The ultimate tradeoff: how red cell adaptations to malaria alter the host response during critical illness

Abstract

The human immune system evolved in response to pathogens. Among these pathogens, malaria has proven to be one of the deadliest and has exerted the most potent selective pressures on its target cell, the red blood cell. Red blood cells have recently gained recognition for their immunomodulatory properties, yet how red cell adaptations contribute to the host response during critical illness remains understudied. This review will discuss how adaptations that may have been advantageous for host survival might influence immune responses in modern critical illness. We will highlight the current evidence for divergent host resilience arising from the adaptations to malaria and summarize how understanding evolutionary red cell adaptations to malaria may provide insight into the heterogeneity of the host response to critical illness, perhaps driving future precision medicine approaches to syndromes affecting the critically ill such as sepsis and acute respiratory distress syndrome (ARDS).

INTRODUCTION

Malaria has threatened human existence for millennia. It is uniquely positioned to be a driver of natural selection, given its propensity to infect children and cause fatality before reproductive age. In part, survival depends on red blood cell (RBC) mutations that confer innate resistance to the parasite (1–3). J.B.S. Haldane first noted in the 1940s that the geographical distribution of sickle cell disease, the most well-studied red blood cell disorder of the time, aligned with the tropical regions where malaria was endemic. He posited his since-termed “Malaria Hypothesis” that this overlap was no coincidence, but rather selection in those regions for heterozygotes who are more resistant to infection with the Plasmodium species that cause malaria (4). A.C. Allison lent credence to this hypothesis less than a decade later in a series of experiments that proved that the sickle cell polymorphism was balanced such that “the heterozygote has an advantage over either homozygote” (5). Since that time, malaria has become one of the most extensively studied and most elegant examples of an infectious force as the agent of natural selection.

Malarial infection originates when female anopheles mosquitoes bite and inject plasmodium parasites (sporozoites) into the bloodstream. These sporozoites travel first to the liver where they undergo asexual multiplication, before emerging as merozoites, which invade and multiply in erythrocytes. Infected erythrocytes then burst, allowing the parasite to invade other neighboring erythrocytes (6). It comes as no surprise that the host target of malarial selection pressures is the same cell at which parasite sequestration and replication occurs—the red blood cell. Decades of work have been dedicated to investigating the link between malaria and known alterations in gas-exchanging properties of the RBC. Emerging evidence suggests further adaptations to virtually every component of the RBC, from membrane proteins to oxidative and glycolytic enzymes to the newly discovered expression of immune receptors on RBCs (Fig. 1; Table 1).

Figure 1.

Schematic overview of known and potential red blood cell adaptations to malaria. Known malaria adaptions include Duffy antigen receptor for chemokines (DARC), which modulates chemokine availability and neutrophil recruitment; hemoglobinopathies (sickle cell trait/disease and thalassemia); and enzymopathies (G6PD and pyruvate kinase deficiency), which can lead to hemolysis and subsequent free heme release, promoting inflammation and increase susceptibility to infection. Probable malarial adaptations include: ABO glycoproteins, which determine major ABO blood group, Toll-like receptor 9 (TLR9), which binds nucleic acids and mediates clearance of RBCs during inflammation, and miRNAs, which are abundant in RBCs but the functions of many remain undefined. G6PD, glucose-6-phosphate dehydrogenase; RBCs, red blood cells. [Image created with BioRender.com and published with permission.]

Table 1.

Non-gas exchanging functions of RBCs and hypothesized role in critical illness

Hypothesized or Proven Adaptation	RBC Feature	Function	Adaptation to Malaria	Consequence to Critical Illness
Proven	Heme	Generates reactive oxygen species: activates TLR4 Amplifies cytokine production in the presence of LPS (7)	Hemoglobinopathies/thalassemia (8)	Increased susceptibility to nonmalarial infection (9, 10), anemia/transfusion dependence (11)
	Duffy Antigen Receptor for Chemokines	Binds chemokines: maintains chemokine gradient (12)	Downregulation: decreased parasite entry (13, 14)	Increased susceptibility to ARDS (15, 16)
		Binds pathogens: entry point for P. vivax (13)
	Glucose-6-Phosphotase	Involved in pathway to consume reactive oxygen species (17)	Deficiency: increased intracellular oxidative stress decreases parasite survival (18)	COVID-19 severity (19)
	Pyruvate kinase	Catalyzes rate-limiting step in glycolysis to produce intracellular energy in nonmitochondrial cells (mature RBCs) (20)	Deficiency: decreased invasion by P. falciparum and increased phagocytosis of ring- and mature-stage-infected RBCs (20, 21)	Hemolytic anemia/transfusion dependency, functional asplenia (20)
Hypothesized	Glycophorins	Binds pathogens: entry point for P. falciparum (22)	Dantu variant: structural variant in chromosome 4 region, which increases RBC surface tension and decreases merozoite invasion (23)	Potential predisposition to lysis/thrombosis
	Complement Receptor 1	Binds P. falciparum-induced ligand (24, 25)	Deficiency: increased cerebral malaria (26)	Increased RBC-bound complement fragments in COVID-19 and non-COVID-19 sepsis (27)
		Proinflammatory regulator abundant on RBCs (27)	Deficiency: reduced rosetting conferring protection (25)
	Toll-like Receptor 9	Binds nucleic acids (28, 29)	Deficiency/gene polymorphisms	Decreased capacity to bind mtDNA to ameliorate lung injury (28)
		Involved in P. yoeli infection (30, 31)
	ABO Glycoproteins	Surface proteins on RBCs responsible for determining ABO blood group (32)	Blood group O demonstrates different propensity to bind to Duffy binding-like regions of infected erythrocytes (33–36)	Blood group A may confer increased risk for ARDS in critically ill patients and associated with increased risk of AKI (37)

AKI, acute kidney injury; ARDS, acute respiratory distress syndrome; COVID-19, coronavirus disease 2019; LPS, lipopolysaccharide; RBCs, red blood cells.

Critical illnesses such as sepsis and acute respiratory distress syndrome (ARDS) portend high morbidity and mortality with a multitude of mechanisms that collectively lead to profound inflammation, tissue injury, and ultimately organ failure. More and more, as we unveil these distinctive mechanisms, we start to recognize the diverse outcomes that stem from patient heterogeneity. For years, RBC dysfunction was perceived to accompany critical illness as a byproduct of excessive inflammation that resulted in RBC membrane distortion, oxidative disequilibrium, and insufficient oxygen delivery (38). However, recent studies have demonstrated that RBCs play an active role in modulating diverse immune functions critical for host defense, including chemokine regulation and nucleic acid scavenging. This long-overlooked erythrocyte contribution to host defense implores further understanding of its role in critical illness, namely, not as mere vessels for gas exchange but as active participants in immunoregulation. The coronavirus disease 2019 (COVID-19) pandemic has highlighted the remarkable heterogeneity of the host response to a specific pathogen. Given the importance of the host response to infection in mediating outcomes and treatment response, it is imperative that a comprehensive understanding of all potential modulators of the innate immune response is obtained. Here, we will review the emerging evidence for alterations in RBC immune function as a byproduct of the evolutionary response to malaria and will highlight some of the major advances over the past century in understanding RBC immunoregulatory biology. Equipped with this knowledge, we will be able to better understand how RBC immune function contributes to heterogeneous host responses in the face of pathogenic or sterile insults that underlie critical illnesses such as sepsis and ARDS.

KNOWN MALARIAL ADAPTATIONS AND THEIR RELEVANCE TO CRITICAL ILLNESS

The Erythrocyte Chemokine Receptor ACKR1—Duffy Antigen Receptor for Chemokines

Perhaps best known as the receptor by which Plasmodium vivax species gain entry into red blood cells, the Duffy Antigen Receptor for Chemokines (DARC) was initially reported as a chemokine binding protein on erythrocytes. Though unnamed and unidentified at that time, it was first described in a 1950 case report of a hemophilic patient who suffered a hemolytic transfusion reaction due to an antibody to an unknown substance on red blood cells (RBCs) (39). Subsequently, RBCs were demonstrated to carry a surface protein that binds the chemokine interleukin- 8 (IL-8), diminishing the concentration of soluble IL-8 in blood plasma samples and thereby dampening immune signaling (12). In 1993, Horuk et al. (13) united these two independent findings and showed that the Duffy antigen receptor, used by Plasmodium vivax for erythrocyte cell entry, was the same RBC receptor that bound IL-8. Since that time, DARC has been found to bind multiple inflammatory chemokines with high affinity (40, 41).

DARC belongs to a family of chemokine receptors that lack a G-coupled protein to mediate intracellular signaling, rendering the chemokine inaccessible to circulating immune cells (40–42). As such, DARC plays a significant immunoregulatory role by sequestering chemokines from immune cells such as neutrophils and acts as a reservoir for future release (43–46). In preclinical studies using lipopolysaccharide (LPS)-induced lung injury, erythrocyte DARC was found to dampen the local chemokine effects by soaking up chemokines in the lung microvasculature for release in the systemic circulation, preventing polymorphonuclear leukocyte infiltration into the alveolar space to cause lung injury (45).

It is thus of no surprise that RBC-derived cytokines play a role in transfusion-related lung injury. Lysis of transfused RBCs that contain chemokines can lead to profound lung inflammation and result in antibody negative-transfusion-related acute lung injury (TRALI) (47). Interestingly, prolonged RBC storage can result in decreased DARC expression on erythrocytes, thereby diminishing its chemokine-binding capacity (15). As such, these DARC-low erythrocytes are less capable of dampening local tissue inflammation following LPS-induced lung injury.

Consistent with the theory of malaria as a potent driver of human evolution, the DARC-null allele is present in as high as 90%–95% of sub-Saharan Africans in regions with the highest threat of P. vivax (14). In the United States, up to 68% of African Americans have the DARC-null allele (14). One observational study showed that patients with the Duffy-null phenotype with acute lung injury were at increased risk of mortality at 60 days, had increased number of days on mechanical ventilation, and had increased organ failure compared with the Duffy-positive counterparts (16). Of note, Duffy-positive African Americans had similar clinical outcomes compared with those of European descent. Though larger studies have yet to validate these findings, and plasma cytokines have yet to confirm the mechanism, these observations complement preclinical data denoting erythrocyte DARC as an essential player that tempers local tissue inflammation. Thus, the same adaptation that provides survival benefits to the threat of malaria infection may confer a higher risk of mortality when facing critical illness in an ICU setting, merely by altering the RBC’s ability to sequester and transport inflammatory chemokines.

Hemoglobinopathies

Hemoglobin has evolved to adapt to malarial pressures, rendering erythrocytes less hospitable to these invading parasites. However, under acute stress, hemoglobinopathies lead to hemolysis and subsequent release of chemokines and free heme into the circulation, promoting inflammation.

HbSS.

Of the hemoglobin adaptations to malaria-inducing pathogens, Hemoglobin SS (HbSS) is the most well-studied. HbSS arises from a single point mutation in the β-globin chain of the hemoglobin molecule. The resultant amino acid substitution of this mutation leads to polymerization of globin chains, which causes numerous and devastating sequelae of sickle cell disease (48). There have been countless studies confirming that the persistence of this hemoglobin mutation is due to the protective advantage against P. falciparum conferred in heterozygotes of the mutation (49).

The sequelae of sickle cell disease are known to impact host immunity indirectly via numerous vaso-occlusive mechanisms and splenectomy. Free heme released from hemolysis during vaso-occlusive crisis is extremely proinflammatory and can lead to endothelial damage (50). Sickled erythrocytes have also been shown to retain mitochondria, which can provide immunologically active mitochondria DNA (mtDNA) that ultimately leads to endothelial damage. Whether mitochondria retention is an advantageous adaption to malaria or a byproduct of abnormal erythroid development remains to be seen (51, 52). Nevertheless, mtDNA is elevated in patients with sickle cell disease and further increases during sickle cell crisis. Notably, mtDNA is also elevated in ARDS and is associated with increased mortality (53). It has long been appreciated that patients with sickle cell disease are at higher risk of severe disease and hospitalization from bacterial and viral infections and confer a higher risk for ICU admissions. Whether the level of mtDNA in patients with sickle cell disease correlates with a higher risk of acute chest syndrome or predisposition to severe ARDS has yet to be determined.

Despite long-held assumptions that the sickle cell trait is a silent protector against malaria, emerging evidence suggests that heterozygotes may have altered susceptibility to critical illness. Sickle cell trait is usually asymptomatic but in rare instances, severe dehydration or hypoxia can lead to erythrocyte sickling, vaso-occlusive crisis, and exertional rhabdomyolysis. Notably, a recent study found increased risk of mortality and acute kidney injury after SARS-CoV-2 infection in patients of African ancestry with sickle cell trait (54). Dedicated investigation into the impact of sickle cell trait on red blood cell immune function would help to better understand the potential consequences of this malarial adaptation on the host’s inflammatory responses.

Thalassemia.

Thalassemias are a heterogeneous group of red blood cell disorders in which the globin-chain units making up normal adult hemoglobin (α2β2) are reduced or absent. As a result, carriers of these mutations show microcytic anemia to varying degrees, and at least in the neonatal period, compensate with elevated levels of circulating fetal hemoglobin (γ4) (8). The prevalence of α- and β-thalassemia have similarly been shown to coincide with malaria-endemic regions, leading to hypotheses that these mutations also confer selective advantage against deadly malaria infections (55).

One proposed mechanism for that protection against malaria is increased susceptibility to P. vivax and P. falciparum during the neonatal period, likely due to increased erythrocytosis, acting as a natural vaccine and providing more enhanced protection in later life (56). Others have shown that parasitized α-thalassemic erythrocytes express higher levels of novel surface antigens (which are currently ill-defined) than wild-type parasitized erythrocytes, with a corresponding increase in antiparasitic antibody binding in vitro (57, 58). This enhanced detection and subsequent enhanced clearance of parasitized RBCs may explain how thalassemia protects against malaria.

Despite protection against malaria, thalassemia carriers are at increased infection risks due to several qualitative and functional defects in the immune system rendering them susceptible to pathogens such as Klebsiella pneumoniae and Yersinia enterocolitica infections (9). These defects range from defective T and B lymphocyte functions to altered chemotactic and phagocytotic processes to complement system deficiencies (9). Indirect consequences of chronic anemia also led to mortality and morbidity from increased exposure to transfusion-transmitted pathogens like Hepatitis C, chronic transfusion-related iron overload, and therapeutic splenectomies (11). Given this increased infection burden and mortality, this genetic adaptation to malaria has resulted in a less fit host if they live in regions with unlikely threat of malaria.

Enzymopathies

G6PD deficiency.

Another well-studied RBC adaptation to malarial infection is glucose-6-phosphate dehydrogenase (G6PD) deficiency. G6PD is an enzyme in the pathway that consumes oxygen-free radicals within RBCs, thereby minimizing oxidative stress. This X-linked, inherited RBC disorder has increased prevalence in regions with the highest malaria burden, and the prevalent form of G6PD deficiency in Africa has been demonstrated to correlate with a 50% reduced risk of severe malaria (17). The hypothesized mechanism of protection is that the increase in oxidative stress seen in G6PD-deficient RBCs makes them a less hospitable host for malaria-causing parasites.

Although this adaptation offers protection from the potentially lethal pathogen, it comes at a cost. The oxidative stress leads to early RBC breakdown and, in situations of stress or exposure, widespread hemolysis, leading to free heme and chemokine release. Those with G6PD deficiency have also been shown to fare worse in the face of critical illness during COVID-19 infection. A retrospective chart review of hospitalized patients with COVID-19 pneumonia requiring supplemental oxygen found that the cohort with G6PD deficiency demonstrated prolonged hypoxia and a longer need for mechanical ventilation (19). Outside of COVD-19, no study specifically addressing whether G6PD deficiency confers a higher risk for ARDS mortality has been done.

Pyruvate kinase deficiency.

Pyruvate kinase (PK) deficiency has similarly been linked to resistance against P. falciparum. Pyruvate kinase catalyzes the rate-limiting step of glycolysis, a process that is crucial to energy production in nonmitochondrial cells like mature erythrocytes. In a study comparing in vitro P. falciparum inoculation of RBCs obtained from homozygous and heterozygous pyruvate kinase-deficient adults and controls, PK-deficient RBCs showed decreased invasion by P. falciparum (20). Furthermore, phagocytosis of ring-stage and mature-stage-infected erythrocytes was increased in samples from patients deficient in PK, indicating enhanced clearance of parasitized cells (20). Additional genotyping studies have indicated a global geographical codistribution between malaria and high frequency of PK deficiency (21).

Much like G6PD deficiency, this adaptation results in wide-ranging clinical consequences from mild hemolytic anemia to complete transfusion dependency. Though high-powered studies have yet to be performed to assess the susceptibility of individuals with PK deficiency to other pathogens and critical illnesses, the propensity toward anemia and the increased risk of functional asplenia are likely deleterious consequences accompanying the protection against P. falciparum.

PROBABLE ADAPTATIONS TO MALARIA

Beyond the RBC mutations that have been proven to provide malaria resistance to their host, there are numerous erythrocyte mutations that follow similar geographical patterns indicating similar evolutionary pressures and have shown some early associations with host resistance to infection.

ABO Blood Type

The ABO histo-blood group refers to a family of glycosyl-transferases encoded by the ABO gene that catalyze specific carbohydrate modifications on glycans and glycoproteins (32). These glycans are present on the surface of RBCs, platelets, endothelium, and epithelium. DNA sequencing has placed the development of the group O allele before human migration out of Africa, and it has been hypothesized that P. falciparum, which was active in Africa before the early migration of humans and was lethal before or during the human reproductive years, was the infectious disease driving the emergence the O allele component of ABO groups (59). This hypothesis is supported by the worldwide distribution of A and O blood groups, the ratio of which correlates with the global distribution of malaria (59).

Several studies have supported the hypothesis that group O is associated with improved outcomes of severe P. falciparum infection in both young adults and the fetuses of pregnant women with placental infections compared with group A (33–36). One study suggested that H antigen may be important for P. falciparum invasion, however, no other studies have addressed this hypothesis (60). The prevailing hypothesis is that the infected RBCs expressing the malarial encoded protein Pfemp contain Duffy binding-like regions that vary in cytoadhesion based on blood group, with group O being the least cytoadhesive and blood group A being the most cytoadhesive and demonstrating increased rosetting (59, 61, 62).

There is a growing body of work investigating ABO blood groups’ role following trauma and in critical illness (63). A series of studies demonstrated that the A group, and specifically the A1 allele, confers increased risk for ARDS in a cohort of critically ill patients (64). In a genome-wide association study of patients with COVID-19, blood group A was associated with a high risk of acquiring COVID-19, though this did not translate to higher rates of intubations in the severely ill patients with COVID-19 in a separate study (65, 66). The A group is also associated with acute kidney injury (AKI) risk in critically ill patients with trauma or severe sepsis (37). Recent evidence suggests that ABO expression on endothelium drives this association, which raises the important question of whether evolutionary adaptations to proteins on RBCs have bystander impacts on nonerythroid cells on which they are also expressed (64). Further work will be important in determining whether other RBC adaptations indirectly affect nonerythroid cells.

CR1

Complement receptor 1 (CR1), a regulatory component of one of the oldest arms of the innate immune system, the complement system, is more abundantly expressed in erythrocytes than anywhere else in the body (27).

The hypothesis that CR1 deficiency may be the result of selection pressures by malaria-causing species comes from cumulative research showing CR1 to be the host receptor for the P. falciparum-induced ligand, PfEMP1 (24, 26, 61). CR1 further participates in malaria pathogenicity by aiding in rosetting, the process by which parasitized erythrocytes grab on to neighboring erythrocytes to spread infection (25). Clinical studies of host susceptibility to malaria show conflicting evidence as to whether CR1 deficiency is beneficial or detrimental, likely attributable to the fact that there are several polymorphisms that alter receptor expression in different ways. One study showed that a CR1 polymorphism resulted in CR1 deficiency, which conferred protection against severe malaria by reducing rosetting of P. falciparum-infected cells (25). Other studies, however, showed that CR1 deficiency polymorphisms are associated with an increase in cerebral malaria (26). Supporting these findings another study demonstrated polymorphisms associated with higher erythrocyte CR1 were associated with protection from severe cerebral malaria (67). Given CR1’s role in dampening complement activation, one can speculate that perhaps the reduction in parasitic load is counterbalanced with increased inflammation leading to blood-brain barrier breakdown and parasitic access to the CNS.

A recent study suggested that increased function of the alternative complement pathway is associated with improved survival during critical illness (68). We have demonstrated that RBC-bound complement activation products are elevated in both COVID-19 and non-COVID sepsis (69). Yet, loss of CR1 density on erythrocytes in ICU patients with COVID-19 correlated with disease severity (27). Whether alterations in erythrocyte-specific CR1 expression drive sepsis or host responses in critical illness remains unknown. Further studies will be necessary to elucidate the role of red cell complement regulation in critical illness.

Toll-Like Receptor 9

Recognition of nucleic acids is a vital component of the innate immune system, which occurs through cytosolic and endosomal Toll-like receptors. Toll-like receptor 9 (TLR9) has been shown to mediate inflammatory responses to malarial DNA (70). TLR9 gene polymorphisms are associated with symptomatic malaria in Ghanaian children, and TLR9 has been shown to play an important role in the development of protective immunity to P. yoelli, especially in the regulation of pro- and anti-inflammatory cytokines (30, 71). TLR9 deficiency has also been shown to confer partial resistance to lethal infection from P. yoelli, likely due to malaria parasites requiring TLR9 signaling for immune evasion via regulatory T cells (31).

We have identified TLR9 presence on RBCs where it plays a role in scavenging cell-free DNA from the circulation during quiescent states (28). During inflammatory states, however, excess DNA binding (including mtDNA as well as CpG-DNA from pathogens) leads to RBC-TLR9-driven anemia and innate immune activation (29). RBCs loaded with TLR9-bound DNA effectively concentrate and deliver the CpG-containing DNA to tissue macrophages, which in turn phagocytose the RBCs, contributing to sepsis-induced anemia.

In a CpG-induced lung injury model in mice, RBC-derived TLR9 binds CpG-DNA to ameliorate lung injury (28). Whether red cell TLR9 alters the host response in human lung injury remains to be seen, but if found, could indicate another mechanism through which RBC adaptation to malaria contributes to its immunomodulatory role to impact host response to critical illness.

miRNAs

RBCs contain abundant micro RNAs (miRNAs), which are noncoding RNAs that act as critical mediators of posttranscriptional regulation to enhance or suppress protein translation of its target. Despite the known abundance of miRNAs in RBCs, the specific roles of these miRNAs remain to be determined. Some have posited that erythrocytes may serve as repositories of miRNAs in the circulatory system, serving not only as markers of disease but as mediators of inflammation (72).

Studies have shown that miRNAs are important for host defense against intraerythrocytic pathogens like plasmodium species (73). At least two human miRNAs (miRNA-451 and miRNA-233) are more abundant in cells from individuals with sickle cell disease and sickle cell trait than normal red blood cells (74). The implication of this abundance in protection against malaria species was proven in in vitro experiments showing that miRNA-451 can be incorporated into P. falciparum mRNAs where it inhibits mRNA translation and parasite growth (74). When normal erythrocytes are manipulated to overexpress miRNA-451 and miRNA-223, these erythrocytes show reduced parasite growth. Conversely, blocking the expression of these miRNAs in erythrocytes from those with sickle cell disease and sickle cell trait led to diminished malaria resistance (74).

The numerous mechanisms by which miRNAs play a role in regulating immune cell function remain broad and under investigation. Both intracellular and extracellular miRNAs have been shown to mediate immune responses. Studies have found that tissue ischemia, like that seen in sepsis, causes release of cellular RNA, including miRNA, into the extracellular space. Once released, it may mediate local and systemic inflammatory responses via activation of pattern recognition receptors (75, 76). Alteration of circulating miRNA levels has even been linked to ARDS prognosis, with miRNA-126 showing the potential to predict 28-day mortality (77). miRNA-223, which is linked to increased resistance to P. falciparum as discussed earlier, also demonstrates a role in ameliorating acute lung injury (ALI) by decreasing IL-1b secretion and neutrophil recruitment (78, 79).

Glycophorin

Glycophorins (GYPs) are another surface protein on human erythrocytes, which exhibit diverse genetic variation. Human GYPs are determinants of the major MNS blood group and Gerbich blood group systems, some of which play a role as RBC receptors for P. falciparum invasion and other pathogens (22). GYPA, in particular, interacts with P. falciparum protein erythrocyte binding ligand 1 (EBL-1). Recent investigation has shown a large structural variant in the chromosome 4 region which gives rise to the Dantu glycophorin variant and is found predominantly in East African populations and has been associated with a 40% reduction in risk for severe malaria (23). This Dantu variant is believed to confer protection by increasing RBC surface tension to a high enough level that merozoite invasion rarely occurs (80).

The impact, if any, on critical illness survival for those with Dantu homozygosity remains unclear. However, it is tempting to speculate that mutations that alter membrane surface tension may predispose to RBC lysis or thrombosis thereby driving inflammatory dysregulation, especially in light of RBCs’ role in chemokine trafficking.

DISCUSSION

The need to better understand the role of RBC adaptions in host immunity is driven by the hypothesis that different populations may respond differently to modern protocols used in treating critical illness. Numerous studies have begun to address social determinants of health and other host factors that may contribute to the host response during critical illness, whereas factors such as RBC-mediated immunoregulation have been overlooked. As reviewed in this paper, we are only beginning to uncover the role RBCs play in mediating immune function. Beyond its gas exchange functions, this ubiquitous cell traverses the distance of the human body to transport chemokines, miRNA, cell-free DNA, and effectively alter, aid, or dampen the immune response. Selective pressures exerted over the millennia in malaria-endemic regions altered the makeup of erythrocytes in select individuals, impacting not only the cargo-carrying capacity but also the type of cargo it transports. Recognizing this previously overlooked compartment in critical illness (along with the selective pressures that alter the genetic makeup) adds another layer of complexity to the already heterogeneous population that makes up patients with ARDS and sepsis. Yet understanding it better may lend insight into why some protocolized therapies failed.

Take, for example, the multiple studies demonstrating that populations in malaria-endemic regions have not responded to protocols developed in cohorts from nonmalaria regions. Perhaps the most drastic example of this is the the Fluid Expansion as Supportive Therapy (FEAST) trial, in which fluid resuscitation strategies that have been widely endorsed by western-society pediatric life-support training programs were applied to children with severe febrile illness and impaired perfusion in Uganda, Kenya, and Tanzania, all regions in which centuries of endemic malaria has exerted its pressures (81). In contrast to original research performed on patients in nonendemic regions, the study found that excess mortality with fluid resuscitation was consistent across all groups, irrespective of physiological derangement or underlying microbial pathogen. A similar study in an adult population in Zambia in 2017 showed increased in-hospital mortality with early resuscitation with intravenous fluids and vasopressors compared with usual care (82). In the United States, a subgroup reanalysis of the Fluids and Catheters Treatment Trial (FACTT) trial, a study that initially concluded that a restrictive fluid resuscitation strategy, was superior to a liberal one in intubated patients with ARDS. Jolley et al. (83) found that although there was a decreased 1-yr mortality in black-identifying participants, the conservative fluid strategy actually led to a higher 1-yr mortality for white-identifying participants.

Despite limitations, this important observation that there is a heterogeneous treatment response calls for ongoing discussions regarding the roles of protocolized versus precision medicine. As Dellinger et al. (84) noted, although protocols are often borne out of necessity for quick action in resource- or time-limited settings, and often improve adherence to best practice standards, precision-based methods will likely be a critical component of future sepsis treatment.

The recent work that has been done in defining subgroups of patients with different host immune responses in sepsis lends credence to the benefits of precision medicine that could enhance well-designed protocols. These distinct phenotypes, derived from transcriptomic analysis of leukocytes in peripheral blood, have revealed a relatively immunosuppressed versus immunocompetent phenotype with differential response to steroid administration—the relatively immunocompetent group showing increased mortality with hydrocortisone use (85–87). Similarly, patients with acute respiratory distress syndrome (ARDS) have been stratified into two subgroups defined by levels of inflammatory biomarkers. Phenotype 1 (the hypoinflammatory subgroup) has lower mortality, more ventilator-free, and more organ failure-free days compared with the hyperinflammatory Phenotype 2 (88, 89). Much like the transcriptionally defined sepsis subgroups described earlier, the importance of stratifying patients in clinical trials by these phenotypes has been demonstrated by the use of steroids: Sinha et al. (90) recently demonstrated that steroids decreased mortality in Phenotype 2 patients with ARDS secondary to COVID-19, but not in Phenotype 1 patients. Emerging evidence demonstrating RBCs’ immunomodulatory roles coupled with millennia of selective evolutionary pressures on RBCs call for a reexamination of RBC heterogeneity as a variable that can modulate the host response to inflammation and infection. For example, one critical question would be how would subgroup analysis of the above studies segregate by RBC adaptations?

The current pandemic provides us with a unique opportunity to a single, ubiquitous pathogen through which we can gain insight into remarkably divergent host responses, including those involving RBC heterogeneity. Several of the RBC adaptations discussed in this review have shown significant differences in host response to coronavirus infection. A genome-wide association analysis of almost 2,000 patients with severe COVID-19 (defined by respiratory failure) found potential involvement of the ABO system in the severity of disease, with blood group A conferring higher risk (66). A large, multicenter retrospective chart review conducted from January 2020 to September 2020 of patients with COVID-19 found that, compared with Black-identifying individuals without sickle cell trait or sickle cell disease, Black-identifying individuals with sickle cell disease were at higher risk for hospitalization and the development of pneumonia, even when controlling for other preexisting conditions (91). RBCs obtained during the acute phase of infection from patients with moderate to severe COVID-19 pneumonia (defined as requiring supplemental oxygen support but not needing intensive care) were shown to induce endothelial injury via RBC-derived reactive oxygen species (ROS) and cytokines (92). In addition, a small but significant study found that, among hospitalized patients with COVID pneumonia, the group with G6PD deficiency was significantly more likely to have a worse P:F ratio, spend a longer period of time on mechanical ventilation, and have decreased hematocrit throughout their hospitalization (19). In a small retrospective study, the DARC polymorphism was suggested to be associated with COVID-19 severity infection though the study was underpowered to make definitive conclusions (93).

Despite the abovementioned studies, the role of RBC heterogeneity remains understudied in critical illness. The collection and biobanking of RBC samples combined with further translational studies will be necessary to elucidate the immunomodulatory functions of RBCs during critical illness. Given the accumulating evidence for RBC immune function and the recognition that host immune heterogeneity underlies differential severity to numerous pathogens, it is crucial that further investigations promoting precision medicine include large-scale exploration of RBC heterogeneity. By examining RBC heterogeneity through the most powerful evolutionary pressure exerted on them—malaria—we may continue to uncover previously unknown complexities of RBC immune function, and therefore host immune diversity. Moreover, given that these adaptations have been shaped by pressures that are unequal across geography, ancestry, and environment, we must account for genetic ancestry and environment as potential interacting or confounding factors in addition to the socioeconomic disparities already under investigation. Collectively, investigation of adaptations to malaria may provide a framework through which to better understand this missing piece of host immune diversity, in the current pandemic and beyond.

GRANTS

This work was supported in part by the NIH Grant R21 AI166813 and by the Penn Center for Precision Medicine at the University of Pennsylvania (to N.S.M.).

DISCLOSURES

No conflicts of interest, financial or otherwise, are declared by the authors.

AUTHOR CONTRIBUTIONS

J.D., L.W., and N.S.M. conceived and designed research; J.D., L.W., and N.S.M. prepared figures; J.D., L.W., and N.S.M. drafted manuscript; J.D., L.W., and N.S.M. edited and revised manuscript; J.D., L.W., and N.S.M. approved final version of manuscript.