STING agonists enable antiviral cross-talk between human cells and confer protection against genital herpes in mice

Abstract

In recent years, there has been an increasing interest in immunomodulatory therapy as a means to treat various conditions, including infectious diseases. For instance, Toll-like receptor (TLR) agonists have been evaluated for treatment of genital herpes. However, although the TLR7 agonist imiquimod was shown to have antiviral activity in individual patients, no significant effects were observed in clinical trials, and the compound also exhibited significant side effects, including local inflammation. Cytosolic DNA is detected by the enzyme cyclic GMP-AMP (2'3'-cGAMP) synthase (cGAS) to stimulate antiviral pathways, mainly through induction of type I interferon (IFN)s. cGAS is activated upon DNA binding to produce the cyclic dinucleotide (CDN) 2'3'-cGAMP, which in turn binds and activates the adaptor protein Stimulator of interferon genes (STING), thus triggering type I IFN expression. In contrast to TLRs, STING is expressed broadly, including in epithelial cells. Here we report that natural and non-natural STING agonists strongly induce type I IFNs in human cells and in mice in vivo, without stimulating significant inflammatory gene expression. Systemic treatment with 2'3'-cGAMP reduced genital herpes simplex virus (HSV) 2 replication and improved the clinical outcome of infection. More importantly, local application of CDNs at the genital epithelial surface gave rise to local IFN activity, but only limited systemic responses, and this treatment conferred total protection against disease in both immunocompetent and immunocompromised mice. In direct comparison between CDNs and TLR agonists, only CDNs acted directly on epithelial cells, hence allowing a more rapid and IFN-focused immune response in the vaginal epithelium. Thus, specific activation of the STING pathway in the vagina evokes induction of the IFN system but limited inflammatory responses to allow control of HSV2 infections in vivo.

Fig 1. STING agonists induces type I IFN and anti-viral effect in human cells.

(A) HaCaT cells were permeabilized with digitonin before stimulated with different STING agonists at a low (L: 10μg/ml) or high (H: 100μg/ml) concentration for 24 hours. Cell lysates were used for Western blotting for pSTAT1, STING and viperin. (B) HaCaT and THP-1 cells were stimulated with 2’3’-cGAM(PS)₂ for 24 hours and type I IFN activity was measured in the supernatant (n = 3, * = p<0,05). (C) HaCaT cells were permeabilized with digitonin and treated with 2’3’-cGAM(PS)₂ before or after infection with HSV2 (MOI 0.1) for 24 hours. Cell lysates were used for Western blotting for VP5, STING and viperin (n = 3). (D, E, F) HaCaT and THP-1 cells were stimulated with 2’3’-cGAM(PS)₂, IFNα or IFNβ for 24 hours and levels of viperin and ISG15 were determined by Western blotting. (G, H, I) THP-1 cells were stimulated with 2’3’-cGAM(PS)₂ for 24 hours before the media were transferred to wild type or IFNAR2^-/- HaCaT cells. HaCaT cells were infected 24 hours later with HSV2 (MOI 0.1) for 24 hours. Cell lysates were used for Western blotting for VP5 and ISG15 and the media were used to determine the HSV-2 virus load. For all Western blots, Vinculin was used as loading control. n = 3, a representative sample is showed. NT, non-treated. Statistics, (B, I) Two-way ANOVA with Šidák’s multiple comparisons test; p(interaction)<0.05.

Fig 2. STING agonists induces type I IFN expressing in vivo.

Equimolar (1.687x10^-7 mol) doses of STING agonists 2’3’-cGAMP (121 μg/mouse), 3’3’c-diAMP (119 μg/mouse), 2’3’-cGAM(PS)₂ (125 μg/mouse), 3’3’-cAIMP (111 μg/mouse) or DMXAA (95 μg/mouse, STING activation by DMXAA requires two molecules (3.374x10^-7 mol)) were administrated to mice i.p. Samples were collected 6 hours later for further analysis. (A) Levels of phosphorylated STAT1 (pSTAT1), STING, viperin and ISG15 were determined by Western blotting of spleen, vagina and brain tissues. Vinculin was used as loading control. n = 5, two representative samples are shown. (B) IFNα and IFNβ in the serum determined by ELISA. n = 3–5. * = p<0.05 compared to mock. (C) Expression for Ifnb and Mx1 mRNA in tissues samples from vagina, spleen and brain, normalized to GAPDH. n = 3–5. * = p<0,05 compared to mock. (D) Mice were perfused prior to isolation of brain samples and gene expressions of Ifnb and Mx1 mRNA were measured. n = 3–5. * = p<0,05 compared to mock. Statistics, (B-D) Kruskal-Wallis test with Dunn’s multiple comparisons test.

Fig 3. HSV2 and STING agonists induce antiviral genes in vaginal epithelium.

Mice were injected i.p. with 2’3’-cGAM(PS)₂ (125 μg/mouse) or infected with HSV2 (6.7×10⁴ p.f.u.). Tissues were isolated from mice 6 h and 24 h after CDN stimulation and HSV2 infection, respectively. Paraffin sections of the vaginal tissues were stained for viperin (red) and HSV2 (green). DAPI (blue) marks the nuclei and the dotted white lines mark basal membrane between the epithelium and stroma. White arrows highlight examples of viperin positive cells, and arrowheads mark examples of HSV2-infected cells. L = lumen, E = epithelium, S = stroma. n = 3. One representative picture is shown for each staining and treatment group.

Fig 4. Systemic treatment with STING agonists confers protection against genital HSV2 infection.

Wildtype and cGas^-/- mice were treated with 2’3’-cGAM(PS)₂ (Rp/Sp) (125 μg/mouse) and infected intravaginally with HSV2 (6.7×10⁴ p.f.u.). (A) Illustration of the timeline for the treatment regimens. (B, C) Overall survival for HSV2-infected and treated wildtype (B) or cGas^-/- (C) mice. n = 6–10. * = p<0,05 compared to mock. (D) HSV2 titer (TCID50) in vaginal washes collected 48 hours post infection. n = 6–10. * = p<0,05 compared to mock in the same genotype. Statistics, (B, C) Log-rank test with Holm-Bonferroni correction. (D) One-way ANOVA of log₁₀-transformed data with Dunnett’s multiple comparisons test.

Fig 5. Local application of STING agonists protects against genital HSV2 infection.

Mice were anesthetized for 30 min and 250 μg 3’3’-cAIMP was applied to the vagina. (A) Tissues were isolated from mice 6 h after CDN stimulation. Paraffin sections of the vaginal tissues were stained for viperin (red) and HSV2 (green). DAPI (blue) marks the nuclei and the dotted white lines mark basal membrane between the epithelium and stroma. White arrows highlight examples of viperin positive cells. L = lumen, E = epithelium, S = stroma. n = 4. One representative picture is shown for each staining. (B) After 6 hours of 3’3’-cAIMP treatment (no infection), gene expression was measured in vaginal and spleen samples. The expression levels were normalized to GAPDH. n = 3–5. * = p<0.05. (C) IFNβ levels. in serum from mice treated with 3’3’-cAIMP for 6 hours. n = 5. (D, E) Overall survival for wildtype (D) or cGas^-/- (E) mice treated with 3’3’-cAIMP and infected with HSV2 12 h later. n = 6–10. * = p<0,05 compared to mock. (F) HSV2 titer (TCID50) in vaginal washes (48 hours p.i.) from wildtype or cGas^-/-mice treated with 3’3’-cAIMP and infected with HSV2 12 h later. n = 6–10. * = p<0,05 compared to mock treated in the same genotype. (G) Tissues were isolated from mice treated intravaginally for 12 h with 3’3’-cAIMP followed by 24 h infection with HSV-2. The samples were prepared and analyzed as in A. (H) Overall survival and (I) virus load for wildtype mice pre-treated with 3’3’-cAIMP 12, 24 or 72 h prior to virus infection. n = 8. * = p<0,05 compared to mock treated group. Statistics, (B) Kruskal-Wallis test with Dunn’s multiple comparisons test. (C, F, I) Mann-Whitney-Wilcoxon U test. (D, E, H) Log-rank test.

Fig 6. STING agonists induce IFN responses in the vaginal epithelium faster and more efficiently than TLR agonists.

(A) HaCaT cells were treated with imiquimod (1μg/ ml), ODN2216 (1μg/ ml), and 3’3’-cAIMP (100μg/ ml) for 24 h. Levels of ISG15 and viperin were determined in the cell lysate by Western blotting. (B) Mice were treated intravaginally with imiquimod or ODN1826 (25 μg per mouse) 12 h prior to infection with HSV2. Vaginal washes were collected 48 h p.i. and viral load was determined. n = 5 per group. (C-D) Mice were anesthetized for 30 min and imiquimod, ODN1826, or 3’3’-cAIMP was applied to the vagina. Tissues were isolated (C) 6 h or (D) 36 h after treatment. Paraffin sections of the vaginal tissues were stained for viperin (red). DAPI (blue) marks the nuclei and the dotted white lines mark basal membrane between the epithelium and stroma. White arrows highlight examples of viperin positive cells. L = lumen, E = epithelium, S = stroma. n = 4. One representative picture is shown for each staining. (E-G) RNA was isolated from vaginal tissue treated as indicated for 6 h, and levels of Ifnb, Mx1, and Tnfa mRNA were determined by RT-qPCR. n = 4–5. mRNA levels were normalized to Gapdh and shown as relative levels of expression compared to mock-treated mice. (B, E-G) Statistics, Kruskal-Wallis test with Dunn’s multiple comparisons test. * = p<0,05 compared to mock treated.