**3. The erythrocyte and virus**

The erythrocyte does not contain a nucleus or other organelles necessary for virus replication and survival. Its interaction with viruses is therefore mainly restricted to adherence of virus to the plasma membrane, transmembrane proteins or glycocalyx carbohydrates like sialic acid (**Figure 2**). Erythroid progenitor cells may however be infected with parvovirus B19 [28] which may lead to anemia. The ongoing Covid-19 pandemic has renewed interest in erythrocyte-virus interactions, due to the hypoxia or acute respiratory distress syndrome (ARDS) often seen in the disease [29]. Erythrocytes from Covid-19 patients showed increased protein degradation and glycolysis [29]. Increased or decreased levels of many lipids were seen [29]. Some signs of oxidative stress was noted, such as increased oxidized glutathione and increased protein levels of peroxiredoxin 2. Interestingly, N-terminal oxidation or degradation of band3 was noted (**Figure 2**). The cytosolic, N-terminal part of band3 has been shown to be of importance in signaling and hemoglobin regulation [30] and its oxidation or degradation may therefore explain some of

**13**

*Erythrocytes as Biomarkers of Virus and Bacteria in View of Metal Ion Homeostasis*

the other noted effects of SARS-CoV-2 on the erythrocyte (**Figure 2**). Based on metabolic and molecular pathway analysis [29], protein degradation pathways, such as proteasome and ubiquitinylation components were identified as significantly affected in erythrocytes of Covid-19 patients (**Figure 2**). In addition, effects were noted on ferroptosis, cyclic-adenosine monophosphate (cAMP) and AMP-activated protein kinase signaling cascades. Lipid metabolism was also affected, especially acyl-carnitine and sphingolipid metabolism [29]. The effect on ferroptosis was based mainly on increased levels of peroxidated lipids and increased levels of heat shock protein beta-1, which inhibits ferroptosis by reducing intracellular levels of iron and maintaining levels of reduced glutathione [31]. The precise mechanism may be through inhibition of transferrin-receptor mediated iron uptake via stabilization of the cytoskeleton [31]. Ferroptosis is an iron-dependent non-apoptotic version of regulated cell death. It is caused by increased levels of iron accompanied by reduced activity of the selenium-dependent enzyme glutathione-peroxidase 4 and mitochondrial changes such as reduced mitochondrial size and mitochondrial membrane rupture. Protein levels of glutathione peroxidase 4 were unchanged in erythrocytes of Covid-19 patients [29]. The absence of mitochondria in the erythrocyte would argue against the existence of ferroptosis in the erythrocyte. In the Covid-19 infected patients, there were no alterations of directly clinically relevant hematological parameters, such as erythrocyte count, hematocrit, or mean corpuscular hemoglobin concentration [29]. The reason for hypoxia or ARDS in Covid-19 patients may therefore not be found in the erythrocyte, possibly with exception of the severe hypoxia without impaired lung function that has been seen in some patients [32]. Respiratory failure is the most common direct cause of death in Covid-19 patients [33, 34]. Studies have reported the nuclear remnants called Howell-Jolly bodies, or seemingly fully nucleated erythrocytes in Covid-19 patients [35, 36]. Nucleated erythrocytes indicate a bad prognosis and often occurs in critically ill patients in several diseases [37]. Mortality risk in Covid-19 seems to be associated with higher erythrocyte distribution width (RDW), a measure of heterogeneity of erythrocyte volume (**Figure 2**). In a meta-analysis of Covid-19 patients, higher RDW was associated with more severe disease [38]. The precise reason for this is unknown, but may involve reduced erythropoiesis or impaired erythrocyte clearance. A more prominent role of erythrocytes in the disease progression can therefore not be ruled out. For instance, in post-mortem analysis of Covid-19 patients with kidney injury, intact erythrocytes were found obstructing peritubular and glomerular capillary lumens [39]. A study of blood samples from Covid-19 patients showed depletion of several immune factors, among them interferon-alpha [40], an interferon that was previously localized in erythrocytes [9]. However, erythrocyte cytokine levels have so far not been analyzed in Covid-19 patients. Anemia in Covid-19 patients may be related to the increased interleukin-6 levels (**Figure 2**) that have been reported in the disease [41]. Interleukin-6 downregulates the iron exporter ferroportin via hepcidin [42]. Lower ferroportin activity makes iron stored in macrophages less available for erythropoiesis and for existing erythrocytes [43, 44]. Increased levels of interleukin-6 have also been implicated as being responsible for the cytokine release syndrome seen in some Covid-19 patients, particularly those affected by ARDS or severe hypoxia [41, 45]. The interleukin-6 receptor blocker tocilizumab showed disappointing results in Covid-19 patients [46, 47] suggesting a more complex pathophysiology of cytokine release syndrome in Covid-19 patients. In HIV-1 patients, anemia often appears together with secondary infections or nutritional deficiencies [44, 48], or is caused by treatment with zidovudine, which is a known cause of bone-marrow suppression [48]. Reduced erythropoietin levels in HIV-1-related anemia may have been caused by upregulation of proinflammatory cytokines like interleukin-1-beta and TNF-alpha [49] or by

*DOI: http://dx.doi.org/10.5772/intechopen.97850*

#### *Erythrocytes as Biomarkers of Virus and Bacteria in View of Metal Ion Homeostasis DOI: http://dx.doi.org/10.5772/intechopen.97850*

the other noted effects of SARS-CoV-2 on the erythrocyte (**Figure 2**). Based on metabolic and molecular pathway analysis [29], protein degradation pathways, such as proteasome and ubiquitinylation components were identified as significantly affected in erythrocytes of Covid-19 patients (**Figure 2**). In addition, effects were noted on ferroptosis, cyclic-adenosine monophosphate (cAMP) and AMP-activated protein kinase signaling cascades. Lipid metabolism was also affected, especially acyl-carnitine and sphingolipid metabolism [29]. The effect on ferroptosis was based mainly on increased levels of peroxidated lipids and increased levels of heat shock protein beta-1, which inhibits ferroptosis by reducing intracellular levels of iron and maintaining levels of reduced glutathione [31]. The precise mechanism may be through inhibition of transferrin-receptor mediated iron uptake via stabilization of the cytoskeleton [31]. Ferroptosis is an iron-dependent non-apoptotic version of regulated cell death. It is caused by increased levels of iron accompanied by reduced activity of the selenium-dependent enzyme glutathione-peroxidase 4 and mitochondrial changes such as reduced mitochondrial size and mitochondrial membrane rupture. Protein levels of glutathione peroxidase 4 were unchanged in erythrocytes of Covid-19 patients [29]. The absence of mitochondria in the erythrocyte would argue against the existence of ferroptosis in the erythrocyte. In the Covid-19 infected patients, there were no alterations of directly clinically relevant hematological parameters, such as erythrocyte count, hematocrit, or mean corpuscular hemoglobin concentration [29]. The reason for hypoxia or ARDS in Covid-19 patients may therefore not be found in the erythrocyte, possibly with exception of the severe hypoxia without impaired lung function that has been seen in some patients [32]. Respiratory failure is the most common direct cause of death in Covid-19 patients [33, 34]. Studies have reported the nuclear remnants called Howell-Jolly bodies, or seemingly fully nucleated erythrocytes in Covid-19 patients [35, 36]. Nucleated erythrocytes indicate a bad prognosis and often occurs in critically ill patients in several diseases [37]. Mortality risk in Covid-19 seems to be associated with higher erythrocyte distribution width (RDW), a measure of heterogeneity of erythrocyte volume (**Figure 2**). In a meta-analysis of Covid-19 patients, higher RDW was associated with more severe disease [38]. The precise reason for this is unknown, but may involve reduced erythropoiesis or impaired erythrocyte clearance. A more prominent role of erythrocytes in the disease progression can therefore not be ruled out. For instance, in post-mortem analysis of Covid-19 patients with kidney injury, intact erythrocytes were found obstructing peritubular and glomerular capillary lumens [39]. A study of blood samples from Covid-19 patients showed depletion of several immune factors, among them interferon-alpha [40], an interferon that was previously localized in erythrocytes [9]. However, erythrocyte cytokine levels have so far not been analyzed in Covid-19 patients. Anemia in Covid-19 patients may be related to the increased interleukin-6 levels (**Figure 2**) that have been reported in the disease [41]. Interleukin-6 downregulates the iron exporter ferroportin via hepcidin [42]. Lower ferroportin activity makes iron stored in macrophages less available for erythropoiesis and for existing erythrocytes [43, 44]. Increased levels of interleukin-6 have also been implicated as being responsible for the cytokine release syndrome seen in some Covid-19 patients, particularly those affected by ARDS or severe hypoxia [41, 45]. The interleukin-6 receptor blocker tocilizumab showed disappointing results in Covid-19 patients [46, 47] suggesting a more complex pathophysiology of cytokine release syndrome in Covid-19 patients. In HIV-1 patients, anemia often appears together with secondary infections or nutritional deficiencies [44, 48], or is caused by treatment with zidovudine, which is a known cause of bone-marrow suppression [48]. Reduced erythropoietin levels in HIV-1-related anemia may have been caused by upregulation of proinflammatory cytokines like interleukin-1-beta and TNF-alpha [49] or by

*Erythrocyte - A Peripheral Biomarker for Infection and Inflammation*

Blood group O favors rosetting less than the A or B blood groups. Interestingly, the same pattern can be seen in Covid-19, where blood groups A or B are risk factors, whereas group O appears to be comparatively protective [26]. Glycophorin A is the most abundant sialo-glycoprotein on erythrocytes (**Figure 1**). Sialic acid residues are often used as receptors for invasion by bacteria and other pathogens [27] (**Figure 2**). Sequence analysis of glycophorin A from several primate species revealed high diversifying selection in the glycophorin A gene [27]. High selective pressure could be explained as a consequence of targeting of different pathogens by the glycophorin A receptor. This led to the pathogen decoy hypothesis, which states that the erythrocyte functions as a "flypaper", using glycophorin A to bind pathogens [27]. Pathogens may then be cleared by macrophages as erythrocytes

*Schematic figure of the erythrocyte and its interactions with viruses. Adenovirus and parvovirus lack a lipid bilayer around the virus capsid. Covid-19 disease affects the erythrocyte without entry of SARS-CoV-2 into the* 

The erythrocyte does not contain a nucleus or other organelles necessary for virus replication and survival. Its interaction with viruses is therefore mainly restricted to adherence of virus to the plasma membrane, transmembrane proteins or glycocalyx carbohydrates like sialic acid (**Figure 2**). Erythroid progenitor cells may however be infected with parvovirus B19 [28] which may lead to anemia. The ongoing Covid-19 pandemic has renewed interest in erythrocyte-virus interactions, due to the hypoxia or acute respiratory distress syndrome (ARDS) often seen in the disease [29]. Erythrocytes from Covid-19 patients showed increased protein degradation and glycolysis [29]. Increased or decreased levels of many lipids were seen [29]. Some signs of oxidative stress was noted, such as increased oxidized glutathione and increased protein levels of peroxiredoxin 2. Interestingly, N-terminal oxidation or degradation of band3 was noted (**Figure 2**). The cytosolic, N-terminal part of band3 has been shown to be of importance in signaling and hemoglobin regulation [30] and its oxidation or degradation may therefore explain some of

**12**

pass through the spleen.

**Figure 2.**

**3. The erythrocyte and virus**

*erythrocyte. Abbreviations are explained in the text.*

autoantibodies to erythropoietin [50]. HIV-1 may infect hematopoietic progenitor cells, but this is not considered a critical cause of anemia in HIV-1 patients [51].

Using biophysical modeling, band3 was suggested to be the point of attachment for SARS-CoV-2 on the erythrocyte surface [32]. The oxidation or degradation noted in band3 [29] may therefore have been caused directly by the binding of the virus. It should be noted that no entry of SARS-CoV-2 into the erythrocyte has as yet been proven (**Figure 2**). The effect of the virus on the erythrocyte may hence be due only to binding to surface receptors. The angiotensin-converting enzyme 2 (ACE2) membrane protein that is considered necessary for entry does not occur in erythrocyte membranes according to the erythrocyte proteome database (http:// rbcc.hegelab.org/) [52], making ACE2 an unlikely entry or attachment point for SARS-CoV-2 on the erythrocyte surface [32]. Basigin, one of the erythrocyte receptors for *P. falciparum*, is not considered a receptor for SARS-CoV-2 [53], although such claims have been advanced [54]. The differential susceptibility conferred by the ABO blood group system to Covid-19 may imply a participation of the ABO glycosylation in attachment of the virus to the erythrocyte or other cells [26]. Band3, the suggested erythrocyte attachment protein for SARS-CoV-2, contains ABO glycosylation on the N-642 asparagine residue in the extracellular loop between transmembrane helices TM7 and TM8 [30]. This does not by itself necessarily imply involvement of the erythrocyte in Covid-19 pathogenesis since the ABO blood group system is expressed also on other membrane proteins and in other cells in the human body [55]. In contrast, the ABO blood group system seems not to be of importance for infection by the HIV-1 virus [56].

The erythrocyte has been suggested to be a hiding place for viruses, although these would not be able to infect the erythrocyte. Adsorption to erythrocytes has been shown for HIV-1, influenza and adenovirus [57–59] (**Figure 2**). Zika virus has been shown to survive in erythrocyte samples [60]. Zika virus could be detected in erythrocytes for a significantly longer time than in serum. Considering the ongoing SARS-CoV-2 pandemic, it would be of interest for applied transfusion and blood bank science to know the persistence of the SARS-CoV-2 virus on erythrocytes.

## **4. Erythrocyte selenium, metal ion and trace element status in relation to virus exposure**

Metal ions, trace elements and other compounds contribute in different hierarchies to complex reactions that maintain blood homeostasis (**Figure 3**) [61]. Erythrocyte redox homeostasis is maintained through ascorbic acid, superoxide dismutase, catalase, glutathione and glutathione peroxidase. Antioxidant activity of selenium-dependent glutathione peroxidase may protect band3 of erythrocytes. Selenium is therefore important even for the basic oxygen and carbon dioxide exchange performed by the erythrocyte. Selenium as organic or inorganic form is metabolized by erythrocytes, platelets and neutrophils in different ways [62]. In one study, selenium was supplied together with plain food as inorganic sodium selenite at 200 microgram/day for one year. Neutrophils accumulated selenium more than platelets followed by erythrocytes [62]. In a similar study where selenium was supplied as 1-selenomethionine at 50 microgram/day for one year, platelets accumulated most selenium followed by erythrocytes and neutrophils [63]. These observations indicate that different cells metabolize inorganic and organic forms of selenium differently. The absence of nucleus and organelles in erythrocytes may partly explain the difference. Selenium supplied as inorganic sodium selenite was associated with decreased iron concentration in platelets and neutrophils.

**15**

spectrometry) [61].

**Figure 3.**

intake may worsen the disease progress.

of microRNA function are still not fully understood.

*Erythrocytes as Biomarkers of Virus and Bacteria in View of Metal Ion Homeostasis*

This result may indicate an iron-regulating function of selenium which may be particularly relevant for the erythrocyte considering its high iron content. Analysis of selenium and other elements was performed by micro-PIXE (micro-particle induced X-ray emission). Increased levels of Cd, Pb and Ag were reported in a study of erythrocytes in patients with Alzheimer's or Parkinson's disease, using the more sensitive and selective ICP-MS (inductively coupled plasma mass

*may be restored or adapted, but large damages will present symptoms and are difficult to restore.*

*The flow of compounds and metal ions between cells is dependent on the three main chemical pathways in a hierarchy. The first path involves free radical-induced production of compounds where hydroxyl radical and solvated electron reactions may be involved. The second path involves electrophilic and nucleophilic compounds forming products adequate to the cells. The third path involves metal ions and ligands dependent on the previous two pathways but adapted to cell demand. In erythroid precursors and in other cells, excluding the erythrocyte, compounds and metal ions may reach DNA and RNA not controlled by evolutionary developed genome adapted to cell demand. Small changes of DNA and RNA may take place, an epigenetic change, which* 

In view of these observations it is interesting that Covid-19 patients with higher selenium and selenoprotein P levels had lower mortality risk [64]. A correlation has also been found between cure rate and hair selenium concentration in Chinese Covid-19 patients [65]. As virus bind to erythrocytes, virus can be transported to different organs e.g. lung, kidney and brain. Viral particles may then find their way through the blood–brain-barrier or enter through the choroid plexus, an important part of the brain filtration system. The SARS-CoV-2 virus may decrease oxygen supply when attached to erythrocytes and impair brain oxygen supply. Low selenium

Erythropoiesis is partly regulated by microRNA and dependent on a high selenium status [66]. For instance, using a mouse loss-of-function allele,

microRNA-142 was found to be required for maintaining typical erythrocyte shape, for normal metabolism of reactive oxygen species, and for overall erythrocyte lifespan [67]. MicroRNAs are differentially expressed depending on selenium status [68]. However, a precise role of selenium in regulating microRNA in relation to erythropoiesis has not been described. In addition, mechanisms underlying much

*DOI: http://dx.doi.org/10.5772/intechopen.97850*

*Erythrocytes as Biomarkers of Virus and Bacteria in View of Metal Ion Homeostasis DOI: http://dx.doi.org/10.5772/intechopen.97850*

#### **Figure 3.**

*Erythrocyte - A Peripheral Biomarker for Infection and Inflammation*

importance for infection by the HIV-1 virus [56].

**to virus exposure**

autoantibodies to erythropoietin [50]. HIV-1 may infect hematopoietic progenitor cells, but this is not considered a critical cause of anemia in HIV-1 patients [51].

for SARS-CoV-2 on the erythrocyte surface [32]. The oxidation or degradation noted in band3 [29] may therefore have been caused directly by the binding of the virus. It should be noted that no entry of SARS-CoV-2 into the erythrocyte has as yet been proven (**Figure 2**). The effect of the virus on the erythrocyte may hence be due only to binding to surface receptors. The angiotensin-converting enzyme 2 (ACE2) membrane protein that is considered necessary for entry does not occur in erythrocyte membranes according to the erythrocyte proteome database (http:// rbcc.hegelab.org/) [52], making ACE2 an unlikely entry or attachment point for SARS-CoV-2 on the erythrocyte surface [32]. Basigin, one of the erythrocyte receptors for *P. falciparum*, is not considered a receptor for SARS-CoV-2 [53], although such claims have been advanced [54]. The differential susceptibility conferred by the ABO blood group system to Covid-19 may imply a participation of the ABO glycosylation in attachment of the virus to the erythrocyte or other cells [26]. Band3, the suggested erythrocyte attachment protein for SARS-CoV-2, contains ABO glycosylation on the N-642 asparagine residue in the extracellular loop between transmembrane helices TM7 and TM8 [30]. This does not by itself necessarily imply involvement of the erythrocyte in Covid-19 pathogenesis since the ABO blood group system is expressed also on other membrane proteins and in other cells in the human body [55]. In contrast, the ABO blood group system seems not to be of

Using biophysical modeling, band3 was suggested to be the point of attachment

The erythrocyte has been suggested to be a hiding place for viruses, although these would not be able to infect the erythrocyte. Adsorption to erythrocytes has been shown for HIV-1, influenza and adenovirus [57–59] (**Figure 2**). Zika virus has been shown to survive in erythrocyte samples [60]. Zika virus could be detected in erythrocytes for a significantly longer time than in serum. Considering the ongoing SARS-CoV-2 pandemic, it would be of interest for applied transfusion and blood bank science to know the persistence of the SARS-CoV-2 virus on erythrocytes.

**4. Erythrocyte selenium, metal ion and trace element status in relation** 

Metal ions, trace elements and other compounds contribute in different hierarchies to complex reactions that maintain blood homeostasis (**Figure 3**) [61]. Erythrocyte redox homeostasis is maintained through ascorbic acid, superoxide dismutase, catalase, glutathione and glutathione peroxidase. Antioxidant activity of selenium-dependent glutathione peroxidase may protect band3 of erythrocytes. Selenium is therefore important even for the basic oxygen and carbon dioxide exchange performed by the erythrocyte. Selenium as organic or inorganic form is metabolized by erythrocytes, platelets and neutrophils in different ways [62]. In one study, selenium was supplied together with plain food as inorganic sodium selenite at 200 microgram/day for one year. Neutrophils accumulated selenium more than platelets followed by erythrocytes [62]. In a similar study where selenium was supplied as 1-selenomethionine at 50 microgram/day for one year, platelets accumulated most selenium followed by erythrocytes and neutrophils [63]. These observations indicate that different cells metabolize inorganic and organic forms of selenium differently. The absence of nucleus and organelles in erythrocytes may partly explain the difference. Selenium supplied as inorganic sodium selenite was associated with decreased iron concentration in platelets and neutrophils.

**14**

*The flow of compounds and metal ions between cells is dependent on the three main chemical pathways in a hierarchy. The first path involves free radical-induced production of compounds where hydroxyl radical and solvated electron reactions may be involved. The second path involves electrophilic and nucleophilic compounds forming products adequate to the cells. The third path involves metal ions and ligands dependent on the previous two pathways but adapted to cell demand. In erythroid precursors and in other cells, excluding the erythrocyte, compounds and metal ions may reach DNA and RNA not controlled by evolutionary developed genome adapted to cell demand. Small changes of DNA and RNA may take place, an epigenetic change, which may be restored or adapted, but large damages will present symptoms and are difficult to restore.*

This result may indicate an iron-regulating function of selenium which may be particularly relevant for the erythrocyte considering its high iron content. Analysis of selenium and other elements was performed by micro-PIXE (micro-particle induced X-ray emission). Increased levels of Cd, Pb and Ag were reported in a study of erythrocytes in patients with Alzheimer's or Parkinson's disease, using the more sensitive and selective ICP-MS (inductively coupled plasma mass spectrometry) [61].

In view of these observations it is interesting that Covid-19 patients with higher selenium and selenoprotein P levels had lower mortality risk [64]. A correlation has also been found between cure rate and hair selenium concentration in Chinese Covid-19 patients [65]. As virus bind to erythrocytes, virus can be transported to different organs e.g. lung, kidney and brain. Viral particles may then find their way through the blood–brain-barrier or enter through the choroid plexus, an important part of the brain filtration system. The SARS-CoV-2 virus may decrease oxygen supply when attached to erythrocytes and impair brain oxygen supply. Low selenium intake may worsen the disease progress.

Erythropoiesis is partly regulated by microRNA and dependent on a high selenium status [66]. For instance, using a mouse loss-of-function allele, microRNA-142 was found to be required for maintaining typical erythrocyte shape, for normal metabolism of reactive oxygen species, and for overall erythrocyte lifespan [67]. MicroRNAs are differentially expressed depending on selenium status [68]. However, a precise role of selenium in regulating microRNA in relation to erythropoiesis has not been described. In addition, mechanisms underlying much of microRNA function are still not fully understood.
