Role of Bioavailable Iron Mediating the Outcome of COVID-Associated Secondary Mold Infection

We tested the hypothesis that excess airway iron released during viral infection promotes secondary Aspergillus fumigatus (A.f) infection by enhancing fungal growth and impairing neutrophil antifungal activity. Using ferrozine colorimetry and spectral deconvolution, bronchoalveolar lavage (BAL) samples from patients with severe SARS-CoV-2 infection were found to contain significantly higher levels of total iron, Fe³⁺, Fe²⁺, and hemoglobin (Hb) compared to those with bacterial pneumonia. Formalin-fixed paraffin-embedded (FFPE) lung tissues from SARS-CoV-2 patients exhibited increased extracellular ferritin, Hb, and lactoferrin in airway spaces. Correspondingly, BAL fluid showed elevated IL-6 and expression of host iron-sequestration genes including hepcidin, transferrin receptor, lactoferrin, ferritin, and Nramp1.

Using mutant fungal strains, we demonstrated that Fe³⁺, Fe²⁺, Hb, and heme enhanced A.f growth, while reductive iron assimilation mediated Hb- and heme-related fungal toxicity. In a neutrophil airway transmigration model, exposure to Fe³⁺ increased reactive oxygen species (ROS) and counterproductive NET production with minimal gene expression changes, whereas Hb and heme suppressed ROS and, at low doses, induced transcriptional regulation of iron metabolism and pro-inflammatory genes. At higher doses, all iron sources impaired neutrophil gene transcription, suggesting dysregulation of pre-formed antifungal effectors as the primary mechanism of dysfunction. Co-culture experiments showed that when both neutrophils and fungi were exposed to iron, Hb, or heme, fungal growth predominated, resulting in uncontrolled hyphal proliferation that was countered by small-molecule inhibitors of siderophore biosynthesis.

Together, these findings show that excess iron availability during SARS-CoV-2 pneumonia enables a permissive airway niche that promotes rapid fungal growth, impairs host immunity, and facilitates secondary mold infection.