Bat Red Blood Cells express Nucleic Acid Sensing Receptors and bind RNA and DNA

Red blood cells (RBCs) demonstrate immunomodulatory capabilities through the expression of nucleic acid sensors. Little is known about bat RBCs, and no studies have examined the immune function of bat erythrocytes. Here we show that bat RBCs express the nucleic acid-sensing Toll-like receptors TLR7 and TLR9 and bind the nucleic acid ligands, single-stranded RNA, and CpG DNA. Collectively, these data suggest that, like human RBCs, bat erythrocytes possess immune function and may be reservoirs for nucleic acids. These findings provide unique insight into bat immunity and may uncover potential mechanisms by which virulent pathogens in humans are concealed in bats.

Human red blood cells express the RNA sensor TLR7 and bind viral RNA

Red blood cells (RBCs) express the nucleic acid-sensing toll-like receptor 9 (TLR9) and bind CpG-containing DNA. However, whether human RBCs express other nucleic acid-sensing TLRs and bind RNA is unknown. Here we show that human RBCs express the RNA sensor, TLR7. TLR7 is present on the red cell membrane and associates with the RBC membrane protein Band 3. RBCs bind synthetic single-stranded RNA and RNA from pathogenic single-stranded RNA viruses. RNA acquisition by RBCs is attenuated by recombinant TLR7 and inhibitory oligonucleotides. Thus, RBCs may represent a previously unrecognized reservoir for RNA, although how RNA-binding by RBCs modulates the immune response has yet to be elucidated. These findings add to the growing list of non-gas exchanging RBC immune functions.

The protein organization of a red blood cell

SUMMARYRed blood cells (RBCs, erythrocytes) are the simplest primary human cells, lacking nuclei and major organelles, and instead employing about a thousand proteins to dynamically control cellular function and morphology in response to physiological cues. In this study, we defined a canonical RBC proteome and interactome using quantitative mass spectrometry and machine learning. Our data reveal an RBC interactome dominated by protein homeostasis, redox biology, cytoskeletal dynamics, and carbon metabolism. We validated protein complexes through electron microscopy and chemical crosslinking, and with these data, built 3D structural models of the ankyrin/Band 3/Band 4.2 complex that bridges the spectrin cytoskeleton to the RBC membrane. The model suggests spring-link compression of ankyrin may contribute to the characteristic RBC cell shape and flexibility. Taken together, our study provides an in-depth view of the global protein organization of human RBCs and serves as a comprehensive resource for future research.

266 AK117, a CD47 blocking antibody with robust macrophage activation without red blood cell hemagglutination

BackgroundAK117 is a humanized monoclonal antibody targeting CD47 which widely expresses on innate immune cells, such as macrophages, and functions as a regulator of phagocytosis. CD47 serves as the ligand for a receptor on these innate immune cells, SIRPα, which in turn delivers an inhibitory signal for phagocytosis. Hematology toxicity is the major concern of an anti-CD47 antibody. As an agent targeting CD47 being investigated as an anti-tumor therapeutic, AK117 is engineered on a human IgG4 scaffold to minimize recruitment of Fc-dependent effector functions, as well as identified with favorable hematology safety profile and robust pro-phagocytosis activity.MethodsActivity of AK117 binding to CD47 to block the interaction between CD47 and SIRPα were determined by FACS, and binding of AK117 to human RBC was also evaluated. Raji cells, HT-29 cells, and HL-60 cells which highly express CD47 were used as target cells to evaluate a pro-phagocytic activity of AK117 as a monotherapy or in combination with anti-EGFR antibody, anti-CD20 antibody or azacitidine. In in-vivo pharmacology studies, anti-tumor activity of AK117 was investigated in SCID/beige mouse Raji tumor model. Effects of AK117 on hemagglutination of human RBC at was tested. Hemoglobin (HGB) and hematocrit (HCT) was evaluated after single dose of 10 mg/kg AK117 or Hu-5F9 in male and female cynomolgus monkeys (n=1/gender).ResultsAK117 could effectively binds to CD47, and competes with SIRPα for binding to the antigen on Raji cells (figure 1). AK117 alone or combines with anti-EGFR antibody, anti-CD20 antibody and azacitidine shows potent phagocytosis of tumor cells in a dose-dependent manner (figure 2). AK117 significantly inhibited tumor growth in these tumor models (figure 3). Favorable hematology safety profile of AK117 was observed. A significant weaker binding to human RBC of AK117 was identified (figure 4), and AK117 does not induce hemagglutination of human RBC up to a concentration of 1050 μg/mL, while Hu-5F9 triggers hemagglutination even at a low concentration of 1.44 μg/mL (figure 5). AK117 has minimal anemia effect in monkey studies compared to hu5F9-G4 after single dose in cynomolgus monkeys (figure 6). AK117 showed a rather superior safety profile to Hu5F9-G4 as a shorter duration of anemia.Abstract 266 Figure 1Binding and Competition activity of AK117 to CD47. (A) FACS binding curves of AK117 and Hu5F9-G4 to CD47 on raji cells. (B) FACS competitive binding curve of AK117 and Hu5F9-G4 with SIRPαECD-mFc to CD47 on raji cells.Abstract 266 Figure 2The pro-phagocytic activity against tumor cells. (A) The phagocytic index of raji cells by macrophages with AK117. (B) The phagocytic index of HL-60 cells by macrophages with AK117 and azacitidine. (C) The phagocytic index of HT-29 cells by macrophages with AK117 and cetuximab. (D) The phagocytic index of raji cells by macrophages with AK117 and rituximab.Abstract 266 Figure 3Anti-tumor activity in raji tumor mouse model. The (A) Tumor growth curves and (B) Body weight curves of different groups in SCID/Beige mice with subcutaneous raji tumor.Abstract 266 Figure 4Binding activity of AK117 to human RBCs. Binding Curves of Hu5F9-G4 and AK117 to CD47 on human RBCsAbstract 266 Figure 5Hemagglutination effect on human erythrocytes. Hemagglutination effect of AK117 on human erythrocytesAbstract 266 Figure 6HGB and HCT in cynomolgus monkeys. The curves of (A) hemoglobin and (B) Hematocrit at different times in cynomolgus monkeys.ConclusionsWith pre-clinical pharmacology activities comparable to Hu5F9-G4 as well as superior safety properties demonstrated in non-clinical pharmacodynamics studies, AK117 has emerged as a promising new treatment for solid tumor.

Co-chaperone involvement in knob biogenesis implicates host-derived chaperones in malaria virulence

The pathology associated with malaria infection is largely due to the ability of infected human RBCs to adhere to a number of receptors on endothelial cells within tissues and organs. This phenomenon is driven by the export of parasite-encoded proteins to the host cell, the exact function of many of which is still unknown. Here we inactivate the function of one of these exported proteins, PFA66, a member of the J-domain protein family. Although parasites lacking this protein were still able to grow in cell culture, we observed severe defects in normal host cell modification, including aberrant morphology of surface knobs, disrupted presentation of the cytoadherence molecule PfEMP1, and a total lack of cytoadherence, despite the presence of the knob associated protein KAHRP. Complementation assays demonstrate that an intact J-domain is required for recovery to a wild-type phenotype and suggest that PFA66 functions in concert with a HSP70 to carry out host cell modification. Strikingly, this HSP70 is likely to be of host origin. ATPase assays on recombinant protein verify a functional interaction between PFA66 and residual host cell HSP70. Taken together, our data reveal a role for PFA66 in host cell modification, strongly implicate human HSP70s as being essential in this process and uncover a new KAHRP-independent molecular factor required for correct knob biogenesis.

Magnetophoretic and spectral characterization of oxyhemoglobin and deoxyhemoglobin: Chemical versus enzymatic processes

A new method for hemoglobin (Hb) deoxygenation, in suspension or within red blood cells (RBCs) is described using the commercial enzyme product, EC-Oxyrase®. The enzymatic deoxygenation method has several advantages over established deoxygenation methodologies, such as avoiding side reactions that produce methemoglobin (metHb), thus eliminating the need for an inert deoxygenation gas and airtight vessel, and facilitates easy re-oxygenation of Hb/RBCs by washing with a buffer that contains dissolved oxygen (DO). The UV-visible spectra of deoxyHb and metHb purified from human RBCs using three different preparation methods (sodium dithionite [to produce deoxyHb], sodium nitrite [to produce metHb], and EC-Oxyrase® [to produce deoxyHb]) show the high purity of deoxyHb prepared using EC-Oxyrase® (with little to no metHb or hemichrome production from side reactions). The oxyHb deoxygenation time course of EC-Oxyrase® follows first order reaction kinetics. The paramagnetic characteristics of intracellular Hb in RBCs were compared using Cell Tracking Velocimetry (CTV) for healthy and sickle cell disease (SCD) donors and oxygen equilibrium curves show that the function of healthy RBCs is unchanged after EC-Oxyrase® treatment. The results confirm that this enzymatic approach to deoxygenation produces pure deoxyHb, can be re-oxygenated easily, prepared aerobically and has similar paramagnetic mobility to existing methods of producing deoxyHb and metHb.

Development of combination vaccine conferring optimal protection against six pore-forming toxins of Staphylococcus aureus

In Gram-positive pathogen Staphylococcus aureus , pore-forming toxins (PFTs) such as leukocidins and hemolysins play prominent roles in staphylococcal pathogenesis by killing host immune cells and red blood cells (RBCs). However, it remains unknown which combination of toxin antigens would induce the broadest protective immune response against those toxins. In this study, by targeting six major staphylococcal PFTs (i.e., HlgAB, HlgCB, LukAB, LukED, LukSF-PV, and Hla), we generated ten recombinant toxins or toxin-subunits, three toxoids, and their rabbit antibodies. Using the cytolytic assay for RBCs and polymorphonuclear cells (PMNs), we determined the best combination of toxin antibodies conferring the broadest protection against those staphylococcal PFTs. Although anti-HlgA IgG (HlgA-IgG) showed low cross-reactivity to other toxin components, it was essential to protect rabbit and human RBCs and human PMNs. For the protection of rabbit RBCs, Hla H35L toxoid-IgG was also required, whereas, for human PMNs, LukS-IgG and LukA E323A B-IgG were essential too. When the toxin/toxoid antigens HlgA, LukS-PV, Hla H35L , and LukA E323A B were used to immunize rabbits, they increased rabbit survival; however, they did not block staphylococcal abscess formation in kidneys. Based on these results, we proposed that the combination of HlgA, LukS, Hla H35L , and LukA E323A B is the optimal vaccine component to protect human RBCs and PMNs from staphylococcal PFTs. We also concluded that a successful S. aureus vaccine requires not only those toxin antigens but also other antigens that can induce immune response blocking staphylococcal colonization.

Pharmacological activation of PIEZO1 in human red blood cells prevents Plasmodium falciparum invasion

An inherited gain-of-function variant (E756 del) in the mechanosensitive cationic channel PIEZO1 was recently shown to confer a significant protection against severe malaria. Here, we demonstrate in vitro that human red blood cell (RBC) infection by Plasmodium falciparum is prevented by the pharmacological activation of PIEZO1. The PIEZO1 activator Yoda1 inhibits RBC invasion, without affecting parasite intraerythrocytic growth, division or egress. RBC dehydration, echinocytosis and intracellular Na+/K+ imbalance are unrelated to the mechanism of protection. Inhibition of invasion is maintained, even after a prolonged wash out of Yoda1. Similarly, the chemically unrelated activators Jedi1 and Jedi2 potently inhibit parasitemia, further indicating a PIEZO1-dependent mechanism. Notably, Yoda1 treatment significantly reduced RBC surface receptors of P. falciparum, and decreased merozoite attachment and subsequent RBC deformation. Altogether these data indicate that the pharmacological activation of Piezo1 in human RBCs inhibits malaria infection by impairing P. falciparum invasion.

Abstract P782: Bioimpedance as a Means to Quantify Clot Composition and Guide Selection of Thrombectomy Devices

Introduction: Recent studies suggest that suction based thrombectomy leads to better recanalization with RBC-rich clots while stent-retrievers perform better with fibrin-rich clots. The ability to determine real-time histology of clots, in the setting of large vessel occlusion (LVO), could potentially lead to improved recanalization and better clinical outcomes while simultaneously reducing procedural costs. There is an unmet need for technology that would allow for accurate assessment of real-time clot histology in LVOs. Hypothesis: Bioimpedance measurements can be used to predict real-time clot histology. Methods: We constructed an in vitro test bed to measure the bioimpedance of clot analogs with different RBC percentages. The test bed includes a cylindrical chamber with a gold electrode embedded at the bottom. A mixture of human RBCs and plasma was injected into the chamber and coagulation was induced by adding calcium chloride solution. After coagulation, another gold electrode was used to sandwich the clot. Both electrodes were connected to an inductance, capacitance, and resistance (LCR) meter and a low-voltage (10 mV) excitation was sent to the clot at 1 to 201 kHz. We fabricated 5 types of clot analogs by mixing human RBCs and plasma with different RBC percentages (10%, 30%, 50%, 70%, and 90%). Clot types were measured 3 times. The Pearson correlation coefficient was calculated and used to evaluate the relationship between clot bioimpedance and RBC percentage. Results and Conclusions: Clot impedance decreases with the excitation frequency and clots with higher RBC percentages had higher impedance at all excitation frequencies (Fig. 1). The impedance was strongly and positively related to the RBC percentage ( r = .996, p = .0004). By minimizing the electrode size and integrating them into catheters, stents, or wires, such technology could inform clinicians of clot composition and guide device selection for optimum recanalization.