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.</p

MMAE enhanced cGAMP-mediated antiviral activity <i>in vitro</i>.

(A-C and E) THP1 cells (WT, STING KO) and L929 cells (WT, STING KO) were infected with VSV-GFP (MOI = 0.1) and HSV-1-GFP (MOI = 1) respectively, and then cultured cGAMP (0.5 μM) and/or MMAE (0.25 μM) for 24 h. The cells were imaged with Olympus IX83 Inverted fluorescence microscope. The fluorescence intensity of viral GFP was determined by ImageJ software, shown on the right of each row of images (n = 15, biological replicates). Scale bars, 100 μm. (D) STING protein were analyzed by immunoblotting in L929 cells (WT and STING KO). (F) L929 cells (STING KO) were infected with HSV-1-GFP (MOI = 1), and then cultured cGAMP (0.5 μM) or MMAE (0.25 μM) for indicated times. Viral propagation was determined by western blot. The results are representative of three independent biological replicates. Bars are the mean ± SEM. Significance was determined by one-way ANOVA; *p p p p (TIF)</p

MMAE amplified the STING signaling cascade by increasing the number of STING puncta induced by CDNs.

(A) Chemical structure of VcMMAE (valine-citrulline (Vc) conjugate to MMAE, a part of ADC). (B) HeLa cells stably expressing hSTING-GFP were treated with 3’3’-cGAMP (2 μM), cyclic-di-AMP (10 μM), HT-DNA (2 μg, transfection with PEI) with or without MMAE (1 μM) for 2 h or 8 h (HT-DNA) in the presence or absence of brefeldin A (BFA 1μM), followed by confocal imaging. Green, STING-GFP. Nuclei were stained with 4’,6-diamidino-2-phenylindole (DAPI; blue). Scale bars, 10 μm. (C and D) THP1-Lucia ISG cells were stimulated with cGAMP (0.5 μM) for 24 h (C) or 6 h (D) in the presence or absence of MMAE (indicated doses or 1 μM) or VcMMAE (indicated doses or 1 μM). Fold changes in luminescent signals were normalized to cGAMP-treated cells (C). The induction of CXCL10, CCL5, IL-6, TNFα, IFITM1, and IFIT3 expression was analyzed by real-time PCR (D). Data are presented as mean ± SEM and analyzed by one-way ANOVA (*p p p p (TIF)</p

MMAE directly enhanced the cGAMP-STING signaling pathway.

(A) A model showing whether the potentiation effect of MMAE is dependent on the direct STING-IRF3 signal axis or the indirect IFNα/β and its receptors (IFNAR) pathway. (B-I) ISRE reporter activities and STING phosphorylation cascades were measured in response to cGAMP, RO8191 (0.25 μM, an IFNAR2 agonist) or combined with indicated MMAE for 24 h or 6 h in THP1-Lucia ISG cells (WT, STAT1 KO, STAT2 KO and STAT3 KO). The fold changes in luminescent signals were normalized to DMSO-treated cells. The activation of STING signaling was assessed by immunoblotting. Data are representative of three independent experiments. Bars are the mean ± SEM of indicated (n) independent experiments. Significance was determined by one-way ANOVA; *p p p p (TIF)</p

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.</p

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.</p

MMAE synergized with cGAMP-mediated STING signaling to show potent and broad antiviral activity <i>in vitro</i>.

(A and C) THP1 and L929 cells were infected with VSV-GFP (MOI = 0.1), HSV-1-GFP (MOI = 1) or PRV-GFP (MOI = 1), and then cultured cGAMP (0.5 μM) and/or MMAE (0.25 μM) for indicated times. Viral propagation was determined by western blot. The results are representative of three independent biological replicates. (B) THP1 cells viability was determined by ATP assay after indicated treatments for 24 h. (D) Donor THP1 cells were treated with cGAMP and/or MMAE for 6h, then washed out to produce 24 h-conditioned media, which was added to recipient Vero cells infected or uninfected with VACV-GFP (MOI = 5) or EV-A71 (MOI = 1). Whole-cell lysates were subjected to immunoblotting with specific antibodies at indicated times. (TIF)</p

MMAE specifically increased cGAMP-mediated immune response in a STING-dependent manner.

(A) THP1-Lucia ISG cells (WT and STING KO) were treated with cGAMP and/or MMAE for 6 h, respectively. Total RNA was harvested and IFNβ, CXCL10, CCL5, TNFα, IL-6, ISG15, IFIT3 and IFITM1 mRNA expression was measured by real-time PCR (n = 3 biological replicates). (B and C) THP1-Lucia ISG cells (WT and MyD88 KO) were stimulated with cGAMP with or without MMAE for 24 h (B) or 6 h (C), and luciferase signals were measured (B). Phosphorylation of STING downstream signal transduction was assessed by immunoblotting with indicated antibodies (C). (D-G) THP1-Lucia ISG cells were stimulated with cGAMP, Sendai virus (SeV, MOI = 0.1), LPS (1 μg), Poly(I:C) (10 μg) or co-treated with indicated concentrations of MMAE for 24 h. ISRE reporter activity was measured and the fold changes in luminescent signals were normalized to DMSO-treated cells. (H and I) THP1-Lucia NF-kB cells were treated with LPS (1 μg), IL-1β (1 μg) or combined with MMAE for 24 h. Fold changes in NF-kB activation were measured by Lucia luciferase signal and normalized to DMSO-treated cells. Bars are the mean ± SEM of indicated (n) independent experiments. Significance was determined by one-way ANOVA; *p p p p (TIF)</p

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).</p

MMAE enhanced the antiviral effects of cGAMP in a STING-dependent manner <i>in vivo</i>.

(A-G and H-J) 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-C and I) Body weight of mice were observed and recorded daily. (D-G, H and J) Six days after virus infection, three C57BL/6 mice (WT and Stinggt/gt) were randomly selected for subsequent experiments. The viral titers of mouse spleens were measured by qRT-PCR assay (n = 3 biological replicates) (D). Expressions of viral genes in spleens were measured by immunoblotting and qPCR analysis (n = 3) (E, F, and J). Expressions of IFNβ and ISGs in spleens were analyzed by qPCR analysis (n = 3) (G and H). (K-P) C57BL/6 mice (WT, n = 4) were infected intravenously with HSV-1 at 1 × 107 pfu per mouse. 16 hours later, the mice were treated with PBS, cGAMP (30 μg/mice), MMAE (0.5 mg/kg), or cGAMP along with MMAE by intraperitoneal injection (i.p.) for 3 days. The viral titers of mouse livers and spleens were measured by qRT-PCR assay (K and N). Expressions of viral genes in livers and spleens were qualified by immunoblotting and qPCR analysis (L, M, O and P). (TIF)</p