ORF8 contributes to cytokine storm during SARS-CoV-2 infection by activating IL-17 pathway

Abstract

Recently, COVID-19 caused by the novel coronavirus SARS-CoV-2 has brought great challenges to the world. More and more studies have shown that patients with severe COVID-19 may suffer from cytokine storm syndrome; however, there are few studies on its pathogenesis. Here we demonstrated that SARS-CoV-2 coding protein open reading frame 8 (ORF8) acted as a contributing factor to cytokine storm during COVID-19 infection. ORF8 could activate IL-17 signaling pathway and promote the expression of pro-inflammatory factors. Moreover, we demonstrated that treatment of IL17RA antibody protected mice from ORF8-induced inflammation. Our findings are helpful to understand the pathogenesis of cytokine storm caused by SARS-CoV-2 and provide a potential target for the development of COVID-19 therapeutic drugs.

Keywords: Biological Sciences; Microbiology; Virology.

Graphical abstract

Figure 1

ORF8 promotes the secretion of inflammatory factors by activating IL-17 pathway (A) HEK293T cells were co-transfected with Myc-IL17RA and HA-NSP2/HA-ORF7a/HA-ORF8 for 24 h, and the interaction of IL17RA with NSP2/ORF7a/ORF8 was detected by immunoprecipitation. (B) GST pulldown analysis of the interaction between GST-ORF8 and Myc-IL17RA. (C) Il17ra^+/+ PMs were treated with 1 μg/mL His-tagged NSP2/ORF7a/ORF8 for 24 h, and immunoprecipitation was performed to detect the interaction of IL17RA with NSP2/ORF7a/ORF8. (D) Il17ra^+/+ PMs were treated with different concentrations of purified His-ORF8 protein for 24 h, and immunoprecipitation was performed to detect the interaction of IL17RA with ORF8. (E) Schematic diagram of IL17RA truncations. (F and G) Co-immunoprecipitation analysis of the interaction between ORF8 and IL17RA truncations in HEK293T cells co-transfected with HA-ORF8 and truncation plasmids for 24 h (F), or in Il17ra^−/− RAW264.7 transfected with IL17RA truncation plasmids for 24 h, and treated with 1 μg/mL His-ORF8 protein for 24 h (G). (H–K) Il17a^−/− RAW264.7 were treated with 50 ng/mL IL-17 or 0.1–1 μg/mL His-ORF8 protein as indicated for 24 h. The interaction between IL17RA and ACT1 was detected by co-immunoprecipitation (H); NF-κB activity was detected by dual luciferase reporter analysis (I); phosphorylation level of IκBα was detected by western blotting (J); and secretion of TNF-α, IL-1β, IL-6, and IL-12 was detected by ELISA analysis (K). Data are representative of three independent experiments (A–D, F–H, and J) or three independent experiments with n = 3 technical replicates (I and K) (shown as mean ± SEM in I and K). Individual data points represent individual technical replicates (I). Data are analyzed by two-tailed Student's t test (I and K). ∗∗p < 0.01.

Figure 2

IL17RA antibody protects mice from ORF8-induced inflammation (A and B) Il17a^−/− RAW264.7 were treated with IL17RA antibody as indicated for 8 h and treated by 1 μg/mL His-ORF8 protein for 24 h. NF-κB activity was detected by dual luciferase reporter analysis (A), and the secretion of TNF-α, IL-1β, IL-6, and IL-12 was detected by ELISA (B). Blank: negative control; IL-17: cells were treated with 50 ng/mL IL-17 for 24 h; His-ORF8: cells were treated with 1 μg/mL His-ORF8 for 24 h; Isotype Ctrl: cells were treated with Isotype antibody of IL17RA for 8 h and further treated by 1 μg/mL His-ORF8 protein for 24 h. (C and D) Il17a-deficient C57BL/6 mice were intraperitoneally injected with 200 μg IL17RA antibody, and the injection was repeated every 3 days. After the second injection, mice were intratracheally infected with the adenovirus expressing ORF8 (10⁸ PFU/mouse). The time was recorded as day 0. Afterward, lung (C) and liver (D) sections were taken every 3 days. The secretion of TNF-α, IL-1, IL-6, and IL-12 was detected by ELISA. (E and F) H&E staining in lung (E) and liver (F) sections on day 9 post-infection. The degree of organ damage was assessed by a scoring system. Scale bar, 400 μm. Data are representative of three independent experiments (E and F) or three independent experiments with n = 3 technical replicates (A–F) (shown as mean ± SEM in A–F). Individual data points represent individual technical replicates (A, B, E, and F). Data are analyzed by two-tailed Student's t test (A–F). ∗∗p < 0.01.