Journal: Frontiers in Immunology

Article Title: Fumarate inhibits the formation of neutrophil extracellular traps (NETs) in a Nrf2-controlled and Annexin-A1-dependent manner associated with mitochondrial fusion

doi: 10.3389/fimmu.2026.1770063

Figure Lengend Snippet: Expression of Nrf2 and ANXA1 in relation to NET release by neutrophils treated with dimethyl fumarate. Neutrophils isolated from mice were either pre-treated with DMF (25 µM), ML385 (Nrf2 inhibitor), WRW4 (Fpr2 antagonist) for 1 h or their combination: DMF→ML385, WRW4→DMF incubated with lipopolysaccharide (LPS) at a concentration of 50 μg/mL (for 6 h) or left unstimulated (CTR). (A) Quantification of Nrf2 expression and (B) NETs (extDNA and citH3) quantification after Nrf2 inhibition. (Ci) Representative images and (Cii) area of NETs formed upon the above treatments with WRW4 and DMF. ExtDNA is shown in green and citH3 is shown in red. To visualize co-localization of extDNA with citH3 expression, the images from each channel were overlaid. (D) Levels of ANXA1 were measured in neutrophils supernatants upon the above treatments with DMF and ML385 by ELISA. The results are expressed as the mean values ± SD; n≥3. Values significantly different between the groups (p < 0.05) according to one-way ANOVA ( post hoc Bonferroni test) are designated by letters, where the same letter indicates no differences between groups (different letters indicate statistical differences). Explanation of abbreviations: Nrf2, nuclear factor erythroid 2-related factor 2; ANXA1, Annexin A1; extDNA, extracellular DNA; citH3, citrullinated histone H3.

Article Snippet: The following inhibitors were used: nuclear factor erythroid 2-related factor 2 (NRF2) inhibitor – ML385 (10 μM, MedChemExpress), formyl peptide receptor 2 (Fpr2) antagonist - WRW4 (10 μM, MedChemExpress), NADPH oxidase (NOX) inhibitor - Diphenyleneiodonium chloride (DPI, 10 μM, Merck), necroptosis inhibitor - Necrostatin-1 (50 μM, Torcis Bioscence, Bristol, UK), pyroptosis inhibitor - Disulfiram (30 μM, MedChemExpress) and mitochondrial complex I inhibitor - Rotenone (5 μM, Sigma-Aldrich, Saint Louis, MO, USA).

Techniques: Expressing, Isolation, Incubation, Concentration Assay, Inhibition, Enzyme-linked Immunosorbent Assay