The Evolving Erythrocyte: Red Cells as Modulators of Innate H. Luke Anderson, Igor E. Brodsky and Nilam S. Mangalmurti This information is current as of September 27, 2021. J Immunol 2018; 201:1343-1351; ; doi: 10.4049/jimmunol.1800565 http://www.jimmunol.org/content/201/5/1343 Downloaded from

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The Journal of Immunology is published twice each month by The American Association of Immunologists, Inc., 1451 Rockville Pike, Suite 650, Rockville, MD 20852 Copyright © 2018 by The American Association of Immunologists, Inc. All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. The Evolving Erythrocyte: Red Blood Cells as Modulators of Innate Immunity x { H. Luke Anderson,* Igor E. Brodsky,†,‡ and Nilam S. Mangalmurti‡, , The field of red cell is undergoing a quiet rev- mitophagy (2, 4). Loss of the erythroid nucleus is thought to have olution. Long assumed to be inert carriers, facilitated the evolution of mammalian endothermy, as it allowed RBCs are emerging as important modulators of the in- erythrocytes to further specialize for efficient gas exchange (4). nate . Erythrocytes bind and scavenge In the classical literature, human erythrocytes have long been , nucleic acids, and in circulation. typecast as mere oxygen transporters. However, mounting evi- Depending on the conditions of the microenvironment, dence suggests that these cells also play an important role in the erythrocytes may either promote immune activation or innate (5–7). From an evolutionary perspective, maintain immune quiescence. We examine erythrocyte the involvement of human erythrocytes in innate immunity Downloaded from immune function through a comparative and evolution- should not be surprising, as the nucleated erythrocytes of , ary lens, as this framework may offer perspective into amphibians, and fishes actively participate in the immune re- newly recognized roles of human RBCs. Next, we re- sponse via production of -like factors, upregulation of viral response , and sequestration of pathogens through view the known immune roles of human RBCs and surface binding or (8–20). Despite their deletion discuss their activity in the context of where of organelles during and subsequent inability to http://www.jimmunol.org/ erythrocyte function may prove important to disease carry out transcription and translation, mammalian erythrocytes pathogenesis. Given the limited success of immuno- retain the ability to interact with endogenous and exogenous modulatory therapies in treating inflammatory diseases, inflammatory molecules in the blood. Research over the past we propose that the immunologic function of RBCs several decades demonstrates that, like their evolutionary provides an understudied and potentially rich area of counterparts, these cells retain the ability to bind and interact research that may yield novel insights into mechanisms with a variety of inflammatory molecules, including chemo- of immune regulation. The Journal of Immunology, kines, nucleic acids, and pathogens, thereby regulating and

2018, 201: 1343–1351. modulating immune responses. Internal components of eryth- by guest on September 27, 2021 rocytes such as and heme are also formidable facets of innate immunity, capable of generating reactive rythrocytes are the most abundant cell type in the oxygen species (ROS) to defend against invading hemolytic human body, numbering between 20 and 30 trillion microbes as well as promoting pathologic inflammatory and and accounting for nearly 70% of the total cell count in E autoimmune responses. Finally, the involvement of erythrocytes the average adult (1). Erythropoiesis begins with the differenti- in host– interactions may render them crucially im- ation of multipotent hematopoietic stem cells in the bone portant in the context of sepsis and , a state of marrow, which then give rise to erythroid-committed precursors. dysregulated host response to where the body is In the final stages of the process, known as terminal erythro- overwhelmed with endotoxins and other inflammatory media- poiesis, the nucleus and other organelles are extruded. These tors. A deeper understanding of erythrocyte function will likely enucleated are then released into the bloodstream to be vital to elucidating the ways in which these cells contribute to complete their maturation process (2). After a lifespan of ∼120 d, the pathogenesis of a variety of acute and chronic diseases. RBCs are cleared by in the spleen and (3). Importantly, mammals are the only whose eryth- rocytes are enucleated in their mature state. The extrusion of Evolutionary perspectives the nucleus and other organelles occurs through a series of Erythrocyte origins. Throughout the course of evolution, the complex stages, including chromatin condensation, budding, and cellular components of the blood have undergone functional

*Department of Biology, University of Pennsylvania, Philadelphia, PA 19104; Health Affairs through the Peer Reviewed Medical Research Program under Contract †Department of Pathobiology, University of Pennsylvania School of Veterinary Medi- W81XWH-15-1-0363 (to N.S.M.). cine, Philadelphia, PA 19104; ‡Institute for Immunology, Perelman School of Medicine, x Address correspondence and reprint requests to Dr. Nilam S. Mangalmurti, Perelman University of Pennsylvania, Philadelphia, PA 19104; Pulmonary, and Critical School of Medicine, University of Pennsylvania, 220 Stemmler Hall, 3450 Hamilton Care Division, Department of Medicine, Perelman School of Medicine, University of { Walk, Philadelphia, PA 19104. E-mail address: [email protected] Pennsylvania, Philadelphia, PA 19104; and Penn Center for Pulmonary Biology, Perel- man School of Medicine, University of Pennsylvania, Philadelphia, PA 19104 Abbreviations used in this article: DAMP, damage-associated molecular pattern; DARC, Duffy Ag for chemokines; GYPA, ; HO-1, heme oxygenase-1; ORCIDs: 0000-0002-4150-0517 (H.L.A.); 0000-0001-7970-872X (I.E.B.); 0000- mtDNA, mitochondrial DNA; NRBC, nucleated RBC; ROS, . 0002-9146-1608 (N.S.M.). Received for publication April 20, 2018. Accepted for publication May 16, 2018. Copyright Ó 2018 by The American Association of Immunologists, Inc. 0022-1767/18/$35.00 This work was supported by National Institutes of Health Award HL126788 (to N.S.M.). This work was also supported by the Office of the Assistant Secretary of Defense for www.jimmunol.org/cgi/doi/10.4049/jimmunol.1800565 1344 BRIEF REVIEWS: IMMUNOMODULATORY EFFECTS OF RBCs specialization. The earliest blood cells are thought to have been behavior was co-opted by the malarial parasite or that it is relative generalists involved in both phagocytosis and nutrient the maladaptive consequence of an evolutionarily conserved delivery. However, they did not serve a function in oxygen mechanism of pathogen sequestration. transport (21, 22). Prior to the emergence of circulatory systems, Mammalian erythrocytes. Mammals are the only vertebrates with small accomplished tissue oxygenation by simple enucleated erythrocytes under homeostatic conditions (Fig. 1). alone; this mechanism is still observed today in prim- Intriguingly, however, nucleated erythrocytes are sometimes itive such as the sponges, cnidarians, and flatworms (21, observed in humans during pathologic states, constituting a 23). Later, the emergence of larger body plans necessitated more striking exception and suggesting that enucleated erythrocytes efficient oxygen delivery mechanisms, leading to the evolution of are not an obligate feature of mammalian physiology (32–34). increasingly elaborate circulatory systems (23) and a variety of Mammalian erythrocytes also lack endoplasmic reticula, ribo- oxygen-carrying respiratory pigments (24, 25). Hemoglobin somes, and mitochondria, as early developmental processes appears to have arisen first among them, followed by the expunge these typically vital organelles (2). It has been hypoth- hemerythrins, the molluscan hemocyanins, and eventually the esized that the deletion of nuclei and organelles in mature hemocyanins (25). Although hemoglobin likely erythrocytes may have allowed for increased surface area– began as a free-floating , the emergence of cells to to-volume ratio, facilitating increased gas exchange efficiency encapsulate the pigment (i.e., erythrocytes) appears to have (35–37). Moreover, the feature allowed for elevated cellular occurred soon after (24, 25). All modern vertebrates [with the hemoglobin capacity (4), increased pliability, and an improved notable exception of white-blooded ice fish from the family Downloaded from ability to traverse small (15, 37). Channichthyidae (26)] accomplish oxygen delivery by means of It is possible that erythrocyte enucleation evolved in response to circulating hemoglobin-rich erythrocytes (24). Similar cells are a reduction in Earth’s atmospheric oxygen concentrations (35) or sporadically observed in numerous groups as well, because of the increased metabolic demands of endothermy (37). including the echinoderms, phoronids, annelids, nemerteans, In line with the latter hypothesis, erythrocytes are comparatively and mollusks (25, 27). Erythrocytes may have arisen separately larger in ectothermic, phylogenetically lower organisms like in multiple lineages or once prior to the divergence of the http://www.jimmunol.org/ fishes and herptiles (36, 38). The extrusion of mitochondria in animal phyla, although the precise evolutionary history remains mammalian erythrocytes may also have reduced production of unresolved (21, 22, 27). damaging ROS (39). This is an attractive hypothesis given that Erythrocytes of nonmammalian vertebrates. The erythrocytes of human erythrocytes generate extensive free radicals even without nonmammalian vertebrates are nucleated, save rare exceptions mitochondria, necessitating their ample arsenal of protective in the fishes (28) and amphibians (29, 30). Furthermore, like antioxidant molecules and (40). However, avian many other hematopoietic cells, nonmammalian erythrocytes erythrocytes exhibit lower levels of blood oxidative and directly participate in the immune response. In fishes (8, 16), similar cellular hemoglobin content in comparison with mam- amphibians (19), and birds (9, 13), erythrocytes actively un- mals, despite their retaining both functional mitochondria and by guest on September 27, 2021 dergo transcription and protein production in response to en- nuclei(4,41),indicatingthatthe presence of mitochondria and vironmental cues. Specifically, Atlantic salmon (Salmo salar L.) nuclei in erythrocytes is not necessarily a barrier to endothermy. erythrocytes increase transcription of Ag-presenting and viral Because endothermy arose separately in the avian and mam- response genes during piscine orthoreovirus infection and malian lineages, it is perhaps to be expected that alternative suites upregulate IFN-a in response to infectious salmon anemia of physiologic adaptations accompanied it. , contributing to host resistance (10, 11). Erythrocytes Regardless of the specific pressures driving the loss of or- in striped beakfish (Oplegnathus fasciatus) appear to coordinate ganelles in mammalian erythrocytes, the change appears to have immune function with leukocytes, upregulating an additional been an evolutionary compromise: opting for greater respiratory 338 genes when incubated with leukocytes and LPS compared specialization, the forfeiture of key cellular components rendered with LPS alone (18). Furthermore, both chicken and trout mammalian erythrocytes more passive actors than those of erythrocytes release cytokine-like factors following exposure to other vertebrates. Without ribosomes or a , mammalian fungal Ags (8, 9) and express functional pattern recognition erythrocytes are incapable of , tran- receptors (including TLR and recognition ) scription, or new protein synthesis and are therefore unable to on their surfaces (14, 31). Remarkably, trout erythrocytes have contribute to the immune response via de novo production of even been observed engaging in phagocytosis of pathogenic yeast immunologically active signaling molecules. However, there is ( albicans) (12). significant evidence that mammalian erythrocytes remain key Finally, the erythrocytes of trout and chickens form cellular contributors to innate immunity. Despite their loss of internal aggregations known as rosettes, which serve to expedite pathogen destruction by macrophages (12, 17). The rosetting organelles, mammalian erythrocytes express a large number of behavior of piscine and avian erythrocytes stands in stark cell surface receptors that mediate their interactions with both contrast to that of human erythrocytes, which is notoriously endogenous and exogenous agents in the blood (42), providing observed in severe cases of . them with an extensive capacity for scavenging or sequestration Rosetting in this context is characterized not as a defense of circulating molecules that impact immune function. mechanism of the human host but rather as a virulence Hemoglobin and heme. Among the respiratory pigments, hemo- mechanism of the malarial parasite (20). Infected erythrocytes globin is the most ancient (25). Circulating hemoglobin appears adhering to uninfected erythrocytes and endothelial cells re- to have arisen from the tissue-localized of primitive sults in a sequestration of blood cells in affected organs, animals, such as the turbellarian platyhelminths (25). Although which, particularly when localized in cerebral tissue, is asso- oxygen carriage is their primary function, hemoglobin and free ciated with mortality (20). It is possible that this rosetting heme also participate in the innate immune response. The Journal of Immunology 1345

FIGURE 1. Comparing the immunologic properties of erythrocytes across orders. The erythrocytes of lower vertebrates (i.e., fishes, amphibians, reptiles, and birds) are nucleated in their mature state. Erythrocytes of fish and birds are known to actively undergo transcription and translation, produce and release , form ro- settes to sequester pathogens, and, in the case of trout, phagocytose pathogens. Although the activities of eryth- rocytes in reptiles and amphibians are less well studied, it is likely that they retain many of these characteristics given Downloaded from that they have been maintained across the phylogenetic distance from fish to birds. Despite lacking nuclei and organelles, mammalian (and human) erythrocytes retain the ability to influence innate immunity. All erythrocytes contain hemoglobin, which participates in host defense by generating antimicrobial ROS. http://www.jimmunol.org/ by guest on September 27, 2021

Because hemoglobin provides a rich source of iron for bac- appropriate fragments of its host’s hemoglobin for use in its terial (43–45), microbial invaders have evolved own gut (54). Beyond their function in gas exchange, the both hemolytic toxins and high-affinity scavenging systems to utility of respiratory pigments as innate immune elements access it (46–48). In response, host hemoglobin provides a has been realized at multiple points throughout evolution counterattack: activated by the extracellular proteases of viru- (12, 25, 53). lent , the peptide emits bactericidal ROS in the mi- Although hemoglobin may confer antimicrobial protection crobial vicinity (49). Studies show that both the a and b under homeostatic conditions, research indicates that it has the subunits of hemoglobin contain high-affinity binding sites for opposite effect during pathologic states. Particularly when the LPS, the immune-stimulating endotoxin present in the mem- system is overwhelmed with endotoxin, as is the case in severe branes of Gram-negative bacteria. When LPS binds to hemo- sepsis, elevated levels of cell-free hemoglobin are associated globin, it stimulates the redox activity of the peptide and leads with increased mortality (55, 56). Interestingly, i.p. injection to the production of antimicrobial free radicals (50). Addi- of globin had protective effects on endotoxemic mice (55), tionally, the presence of hemoglobin has been shown to amplify whereas i.v. injection of hemoglobin into endotoxemic mice cytokine production in the presence of LPS as well dramatically increased rates of mortality (56). And although as enhance macrophage binding of LPS (51). Finally, micro- globin alone reduces macrophage TNF production in the molar concentrations of hemoglobin fragments are sufficient to presence of LPS, thereby dampening immune response to inhibit the growth of yeast and bacteria (both Gram-positive endotoxin, hemoglobin actually stimulates macrophage TNF and Gram-negative) via ROS production, while demonstrating production and triggers the release of proinflammatory no toxic effects on human blood cells (52). cytokines under the same conditions (55). The conflicting The immunologic functions of respiratory globins are nature of the hemoglobin literature appears to be due to the widespread across the animal kingdom. Invertebrate horseshoe dualistic properties of the hemoglobin molecule itself in (Tachypleus tridentatus) contain blue-tinted blood rich in which, broadly speaking, globins attenuate immune response hemocyanin, a cell-free protein that carries oxygen throughout (55, 57), whereas heme exacerbates it (58, 59). These effects the blood of many mollusks and . In these animals, are likely attributable to the ability of globin to scavenge ex- hemocyanin similarly generates ROS in response to microbial cess free heme during endotoxemia, resulting in attenuated challenge (49, 53). The antimicrobial properties of globins immune activation. In contrast, if heme is inflammatory, are so universal that the cattle tick (Boophilus microplus) can injecting hemoglobin could serve to exacerbate this effect. 1346 BRIEF REVIEWS: IMMUNOMODULATORY EFFECTS OF RBCs

Table I. The dualistic nature of RBCs in innate immune function, and its relevance to human disease states

Innate Promotes Immune Maintains Immune RBC Component Immune Function Response Quiescence Relevant Disease States DARC Binds chemokines Maintains Scavenges and sequesters Critical illness (103) (6, 7, 63) gradient (64) chemokines (7) Binds pathogens Serves as entry point Malaria (83285) (83, 91) for P. vivax (83) Binds HIV-1 (91) HIV (91) TLR9 Binds nucleic ?a Presents mtDNA to immune Scavenges and sequesters Trauma (5) acids (5) or endothelial cells (5) inflammatory nucleic acids (5) Results in RBC crenation (5) GYPA Binds pathogens ?a Facilitates trans- Facilitates pathogen destruction Malaria (86, 87) (42, 86, 87) Serves as attachment site via decoy receptor binding (42) for P. falciparum (86, 87) Heme/hemoglobin Generates ROS Activates TLR4 (62) Prevents the growth of yeast Sepsis (58) (49, 50) Amplifies cytokine production and bacteria (52) infection (46) in the presence of LPS (51, 55) Depending on the conditions of the local and systemic microenvironment, immunomodulatory components of human RBCs may function to either repress or promote inflammatory responses. aThe ? refers to speculated function. Downloaded from

Heme has also been characterized as a potent danger signal in rather than being cleared. In support of this view, Duffy wild type vitro, capable of activating NF-kB–mediated expression of mice contain significantly higher blood chemokine concentrations proinflammatory proteins (including IL-1 and TNF-a), than Duffy knockout mice (64). Darbonne’s aforementioned generation of intracellular ROS, 100-fold increase in tran- experiments, which concluded that chemokine binding is super- scription of proinflammatory genes, and macrophage release ficial and easily reversible, may also hint at the potential for sus- http://www.jimmunol.org/ of TNF-a and IL-1 (60). It has been suggested that free heme tained immune activation, as erythrocyte receptors could alternately is unable to cause damage on its own but rather produces bind and release their substrates in the tissue microenvironment. deleterious effects only in the presence of other immune These two models for erythrocyte regulation of blood che- modulators (61). However, others have demonstrated that mokine levels can be termed the sink hypothesis (i.e., erythrocyte free heme is an independent activator of TLR4 (62), stimu- chemokine binding serves to diminish immune-activating signals) lating secretion of TNF-a, ROS, and B4 from and the reservoir hypothesis (i.e., erythrocyte binding prevents macrophages (59, 62) and release of high mobility group box chemokine clearance, thereby prolonging chemokine half-life in

1 (HMGB1) from nuclei (58). the blood). Although these models appear contradictory at first, by guest on September 27, 2021 they may not be mutually exclusive: Fukuma et al. (64) suggested Human erythrocytes and the that erythrocytes do scavenge chemokines from sites of inflam- Chemokine binding. A number of studies have revealed that RBCs mation but eventually release them in response to decreases in serve a variety of functions beyond oxygen transport (6, 7, 63, 64) (Table I). One important immunomodulatory property of human erythrocytes is their propensity to bind a wide variety of chemokines (Fig. 2). One major for binding is the Duffy Ag receptor for chemokines (DARC), which was first recognized by Darbonne and colleagues (7). This work demonstrated that erythrocytes act as a sink for CXCL8, thereby inactivating the CXCL8-dependent chemokine gradient and preventing recruitment (7). Duffy binds other immunomodulatory proteins at high affinity as well, including additional CXC and CC chemokines besides CXCL8 (6, 63). In vitro experiments by Lee et al. (63) demonstrated that blood lacking erythroid Duffy exhibited higher plasma chemokine levels following LPS exposure, further implicating Duffy in chemokine scavenging. This is an evolutionarily conserved feature of DARC, as Duffy knockout mice exhibit higher FIGURE 2. chemokine (i.e., MIP-2 and KC) concentrations in the An overview of the binding activities of human erythrocytes. Clockwise from top left. Erythrocytes bind chemokines through DARC, thereby vascular space (63). Some have proposed that the sequestration modulating neutrophil recruitment and immune activation. Recent studies have of inflammatory molecules by red cell surface receptors functions shown that erythrocytes are capable of binding and scavenging mtDNA via TLR9. to dampen immune response via attenuation of neutrophil It is not yet known whether erythroid TLR9 can also scavenge other CpG-rich signaling (7). Such an inhibitory system could confer a selective DNA fragments (e.g., bacterial DNA). Lysed erythrocytes can release heme and advantage, as overstimulation of the immune system can generate hemoglobin into the extracellular environment. In the vicinity of invading mi- excessive inflammation and lead to tissue damage. crobes, hemoglobin can bind LPS and generate toxic ROS that function in host defense. However, the release of free heme during states of inflammation can result However, an alternative model for widespread binding of chemo- in excessive immune activation, leading to cellular injury or death. GYPA has been kines by DARC may actually result in the maintenance of blood identified as a potential decoy receptor on the surface of human erythrocytes, chemokine levels, as chemokines would remain in circulation functioning to sequester pathogens and facilitate their removal from circulation. The Journal of Immunology 1347 plasma chemokine concentration, effectively maintaining ho- or endothelial cells. Furthermore, mtDNA binding causes meostasis. crenation of the erythrocyte membrane, which could result in Nucleic acid binding. In addition to regulating chemokines, accelerated clearance of abnormal RBCs or entrapment of the recent research demonstrates that erythrocytes are also capable RBCs in the microvasculature. of binding another class of molecules with the potential Although the recent results are consistent with a model in to mediate inflammatory responses: nucleic acids. Specifically, which RBCs scavenge mitochondrial DAMPs from the lung our recent work has focused on the ability of erythrocytes compartment, the dynamics of mtDNA scavenging may be to bind DNA derived from mitochondria (mtDNA) (5). more nuanced. It is possible that, similarly to findings with Mitochondria comprise a sizable endogenous reservoir of Duffy Ag and the chemokines that it binds, mtDNA binding potentially inflammatory nucleic acids, which are released could act either as a protective or pathologic mechanism, with into circulation during normal cell turnover by activated the precise impact being largely dependent on the conditions of immune cells and during cell death. Similar to the DNA the local microenvironment. of their bacterial ancestors, mtDNA is rich in immunosti- It is not yet known whether RBCs are also capable of mulatory CpG motifs and initiates proinflammatory signaling scavenging other types of nucleic acids. However, recent data through direct binding of CpG-containing DNA regions suggest that Zika RNA may persist on red cells during storage to TLR9. Furthermore, mtDNA is known to activate the (80). The recent discovery of numerous TLRs (including inflammasome (65, 66), and cytosolic mtDNA is a potent TLR3) in the transcriptome of mammalian reticulocytes of- inducer of STING (stimulator of IFN genes), an ER protein fers a potential mechanism by which this binding could occur Downloaded from that senses a cellular second messenger produced in response and intimates that still other TLRs and nucleic acid scavengers to cytosolic dsDNA (67–70) and bacterially derived cyclic may yet be found on the surface of mature human erythro- dinucleotides (67, 71). A growing body of literature implicates cytes (81). Furthermore, RBC binding of viral material may these mitochondrial damage-associated molecular patterns be important for disease pathogenesis and may also have (DAMPs) in the pathogenesis of a variety of diseases, including implications regarding safe blood storage and transfusion autoimmunity, cancer, cardiovascular disease, trauma, and practices. http://www.jimmunol.org/ critical illness (72–76). Pathogen binding. In addition to their affinity for endogenous We recently reported that human erythrocytes express molecules like mtDNA and chemokines, human erythrocytes TLR9, potentially providing human erythrocytes with the are also known to bind pathogens. Undoubtedly the most capacity to scavenge or buffer potentially inflammatory nucleic infamous and evolutionarily consequential erythrocyte path- acids in the circulation (5). Although in nonerythroid cells, ogen belongs to the genus Plasmodium, a group of protists TLR9 is trafficked to endolysosomal compartments and responsible for causing malaria. These pathogens have exerted cleaved by lysosomal cathepsins to initiate innate immune the strongest evolutionary pressures on the in signaling (77–79), it remains unknown whether erythroid recent millennia (82). by guest on September 27, 2021 surface TLR9 undergoes cleavage and signaling after binding Each Plasmodium species has evolved distinct mechanisms mtDNA. We found that TLR9-positive erythrocytes bind and for adhering to human erythrocytes. P. vivax and P. knowlesi sequester circulating cell-free mtDNA, and in vivo mouse attach via the aforementioned DARC (83, 84), rendering the studies demonstrated that loss of this function results in en- presence of Duffy so maladaptive in high-malaria regions that hanced lung injury during states of inflammation (5). Addi- nearly all West Africans and 68% of African Americans exhibit tionally, RBCs scavenged CpG DNA from human microvascular a Duffy-negative phenotype (85). Clearly, the benefits con- endothelial cells in vitro, which may serve to attenuate endo- ferred by Duffy’s ability to scavenge chemokines were dwarfed thelial TLR9 activation (5). by its deleterious effects in the presence of malaria, resulting in Notably, there exists significant heterogeneity in erythroid the spread of the allele conferring Duffy negativity. TLR9 expression both between and within individuals (5). The most virulent of the human malarial parasites, Some individuals express relatively high percentages of ery- P. falciparum, invades erythrocytes after adhering to glyco- throid TLR9, whereas others express virtually none. Even in phorin A (GYPA), B, or C (86, 87). Although individuals those who do express erythroid TLR9, the receptor is only deficient in any of the glycophorin types exhibit increased present in a fraction of the total RBC population. Never- resistance to P. falciparum, GYPA paradoxically remains the theless, because of the vast number of erythrocytes in circu- most common erythrocyte surface protein in humans (12, 42, lation [between 20 and 30 trillion in the average adult (1)], 88). Complete deficiency of erythrocyte GYPA is a viable but even a minority of TLR9-positive cells would have incredible extremely rare phenotype, suggesting that the absence of potential to influence systemic function or modulate organ GYPA must come at a cost (42) or that GYPA’s presence must injury. provide sufficient benefit to override the selective pressure We are just beginning to understand the ways in which exerted by P. falciparum. Otherwise, P. falciparum would erythrocyte binding of nucleic acids influences systemic presumably have driven the widespread disappearance of function. As in the case of RBC chemokine binding, nucleic GYPA in malarial regions. acid scavenging may have discrepant effects on inflammation. GYPA may act as a decoy receptor, chaperoning pathogens In some instances, RBCs may attenuate inflammation in the away from important tissues and into the spleen to facilitate lung compartment by scavenging mtDNA from endothelial their destruction by macrophages (42). In support of this cells; however, given the close mechanical contact between hypothesis, many of the pathogens that bind human glyco- erythrocytes and the microvascular , RBCs phorins (e.g., reovirus, influenza C, Sendai, Mycoplasma binding mtDNA could also exacerbate inflammation by in- pneumoniae, , and Ureaplasma urealyticum)do cidentally presenting the inflammatory molecules to immune not infect erythrocytes themselves (42). This could also help 1348 BRIEF REVIEWS: IMMUNOMODULATORY EFFECTS OF RBCs

FIGURE 3. A systemic model of erythrocyte involvement in critical illness. Under basal conditions, erythrocytes contribute to immune homeostasis by binding and scavenging mtDNA via TLR9. During states of trauma, extensive cell death leads to increased cell-free mtDNA levels. TLR9-positive erythrocytes scavenge these nucleic acids. Erythrocytes binding mtDNA become crenated (i.e., deformed), which could either expedite their clearance from the circulation or trigger the activation of endothelial cells. Unbound mtDNA may engage immune cells, leading to increased inflammation. In sepsis, the circulation is overwhelmed with both Downloaded from bacterial and mtDNA as well as free heme (not shown). It remains unknown whether mtDNA-binding capacity of erythrocytes is reduced during sepsis. explain why GYPA persists on the erythrocyte surface despite crenation may expedite RBC clearance and turnover, potentially selective pressure from P. falciparum. Engineering decoy viral leading to reduced RBC counts and anemia. Finally, crenated receptors on erythrocytes to facilitate adsorption and prevent RBCs may also be involved in inflammation, organ failure, invasion of nucleated cells therefore provides a potentially tissue ischemia, and vascular occlusion, all of which are central http://www.jimmunol.org/ promising approach to clear pathogens from circulation. In- to the sepsis phenotype. deed, such an approach has been shown to attenuate cox- Furthermore, erythrocytes of critically ill trauma patients sackievirus B infection in murine models (89). demonstrate diminished mtDNA scavenging ability (5). This Unfortunately, not all decoy adsorption has desirable effects. may be highly relevant to the disease pathogenesis of severe Notably, human erythrocytes bind HIV-1 virions, and this sepsis, as recent studies have concluded that elevated levels of binding results in the 100-fold enrichment of viral infectivity plasma mtDNA during critical illness predict mortality (99, (90). Additionally, HIV-1 binding to DARC on erythrocytes 100). The loss of scavenging ability could be attributable to maintains virus viability and presents viral particles to CD4- saturation of erythroid TLR9 receptors due to the sheer amount by guest on September 27, 2021 positive T cells, although these findings have been debated in of excess nucleic acids in circulation or to cellular damage in- the literature (91–95). In some cases, erythrocyte binding curred by erythrocytes during inflammatory states. Whether activity may therefore facilitate trans-infections of other defective nucleic acid sequestration by RBCs contributes to the as well. As in the case of chemokine and mtDNA development of lung or other organ injury during sepsis remains binding, local conditions in the bloodstream and the specific unknown. Additionally, future research should aim to deter- interactions between viral ligands and their host receptors may mine whether erythroid TLR9 has the ability to bind bacterial ultimately determine whether erythrocyte pathogen binding CpG DNA in addition to mtDNA, as this could potentially proves advantageous or detrimental to the host. illuminate components of disease pathogenesis. RBCs in the context of modern medicine Cell-free heme is also an important factor in the context of critical illness. In states of infection or inflammation, free heme Critical illness. Given their capacity for interaction with both exogenous and endogenous inflammatory molecules, eryth- can act as a DAMP, contributing to excess inflammation, tissue rocytes likely play a major role in the context of critical illness damage, and even death in susceptible hosts (58, 59). Various (Fig. 3). During sepsis, the circulation is inundated with ex- scavengers of free heme have been identified, and reduced cess cell-free nucleic acids, pathogens, and chemokines, all of levels of these heme scavengers are also associated with in- which have been shown to bind to erythrocytes. Thus, any creased mortality (58). One such heme scavenger, heme loss of erythrocyte function has the potential to dramatically oxygenase-1 (HO-1), catabolizes free heme and is upregulated alter host response and contribute to the pathogenesis of these following LPS exposure (101). Although the carbon monox- conditions. ide produced by HO-1 catabolism of heme can be a source of Research indicates that physical and chemical changes do ROS signaling and oxidative stress, the overall effects of the occur to erythrocytes during periods of sepsis and endotoxemia HO-1/CO system are anti-inflammatory because of its re- (96). Septic shock decreases erythrocyte 2,3-bisphosphogly- duction of TNF-a, IL-8, and MIP-1b levels and upregulation cerate levels, renders them less deformable, and reduces their of IL-12 (101). HO-1 has been shown to be protective in ability to traverse microvessels (96–98). individuals with severe sepsis, suggesting that elevated levels of Crenation of the RBC membrane is another potentially circulating free heme are central to the pathogenesis of the important component of sepsis pathology. This cellular de- condition (58). Additionally, low levels of the heme seques- formation appears to be a consequence of mtDNA binding (5), tering protein hemopexin (HPX) are associated with increased and elevated levels of cell-free mtDNA during sepsis likely mortality during sepsis, and administration of HPX during increase the incidence of RBC crenation. We speculate that septic shock reduced probability of mortality (58). The Journal of Immunology 1349

Critically ill patients are prone to developing acute respiratory metabolism may contribute to alterations in immune function. distress syndrome, a life-threatening syndrome characterized by In addition, further investigation into the dualistic nature of disruption of the alveolar endothelial–epithelial barrier resulting erythrocyte immune modulation is required. Research thus far in leakage of proteinaceous fluids into the airspaces, loss of lung suggests that the utility of RBC binding activities is context compliance, hypoxia, and respiratory failure (102). In pre- dependent, sometimes exacerbating the immune response and clinical models of lung injury, loss of erythrocyte DARC in- sometimes maintaining immune quiescence, but the precise creased lung inflammation and airspace neutrophil recruitment mechanisms and environmental factors moderating this (103, 104). Consistent with these findings, Kangelaris et al. dualism remain enigmatic. Finally, it remains unresolved (105) reported that African American patients possessing a gene whether RBC alterations during disease states are the product polymorphism that abrogates erythroid DARC expression was or the cause of disease pathogenesis. associated with a 17% increased risk of mortality from acute Further research into the immune functions of erythrocytes respiratory distress syndrome compared with patients of Eu- will be crucial to understanding not only diseases that affect ropean ancestry. African American patients with erythroid RBCs directly (e.g., malaria and sickle cell disease) but also DARC, however, fared similarly to European American pa- chronic and acute inflammatory states where regulation of tients (105). Thus, it is possible that reduced erythrocyte DAMPs and other mediators is essential for disease control chemokine scavenging function during critical illness may (e.g., sepsis, trauma, and autoimmunity). Given the immune compound inflammation in susceptible hosts. functions of erythrocytes in other vertebrate orders, compar-

Malaria. In the context of malaria, free heme is generated ative evolutionary approaches may yield insights into undis- Downloaded from following parasite of hemoglobin. To protect them- covered functions of erythrocytes in humans. In of recent selves from the cytotoxic effects of heme, P. falciparum parasites findings demonstrating that Zika RNA persists on stored RBCs convert the compound into an insoluble crystal known as (80) and the presence of TLR on erythrocytes (5), we stress hemozoin. Hemozoin itself is immunologically inert; however, the importance and urgency of continued research into the it becomes coated with malarial DNA and subsequently pre- immunologic functions of human RBCs. sents the DNA to host TLR9, thereby magnifying the innate http://www.jimmunol.org/ immune response (106). Whether erythroid TLR9 is important Acknowledgments in the pathogenesis of malaria remains to be investigated. We thank Dr. Caitlin Clancy for work designing Figs. 1–3 as well as Appearance of nucleated erythrocytes in disease states. Finally, it is Dr. Stefano Rivella for constructive feedback during the writing process. worth noting that there have been numerous documented cases of nucleated RBCs (NRBCs) circulating in adult humans with Disclosures severe diseases (32, 33). This is a highly abnormal phenotype, The authors have no financial conflicts of interest. as NRBCs are typically only observed in the peripheral bloodstream of fetuses and neonates (32), yet it is observed in References by guest on September 27, 2021 10 to 30% of critically ill patients (34). Patient prognosis is 1. Bianconi, E., A. Piovesan, F. Facchin, A. Beraudi, R. Casadei, F. Frabetti, poor in these cases, with over 50% of individuals dying in the L. Vitale, M. C. Pelleri, S. Tassani, F. Piva, et al. 2013. An estimation of the number of cells in the human body. Ann. Hum. Biol. 40: 463–471. hospital. 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