Fig 2: Efficacy and reactogenicity of A-910823 induced via IL-1 receptor/MyD88 signaling pathway.A Study schedule. Mice were injected IM with Ag mixed with A-910823 on days 0 and 14 (n = 4–10/group), using Myd88 (B), Il1r1 (C), and Il1rn KO mice (D), as well as Myd88flox/flox (f/f) and Cd11ccre (E) or LysMcre mice (F). B–F Serum titers of neutralizing antibodies against SARS-CoV-2 on day 28. Bars represent geometric mean titer (GMT); error bars indicate 95% confidence intervals; circles represent neutralizing antibody titers in individual mice. Percentages of T follicular helper (Tfh; PD1+CXCR5+) cells in TCRb+CD4+ cells and percentages of germinal center B (GCB; FAS+GL7+) cells in CD19+ cells in draining LNs on day 28. Bars represent means; error bars indicate standard error of the mean; circles represent percentages of Tfh and GCB cells in individual mice. Injection-site swelling scores on day 15. Bars represent means; error bars indicate standard error of the mean; circles represent swelling scores in individual mice. Average body temperature was assessed on Day 12 (08:00–19:59) and on Day 15 (08:00–19:59). Vaccination at day 14 was performed at 18:00. Bars represent means; error bars indicate standard error of the mean. The data were derived from independent experiments: (1) Neutralizing titer/Tfh/GCB measured in the same cohort, (2) Swelling measured in an independent cohort, and (3) Fever measured in an independent cohort. Statistical significance determined using two-tailed Student’s t-test for two-group comparisons or two-way ANOVA. ns, P ≥ 0.05, *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001.

Fig 3: ACOD1 regulates arginine metabolism via ACLY. (A) Citrate amounts in Acod1-/- and wt microglia treated for 24 h with PBS or LPS+IFN-γ (n=6). (B) Western blot for phosphorylated ACLY in Acod1-/- and wt microglia treated for 24 h with LPS+IFN-γ using β-actin as a loading control and band intensity quantification (n=3-5, 2 experiments). (C) Acod1-/- and wt primary microglia were incubated with 13C-citrate for the indicated times, 13C-acetyl-CoA was measured in the cell lysates by LC–MS/MS and normalized to the protein content (n=3-5). (D) Acetyl-CoA levels in Acod1-/- and wt primary microglia treated for 24 h with LPS+IFN-γ (n=6). (E) Acly expression in microglia sorted from Acod1-/- and wt mice treated for 16 h with LPS (3 mg/kg) (n=8 mice per group). (F) Acly and Slc25a1 mRNA expression in Acod1-/- and wt microglia treated for 24 h with LPS+IFN-γ (n=14-16). (G) Ass1, Nos2, Odc1, Il-1b and Il-6 expression in Acod1-/- microglia treated for 24 h with BMS303141 (20 μM), LPS+IFN-γ or respective controls (n=8-14). (H) Citrulline, spermidine and spermine amounts in Acod1-/- microglia treated for 24 h with LPS+IFN-γ and BMS303141 (20 μM) or carrier (n=6). (I) Abundance of H3K9ac marks in the Ass1 gene promoter in microglia sorted from LPS-treated Acod1-/- and wt mice, shown by Cut&Tag (n=4-5). (J) Abundance of H3K9ac marks in the Ass1 gene promoter in Acod1-/- and wt primary microglia treated for 24 h with LPS+IFN-γ and BMS303141 (20 μM) or carrier, shown by Cut&Tag (n=4-5). (K) Schematic presentation of the hypothesis. *p < 0.05, **p < 0.01, ****p<0.0001.

Fig 4: A-910823 induced alterations in gene expression associated with IL-1 signaling dependent on α-tocopherol.Mice were immunized IM with antigen (Ag), Ag combined with A-910823 (Ag + A-910823), or Ag combined with A-910823 without α-tocopherol (Ag + A-910823 w/o α-Toc), and the draining LNs and muscle tissues were harvested 30 min or 4 h post-immunization for gene expression analysis via RNA sequencing. The data set for each group comprised three independent pooled biological replicates. A Principal component analysis (PCA) of gene expression in the indicated tissues and groups. Each dot represents a biological replicate. B Volcano plots showing differentially expressed genes in the indicated tissues at 4 h after immunization. Red and blue dots represent upregulated and downregulated genes, respectively, in the Ag + A‑910823 group compared with the Ag group. C Heatmaps showing Z-score normalized expression of differentially expressed genes associated with cytokines, including chemokines, ILs, IFNs, TNF, TGF-β, and their respective receptors in the indicated tissues at 4 h post-immunization. Value in parentheses represents number of differentially expressed genes. D Over-representation analysis using KEGG signaling pathways with upregulated genes in the Ag + A-910823 group compared with the Ag group in the indicated tissues at 4 h post-immunization. E Relative expression of genes associated with IL-1 signaling in draining LNs at 4 h post-immunization. Red and blue dots represent differentially upregulated and downregulated genes, respectively, in the Ag + A-910823 group compared with the Ag + A-910823 w/o α-Toc group. Statistical significance calculated using Wilcoxon’s matched-pairs signed rank test. ***P < 0.001.

Fig 5: ACOD1 deficiency reprograms arginine metabolism. (A) Argininosuccinate amounts in primary Acod1-/- and wt microglia treated for 24 h with LPS+IFN-γ or carrier (PBS) (n=6-7). (B) Ass1 expression in microglia sorted from Acod1-/- and wt mice treated for 16 h with PBS or LPS (3 mg/kg) (n=6 mice per group). (C) Ass1 and Il-1b expression in primary Acod1-/- microglia transfected for 24 h with siAss1 or siCtrl and treated with LPS+IFN-γ for 24 h (n=8-10). (D) mRNA expression of Il-1b, Sall1, Tmem119, Cx3cr1, Trem2 and Mertk in microglia sorted from wt mice treated for 3 h with argininosuccinate (ASA) (50 mg/kg) and then for 4 h with LPS (1 mg/kg) (n=7–8 mice per group). (E) Spermidine and spermine amounts in wt primary microglia transfected with 30 nM siAss1 or siCtrl and treated with LPS+IFN-γ for 24 h (n=3). (F) GSEA for polyamine metabolism-related genes based on bulk RNAseq analysis in microglia sorted form LPS-treated Acod1-/- and wt mice (n=3 mice per group). (G) Spermidine amounts in Acod1-/- and wt primary microglia treated for 24 h with LPS+IFN-γ (n=9-10). (H) Ornithine and spermidine amounts in primary Acod1-/- and wt microglia treated for 24 h with LPS+IFN-γ (n=3-6). (I) Odc1 and Il-1b expression in primary wt microglia transfected for 48 h with 30 nM siOdc1 or siCtrl and then treated for 4 h with LPS+IFN-γ (n=9). (J) Il-1b expression in primary Acod1-/- microglia treated for 24 h with spermidine (SPD), LPS+IFN-γ or respective controls (n=11). *p < 0.05, **p < 0.01, ***p<0.001, ****p<0.0001. n.d.: non-detectable.