The emerging role of red blood cells in cytokine signalling and modulating immune cells

Journal Pre-proof The emerging role of red blood cells in cytokine signalling and modulating immune cells

Elisabeth Karsten, Benjamin R. Herbert PII:

S0268-960X(19)30158-4

DOI:

https://doi.org/10.1016/j.blre.2019.100644

Reference:

YBLRE 100644

To appear in:

Blood Reviews

Please cite this article as: E. Karsten and B.R. Herbert, The emerging role of red blood cells in cytokine signalling and modulating immune cells, Blood Reviews(2019), https://doi.org/10.1016/j.blre.2019.100644

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Journal Pre-proof The emerging role of red blood cells in cytokine signalling and modulating immune cells Elisabeth Karsten1,2,3,*; Benjamin R. Herbert1,2

* Correspondence to: Elisabeth Karsten Address: PO Box 4054, Royal North Shore Hospital, Sydney, NSW 2065 Email: [email protected]

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Telephone number: +61 405 299 536 Degree: PhD

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Affiliations: 1 Translational Regenerative Medicine Laboratory, Kolling Institute, Royal North Shore

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Hospital, Sydney, Australia; 2Sangui Bio Pty Ltd, Sydney, Australia; 3 Faculty of Medicine, University of

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Sydney, Sydney, Australia

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Benjamin R. Herbert

Email: [email protected]

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Address: PO Box 4054, Royal North Shore Hospital, Sydney, NSW 2065

Degree: PhD

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Telephone number: +61 411 849 118

Affiliations: 1 Translational Regenerative Medicine Laboratory, Kolling Institute, Royal North Shore Hospital, Sydney, Australia; 2Sangui Bio Pty Ltd, Sydney, Australia

Journal Pre-proof Abstract For many years red blood cells have been described as inert bystanders rather than participants in intercellular signalling, immune function, or inflammatory processes. However, studies are now reporting that red blood cells from healthy individuals regulate immune cell activity and maturation, and red blood cells from disease cohorts are dysfunctional. These cells have now been shown to bind more than 50 cytokines and have been described as a sink for these molecules, and the loss of this activity has been correlated with disease progression. In this review, we summarise what is

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currently understood about the role of red blood cells in cytokine signalling and in modulating the activity of immune cells. We also discuss the implications of these findings for transfusion medicine

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and in furthering our understanding of anaemia of chronic inflammation. By bringing these disparate

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units of work together, we aim to shine a light on an area that requires significantly more

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investigation.

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Keywords: red blood cells; transfusion; cytokines; chemokines; DARC; inflammation.

Journal Pre-proof 1.

Introduction

For many years, red blood cells have been described as inert bystanders rather than participants in intercellular signalling, immune function, or inflammatory processes. Advances over the last 30 years have revealed that intercellular signalling is a more complex system than initially appreciated, with components such as exosomes and microRNA taking their places as distinct classes of biological regulators [1]. Whilst there has been driven widespread progress in the signalling area, an unintended consequence has been the lack of focus on cells, such as red blood cells, that are

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functionally outside these classical pathways.

Red blood cells are the most abundant cell type in the body and are directly exposed to a wide

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variety of tissues and cells on their journey through the circulatory system. The 25 trillion red blood

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cells in an average human collectively present a very large surface area in the body, of approximately

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3500 m2 [2]. These cells are further covered with a network of membrane-bound proteoglycans and glycoproteins known as the glycocalyx, that interact with the similar glycocalyx covering the 4000-

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7000 m2 of the endothelium luminally [3,4]. Amongst the myriad of established functions, there is

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evidence that the endothelial glycocalyx can also bind cytokines, which can then affect glycocalyx synthesis and composition, or modulate the inflammatory response [5]. The structure and

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composition of the endothelial glycocalyx shares similarity with the red blood cell glycocalyx [3], lending to the hypothesis that these red blood cells can too bind cytokines and subsequently modulate the inflammatory response. Although it is now clear that red blood cells are involved in many pathologies, the specific nature of their involvement for the most part has not yet been elucidated. Red blood cells themselves have also been shown to be immuno-stimulatory to specific cellular populations and can cause cytokine storm events following their transfusion [6]. It appears that we are only just scratching the surface of red blood cells and their role in inflammation, disease, and repair.

2.

Red blood cells in the clinic today

Journal Pre-proof 2.1

Diagnostics

Although red blood cells are largely believed to be inert, in regular clinical practice, red blood cell characteristics are collected and are used in diagnostics. These diagnostic processes are, however, entirely biased towards evaluating the oxygen binding capacity of these cells with a focus on diagnosing different types of anaemia. A low red blood cell count, variations in the average size of the cells (mean corpuscular volume, MCV), or variations in the average amount of haemoglobin in

2.1.1

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are all collected as part of the standard full blood count panel.

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each cell (mean corpuscular haemoglobin concentration, MCHC) can all be indicative of anaemia and

Measures of inflammation

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There is only one test used regularly that assesses re d blood cells in the context of non-specific

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inflammation. This simple test has been used since the 19 th century and it assesses the rate of

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erythrocyte sedimentation. A very high rate of sedimentation (compared to a normal range) indicates broad spectrum inflammation such as an infection or an auto-immune condition [7,8].

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However, this is not an optimal biomarker of inflammation, as other plasma-derived markers such as

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C-reactive protein have been shown to be more reliable and more respons ive to changes in the inflammatory microenvironment [9].

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Recently there has been investigation into the analysis of another re d blood cell characteristic as a diagnostic marker for inflammation [10]. In an extensive study of almost 4000 participants, Lippi et al. identified that red blood cell distribution width (RDW) was closely correlated with inflammation as indicated by erythrocyte sedimentation rate and level of C-reactive protein [10]. This correlation of RDW with inflammation has received a bit of attention over the last decade. High RDW values have been reported as a risk factor for increased mortality in cardiovascular patients [11], diabetics [12], and patients with chronic lymphocytic leukemia [13]. Curiously, this increased risk of mortality is not isolated to specific diseases. Patel et al. demonstrated that high RDW values were a strong predictor of mortality in people over the age of 45 and was independent of anaemia and the presence of other diseases [14]. They hypothesised that this observation was a result of increased

Journal Pre-proof inflammation and oxidative stress [14]. In light of these results, analysis of red blood cells as an indicator of inflammation or overall health may be a valuable addition to the standard blood analysis panels in clinic. 2.2

Red blood cell transfusions

Red blood cell transfusions are the most common blood derived therapeutic and have been used widely over the last century. As a transplant, they are generally well tolerated as donor matching only needs to be met at the blood type level. Due to its extensi ve history, the science of transfusion

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medicine has now moved past optimising the treatment for the patient and into optimising the storage parameters for the blood products to extend their use. At the moment, red blood cells are

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stored at 4 °C for up to 42 days before use [15]. Although there is a lot known about the cause of

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most of the adverse reactions that can occur with red blood cell transfusion, there remains the

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controversial issue of the storage lesion. The storage lesion refers to the red blood cell deformability that can occur during long term storage. The debate regarding the implications of usin g these

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altered cells as a treatment is ongoing [16]. Whilst there are many studies that report a correlation

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between the transfusion of older (deformed) red blood cells and the occurrence of adverse events [6,17], there are also studies that report that the length of storage has no effect at all [18]. This

time soon.

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debate has been going on now for decades, and it doesn’t appear that an answer will be reached any

Many analyses have been done in an attempt to determine exactly what occurs in red blood cells during storage. The morphology of these cells is known to change substantially over time, wherein they become more rigid and are more susceptible to haemolysis [19]. There are also a number of reports that suggest that these cells undergo oxidative damage during storage and begin to take on the qualities of senescent red blood cells [20]. Whilst others have focused more on the issue of cytokine release from stored blood [21,22]. Some adverse events following red blood cell transfusion were hypothesised to be a result of contaminating white blood cells which secrete a range of inflammatory proteins during storage [23]. Thus, transfusion of a blood packs containing both the

Journal Pre-proof contaminating white blood cells and the resulting cytokines could elicit a detrimental immune reaction. However, the evidence now suggests that leukodepletion does not entirely abolish the cytokine release [21] and that in a number of situations it is no different to non-leukodepleted red blood cells [24,25]. Weisbach et al. demonstrated that stored leukodepleted red blood cells actually produced significantly more IL-8 than the non-leukodepleted red blood cells [21].

3.

Discovery of inflammatory signalling molecules in red blood cells

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Cytokines are key mediators and signalling molecules in inflammation. There are a handful of reports that have identified that red blood cells interact directly with inflammatory molecules including

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cytokines, but the majority of those studies are restricted to the investigation of the Duffy antigen

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receptor for chemokines (DARC). This receptor is present on red blood cells and its described as a

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chemokine scavenger, however only eleven red blood cell associated chemokines have been identified as ligands of this receptor (Figure 1). This is not the first instance of describing red blood

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cells as scavengers. They have also been described as scavengers of cholesterol and other lipids [26]

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and for this reason have been implicated in the pathogenesis of cardiovascular events such as atherosclerosis [27]. These scavenging processes of red blood cells may share a relationship, but that

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investigation remains to be completed.

In light of the limited investigation into red blood cells and cytokines or chemokines, our laboratory sought to identify any additional signalling proteins associated with the cells, and subsequently discovered that the red blood cells are associated with up to 45 different signalling molecules [28,29] Outside of these areas, there is minimal other literature. Although there are some reports on cytokines in red blood cells, no study other than ours has attempted to quantify these proteins in a unit of these cells. This information is crucial for understanding the role of cytokine interactions with red blood cells in blood and the implications of haemolysis in sample collection and preparation. A summary of the known literature regarding the relationship between red blood cells and cytokines is presented in Table 1. These studies are expounded in further detail in the subsequent sections.

Journal Pre-proof 3.1

Duffy antigen receptor for chemokines (DARC)

DARC was first identified on red blood cells in 1950 and was describe d as a new blood group [30]. Since then, it has been reported that DARC is not only present on red blood cells but is also expressed on some endothelial cells [31]. Subsequent to its identification, the majority of research on this receptor on red blood cells has focused on its role in malarial pathogenesis. Plasmodium species, the parasites that causes malaria, have been shown to use DARC on red blood cells to invade the cells and thus progress the infection [32]. More than 95 % of West Africans [33], and 68 % of

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African Americans [34] are negative for DARC on their red blood cells, but are still positive for the receptor on their endothelial cells [35]. Chemokine binding to red blood cells

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3.1.1

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In 1991, Darbonne et al. reported the presence of a receptor on the surface of red blood cells that

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could readily bind IL-8 and implicated red blood cells as a biological sink for this chemokine [36]. Following this report, the same receptor was then described to be promiscuous as it was able to

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bind not only IL-8 but also some C-X-C (GRO-α, NAP-2) or C-C (RANTES, MCP-1) chemokines [37].

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Further analysis of this receptor identified that it was in fact DARC [38,39]. In the 20 years following these reports, few additional molecules have been identified as additional ligands for DARC [38,40–

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42]. Due to the broad binding capacity of DARC on red blood cells, they have been implicated as sinks for circulating inflammatory markers [36,43,44]. Although this receptor is structurally similar to G-protein-coupled chemokine receptors with its seven transmembrane domains, unlike these receptors, ligation of DARC does not appear to activate intracellular chemokine signal transduction pathways [38]. In 1993, Neote et al. reported that RBC-bound IL-8 had no effect on mobilising Ca2+ ion stores, which is indicative of signal transduction with G-protein-coupled receptors [37]. Since that report, no other evidence of signal transduction has been identified. Similarly, no reference to phosphorylation of the receptor in the membrane or its ligand(s) have been reported in the literature. These results have been used to support the theory that the primary function of chemokine ligation of DARC is not in chemokine signalling, but instead as an inflammatory sink for

Journal Pre-proof these markers [38,45]. Investigation into the concentration of these cytokines in the red blood cells of healthy individuals or individuals with inflammatory conditions may provide valuable information regarding disease state and in the identification of biomarkers. 3.1.2

DARC in disease and inflammatory conditions

Evidence is gathering that DARC on red blood cells, and cytokine ligation of this receptor plays a role in inflammatory disease. Whilst DARC negative individuals are less susceptible to malarial infection, they have been reported to have a higher risk of developing inflammatory conditions such as graft

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rejection [46] or prostate cancer [47]. This is theorised to be a result of the reduced availability of a DARC mediated inflammatory sink [48]. Although the cytokine binding capacity of red blood cells in

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DARC negative individuals is lost, some capacity to bind excess inflammatory markers is retained by

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the DARC positive endothelial cells. This has been evidenced in animal knockout models . In mice

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lacking DARC from the red blood cells only, the overall level of CXCL1 (mouse homologue of IL-8) and MCP-1 being bound was reduced compared to the DARC positive control [43]. However, binding was

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still higher than that observed in the complete DARC knockout model (no DARC on the red blood

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cells or the endothelial cells) [43]. In addition to altered binding capacity of the cells, the inflammatory response to stimuli was exaggerated when DARC was not present in the animals [43].

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Although interesting, it is important to note that it is currently unknown if mouse CXCL1 performs the same function(s) as IL-8 in humans despite both molecules being homologues as well as ligands of DARC [49].

The progression of polycythermia vera to myelofibrosis may be another interesting condition to investigate the sequestering role of DARC on red blood cells. Polycythermia vera is characterised by an initial overproduction of red blood cells that can then lead to the development of fibrosis in the bone marrow (myelofibrosis) which can restrict red blood cell production and often results in anaemia [50]. This shift from overproduction to underproduction of red blood cells would have a significant effect on this hypothesised inflammatory sink. As an example of this, IL-8 (a DARC ligand) is reported to be higher in patients with primary myelofibrosis [51] and increased levels are

Journal Pre-proof predictive of an inferior anaemia response and survival [51,52]. This may be due to a decreased capacity to bind the excess IL-8, leading to further disease progression. Investigation into the incidence of these conditions in DARC negative populations may be a useful target for this analysis. Fukuma et al. sought to further investigate the role of DARC on red blood cells as an inflammatory sink for cytokines [40]. In this study, they treated DARC positive and negative mice with recombinant protein and monitored the protein half-life. They reported that in DARC negative mice, MCP-1 and Eotaxin were rapidly cleared upon administration. However, the half -life of the proteins in the

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peripheral circulation was significantly higher in the DARC positive animals due to red blood cell sequestration. With these results, they hypothesised two roles for cytokine binding to DARC on red

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blood cells in managing inflammation. First, that DARC on red blood cells acts as an inflammatory

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sink to remove excess chemokines, and second, that it acts as a reservoir o f these cytokines to

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release when they are needed [40]. The mechanisms behind chemokine release from DARC have since been investigated. Release of MCP-1 (a C-C chemokine) from DARC is activated by two

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identified mechanisms, (1) clotting or (2) treatment with unfractionated heparin [53]. This discovery

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supports the hypothesis of Fukuma et al., that DARC is involved with both binding and releasing cytokines as needed [40]. Release of chemokines in response to clotting identifies a key role of DARC

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in inflammation. In vivo, clotting occurs at the site of injury or in the case of thrombi, and red blood cells are key to both of these processes. Thus, a bolus of chemokines released from red blood cells at the site of injury or thrombi may play a role in the resulting inflammatory processes. Humphries et al. identified that in a thrombus, MCP-1 levels increase and remain high until resolution and that treatment with MCP-1 leads to accelerated thrombus clearance [54]. Red blood cells in the thrombus are likely to be a major source of this chemokine. Similarly, lung inflammation in response to lipopolysaccharide stimulation has been shown to be modulated by DARC. Neutrophil migration into the lungs in response to inflammatory stimuli is attenuated and the migration of mononuclear inflammatory cells is increased in DARC knockout models [43,55,56]. Local levels of DARC ligand chemokines are also reported to be higher in the lungs of these stimulated mice [55,56]. The results

Journal Pre-proof of these studies propose a notable role for cytokines binding to red blood cells in modulating immune responses. 3.1.3

DARC on red blood cell microparticles

Red blood cells produce microparticles as they age and these have been well documented in the literature. These microparticles have been found to stimulate a variety of disease processes including immunomodulation [57], particularly following transfusion [58]. Although various mechanisms of action have been investigated such as the activation of antigen presenting cells [58],

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the cytokine contribution from these microparticles has been largely overlooked. In 1992, DARC was identified on the surface of red blood cell microparticles, and its presence has since been confirmed

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[59,60]. This chemokine binding receptor is not only present on the membranes of these

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microparticles, but it also retains its capacity to bind GRO-α out of solution, although the binding

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affinity is reduced [42]. The number of microparticles released from red blood cells increases over time with storage and the presence of these microparticles have been implicated as a potential risk

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for red blood cell transfusion [61]. Interaction with platelets has been demonstrated to stimulate the

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release of GRO-α from allogenic microparticles [42]. This models a biological interaction that may

reactions. 3.1.4

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occur in vivo following red blood cell transfusion, which may play a role in the observed adverse

Gardos channel

The Gardos channel, present on red blood cells, regulates cell volume by transporting potassium ions across the cell membrane [62] and plays an important role in promoting red blood cell dehydration in sickle cell anaemia [63]. Haemoglobin concentration in red blood cells increases as a result of cell dehydration which subsequently enhances the rate of haemoglobin polymerisation, a key characteristic of sickled red blood cells [64,65]. High levels of cytokines have been observed in the plasma of sickle cell anaemia patients, and these cytokines have been demonstrated to play a role in its pathogenesis [66]. Rivera et al. reported that the presence of select cytokines (including IL-10 and RANTES) at high levels increased the Gardos channel activity by up to 80 % [67]. Further

Journal Pre-proof investigation into this interaction identified that this activity was mediated, at least in part, by cytokine binding to DARC on red blood cells [67,68]. DARC positive red blood cells were found to be 17x more dense (and thus more dehydrated) than DARC negative red blood cells and the density of the DARC positive cells could be modulated with the addition of RANTES or IL-8 [68]. Whilst there are no reported downstream effects of cytokine binding to DARC on red blood cells, these studies suggest a functional role of cytokines on red blood cells that is additional to its role as a chemokine sink.

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Macrophage migration inhibitory factor (MIF)

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3.2

Macrophage migration inhibitory factor (MIF) is a pro-inflammatory cytokine and an oxido-reductase

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enzyme that was first identified in 1966 [69]. Since its identification, MIF has been shown to play a

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critical role in a wide range of inflammatory conditions. These conditions include, but are not limited

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to, sepsis, rheumatoid arthritis, and preeclampsia, and patients with these conditions typically have higher levels of the protein in their serum or plasma [70–72]. As such, considerable research has

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focused on MIF as a potential disease biomarker. However, MIF as a biomarker is complicated by the

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fact that many cells have been shown to produce and secrete MIF. This protein was originally identified in lymphocytes [69], and has since been identified in macrophages and other

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inflammatory cells [70,73], adipocytes [74], liver cells [75], and many other cell types. It is even secreted from the pituitary gland in response to infection [76]. There are reports, although few, that have suggested that another source of MIF is mature red blood cells. Shortly after its identification, Fox et al. reported in 1974 that the activity of MIF from fetal calf serum was attenuated when red blood cells were present [77]. They hypothesised that this loss of activity was indicative of MIF being bound to the red blood cells and becoming inactivated. Since that report, red blood cells have been postulated to not only bind, but also harbour MIF. A strong correlation between haemoglobin concentration and the concentration of MIF in plasma or serum has been observed [78,79]. This rise in MIF concentration in the plasma has been attributed to red blood cell derived MIF. However, this has been the extent of the investigation. Up until very recently

Journal Pre-proof no study has sought to quantify the concentration of MIF in a unit of red blood cells nor to investigate its activity. In fact, it had been recommended to simply avoid analysis of MIF where red blood cells may be present in order to minimise inference [79], but this view is particularly shortsighted. Determining the concentration of MIF in red blood cells and its chemokine activity is a crucial step in understanding the inflammatory processes following haemolysis in vivo. To address this, our laboratory quantified the level of red blood cell derived MIF per volume of whole blood and determined that red blood cells were actually the largest reservoir for this protein in blood,

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contributing 25 µg of this protein per millilitre [28]. This is 1000-fold higher than the average levels detected in plasma [28]. MIF has two major documented functions that appear to be dependent on

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its cellular location. Secreted or free MIF has a primary role as a pro-inflammatory cytokine which

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modulates the migration of inflammatory cells [80], and is dependent on its tautomerase active site

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[81]. Whilst the secondary role of MIF is largely intracellular and is a specific oxidoreductase activity which has been demonstrated to regulate cellular oxidative stress [82,83], this is likely the primary

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role that this protein is playing in red blood cells. Notably, in our analysis we discovered that the MIF

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present in the red blood cell cytosol was enzymatically active, indicating that it is also likely to be chemotactically active upon haemolysis [28].

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The value in this analysis is made clearer by looking at a condition where MIF plays a key role, such as sepsis. MIF has been reported to be significantly elevated in the plasma of septic patients, and high levels of this protein have been correlated with early death in patients with severe sepsis [84]. Similarly, by inhibiting MIF, the survival rate of patients with severe sepsis improves significantly [85,86]. Haemolysis is a common complication of sepsis, and haemoglobin has even been proposed as a potential biomarker for the condition [87]. In fact, Adamzik et al. reported that free haemoglobin levels were twice as high in the non-survivors of severe sepsis compared to the survivors [87]. It is likely that at least some of the MIF detected in the plasma of severe septic patients is a result of haemolysis and red blood cell release. Thus, understanding of where the MIF has come from and determining what concentration of MIF can be attributed to red blood cells

Journal Pre-proof could lead to improved therapeutic targets for treating sepsis, a condition with very high mortality (approximately 30 %). At the very least, this warrants further investigation. 3.2.1

D-dopachrome tautomerase (DDT)

D-dopachrome tautomerase (DDT), also known as MIF-2, has been described as a functional homologue of MIF [88]. Although DDT cannot replicate the same oxido-reductase activity of MIF, it shares its tautomerase and cytokine activity [89,90]. MIF and DDT bind to the same cell surface receptor complex formed between CD44-CD74, and ligation of this receptor leads to the same

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downstream effects [88]. These proteins tend to be localised to the same tissues and areas of the body, in fact, DDT has even been identified in red blood cells. Bjork et al. reported that red blood

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cells were responsible for approximately 99 % of the D-dopachrome tautomerase activity in whole

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blood and identified the presence of DDT in these cells [91]. The reason for its localisation in red

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blood cells is unknown, particularly since it does not share the same oxido-reductase activities of MIF.

Other cytokines in red blood cells

3.3.1

Interleukin 33 (IL-33)

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3.3

IL-33 has also been identified in the red blood cell lysates of healthy individuals [92]. Notably, IL-33 is

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a nuclear protein that is released from dying or dead cells. As such, its presence in red blood cells, an enucleate cell, is unexpected. Wei et al. identified that IL-33 was expressed in erythroid progenitor cells, thus hypothesising that the protein had been present in the cells since they were nucleated [92]. IL-33 can be released from apoptotic or necrotic cells [93] and its release has been shown to stimulate the secretion of TH2 cytokines and play a key role in promoting allergic inflammation [94]. Haemolysis of red blood cells would thus result in a release of this cytokine into the soluble fraction of blood. In sickle cell anaemia patients, higher levels of IL-33 in plasma were observed with increased levels of haemolysis [92]. 3.3.2

Cytokine screening

Journal Pre-proof In light of the narrow, single cytokine studies that have occurred previously, our laboratory sought to screen red blood cell lysates from healthy participants to determine what, if any, other cytokines were associated with these cells. This analysis revealed that 45 cytokines were present in the red blood cell lysates [29]. These red blood cells were isolated using a rapid yet gentle technique (dextran sedimentation) to minimise the loss of surface bound proteins . These spanned C-C chemokines, C-X-C chemokines, cytokines in the CSF family, GM-CSF/IL-15/IL-3 family, IL-1 superfamily, IL-6 family, IL-12 family, TNF superfamily, and PDGF growth factors, amongst others.

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The full list of cytokines identified in this study can be found in Table 1 and Figure 2. This is the broadest cytokine analysis that has been performed to date. Of the 45 cytokines that were detected,

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9/45 were detected in every participant [29]. These were Eotaxin-1, bFGF, HGF, IL-3, IL-18, MIF,

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RANTES, SDF-1α, and VEGF. Furthermore, 33/45 of the cytokines were detected in 50 % or more of

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the participants [29]. This study also demonstrated that intact red blood cells retained the capacity to bind and release these cytokines, and that vigorous washing of the cells stimulated a loss of the

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molecules [29].

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These results provide evidence that the interaction between red blood cells and signalling molecules is much greater than what was once thought. Much remains unknown at this stage such as what the

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cytokines are binding to on the cells and what mechanisms are behind the binding and release of these molecules. This study merely scratches the surface of the complexities of red blood cells. Whilst the results of all the studies outlined in Table 1 clearly demonstrate a relationship between red blood cells and signalling molecules, an understanding of how or if red blood cells communicate and interact with other cells is less clearly defined.

4.

Red blood cells and their interactions with other cells

Most of the investigation into the activity of immune cells was, and still is for the most part, done in single cell cultures containing the target immune cell. The dogma of immunology teaches that the other cells of the blood (such as red blood cells or platelets) are inert and would not contribute to

Journal Pre-proof the overall activity of the immune system. However, all of the recent research into red blood cells suggests that this is unlikely to be the case. In response to the changing climate around red blood cells and their involvement in inflammatory conditions, a few laboratories across the world have started to investigate the specific interaction between red blood cells and other cell types. The majority of this investigation has focused on the role red blood cell s play in altering the function of immune cells. 4.1

T lymphocytes

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One of the first reports in this area documented the effect of red blood cells on freshly isolated T cells in vitro [95]. T cells are a subset of lymphocytes, and any increase in proliferation of these cells

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in vitro following stimulation is used as a model of immune activation. This initial paper reported

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that by stimulating T cells and treating them with fresh, autologous red blood cells their activity was

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significantly altered [95]. Red blood cells stimulated these T cells to proliferate more than the untreated control, and were also found to be protective for these cells against apoptosis [95]. These

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results have since been replicated under similar conditions and does not appear to be dependent on

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the ratio of red blood cells to T cells, but is dependent on the red blood cell being intact [96–98]. These studies have led to the question of whether or not this state of T cell activation is actually its

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natural state in vivo, as they are always in contact with red blood cells. In particular diseases or inflammatory states, red blood cells have been shown to be dysregulated in this activity in that they do not have the same effect on T cells that healthy red blood cells do [97,99,100]. As an example, red blood cells isolated from patients with carotid atherosclerosis were found to be no longer protective against T cell apoptosis [97], and red blood cells isolated after hip arthroplasty were no longer immunogenic to the T cells unlike the cells i solated prior to the surgery [99]. It has been hypothesised that these results are representative of a dysregulation of T cell homeostasis that occurs in vivo, and may be involved in potentiating the conditions [97]. 4.2

Dendritic cells

Journal Pre-proof Red blood cells from healthy donors are now known to restrict the maturation of dendritic cells, and in the process, the secretion of IL-12(p40) [101,102]. This is proposed to be a mechanism for controlling the overstimulation of dendritic cells and the hyper release of pro-inflammatory cytokines in vivo [102]. However, in carotid atherosclerosis this system is similarly dysfunctional to the observed effects on T cell survival. Red blood cells collected from patients with carotid atherosclerosis were less able to prevent the maturation of dendritic cells, and thus the secretion of pro-inflammatory cytokines [101].

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Other immune cells

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4.3

There is minimal other literature on the interaction between red blood cells and immune cells other

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than T cells or dendritic cells. A 30 year old study reported that natural killer cells, when in the

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presence of red blood cells, were more cytotoxic towards tumour cells [103]. Virella et al.

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investigated the effect of autologous red blood cells on B cell activity and they reported a significant increase in the secretion of immunoglobulins and of the pro-inflammatory cytokine, IFN-γ [104].

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Finally, a recent study reported that red blood cells promoted eosinophil migration through

allergic inflammation [105]. Fibroblasts

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4.4

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endothelial cells by scavenging the chemokine RANTES, thus suggesting a role for red blood cells i n

The interaction between red blood cells and fibroblasts, a non-immune cell type, has been evaluated extensively by one laboratory. This group demonstrated that the presence of red blood cells in a culture of fibroblasts alter the function and secretome of these cells. Unlike the effect of red blood cells on T cell proliferation and survival, red blood cells suppressed the proliferation of fibroblasts and promoted staurosporine-induced apoptosis [106]. Additionally, this effect was only achieved using intact red blood cells. The red blood cell conditioned media had no influence on fibroblast apoptosis, suggesting that soluble factor(s) are unlikely to be responsible [106]. Red blood cells also promoted fibroblast mediated contraction of collagen [107], the secretion of the chemokine IL-8

Journal Pre-proof [108], and also the secretion of matrix metalloproteinases [109]. This modulation of fibroblast activity illustrates a role for red blood cells in the remodelling process of wound healing. 4.5

Endothelial glycocalyx

The endothelial glycocalyx is a complex network of membrane-bound proteoglycans and glycoproteins and is structurally similar to the glycocalyx attached to the red blood cell membrane [3]. Under inflammatory conditions, activated polymorphonuclear leukocytes including macrophages and mast cells secrete enzymes and cytokines such as matrix metalloproteinases and TNF-α, which

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contributes to degradation of the endothelial glycocalyx [110]. As the effects of inflammation occur in the capillaries where red blood cells are slow moving, these cells interact and their glycocalyx is

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also subject to the same degradation as the endothelium [3,111]. This enzymatic and cytokine driven

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degradation of the glycocalyx on the endothelium and red blood cells reduces its thickness, alters

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permeability and binding capacity for macromolecules, and increases exposure to receptors [110,112]. This response may be a functional mechanism employed in response to acute

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inflammation, which can become problematic upon chronic inflammatory stimulus. Degradation of

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the red blood cell glycocalyx has been observed as a result of cellular ageing and during long term storage at 4 °C similar to the conditions used to store blood packs prior to transfusion [113,114].

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The endothelial glycocalyx is known to bind and interact with a variety of C-X-C and C-C chemokines [115]. Considering its structural resemblance to the red blood cell glycocalyx, there is evidence to suggest that the red blood cell glycocalyx may also be responsible for binding and releasing a proportion of these inflammatory cytokines. As such, degradation of the glycocalyx is likely to lead to a loss of bound chemokines and the level of this degradation may be associated with altered inflammatory properties of red blood cells following storage. 4.6

Mechanism of action

The mechanism of action for this modulation of cellular function by red blood cells has not yet been investigated in detail but there are multiple theories. Profumo et al. investigated the cause of the dysfunction in the red blood cells from carotid atherosclerosis patients that had altered T cell activity

Journal Pre-proof [97]. They reported that the result could be replicated in healthy red blood cells by subjecting them to oxidative stress, as such they concluded that it was a result of oxidative imbalance in the red blood cells [97]. Similarly, it was reported that treatment with healthy red blood cells reduced the level of oxidative stress in the target T cell population [95]. A publication by Antunes et al. has been the only study thus far to perform an investigation into the factors responsible for the activity of healthy red blood cells on T cells [98]. In a series of experiments, they identified that the causative agent(s) were present in the red blood cell conditioned media, and were soluble protein factors that

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were sensitive to heat [98]. However, they were not able to identify the specific molecule(s) responsible for the activity. In the promotion of eosinophil migration, Kanda et al. attributed their

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results to red blood cells being able to bind the chemokine RANTES out of the media and thus play a

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role in chemotaxis [73]. There is growing evidence that these signalling molecules (cytokines and

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chemokines) at the centre of inflammatory processes may also be interacting with red blood cells,

Clinical implications

5.1

Transfusion medicine

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5.

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thus giving them a role in inflammation but more investigation is required.

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Overall, the literature on red blood cell transfusions, the storage lesion, and optimising storage parameters has very little agreement [116]. One reason for this may be that the entire area of red blood cell interactions with cytokines has been underappreciated until this point, particularly in the field of transfusion medicine. Whilst there is some literature that has evaluated the cytokine level of the red blood cell storage medium over time [21,23,117], there is no literature that thoroughly investigates the cytokine load of red blood cells in these conditions. Even if no significant increase in cytokine concentration in the storage media is observed, the red blood cells may still be involved in a continual process of binding and releasing cytokines to maintain a particular extracellular cytokine load, the process of which may result in potentially detrimental cellular alterations. Any cytokine changes that are likely occurring in the red blood cells during storage may be responsible, at least in

Journal Pre-proof part, for the progression of this ‘storage lesion’. By ignoring the red blood cells in the analysis of cytokines in the storage lesion, the issues presented by stored red blood cells may never be totally resolved. The primary intention of our red blood cell screening study was to identify previously unknown cytokines associated with red blood cells [29], but what was also realised was the vast donor variability in cytokine concentrations. Although nine of these markers were detected in the red blood cells of every participant, the levels of these in each participant varied significantly. For

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example, HGF had a mean of 831 pg/mL in the red blood cell fraction but had a standard deviation of 423 pg/mL, indicating a vast spread of data from the biological replicates. Furthermore, there were

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36 other cytokines that were reported were not detected in every participant. Highly inflammable

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molecules such as TNF-α and IFN-γ were identified in only 50 % and 70 % of the participants

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respectively. Most of the studies that have analysed the red blood cell pack supernatant for cytokine levels do so using a small number of donors, typically less than 20, and focusing on only 2 or 3

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cytokines [21,23,117]. Considering the limited nature of these studies, there is a risk that they have

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missed some potentially inflammatory donors. Further studies with larger participant numbers and broader cytokine screens are required to better capture the cytokine variability of the red blood cell

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fraction and may be informative in determining optimal donor selection criteria. These findings are likely to have ongoing implications in understanding the storage lesion. 5.1.1

Inflammatory events following transfusion

Regardless of the differences observed during red blood cell storage, red blood cell transfusions can have longer term effects. For example, following hip replacement surgery (arthroplasty), 31 % of people who received red blood cell transfusions experienced wound healing delays and disruptions; this was significantly higher than the non-transfused group [118]. Furthermore, people receiving multiple transfusions typically have higher levels of circulating CD8+ cytotoxic T cells which has been suggested to be the cause of the immunosuppression observed following transfusions [119,120]. In fact, in a systematic review of the literature on the use of red blood cell transfusions, the authors

Journal Pre-proof concluded that these transfusions were strongly correlated with increased morbidity and mortality, and that on average, the risks of transfusion outweighed any potential benefit [121]. Red blood cell transfusion was identified as a primary, independent, risk factor for infection. Of note, this risk of infection was not correlated with transfusion-transmitted infections, but instead with transfusiondependent immunomodulation resulting in increased susceptibility to infection [121,122]. There is also now evidence that intraoperative red blood cell transfusion in cardiac surgery has a significantly increased risk of mortality in high risk groups [123,124]. As a result of these recent

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studies, there has been a push to use a restrictive red-cell transfusion threshold, wherein transfusions are only given when the haemoglobin concentration gets below 7.5 g/dL, as opposed to

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the original level of 9.5 g/dL. The evidence suggests that anaemia at or above 7.5 g/dL does not

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contribute to any additional adverse outcomes following surgery, but that receiving a transfusion at

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that point may propagate negative outcomes particularly if the patient is already high risk of death [123,124]. Similarly, in a study of transfusion in extremely premature infants, restrictive transfusion

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was not only found to be safe, but also to be associated with significantly lower rates of infectious

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disease including sepsis and necrotising enterocolitis [125]. These results and other similar studies demonstrate that the red blood cells that are transfused have long term and potentially lethal

5.2

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immunological effects, although the mechanisms of which are not well understood. Anaemia of chronic inflammation

Anaemia of chronic inflammation is defined as normocytic, normochromic anaemia, where the size and the haemoglobin load of the red blood cells is normal, but the anaemia is caused by there being fewer red blood cells in the circulation. The reduced number of red blood cells i n this condition is a result of both increased red blood cell destruction and impaired differentiation of these cells [126]. Anaemia of chronic inflammation presents as a secondary condition to other chronic inflammatory diseases such as infection, cancer, or auto-immune conditions [127,128]. Whilst there is a lot left to understand about this condition, a key molecule in its pathogenesis has now been identified [129]. High levels of the protein hepcidin slows iron export into local tissue, thus resulting in lower levels of

Journal Pre-proof available iron [130]. This activity is in turn regulated by the presence of pro-inflammatory cytokines such as IL-6 [131]. This condition is then driven in two ways, first, the production of new red blood cells from the bone marrow is restricted by low levels of the red blood cell differentiation hormone, erythropoietin [132], and second, the maturation of red blood cell progenitors in the bone marrow is inhibited in response to inflammatory cytokines signalling [126]. Adequate treatment for the anaemia of chronic inflammation does not yet exist, and the recommendation is to focus on treating the underlying condition, as the anaemia typically improves

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once the primary condition is alleviated [133]. It is not yet understood if this anaemia is a result of the chronic inflammation and is a biological response to mediate the inflammation, or if the chronic

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inflammation is in part a result of the anaemia itself, and if the red blood cells are playing a role in

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the modulation of the levels of inflammatory molecules during disease progression.

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The reduced number of red blood cells observed in this anaemic condition may well contribute to a dysregulated red blood cell-dependent cytokine buffering system, and as such, the chronic

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inflammation may be closely related to the progression of the anaemia. Chronic inflammation can be

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damaging to the normal function of cells. A recent study identified that chronic exposure of the proinflammatory cytokine, IL-1, on haematopoietic stem cells severely restricted the cellular capacity to

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self-renew, but the self-renewal was restored upon cessation of IL-1 exposure [134]. Cytokines from the IL-1 superfamily have now been reported to be associated with red blood cells [29]. Thus, red blood cells may be involved in the mechanism behind managing the over-exposure of IL-1 molecules in vivo, and the dysregulation of which could have detrimental down-stream effects. An example of the effect of attenuating cytokine binding has been observed using a DARC knockout model. Interference with this chemokine sink system has been shown to be involved in the pathogenesis of some inflammatory conditions including prostate cancer [47] and graft rejection [46]. Shen et al. demonstrated that mice lacking DARC had increased prostate cancer tumour growth and tumour vessel density when compared to DARC positive mice [47]. Notable, the concentration of pro-angiogenic chemokines – that are known ligands for DARC – were significantly higher in the

Journal Pre-proof tumour of DARC negative mice [47]. With these results, the authors concluded that the chemokine binding activity of red blood cells was directly related to tumour management by clearing the tumour of pro-angiogenic chemokines and thus restricting angiogenesis. This binding and release of cytokines, chemokines, and growth factors from red blood cells is likely to have even more widespread effects.

6.

Conclusion and future considerations

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There is a small, but growing body of literature that illustrates that mature red blood cells have the capacity to bind and release a range of chemokines, cytokines, and growth factors, and also interact

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directly with a variety of different cells. In this review, we have highlighted a potential relationship

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between the microscopic and macroscopic inflammatory roles of red blood cells. These cells are

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associated with a wide variety of inflammatory molecules which may in part be regulating the levels of inflammatory molecules in blood through a buffering system. But additionally, these cells have

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been shown to directly affect the activity of immune cells and are likely to be directly

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communicating with these cells. Considering how abundant red blood cells are in the body, improved understanding into the role that red blood cells play in the immune system is likely to be

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hugely valuable. This is likely to have larger implications on understanding how red blood cellassociated cytokines affect red blood cell transfusion outcomes or for treating anaemia of chronic inflammation and may be useful in the development of improved storage and transfusion delivery methodologies.

Research agenda 

Although a number of inflammatory proteins have been identified on red blood cells, t he knowledge of the molecular processes behind the binding and release of many of these cytokines from red blood cells remains superficial. More understanding here may help direct developments in red blood cell diagnostics or therapeutics.

Journal Pre-proof 

To date, no studies have looked at the intracellular cytokine levels of red blood cells from stored blood packs. Only extracellular, secreted, cytokines have been quantified. This additional analysis of cytokine levels in donor red blood cells may provide more understanding around the incidence of inflammatory adverse events and may be correlated with transfusion outcomes.



Research into the effect of red blood cells on immune cell function is still preliminary. This research should be repeated with a greater focus on determining the mechanism of action.

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There is evidence that the red blood cell glycocalyx mimics the endothelial glycocalyx which

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binds chemokines and can alter the function of the cells. It is possible that the red blood cell

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glycocalyx may play a role in the binding and release of red blood cell associated cytokines.

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Further investigation into this may be beneficial.

Practice points

Red blood cells are a reservoir of inflammatory cytokines, chemokines, and growth factors.



Red blood cell transfusion can stimulate the occurrence of inflammatory events.



Long term red blood cell transfusions can have on-going adverse effects, including

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

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immunosuppression and increased morbidity and mortality. As such, implementation of a restrictive red-cell transfusion threshold may be beneficial.

Conflict of interest statement E.K. is a shareholder and employee of Sangui Bio Pty Ltd. B.H. is a shareholder and director of Sangui Bio Pty Ltd.

Role of funding source E.K. was a PhD candidate at the University of Sydney and this work was submitted in partial fulfillment of the requirement for the PhD. The sponsor of this study was Sangui Bio Pty Ltd.

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Journal Pre-proof Table 1.

Summary of the literature that has reported a relationship between red blood cells

and cytokines.

Competitive binding assay for DARC

CC chemokines CXC chemokines

Competitive binding assay for DARC

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CC chemokines CXC chemokines

CC chemokines CXC chemokines

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GROa (CXCL1) IL-8 (CXCL8) MCP-1 (CCL2) NAP-2 (CXCL7) RANTES (CCL5) Chaudhuri, A. et GROa (CXCL1) al. (1994) [39] IL-8 (CXCL8) MCP-1 (CCL2) Platelet factor 4 (CXCL4) RANTES (CCL5) Neote, K. et al. GROa (CXCL1) (1994) [38] IL-8 (CXCL8) MCP-1 (CCL2) MIP-1a (CCL3) RANTES (CCL5) Szabo, M. et al. GROa (CXCL1) (1995) [41] I-309 (CCL1) MCP-1 (CCL2) Platelet factor 4 (CXCL4) RANTES (CCL5) Bjork, P. et al. DDT (MIF-2) (1996) [91]

CXC chemokines

Methods used in analysis In vitro loss of MIF activity in the presence of RBCs Radioimmunoassay

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IL-8 (CXCL8)

Cytokine families MIF family

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Cytokines identified MIF

CC chemokines CXC chemokines

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Rivera, A. et al. (2002) [67]

Fukuma, N. et al. (2003) [40]

Shen, H. et al. (2006) [47]

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Mizue, Y. et al. (2000) [78]

MIF

Competitive binding assay for DARC

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Reference Fox, R. A. et al. (1974) [77] Darbonne, W. C. et al. (1991) [36] Neote, K. et al. (1993) [37]

MIF family

MIF family

Endothelin-1 IL-10 Platelet activating factor RANTES (CCL5) Eoxtain (CCL11) MCP-1 (CCL2)

CC chemokines Endothelin family IL-10 family PAF family CC chemokines

CXCL2 GROa (CXCL1) IL-8 (CXCL8)

CXC chemokines

Competitive binding assay for DARC on mouse erythrocytes

Assessment of DDT enzymatic activity as well as protein isolation and sequencing Correlation between haemolysis and MIF concentration according to ELISA quantification and subsequent protein purification and identification Activity of Gardos channel in the presence of cytokines Clearance of chemokines from plasma in DARC knockout and wildtype mice In vitro clearance of chemokines using erythrocytes from DARC

Journal Pre-proof Cytokine families

IL-8 (CXCL8) MCP-1 (CCL2)

CC chemokines CXC chemokines

Reutershan, J. et al. (2009) [56]

CXCL2 GROa (CXCL1)

CXC chemokines

Durpès, M. C. et al. (2010) [68]

IL-8 (CXCL8) RANTES (CCL5)

CC chemokines CXC chemokines

Schnabel, R. B. et al. (2010) [53]

MCP-1 (CCL2)

CC chemokines

Xiong, Z. et al. (2011) [42]

CXCL2 CXCL5 GROa (CXCL1) IL-8 (CXCL8) MCP-1 (CCL2) NAP-2 (CXCL7) MIF

Karsten, E. et al. (2018) [28]

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CC chemokines CXC chemokines

MIF family

Correlation between haemolysis and MIF levels according to ELISA quantification IL-1 cytokine Correlation between superfamily haemolysis and IL-33 using immunoblotting and densitometry

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Wei, J. et al. (2015) [92]

IL-33

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Sobierajski, J. et al. (2013) [79]

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Dawson, T. C. et al. (2009) [43]

Methods used in analysis knockout and wildtype mice and quantification of tumour concentration of chemokines in DARC knockout and wildtype mice Competitive binding assay for DARC using DARC knockout mice Quantification of cytokines in red blood cell lysates and plasma in DARC knockout and wildtype mice Activity of Gardos channel channel in the presence of cytokines Genetic correlation between MCP-1 concentration and DARC polymorphism Competitive binding assay for DARC using both intact red blood cells and red blood cell microparticles

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Cytokines identified

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Reference

MIF

MIF family

Quantification of MIF in red blood cell lysates by ELISA and subsequent confirmation enzymatic activity

Journal Pre-proof

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Methods used in analysis Quantification of cytokines in red blood cell lysates and conditioned media using Luminex®

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Cytokine families CC chemokines CSF family CXC chemokine FGF growth factors GM-CSF/IL-15/IL-3 family IFN family (Type I) IFN family (Type II) IL-1 superfamily IL-6 family IL-12 family IL-17 family MIF family PDGF growth factors TNF superfamily

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Cytokines identified CTACK IL-16 (CCL27) IL-17 Eotaxin-1 IL-18 (CCL11) IP-10 bFGF (CXCL10) G-CSF LIF GM-CSF MCP-1 GROa (CCL2) (CXCL1) MCP-3 HGF (CCL7) IFN-a2 M-CSF IFN-y MIF IL-1b MIG IL-1ra (CXCL9) IL-2 MIP-1a sIL-2ra (CCL3) IL-3 MIP-1b IL-4 (CCL4) IL-5 b-NGF IL-7 PDGF-bb IL-8 (CXCL8) RANTES IL-9 (CCL5) IL-12(p40) SCF IL-12(p70) SCGF-b IL-13 SDF-1a IL-15 (CXCL12) TNF-a TNF-b TRAIL VEGF

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Reference Karsten, E. et al. (2018) [29]

Summary schematic of the understanding of the interaction between cytokines and

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Figure 1.

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Journal Pre-proof

red blood cells until 2019. At this point, only 17 cytokines had been demonstrated to have a

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relationship with (1) red blood cells or (2) their microparticles. Most of these cytokines were C-X-C or

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C-C chemokines that were found to bind to the Duffy antigen receptor for chemokines (DARC) on the surface of the cells or on the surface of red blood cell microparticles. The remaining seven cytokines

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were found to influence red blood cell dehydration via activation of the Gardos channel or to primarily reside inside these cells (MIF, DDT, and IL-33).

Karsten et al. [29] expanded the list of cytokines found to be associated with red

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Figure 2.

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blood cells - up to 45 cytokines - by using Luminex® technology to screen red blood cell lysates. It

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was also determined that a number of these factors could bind to and be released from these cells.

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Although these additional cytokines were identified, the receptor(s) they are associated with on red

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blood cells are yet to be determined.

Figure 1

Figure 2