**7. Protein interactions and signaling mechanisms of possible immunological relevance**

Band3 is the most abundant protein of the erythrocyte plasma membrane. The function of band3 is the excretion of bicarbonate formed from CO2 by carbonic anhydrase. A chloride ion is simultaneously imported. The process is reversed in the lungs where CO2 is released. Band3 has been implicated in intracellular signaling based on its cytosolic N-terminal domain, which consists of the first 360 of the 911 amino acid residues of the protein [30]. It was shown to interact with deoxyhemoglobin and enzymes of the glycolytic pathway. The much shorter C-terminal tail (amino acid residues 873–911) interacts with carbonic anhydrase II (CA) (**Figure 4**), forming a metabolon [102]. Band3 also interacts with ankyrin and spectrin [103], which stabilizes the band3 tetramer structure, calnexin during its maturation [30] and stomatin (**Figure 4**), which modulates the anion exchange activity of band3 [104]. Band3 often occurs in complex with glycophorin A presumably contributing to stabilization of the band3 tetramer in the plasma membrane [30]. In stored blood, band3 was found to interact also with flotillin-2, alphaadducin and adenylosuccinate lyase [15].

#### **Figure 4.**

*Schematic figure of protein interactions and signaling mechanisms of the erythrocyte. Abbreviations are explained in the text.*

**19**

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

regulate coagulation by supporting activated Protein C [110].

exposure and subsequent clearance by macrophages [112].

**8. Senescence, aging, eryptosis and hemolysis of the erythrocyte**

also comes from mutations in PIEZO1 [113] and the Gardos channel.

The erythrocyte has an average lifespan of around 120 days. As erythrocytes age they become less deformable, shrink in size and display dysfunctional hemoglobin accumulations. Calcium regulation may be part of what determines erythrocyte lifespan (**Figure 5**). Normally calcium concentrations are much lower in erythrocyte cytosol than in the surrounding fluid. During erythrocyte aging, intracellular levels of calcium increase. The events leading to this increase are not fully understood. One proposed mechanism is activation of the mechanosensitive PIEZO1 ion channel [113] through decreased deformability of the aging erythrocyte (**Figure 5**). PIEZO1 is a rather unspecific ion channel and can therefore cause general dissipation of ion gradients. In the case of calcium this would mean higher intracellular concentration. Gradually dysfunctional plasma membrane calcium efflux channels may also contribute to the higher calcium levels. Higher intracellular calcium concentrations are known to activate the Gardos channel, a potassium efflux channel in the plasma membrane involved in the regulation of cell volume in erythrocytes (**Figure 5**) and some other cells. Activation of the Gardos channel leads to potassium export and concomitant loss of water and chloride, together known as the Gardos effect. As they grow older, erythrocytes accordingly become more dehydrated. Support for this

A connection between blood coagulation and the immune system was shown by the finding that macrophages produce tissue factor, a transmembrane protein that starts the extrinsic coagulation cascade [105]. A connection between erythrocytes and coagulation is suggested by a correlation between decreased hematocrit and longer bleeding times. Erythrocytes receive signals in the form of lysophosphatidic acid and prostaglandin E2 from activated platelets [106] (**Figure 4**). Increasing cytosol calcium concentration then increases interaction forces between erythrocytes, thereby contributing to thrombus formation [107]. Increasing intracellular calcium levels also lead to phosphatidylserine exposure on the plasma membrane of the erythrocyte (**Figure 4**). Phosphatidylserine then acts as a binding surface for gamma-carboxyglutamyl residues of the vitamin K-dependent carboxylation/ gamma-carboxyglutamic (Gla) domains of coagulation factors [108] (**Figure 4**). This initiates the formation of thrombin from prothrombin [109]. Contribution to coagulation also comes from erythrocyte micro-vesicles that similarly expose phosphatidylserine on the surface. However, erythrocyte micro-vesicles negatively

Erythrocytes respond to adenosine nucleotides by the adenosine tri-phosphate (ATP)-gated P2X and P2Y cation channel receptors in the erythrocyte plasma membrane. ATP is released from erythrocytes in an autocrine or paracrine mechanism of action in response to bacterial exotoxins called hemolysins [111]. They are produced by many bacteria and display several mechanisms of action. Alpha-hemolysin of *Escherichia coli* cause an increase in cellular calcium levels and opening of potassium and chloride ion channels [112]. ATP release then occurs either through pannexin channels (**Figure 4**) or through pores formed by hemolysins in the erythrocyte plasma membrane [111]. The subsequent activation of P2X receptors leads to exposure of phosphatidylserine on the surface of erythrocytes, making possible clearance by macrophages. Phosphatidylserine exposure can be prevented by P2X receptor blockers [112]. Purinergic signaling may be a way to avoid the damaging effects of intravascular hemolysis that would result without phosphatidylserine

*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*

A connection between blood coagulation and the immune system was shown by the finding that macrophages produce tissue factor, a transmembrane protein that starts the extrinsic coagulation cascade [105]. A connection between erythrocytes and coagulation is suggested by a correlation between decreased hematocrit and longer bleeding times. Erythrocytes receive signals in the form of lysophosphatidic acid and prostaglandin E2 from activated platelets [106] (**Figure 4**). Increasing cytosol calcium concentration then increases interaction forces between erythrocytes, thereby contributing to thrombus formation [107]. Increasing intracellular calcium levels also lead to phosphatidylserine exposure on the plasma membrane of the erythrocyte (**Figure 4**). Phosphatidylserine then acts as a binding surface for gamma-carboxyglutamyl residues of the vitamin K-dependent carboxylation/ gamma-carboxyglutamic (Gla) domains of coagulation factors [108] (**Figure 4**). This initiates the formation of thrombin from prothrombin [109]. Contribution to coagulation also comes from erythrocyte micro-vesicles that similarly expose phosphatidylserine on the surface. However, erythrocyte micro-vesicles negatively regulate coagulation by supporting activated Protein C [110].

Erythrocytes respond to adenosine nucleotides by the adenosine tri-phosphate (ATP)-gated P2X and P2Y cation channel receptors in the erythrocyte plasma membrane. ATP is released from erythrocytes in an autocrine or paracrine mechanism of action in response to bacterial exotoxins called hemolysins [111]. They are produced by many bacteria and display several mechanisms of action. Alpha-hemolysin of *Escherichia coli* cause an increase in cellular calcium levels and opening of potassium and chloride ion channels [112]. ATP release then occurs either through pannexin channels (**Figure 4**) or through pores formed by hemolysins in the erythrocyte plasma membrane [111]. The subsequent activation of P2X receptors leads to exposure of phosphatidylserine on the surface of erythrocytes, making possible clearance by macrophages. Phosphatidylserine exposure can be prevented by P2X receptor blockers [112]. Purinergic signaling may be a way to avoid the damaging effects of intravascular hemolysis that would result without phosphatidylserine exposure and subsequent clearance by macrophages [112].

### **8. Senescence, aging, eryptosis and hemolysis of the erythrocyte**

The erythrocyte has an average lifespan of around 120 days. As erythrocytes age they become less deformable, shrink in size and display dysfunctional hemoglobin accumulations. Calcium regulation may be part of what determines erythrocyte lifespan (**Figure 5**). Normally calcium concentrations are much lower in erythrocyte cytosol than in the surrounding fluid. During erythrocyte aging, intracellular levels of calcium increase. The events leading to this increase are not fully understood. One proposed mechanism is activation of the mechanosensitive PIEZO1 ion channel [113] through decreased deformability of the aging erythrocyte (**Figure 5**). PIEZO1 is a rather unspecific ion channel and can therefore cause general dissipation of ion gradients. In the case of calcium this would mean higher intracellular concentration. Gradually dysfunctional plasma membrane calcium efflux channels may also contribute to the higher calcium levels. Higher intracellular calcium concentrations are known to activate the Gardos channel, a potassium efflux channel in the plasma membrane involved in the regulation of cell volume in erythrocytes (**Figure 5**) and some other cells. Activation of the Gardos channel leads to potassium export and concomitant loss of water and chloride, together known as the Gardos effect. As they grow older, erythrocytes accordingly become more dehydrated. Support for this also comes from mutations in PIEZO1 [113] and the Gardos channel.

*Erythrocyte - A Peripheral Biomarker for Infection and Inflammation*

sepsis-specific therapy exists despite many clinical trials.

**immunological relevance**

adducin and adenylosuccinate lyase [15].

**7. Protein interactions and signaling mechanisms of possible** 

Band3 is the most abundant protein of the erythrocyte plasma membrane. The function of band3 is the excretion of bicarbonate formed from CO2 by carbonic anhydrase. A chloride ion is simultaneously imported. The process is reversed in the lungs where CO2 is released. Band3 has been implicated in intracellular signaling based on its cytosolic N-terminal domain, which consists of the first 360 of the 911 amino acid residues of the protein [30]. It was shown to interact with deoxyhemoglobin and enzymes of the glycolytic pathway. The much shorter C-terminal tail (amino acid residues 873–911) interacts with carbonic anhydrase II (CA) (**Figure 4**), forming a metabolon [102]. Band3 also interacts with ankyrin and spectrin [103], which stabilizes the band3 tetramer structure, calnexin during its maturation [30] and stomatin (**Figure 4**), which modulates the anion exchange activity of band3 [104]. Band3 often occurs in complex with glycophorin A presumably contributing to stabilization of the band3 tetramer in the plasma membrane [30]. In stored blood, band3 was found to interact also with flotillin-2, alpha-

*Schematic figure of protein interactions and signaling mechanisms of the erythrocyte. Abbreviations are* 

diseases, although more so in the early stages of sepsis [97]. Lymphopenia has been reported in Covid-19 and is a typical feature of the immunosuppressed later stage of sepsis. Activation of the coagulation cascade occurs in both diseases, including reports of disseminated intravascular coagulation [98]. Similarities in pathophysiology implies that the same therapeutics may be relevant for both diseases. A murine model of sepsis based on cecal ligation and puncture recently showed increased survival with subcutaneous human interleukin-7 [99]. Positive results were later reported from a phase 2 clinical trial [100] of sepsis patients. Based on these results, interleukin-7 therapy has also been suggested for Covid-19 [97]. Interleukin-7 stimulates hematopoietic stem cells to differentiate into the common lymphoid progenitor. The rationale for interleukin-7 therapy is therefore a rebalancing of lymphoid cells, which is implicated by lymphopenia in both diseases. Interleukin-7 may also prevent apoptosis of immune cells [101]. Unfortunately, no approved

**18**

**Figure 4.**

*explained in the text.*

#### *Erythrocyte - A Peripheral Biomarker for Infection and Inflammation*

#### **Figure 5.**

*Schematic figure of senescence, aging, eryptosis and hemolysis of the erythrocyte. Abbreviations are explained in the text.*

The sequence of events from dehydration to erythrocyte clearance from circulation have not been fully elucidated. In one recently proposed model, erythrocytes may respond to dehydration by shedding Glycophorin C-containing vesicles, thereby losing membrane sialic acid [13]. Less sialic acid would result in activation of the basal cell adhesion molecule (BCAM, also known as Lutheran antigen) and CD44 membrane proteins of the erythrocyte. Ligands of BCAM and CD44 are laminin-alpha-5 and hyaluronan (**Figure 5**), known as parts of the extracellular matrix. Binding to laminin-alpha-5 or hyaluronan could possibly delay erythrocyte passage through spleen and liver making encounters with macrophages and clearance from circulation more likely.

Activation of the Gardos channel and dehydration are thus effects of increased erythrocyte calcium levels. Another consequence of increased calcium levels is phosphatidylserine exposure on the erythrocyte plasma membrane (**Figure 5**). This effect is likely to occur because of calcium-caused activation of scramblase, a membrane enzyme that equilibrates membrane lipids between the two plasma membrane leaflets. Further to this effect, calcium inactivates flippase, a membrane enzyme that moves lipids from the outer plasma membrane to the inner plasma membrane leaflet. Exposed phosphatidylserine can then bind to several phosphatidylserine receptors exposed on phagocytic cells [114], facilitating clearance of erythrocytes from circulation by spleen or liver macrophages. In young and healthy cells, phosphatidylserine occurs mainly on the inner leaflet of the plasma membrane. Exposure of phosphatidylserine is a marker for cell stress and effectively functions as an "eat-me" signal.

Yet another contribution to erythrocyte aging comes from the membrane protein CD47, a heavily glycosylated protein with five transmembrane domains and an extracellular N-terminal immunoglobulin-like domain (**Figure 5**). CD47 is a "don't eat me" signal that protects the cell from being phagocytosed by macrophages or other phagocytes. CD47 binds the macrophage signal-regulatory protein alpha (SIRPalpha), a transmembrane protein with three immunoglobulin-like extracellular domains. CD47-SIRP-alpha interaction leads to phosphorylation of immunoreceptor tyrosine-based inhibition (ITIM) motifs in the cytoplasmic tail of SIRP-alpha. This leads to the recruitment of Src homology phosphatases (SHP) preventing

**21**

spleen or liver macrophages.

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

an "eat me" signal when binding to SIRP-alpha of the macrophage [115].

myosin-IIA accumulation and phagocytosis by the macrophage. As erythrocytes grow older they display less CD47 on their surfaces, thereby increasing the likelihood for being eaten by macrophages. However, the role of CD47 in erythrocyte clearance may not be limited to a reduced "do not eat me" signal. CD47 can also bind thrombospondin-1, a multifunctional protein of the extracellular matrix. Evidence suggests that conformational changes in CD47, perhaps caused by oxidation as the cell ages, promotes binding of thrombospondin. The resulting complex then instead becomes

Senescence and aging has been suggested to be regulated through band3 acting as a "molecular clock" for the erythrocyte [116]. Partial proteolysis, oxidation, phosphorylation and binding of methemoglobin or hemichrome to band3 leads to protein clustering and conformational changes that are recognized by anti-band3 autoantibodies (**Figure 5**). Binding of C3 complement then leads to phagocytosis of the aging erythrocyte when it encounters red pulp spleen or liver macrophages (**Figure 5**). Presumably, antibodies and complement must reach a threshold of opsonization for clearance by macrophages to take place. An appealing aspect of this "clock" is that it gauges the content of dysfunctional hemoglobin inside each erythrocyte, thus weeding out erythrocytes that have become unacceptably dysfunctional in their crucial role as oxygen carriers. If erythrocytes are ruled by a "clock", another candidate is the period circadian protein homolog 2 (PER2) of the circadian clock, a physiological system that determines the biological day-length of the organism. Erythrocytes of PER2-deficient mice showed morphological changes

Eryptosis is the regulated cell death associated with erythrocytes. Eryptosis can be induced by oxidative stress, pathogen infection, certain diseases like sepsis and sickle-cell disease and certain drugs and chemicals. Eryptosis can also be protected against by several means, notably some plant-derived substances like ascorbate, caffeine and resveratrol [118]. Signaling leading to eryptosis involves ceramide, prostaglandin E2 and increased intracellular calcium levels. Similar to what happens in senescence and aging, this induces scramblase and inhibits flippase leading to phosphatidylserine exposure on the plasma membrane. Eryptosis is largely seen as a means for the organism to avoid the serious consequences of hemolysis and may even prevent pathogen growth [118]. Still, phosphatidylserine receptors on vascular endothelial cells can bind eryptotic erythrocytes leading to impaired microcirculation [119]. Regulation of eryptosis involves several kinases like the p21-activated kinase PAK2 and mitogen-and stress-activated kinase MSK1/2 [119]. Experiments with knock-out mice further suggested involvement of the beta-glucosidase-like protein Klotho. In healthy human and mice, Klotho is a co-receptor for fibroblast growth factor 23, regulating phosphate metabolism and vitamin-D synthesis. Klotho deletion ultimately leads to premature aging and death. Whether Klotho is involved in regulation of physiological eryptosis in humans remains unknown. Both eryptosis and senescence involve erythrocyte-macrophage interactions. The close interactions between erythrocytes and macrophages follow throughout the erythrocyte lifespan. To begin with, macrophages participate in erythropoiesis, the formation and development of erythrocytes in the erythroblastic islands of the bone marrow. During the lifetime of the erythrocyte, passage through spleen and liver generates new macrophage encounters. In these encounters erythrocytes are screened for healthiness and may be repaired by the macrophages [120], for instance by removal of the inclusion bodies containing damaged hemoglobin, called Heinz bodies. Erythrocytes are the main source of blood MIF1 (macrophage migration inhibitory factor 1), although the physiological significance of this remains to be established [8]. At last, the senescent or eryptotic erythrocyte is phagocytosed by

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

and impaired oxygen transport [117].

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

myosin-IIA accumulation and phagocytosis by the macrophage. As erythrocytes grow older they display less CD47 on their surfaces, thereby increasing the likelihood for being eaten by macrophages. However, the role of CD47 in erythrocyte clearance may not be limited to a reduced "do not eat me" signal. CD47 can also bind thrombospondin-1, a multifunctional protein of the extracellular matrix. Evidence suggests that conformational changes in CD47, perhaps caused by oxidation as the cell ages, promotes binding of thrombospondin. The resulting complex then instead becomes an "eat me" signal when binding to SIRP-alpha of the macrophage [115].

Senescence and aging has been suggested to be regulated through band3 acting as a "molecular clock" for the erythrocyte [116]. Partial proteolysis, oxidation, phosphorylation and binding of methemoglobin or hemichrome to band3 leads to protein clustering and conformational changes that are recognized by anti-band3 autoantibodies (**Figure 5**). Binding of C3 complement then leads to phagocytosis of the aging erythrocyte when it encounters red pulp spleen or liver macrophages (**Figure 5**). Presumably, antibodies and complement must reach a threshold of opsonization for clearance by macrophages to take place. An appealing aspect of this "clock" is that it gauges the content of dysfunctional hemoglobin inside each erythrocyte, thus weeding out erythrocytes that have become unacceptably dysfunctional in their crucial role as oxygen carriers. If erythrocytes are ruled by a "clock", another candidate is the period circadian protein homolog 2 (PER2) of the circadian clock, a physiological system that determines the biological day-length of the organism. Erythrocytes of PER2-deficient mice showed morphological changes and impaired oxygen transport [117].

Eryptosis is the regulated cell death associated with erythrocytes. Eryptosis can be induced by oxidative stress, pathogen infection, certain diseases like sepsis and sickle-cell disease and certain drugs and chemicals. Eryptosis can also be protected against by several means, notably some plant-derived substances like ascorbate, caffeine and resveratrol [118]. Signaling leading to eryptosis involves ceramide, prostaglandin E2 and increased intracellular calcium levels. Similar to what happens in senescence and aging, this induces scramblase and inhibits flippase leading to phosphatidylserine exposure on the plasma membrane. Eryptosis is largely seen as a means for the organism to avoid the serious consequences of hemolysis and may even prevent pathogen growth [118]. Still, phosphatidylserine receptors on vascular endothelial cells can bind eryptotic erythrocytes leading to impaired microcirculation [119]. Regulation of eryptosis involves several kinases like the p21-activated kinase PAK2 and mitogen-and stress-activated kinase MSK1/2 [119]. Experiments with knock-out mice further suggested involvement of the beta-glucosidase-like protein Klotho. In healthy human and mice, Klotho is a co-receptor for fibroblast growth factor 23, regulating phosphate metabolism and vitamin-D synthesis. Klotho deletion ultimately leads to premature aging and death. Whether Klotho is involved in regulation of physiological eryptosis in humans remains unknown.

Both eryptosis and senescence involve erythrocyte-macrophage interactions. The close interactions between erythrocytes and macrophages follow throughout the erythrocyte lifespan. To begin with, macrophages participate in erythropoiesis, the formation and development of erythrocytes in the erythroblastic islands of the bone marrow. During the lifetime of the erythrocyte, passage through spleen and liver generates new macrophage encounters. In these encounters erythrocytes are screened for healthiness and may be repaired by the macrophages [120], for instance by removal of the inclusion bodies containing damaged hemoglobin, called Heinz bodies. Erythrocytes are the main source of blood MIF1 (macrophage migration inhibitory factor 1), although the physiological significance of this remains to be established [8]. At last, the senescent or eryptotic erythrocyte is phagocytosed by spleen or liver macrophages.

*Erythrocyte - A Peripheral Biomarker for Infection and Inflammation*

The sequence of events from dehydration to erythrocyte clearance from circulation have not been fully elucidated. In one recently proposed model, erythrocytes may respond to dehydration by shedding Glycophorin C-containing vesicles, thereby losing membrane sialic acid [13]. Less sialic acid would result in activation of the basal cell adhesion molecule (BCAM, also known as Lutheran antigen) and CD44 membrane proteins of the erythrocyte. Ligands of BCAM and CD44 are laminin-alpha-5 and hyaluronan (**Figure 5**), known as parts of the extracellular matrix. Binding to laminin-alpha-5 or hyaluronan could possibly delay erythrocyte passage through spleen and liver making encounters with macrophages and clear-

*Schematic figure of senescence, aging, eryptosis and hemolysis of the erythrocyte. Abbreviations are explained* 

Activation of the Gardos channel and dehydration are thus effects of increased erythrocyte calcium levels. Another consequence of increased calcium levels is phosphatidylserine exposure on the erythrocyte plasma membrane (**Figure 5**). This effect is likely to occur because of calcium-caused activation of scramblase, a membrane enzyme that equilibrates membrane lipids between the two plasma membrane leaflets. Further to this effect, calcium inactivates flippase, a membrane enzyme that moves lipids from the outer plasma membrane to the inner plasma membrane leaflet. Exposed phosphatidylserine can then bind to several phosphatidylserine receptors exposed on phagocytic cells [114], facilitating clearance of erythrocytes from circulation by spleen or liver macrophages. In young and healthy cells, phosphatidylserine occurs mainly on the inner leaflet of the plasma membrane. Exposure of phosphatidylserine is a marker for cell stress and effectively functions as an "eat-me" signal. Yet another contribution to erythrocyte aging comes from the membrane protein CD47, a heavily glycosylated protein with five transmembrane domains and an extracellular N-terminal immunoglobulin-like domain (**Figure 5**). CD47 is a "don't eat me" signal that protects the cell from being phagocytosed by macrophages or other phagocytes. CD47 binds the macrophage signal-regulatory protein alpha (SIRPalpha), a transmembrane protein with three immunoglobulin-like extracellular domains. CD47-SIRP-alpha interaction leads to phosphorylation of immunoreceptor tyrosine-based inhibition (ITIM) motifs in the cytoplasmic tail of SIRP-alpha. This leads to the recruitment of Src homology phosphatases (SHP) preventing

**20**

ance from circulation more likely.

**Figure 5.**

*in the text.*

Hemolysis is the lysis of erythrocytes and release of their cellular contents in surrounding fluids. Intravascular hemolysis occurs mainly as a consequence of bacterial infection and production of endotoxins. Hemolysis may also occur as a consequence of shear stress when erythrocytes pass through narrow capillaries. The mechanism underlying hemolysis is overhydration due to defective ion efflux channels, particularly sodium/potassium ATPase. Cell volume increase will lead to rupture of the cell membrane and release of cytosolic contents. Hemolysis is typical of some diseases such as sickle-cell disease or glucose-6-phosphate deficiency. The fate of hemoglobin is an important aspect of hemolysis. Free hemoglobin typically binds to haptoglobin and the complex is then internalized through binding to the CD163 receptor on macrophages (**Figure 5**). If this does not occur, and if the iron has been oxidized to the ferric Fe(III) state, plasma hemoglobin will be separated into globin and heme. Being hydrophobic, free heme makes contact to plasma proteins such as albumin, alpha-1 antitrypsin, alpha-2 macroglobulin, lipoproteins and hemopexin. Binding to hemopexin effectively sequesters heme, making the complex largely safe for further circulation in the vascular compartment. The complex will eventually be internalized through binding to the low density lipoprotein receptor-related protein 1 (LRP1) receptor (**Figure 5**) on hepatocytes or macrophages [121]. If bound to albumin, heme can be transported into cells by binding to transferrin receptor 1. If cellular uptake does not happen by these routes, for instance due to hemopexin saturation, free heme can contribute to inflammation by binding the TLR4 receptor (**Figure 5**). Heme is in this context acting as a danger-associated molecular pattern (DAMP). Since TLR4 occurs also on endothelial cells, the vasculature is able to directly respond to increased heme levels. The effect of TLR4 stimulation is activation of NF-kB (nuclear factor kappa-light-chain-enhancer of activated B cells), inflammasome activation leading to activation of caspase-1 and release of pro-inflammatory interleukins like interleukin-1-beta, and interleukin-18. Recently, binding of heme to TLR4 was shown to be dependent on myeloid differentiation factor 2 (MD-2) and the glycosylphosphatidyl inositol (GPI)-anchored leucine-rich repeat protein CD14 [122]. As a comparison, in eryptosis and normal senescence and aging, macrophages typically phagocytose the whole erythrocyte and free heme does not appear.

Heme can also activate the alternative pathway of complement, and activate platelets through C-type-lectin-like receptor 2 [123]. In sickle cell disease patients and murine models of sickle-cell disease, hemolysis caused kidney deposits consisting of complement C3. The condition was probably caused by heme activation of complement, since it was possible to counteract by addition of hemopexin [124]. Free heme and its iron is available for use by bacterial pathogens and this is considered as an important aspect of hemolysis, for instance in hemolytic anemia. Sequestering of heme and hemoglobin is thus an important protection against damage caused by hemolysis. Inside the cell, heme is catabolized by heme oxygenase-1, producing biliverdin, free iron and carbon monoxide. The expression of heme oxygenase-1 can be induced by heme relieving gene repression through the BACH-1 heme-sensing repressor (**Figure 5**). The net effect of the catabolism is largely anti-inflammatory due to lower levels of TNF-alpha, interleukin-1-beta, and macrophage inflammatory protein (MIP) and upregulation of interleukin-10 [125]. The carbon monoxide produced as a by-product may however contribute to oxidative stress due to reactions with reduced transition metals [125]. Heme oxygenase-1 is itself a heme-containing enzyme of the heat shock protein family, present in the endoplasmic reticulum membrane oriented to the cytosol and expressed throughout the body (**Figure 5**). The globin part of hemoglobin is regarded as largely noninflammatory [3]. Heme and its consequences are the most well-studied aspects of hemolysis. Less well-studied effects of hemolysis include release of intracellular or surface-bound cytokines from erythrocytes.

**23**

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

**9. Some genetic diseases particularly relevant in the erythrocyte context**

Glucose-6-phosphate dehydrogenase is an enzyme in the glycolysis and pentose phosphate pathways. Its deficiency particularly effects the erythrocyte since the erythrocyte depends on glycolysis and the pentose phosphate pathway for its energy needs. Erythrocyte irregularities called Heinz bodies are frequent in glucose-6-phosphate deficiency. The deficiency is particularly common in sub-Saharan Africa due to a survival advantage in malaria-afflicted areas. Interestingly, deficiency for glucose-6-phosphate dehydrogenase seems to be associated with hemolysis in Covid-19 patients [126]. Patients with glucose-6-phosphate deficiency may react with hemolysis as a response also to other infections and treatment with primaquine or hydroxychloroquine [127, 128], suggesting a more general susceptibility to hemolysis associated with glucose-6-phosphate deficiency. Some other diseases involving the erythrocyte are sickle-cell disease, thalassemia,

stomatocytosis, spherocytosis, ovalocytosis, paroxysmal nocturnal hemoglobinuria (PNH), polycythemia vera, acute erythroid leukemia and lecithin-cholesterol acyltransferase deficiency (LCAT). Paroxysmal nocturnal hemoglobinuria (PNH) is an intravascular hemolytic anemia, most often caused by somatic mutations in the gene encoding phosphatidylinositol glycan A (PIGA), leading to dysfunctional GPI-dependent anchoring of membrane proteins. One such erythrocyte membrane protein is decay accelerating factor (DAF) which acts to limit action of the alternative complement pathway. The resulting complement-driven hemolysis can be counteracted by the humanized monoclonal antibody eculizumab directed against

The Duffy antigen may be of diagnostic value for some diseases, particularly malaria, but may perhaps also prove to be a druggable target, since G-protein coupled receptors are among the most common drug targets. The signaling mechanisms discovered recently particularly regarding band3 may also be of pharmacological interest. The mechanism underlying eryptosis may be used to kill off infected erythrocytes [129]. It is known that antioxidants like ascorbic acid and catechin, provided through the diet can have protective effects on the erythrocyte. Refined erythrocyte related diet recommendations could perhaps be expected. The erythrocyte has previously been used in various physical or biochemical ways as a diagnostic tool. Such tests are the erythrocyte sedimentation rate, hematocrit, erythrocyte distribution width, erythrocyte count, mean corpuscular hemoglobin concentration and mean corpuscular volume. New diagnostic or treatment developments can be expected based on molecular, biotechnological and genetic techniques. One example would be ICP-MS to detect element profile changes of genetic,

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

terminal complement component C5.

epigenetic or environmental origin.

**10. Outlook for new treatments and diagnosis**

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