Am J Physiol Lung Cell Mol Physiol 324: L169–L178, 2023.

First published January 3, 2023; doi:10.1152/ajplung.00127.2022

PERSPECTIVES

The ultimate tradeoff: how red cell adaptations to malaria alter the host
response during critical illness
Jane Dobkin,1
Ling Wu,2,3
and Nilam S. Mangalmurti2,3
1
Perelman School of Medicine, University of Pennsylvania, Philadelphia, Pennsylvania; 2Division of Pulmonary, Allergy and
Critical Care, Perelman School of Medicine, University of Pennsylvania, Philadelphia, Pennsylvania; and 3Lung Biology
Institute, Perelman School of Medicine, University of Pennsylvania, Philadelphia, Pennsylvania

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 re-
silience 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).

ARDS; COVID-19; malaria; red blood cell; sepsis

INTRODUCTION in erythrocytes. Infected erythrocytes then burst, allowing

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 geo-
graphical 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 heterozy-
gote 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 (sporo-
zoites) into the bloodstream. These sporozoites travel first
to the liver where they undergo asexual multiplication,
before emerging as merozoites, which invade and multiply
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 sug-
gests further adaptations to virtually every component of
the RBC, from membrane proteins to oxidative and glyco-
lytic enzymes to the newly discovered expression of
immune receptors on RBCs (Fig. 1; Table 1).
Critical illnesses such as sepsis and acute respiratory dis-
tress syndrome (ARDS) portend high morbidity and mortal-
ity 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 mech-
anisms, 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 ex-
cessive inflammation that resulted in RBC membrane distor-
tion, 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 reg-
ulation and nucleic acid scavenging. This long-overlooked
erythrocyte contribution to host defense implores further

J. Dobkin and L. Wu contributed equally to this work.

Correspondence: N. S. Mangalmurti (nspatel@pennmedicine.upenn.edu).
Submitted 18 April 2022 / Revised 9 December 2022 / Accepted 22 December 2022

http://www.ajplung.org 1040-0605/23 Copyright © 2023 the American Physiological Society. L169

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 RED CELL ADAPTATIONS AND CRITICAL ILLNESS

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 re-
ceptor 9 (TLR9), which binds nucleic acids and mediates clearance of RBCs during inflammation, and miRNAs, which are abundant in RBCs but the func-
tions of many remain undefined. G6PD, glucose-6-phosphate dehydrogenase; RBCs, red blood cells. [Image created with BioRender.com and
published with permission.]

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 impor-
tance of the host response to infection in mediating out-
comes 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 interleu-
kin- 8 (IL-8), diminishing the concentration of soluble IL-8
in blood plasma samples and thereby dampening immune

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 RED CELL ADAPTATIONS AND CRITICAL ILLNESS

Table 1. Non-gas exchanging functions of RBCs and hypothesized role in critical illness
Hypothesized or
Proven
Adaptation RBC Feature Function
Proven Heme Generates reactive oxygen
species: activates TLR4
Amplifies cytokine produc-
tion in the presence of
LPS (7)
Duffy Antigen Receptor Binds chemokines: maintains
for Chemokines chemokine gradient (12)
Binds pathogens: entry point
for P. vivax (13)
Glucose-6-Phosphotase Involved in pathway to con-
sume reactive oxygen spe-
cies (17)
Pyruvate kinase Catalyzes rate-limiting step in
glycolysis to produce intra-
cellular energy in nonmito-
chondrial cells (mature
RBCs) (20)
Hypothesized Glycophorins Binds pathogens: entry point
for P. falciparum (22)
Complement Receptor 1 Binds P. falciparum-induced
ligand (24, 25)
Proinflammatory regulator
abundant on RBCs (27)
Toll-like Receptor 9 Binds nucleic acids (28, 29)
Involved in P. yoeli infection
(30, 31)
ABO Glycoproteins Surface proteins on RBCs re-
sponsible for determining
ABO blood group (32)
AKI, acute kidney injury; ARDS, acute respiratory distress syndrome; COVID-19, coronavirus disease 2019; LPS, lipopolysaccharide;
RBCs, red blood cells.
Adaptation to Malaria
Hemoglobinopathies/thalasse-
mia (8)
Downregulation: decreased par-
asite entry (13, 14)
Deficiency: increased intracellu-
lar oxidative stress decreases
parasite survival (18)
Deficiency: decreased invasion
by P. falciparum and
increased phagocytosis of
ring- and mature-stage-
infected RBCs (20, 21)
Dantu variant: structural variant
in chromosome 4 region,
which increases RBC surface
tension and decreases mero-
zoite invasion (23)
Deficiency: increased cerebral
malaria (26)
Deficiency: reduced rosetting
conferring protection (25)
Deficiency/gene polymorphisms
Blood group O demonstrates dif- Blood group A may confer
ferent propensity to bind to
Duffy binding-like regions of
infected erythrocytes (33–36)
Consequence to Critical Illness
Increased susceptibility to nonma-
larial infection (9, 10), anemia/
transfusion dependence (11)
Increased susceptibility to ARDS
(15, 16)
COVID-19 severity (19)
Hemolytic anemia/transfusion de-
pendency, functional asplenia
(20)
Potential predisposition to lysis/
thrombosis
Increased RBC-bound complement
fragments in COVID-19 and non-
COVID-19 sepsis (27)
Decreased capacity to bind mtDNA
to ameliorate lung injury (28)
increased risk for ARDS in crit-
ically ill patients and associated
with increased risk of AKI (37)

signaling (12). In 1993, Horuk et al. (13) united these two in-
dependent findings and showed that the Duffy antigen re-
ceptor, 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 inflam-
matory 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 immunore-
gulatory role by sequestering chemokines from immune
cells such as neutrophils and acts as a reservoir for future
release (43–46). In preclinical studies using lipopolysac-
charide (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, pro-
longed 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 clini-
cal 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 pro-
vides 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.

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 RED CELL ADAPTATIONS AND CRITICAL ILLNESS
Hemoglobinopathies
Hemoglobin has evolved to adapt to malarial pressures,
rendering erythrocytes less hospitable to these invading par-
asites. 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 patho-
gens, Hemoglobin SS (HbSS) is the most well-studied. HbSS
arises from a single point mutation in the b-globin chain of
the hemoglobin molecule. The resultant amino acid substi-
tution 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 con-
ferred in heterozygotes of the mutation (49).
The sequelae of sickle cell disease are known to impact host
immunity indirectly via numerous vaso-occlusive mecha-
nisms 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 pro-
vide immunologically active mitochondria DNA (mtDNA)
that ultimately leads to endothelial damage. Whether mito-
chondria 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 associ-
ated with increased mortality (53). It has long been appreci-
ated 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 dis-
ease 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 erythro-
cyte sickling, vaso-occlusive crisis, and exertional rhabdo-
myolysis. Notably, a recent study found increased risk of
mortality and acute kidney injury after SARS-CoV-2 infec-
tion 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 under-
stand the potential consequences of this malarial adaptation
on the host’s inflammatory responses.
Thalassemia.
Thalassemias are a heterogeneous group of red blood cell dis-
orders in which the globin-chain units making up normal
adult hemoglobin (a2b2) are reduced or absent. As a result,
carriers of these mutations show microcytic anemia to vary-
ing degrees, and at least in the neonatal period, compensate
with elevated levels of circulating fetal hemoglobin (c4) (8).
The prevalence of a- and b-thalassemia have similarly been
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shown to coincide with malaria-endemic regions, leading to
hypotheses that these mutations also confer selective advant-
age against deadly malaria infections (55).
One proposed mechanism for that protection against
malaria is increased susceptibility to P. vivax and P. falcipa-
rum 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 a-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 func-
tional defects in the immune system rendering them suscepti-
ble to pathogens such as Klebsiella pneumoniae and Yersinia
enterocolitica infections (9). These defects range from defec-
tive 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-trans-
mitted pathogens like Hepatitis C, chronic transfusion-related
iron overload, and therapeutic splenectomies (11). Given this
increased infection burden and mortality, this genetic adapta-
tion 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 radi-
cals 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 poten-
tially 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 hospital-
ized patients with COVID-19 pneumonia requiring supple-
mental oxygen found that the cohort with G6PD deficiency
demonstrated prolonged hypoxia and a longer need for me-
chanical ventilation (19). Outside of COVD-19, no study spe-
cifically 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
 RED CELL ADAPTATIONS AND CRITICAL ILLNESS
energy production in nonmitochondrial cells like mature
erythrocytes. In a study comparing in vitro P. falciparum
inoculation of RBCs obtained from homozygous and hetero-
zygous pyruvate kinase-deficient adults and controls, PK-defi-
cient 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 sus-
ceptibility of individuals with PK deficiency to other patho-
gens and critical illnesses, the propensity toward anemia
and the increased risk of functional asplenia are likely dele-
terious 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 erythro-
cyte mutations that follow similar geographical patterns indi-
cating 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 car-
bohydrate modifications on glycans and glycoproteins (32).
These glycans are present on the surface of RBCs, platelets, en-
dothelium, and epithelium. DNA sequencing has placed the de-
velopment 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 com-
ponent 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 impor-
tant for P. falciparum invasion, however, no other studies
have addressed this hypothesis (60). The prevailing hypothe-
sis 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 cytoad-
hesive 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
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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 im-
portant in determining whether other RBC adaptations indi-
rectly 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 eryth-
rocytes 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 cu-
mulative 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 neighbor-
ing 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 at-
tributable 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 rosett-
ing 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 associ-
ated with higher erythrocyte CR1 were associated with protec-
tion 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 al-
ternative complement pathway is associated with improved
survival during critical illness (68). We have demonstrated
that RBC-bound complement activation products are ele-
vated 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 stud-
ies will be necessary to elucidate the role of red cell comple-
ment 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 ma-
larial DNA (70). TLR9 gene polymorphisms are associated
with symptomatic malaria in Ghanaian children, and TLR9
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 RED CELL ADAPTATIONS AND CRITICAL ILLNESS
has been shown to play an important role in the develop-
ment 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 resist-
ance 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 circula-
tion 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 sep-
sis-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 posttranscrip-
tional 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 protec-
tion against malaria species was proven in in vitro experi-
ments showing that miRNA-451 can be incorporated into P.
falciparum mRNAs where it inhibits mRNA translation and
parasite growth (74). When normal erythrocytes are manipu-
lated to overexpress miRNA-451 and miRNA-223, these eryth-
rocytes 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 extracellu-
lar 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 show-
ing the potential to predict 28-day mortality (77). miRNA-223,
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which is linked to increased resistance to P. falciparum as dis-
cussed 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. falcipa-
rum 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 glyco-
phorin 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 popu-
lations 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 che-
mokines, 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 compart-
ment 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 per-
fusion in Uganda, Kenya, and Tanzania, all regions in which
 RED CELL ADAPTATIONS AND CRITICAL ILLNESS
centuries of endemic malaria has exerted its pressures (81). In
contrast to original research performed on patients in nonen-
demic regions, the study found that excess mortality with
fluid resuscitation was consistent across all groups, irrespec-
tive 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 resus-
citation with intravenous fluids and vasopressors compared
with usual care (82). In the United States, a subgroup reanaly-
sis of the Fluids and Catheters Treatment Trial (FACTT) trial,
a study that initially concluded that a restrictive fluid resusci-
tation 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 par-
ticipants, 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 dis-
cussions 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 periph-
eral blood, have revealed a relatively immunosuppressed ver-
sus 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 syn-
drome (ARDS) have been stratified into two subgroups defined
by levels of inflammatory biomarkers. Phenotype 1 (the hypo-
inflammatory subgroup) has lower mortality, more ventilator-
free, and more organ failure-free days compared with the
hyperinflammatory Phenotype 2 (88, 89). Much like the tran-
scriptionally 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 infec-
tion. 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 opportu-
nity 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 signifi-
cant 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
AJP-Lung Cell Mol Physiol  doi:10.1152/ajplung.00127.2022  www.ajplung.org
Downloaded from journals.physiology.org/journal/ajplung (189.203.131.199) on March 2, 2024.
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-identify-
ing 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 in-
tensive 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 associ-
ated with COVID-19 severity infection though the study was
underpowered to make definitive conclusions (93).
Despite the abovementioned studies, the role of RBC het-
erogeneity remains understudied in critical illness. The col-
lection and biobanking of RBC samples combined with
further translational studies will be necessary to elucidate
the immunomodulatory functions of RBCs during critical ill-
ness. Given the accumulating evidence for RBC immune
function and the recognition that host immune heterogene-
ity underlies differential severity to numerous pathogens, it
is crucial that further investigations promoting precision
medicine include large-scale exploration of RBC heterogene-
ity. By examining RBC heterogeneity through the most
powerful evolutionary pressure exerted on them—malaria—
we may continue to uncover previously unknown complex-
ities 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 con-
founding 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.
ACKNOWLEDGMENTS
The authors acknowledge Kaitlyn Eckart and Julia Yuen for
administrative assistance. Graphical abstract image created with
BioRender.com and published with permission.
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.
L175
 RED CELL ADAPTATIONS AND CRITICAL ILLNESS
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.
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