**6. The erythrocyte in sepsis**

*Erythrocyte - A Peripheral Biomarker for Infection and Inflammation*

Reactive oxygen species (ROS) are a set of oxygen containing compounds that easily react with and damage other constituents of the cell. Typical ROS in biological systems are superoxide, hydrogen peroxide and hydroxyl radical. ROS can be thought of as forming a hierarchy in the living organism (**Figure 3**). In the erythrocyte, superoxide is produced when Fe(II) of hemoglobin donates an electron to an oxygen molecule, generating the anionic superoxide and the oxidized Fe(III). This reaction is unavoidable due to the existence of iron and oxygen in close contact in the erythrocyte [69]. Superoxide can also be generated in the erythrocyte by nitric oxide synthase, an enzyme identical to the endothelial nitric oxide synthase (e-NOS). The superoxide is converted to water and hydrogen peroxide by copper and zinc-dependent superoxide dismutase. Hydrogen peroxide needs to be further metabolized to avoid the Fenton reaction which otherwise would generate the hydroxyl radical, a very reactive form of ROS. To defuse hydrogen peroxide the erythrocyte contains several enzymes, the most important being catalase, peroxiredoxin 2 and glutathione peroxidase 4. Peroxiredoxin 2 is one of the most abundant proteins in the erythrocyte cytosol [70]. In the oxidized form, peroxiredoxin 2 is recycled by thioredoxin using reduced nicotinamide adenine dinucleotide phosphate (NADPH) and thioredoxin reductase. Peroxiredoxin 2 is suggested to function as a non-catalytic scavenger of low-level hydrogen peroxide generated from autoxidation of hemoglobin [71]. However, a fraction of peroxiredoxin 2 is found attached to the erythrocyte plasma membrane and may be particularly important in detoxifying membrane lipid peroxides [72]. Catalase and the selenium-dependent glutathione peroxidase are important for the detoxification of hydrogen peroxide received by the erythrocyte from other parts of the vascular compartment. In plasma, superoxide is generated by xanthine oxidase, NADPH oxidase and nitric oxide synthase [73], enzymes that can occur in association to glycosaminoglycans of the endothelial cells of the vascular wall. Superoxide generated by these means can enter the erythrocyte by anion channels like band3. Alternatively, superoxide in plasma may be converted to hydrogen peroxide by extracellular superoxide dismutase. Hydrogen peroxide can then enter the erythrocyte by diffusion through the plasma membrane. Under stress conditions the erythrocyte receives much ROS generated by other cells inside or outside of the vascular compartment. Such ROS may have been generated by mitochondrial stress as a result of bacterial and viral infections (**Figure 3**). For instance, mitochondrial generation of ROS has been proposed as an important part of Covid-19 disease [74]. Antioxidant defense in erythrocytes also depends on small-molecule antioxidants like alpha-tocopherol, glutathione, ascorbate and reductants like NADPH generated in the pentose phosphate pathway. Alpha-tocopherol is present in erythrocyte plasma membrane where it may prevent lipid peroxidation. Glutathione participates as reductant in enzymecatalyzed reactions. Ascorbate then recycles oxidized forms of alpha-tocopherol and glutathione back to the reduced forms. Superoxide can also react with nitric oxide generating peroxynitrite, a reactive nitrogen species [73]. The extent to which this reaction occurs depends on the presence of nitric oxide inside the erythrocyte. Nitric oxide is produced by nitric oxide synthase but also scavenged by oxyhemoglobin in the erythrocyte. Peroxynitrite can also be formed in plasma and may enter the erythrocyte as anion through band3. In conditions of stress, such as infection with SARS-CoV-2, ROS overload damage the erythrocyte plasma membrane which can lead to hemolysis. The necessity of the antioxidant system of erythrocytes has been proven in gene knock-out studies in mice. For instance, deletion of peroxiredoxin 1 or peroxiredoxin 2 led to increased plasma ROS, hemolytic anemia and

**5. The erythrocyte and reactive oxygen species**

**16**

shortened life and erythrocyte lifespan [75, 76].

Sepsis is a strong and dysfunctional inflammatory response to an infection. In later stages it proceeds to immunosuppression, decreased pro-inflammatory cytokine levels and increased apoptosis of immune cells. The immunosuppressed stage can last for many months or even years and comes with increased risk for death due to secondary infections. Many aspects of sepsis have been highlighted recently like the presence of exosomes [77], complement and histones [78], metabolic changes [79] and ROS and inflammasomes [80]. Gene expression analysis of sepsis patients revealed CXCL8, tumor protein P53 and TNF as particularly important based on a subsequent pathway analysis [81, 82]. The chemokine CXCL8 binds to chemokine receptors on neutrophils, ultimately leading to attachment of neutrophils at sites of infection. Neutrophils have an important function in phagocytosis of bacteria through neutrophil extracellular traps (NET). Analysis of plasma protein levels of sepsis patients identified the cytokines interleukin-17 and interleukin-27 as being elevated early in sepsis, whereas interleukin-33 protein levels increased later in the immunosuppressive phase [83]. The three interleukins form an axis in sepsis pathophysiology with interleukin-27 having a inhibitory effect on the proinflammatory interleukin-17. The effect of interleukin-27 on interleukin-33 may depend on the stage of disease. Interleukin-33 has been shown to improve survival in murine sepsis models, which can be explained by increased neutrophil presence and more bacterial clearance at the site of infection. Interleukin-33 may have a protective effect by rebalancing different types of immunity [83]. Interleukin-33 has also been reported to suppress and modulate lymphocytes of the innate immune system such as type 2 innate lymphoid cells [84], and induce regulatory T cells. The contribution of erythrocytes to the pool of cytokines in sepsis has not been investigated. However, some of the cytokines implicated in sepsis pathophysiology like interleukin-17, interleukin-33 and CXCL8 have been reported to occur on erythrocytes from healthy individuals [9]. Pathophysiology of sepsis in individuals negative for the CXCL8 receptor Duffy or Duffy knock-out mice has not been reported. Apoptosis is a contributing factor to immunosuppression in sepsis [85]. A first hint of this was the finding that overexpression of B-cell lymphoma 2 (Bcl-2) protected against sepsis [86]. Bcl-2 is a mitochondrial protein that counteracts apoptotic processes. However, in the inflammatory phase of sepsis, some apoptosis of neutrophils is necessary [87].

Changed erythrocyte features have been reported in sepsis, such as the thorny erythrocytes called echinocytes [88] and increased RDW [89]. Reports have also claimed RDW to be of prognostic value in sepsis [90], but this has not been confirmed in other studies [91]. Erythrocytes seem to experience oxidative stress in sepsis [88, 89, 92]. Malondialdehyde, a product of lipid peroxidation, was increased by 3-fold in erythrocytes of sepsis patients [92]. Peroxynitrite has been reported as the major ROS in plasma of sepsis patients [88]. Sepsis patients often suffer from a moderate level of anemia [93]. Anemia in sepsis can have multiple causes such as anemia of chronic disease or hemolysis by bacterial toxins [43]. Hemolysis has often been reported in sepsis caused by *Clostridium perfringens* [94]. It is caused by disruption of the erythrocyte plasma membrane by phospholipase C activity of the *Clostridium* alpha-toxin [94]. A murine cecal ligation and puncture model of sepsis showed that sepsis is lethal in mice deficient for the heme-degrading enzyme heme oxygenase-1 [95]. The same study also showed that administration of the heme-sequestering protein hemopexin could prevent lethality. This suggests the importance of hemolysis in the pathogenesis of sepsis.

Sepsis can also be induced by virus [96]. For instance, COVID-19 and sepsis share similarities but also show some differences. Cytokine storm occurs in both 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 sepsis-specific therapy exists despite many clinical trials.
