Fig 2: The deficiency of IRG1 aggravated the pain hypersensitivity after nerve injury. (A) Schematic diagram of constructing Irg1 -/- mice. (B) At 14th days post injury, the protein level of IRG1 in L3-L5 segments of the ipsilateral spinal cords from control CCI and Irg1 -/- CCI mice. (C, D) At 14th days post injury, the PWT of ipsilateral and contralateral hind paw to von Frey test in Sham and CCI mice. (E, F) At 14th days post injury, the PWL of ipsilateral and contralateral hind paw to Hargreaves test in Sham and CCI mice. Data are presented as mean ± SEM. **p<0.01, *p<0.05 vs. Control CCI, followed by two-way ANOVA with Tukey’s multiple comparisons test (n = 10).

Fig 3: STING1 and MYD88 selectively mediate TLR signaling to induce ACOD1 expression(A) Indicated THP1 cells were stimulated with pam3CSK4 (1 ng/mL), HKLM (107 cells/mL), poly (I:C) (10 µg/mL), LPS (500 ng/mL), FLA-ST (100 ng/mL), FSL1 (0.1 ng/mL), imiquimod (5 µg/mL), ssRNA40 (5 µg/mL), or ODN2006 (10 µg/mL) for 6 h and the mRNA level of ACOD1 was assessed (Data are presented as mean ± SD; n = 3 biologically independent samples; two-way ANOVA with Tukey’s multiple comparisons test).(B) Wild-type THP1 cells were stimulated with indicated TLR ligands for 3 h and the mRNA level of TNF was assessed (Data are presented as mean ± SD; n = 3 biologically independent samples).(C) Wild-type THP1 cells were stimulated with indicated TLR ligands in the absence or presence of 3'3'-cGAMP (10 µg/mL) for 6 h and the mRNA level of ACOD1 was assessed (Data are presented as mean ± SD; n = 3 biologically independent samples; two-way ANOVA with Tukey’s multiple comparisons test).(D) Wild-type and V115M THP1 cells were stimulated with indicated TLR ligands for 6 h and the mRNA level of ACOD1 was assessed (Data are presented as mean ± SD; n = 3 biologically independent samples; two-way ANOVA with Tukey’s multiple comparisons test). The TLR ligand concentration used in panels B-D is the same as for panel A.

Fig 4: The MYD88-STING1 protein complex prevents autophagic degradation of STING1(A) IP analysis of the MYD88-STING1 protein complex in THP1 cells following treatment with LPS (500 ng/mL) and 3'3'-cGAMP (10 µg/mL) for 6 h.(B) Representative colocalization images of MYD88 and STING1 in RAW264.7 cells in the presence or absence of LPS (500 ng/mL) and 3'3'-cGAMP (10 µg/mL) for 6 h. Bar = 10 µm.(C–F) Western blot analysis of protein expression in indicated THP1 cells following treatment with LPS (500 ng/mL) and 3'3'-cGAMP (10 µg/mL) in the absence or presence of chloroquine (50 µM) or MG132 (5 µM) for 6 h.(G) qPCR analysis of ACOD1 mRNA expression in indicated THP1 cells following treatment with LPS (500 ng/mL) and 3'3'-cGAMP (10 µg/mL) in the absence or presence of chloroquine (CQ; 50 µM) for 6 h (Data are presented as mean ± SD; n = 3 biologically independent samples; two-way ANOVA with Tukey’s multiple comparisons test).

Fig 5: STING1 is required for LPS-induced ACOD1 expression(A–D) RAW264.7 and THP1 cells were treated with LPS (50-5000 ng/mL) in the absence or presence of 3'3'-cGAMP (10 µg/mL) for 6 h, and then Acod1/ACOD1 mRNA and intracellular itaconate concentration were assayed (Data are presented as mean ± SD; n = 3 biologically independent samples; two-way ANOVA with Tukey’s multiple comparisons test).(E and F) RAW264.7 and THP1 cells were treated with LPS (500 ng/mL) in the absence or presence of 2'2'-cGAMP, 2'3'-cGAMP, c-di-AMP, or c-di-GMP at 10 µg/mL for 6 h, and then Acod1/ACOD1 mRNA and intracellular itaconate concentration were assayed (Data are presented as mean ± SD; n = 3 biologically independent samples; p < 0.01 versus LPS along group; two-way ANOVA with Tukey’s multiple comparisons test).(G–J) WT and V155M-THP1 cells were treated with indicated LPS for 6 h, and then IFNA1 mRNA, IL6 mRNA, ACOD1 mRNA, and intracellular itaconate concentration were assayed (Data are presented as mean ± SD; n = 3 biologically independent samples; two-way ANOVA with Tukey’s multiple comparisons test).(K) Western blot analysis of protein expression in indicated THP1 cells following treatment with LPS (500 ng/mL) for 6 h.(L) In parallel, intracellular itaconate concentration was assayed (Data are presented as mean ± SD; n = 3 biologically independent samples; two-way ANOVA with Tukey’s multiple comparisons test).