Angiopoietin-like protein 8 orchestrates macrophage glycogen metabolism and polarization via the JNK signaling pathway in cytokine storm syndrome

Figures

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  Fig. 1. Systemic Angptl8 is increased in response to CSS signals. Mice were subjected with 25 mg/kg LPS for 6 h. A Serum Angptl8 levels. B The relative mRNA of Angptl8 in mouse liver. **P < 0.01 v.s. Saline group, one-way ANOVA followed by Bonferroni’s posthoc test, n = 6. C The protein expression of Angptl8 in mouse liver. **P < 0.01 v.s. Saline group, one-way ANOVA followed by Bonferroni’s posthoc test, n = 3. (D) The relative mRNA and protein expression of Angptl8 in mouse PHs treated with indicated doses and time-points of LPS. E The relative mRNA and protein expression of Angptl8 in mouse BMDMs treated with indicated doses and time-points of LPS. F Supernatant Angptl8 levels in mouse PHs treated with indicated dose and time-points of LPS. G Supernatant Angptl8 levels in mouse BMDMs treated with indicated dose and time of LPS. *P < 0.05 and **P < 0.01 v.s. No stimulus group, one-way ANOVA followed by Bonferroni’s posthoc test, n = 3. All values are presented as the mean ± SD

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  Fig. 2. Angptl8 deficiency improves survival and inhibits CSS progression in LPS-induced endotoxemic mice. A The survival of the WT and Angptl8−/− mice stimulated with LPS (25 mg/kg) (i.p.). ##P < 0.01 v.s. WT + LPS group, one-way ANOVA followed by Bonferroni’s posthoc test, n = 10. B Measurement of TNF-α, IL-6, IFN-γ secretion in mouse serum by ELISA after treatment with LPS for 6 h. C–E The relative mRNA expression of Tnf-α, Il-6, Il-1β and Inos in mouse C liver, D spleen and E bone marrow. **P < 0.01 v.s. WT + Saline group, #P < 0.05 and ##P < 0.01 v.s. WT + LPS group, one-way ANOVA followed by Bonferroni’s posthoc test, n = 6. F Representative images of liver by H&E staining. The black arrows point to hepatocyte necrosis, and the red arrows point to inflammatory cells infiltration. G TUNEL assay for liver. H Representative images of spleen by H&E staining. The yellow arrows point to fragmentation of spleen cells. All values are presented as the mean ± SD

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  Fig. 3. Angptl8 activates macrophage M1 polarization. A White blood cell composition and proportion in mice. *P < 0.05 and **P < 0.01 v.s. WT + Saline group, #P < 0.05 and ##P < 0.01 v.s. WT + LPS group, one-way ANOVA followed by Bonferroni’s posthoc test, n = 6. B, C Percentages of CD86+ and CD206+ cells detected by flow cytometry in the B bone marrow and C spleen. D Percentages of CD86+ cells detected by flow cytometry in BMDMs isolated from WT and Angptl8−/− mice. E The relative mRNA expression of Tnf-α, Il-6, Il-1β and Inos in BMDMs isolated from WT and Angptl8−/− mice. **P < 0.01 v.s. WT + Saline group, #P < 0.05 and ##P < 0.01 v.s. WT + LPS group, one-way ANOVA followed by Bonferroni’s posthoc test, n = 3. F Percentages of CD86+ cells detected by flow cytometry in BMDMs treated with recombinant Angptl8 (0, 10, 20, 40 nM) for 24 h. G The relative mRNA and H protein expression of Tnf-α, Il-6, Il-1β and Inos in BMDMs treated with recombinant Angptl8. *P < 0.05 and **P < 0.01 v.s. No stimulus group, one-way ANOVA followed by Bonferroni’s posthoc test, n = 3. All values are presented as the mean ± SD

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  Fig. 4. Angptl8 inhibits macrophage M2 polarization in BMDMs. A Percentages of CD206+ cells detected by flow cytometry in BMDMs isolated from WT and Angptl8−/− mice treated with IL-4. B The relative mRNA expression of Cd206, Arg1, Ym1 and Fizz1 in BMDMs. **P < 0.01 v.s. No stimulus group, #P < 0.05 and ##P < 0.01 v.s. WT + IL-4 group, one-way ANOVA followed by Bonferroni’s posthoc test, n = 3. C–E The relative mRNA expression of Cd206, Arg1, Ym1 and Fizz1 in mouse C liver, D spleen, E bone marrow. *P < 0.05 and **P < 0.01 v.s. WT + Saline group, #P < 0.05 and ##P < 0.01 v.s. WT + LPS group, one-way ANOVA followed by Bonferroni’s posthoc test, n = 6. F Percentages of CD206+ cells detected by flow cytometry in BMDMs treated with IL-4 and recombinant Angptl8. G The relative protein expression of Cd206 and Arg1 in BMDMs detected by Western Blot. H The relative mRNA expression of Cd206, Arg1, Ym1 and Fizz1 in BMDMs treated with IL-4 (20 nM) and recombinant Angptl8 (0, 10, 20, 40 nM). **P < 0.01 v.s. No stimulus group, ##P < 0.01 v.s. IL-4 group, one-way ANOVA followed by Bonferroni’s posthoc test, n = 3. All values are presented as the mean ± SD

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  Fig. 5. Angptl8 modulates macrophage polarization in BMDMs through regulation of glycogen metabolism. A Volcano plot showing differentially expressed genes (DEG) between Angptl8−/−+LPS and WT + LPS group. The up-regulated genes (red) with log2_fold change > 1, and the down-regulated genes (blue) with log2_fold change < − 1, and with P < 0.05. B GO enrichment analysis of down and up regulations for key targets. C GSEA of key pathways. D Intracellular glycogen levels in BMDMs isolated from WT and Angptl8−/− mice treated with LPS (100 ng/mL) were detected and observed by PAS staining and E TEM (The arrows point to intracellular glycogen deposits). F The relative mRNA expression of Pgm1, Ugp2 and Gys1 (glycogen biosynthesis) and Pygm (glycogenolysis) in BMDMs isolated from WT and Angptl8−/− mice. **P < 0.01 v.s. No stimulus group, ##P < 0.01 v.s. WT + LPS group, one-way ANOVA followed by Bonferroni’s posthoc test, n = 3. G Intracellular glycogen levels in BMDMs treated with LPS (100 ng/mL) and recombinant Angptl8 (40 nM) were detected and observed by PAS staining and H TEM (The arrows point to intracellular glycogen deposits). I The relative mRNA expression of Pgm1, Ugp2 and Gys1 and Pygm in…

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  Fig. 6. Phosphorylated JNK mediates Angptl8-induced activation of glycogen metabolism and M1 polarization in macrophages. A KEGG pathway enrichment analysis of key targets. B The protein expression of JNK phosphorylation (p-JNK) and JNK in BMDM treated with recombinant Angptl8 (40 nM) for 0, 5, 15, 30 min, detected by western blot. C The quantitative analysis of Relative JNK phosphorylation analyzed by Image J. *P < 0.05 and **P < 0.01 v.s. No stimulus group, one-way ANOVA followed by Bonferroni’s posthoc test, n = 3. D Intracellular glycogen levels in BMDMs treated with recombinant Angptl8 and SP600125 (a JNK inhibitor 10 µM) were detected and observed by PAS staining and E TEM (The arrows point to intracellular glycogen deposits). F The relative mRNA expression of Pgm1, Ugp2, Gys1 and Pygm in BMDMs. G Percentages of CD86+ cells detected by flow cytometry. H The relative mRNA expression of Tnf-α, Il-6, Il-1β and Inos in BMDMs. **P < 0.01 v.s. No stimulus group, #P < 0.05 and ##P < 0.01 v.s. Angptl8 group, one-way ANOVA followed by Bonferroni’s posthoc test, n = 3. I Percentages of CD206+ cells in BMDMs treated with IL-4, recombinant Angptl8 and SP600125, detected by flow cytometry. J…

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  Fig. 7. Angptl8 neutralization alleviated LPS-triggered CSS. A The survival of mice treated with LPS (25 mg/kg) (i.p.) and neutralized Angptl8 antibody (low: 10 µg/25 g; high: 50 µg/25 g) (i.v.). *P < 0.05 and **P < 0.01 v.s. LPS group, one-way ANOVA followed by Bonferroni’s posthoc test, n = 10. B Measurement of TNF-α, IL-6, IFN-γ secretion in mouse serum by ELISA. C–E The relative mRNA expression of Tnf-α, Il-6, Il-1β and Inos in mouse C liver, D spleen and E bone marrow. **P < 0.01 v.s. Saline group, #P < 0.05 and ##P < 0.01 v.s. LPS group, one-way ANOVA followed by Bonferroni’s posthoc test, n = 6. F Representative images of liver by TUNEL. G Representative images of liver by H&E staining (The black arrows point to hepatocyte necrosis, and the red arrows point to inflammatory cells infiltration.) and H spleen (The yellow arrows point to fragmentation of spleen cells). I White blood cell composition and proportion in the serum of mice. n = 6. J Percentages of CD86+ and CD206+ cells detected by flow cytometry in the bone marrow and K spleen. **P < 0.01 v.s. Saline group, #P < 0.05 and ##P < 0.01 v.s. LPS group, one-way ANOVA followed by Bonferroni’s posthoc test, n = 3. All values are…

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  Fig. 8. A schematic model illustrating that targeting hepatokine Angptl8 alleviates cytokine storm via macrophage metabolic reprogramming in mice