Since the outbreak of coronavirus disease 2019 (COVID-19) caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) (1), the number of confirmed cases has increased rapidly around the world (2, 3). Acute respiratory distress syndrome (ARDS) and multiple organ dysfunction are the main clinical manifestations and leading causes of mortality in severe COVID-19 cases (4). Iron homeostasis is crucial for host immune defense and inflammatory response. Disorders of iron metabolism are mainly manifested as iron deficiency and/or overload, both of which can cause cellular and organ dysfunction (5). Low serum iron (SI) concentration restricts hemoglobin synthesis and causes anemia and systemic hypoxemia. Emerging data have found that hypoferremia is an independent risk factor for hypoxic respiratory failure and death in COVID-19 patients (6, 7). The rapid and excessive accumulation of intracellular iron, especially in macrophages, causes cell and tissue damage, presumably through the production of reactive oxygen species (ROS) catalyzed by iron. In addition, iron overload may also trigger a unique iron-dependent form of non-apoptotic cell death termed ferroptosis (8). Serum ferritin concentration has been proven to reflect the individual's iron storage status. Studies have found that increased serum ferritin levels are associated with adverse outcomes (9–11). Although studies on transferrin saturation (TSAT) have been far fewer than that of ferritin and hypoferritemia, low TSAT has been consistently reported in severe COVID-19, and significantly correlated to lung aeration loss, inflammatory markers and worse outcomes (7, 12–14).

The hepatic peptide hepcidin has been identified as the systemic iron-regulatory hormone and plays an important role in maintaining iron homeostasis (15). Hepcidin regulates intestinal iron absorption, SI concentrations, and tissue iron redistribution by inducing the degradation of its receptor, the sole iron exporter ferroportin (14, 16), and blocking ferroportin export activity (17). Serum hepcidin measurement may be a promising tool for assessing the status of iron metabolism (18), but has not been widely implemented in clinical practice (14, 19, 20). Notably, unlike serum ferritin testing that has been documented in guidelines, hepcidin has been rarely measured in COVID-19 patients (13, 21). As expected in severe inflammatory diseases, recent studies have reported that most COVID-19 patients showed varying degrees of upregulation of hepcidin levels (16, 21–25). More importantly, hepcidin levels have significantly negatively correlated with the ratio of arterial oxygen partial pressure (PaO₂) and fraction of inspired oxygen (FiO₂), which can be used to predict COVID-19 severity and mortality (22). To adopt the results for clinical use, these interesting findings need to be validated in wider cohorts, and the relationship between hepcidin-mediated iron dysmetabolism and COVID-19 severity also needs to be further analyzed. In view of the particularity of the prevention and control of the COVID-19 epidemic and the randomness of its incidence, large-scale, multi-center, prospective cohort studies are very difficult to achieve. Here we classify mild and moderate as non-severe, severe, and critical as severe, and conduct a meta-analysis to re-evaluate the hepcidin-related iron metabolism parameters (hepcidin, ferritin, iron, and TSAT) in COVID-19 patients and explore the relationship between iron dysmetabolism and severity.

To our knowledge, this study is the first meta-analysis of the relationship between hepcidin-mediated iron dysmetabolism and COVID-19 severity. However, there is significant heterogeneity among six studies included in the data synthesis of hepcidin and ferritin. The possible reasons are analyzed as follows: (a) The time point and time course of laboratory testing were different. Some studies collected blood samples at admission (21–23), another 2 months after onset (16), and others throughout the hospital stay (24, 25). In addition, the detection methods and the reagents used were not the same. Enzyme-linked immunosorbent assay (ELISA) kits (16, 21–24) or competitive enzyme immunoassay (EIA) kits (25) were used to detect hepcidin. (b) Iron metabolism is strongly influenced by gender, with females generally having lower SI and hepcidin levels than males (22). Also, males have a higher ratio of severe COVID-19 cases. There were potential effects on heterogeneity due to differences in the composition of males and females between studies. (c) Our study classified mild and moderate as non-severe, severe and critical as severe. Differences in the proportions and sample sizes of cases of distinct severity between studies contributed to heterogeneity. Unfortunately, hepcidin and its related iron metabolism parameters are rarely tested simultaneously in COVID-19, and there are even fewer original studies analyzing the relationship between hepcidin and disease severity. Also, subgroup analysis could not be performed due to the lack of access to raw data. Furthermore, only the Sonnweber 2020 study tested hepcidin and ferritin 2 months after onset. We removed this study and performed a new meta-analysis and found no significant change in the final overall effect (Supplementary Figure 1). Therefore, from the results of the meta-analysis, differences in blood collection at different stages of COVID-19 between studies may not affect the fact that severe cases have higher levels of hepcidin and ferritin. This also suggests that hepcidin-mediated iron dysmetabolism throughout the course of COVID-19 may be some very promising therapeutic targets. Comprehensively, these studies met rigid criteria for inclusion and were homogeneous in terms of study design and objectives, as well as cohort settings. Therefore, the final overall effect generated by the random effects model brings novel insights into the biological effects of COVID-19.

Hepcidin, the key iron metabolism regulatory hormone, sequesters iron and prevents iron efflux in enterocytes and macrophages, resulting in increased intracellular ferritin and hypoferremia (30, 31). Physiologically, hepcidin synthesis by hepatocytes is reactively upregulated or downregulated by high or low SI, respectively. Other hepcidin agonists are inflammatory cytokines represented by interleukin-6 (IL-6). Conversely, hepcidin is antagonized by hypoxemia, with hypoxia induced factors (HIF) release (15, 32). Our study found that severe COVID-19 cases had higher serum levels of hepcidin and ferritin, and lower SI, without significant differences in TSAT.

These findings were consistent with most of other studies. Unexpectedly, hepcidin levels in severe COVID-19 cases did not appear to be downregulated by hypoferremia in a feedback manner and antagonized by systemic hypoxemia. Hyperferritinemia inevitably promotes the production of ROS and lipoperoxidation, which ultimately leads to extensive cell and tissue damage possibly through the ferroptosis pathway, and cascade-amplified inflammation (32, 33). In turn, hyperinflammation will transcriptionally upregulate hepcidin, further exacerbating iron dysmetabolism. We speculate that the potential intervention targets for this vicious circle may lie in the hepcidin-mediated iron metabolism axis.

Cytokine storm is a hallmark of the hyperinflammatory state of COVID-19 and is closely linked to disturbances in iron metabolism (30, 34). A growing body of studies have suggested that several components of elevated inflammatory status may be effective therapeutic targets, notably the administration of IL-6 receptor antagonists such as tocilizumab, which significantly reduces the mortality of severe COVID-19 cases (35, 36). However, the effective suppression of the excessive inflammatory response has also raised concerns about prolonged SARS-CoV-2 clearance. Furthermore, data on whether IL-6 receptor antagonists can correct hepcidin-mediated iron dysmetabolism in COVID-19 cases have been lacking to date.

Ehsani highlighted the striking similarity between the amino acid sequence of the SARS-CoV-2 spike glycoprotein and the hepcidin protein (37). This observation provides ideas for vaccine design and bioengineered antibody development for SARS-CoV-2. Hepcidin-mimetic action of SARS-CoV-2 may markedly increase circulating and tissue ferritin, while inducing SI deficiency (32, 33). However, whether SARS-CoV-2 utilizes ferroportin on the cell membrane as another binding receptor for the spike protein to invade host cells requires further analysis. Moreover, whether SARS-CoV-2 directly degrades ferroportin through mimicking hepcidin or indirectly upregulates hepcidin through inflammation to mediate iron dysmetabolism also needs to be carefully investigated. In animal models, hepcidin-neutralizing monoclonal antibodies have been shown to reverse inflammatory anemia (38). Recently, an antibody targeting ferroportin has been described and hypothesized to reduce ferroportin degradation by interfering with hepcidin binding, thereby increasing SI (39). The application of hepcidin antibodies to block viral entry and correct iron metabolism disturbances in COVID-19 cases may be a very promising therapeutic approach in the future.

Hyperferritinemia in COVID-19 lies downstream of the hepcidin-mediated iron metabolism axis. Iron chelation has been shown to reduce viral replication and exert anti-inflammatory effects in viral infections (32, 40, 41). Multiple evidences have shown that deferoxamine reduces the levels of IL-6, the main inflammatory mediator that triggers cytokine storm, mimics HIF, and downregulates hepcidin (40, 42). However, the application of iron chelators in COVID-19 has brought great controversy (42–44). The iron dysmetabolism of COVID-19 is essentially abnormal iron distribution; the coexistence of low SI and local intracellular iron overload, and total iron in the body may not increase. Therefore, the safety and efficacy of systemic iron chelator administration has been challenged (43), and hepcidin antagonists may be preferred as supportive treatments for COVID-19 compared with iron chelators. Four clinical trials of iron chelation are currently underway (NCT04333550, NCT04361032, NCT04389801, IRCT20200506047323N4) and the results are pending.

There are some limitations to this study that need to be noted. First, most of the studies were single-center, retrospective, with small sample sizes, and may be subject to confounding and bias. Second, the six studies used for hepcidin and ferritin data synthesis were heterogeneous, subgroup analyses were not performed, and the results from random-effects models may be inaccurate. Third, the literature search may not be completely comprehensive, resulting in the omission of a few relevant studies. Fourth, this included preprint is a preliminary manuscript version. There are certain risks to the validity and applicability of the data it provides.

Severe COVID-19 cases had higher serum levels of hepcidin and ferritin, and lower SI, without significant differences in TSAT. Compared with other clinically applied therapeutic options targeting the iron metabolism axis, such as IL-6 receptor antagonists and iron chelators, hepcidin antibody may be more promising, but further studies are needed to verify whether targeting the hepcidin-mediated iron metabolism axis may influence the outcome and treatment of COVID-19.

The original contributions presented in the study are included in the article/Supplementary Material, further inquiries can be directed to the corresponding author/s.

DP was responsible for methodology, investigation, formal analysis, data curation, writing the original draft, and visualization. YG and LZ for investigation, formal analysis, and data curation. YL for conceptualization, investigation, review and editing, and supervision. ZL and HW worked on conceptualization, formal analysis, investigation, and data curation. All authors have read and approved the final manuscript version to be submitted.

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

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