COVID-19: Hemoglobin/iron dysmetabolism is likely to be the leading process causing multiorgan disease and hypoxia


The COVID-19 pandemic caused by the SARS-CoV-2 virus has resulted in significant morbidity and mortality worldwide. While much has been learned about the clinical course of the disease, its underlying mechanisms and pathogenesis remain unclear. Recent studies have suggested a potential role for hemoglobin iron dysmetabolism in the development of multiorgan disease and hypoxia in COVID-19.

Hemoglobin is the protein in red blood cells that carries oxygen from the lungs to the body’s tissues. Iron is an essential component of hemoglobin and is critical for oxygen transport and cellular metabolism. In COVID-19, the virus has been shown to bind to heme, the iron-containing molecule in hemoglobin, and disrupt its metabolism. This disruption can lead to the release of free iron, which can cause oxidative damage and inflammation in tissues throughout the body.

Several studies have reported evidence of hemoglobin iron dysmetabolism in COVID-19. For example, a study of 10 patients with COVID-19 found that all had elevated levels of plasma-free hemoglobin, indicating hemolysis, or the breakdown of red blood cells. Another study of 21 patients with severe COVID-19 found that all had evidence of hemoglobin oxidation and depletion, as well as elevated levels of ferritin, a marker of iron overload.

The mechanisms underlying the potential role of hemoglobin iron dysmetabolism in COVID-19 are not yet fully understood. However, several hypotheses have been proposed.

First, the release of free iron can lead to the formation of reactive oxygen species, which can damage cells and tissues and contribute to the development of multiorgan disease.

Second, the disruption of hemoglobin metabolism can lead to the formation of abnormal heme molecules that may be toxic to cells and tissues. Third, the release of heme from hemoglobin can lead to the activation of the immune system and contribute to the development of inflammation.

In addition to its potential role in the pathogenesis of COVID-19, hemoglobin iron dysmetabolism may have broader implications for the development of other diseases. For example, hemolysis and iron overload have been associated with a range of conditions, including sickle cell disease, thalassemia, and hemochromatosis.

Overall, the evidence suggests a potential role for hemoglobin iron dysmetabolism in the development of multiorgan disease and hypoxia in COVID-19. Further research is needed to fully understand the mechanisms underlying this association and to explore the potential for interventions targeting hemoglobin iron dysmetabolism as a therapeutic approach for COVID-19 and related diseases.

In conclusion, hemoglobin iron dysmetabolism may play a significant role in the pathogenesis of COVID-19, leading to the development of multiorgan disease and hypoxia. Further research is needed to fully understand the mechanisms underlying this association and to explore potential interventions targeting hemoglobin iron dysmetabolism as a therapeutic approach for COVID-19 and other diseases.

New study shows iron dyshomeostasis in COVID-19

Dysregulated levels of iron-related biomarkers are associated with various pathological conditions [62,76], including pulmonary and respiratory disease [77,78], and are also reported in hyperferritinemic syndromes (HFS) [79,80]. HFS comprise a spectrum of diseases characterized by high levels of serum ferritin (ferritin > 300 ng/mL) [81], such as macrophage activation syndrome (MAS), Gaucher disease, adult-onset Still’s disease (AOSD), rheumatoid arthritis, and catastrophic antiphospholipid syndrome (CAPS) [80,82,83].

Hyperferritinemia has been shown to be relevant for viral diseases [33,84] and predicts poor outcomes in patients affected by influenza A [85] and Crimean–Congo hemorrhagic fever [86]. The cytokine profiles and the extremely high levels of ferritin reported in HFS are in line with the results reported here for COVID-19 patients (Table 1), confirming that COVID-19 is an HFS [87,88,89].

During viral infection, two principal mechanisms occur to modulate ferritin levels, iron availability, and inflammatory cytokines levels: IL-1β and IL-6 [39,84]. Elevated ferritin levels can persist several months after the onset of COVID-19 in some cases [90,91], as also reported in our cohort of long-COVID patients (Table 1).

Raised serum ferritin levels can induce hepatic cell death, triggering an increase in free iron systemic levels after iron release from ferritin [92,93]. Notably, in hospitalized COVID-19 patients, high ferritin levels and iron dysregulation were associated with an adverse clinical course [64,91,94].

To date, it is not clear if disturbances of iron handling are just an adaptation response to SARS-CoV-2 infection or are involved in the pathophysiological mechanism, and further studies are required [95,96]. In the immune response to COVID-19, ferritin levels increase dramatically leading to the condition of hyperferritinemia [87,88,89].

This iron-based resistance mechanism to pathogens involves a systemic reduction in circulating iron due to the inhibition of cellular iron export and the induction of cellular iron import [33,41]. In particular, this mechanism is activated by the synthesis and secretion of hepcidin by hepatocytes, which are in turn influenced by iron levels in the body as well as inflammation, high levels of HFE, erythropoiesis, and hypoxia [38,41].

Thus, under these inflammatory conditions, the hepatic hormone hepcidin is upregulated, reducing cellular iron efflux into the blood lumen by binding to FPN [37,39,41]. The blockage of FPN decreases iron absorption from the intestine and results in iron being retained in the macrophages, which might explain the low serum iron concentration and hypoferremia as a reaction to the infection [33,97].

On the other hand, an increase in intracellular iron can lead to an increase in inflammation via the iron-dependent intracellular post-translational activation of 5-LOX and its LTB4 products (Figure 4A) without any increase in protein levels (Figure 3). In our study, we individuated iron-related metabolism proteins differentially expressed in COVID-19 and long-COVID patients in comparison to healthy donors (Figure 1 and Figure 2).

We found that the amounts of Cp, Tf, HPX, LCN2, and SOD1 increased in PBMCs isolated from COVID-19 patients, compared to both the long-COVID group and the control group (Figure 2). The functional interplay between Tf and Cp has an important role in intestinal iron absorption and in iron systemic transport; the bilobate Tf can bind to ferric iron (Fe3+) and represents the major transporter of the metal in plasma, while Cp modulates the loading of iron into Tf by catalyzing the oxidation of the ferrous form (Fe2+) into Fe3+ [40,98,99,100].

Our data reported increased levels of both proteins in COVID-19 patients (Figure 2), suggesting the activation of a possible protective mechanism against the iron overload reported during SARS-CoV2 infection [92,95,101] by binding Fe3+ and oxidating Fe2+ to less toxic ferric forms [40,102].

Excessive Fe2+ catalyzes the Fenton and Haber–Weiss reactions and promotes the accumulation of reactive oxygen species (ROS) and, in particular, the formation of hydroxyl radical (⋅OH) [103,104]. This condition promotes oxidative stress, and can also be responsible for hemolysis, a common complication reported in COVID-19 cases [105,106,107]. During the acute phase of SARS-CoV-2 infection, and in some cases after the resolution of infection, a spectrum of hematological/hemolytic complications has been reported, characterized by the increased destruction of red blood cells (RBCs) [108,109].

The hemolytic process induces an increase in the free hemoglobin, heme, and Fe2+ described in COVID-19 patients, and concurs with increasing inflammation and oxidative stress [108,109]. Our results reported increased HPX levels in COVID-19 and long-COVID patients (Figure 2); this protein is known to protect cells from free heme toxicity occurring during hemolysis [48,49].

Indeed, in animal models of ARDS and chronic pulmonary disease, the administration of HPX attenuates inflammation and lung fibrosis [110,111]. We speculate that the increased HPX levels reported here could act protectively by binding not only free heme, but also the RBC membrane, preventing further hemolysis [48,110].

Furthermore, the induction of ROS formation could also be responsible for enhanced SOD1 levels (Figure 2), one of the most powerful antioxidant enzymes in the first line against ROS [51,112]. Previous works have reported an increase in antioxidant enzyme activities, such as SOD and catalase, during SARS-CoV2 infection [113,114], and animal studies have shown that the use of a synthetic and stable SOD has a protective effect in pulmonary fibrosis, lung inflammation, and ARDS [115,116,117].

However, the redox status and complete profiling of oxidative stress markers in COVID-19 and in long-COVID patients are not yet described. Some studies reported no changes between the serum activities of SOD and CAT enzymes in COVID-19-infected patients [118], or an inhibition of antioxidant systems, leading to a decrease in the overall antioxidant capacity [119,120].

In addition, the ingenuity pathway analysis (IPA) performed with our proteomic data highlighted the upregulation of oxidative stress-related biological functions such as the “metabolism of hydrogen peroxide” in COVID-19 patients versus healthy controls, driven by the pool of proteins as reported in Supplementary Figure S3.

Oxidative stress pathways could also be responsible for somatic and mental symptoms of long-COVID. Depression, generalized anxiety disorder, and chronic fatigue are manifestations of oxidative stress-activated pathways and are coupled with lowered antioxidant defenses [121,122,123]. Previous studies have suggested connections between redox imbalance/oxidative damage and long-COVID symptoms caused by a reduction in antioxidant defense mechanisms [124,125].

In line with this theory, in our cohort of long-COVID patients, we found a decreasing trend of antioxidant proteins, such as Cp, Tf, and SOD1 (Figure 2) [42,51,126], in comparison to COVID-19 patients and healthy controls, and a significant increase in HPX levels compared to the healthy controls (Figure 2).

These results suggest that oxidative stress is a pivotal point in the pathophysiology of COVID-19, as well as in long-COVID. Indeed, in patients affected by long-COVID, the imbalance between the low concentration/activity of antioxidant proteins Cp, Tf, and SOD1 and the increased hemolytic crisis, suggested by the upregulated HPX levels (Figure 2), could be responsible for increased levels of ROS and the aberrant oxidative stress reported after recovery from SARS-CoV2 infection [127,128].

Our data show a possible connection between the redox imbalance reported in COVID-19 and long-COVID and altered iron metabolism. Interestingly, it has been reported that susceptibility to viral infection with HIV, H1N1, SARS, and COVID-19 is associated with iron levels [129,130,131], and increased plasma levels of free iron correlate with adverse outcomes for COVID-19 patients [132,133].

Previous studies have demonstrated the anti-viral effects of iron-chelators, such as deferoxamine (DFO) or deferiprone, for HIV, HSV-1, and CMV [56,57,134,135], and iron chelation therapies are shown to be effective in the management of COVID-19 patients by decreasing the production of free radicals and reducing IL-6 levels [129,136].

Furthermore, iron excess has been reported to be involved in the increased lipoxygenase activity described in immune cells during inflammation [61] and in the cell death mechanism triggered by iron-catalyzed lipid peroxidation, known as ferroptosis [61,79]. Here, we report a significant increase in the gene expression levels of 5-LOX in COVID-19 patients (Supplementary Figure S2), in line with previously published data from other groups [137], and a downregulation of protein amounts in COVID-19 and long-COVID patients in comparison to healthy donors (Figure 3A,B).

Concerning the 5-LOX activation state, we report a significant increase in LTB4 plasma levels in COVID-19 and long-COVID patients versus healthy donors (Figure 4A), which seems to be in line with the previously described post-transcriptional activation of an apo-form of 5-LOX, which leads to an active holo-5-LOX able to produce LTB4 in iron overloading conditions [61].

It is fundamental to underline that 5-LOX expression is complex and its modulation is associated with the cytokine profile. The important link between IL-4 and LOXs during the inflammatory/immune response has been well described [138,139]. Furthermore, Spanbroek and colleagues described a cytokine-specific modulation of 5-LOX, reporting that prolonged stimulation with IL-4 downregulates 5-LOX protein levels in dendritic cells and other leukocytes [140].

Thus, we speculate that the persistent high levels of IL-4 reported during SARS-CoV-2 infection and in the post-acute phase [29,141] are responsible for the downregulation of the 5-LOX protein in COVID-19 and long-COVID patients, compared with healthy donors (Figure 3A,B).
LTB4 is one of the most important candidates responsible for the hyperimmune/inflammatory response in the progression of COVID-19 due to its chemoattractant properties and capability to carry lymphocytes out to airways [142,143,144].

During acute COVID-19 infection, LTB4 could act protectively by suppressing viral replication [145,146] and inducing leukocyte recruitment. This occurs in other viral infections, including the herpes virus, CMV, and influenza [145,147]. However, it has been reported that aberrant and chronic LTB4 production can induce an uncontrolled release of chemokines and cytokines, causing blood lymphocytopenia, as described in COVID-19, and could be detrimental to host defense [148,149].

In different chronic inflammatory diseases, including autoimmune diseases, allergy, obesity, and chronic infection, excessive plasma LTB4 levels could propagate pathological inflammation in affected tissues, thus contributing to tissue injury [148,150,151,152]. In long-COVID patients, a low and continuous grade of inflammation has been reported [153,154], mimicking the conditions occurring in chronic disease [155]. In line with these data are the elevated LTB4 plasma levels reported here in relation to long-COVID patients (Figure 4A).

Furthermore, LTB4 systemic levels were found to be associated with the severity grade of COVID-19 in patients with diabetes [137], and increased LTB4 production has been reported in immune cells after SARS-CoV2 infection [156].
LCN2 is a multifaceted protein member of the adipocytokines with a well-characterized bacteriostatic role [157,158], and its association with viral infections has been described [159,160]. LCN2, also known as neutrophil gelatinase-associated lipocalin (NGAL) or siderocalin [47,161], is upregulated in several immune disorders [162,163,164].

It is measurable in biological fluids during viral infection and inflammation states [45,158,165,166], and its role as an iron regulatory protein has recently emerged [45,158,167]. LCN2 protects cells against oxidative stress [50], and during iron overload conditions, its expression is upregulated both at the cellular and systemic level [168,169].

The defensive role of LCN2 against iron excess and oxidative stress is related to its ability to indirectly bind iron [170,171,172], to induce the expression of antioxidant molecules (including SOD1 [173,174]), and its intrinsic antioxidant properties [169]. In our work, we measured an increase in circulating plasma LCN2 levels in the cohort of COVID-19 versus long-COVID patients and healthy controls (Figure 4B), parallel to the data reported here concerning the protein amounts in PBMCs (Figure 2). Given the observation that the cellular and systemic LCN2 forms were significantly elevated in COVID-19 patients, we propose that during SARS-CoV2 infection, an induction of LCN2-mediated protection against iron-induced toxicity can occur.

Finally, SARS-CoV-2 infection induces the well-described cytokine storm responsible for, among other things, high ferritin levels and mitochondrial dysfunction, leading to oxidative stress [87,88,89]. These events contribute to modulating iron levels and induce significant changes in the proteins responsible for iron metabolism.

Overall, our findings suggest that iron dyshomeostasis causes an increase in ROS levels, oxidative stress, and the hemolytic process, which in turn can increase free iron and heme levels, as well as cellular iron overloading and the post-translational activation of 5-LOX (see Figure 5).

Here, we speculate on the presence of the following defensive mechanisms that could occur against free iron/heme toxicity and oxidative stress during COVID-19: (i) Tf, Cp, and LCN2 are increased to protect against free iron by reducing Fe3+; (ii) the antioxidant enzyme SOD1 is upregulated to reduce ROS levels and modulate LCN2 levels; (iii) HPX protein levels are enhanced to protect against free heme toxicity and prevent further hemolysis (Figure 5).

Figure 5. Schematic representation of pathogenesis of SARS-CoV-2 infection and possible defense mechanisms.

These data suggest further investigations are needed to evaluate the role of different iron-related proteins and 5-LOX activation in an increased number of COVID-19 and long-COVID patients in order to better assess the correlation between disease progression and severity. Indeed, a limitation of this study is the number of subjects included in the two cohorts of patients, particularly for long-COVID.

The small size cohort for long-COVID analyzed in this study determines the exploratory and preliminary nature of our results. Further analyses are required in a larger cohort of patients in order to better validate LTB4 and LCN2 as a potential biomarker for COVID-19 and long-COVID. In conclusion, these data strongly suggest the need to extend the clinical analyses of COVID-19 patients in the context of the iron-related proteins reported here for a better evaluation of the inflammatory state and disease progression of the patients, and to develop innovative therapeutical approaches.

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