The infection of SARS-CoV-2 can cause severe pneumonia as well as other complications with significant morbidity and mortality. Currently, there is no suitable cure or appropriate medication for this fatal lung involvement. Our knowledge about the host immune interaction with SARS-CoV-2 is restricted, making it more difficult for management and developing new therapies. Viral infection typically induces massive changes in the transcriptome of the host, resulting in aberration of host cells’ metabolism and modulation of immune response, toward enhancing viral replication [16, 17]. Using several samples expression data from human lung epithelial cells including independent biological triplicates of primary human lung epithelium (NHBE) and transformed lung alveolar (A549) cells, we performed transcriptome analysis to better understand the molecular basis of the COVID-19 and identify putative markers. We found that 62 and 167 genes were differential expressed (p-value<0.05, and |log2FC|≥2) when A549 and NHBE samples of COVID-19 cells were compared to mock treated control, respectively. As expected for GO functional and KEGG pathway enrichment analysis, these genes belongs to type I interferon signaling pathway, IL-17 signaling pathway, cytokine mediated signaling pathway, and defense response to virus categories which all play significant roles in restricting viral infection.
We found that the expression level of IL-6 was increased several folds in both studies cells’ lines. In parallel, Systemic inflammation and cytokine storms with elevated levels of CXCL family, IL-8, and IL-6 are reported among SARS patients [16, 18]. Latest laboratory findings from Wuhan patients also showed that COVID-19 mild patients had elevated levels of IL-1B, Interferon gamma (IFNγ), CXCL10 and CCL2, while in severe cases, Granulocyte-colony stimulating factor (G-CSF), CXCL10, CCL2 and CCL3 were elevated [2]. Consistent with our results, a significant elevated expression of a large number of cytokines was observed in SARS-CoV-2 treated samples compared to mock-treated control. These findings show that infection with the SARS-CoV-2 virus can resulted to a cytokine storm that associated with severity of the disease. In another study, IL-6 was increased in patients with SARS disease, which is needed to regulate inflammatory response, B-cell differentiation and antibody development [19].
Use of WGCNA for co-expression analysis does not depend on particular contrasts (differential expression) and may establish associations in the study design between the co-expressed genes and important factors. Based on our analysis of data in the convenient models of bronchial epithelium cells (NHBE and A549 cells), we created gene co-expression network using WGCNA and recognized two associated modules. The Turquoise module from A549 samples with 196 gene and Yellowgreen module from NHBE samples with 194 gene were the most correlated modules with SARS-CoV-2 infection. KEGG and GO enrichment analysis of genes of these two modules further confirmed multiple viral infection-related processes both enriched in acute inflammatory response and defense response to virus categories which are biologically rational and associated with COVID-19 pathogenicity. One of remarkable pathways enriched in interested modules is regulation of viral life cycle. The genes in this category have a wide range of roles in SARS-CoV-2 life cycle like facilitating survival, attachment, entry as well as replication of the virus particles are so clear as candidate targets of antiviral drugs [20].
As mentioned in results section, the first 10 Hubgenes of Turquoise and Yellowgreen modules with GS and MM > 0.9915 and 0.94 respectively with |log2FC| ≥ 2 were selected including MX1 (Myxovirus (Influenza) Resistance 1), IFIT1, ISG15, DDX60 (DExD/H-Box Helicase 60), IRF9, PARP9 (Poly(ADP-Ribose) Polymerase Family Member 9), PGLYRP4, IL36G, SAA2 and IL-8 which had a high significance for immune related COVID-19 response. The important role of these genes in host response to various viruses has been described by many studies [21-31]. In line with this, some of these Hubgenes has been recently reported to be greatly related to COVID-19. For example, functional overexpression of MX1 was significantly mediates antiviral response to SARS-CoV as well as probably SARS-CoV-2 [21, 32]. Elevated expression levels of IL-8 and IL-36G have been declared to be closely related to COVID-19 severity [33, 34].
Transcriptional changes of well-characterized Hub-genes of Turquoise module in table 3 shows direct effectors of the IFN-stimulated antiviral response including MX1, IFIT1, ISG15, IFI6, DDX60, OAS1-3 and Signal transducer and activator of transcription 1 (STAT1) in response of SARS-Co-2.
In order to compare differences between SARS-CoV-2 and RSV molecular pathogenicity, we performed DEG analysis on A549 cell which infected with RSV. Interestingly, the pattern of gene expression between two lists revealed diminished antiviral response to SARS-CoV-2 discriminating this response from common robust interferon induction of antiviral genes which reported in other studies after viral infections [35]. However, considering obvious suppressed expression levels of IFN-1 and IFN-3 by means of SARS-CoV-2, the underling mechanism of this unexpected antiviral response remains to be ascertained. In line with this, IRF9 and IFI6, amongst these hub genes are so attracting due to unabated transcriptional response to SARS-Co-2 as compared to RSV.
IRF9 is a well-known essential IFN regulatory factor acts as a part of interferon-stimulated gene factor 3 (ISGF3) complex consisting of STAT1, STAT2 and IRF9 that mediates expression of several IFN-inducible genes including MX1, OAS1-3, ISG15, IFIT1, IFI6 as well as transcription factors of IFN-1 expression, interferon regulatory factor 3 and 7 (IRF3 and IRF7) [35, 36]. The pivotal role of IRF9 in antiviral responses against various common viruses including respiratory viruses has been well demonstrated [36, 37]. Importantly, emerging data suggest that various viruses such as human papillomavirus (HPV), reovirus, hepitis B virus, adenovirus, human cytomegalovirus and RSV evolved mechanisms to evade from IRF9-mediated antiviral response [38-42]. Strikingly, IRF9 overexpression amplifies the interferon response to viral infections. Interestingly, more recent study declared that initial concentration of IRF9 is a substantial determinant regulating amplitude, speed as well as strength of IFNα-mediated antiviral signaling [38].
Taking together, these findings strengthen the idea that unique unmuted expression of IRF9 in response of SARS-Co-2 infection would responsible for observed significant up-regulation of at least a subset of ISGs to escape from robust control over IFN-1 and IFN-3 expression. Therefore, the early increase of IRF9 expression levels during early stages of SARS-CoV-2 infection is a crucial regulator of early, enhanced and accelerated initiation immune response despite of suppressed expression of IFN avoiding presumed route used by coronavirus to escape from immune response during early phase of infection. Thus, variations in either IRF9 basal levels or the amount of IRF9 induction rate may has a robust predictive value not only for disease severity and clinical outcomes providing a putative potential target for COVID19-related therapy, but also may be a reason for patient-to-patient differences in disease development.
Whereas, IRF9 hub gene is a pathway activator/regulator, IFI6 is known as an antiviral effector and its expression can be induced by IFN-1 [43]. Recently, IFI6 has been purposed as a gene expression signature distinguishes between viral and bacterial infections of respiratory tract [25]. Importantly, some studies reported an essential role of IFI6 overexpression in pro-survival signaling by subverting TRAIL-mediated mitochondrial loss, cytochrome C release and subsequent apoptosis [44]. In this regard, RSV has been revealed to suppressed IFN-induced up-regulation of IFI6 in a time dependent manner at early phase of infection to escape from IFN-mediated antiviral response [45].
Remarkably, recent study suggests that diffuse alveolar damage and selective viral-mediated type 2 pneumocytes apoptosis would be as the major pathological basis of COVID-19, culprit for both decreased capacity of air exchange and fluid leakage result in acute respiratory distress syndrome (ARDS) and even death [46]. These findings support a model in which IFI6 up-regulation could serve as a possible antiviral strategy to protect healthy respiratory epithelial cells from early apoptosis elicited by surrounding pro-apoptotic cytokines leading to enhanced IFN production. Therefore, IFI6 could serve as an appealing target for innovate new therapies in combating COVID-19.
On the other hand, it is identified that IFI6 up-regulation potentiates the antiviral activity of IFN-α and restricts replication of several RNA viruses including dengue, Zika, West Nile and yellow fever viruses (Dengue virus (DENV), Zika virus (ZIKV), West Nile virus (WNV) and Yellow fever virus (YFV) respectively) as shown by its ability to interfere in YFV replication indirectly via preventing viral invagination into endoplasmic reticulum (ER) [47-49]. However, this antiviral mechanism of IFI6 localized in ER seem to be specific to flaviviruses due to lack of inhibitory effect on neither HCV nor coronavirus replication in COS-7 cells that organized in double-membrane ER structures [48]. Moreover, IFI6 is also identified to improve IFN-induced antiviral response as a result of activation of STAT3 signaling [45]. Given this findings, one can speculate that perhaps localization of IFI6 at either mitochondria or ER membrane in various cell types might be an important determinant of IFI6 activity against viruses.
As depicted in figure 5, further network-based drug repurposing is calculated and identified novel and potent drugs targeting SARS-CoV-2. However, some of predictions have been showed by recent literature to be beneficial for COVID-19. For instance, recent computational studies offer Cidofovir, Deferoxamine, Cytarabine and Clarithromycin as potent drugs for COVID-19 treatment [50-53]. ACE2-associated network analysis suggest a crucial role of Fluticasone propionate for COVID-19 treatment [54]. Moreover, Cyclosporine exhibited antiviral efficacy (with the half maximal inhibitory concentration (IC50) value of 5.82 µM) against SARS-CoV-2 [55]. Notably, considering immunomodulatory and anti-oxidative properties as well as beneficial roles of melatonin in reducing vessel permeability, anxiety, and ameliorating sleep quality, strongly suggest potential adjuvant use of melatonin for critical care patients infected by COVID-19 [56]. Importantly, emerging data supporting the beneficial role of Dipyridamole, a convenient antiplatelet agent, against broad spectrum of viruses mainly the positive-stranded RNA viruses as well as COVID-19 which stimulate type I interferon responses, decrease lung injury and suppressed in vitro replication of COVID-19 [46]. Moreover, corticosteroids such as Hydrocortisone, Beclomethasone and Fluticasone have been suggested to might have beneficial effects for the sever patients with pulmonary edema in combination with ventilator to prevent COVID-19 associated-ARDS development [46].
Additionally, Naproxen is a familiar non-steroid anti-inflammatory drug (NSAID) currently used as a part of triple combination therapy (Azithromycin, Naproxen and Prednisolone) that largely administered by Iranian’s physicians to treat patients with COVID-19 that supposed to reduce duration of hospitalization [57].
According to figure 5, Ribavirin and PEG interferon α-2b are shown to interact with IFIT1 which its vital role in the IFN-induced host defense against RSV, IAV, HBV and vesicular stomatitis virus (VSV) has been reported by several studies [23, 24]. Ribavirin is a well-known directly acting antiviral drug against hepatitis C virus (HCV) with a broad-spectrum of antiviral [58]. It has been reported that ribavirin exhibits an inhibitory effect against zoonotic paramyxoviruses leading to severe respiratory infection such as Hendra virus (HeV) and Nipah virus (NiV) [59]. Moreover, ribavirin has been reported to effectively prevents RSV reproduction [60]. Surprisingly, recent modeling and docking analysis suggests the effectiveness of ribavirin as a potent drug for COVID-19 treatment [58]. PEG interferon α-2b is another broad-acting antiviral drug used for hepatitis treatment that its inhibitory effect on coronaviruses has been demonstrated by in vitro studies [61]. Interestingly, both IFN-α and ribavirin therapy of HCV infection could lead to up-regulation of IFI6 [62]. Consistent with our results, Ribavirin and PEG interferon α-2b have been suggested in the guidelines for the Prevention, Diagnosis, and Treatment of COVID-19 issued by National Health Commission (NHC) of the People’s Republic of China [61]. Taking together, Ribavirin and PEG interferon α-2b may offer a potential combination therapy for COVID-19 that emerged with promising results by synergistically targeting IFIT1 and IFI6 hub genes.