The multi-systemic involvement associated with rapid clinical deterioration is among the hallmarks of COVID-19-related mortality. Blood components are deeply involved with widespread virus dissemination and disease aggressiveness; recent approaches uncovered that coronavirus’s immune responses persist beyond six months [34]. To better understand what happens with the blood of patients over aging, we investigated the virus-host interactions using whole blood samples of a young adult compared with older adult individuals. In general, a distinct profile of blood targets was clustered with younger ages (20–49) and older ages (50–79), in which the most distant groups (20–29 years old versus 70–79 years old) displayed an inverse gene expression pattern. The most pronounced effects were observed over the age of 50 and included higher expression of SARS-CoV-2-related genes (e.g., genes involved in immune response, inflammation, cell component and adhesion, biological process, and platelet activation/aggregation). The increase in inflammatory mediators strongly correlates with disease severity within the conception of cytokine storm [35, 36]. 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 [37]. 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 [38, 39].
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 [40]. Although Fas-FasL signaling has shown involvement in apoptosis of immune cells and virus-infected-target cells [41], 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) [42, 43]. FasL promotes T cell activation in humans by recruiting cFLIP to the DISC, thereby activating NF-kB and ERK/AP-1 transcription factors [44]. 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 [45] without triggering apoptotic signaling. Consistently with these findings, we detected by single-cell analysis that FasL is mainly secreted at low levels by CD8 + T and NK cells in the peripheral blood of SARS-CoV-2-infected patients. More importantly, a possible interaction of viral Orf8 with a variety of host proteins including FasL may explain the rapid spread of the coronavirus and immune evasion, since Orf8 of SARS-CoV-2 is highly secreted and downregulated MHC-I in cells [46]. A recent study by Sorbera et al. [47] on specific SARS-CoV-2-induced targets reported Fas-FasL signaling involved with endothelial function and neutrophil lifespan and related SARS-CoV-induced apoptosis with potential viral replication. Thus, inhibiting Fas/FasL interaction may be useful in the treatment of COVID-19.
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 γδ T-cells count, which is likely due to its differentiation and activation [48]. Based on this fact, we believe that the low count of FasL-associated CD8 + T cells could result from its activation. Although the role of Fas/FasL system in lymphocytes is unclear, patients suffering from SARS-CoV-2 infection and COVID-19 disease present T-cell dysfunction and diminished number of T and NK cells in peripheral blood [49]. The immune responses to SARS-CoV-2 infection are often characterized by hyperactivation of CD4 + and CD8 + T cells [50, 51] and macrophages [52], 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 [36]; in addition to the infiltration of inflammatory cells, immune cells have been found in patients’s lung tissues. Notably, disease-associated transcriptional change in aged whole blood had a more pronounced overlap with control blood in comparison to lung tissue transcriptome [53]. By interacting host genes with SARS-CoV-2 and blood transcriptome, Bhattacharyya and Thelma [53] 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 [54–56]. 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 [57]. Otherwise, CTSE is expressed in immune cells being implicated in antigen processing MHC class II pathway [58]. 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 [59]. There are several pathological evidence of thromboembolism, diffuse endothelial inflammation, and viral infection of endothelial cells [60, 61] 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 BCL2-associated 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 [62]. In this context, BAG3 inhibition seems to promote a significant suppresion of viral replication. Like VCAM1, the BAG3 is predicted to interact with spike (S) glycoproteins. These surface molecules favor virus attachment, fusion, and entry into host cells as a direct target involved in immune responses, being lately evaluated for design and development of the S protein-based vaccines [63]. After SARS-CoV‐2 enters the bloodstream, a cascade of events occurs resulting in blood clots and strokes [64]. We verified upregulation of proto-oncogene (SRC) and heat-shock protein 70 member 5 (HSPA5) genes in aged individuals (70–79 years old); these are linked to platelet aggregation, activation, and signalings. The other upregulated targets showed involvement in adaptive immunity, immunoglobulin domain, T cell receptor signalings, and cell adhesion.
Caveats and limitations of the study is that this is an observational interactive study, and facing the absence of a specific validation targeting the main component of our model, our results are hypothesis-generating. Despite these limitations, our dataset-based model to understand SARS-CoV-2 pathophysiology in whole blood in elderly matched with SARS-CoV-2-infected patients with high viral load offering a predictive model that can serve as a template for future intervention design in older patients with severe disease. Future approaches are needed to evaluate the role of these blood targets considering COVID-19-related comorbidities and individual physical conditions over aging.
In summary, we demonstrated that blood gene expression of FASLG, a Fas receptor-ligand triggering non-apoptotic inflammatory activities, is overexpressed in males and females after the age of 50. Through single-cell analysis using COVID-19 patients samples, we suggested the involvement of FASLG mainly associated with NK and CD8 + T cells. The FASLG is overexpressed in SARS-CoV-2-infected female and male patients with high viral load. 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.