COVID-19, which was caused by SARS-CoV2 had brought great disasters to human life. Up to now, COVID-19 has caused billions of infections and millions of deaths worldwide. Our study revealed that significant change has taken place in COVID-19 patients from the blood sample. Although nasopharynx swab nucleic acid examination can almost identify all patients early, we can't merely accurately classify patients according to the nucleic acid report, not to mention that most patients are asymptomatic infected. There are also patients who have severe conditions that require intensive care units (ICUs) despite the rapid development of vaccines and antiviral treatments 16. Meanwhile, some authoritative reports indicate that patients who recovered from severe COVID-19 may have suffered long-term sequelae that may last for a long time17. Therefore, discovering more ideal markers that can differentiate severity and prognosis of the disease will have a profound impact on its prevention and treatment.
In line with the previous report, the inflammatory response induced by viremia sepsis is an important determinant of the systemic multiple organ injury caused by SARS-CoV2. Early studies also reported that the extent of inflammation is proportion to the disease severity, and the inhibitor or antagonist of cytokines and inflammatory factors can effectively slow down the disease progress. The bioinformatics analysis of COVID-19 in the early stage has helped us to understand and specific mechanism of the disease 18, 19. Ghanem M had identified 17 different expressed proteins which showed a consistent change in other datasets and these genes were mainly enriched in inflammatory responses, platelet degranulation and neutrophil chemotaxis20.
Although studies have reported that inflammatory factors are proportion to the severity of the disease, we still lack specific diagnostic markers related to virus replication. Our study also demonstrated that significant different genes existed between normal and COVID-19 patients. Additionally, these DEGs were mainly enriched in cellular senescence, complement and coagulation cascades, organelle fission, DNA replication, and P53 signaling pathways.
Pakorn Sagulkoo illustrated that upregulated DEGs in COVID-19 were mainly concentrated in cell division, cell cycle, and innate immune signaling pathways, which was consistent with our study21. Lucy Kundura discovered that the monocyte of COVID-19 spontaneously released ROS (reactive oxygen species, ROS) able to result in DNA damage and apoptosis in neighboring cells. Moreover, they also observed the presence of DNA damage in up to 50% of their PBMCs and T-cell apoptosis. At the same time, ICU patients exhibit more T-cell apoptosis and lymphopenia than non-ICU patients22. Alexandra Ioana Moatar reported that differentially expressed microRNA are primarily enriched in mitotic cell cycle in the heart, lung, and kidney23. The gene ontology and enrichment results of the whole-blood dataset from Samaneh Maleknia also reported an enhancement in the inflammatory host response, cell cycle, and interferon signatures among COVID-19 patients 24.
The dysregulated host response signaling pathways play a critical role in SARS-CoV-2 infection and viral pathogenesis. Our study demonstrated that P53 was closely associated with COVID-19 infection. The role of P53 was mainly involved in the cell cycle, DNA repair, cellular stress responses and apoptosis 25. P53 also stimulate type I IFN response during the innate immune responses and regulate virus infection in both directions26, 27. Abhishek Kumar demonstrated that activation of P53 can directly restrain virus replication implying the underline host restriction factor of SARS-CoV-2. While the main viral protease nsp5 can inversely repress the transcriptional activity of P5328. They discovered four activators and four inhibitors in SARS-CoV-2 proteins for P53-mediated activation. They confirmed the result in an independent luciferase reporter assay that nsp5, nsp15, orf3a, and orf6 could significantly repress the activity of a P53-regulated promoter, while the C145A mutant of nsp5 could not.
Our study also revealed the dynamic change of immune cell infiltration. The fraction of B cells memory, plasma cells, and T cells CD4 memory activated, monocytes, macrophages M0 were significantly higher in COVID-19. A previous study has illustrated that T cell responses remain stable for up to 9 months during SARS-CoV2 infection, whereas the amplitude of early CD4 + T cell immune responses was proportion to the severity of initial infection. They also found that neutralizing antibody (NAb) levels were strongly correlated with SARS-CoV-2-specific CD4+, but not CD8+, during T cell responses. The N-, N.361-, S-, and RBD-specific antibody levels were significantly correlated with N- and S-specific CD4 + T cell responses, but much less with CD8 + T cell responses 29. Several studies had revealed the aberrant B-cell responses especially in critical-ill patients. Taeseob Lee demonstrated the increased level of acute B cells differentiate into plasma cells in several patients, moreover, the diversity of increased B-cell receptor (BCR) were proportion to the higher levels of anti-SARS-CoV-2) spike antibodies in plasma30.
Cytokine release syndrome (CRS) is a potentially fatal complication of infectious diseases after SCRS-CoV2. At the same time, the activation and transformation of macrophages is also an effective mechanism of SARS-CoV-2 mediated multiple organ injury. Huirong Jiang discovered that M0 macrophage membrane-derived biomimetic nanoparticles could neutralize pro-inflammatory cytokines during the CRS and alleviate multi-damage in a mouse model31. Qizhou Lian reported that both classically polarized macrophages (M1) and M2 displayed the inhibitory effects upon SARS-CoV-2 infection, while M1 and non-activated (M0) macrophages, instead of M2 macrophages can directly trigger the inflammatory response which was consistent with our studies 32.
In addition, the ratio of T cells CD8, and NK cells were down-regulated in COVID-19 patients. Previous study also demonstrated that CD8 + T-cell and NK cell loss due to programmed cell death might contribute to a cytokine storm33. Indeed, these cytotoxic lymphocytes have been involved in the downregulation of immune activation via their ability to kill T cells, NK cells, and antigen-presenting cells34. The reduced level of CD8 + T cells and NK cells could impair negative feedback on immune activation in several COVID-19 patients22.In one report, T cells unique to specific SARS-CoV-2 peptides were enriched in patients with severe disease, raising the intriguing possibility that the immunological response to specific epitopes may contribute to disease severity 35.
Unlike previous studies that used inflammatory factors and pro-inflammatory release factors as diagnostic markers, our study identified novel markers related to virus replication and immune infiltration. The expression level of CDC25A, PDCD6, and YWAHE maybe serve as potential diagnostic markers for COVID-19, and their values were strongly related to the immune cells.
Cell division cycle-25a (CDC25A)
Cell division cycle-25a (CDC25A), a member of the CDC25A family, plays an essential role in cell cycle by dephosphorylating its substrate. Pakorn Sagulkoo identified CDC25A as key gene in severe COVID-19 and can regulate cell cycle and apoptosis in many disease progression36. Furthermore, there was also a study that up-regulated CDC25A after Sendai virus infection inhibited the release of IFN-β, while knockdown of CDC25A offset the protective effects.37. Hang Deng discovered that CDC25A can respond to poly (I:C) and the level of CDC25A can down-regulate the expression of IFN-1 with or without poly (I:C) stimulation38. CDC25A was also involved in other pathologic processes such as fibrosis and inflammatory cell infiltration as well. Malte Kölling revealed that miR-21 promotes a G1-phase arrest in mesangial cells (MCs) by targeting the novel targets CDC25A, culminating in MC hypertrophy39. Our study also revealed that CDC25A was up-regulated in COVID-19 and was closely associated with plasma cells, macrophages M0, monocytes, T cells CD4 memory resting, T cells CD8, and dendritic cells resting, which may imply that CDC25A was closely associated with inflammatory immune response.
Programmed cell death 6 (PDCD6)
PDCD6, known as apoptosis-linked gene-2 (ALG-2), belong to a well-known apoptosis regulator in mammals40, 41. PDCD6 can directly interact with the human death-associated protein kinase 1 (DAPk1) and trigger apoptosis via caspase-3 dependent pathway during the apoptotic process 42. There were also studies that indicated PDCD6 was redundant and involved in the cell migration and invasion during the tumorigenesis 43, 44. In addition, PDCD6 can also act as a significant prognostic biomarker in gastric cancer 45. In addition to being an apoptosis regulator,PDCD6 also take part in endocytosis and signal transduction 46. Yi Gong demonstrated that PDCD6 could inhibit virus infection via apoptosis promotion 47. Our study has discovered that the obvious difference in PDCD6 between normal and COVID-19 patients and the PDCD6 can be considered as a proper biomarker for the diagnosis of COVID-19. Furthermore, in response to DNA damage, PDCD6 also mediated the pro-apoptosis effect via the P53 signaling pathway which was in accordance with our enrichment analysis 48. Besides, PDCD6 also exert the pro-apoptotic activity of cisplatin or TNFα via the downregulation of NF-κB transcription in ovarian carcinoma cells 49. SARS-CoV2 infection was also correlated with PDCD6, which is known to regulate apoptosis and participate in antiviral immunoregulation.
YWAHE
YWAHE, also known as 14-3-3ε, belongs to a protein family known as 14-3-3, which are abundant throughout the body, including the central nervous system (CNS). The primary function of YWAHE can interact with other proteins in both cell cycle and apoptotic pathways in CNS. Previous study revealed that YWHAE were upregulated in several neurological diseases and loss or duplication of the YWHAE leads to Miller-Dieker Syndrome (MDS). Moreover, they also discovered that YWAHE could serve as a marker of human immune deficiency virus (HIV)-dependent neurodegeneration, and as a tool to assess disease progression. They suggested that 14-3-3 proteins may be released from destroyed neurons and acting a marker of cellular destruction. The 14-3-3ε levels also correlated with the viral load of HIV-1 in the brain and CSF 50. In addition, gp120 levels, which regulate cell cycle and apoptosis, are inversely related to those of 14-3-3ε51. In human 293T cells, cleavage of 14-3-3ε releases BAD, facilitating its translocation and subsequent interaction with Bcl-2 to promotes cell death 51. George D. Vavougios proposed that the dysregulation of YWAHE promote the incidence of neurodegeneration after SARS-CoV-2 infection 52. Shujuan Jiang discovered that human cytomegalovirus microRNA miR-US25-1-5p could repress viral replication by interacting with YWHAE53. Despite being involved in the central nervous system and cell cycle regulation, YWAHE could serve as a component of TNFR2 complexes in response to progranulin stimulation in macrophages. YWAHE was essential for TNFR2 signaling-mediated regulation of macrophage polarization and switch. Both global and myeloid-specific deletion of YWAHE resulted in exacerbated inflammatory arthritis and counteracted the protective effects of progranulin-mediated TNFR2 activation against inflammation and autoimmunity 54. The pro-inflammatory effects of YWAHE and release of IL6 and MCP1 in the treated cells were significantly reduced under the treatment of TLRs inhibition or in TLRs KO chondrocytes 55.
DNA polymerase theta (POLQ)
POLQ is a principal component of the alternative non-homologous end-joining (ANHEJ) DNA repair pathway that ligates DNA double-strand breaks56. The POLQ gene encodes DNA polymerase θ, a 2590 amino acid protein product harboring DNA-dependent ATPase, template-dependent DNA polymerase, dNTP-dependent endonuclease, and 5'-dRP lyase functions. 57. In addition, Sitong Zhou also proved that VASP and POLQ can serve as diagnostic markers in burn processes 58. Our study also revealed that POLQ was significantly elevated after SARS-CoV2 infection, however, the diagnostic value was not satisfactory in the end.