Fig 2: TIPE2 regulates ferroptosis-related pathways in THP-1 macrophages following SARS-CoV-2 spike protein stimulation. a, b Expression of ferroptosis-related genes in THP−1 macrophages by qRT-PCR and Western blotting. the ferroptosis-related proteins including GPx4, SLC7A11,FSP1 and ACSL4. Cropped blot is shown for clarity, retaining relevant bands. The uncropped, full-length blot with visible edges is included in Supplementary Figure S3, 4. c Cell viability was evaluated by CCK8 assay in TIPE2-transfected or control THP-1 macrophages treated with S protein alone or in combination with Fer-1 (2µM). d-h Quantification of intracellular levels of ferroptosis-associated metabolic indicators, including ferrous iron (Fe²+), Coenzyme Q10 (CoQ10), NADP⁺/NADPH ratio, malondialdehyde (MDA), and GSH/GSSG ratio in THP-1 macrophages under the indicated treatments. i Representative fluorescence microscopy images showing lipid ROS accumulation in THP-1 macrophages labeled with specific probes. Nuclei are stained blue (Hoechst), lipid ROS appear in green (FITC channel), and merged images are presented. Data are representative of at least three independent experiments. Student’s t-test evaluated differences between two groups, and one-way ANOVA with post-hoc testing (LSD or Tamhane’s T2) was used for comparisons among three groups. (*P < 0.05, **P < 0.01, ***P < 0.001)

Fig 3: SARS-CoV-2 spike protein suppresses TIPE2 and GPx4 expression and induces mitochondrial morphological changes in THP-1 macrophages. a TIPE2 and GPx4 mRNA expression levels in THP-1 macrophages were altered following treatment with different concentrations (0, 10, 20, 50, 100 ng/ml) of the SARS-CoV-2 spike protein for 12–24 h. b Protein expression levels of TIPE2 and GPx4 in THP-1 macrophages were evaluated by Western blotting after 24 h of stimulation with 20 ng/mL S protein. Cropped regions from the same blot are shown. The full-length blot, including molecular weight markers and uncropped lanes, is presented in Supplementary Fig. S1. c The mitochondrial morphology of phorbol-12-myristate-13-acetate (PMA) -induced THP-1 macrophages under transmission electron microscopy, both untreated and treated with the spike protein. Black arrows denote normal mitochondria, whereas red arrows indicate pathological changes characterized by elevated membrane density, membrane rupture, and the reduction or disappearance of mitochondrial cristae (scale bar = 500 nm). Student’s t-test evaluated differences between two groups, and one-way ANOVA with post-hoc testing (LSD or Tamhane’s T2) was used for comparisons among three groups.(*P < 0.05, **P < 0.01, ***P < 0.001).

Fig 4: Critical COVID−19 Patients Exhibit Defects in TIPE2 and GPx4 Expression, Accompanied by Iron Dysregulation and Elevated Inflammation. a, b Plasma concentrations of inflammatory cytokines (IL-6, -1β, -10 and TGF-β) and iron metabolism markers (ferritin and total iron) in COVID-19 patients and controls. c Plasma concentrations and PBMC transcript levels of TIPE2 and GPx4 among the three groups. d Correlations between TIPE2 and clinical disease severity, GPx4, TGF-β and ferritin. P values were adjusted for multiple comparisons using the FDR method. Data are presented as mean ± SD or correlation coefficients. Statistical significance was determined using one-way ANOVA with Tukey’s multiple comparisons test for group comparisons, and Spearman’s correlation for association analyses.(*P < 0.05, **P < 0.01, ***P < 0.001.)