminophospholipid translocase, or flipase, which actively transports PS (and phosphatidylethanolamine) from the outer to the inner monolayer. 9 In addition to inhibition of the flipase, active phospholipid scrambling is needed to expose PS on the membrane surface. 10 In RBCs, this process can be initiated by activation of a Ca ϩϩ -dependent scramblase. 11,12 There is independent evidence that many sickle RBCs experience periods of increased cytoplasmic Ca ϩϩ during deoxygenation-induced sickling. 13 Repeated sickling of sickle cells has been shown to lead to formation of dense cells and irreversibly sickled cells (ISCs) that are present in the mature RBC population. [14][15][16] Sickle cell dehydration is mediated in part by activation of the Ca ϩϩ -dependent K ϩ (Gardos) channels. 13,17 On the basis of these findings, we hypothesized that the cell membrane damage and calcium influx accompanying repeated sickling in vivo could directly lead to induction of membrane phospholipid scrambling. This idea was supported by the finding that high hemoglobin F, which prevents sickling, resulted in fewer PS-exposing cells. 18 We anticipated that PS-exposing cells would be found mainly in the dense cell fraction, representing the cells that are damaged possibly owing to multiple sickling events, and would share common denominators, such as ISC morphology, increased cytosolic Ca ϩϩ , a decreased flipase activity, and an increased scramblase activity. We therefore analyzed the density distribution of PS-exposing sickle cells, using labeling with fluorescent annexin V (AV), and tested the correlation of PS exposure with measurements of RBC morphology, cytosolic Ca ϩϩ , and fl...

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Phospholipid asymmetry in the red blood cell (RBC) lipid bilayer is well maintained during the life of the cell, with phosphatidylserine (PS) virtually exclusively located in the inner monolayer. Loss of phospholipid asymmetry, and consequently exposure of PS, is thought to play an important role in red cell pathology. The anemia in the human thalassemias is caused by a combination of ineffective erythropoiesis (intramedullary hemolysis) and a decreased survival of adult RBCs in the peripheral blood. This premature destruction of the thalassemic RBC could in part be due to a loss of phospholipid asymmetry, because cells that expose PS are recognized and removed by macrophages. In addition, PS exposure can play a role in the hypercoagulable state reported to exist in severe β-thalassemia intermedia. We describe PS exposure in RBCs of 56 comparably anemic patients with different genetic backgrounds of the α- or β-thalassemia phenotype. The use of fluorescently labeled annexin V allowed us to determine loss of phospholipid asymmetry in individual cells. Our data indicate that in a number of thalassemic patients, subpopulations of red cells circulate that expose PS on their outer surface. The number of such cells can vary dramatically from patient to patient, from as low as that found in normal controls (less than 0.2%) up to 20%. Analysis by fluorescent microscopy of β-thalassemic RBCs indicates that PS on the outer leaflet is distributed either over the entire membrane or localized in areas possibly related to regions rich in membrane-bound α-globin chains. We hypothesize that these membrane sites in which iron carrying globin chains accumulate and cause oxidative damage, could be important in the loss of membrane lipid organization. In conclusion, we report the presence of PS-exposing subpopulations of thalassemic RBC that are most likely physiologically important, because they could provide a surface for enhancing hemostasis as recently reported, and because such exposure may mediate the rapid removal of these RBCs from the circulation, thereby contributing to the anemia.