Fig 1.
MDAs including MMAE enhanced the cGAMP-mediated STING pathway.
(A, B, E, and G) THP1-Lucia ISG (WT and STING KO) and RAW-Lucia ISG cells were treated with 2’3’-cGAMP (cGAMP, 0.5 μM) and/or indicated doses of MDAs or MMAE for 24 h, and the fold change of luminescence was normalized to DMSO or cGAMP-treated cells. (C, D, F, and H) THP1-Lucia ISG (WT and STING KO) and MEF cells were treated with cGAMP (0.5 μM) and/or indicated concentrations of MMAE for indicated times (C, D and F) or 6 h (H), and activation of the STING pathway were analyzed by immunoblotting. (I-L) THP1-Lucia ISG cells (WT and STING KO) and BMDMs (WT, Stinggt/gt, or Myd88-/- mice) were stimulated with cGAMP and/or indicated concentrations of MMAE for 12 h (I, K) or 6 h (J, L). IFNβ production was measured by ELISA analysis (I and K). the activation of STING pathway was analyzed by immunoblotting (J). mRNA expression levels of IFNβ, CCL5 and CXCL10 in BMDMs (WT, Stinggt/gt, or Myd88-/- mice) (n = 3 biological replicates) (L). cGAMP was used at 0.5 μM for all experiments unless otherwise noted.
Fig 2.
MMAE specifically enhanced the cGAMP-STING-mediated NF-kB immune response.
(A) A diagram of MMAE promoting STING-mediated NF-kB signaling. (B-E) THP1-Lucia NF-kB (WT, p50 KO and p65 KO) and THP1-Lucia ISG (WT and p65 KO) were treated with cGAMP (0.5 μM) and/or indicated doses of MMAE for 24 h. The fold change of luminescence was normalized to DMSO-treated cells. (F, I and J) HEK293T cells were transiently transfected with STING plasmids (WT, Flag-hSTING (S366A)) for 24 hours. Cells were stimulated with cGAMP (0.5 μM) and/or MMAE (0.1 μM) for 6 h, and cell lysates were analyzed by immunoblotting for the indicated proteins (F and I). Quantification of LC3II/tubulin ratio from three independent experiments (J). (G) THP1-Lucai ISG (STING KO) cells stably expressing (hSTING WT, Flag-hSTING (S366A)) were stimulated with cGAMP and/or MMAE (1 μM) for 12 h. IFNβ production was measured by ELISA analysis. (H) STING was analyzed by immunoblotting in THP1-Lucai ISG (STING KO) cells.
Fig 3.
MMAE enhanced the activation of STING pathways mediated by distinct CDNs in a direct STING-IRF3 dependent manner.
(A-L) THP1-Lucia ISG (WT and STING KO) cells were treated with cGAMP with or without indicated doses of MMAE for 24 h (A-F) or 6 h (J-L), and the fold change of luminescence was normalized to DMSO-treated cells. The activation of STING pathway was analyzed by immunoblotting (G-L). (M) THP1-Lucia ISG cells were pretreated for 12 h with or without anti-IFNAR2 antibody (20 μg/ml), and then stimulated with cGAMP or IFNβ (200 pg/ml) for 24 h in the absence or presence of MMAE (1 μM). Fold change of luminescence was normalized to DMSO-treated cells. (N and O) THP1-Lucia ISG (WT and IRF3 KO) cells were treated with cGAMP and/or indicated doses of MMAE for 12 h (N) or 6 h (O). IFNβ production was measured by ELISA analysis (N). Expression of IRF3 and the activation of STING pathway was analyzed by immunoblotting (N and O).
Fig 4.
MMAE boosted the cGAMP-mediated STING pathway by increasing STING-containing membrane puncta numbers and the extent of STING oligomerization.
(A) Chemical structure of Monomethyl auristatin E (MMAE). (B) HeLa cells stably expressing human STING-GFP were stimulated with cGAMP (8 μM) and/or MMAE (1 μM), or VcMMAE (1 μM) for 2 h in the absence or presence of brefeldin A (BFA, 1 μM). Fluorescent images of cells were acquired on a Zeiss LSM980 Airyscan2 Confocal microscope using a 63× (NA 1.45) objective and processed in Zen Blue 3.1 software. STING (green), nuclei were stained with Hoechst (blue). Scale bars, 10 μm. (C-E) THP1-Lucia ISG cells were stimulated with cGAMP with or without MMAE (0.5 μM) or VcMMAE (0.5 μM) for 4 h (C and E). STING oligomerization was analyzed by native PAGE, and indicated proteins were detected by immunoblotting. The results are representative of three independent biological replicates (C). The activation of STING pathway was analyzed by immunoblotting (D). cGAMP quantification by LC-MS/MS in THP1-Lucia ISG cell lysates (E). (F) HeLa cells are stimulated similarly as in B. The STING (green) puncta are shown as 3D projections of Z-stack images. Scale bar, 5 μm. The STING puncta volume was quantitated by Imaris software (version 9.7) (n = 20). (G) THP1-Lucia ISG and RAW-Lucia ISG cells were treated with cGAMP with or without MMAE (1 μM), or VcMMAE (1 μM) for 24 h. Fold change of luminescence was normalized to DMSO-treated cells. (H and I) THP1-Lucia ISG cells were stimulated by cGAMP with or without MMAE (1 μM), or VcMMAE (1 μM) for 12 h (H), or 6 h (I). IFNβ induction was measured by ELISA and qPCR analysis.
Fig 5.
MMAE changed STING trafficking routes and promoted cGAMP-mediated STING activity.
(A-C and F) HeLa cells stably expressing human STING-GFP were stimulated with cGAMP (8 μM) and/or MMAE (1 μM), or VcMMAE (1 μM) for 2 h. (A and F) Live cells were stained by ER-Tracker Blue-White DPX and LysoTracker Deep Red. (B and C) Cells were fixed, permeabilized, and stained for GM130 (a Golgi protein, red) or tubulin (red). Nuclei were stained with DAPI (blue). All structured illumination microscope (3D-SIM) images are z-stack images. Scale bars, 10 μm. 3D-SIM images was acquired and processed using the Highly Intelligent and Sensitive SIM (HIS-SIM), and Wiener deconvolution was used in reconstructed images. Dashed white boxes in each main image indicate enlarged areas of interest shown below. Co-localization was quantified using Pearson’s correlation coefficient (r), shown on the right of each row of images (n = 50). (D and E) BJ-5ta cells were stimulated with cGAMP (8 μM) with or without MMAE (pre-treatment for 30 min), bafilomycin A1 (BafA1, 100 nM), or brefeldin A (BFA, 1 μM) for 2 h. Total STING protein was quantified by image J software (n = 3 biological replicates). (G and H) STING stability was analyzed by immunoblotting in the absence or presence of cycloheximide (CHX, 50 μg/ml). BJ-5ta cells were treated and analyzed as in (D and E).
Fig 6.
MMAE alone had antiviral effects and also further amplified the cGAMP-mediated antiviral immune response.
(A and C-E) THP1-Lucia ISG cells (WT and STING KO) were infected with HSV-1-GFP (MOI = 1) or VSV-GFP (MOI = 0.1), and then cultured with cGAMP and/or MMAE (0.25 μM) for 24 h. The cells were then imaged with Olympus IX83 Inverted fluorescence microscope (A). The fluorescence intensity of virus-GFP was determined by ImageJ software, shown on the right of each row of images (n = 15, biological replicates). Scale bars, 100 μm. Cell viability was determined by ATP assay after indicated treatments (C). ISRE reporter activity was measured, and the fold change of luminescence was normalized to DMSO-treated cells (D and E). Bars are the means ± SEM. Significance was determined by one-way ANOVA; *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001, n.s. means non-significant. (B) THP1-Lucia ISG cells were infected with HSV-1-GFP (MOI = 1), and then cultured with cGAMP and/or MMAE (0.25 μM) with or without pretreatment of HSV-1-GFP (MOI = 1) or anti-IFNAR2 antibody (20 μg/ml) for indicated times. Expression of viral gene was determined by immunoblotting. (F) HeLa (STING deficient) and HeLa hSTING cells stably expressing human mCherry-TUBA1B were infected with HSV-1-GFP (MOI = 1), and then cultured with or without MMAE (0.1 μM) for 18–24 h. Representative confocal images of virus transport along microtubules were shown. Enlarged insets highlighted the co-localization of the viral particles (dashed white boxes) with intracellular microtubules. mCherry-TUBA1B (red), HSV-1-GFP (green). Scale bars, 10 μm. (G) Schematic diagram of microtubule-based transport of virus entry, replication, assembly, and egress from the host cell. MMAE-mediated microtubule network disruption seriously affects every process of viral replication and reproduction.
Fig 7.
MMAE relied on the host STING pathway to enhance cGAMP antiviral immune response.
(A-H and I-P) WT and Stinggt/gt C57BL/6 mice (n = 10) were treated with PBS, cGAMP (30 μg/mice), MMAE (0.5 mg/kg), or cGAMP along with MMAE by intraperitoneal injection (i.p.) for 2 h. Then, the mice were infected intravenously with HSV-1-GFP at 2 × 108 pfu per WT mouse or at 1 × 107 pfu per Stinggt/gt mouse. (A and I) Survival curves of virus-infected mice after treatments were analyzed using the log-rank (Mantel-Cox) test. (B, C and J, K) Body weight and body condition score of mice were observed and recorded daily. Body condition score was measured and calculated as in previous research with minor modifications [52] (normal = 0). (D-H and L-P) Six days after virus infection, three C57BL/6 mice (WT and Stinggt/gt) were randomly selected for subsequent experiments. (D and L) The viral titers in mouse brains were measured by qRT-PCR assay (n = 3 biological replicates). (E, F, M, and N) Expressions of viral genes in brains were measured by immunoblotting and qPCR analysis (n = 3). (G and O) Expressions of IFNβ and ISGs in brains were analyzed by qPCR analysis (n = 3). (H and P) IFNβ production in brains were qualified by ELISA assay.
Fig 8.
A graphic model for MMAE enhanced cGAMP-mediated antiviral immunity.
MMAE changed cGAMP-mediated STING trafficking routes from ER to Golgi apparatus by disrupting the microtubule network, and delayed the trafficking-mediated STING degradation. MMAE dispersed the cGAMP-mediated STING perinuclear puncta into large number of tiny vesicles throughout the cytoplasm. The accumulated STING vesicles further amplified the cGAMP-mediated TBK1-STING-IRF3 signaling cascade, and promoted the production of IFNs and ISGs expression. MMAE alone restricted viral replication and infection by destroying microtubule networks, while MMAE combined with cGAMP exerted potent and broad-spectrum antiviral activity in vitro and in vivo in a STING-dependent manner.