E. fischeriana Root Compound Dpo Activates Antiviral Innate Immunity

Abstract

E. fischeriana has long been used as a traditional Chinese medicine. Recent studies reported that some compounds of E. fischeriana exhibited antimicrobial and immune enhance activity. Innate immune system is essential for the immune surveillance of inner and outer threats, initial host defense responses and immune modulation. The role of natural drug compounds, including E. fischeriana, in innate immune regulation is largely unknown. Here we demonstrated that E. fischeriana compound Dpo is involved in antiviral signaling. The genome wide RNA-seq analysis revealed that the induction of ISGs by viral infection could be synergized by Dpo. Consistently, Dpo enhanced the antiviral immune responses and protected the mice from death during viral infection. Dpo however was not able to rescue STING deficient mice lethality caused by HSV-1 infection. The enhancement of ISG15 by Dpo was also impaired in STING, IRF3, IRF7, or ELF4 deficient cells, demonstrating that Dpo activates innate immune responses in a STING/IRFs/ELF4 dependent way. The STING/IRFs/ELF4 axis is therefore important for Dpo induced ISGs expression, and can be used by host to counteract infection.

Keywords: RLR signalling; STING; compound; innate immunity; viruses.

Figure 1

Dpo is an antiviral compound from Euphorbia fischeriana Steud. (A–C) Bone marrow derived macrophages were treated with indicated compounds (1 ng/ul). Four hours later, these cells were infected with VSV (A), HIV-1 (B), and HSV-1 (C). Twenty-four hours later, the viral load were measured by plaque assay. (D,E) Bone marrow derived macrophages were treated with increasing amount of Dpo. Four hours later, these cells were infected with VSV (D) and HSV-1 (E). Twenty-four hours later, the viral load were measured by plaque assay. (F) Wild type C57BL/6 mice were treated with Dpo, 24 h later infected with HSV-1, and monitored daily for 15 days. ^*P < 0.05. (A–E) The data represent mean values ± SEM (n = 3); ^*P < 0.05, ^**P < 0.01, significant compared to control, Student's t-test. The data represent mean values ± SEM (n = 6 mice per group); (F) ^*P < 0.05, significance compared to the control group, non-parametric Mann–Whitney analysis.

Figure 2

Dpo induces expression of interferon-stimulated genes. (A–C) Bone marrow derived macrophages were treated with indicated compounds (1 ng/ul). Four hours later, the mRNA level of IFNβ, CCl5, and IL-β were measured by qPCR. (D–J) Bone marrow derived macrophages were treated with Dpo. Four hours later, these cells were infected with VSV. Twenty-four hours later, the indicated cytokines and genes were measured by qPCR. (A–J) The data represent mean values ± SEM (n = 3); ^*P < 0.05, ^**P < 0.01, significant compared to control, Student's t-test.

Figure 3

STING is required for Dpo mediated anti-viral immune responses. (A,B) WT, Trif ^−/−, Myd88^−/− peritoneal macrophages were treated with Dpo. Four hours later, these cells were infected with HSV-1. Twenty-four hours later, the mRNA level of ISG15 were measured by qPCR (A), and the viral load were measured by plaque assay (B). (C) Tlr4^−/− C57BL/6 mice were treated with Dpo, 24 h later infected with HSV-1, and monitored daily for 15 days. (D,E) WT, Ddx58^−/− (D), Mavs^−/− (E) peritoneal macrophages were treated with Dpo. Four hours later, these cells were infected with VSV. Twenty-four hours later, and the viral load were measured by plaque assay (F). Wild type or Sting^gt/gt peritoneal macrophages were treated with Dpo, 4 h later infected with HSV-1, and the mRNA level of ISG15 were measured by qPCR. (G) Sting^gt/gt C57BL/6 mice were treated with Dpo, 24 h later infected with HSV-1, and monitored daily for 15 days. (A,B,D–F) The data represent mean values ± SEM (n = 3); ^*P < 0.05, ^**P < 0.01 significant compared to control, Student's t-test. The data represent mean values ± SEM (n = 6 mice per group); (C–G) ^*P < 0.05, significance compared to the control group, non-parametric Mann–Whitney analysis.

Figure 4

Dpo induces ISGs via IRFs/ELF4. (A,B) Bone marrow derived macrophages were treated with Dpo. Four hours later, these cells were infected with HSV-1. Twenty-four hours later, the mRNA level of ISG15 (A) and CCl5 (B) were measured by qPCR. (C) Bone marrow derived macrophages were treated with increasing amount of Dpo. Twenty-four hours later, the mRNA level of ISG15 was measured by qPCR. (D,E) DC2.4 cells and peritoneal macrophages were treated with Dpo or infected with VSV. Twenty-four hours later, the mRNA level of ISG15 were measured by RT-PCR. (F–H) WT, Irf3^−/− or Irf7^−/− iBMDM were treated with Dpo. Four hours later, these cells were infected with VSV. Twenty-four hours later, and the viral load were measured by plaque assay (F,G), the mRNA level of ISG15 was measured by qPCR (H). (I,J) WT or Elf4^−/− iBMDM were treated with Dpo. Four hours later, these cells were infected with HSV-1. Twenty-four hours later, and the viral load were measured by plaque assay (I), the mRNA level of ISG15 was measured by qPCR (J). (A–C,F–J) The data represent mean values ± SEM (n = 3); ^*P < 0.05, ^**P < 0.01 significant compared to control, Student's t-test. WT, Tbk1^−/−(K) or Cgas^−/− (L) iBMDM were treated with Dpo. Four hours later, these cells were infected with HSV-1. Twenty-four hours later, the mRNA level of ISG15 was measured by qPCR.