Fig 2: Expression level of AIM2 inflammasome is upregulated by HMGB1. Effects of (A) anti-HMGB1 and (B) rHMGB1 on the expression level of AIM2 in mouse lung tissue was detected by immunohistochemistry (magnification, ×200), and AOD was analyzed in different groups. In the in vivo experiment, the expression levels of AIM2 inflammasome and GAPDH were detected by (C and E) western blotting with (C) anti-HMGB1 and (E) rHMGB1 and RT-qPCR with (D) anti-HMGB1 and (F) rHMGB1. All experiments were repeated more than three times (n=4-6 mice per each group). Data presented is from a representative experiment. All data are expressed as the mean ± standard deviation. *P<0.05, **P<0.01 and ***P<0.001 vs. LPS group. Expression level of AIM2 inflammasome is upregulated by HMGB1. In an experiment with BMMs, the expression levels of the AIM2 inflammasome and GAPDH were also detected by (G) western blotting and (H) RT-qPCR. (I) The expression levels of IL-1ß and IL-18 in culture supernatant of BMMs were measured by ELISA. All experiments were repeated more than three times (n=4-6 mice per each group). Data presented is from a representative experiment. All data are expressed as the mean ± standard deviation. *P<0.05, **P<0.01 and ***P<0.001 vs. LPS group. LPS, lipopolysaccharide; IL, interleukin; rHMGB1, recombinant High mobility group box 1; RT-q, reverse transcription-quantitative; AIM2, absent in melanoma 2; AOD, average optical density; BMMs, bone-marrow derived macrophages; ASC, apoptosis-associated speck-like protein containing a CARD.

Fig 3: GBP1 Mediates Access to PAMPs During Toxoplasma and Salmonella Infection(A) Left: representative immunofluorescence image of type I Tg grown in human foreskin fibroblasts (HFF WTs) in the presence of EdU DNA label. Labeled type I Tg were harvested from the HFFs and used to infect (inf.) THP-1 ?GBP1+Tet-mCH-GBP1 or THP-1 +myc-AIM2 for 6 h. THP-1 cells were pre-treated with IFN? and Dox to induce mCH-GBP1 expression Middle: parasite DNA released into the cytoplasm was visualized by click-chemistry to label the incorporated EdU. Right: quantification of proportion of infected cells with cytosolic Tg-DNA based grouped based on whether the cells contain a GBP1-targeted (GBP1+) Tg PV. Red: mCH-GBP1 or immune-stained myc-AIM2; white: EdU-DNA; green: Tg; blue: nuclei. Some released, cytosolic (and additionally AIM2-bound, lower panels) Tg-DNA indicated by yellow arrowheads. Scale bar, 10 µm.(B and C) Representative immunofluorescence images (B) and quantification (C) of LPS staining intensity of STm from IFN? and Dox-treated THP-1 ?GBP1+Tet-mCH-GBP1+YFP-CASP4C258S cells infected with STm SL1344 (MOI = 30) for 2 h. Red: mCH-GBP1; gray: STm-LPS; green: YFP-caspase-4; blue: nuclei. Scale bar, 10 µm. Contrast enhanced in the nuclei inset, to visualize STm-DNA used for detection of bacteria that do not stain for LPS.(D) Representative immunofluorescence images at 1 h p.i. and quantification of Gal-8 and caspase-4 (CASP4) recruitment to STm in IFN?-primed and Dox-treated THP-1 ?GBP1+Tet-mCH-GBP1+YFP-CASP4C258S infected with STm SL1344 (MOI = 30) at indicated times p.i. Red: mCH-GBP1; gray: Gal-8; green: YFP-caspase-4; blue: nuclei. Scale bar, 10 µm.(E) Left: silver stain of SDS-PAGE gel showing immunoprecipitation (IP) of FLAG-GBP1 from THP-1 ?GBP1+Tet-FLAG-GBP1 treated with IFN? and Dox and infected with STm for 2 h, left UT. Right: volcano plot of mass spectrometry hits comparing IFN? treated cells with IFN? treated and STm-infected cells. Plotted as Welch difference of mass spectrometry intensities versus -log10(p). Significant hits shown in orange/red circles.(F) Representative immunoblots of IP of endogenous GBP1 from IFN?-primed or naive THP-1 WT infected with STm for 2 h as indicated, showing co-precipitation of endogenous caspase-4 identified as a hit using mass spectrometry and no stable interaction with caspase-1.Graphs in (A) and (D) show mean ± SEM from n = 3 independent experiments. Graph in (C) representative of n = 3 independent experiments. *p = 0.05 and ****p = 0.0001 in (C) from nested one-way ANOVA comparing to undecorated vacuoles for the means of the n = 3 independent experiments; ns, not significant.See also Figure S4 and Data S1.

Fig 4: STING pathway contributes to the cytotoxicity arising from the inhibition of autophagosome formation.a Determination of cytotoxicity of SBI-0206965 against shRNA control or STING KD HL-60 cells (n = 4). b Determination of cytotoxicity of SBI-0206965 against shRNA control or AIM2 KD HL-60 cells (n = 4). c Relative mRNA expression of the indicated genes in control or STING shRNA-transfected HL-60 cells at 18 h after treatment with 5 µM SBI-0206965. As a control, cells were treated with DMSO. Data represent mean ± SD from three independent experiments. d Total ROS induction in control or STING shRNA-transfected HL-60 cells at 20 h after treatment with 5 µM SBI-0206965. Cells treated with DMSO were used as a control. MFI of cells stained using a total ROS/superoxide detection kit is shown. Data represent mean ± SD from three independent experiments. e Cytotoxic assay when HL-60 cells were co-cultured with 5 µM SBI-0206965 in the presence or absence of N-acetyl-L-cysteine (NAC, Sigma-Aldrich) (n = 4). f Colony formation of WT mice or STING-/- mice AML cells when they were co-cultured with 5 µM SBI-0206965. Cells treated with DMSO were used as a control. g, h Suppression of colony formation of mouse AML cells by SBI-0206965 was calculated in terms of colony (g) and total cell number (h) by normalization with DMSO control for each. Data represent mean ± SD from three independent experiments. **P < 0.01; *P < 0.05; N.S., no significant difference using Tukey–Kramer test or two-sided Student’s t-test.

Fig 5: The regulation of inflammasomes by L. plantarum K8 lysates and LPS treatment. THP-1 cells were treated with 1 × 109 CFU/mL of L. plantarum K8 lysates for 18 h and/or 500 ng/mL of LPS for 4 h. mRNA levels of absent in melanoma 2 (AIM2) (A), caspase-1 (B), apoptosis-associated Speck-like protein containing a CARD (ASC) (C), NLR family pyrin domain containing (NLRP) 1 (D), and NLRP3 (E) were measured by real-time PCR. (F) Protein levels of AIM2, caspase-1, and ß-actin were measured by western blot. (G) THP-1 cells were transiently transfected with control siRNA, AIM2 siRNA (GS9447), and caspase-1 siRNA (GS834) purchased from Qiagen. After 36 h transfection, cells were stimulated with L. plantarum K8 lysates and/or LPS. The IL-1ß level in the culture supernatants was determined using ELISA. Statistical analysis was conducted with one-way ANOVA followed by Tukey’s HSD post hoc test. * p < 0.05; ** p < 0.01; *** p < 0.001.