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

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, epigenetic or environmental origin.

*Erythrocyte - A Peripheral Biomarker for Infection and Inflammation*

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

**22**

surface-bound cytokines from erythrocytes.

*Erythrocyte - A Peripheral Biomarker for Infection and Inflammation*
