In this study, we identified genes and pathways significantly altered in the severe form of COVID-19. Optimizing the co-expression value in each cluster by the fast greedy algorithm and the subsequent gene sets net correlation analysis (GSNCA) enabled us to detect differentially highly co-expressed clusters of genes in each group of comparison (normal vs. mild, normal vs. severe, and mild vs. severe), which resulted in more precise enrichment analysis.
As the primary goal of this study was to shed light on distinct gene expression profiles that determine the severity of the disease, we analyzed the transcriptional dataset from a comparative perspective. Enrichment analysis of the up and down-regulated genes with different patterns of expression among the mild and severe conditions highlighted potential genes and signaling pathways responsible for the progression of the disease. GO and pathway enrichment analysis of genes with lower levels of expression in severe cases show that 30 genes, including CCL5, CCR7, CDC37, CTDP1, CXCL10, CYLD, EIF2AK2, HLA-DPB1, HLA-DQB1, HLA-DQB2, IFI44L, IFIH1, IFIT1, IFNGR2, IKBKE, ISG15, KPNA2, LGALS9, OAS2, OAS3, RAD23A, RASGRP1, RNF125, TIRAP, TLR9, TNF, UBA7, UBE2G2, USP18, and XAF1 are involved in defensive mechanisms against viruses. Impaired production of interferons and the delay in their onset is reported to be the key factor of severe disease 15. In agreement with our results, a recent comprehensive study on the common gene expression profile of pulmonary tissue and nasal mucosa of COVID-19 patients reported a highly-preserved module of genes, including BST2, IFIT1, IFIT2, IFIT3, IFITM1, ISG15, MX1, MX2, OAS1, OAS2, OAS3, OASL, RSAD2, and STAT1 between two mentioned tissue cells that regulates IFN-I signaling, in addition, level of expression of these 14 genes was discovered to be indicative of disease severity 16. On the other hand, the up-regulated genes were mostly related to cytokines and inflammatory responses. Notably, various pathways related to apoptosis through both intrinsic and extrinsic cascades were enriched by a group of 39 genes, namely BCL2A1, CAPN1, CD70, COPS5, CSF2, CXCR4, DDIT3, EGR3, FAS, FLT3, FZD9, HIPK1, ICAM1, IER3, IFNB1, IL1A, IL1B, IRS2, LYN, MPO, NF1, NFE2L2, NGF, NLRP3, PDPK1, PLAUR, PLK3, PMAIP1, PPP1R15A, PRKDC, PSEN2, SFN, SIAH1, SIAH2, SOS2, TFDP1, TFPT, TUBA4A, and UBC. Lymphopenia is a decisive feature of critical COVID-1917. Several mechanisms can result in the decrease of the Lymphocyte population and particularly lymphoid T cells, including apoptosis. A recent study discovered highly suggestive evidence of the involvement of apoptosis in T-cell depletion and also, based on this information, proposed a new promising treatment option 18. Also, a positive relation between apoptosis and the severity of the disease is reported in other studies 19-21. Considering the significantly different patterns of expression of these two groups of genes across mild and severe cases and their key functions revealed by enrichment analysis, they can potentially play central roles development of the severe condition.
Pathway and GO analysis of DEGs of normal vs. severe and mild vs. severe groups shows the involvement of these genes in inflammation, immune regulation, and infection-related pathways, as expected. On the other hand, results show that there is also a considerable relation between these genes and pathways involved in cancer pathogenesis and neurological disorders. Cancer-related signaling pathways include Proteoglycans in cancer, JAK-STAT signaling pathway, ErbB signaling pathway, PI3K-Akt signaling pathway, MAPK signaling pathway, VEGF signaling pathway, Ras signaling pathway, Pathways in cancer, Central carbon metabolism in cancer, Chemical carcinogenesis, MicroRNAs in cancer, mTOR signaling pathway, Autophagy, GnRH secretion, Choline metabolism in cancer, Signaling pathways regulating pluripotency of stem cells, Estrogen signaling pathway, Prostate cancer, Endometrial cancer, Colorectal cancer, Non-small cell lung cancer, Glioma, Growth hormone synthesis secretion and action, Chronic myeloid leukemia, Breast cancer, Gastric cancer, Renal cell carcinoma, Pancreatic cancer, Melanoma, Bladder cancer, Hepatocellular carcinoma, Acute myeloid leukemia. This evidence demonstrates notable relations between core signaling pathways implicated in cancer pathogenesis and COVID-19, which can be considered in treatment options.
It was observed that patients with cancer, among other groups of vulnerable people, are significantly prone to develop COVID-19 complications 22. Malignancies and COVID-19 have common features like the uncontrolled overexpression of cytokines, in addition to functions of IFN-I, androgen receptor, and regulation of immune signaling checkpoints 23. One of the key cytokines involved in inflammation mediated by COVID-19 is IL-6, which is also reported to be dysregulated in many types of cancers 24. IL-6 can activate the IL-6/JAK/STAT pathway. Hyperactivation of JAK/STAT is considered to play a central role in the cytokine storm 25 and also is mentioned to be present in various types of malignancies and diseases with chronic inflammatory conditions 23. Toshifumi M. et al. proposed that SARS-CoV-2 via two of its products, namely NSP1 and ORF6, impairs STAT1 function and subsequently hyperactivation of STAT3, which results in chains of events ending up with overexpression of pro-inflammatory cytokines and also hypercoagulopathy 26. Xiao Li et al. identified 112 co-expressed genes among COVID-19 and lung adenocarcinoma and reported IL-17 signaling and MAPK signaling as top pathways altered by hub co-expressed genes 27. Another study on the subject, highlighted ALB, CXCL8, FGF2, IL6, INS, MMP2, MMP9, PTGS2, STAT3, and VEGFA as hub differentially expressed genes among COVID-19 and lung cancer and reported EGFR tyrosine kinase inhibitor resistance, IL-17, AGE-RAGE signaling pathway in diabetic complications, HIF-1 signaling pathways and pathways in cancer to be common in both conditions 28. Altogether more investigations are needed to uncover these similarities and to examine the possible benefits of treatment used in cancers for COVID-19 patients.
Signaling pathways related to the nervous system are Long-term depression, Dopaminergic synapse, Serotonergic synapse, Glutamatergic synapse, Circadian entrainment, Retrograde endocannabinoid signaling, Morphine addiction, Cholinergic synapse, Gap junction, Oxytocin signaling pathway, Axon guidance, Alcoholism. Regarding various complications of the nervous system observed in COVID-19 patients and mainly those with severe disease, these results could be informative about how the nervous system is affected by COVID-19.
The presence of ACE2 receptors on glial cells and neurons makes them potential targets for SARS-COV-2 infection 29. The fact that it is also present in the capillary epithelium raises the possibility of direct infection of the brain 30. In addition, the high incidence rate of various types of olfactory dysfunction indicates that the virus can move through the cribriform plate, trigeminal or vagal nerve, or penetrate the olfactory mucosa 31. Apart from direct infection, hyperinflammation in COVID-19 patients can affect normal regulations of CNS through elevated levels of cytokines. Sustaining normal levels of cytokines in the brain is vital to maintain the physiological state needed for normal cognitive behaviors 32. High levels of cytokines can disturb the neuroendocrine system, sleep-wake cycle, and regulation of appetite, and also is known to hinder the effectiveness of antidepressant drugs, potentially by interfering with the metabolism of neurotransmitters (by blocking pumps used to reuptake neurotransmitters like dopamine, norepinephrine, and serotonin), neural proliferation, and disrupting neurocircuitry of mood and anxiety 33. Co-expression and co-regulation of ACE2 and Dopa Decarboxylase (DDC) are another possible mechanism of nervous system disorders observed in COVID-19 patients, as DDC is involved in the biosynthesis of neurotransmitters, including serotonin and dopamine 34. Considering the anti-inflammatory effects of serotonin 35, several studies evaluated the hypothesis of the positive effects of selective serotonin reuptake inhibitors (SSRIs) on COVID-19 treatment, which turned out to be true 36. Despite many efforts, the exact molecular mechanisms behind these manifestations are lacking. It is worth noting that the fact that many pathways related to the nervous system are altered in PBMCs indicates that not only researchers should study the effects of general disease complications like hyperinflammation and hypoxia on the brain but also the direct influences of the virus on neural signaling pathways and gene expressions should be investigated. Our results can highlight the main genes and signaling pathways responsible for forming nervous system complications.
The results of this study identified genes with altered expression and signaling pathways involved in the establishment of different aspects of severe COVID-19. Further studies are needed to evaluate their possible benefits in terms of treatment targets, diagnosis biomarkers, and predictors of outcomes and complications.