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

Summary 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.


INTRODUCTION
Coronavirus disease 2019 (COVID-19) is a respiratory infectious disease caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). At present, it is still a worldwide epidemic with nearly 24 million people infected, which has brought severe challenges to global public health. The clinical course of patients remains to be fully characterized, and no pharmacological therapies of proven efficacy yet exist (Russell et al., 2020). To open up a new breakthrough for clinical therapy, it is necessary to uncover the pathogenesis from a perspective of host-microbe interaction. However, except for a certain understanding of Spike protein (Walls et al., 2020), other proteins' functions have not been extensively studied. In this study, we demonstrated that open reading frame 8 (ORF8) could activate IL-17 signaling pathway and promote the expression of pro-inflammatory factors by interacting with host IL17RA. We also found that inhibition of this interaction by IL17RA antibody was helpful to control the cytokine storm in SARS-CoV-2 infection. Our findings not only made an important contribution to the understanding of how various effectors of the immune system initiate the cytokine storm but also provided a potential target for the development of COVID-19 therapeutic drugs.

ORF8 promotes the secretion of inflammatory factors by activating IL-17 signaling pathway
According to clinical data analysis, patients with severe COVID-19 showed cytokine storm, resulting in acute respiratory distress syndrome (ARDS) and multiple organ failure (Mangalmurti and Hunter, 2020;Vaninov, 2020). Cytokine storm refers to the rapid production of many cytokines, such as TNF-a, IL-1, IL-6, IL-12, and IFN-a. ARDS caused by cytokine storm in the late stage of infection is an important node in the transition from mild to severe illness, and it is also an important cause of death (Wu et al., 2020). In the current treatment of COVID-19, antibodies targeting IL-6 are commonly used to inhibit cytokine storm. However, suppression of IL-6 has not achieved a desired effect in clinical treatment (Hermine et al., 2021;Salama et al., 2021;Stone et al., 2020). As a proinflammatory cytokine, IL-17 has been reported to be related to cytokine storm (Crowe et al., 2009;Kolls and Lindé n, 2004). Targeting IL-17 is immunologically plausible as a strategy to prevent ARDS in COVID-19 (Orlov et al., 2020;Pacha et al., 2020). In this study, yeast two-hybrid system was used to screen the SARS-CoV-2 proteins that interacted with IL-17 receptors. Three candidates (NSP2, ORF7a, and ORF8) were obtained through yeast two-hybrid experiment ( Figure S1), and  iScience Article the interaction was further examined by immunoprecipitation experiments. As a result, only the interaction between ORF8 and IL17RA was successfully verified ( Figure 1A), which is consistent with the predicted SARS-CoV-2 protein interaction map (Gordon et al., 2020). It has been reported that ORF8 is associated with COVID-19 severity (Young et al., 2020). Patients infected with the ORF8 mutant (D382-variant) of SARS-CoV-2 had lower concentrations of pro-inflammatory cytokines and chemokines (Young et al., 2020), indicating an important role of ORF8 in the study of cytokine storm caused by COVID-19. We further validated the interaction between ORF8 and IL17RA using GST pulldown assay and proved an in vitro interaction of ORF8 and IL17RA ( Figure 1B). As IL17RA is an important receptor mainly expressed in immune cells (Lore et al., 2016), in vitro purified His-ORF8 protein was supplemented into wild-type mouse peritoneal macrophages (Il17ra +/+ PMs) to further validate its interaction with IL17RA. The results showed that ORF8 interacted with endogenous IL17RA, and this interaction was in a dose-dependent manner ( Figures  1C and 1D). These evidences indicated that SARS-CoV-2 ORF8 protein interacted with host receptor IL17RA.
IL-17 pathway is an important pro-inflammatory signaling in mammals (McGeachy et al., 2019). IL-17 ligand binds to and activates the corresponding receptor, and then the complex recruits ACT1 from the cytoplasm through the SEFIR domain. ACT1 initiates TNF receptor-associated factor 6 (TRAF6) to activate NF-kB signaling pathway, thus improving the expression levels of pro-inflammatory factors (Schwandner et al., 2000). Given the fact that ORF8 interacts with IL17RA, we investigated the effect of ORF8 on IL-17 pathway.
To eliminate the possibility that ORF8 directly influences the expression of IL-17, we generated Il17a-deficient RAW264.7 cells (Il17a À/À RAW264.7) ( Figure S3) and Il17a-deficient mouse models ( Figure S4). After ORF8 treatment, it was found that ACT1 was recruited by IL17RA, and the recruitment effect was not significantly affected by ORF8 concentrations ( Figure 1H). However, a dose-dependent activation in NF-kB signaling pathway was observed in Il17a À/À RAW264.7 ( Figures 1I and 1J). In addition, a dose-dependent manner in cytokine TNF-a, IL-1b, IL-6, and IL-12 release was identified ( Figure 1K). Taken together, these results implied that ORF8 could bind to IL17RA receptor, leading to IL-17 pathway activation and an increased secretion of pro-inflammatory factors.

Inhibition of IL-17 pathway protects mice from ORF8-induced inflammation
We further explored methods for blocking the ORF8-induced IL-17 pathway activation using IL17RA antibody. Compared with the isotype control, the activity of NF-kB signaling pathway was significantly inhibited after IL17RA antibody treatment (Figure 2A). Similarly, the secretion of cytokines, such as TNF-a, (C) Il17ra +/+ PMs were treated with 1 mg/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 À/À RAW264.7 transfected with IL17RA truncation plasmids for 24 h, and treated with 1 mg/mL His-ORF8 protein for 24 h (G).
(H-K) Il17a À/À RAW264.7 were treated with 50 ng/mL IL-17 or 0.1-1 mg/mL His-ORF8 protein as indicated for 24 h. The interaction between IL17RA and ACT1 was detected by co-immunoprecipitation (H); NF-kB activity was detected by dual luciferase reporter analysis (I); phosphorylation level of IkBa was detected by western blotting (J); and secretion of TNF-a, IL-1b, 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 G 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.

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iScience 24, 102293, April 23, 2021 3 iScience Article Figure 2. IL17RA antibody protects mice from ORF8-induced inflammation (A and B) Il17a À/À RAW264.7 were treated with IL17RA antibody as indicated for 8 h and treated by 1 mg/mL His-ORF8 protein for 24 h. NF-kB activity was detected by dual luciferase reporter analysis (A), and the secretion of TNF-a, IL-1b, IL-6, and IL-12 was detected by ELISA (B iScience Article IL-1b, IL-6, and IL-12, was also reduced to varying degrees according to concentration gradient of IL17RA antibody ( Figure 2B). To study the effect of ORF8 on inflammation, we packaged a pseudovirus expressing ORF8 by using adenovirus. Il17a-deficient mice were infected with 10 8 plaque-forming unit (PFU) pseudoviruses through intratracheal infection. qRT-PCR results of lung and liver showed that ORF8 was stably expressed in mice within 9 days after injection ( Figure S5). Meanwhile, IL-17 receptors were blocked by intraperitoneal injection with IL17RA antibody. As a result, the secretion of pro-inflammatory factors in lung continued to increase after ORF8 pseudovirus infection in the isotype control groups ( Figure 2C). However, for the mice injected with IL17RA antibody, although the secretion of pro-inflammatory factors increased, the total amount was much lower compared with that of the isotype control ( Figure 2C). Liver, another organ with a high rate of impairment in patients with severe COVID-19 (Zhang et al., 2020a), showed a similar trend as lung during ORF8 pseudovirus infection ( Figure 2D). In addition, by using H&E staining and a scoring system, we observed a histological damage on day 9 post-infection. Lungs and livers of the mice injected with IL17RA antibody underwent a slight inflammation compared with those of the untreated mice ( Figures 2E and 2F). Collectively, our study indicated that SARS-CoV-2 coding protein ORF8 might be a contributing factor to the cytokine storm during COVID-19 and treatment with IL17RA antibody could protect organs from inflammation and damage.

DISCUSSION
The control of cytokine storm has always been a difficulty in clinical therapy. At present, studies on SARS-CoV-2 have basically clarified the mechanisms of viral invasion (Hoffmann et al., 2020;Shang et al., 2020), whereas the process of viral replication, viral release, and host immune regulation still needs in-depth exploration. Here, we identified that SARS-CoV-2 ORF8 emulated the function of IL-17 by interacting with host IL17RA, and then promoted the secretion of pro-inflammatory factors by activating NF-kB signaling pathway. To eliminate the possibility that ORF8 stimulates the expression of endogenous IL-17, we generated Il17a À/À cells and mouse models. Supplementation of either IL-17 or ORF8 to Il17a À/À RAW264.7 activated NF-kB pathway, indicating an independent role of ORF8 in promoting inflammation.
In this study, we found that ORF8 protein acted as a contributing factor to the cytokine storm by inducing IL-17 signaling pathway, and the interaction between ORF8 and IL17RA was pivotal in the progress of inflammation. (C and D) Il17a-deficient C57BL/6 mice were intraperitoneally injected with 200 mg IL17RA antibody, and the injection was repeated every 3 days. After the second injection, mice were intratracheally infected with the adenovirus expressing ORF8 (10 8 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-a, 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 mm. Data are representative of three independent experiments (E and F) or three independent experiments with n = 3 technical replicates (A-F) (shown as mean G 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.

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iScience 24, 102293, April 23, 2021 5 iScience Article receptors into rafts and triggers inflammation (Mukhamedova et al., 2019). In a recent study, ORF8 has been shown to interact with MHC-I (Zhang et al., 2020b), which is one of the marker proteins located on the surface of exosomal membranes (Becker et al., 2016). If ORF8 interacts with MHC-I and appears on the surface of exosomal membranes, it will increase the possibility that ORF8 protein interacts with the extracellular domain of IL-17 receptor and subsequently activates the NF-kB signaling pathway and increases the transcription of cytokines. In this way, ORF8 protein achieves being transmitted from alveolar epithelial cells to monocytes/macrophages, thereby leading to the outbreak of cytokine storm. Transwell system has been reported to study cellular communications in different studies, such as the communication between dendritic cells and endothelial cells (Gao et al., 2016), nerve cells and microglial cells (Yin et al., 2020), and even the triple interaction of epithelial cells, endothelial cells, and THP-1 cells (Costa et al., 2019). It would be interesting to construct an epithelial cell-macrophage co-culture system using a Transwell model, so as to study the transmission process of ORF8 from epithelial cells to macrophages.
ORF8 also has an inhibitory effect on the interferon pathway Rao et al., 2021), and Blanco-Melo et al. have reported that reduced interferon pathway coupled with exuberant inflammatory cytokine production are the defining and driving features of COVID-19 (Blanco-Melo et al., 2020). In a recent study, Miorin et al. have showed that ORF6 has the effect of antagonizing interferon signaling (Miorin et al., 2020), whereas we have found that ORF8 has the effect of promoting inflammatory cytokine production. This is consistent with the study of Blanco-Melo et al. Therefore, we speculate that the roles of these ORFs may be opposing, which also makes the pathogenesis of SARS-CoV-2 more complicated than that of common respiratory viruses. In our current work, we have proved that the binding of IL17RA with ORF8 depends on the fnIII_D2 domain of IL17RA and the binding site in ORF8 has not been determined. Young et al. found that D382-variant infection tended to be milder compared with those caused by the wild-type virus, with less pronounced cytokine release during the acute phase of infection (Young et al., 2020). Considering that the interaction between ORF8 and IL17RA has an important contribution in improving the expression of pro-inflammatory factors, we speculate that D382 variant might show a reduced ability to interact with IL17RA, which, however, needs to be verified with further experiments.
As a universal subunit of the IL-17 receptor family, IL17RA participates in the assembly of almost all the receptor complexes (Li et al., 2019), providing a broader site for ORF8 binding. However, it is worth considering that the other members of IL-17 receptor family have a similar structure to IL17RA, which may also be potential binding targets of ORF8. It has been reported that orphan receptor IL17RD can regulate various pathways employed by IL-17A in different ways (Mellett et al., 2012). The lack of IL17RD in cells leads to an enhancement in pre-inflammatory signals (Mellett et al., 2015). If ORF8 interacts with orphan IL17RD, the ORF8-IL17RD complex could disrupt the interaction between ACT1 and TRAF6. In this way, there will be a different regulatory mechanism existed, and further studies on this could be interesting.

Limitations of study
In this study, we found that ORF8 protein of SAR-CoV-2 can activate IL-17 signaling pathway by interacting with IL17RA, thereby up-regulating the secretion of inflammatory factors. Treatment with IL17RA antibody can protect mice from inflammatory damages caused by ORF8. However, as we have discussed in the article, IL17RA is a transmembrane protein, and the way in which ORF8 interacts with its extracellular domain is unclear. In addition, the main findings of this article should be further clarified using SARS-CoV-2 live virus instead of pseudovirus.

Resource availability Lead contact
Further information and requests for resources should be directed to and will be fulfilled by the lead contact, Haibo Wu (hbwu023@cqu.edu.cn).

Materials availability
This study did not generate new unique reagents.   immunoprecipitation. In addition, purified ORF8 was added to culture media of antibodies. Co-immunoprecipitation was performed according to previous studies