key: cord-0785940-54ox6qk8
authors: de Almeida Chuffa, Luiz Gustavo; Souza, Jeferson dos Santos; de Mello, Mariana Costa; de Oliveira Neto, Mario; Carvalho, Robson Francisco
title: The aging whole blood transcriptome reveals a potential role of FASLG in COVID-19
date: 2020-12-06
journal: bioRxiv
DOI: 10.1101/2020.12.04.412494
sha: d9a77c4b8a21533b2791c81d1f673b52887752e6
doc_id: 785940
cord_uid: 54ox6qk8

The risk for severe illness from COVID-19 increases with age as older patients are at the highest risk. Although it is still unclear whether the virus is blood-transmitted, the viral RNA is detected in serum. Identifying how Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) interacts with specific blood components during aging is expected to guide proper therapies. Considering that all human coronavirus require host cellular molecules to promote infection, we investigated the aging whole blood transcriptome from the Genotype-Tissue Expression (GTEx) database to explore differentially expressed genes (DEGs) translated into proteins potentially interacting with viral proteins. From a total of 22 DEGs in aged blood, five genes (FASLG, CTSW, CTSE, VCAM1, and BAG3) changed expression during aging. These age-related genes are involved in immune response, inflammation, cell component and cell adhesion, and platelet activation/aggregation. Both males and females older than 50 overexpress FASLG compared with younger adults (20-30 years old), possibly inducing a hyper-inflammatory cascade that activates specific immune cells. Furthermore, the expression of cathepsins (CTSW and CTSE) and the anti-apoptotic co-chaperone molecule BAG3 was significantly increased throughout aging in both gender. By exploring publicly available Single-Cell RNA-Sequencing (scRNA-Seq) data on peripheral blood of SARS-CoV-2-infected patients, we found FASLG and CTSW expressed mainly in natural killer (NK) cells and CD8+ (cytotoxic) T lymphocytes whereas BAG3 was expressed in CD4+ T cells, naive T cells, and CD14+ monocytes. The increased expression of FASLG in blood during aging may explain why older patients are more prone to severe acute viral infection complications. These results indicate FASLG as a prognostic candidate and potential therapeutic target for more aggressive clinical manifestation of COVID-19.

Coronavirus disease 2019 (COVID-19) is a pandemic infection caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) [1] . This virus is associated with a broad spectrum of respiratory disturbances, varying from upper airway symptoms to aggressive pneumonia [2] . In lung parenchyma, it produces alveolar edema, fibrin deposition, and hemorrhage [2] . Notably, vascular changes are one of the distinctive features of COVID-19. Many patients have demonstrated clinical signs of thrombotic microangiopathy [3] , with intravascular coagulation and thrombosis associated with multisystem organ failure [4] .

Although SARS-CoV-2 affects the lungs primordially, there is convincing evidence that it alters the coagulation processes in severe cases [5] . The formation of blood clots disrupts circulation due to thrombosis, pulmonary embolism, and heart attacks [3, 4] . The association of these changes in coagulation and protein aggregates with alterations in inflammatory parameters results in an increase of COVID-19 mortality at alarming proportions. The micro-thrombotic environment arises from hyperactivation of the coagulation cascade associated with hyper-intense inflammation and immune activities [6] . Of note, the clotting process is complex and likely orchestrated by the massive release of pro-inflammatory mediators, cytokines, and tumor necrosis factor (TNF), mainly released from monocytes and endothelial cells [6] .

The fact that the mortality rate of patients aged 60 years and over is higher than those under 60 years is indisputable [7] . Most of these critical cases are associated with the "cytokine storm," as these exacerbated immune reactions may lead to an early death of elderly people regardless of comorbidities related to more severe cases [8] .

Recent findings are correlating the blood subtype with viral susceptibility and infection [9, 10] . However, the individual molecular machinery of blood cells may be detrimental for the grade and type of response, such as exacerbated or reduced inflammation. Considering that aging is one of the most significant risk factors for severe cases of COVID-19, it is essential to determine blood host genetic variation throughout the aging process. The evolutionary conservation between the 2019 novel SARS-CoV-2 and SARS-CoV [11] allows the possibility to understand similarities and differences between these coronaviruses into public databases. Using computational predictions of SARS-CoV-human protein-protein interactions (PPIs), we can identify possible mechanisms behind the viral infection and identify potential drug targets [12, 13] .

Considering that older individuals, with or without comorbidities, are more prone to develop more severe cases of COVID-19, including those related to blood perturbations, we investigated the whole blood transcriptome data during aging using the Genotype-Tissue Expression (GTEx) database [14, 15] . This strategy provided significant insights into age-associated target genes and how they can predict SARS-CoV-2-interactions in aged blood components.

We used whole blood RNA-Seq data from 670 males and females available at (Supplementary Tables 1-5 ). The DEGs were used to identify protein-protein interaction networks and perform gene ontology enrichment analyses (Supplementary Figure 1) .

We first selected SARS-CoV-2 cell entry mediators using the human proteins available in the COVID-19 Cell Atlas (https://www.covid19cellatlas.org/). In addition to this screening, we compared upregulated and downregulated DEGs in the whole blood during aging with the corresponding proteins that interact with HCoVs (Supplementary Table 9 ) by using publicly available databases. To uncover HCoVshuman PPIs, we used data from the Pathogen-Host Interactome Prediction using Structure Similarity (P-HIPSTer, http://phipster.org/) database. P-HIPSTer is a broad protein catalog of the virus-human interactions upon structural information with an experimental validation rate of approximately 76% [18]. Our research has used P-HIPSTer to identify proteins potentially interacting with SARS-CoV-2 in other tissues [19, 20] .

The relative expression of FASLG, CTSW, CTSE, VCAM1, and BAG3 (TMM normalized; V8 cohort) was also performed independently in male and female samples using One-way ANOVA followed by Tukey's test. The results were analyzed with GraphPad Prism v. 6.00 for Windows (GraphPad Software, La Jolla, California, USA).

Significant differences were set at P < 0.05.

The HumanBase tool (https://hb.flatironinstitute.org) [21] was used to provide whole blood-specific networks based on upregulated genes during aging that code for proteins potentially interacting with SARS-CoV according to P-HIPSTer. We considered five functional modules: co-expression, interaction, transcription factor binding, gene set enrichment analysis perturbations and microRNA targets as active interaction sources, applying a minimum interaction confidence of 0.12. The maximum number of genes was set as 15.

We performed the Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analysis and Gene Ontology enrichment analysis (Biological Processes) to identify the functions of DEGs by using the EnrichR database (http://amp.pharm.mssm.edu/Enrichr/) [22] . Top enriched terms were generated according to the lowest p-value < 0.05 (Fisher's exact test). The molecular function and protein class related to the blood components during aging were performed in the PANTHER classification system v. 11.0 (http://www.pantherdb.org/) [23] . We used the UniProtKB database (http://www.uniprot.org/) to obtain functional information of the identified proteins.

The genes that appeared overexpressed in aged blood samples were analyzed by STRING online tool (https://string-db.org/) [24] . The metasearch STRING database (Search Tool for Retrieval of Interacting Genes, v. 10.5) was used for mapping PPI enrichment. We considered the following settings: text mining, databases, experiments, and co-occurrence as sources of interaction. The minimum interaction score was 0.900 (highest confidence); in the networks, the disconnected nodes were hidden to show reliable interactions exclusively. The PPI enrichment P-value indicates the statistical significance registered by STRING. (access in October 2020).

We investigated the expression of selected genes (FASLG, CTSW, CTSE, VCAM and BAG3) in distinct blood cell populations based on previously published human single-cell RNA-seq data [25] . This single-cell dataset is available at the COVID-19

Cell Atlas (https://www.covid19cellatlas.org/) and was explored using the cellxgene interactive viewer (https://cellxgene.cziscience.com). The dataset includes peripheral blood mononuclear cells (PBMCs) populations from patients hospitalized with COVID-19 (n=7); patients with acute respiratory distress syndrome (n = 4), and healthy controls displaying no diasease (n = 6).

The genes Fas (genbank code: XP_006717882.1) and FasL (genbank code: NP_000630.1) were obtained on NCBI GenBank, and protein structure blast of these sequences were performed using NCBI blastp suite [26] . Next, PDB structures and FASTA sequences were downloaded on protein data Bank [27] , and the sequences obtained from GenBank and PDB were aligned on LALIGN [28] . Finally, sequence molecular mass calculation was evaluated using ProtParam expasy [29] .

The comparison of blood candidate genes was based on Venn diagrams using the Venny2.0 tool (https://bioinfogp.cnb.csic.es/tools/venny/index.html) [30] . Heatmaps and Scatter plots for clustering analyses were performed using the web tool Morpheus (https://software.broadinstitute.org/morpheus) [31] . Metascape was used to provide GO terms enrichment [32] obtained from aged blood genes that potentially interact with SARS-CoV-2.

The number of DEGs varied significantly throughout aging (Log2 FC ≥ |1| ≤ |1| and FDR < 0.05; Tables S1-S5). We detected an increased number of DEGs in subjects 

Next, we used the list of DEGs during aging to identify transcripts translated into proteins that potentially interact with SARS-CoV proteins based on P-HIPSTer database. This analysis identified 22 genes with an age-dependent expression profile (Table S9; CTSW and CTSE proteins are related to viral endosomal escape, FASLG, VCAM1, and BAG3 interact with viral Orf8 and protein sars7a (Tables S10 and S11). Also, VCAM1 and BAG3 showed potential interaction with spike glycoprotein, E2 glycoprotein precursors, Excised polyprotein 1..4369 (gene: orf1ab), and Full_polyprotein 1..4382.

We further compared mean gene expression in a cohort with female and male samples to identify gender-dependent responses (GTEx, release V8). When we compared male and female blood samples together, we found that expression of showed an increased expression over the age of 50 in the blood of males and females in a similar manner ( Figures S2 and S3 ). Among all DEGs, CTSW presented a similar expression profile in males and females. The CTSE was higher in males > 50 years old compared to females. Specifically, VCAM1 and BAG3 showed age-dependent expressions when men and women were analyzed individually ( Figure S2 and S3).

We analyzed the expression profile of our candidate genes (FASLG, CTSW, with disease severity within the conception of cytokine storm [34, 35] . Since blood diffuses through the capillaries to guarantee tissue perfusion, the multiple organ dysfunction syndrome may be likely due to essential elements arising from cytokine storm; therefore, monitoring cytokines reduce mortality in other viral diseases such as SARS, MERS, and influenza [36] . Unlike an early infection, the advanced disease is associated with low levels of the antiviral interferons (IFNs) and high levels of interleukin (IL)-1β, IL-6, and tumor necrosis factor (TNF) and chemokines (CCL-2, CCL-3, and CCL-5) secreted by coronavirus-infected immune cells [37, 38] .

We provided herein a transcriptomic investigation to identify a possible signature of reliable candidates involved with aged blood. While FASLG was found to be overexpressed in the three highest age ranges of 50-59, 60-69, 70-79 years, some cathepsins (CTSW and CTSE), adhesion-related molecule (VCAM1), and chaperone regulator molecule (BAG3) were commonly increased after the age of 60. FasL is a type-II transmembrane and homotrimeric protein belonging to the tumor necrosis factor (TNF) family After binding to its Fas receptor, a type-I transmembrane TNF receptor, FasL triggers apoptotic and highly inflammatory activities [39] . Although Fas-FasL signaling has shown involvement in apoptosis of immune cells and virus-infected-target cells [40] , emerging evidence highlights the apoptosis-independent role of Fas-FasL on the induction of active pro-inflammatory signals in severe pathological conditions (e.g., viral infection) [41, 42] . FasL promotes T cell activation in humans by recruiting cFLIP 1 to the DISC, thereby activating NF-kB and ERK/AP-1 transcription factors [43] . These activations were responsible for the secretion of IL-2 and T cell proliferation; IL-8 was also associated with NF-kB transactivation in bronchiolar epithelial cells, whereas macrophages secreted TNF-α after Fas ligation [44] without triggering apoptotic signaling. Consistently with these findings, we detected by single-cell analysis that A comprehensive study using whole blood samples from 54 COVID-19 patients documented a dramatic increase in immature neutrophils in parallel with a decrease in CD8+ T and VD2 γ δ T-cells count, which is likely due to its differentiation and activation [47] . Based on this fact, we believe that the low count of FasL-associated CD8+ T cells could result from its activation. The immune responses to SARS-CoV-2 infection are often characterized by hyperactivation of CD4+ and CD8+ T cells [48, 49] and macrophages [50] , which produce massive levels of pro-inflammatory cytokines.

Current evidence reports that critically ill patients have elevation in IL-6 levels compared to moderately ill patients [35] ; in addition to the infiltration of inflammatory cells, immune cells have been found in patients's lung tissues. Notably, diseaseassociated transcriptional change in aged whole blood had a more pronounced overlap with control blood in comparison to lung tissue transcriptome [51] . By interacting host genes with SARS-CoV-2 and blood transcriptome, Bhattacharyya and Thelma [51] further suggested that viral infection only alters expression profile already dysregulated in the elderly, thereby resulting in poor prognosis; these altered blood genes may reinforce the appearance of severe clinical manifestations including strokes, blood clots, and heart failures.

The expression of CTSW, CTSE, VCAM1, and BAG3 was further shared in the two last age ranges compared to young individuals. Cathepsins SW (CTSW) and SE (CTSE) are papain-like cysteine protease and intracellular aspartic protease, respectively. These molecules were mainly overexpressed in males compared to females. The cathepsins B/L have been described to mediate viral entry into host cells via the endosomal pathway, participating in cell death, protein degradation, autophagy, and immune activities [52] [53] [54] . Although CTSB and CTSL are mainly associated with SARS-CoV-2 infection, CTSW is involved in the escape of viral particles from late endosomes during influenza A virus (IAV) replication [55] . Otherwise, CTSE is expressed in immune cells being implicated in antigen processing MHC class II pathway [56] . Targeting CTSW and CTSE may also be a promising alternative to treat COVID-19.

Serum levels of VCAM1 are elevated in mild COVID-19 and highly increased in severe cases [57] . There are several pathological evidence of thromboembolism, diffuse endothelial inflammation, and viral infection of endothelial cells [58, 59] that are strictly related to disease severity and dysfunctional coagulation. Importantly, it is of great value to investigate the expression of endothelial cell adhesion molecules in aged COVID-19 patients.

The BAG3, an anti-apoptotic co-chaperone molecule referred to as BCL2associated athanogene 3, was upregulated in naive T cells, CD4+ T cells, and CD14+ T cells from aged individuals. BAG3 is involved in a variety of biological processes such as cell survival and apoptosis, cellular stress response, and cell migration, and is suggested to be part of the SARS-CoV machinery for replication [60] . In this context, BAG3 inhibition seems to promote a significant suppresion of viral replication. Like 

In summary, we demonstrated that blood gene expression of FASLG, a Fas receptor-ligand triggering non-apoptotic inflammatory activities, is overexpressed in NK cells and CD8+ T cells of males and females after the age of 50. Our in-depth study further evidenced the increase in some cathepsins, cell adhesion, and co-chaperone molecules potentially involved in host cell entry, replication, and vascular dysfunction during aging. Because hypercytokinemia is described as the framework for disease severity and high-risk death, we highlight FASL as a prognostic biomarker and a therapeutic proposal to modulate inflammation in elderly patients with COVID-19.

Additional studies are encouraged to test the presence of this biomarker in different disease modalities.

Supplementary Materials: Supplementary materials can be downloaded directly at the site.

The authors declare no conflicts of interest. Figure Legends   Table S1 . Differential gene expression in whole blood transcriptome of individuals aged 20-29 vs 30-39. Table S9 . DEGs in whole blood of aged individuals before and after matching with P-HIPSTer. Table S10 . Human-SARS-CoV Interactome based on in silico computational framework P-HIPSTer (http://phipster.org/). Table S11 . Interactome of human-SARS-CoV performed in the P-HIPSTer for aged whole blood. 

A Randomized Trial of Hydroxychloroquine as Postexposure Prophylaxis for Covid-19

Coronavirus Disease 2019-COVID-19

The science underlying COVID-19: implications for the cardiovascular system

Venous thromboembolism in patients with COVID-19: systematic review and meta-analysis

COVID-19 and coagulation: bleeding and thrombotic manifestations of SARS-CoV-2 infection

COVID-19: The crucial role of blood coagulation and fibrinolysis

Clinical features of COVID-19 in elderly patients: A comparison with young and middle-aged patients

Clinical features of patients infected with 2019 novel coronavirus in Wuhan

Testing the association between blood type and COVID-19 infection, intubation, and death

Association between ABO blood groups and COVID-19 infection, severity and demise: A systematic review and meta-analysis

Network-based drug repurposing for novel coronavirus 2019-nCoV/SARS-CoV-2

A single-cell atlas of the peripheral immune response in patients with severe COVID-19

Database resources of the National Center for Biotechnology Information

Announcing the worldwide Protein Data Bank

A Time-Efficient, Linear-Space Local Similarity Algorithm

Protein identification and analysis tools in the ExPASy server

Venny. An interactive tool for comparing lists with Venn's diagrams

Morpheus: a user-friendly modeling environment for multiscale and multicellular systems biology

Metascape provides a biologist-oriented resource for the analysis of systemslevel datasets

COVID research updates: Immune responses to coronavirus persist beyond 6 months

COVID-19-Immunopathological Mechanisms, Clinical Considerations, and Therapeutic Approaches: The REPROGRAM Consortium Position Paper. Front Immunol

Cytokine Storm in COVID-19: The Current Evidence and Treatment Strategies

Cytokine storm intervention in the early stages of COVID-19 pneumonia

Cytokine responses in severe acute respiratory syndrome coronavirus-infected macrophages in vitro: possible relevance to pathogenesis

Chemokine upregulation in SARS-coronavirus-infected, monocyte-derived human dendritic cells

A domain in TNF receptors that mediates ligand-independent receptor assembly and signalings

Fas/FasL Pathway Participates in Regulation of Antiviral and Inflammatory Response during Mousepox Infection of Lungs

Intrahepatic mRNA Expression of FAS, FASL, and FOXP3 Genes Is Associated with the Pathophysiology of Chronic HCV Infection

Role of Fas/FasL in regulation of inflammation in vaginal tissue during HSV-2 infection

The caspase-8 FLIP pro-motes activation of NF-kB and Erk signaling pathways

Induction of IL-8 secretion and apoptosis in bronchio-lar epithelial cells by Fas ligation

Structure of SARS-CoV-2 ORF8, a rapidly evolving coronavirus protein implicated in immune evasion

Taking aim at a fast-moving target: targets to watch for SARS-CoV-2 and COVID-19

Whole blood immunophenotyping uncovers immature neutrophil-to-VD2 T-cell ratio as an early marker for severe COVID-19

T cell responses in patients with COVID-19

Immunological and inflammatory profiles in mild and severe cases of COVID-19

Pathological inflammation in patients with COVID-19: a key role for monocytes and macrophages

Age-related gene expression alterations by SARS-CoV-2 infection contribute to poor prognosis in elderly

SARS-CoV-2 Cell Entry Depends on ACE2 and TMPRSS2 and Is Blocked by aClinically Proven Protease Inhibitor

Cathepsin L-selective inhibitors: A potentially promising treatment for COVID-19 patients

Inhibitors of cathepsinL prevent severe acute respiratory syndrome coronavirus entry

Cathepsin W Is Required for Escape of Influenza A Virus from Late Endosomes. mBio

Cathepsin E: A mini review

Elevated Expression of Serum Endothelial Cell Adhesion Molecules in COVID-19 Patients

Endothelial cell infection and endotheliitis in COVID-19

Autopsy findings and venous thromboembolism in patients with COVID-19: a prospective cohort study

Quantitative proteomics analysis reveals BAG3 as a potential target to suppress severe acute respiratory syndrome coronavirus replication