News Release

Fatal COVID-19 pulmonary disease involves ferroptosis
Key Points• In COVID-19, severe pulmonary lesions can lead to mortality.
• We analyzed severe pulmonary lesions in COVID-19 using post-mortem
autopsy samples from patients in the United States and an experimental animalmodel with hamsters.
• In severe pulmonary lesions of COVID-19, involvement of ferroptosis, aniron-dependent form of regulated cell death, has been demonstrated,suggesting its potential as a new drug target.
SummarySARS-CoV-2 infection leads to severe lung damage, with ferroptosis, aniron-dependent cell death, playing a significant role. Hyperferritinemia anddisrupted

ironhomeostasis

inCOVID-19

patientssuggestferroptosis

involvement. Autopsy analysis revealed increased ferroptosis markers, lipid
dysregulation, and ferritin accumulation in severe cases. In a hamster model,
ferroptosis markers correlated with lung injury severity. These findings
highlight ferroptosis as a key factor in COVID-19 lung pathology, suggestingthat inhibiting ferroptosis could help prevent lung damage during infection.
Research Background
Coronavirus disease-2019 (COVID-19), caused by SARS-CoV-2, leads to severelung conditions like pneumonia and acute respiratory distress syndrome (ARDS),
which are major contributors to high mortality rates. Acute lung injury (ALI),
including diffuse alveolar damage (DAD), and non-acute lung injury (non-ALI),
such as pulmonary vascular congestion and microthrombi, are common in
COVID-19 patients. These lung pathologies are driven by both viral damage andhost inflammatory responses, including cytokine storms and oxidative injury
from immune cells. Despite treatments like intubation, mechanical ventilation,
and a combination of antiviral and anti-inflammatory medications, effective
therapies for COVID-19 lung disease remain limited.
Ferroptosis, an iron-dependent form of cell death driven by phospholipid
peroxidation, plays a significant role in various diseases. In COVID-19, several
pro-ferroptotic changes have been observed. SARS-CoV-2 infection decreases
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GPX4 expression, reducing the cell's ability to repair lipid peroxidation, whilethe virus also inhibits the NRF2 antioxidant response, increasing susceptibility
to oxidative damage. Additionally, elevated serum ferritin in critically ill
COVID-19 patients supplies iron that drives ferroptosis, disrupting ironmetabolism in the lungs.
This study identifies ferroptosis as a major mechanism underlying COVID-19lung pathology. By examining human COVID-19 lung tissue, we found distinct
molecular markers of ferroptosis, particularly in severe ALI and non-ALI cases.
Lipidomic analysis of lung autopsies revealed changes in lipids associated withinflammation, metabolism, and ferroptosis. Further investigation using a Syrian
hamster model of COVID-19 showed a strong correlation between ferroptosis
and lung pathology.
Research Results
The study explores the role of ferroptosis, a form of iron-dependent cell death,in the lung pathologies of COVID-19 patients. We analyzed lung tissue fromsevere COVID-19 cases, distinguishing between acute lung injury (ALI) andnon-ALI types, as well as from non-affected and non-COVID-19 ALI controls.
Microscopic examination identified hallmark features of ALI and non-ALI in
COVID-19 lungs. The study aimed to determine the cell death mechanisms
involved, focusing on ferroptosis, necroptosis, apoptosis, and pyroptosis.
Using immunofluorescence and immunohistochemistry, the study found thatferroptosis markers, transferrin receptor 1 (TfR1), and malondialdehyde adduct(MDA), were elevated in COVID-19 ALI and non-ALI lungs compared to control
samples. In contrast, markers for necroptosis, apoptosis, and pyroptosis did notshow increased levels, suggesting that ferroptosis is the predominant cell deathmechanism in fatal COVID-19 lung disease. Validation using positive andnegative controls further supported the specificity of these markers forferroptosis. The study also examined the role of tumor suppressor p53 and E3
ligase MDM2, which are known to promote ferroptosis. However, these proteins
were not elevated in COVID-19 lungs, indicating they do not drive the observed
ferroptosis.
We then explored the connection between iron dysregulation and ferroptosis,
noting that COVID-19 patients exhibited elevated serum ferritin, a marker ofiron overload. In severe COVID-19 cases, both TfR1 and ferritin light chain (FTL)
were significantly elevated, indicating iron dysregulation's role in promoting
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ferroptosis. Analysis of gene expression in post-mortem COVID-19 lungs
revealed that iron regulatory genes, especially FTL and TfR1, were highly
expressed in macrophages, fibroblasts, and epithelial cells. In vitro experimentswith lung epithelial cells treated with ferric ammonium citrate (FAC) showedthat iron overload induces ferroptosis and lipid peroxidation, consistent withfindings in human lung tissue.
Lipidomics analysis of COVID-19 lung autopsies further confirmed the presenceof ferroptosis. The study identified significant alterations in 130 lipid species,
with a depletion of phospholipids containing polyunsaturated fatty acids(PL-PUFAs) and an accumulation of lysophospholipids, both hallmarks offerroptosis. Additionally, COVID-19 lungs showed depletion of surfactant
phospholipids and an increase in free fatty acids and storage lipids, indicating
disrupted lung function and cellular lipotoxicity.
Finally, the study used a Syrian hamster model of COVID-19 to assess
ferroptosis during disease progression. The hamsters developed lung injury
that correlated with increased ferroptosis markers such as TfR1 and4-hydroxynonenal (HNE). Inhibition of ferroptosis with liproxstatin-1 reduced
lung injury, suggesting that targeting ferroptosis could be a potential
therapeutic approach.
Overall, the study provides strong evidence that ferroptosis is a key contributor
to COVID-19 lung pathology, driven by iron overload and lipid peroxidation.
These findings suggest that ferroptosis inhibition could be a valuable strategy
in mitigating lung injury in severe COVID-19 cases.
Research Summary and Future Perspective
This study investigates the molecular mechanisms behind COVID-19-related
lung damage, focusing on ferroptosis as the primary form of cell deathcontributing to this damage. Ferroptosis is characterized by iron dysregulation,
lipid peroxidation, and depletion of polyunsaturated fatty acids (PUFAs). The
study examined lung tissues from deceased COVID-19 patients and a hamster
model of the disease, finding significant markers of ferroptosis, particularly inareas of severe lung damage. This suggests that ferroptosis plays a crucial rolein the progression of lung disease in COVID-19.
In COVID-19 patients, lung damage includes diffuse alveolar damage (DAD),
congestion, and blood clotting, with blood accumulation in lung tissues
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contributing to disease severity. High levels of serum ferritin and ferritin lightchain in the lungs of severe cases indicate iron overload, which exacerbates
tissue damage through ferroptosis. The study found that primary lung epithelial
cells, when exposed to iron overload, became more susceptible to ferroptosis,
particularly when combined with other stressors, highlighting the role of iron inpromoting this destructive process during SARS-CoV-2 infection.
The research also used advanced bioinformatics, including single-nucleus
transcriptomic profiling, to identify key genes and pathways linked to
COVID-19-induced lung injury. These included oxidative stress responses andsignaling pathways related to ferroptosis. Additionally, lipidomics analysis
revealed significant changes in lung lipid composition, particularly thedepletion of phospholipids and surfactant components essential for lungfunction.

Thesechanges

reflectincreasedoxidative

stressandlipidperoxidation, further contributing to lung damage.
The study also examined the potential for ferroptosis inhibitors to reduce lunginjury. While these inhibitors partially mitigated lung damage in a hamster model,
their effectiveness was limited by poor lung accumulation. This suggests that
direct pulmonary delivery methods, such as inhalation, might be more effective
in targeting lung lesions and preventing ferroptosis.
In conclusion, ferroptosis plays a significant role in COVID-19 lung pathology,
driven by iron overload and oxidative stress. Targeting ferroptosis may offer atherapeutic approach to preserve lung function in COVID-19 patients, andferroptosis markers could potentially be used to assess disease severity.

Publication
Fatal COVID-19 pulmonary disease involves ferroptosis.
Qiu B, Zandkarimi F, Saqi A, Castagna C, Tan H, Sekulic M, Miorin L, Hibshoosh H,Toyokuni S, Uchida K, Stockwell BR. Nat Commun. 2024 May 20;15(1):3816. doi:
10.1038/s41467-024-48055-0. PMID: 38769293

Japanese ver.
https://www.med.nagoya-u.ac.jp/medical_J/research/pdf/Nat_240823.pdf

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