Stimulator of Interferon Gene Agonists Induce an Innate Antiviral Response against Influenza Viruses

Abstract

The devastating effects of COVID-19 have highlighted the importance of prophylactic and therapeutic strategies to combat respiratory diseases. Stimulator of interferon gene (STING) is an essential component of the host defense mechanisms against respiratory viral infections. Although the role of the cGAS/STING signaling axis in the innate immune response to DNA viruses has been thoroughly characterized, mounting evidence shows that it also plays a key role in the prevention of RNA virus infections. In this study, we investigated the role of STING activation during Influenza virus (IFV) infection. In both mouse bone marrow-derived macrophages and monocytic cell line THP-1 differentiated with PMA, we found that dimeric amidobenzimidazole (diABZI), a STING agonist, had substantial anti-IFV activity against multiple strains of IFV, including A/H1N1, A/H3N2, B/Yamagata, and B/Victoria. On the other hand, a pharmacological antagonist of STING (H-151) or the loss of STING in human macrophages leads to enhanced viral replication but suppressed IFN expression. Furthermore, diABZI was antiviral against IFV in primary air-liquid interface cultures of nasal epithelial cells. Our data suggest that STING agonists may serve as promising therapeutic antiviral agents to combat IFV.

Keywords: STING agonists; air–liquid interface cultures; diABZI; influenza virus; macrophages.

Figure 1

STING agonists are antiviral against IFV in mouse bone marrow-derived macrophages (BMDMs) (A) A schematic view showing that STING agonists were added to BMDMs before or after A/PR8 (MOI 5) or A/H3N2 (MOI 10) (B) BMDMs were exposed to DMSO control (Ctl), 25 μg/mL DMXAA or 3 μM diABZI for 4 h. Phosphorylation of IRF3 was measured by immunoblot analysis. Actin was used as the loading control. The blot shown is representative of three independent experiments. (C,D) BMDMs were treated with DMXAA or diABZI before A/PR8 (MOI 5) infection (Pre) or after A/PR8 infection (Post). RT-qPCR analysis of Interferon-stimulated gene 15 (ISG15), MX Dynamin Like GTPase 1 (Mx1), and O2′-5′-Oligoadenylate Synthetase 1 (OAS1) mRNA were shown. Data are expressed the means ± SD of at least three independent experiments. * p < 0.05; ** p < 0.01; *** p < 0.001, versus control (Ctl; DMSO-treated) cells. (E) Protein levels of IFV NP, phospho-TBK1, and IRF3 were measured, and a representative blot is shown. (F) Secretion levels of mIFN-β and mIL-6 were measured by ELISA following DMXAA or diABZI pre- or post-treatment followed by A/PR8 infection. * p < 0.05; ** p < 0.01; *** p < 0.001, versus mock or A/PR8 infected cells. (G) Antiviral activities of STING agonists in BMDMs were determined by TCID₅₀ assay. Data are expressed the means ± SD of at least three independent experiments. * p < 0.05; ** p < 0.01; *** p < 0.001, versus control (Ctl; DMSO-treated) cells.

Figure 2

STING agonists inhibit IFV replication in human macrophages. (A) THP-1 cells were treated with various doses of STING agonist (diABZI, 2′3′-cGAMP) for 4h. % cell viability was measured. (B,C) THP-1 was treated with 3 M diABZI before A/PR8 (MOI 5) infection (Pre) or after A/PR8 infection (Post) for 16 h. Viral or host gene expression was quantitated, normalized, and analyzed by RT-qPCR (mean ± SD; n = 3). Interferon-stimulated gene 15 (ISG15), O2′-5′-Oligoadenylate Synthetase 1 (OAS1), and interferon-beta (IFN-β) mRNA levels were shown, versus control (Ctl; non-treated) cells. (D) THP-1 was treated with 3 μM diABZI after A/PR8 (MOI 0.1) for 48 h. Viral gene expression was quantitated, normalized, and analyzed by RT-qPCR (mean ± SD; n = 3). (E) Protein levels of IFV NP, M1, phospho-STING/STING, p-TBK1/TBK1, and p-IRF3/IRF3 were measured, and a representative blot was shown. (F) Antiviral activities of STING agonists in THP-1 were determined by TCID₅₀ assay. Data are expressed the means ± SD of at least three independent experiments. * p < 0.05; ** p < 0.01; *** p < 0.001, versus DMSO treatment (Ctl) cells.

Figure 3

STING antagonist upregulates IFV replication. (A) THP-1 cells were treated with various doses of STING antagonist (H-151) for 4 h and infected with A/H1N1 or A/H3N2 (MOI 10). (B) Cell viability of various doses of H-151 treatment in THP-1 cells is shown. (C) THP-1 was exposed to 0, 0.1, 0.25, 1 μM of H-151 for 4 h and infected with A/H1N1 or A/H3N2. Viral PA expression level was quantitated, normalized, and analyzed by RT-qPCR (mean ± SD; n = 3). (D) Viral titer was measured by TCID₅₀ assay (E) Interferon-stimulated gene 15 (ISG15), O2′-5′-Oligoadenylate Synthetase 1 (OAS1), and interferon-induced transmembrane protein 3 (IFITM3) mRNA levels were quantitated, normalized, and analyzed by RT-qPCR (mean ± SD; n = 3). * p < 0.05; ** p < 0.01; *** p < 0.001, versus control (Ctl; non-treated) cells.

Figure 4

STING restricts IFV replication (A) Wild-type (WT), and STING knockout (KO) THP-1 cells differentiated with PMA for 2 days were infected with A/H1N1 or A/H3N2 (MOI 10). (A) Viral titer was measured by TCID₅₀ assay. (B,C) RT-qPCR analysis of viral PA and host ISG15 mRNA was performed. Data are expressed as the means ± SD of at least three independent experiments. (D) Cell-free supernatant was tested for IFN-β by an ELISA method, versus THP-1 WT cells. (E) WT and STING KO THP-1 cells were infected with either A/H1N1 or A/H3N2 for indicated times. The protein levels of viral NP, phospho-STING/STING, phosho-TBK1, phospho-IRF3, Mx1 and RNaseL were analyzed by immunoblot analysis. The blot shown is representative of three independent experiments. (F) A549 cells were transfected with HA-STING plasmid for 24 h and then infected with A/H1N1 (MOI 10) for 24 h, and subsequently treated with 200 nM bafilomycin A (BAF) or 10 μM MG132 (MG) for 4 h. IFV NP and autophagy-related protein (LC3B) were detected by immunoblotting. (G) THP-1 cells were infected with A/H1N1 or A/H3N2 and treated with STING agonist (diABZI, 3 μM) for 16 h and PI/Hoechst staining was performed to measure cell death and examined using a double staining apoptosis assay (Hoechst33342/PI), * p < 0.05; ** p < 0.01; *** p < 0.001.

Figure 5

Antiviral effect of STING agonists was confirmed by the ALI culture model. (A) Schematic representation of the experimental design. HNECs of the ALI culture were treated with IFV (A/H1N1, MOI 10) at the apical side. STING agonist (3 μM diABZI, 1 μg/mL 2′3′-cGAMP)-containing media was also added in the basolateral side. After 90 min incubation, apical side media was removed, and basolateral side media was changed with fresh media containing STING agonists. After that, cells were incubated for 4 days and supernatants from basolateral side were subject to further analysis. (B) Virus titer was measured by TCID₅₀ assay. (C) IL-6 and IL-8 cytokine secretion level was measured by ELISA. Mean + SD, n = 4, when compared to the control. (D) Trans-epithelial electrical resistance (TEER) plotted over time for HNECs of the ALI culture, Results in Ω are mean values of quintuplicate. Data was analyzed using a Mann–Whitney test. * p < 0.05, ** p < 0.01, and *** p < 0.001, versus m (mock infection only), ## < 0.01, ### < 0.001, versus Ctl (Virus infection only).