Introduction

The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has triggered the largest pandemic in recent history, with more than 6.9 million confirmed deaths identified as the etiologic agent of Coronavirus Disease 2019 (COVID-19) (World Health Organization, <https://covid19.who.int>. Accessed on 03 July 2023), before SARS-CoV-2, two other epidemics were caused by betacoronaviruses. The severe acute respiratory SARS-CoV in 2002 and the Middle East respiratory syndrome in 2012 demonstrated the potential of this virus family for human-to-human transmission. The recurrence of these viruses makes understanding their pathogenesis essential (Chen et al, 2020b).

COVID-19 primarily affects the respiratory system but also leads to other symptoms, including taste and olfactory disturbances, gastrointestinal issues, neurological complications, cardiovascular problems, ocular symptoms, coagulation disorders, or even present as asymptomatic (Baj et al, 2020; Giannis et al, 2020; Wang et al, 2020). The COVID-19 disease is categorized into four groups: mild, moderate, severe, and critical, with the critical manifestation characterized by acute respiratory distress syndrome (ARDS) and sepsis or septic shock (Yang et al, 2020; Zhou et al, 2020). Furthermore, several risk factors have been associated with the disease, including advanced age, male sex, overweight, hypertension, diabetes, and preexisting respiratory and cardiovascular conditions (Zhou et al, 2020; Hu et al, 2021).

SARS-CoV-2 requires the host’s cellular apparatus for replication, assembly, and egress from cells. Because of this, the COVID-19 disease is associated with dysregulation of several genes and signaling pathways (V’kovski et al, 2021). The pathogenic mechanism of SARS-CoV-2 involves dysregulation of the renin-angiotensin-aldosterone system, hypoxia, hyperinflammation, cytokine and chemokines storms, and sepsis, resulting in a dysfunctional inflammatory response with persistent fevers and high levels of inflammatory markers (Tay et al, 2020). Sudden storms of cytokines and chemokines may lead to lung injury and ARDS. In addition, viral replication may result in cellular apoptosis, leading to diffuse alveolar damage and ARDS (Mason, 2020; Wu & McGoogan, 2020).

Omics technologies are instrumental in unraveling the molecular mechanisms underlying the physiological and metabolic changes in COVID-19. Despite the high cost, RNA-seq studies provide valuable information about gene expression changes in host tissues and cells after infection by SARS-CoV-2, including altered processes and pathways, favoring the understanding of pathogenesis and host–pathogen interaction (Jain et al, 2021; Yang et al, 2021; Samy et al, 2022). Transcriptomic analysis of blood samples from COVID-19 patients allows the identification of differentially expressed genes (DEGs) associated with the host’s immune or inflammatory response and regulatory networks (Kwan et al, 2021; Daamen et al, 2022). Such analysis can help predict prognostic and diagnostic biomarkers and potential drug targets for pathophysiology groups, supporting personalized treatment decisions (Rodriguez et al, 2021; Iqbal & Kumar, 2022). During the 3 yr of the COVID-19 pandemic, several RNA-seq data were generated and are publicly available for analysis. Different analyses of these data cost little and can guide future research. The integrated analysis of available data can increase the reliability of the results found and reduce bias because of the genetic variability of the participants.

In this work, we reanalyzed previously published transcriptome data from blood samples and leukocytes isolated from COVID-19 patients. We compared them with non-COVID-19 individuals to find potential biomarkers of disease severity and targets to improve the understanding of COVID-19 molecular pathogenesis. Using bioinformatics tools, we identify overexpressed genes related to the endomembrane system and neutrophil activation in hospitalized patients, and down-regulated genes are associated with the T cell receptor complex. In addition, SPI1 and other ETS family transcription factors are the predicted regulators with the most enriched binding motifs between the overexpressed genes in severe COVID-19 patients. At the same time, MAX, MYC, and TP53 were appointed as the central regulators of down-regulated genes. Our results indicate that these transcription factors are essential to gene expression control during COVID-19. These findings can potentially contribute valuable insights to understanding, diagnosing, and treating severe COVID-19.

Results

Discussion

Here, we employed an integrated and reproducible analysis of RNA-seq whole-blood data published during the COVID-19 pandemic to identify critical molecular targets in the progression of the infection by SARS-CoV-2. The integrated analysis of multiple datasets increases the number of patients evaluated and the genetic variability, including patients from different backgrounds. In this analysis, we highlight the increase of three classes of genes: (1) genes that code proteins located in the endomembrane system (including endoplasmic reticulum exit site until lysosomes), (2) extracellular exosome, and (3) genes related to specific granule and secretory granules (some of them related to neutrophils). The enrichment of these components was also seen in non-COVID-19 patients, showing the importance of this process in severe infections. However, specific genes showed increased expression exclusively in severe COVID-19 patients. SARS-CoV-2 intersects with the endomembrane system in various steps of the viral lifecycle (Sicari et al, 2020; Chen et al, 2022a). The virus–host interactome shows that SARS-CoV-2 proteins interact with many proteins of the host secretory pathway (Gordon et al, 2020; Stukalov et al, 2021). Furthermore, genome-wide CRISPR/Cas9 screens identified host factors in the endomembrane system limiting the infection (Wang et al, 2021a; Daniloski et al, 2021; Schneider et al, 2021). For example, the loss of RAB7A reduces viral entry in the cell by altering endosomal trafficking (Daniloski et al, 2021).

Our work highlighted an increased expression of genes related to the endomembrane system, such as RAB1B, RAB24, RAB32, JAK3, and SELP, in severe COVID-19 patients’ blood samples. RAB1B gene encodes a monomeric GTPase that controls ER–Golgi traffic and plays a central role in networks constructed by DEGs, interacting with many other proteins and connected pathways. Previous studies identified that RAB1B inhibition impairs the maturation of the SARS-CoV-2 spike protein (Veeck et al, 2023). The CDC42 up-regulated gene also encodes a central protein in PPI. This gene encodes a small GTPase from the Rho family involved in endocytosis (Gundu et al, 2022). We did not find works analyzing the role of CDC42 during a SARS-CoV-2 infection. However, its role in the entry of other RNA viruses has already been evaluated previously (Swaine & Dittmar, 2015).

In addition, lysosomal membrane-related genes also were up-regulated in the blood of severe COVID-19 patients. Differently from other RNA viruses, it has been demonstrated that β-coronaviruses also egress from cells using lysosomal organelles (Ghosh et al, 2020). In this way, the SARS-CoV-2 ORF3a protein facilitates lysosomal transport in a BORC, ARL8b, VAMP7, and STX4-dependent manner (Chen et al, 2021). In this work, we identified an increased expression of lysosomal genes such as LAMTOR1, NEU1, GBA1, BORCS8, and BLOC1S1 in severe COVID-19 patients. The proteins produced by BORCS8 and BLOC1S1 are part of the BORC complex and regulate lysosome positioning and movement with ARL8b (Pu et al, 2015). These findings indicate that these genes may be essential in disease promotion because of increased expression in patients with severe COVID-19.

In addition, recent articles have highlighted the potential of extracellular vesicles in promoting virus egress from cells. One of these articles demonstrated that VeroE6 cells infected with SARS-CoV-2 produce extracellular vesicles larger than exosomes and originate from the Golgi complex. These vesicles contain viral particles that promote virus escape from neutralizing antibodies (Xia et al, 2023). Another study identified that extracellular vesicles of different sizes were detected in the blood of COVID-19 patients and can be related to the severity of the disease (Lam et al, 2021). Our analysis identified target genes that encode proteins located in exosomes that were crucial to vesicle transport, such as CD63, ATP6V1D, and CHMP1A. CD63, commonly used as an exosome marker, increased protein expression, and correlated to higher exosome levels in severely ill COVID-19 patients (Aharon et al, 2023). In addition, CD63 expression was increased in plasma cell-free RNA sequences of severe COVID-19 patients (Wang et al, 2022). Corroborating with these data, we also observed an increase in mRNA expression of CD63 in whole blood and leukocytes of severe COVID-19 patients.

The group of genes related to the endomembrane and vesicle is essential at the beginning of the infection for endocytosis, replication, and exit of the virus in the cell. In this work, we explored the hypothesis that individuals with greater expression of these specific genes are susceptible to infection by SARS-CoV-2. However, we acknowledge that our analysis did not verify whether this increased expression also occurs at the primary site of infection. In addition, the expression of these genes in immune cells can contribute to the disease progression. For example, immune cells release exosomes with altered contents during inflammation, and these exosomes play an essential role in mediating inflammation (Chan et al, 2019). Notably, plasma exosomes isolated from COVID-19 patients have already been observed to stimulate cytokine production by PBMC cells from healthy donors (Chen et al, 2022b).

Furthermore, our analysis revealed an increase of granules-located genes, which corroborates the increased expression of genes related to neutrophil activation in datasets WB02, WB03, and LEU01. Neutrophil activation is one of the hallmarks of COVID-19 severity. However, it has been observed that COVID-19 shares neutrophil activation with other inflammatory states (Schimke et al, 2022). This work identified specific overexpressed genes related to neutrophils only in severe COVID-19 patients, such as SPI1, SLPI, S100A9, and BRI3, which indicates a specific expression regulation that occurs only in SARS-CoV-2 infection. In this work, the increased expression of SPI1 and its target was explicitly observed in patients with severe COVID-19, indicating that SPI1 has a vital role in transcriptional regulation during COVID-19. It has been observed that transcription factors like SPI1, which is related to granule–progenitor differentiation, were up-regulated in COVID-19 patients (Wang et al, 2021b). However, Wang and colleagues did not identify significant differences in SPI1 expression between mild and severe patients, which may be because of a limited number of participants in the study (n = 6).

In contrast, our integrated analysis involved multiple datasets, enabling us to identify the specific up-regulation of SPI1 in severe COVID-19 cases. We also identified predicted targets of SPI1 with differential expressions between severe and mild patients. Among these genes, S100A9 has already been suggested as a potential biomarker of COVID-19 severity (Chen et al, 2020a; Silvin et al, 2020). This gene encodes a protein that, together with S100A8, forms an alarmin known as calprotectin (S100A8/A9). This alarmin is released after the activation of neutrophils and binds to TLR4 to promote signaling activation via NF-kB and the secretion of inflammatory cytokines such as IL-6 (Mellett & Khader, 2022). In this work, we showed that the increase of S100A9 occurs not only in the release of alarmin but also in gene expression levels. Furthermore, S100A9 expression increases in severe COVID-19 patients before 10 d of symptom onset, suggesting that this increase is not just a consequence of prolonged inflammation.

In addition, the LY96 gene, also known as MD-2, exhibits an expression pattern similar to S100A9, suggesting that this gene also could be a good marker of disease severity. MD-2 is an accessory protein required for LPS and HMGB1 inflammatory signaling through TLR4 (Visintin et al, 2006; Yang et al, 2015; Chen et al, 2018; He et al, 2018). However, to the best of our knowledge, there are no articles that associate LY96 expression with COVID-19 severity.

Among the down-regulated genes, we found enrichment of (1) genes encoding T cell receptor complex proteins only in COVID-9 patients, (2) genes encoding proteins located in the nucleoplasm, and (3) TP53, MYC, and MAX among the master regulators of down-expressed genes. Lymphopenia is a well-known symptom in patients hospitalized for COVID-19. The decrease of T lymphocytes in severe patients may be why we observed a decrease in the expression of specific genes of T cells because the RNA extraction was probably performed with proportionally fewer T cells concerning other leukocytes. However, in addition to genes encoding subunits of the T cell receptor complex, genes related to the T cell-mediated immune response also have decreased expression in patients with severe COVID-19. The IL2RB gene, for example, which encodes an IL-2 receptor (IL-2R) chain, emerged as one of the central genes in the PPI network formed by down-regulated COVID-19 gene, indicating that IL2RB may be an essential target for understanding the COVID-19 disease progression and lymphopenia. IL-2R responds to IL-2 cytokine and is expressed in T and NK cells (Fernandez et al, 2019). IL-2 is critical to T cells’ proliferation, differentiation, and function (Ross & Cantrell, 2018). A study by Zang and colleagues (2020) observed that the increase of soluble IL-2R is correlated to lymphopenia in COVID-19 patients’ plasma (Zhang et al, 2020). Because of the increase in circulating IL-2R, the cellular response to IL2 decreases (Gooding et al, 1995).

Furthermore, plasma IL-2R concentration increased with symptom days (Zhang et al, 2020). Our work further reveals that the expression of IL2RB mRNA is mainly reduced in patients with more than 10 d of symptoms. In addition, GSEA analysis identified a negative relationship between KLRB1 and CD1C expression and the severe phenotype of COVID-19 patients. Both genes are related to NK and T cell antigen presentation. KLRB1 encodes a transmembrane protein expressed in NK cells and T cells named CD161, and CD1C encodes a member of the CD1 family related to MHC proteins (Moody & Cotton, 2017; Wyrożemski & Qiao, 2021).

The second key finding from the integrated analysis of COVID-19 transcriptome data showed decreased expression of nucleoplasm-related genes, including transcription factors, such as ETS1, MYCL, NFATC2, and TP53, and genes that encode proteins related to chromatin regulation such as NCL, KAT6B, and NAT10. The NCL gene encodes nucleolin, a multifunctional nucleolar protein with many interactions in the network of down-regulated genes. NCL is already related to the entry and replication of several viruses (Jia et al, 2017). Furthermore, it interacts with SARS-CoV-2 proteins, such as N and S genes and Nsp1 and Nsp12 proteins. This interaction was suggested to mediate SARS-CoV-2 induction of p53-dependent apoptosis (Merino et al, 2023). We observed that NCL expression was significantly decreased in all datasets from patients with severe COVID-19. In our study, the p53 gene (TP53) expression considerably decreased, particularly after 10 d of symptoms. However, we observed that the expression was similarly diminished in ICU patients who did not have COVID-19.

Furthermore, p53, MYC, and MAX showed binding sites on several down-regulated genes, indicating that these proteins have a potential role in the transcriptional regulation of other genes. P53 is an essential transcriptional regulatory factor well known for its involvement in the cell cycle and apoptosis. However, research has highlighted its role in antiviral immunity, as P53 knockout mice are sensitive to viral infections (Muñoz-Fontela et al, 2005, 2016). In addition, P53 regulates the expression of essential targets for immunity and inflammation, including IRF5 and TLR3 (Mori et al, 2002; Taura et al, 2008). A study by Bordoni et al (2021) observed increased p53 expression in PBMC from COVID-19 patients compared with healthy patients. Therefore, decreased TP53 expression in patients with severe COVID-19 may contribute to the disease’s development.

The binding motif for Myc/Max is required for TP53 transcription (Kirch et al, 1999). Our analysis demonstrates decreased expression of several MYC and MAX targets in patients with severe COVID-19. However, whereas MYC expression was significantly decreased in WB01 and WB03 datasets, MAX expression showed no significant change. These findings indicate that the dysregulation of Myc/Max targets is not solely because of the changes in the expression of transcription factors. In other studies, the SARS-CoV-2 Nsp6 protein has already been observed to interact with components of the MGA/MAX complex in fly cardiomyocytes. This interaction diminishes the antagonistic role of this complex in regulating the expression of MYC/MAX targets and increases the expression of genes encoding glycolysis pathways proteins (Zhu et al, 2022). Although we did not observe an enrichment of glycolysis-related DEGs in any gene ontology classifications in blood, it is crucial to consider that MGA gene expression was decreased in all severe patients, potentially contributing to the up-regulation of MYC targets in these patients. Nonetheless, MYC has diverse roles in regulating the expression of multiple targets and has many partners, warranting further investigation of its function during the inflammatory process triggered by SARS-CoV-2.

In our study, we have identified potential targets whose expression may be crucial in determining susceptibility to severe forms of COVID-19. Furthermore, assessing their cellular interactions could provide insights into the molecular pathophysiology of SARS-CoV-2 infection. In addition, identifying transcriptional factors that regulate the expression of genes involved in infection and inflammatory response could be pivotal in advancing our understanding of the disease. By comprehending cellular interactions and responses to viruses such as SARS-COV-2, we can better equip ourselves to respond effectively to future pandemics.