Fig 2: Inhibitory effect of BAPC‐SME on local cytokine storm in the lung. (a) The schematic of experimental process. (b) Relative mRNA level of TNF‐α, MCP‐1, IL‐1β, IL‐6, IL‐10, and IFN‐γ in the lung. The data are presented as the mean ± standard error of the mean (n = 4). *p < 0.05, **p < 0.01, ***p < 0.001 compared with model (Table S8). (c) The histopathological assays (magnification, ×100) in the lungs were performed at 6 h after LPS instillation. Alveolar collapse (black arrow), Inflammatory cell infiltration (red arrow). (d) Representative image (magnification, ×400) of immunohistochemically identified (brown) T cells and macrophages in lung tissues detected with murine T cells marker protein (CD3) and macrophage marker protein (CD68)

Fig 3: The establishment of systemic cytokine storm model. (a) The schematic of treatment schedule. (b) Mean plasma concentration of TNF‐α, MCP‐1, IL‐27, IL‐17A, IL‐6, IL‐12p70, IL‐10, IL‐1α, MIG, IFN‐β, IFN‐γ, IL‐23, IL‐4, MIP‐1α, and MIP‐1β in C57BL/6 mice at different time after intraperitoneal administration of lipopolysaccharide (5 mg/kg). n = 4–5, *p < 0.05, **p < 0.01, ***p < 0.001 compared with 0 h (Table S4).

Fig 4: An IRG1‐itaconate‐NF2L2 axis regulates LPS‐induced pro‐inflammatory reactivity in microglia. (A) Western blot analysis of IRG1, NF2L2/NRF2, and ACTB protein expressions in BV2 microglia treated with LPS and IL4 for 6 h. Control cells are treated with PBS for 6 h. (B) RT‐qPCR analysis of Acod1 expression in BV2 microglia transfected with siRNAs pool targeting Acod1 (siAcod1) or siRNA nontargeting control (siNTC). (C) RT‐qPCR analysis of Nfe2l2 expression in BV2 microglia transfected with siAcod1, siNfe2l2, or siNTC. (D) Western blot analysis of IRG1, NF2L2/NRF2, and ACTB protein expressions in LPS‐treated BV2 microglia transfected with siAcod1, siNfe2l2, or siNTC. (E–G) RT‐qPCR analysis of Nos2, Il1β and Il6 gene expression in LPS‐treated BV2 microglia transfected with siAcod1 or siNTC. (H) Western blot analysis of NOS2 and ACTB protein expressions in 24 h LPS‐treated BV2 microglia transfected with siAcod1 or siNTC. Graph depicts quantification of NOS2 expression as a ratio to ACTB expression. (I) Secreted IL1β expression levels measured by ELISA in supernatant of 24 h LPS‐treated BV2 microglia transfected with siAcod1 or siNTC. (J) Secreted IL6 expression levels measured by flow cytometry multi‐ELISA LEGENDplex in supernatant of 24 h LPS‐treated BV2 microglia transfected with siAcod1 or siNTC. (K) Secreted IL1β expression levels measured by ELISA of 24 h LPS‐treated BV2 microglia transfected with siNfe2l2 or siNTC. (L) Secreted IL6 expression levels measured by flow cytometry multi‐ELISA LEGENDplex in 24 h LPS‐treated BV2 microglia transfected with siNfe2l2 or siNTC. Data are mean ± SEM from multiple independent biological replicates (passages) N = 5 (B, E, F, G, I, J, K, L) and N = 3 (A, C, D, H) for untreated, LPS (100 ng/mL), or IL4 (20 ng/mL)‐treated groups. Statistical annotations *p < 0.05; **p < 0.01; ***p < 0.001; for the indicated comparison. More detailed information about sample size and statistical tests can be found in Table S4.

Fig 5: LPS treatment shifts microglial metabolism from oxidative phosphorylation to glycolysis. Two experimental approaches employing the Seahorse metabolic analyzer were used to study the effect of IL4 (20 ng/mL) and LPS (100 ng/mL) treatment in BV2 microglia. Panels A–E show 6‐h long treatment at standard culture conditions with subsequent metabolic analysis representing (A) ECAR analysis curve with (B) ECAR quantification, (C) OCR analysis curve with (D) quantification, and (E) OCR/ECAR ratio. Panels F–J show results from a second experimental approach using BV2 cells treated with the same concentrations of IL‐4 and LPS, measured continuously over 6 h using the Seahorse analyzer. Panel (F) shows representative ECAR analysis curve over 6‐h long measurement with (G) quantification, (H) OCR analysis curve with (I) quantification, and (J) OCR/ECAR ratio for 6‐h long ongoing measurement in the Seahorse analyzer. Data are mean ± SEM from n = 16 independent points of measurement from N = 2 different passages (A–E); n = 13 points of measurement from N = 2 different passages (F–J). Statistical annotations *p < 0.05; **p < 0.01; ***p < 0.001; for the indicated comparison. More detailed information about sample size and used statistical tests can be found in Table S4.