To get insight into the interplay between genes consistently deregulated in DS and SARS- cov2 infection and pathogenesis we built a DS-SARS-CoV–2 network. We considered “DS genes”, HSA21 genes, HSA21 neighbors, and non-HSA21 genes found consistently deregulated in at least 4 DS transcriptomic studies (Supplementary Table 1). We then mapped these genes on COVID–19 related pathways (as reported in WikiPathways) and on the interactome(17) coming from a mass-spectrometry affinity study with proteins of SARS-CoV–2(17) ending up with 118 nodes that we connected based on their functional and physical annotated interactions (Figure 1, Supplementary Figure 1).
Approximately half of the nodes in the DS-SARS-CoV–2 network (55 nodes) contained proteins involved in host Covid–19 related pathways, while the other half (59 nodes) contained host proteins known to interact with viral proteins. Six of these nodes were HSA21 genes, and 92 HSA21 neighbor genes. Overall, 26 genes were differentially expressed in at least 4 DS transcriptomic studies, 16 of which were neither a HSA21 gene nor a HSA21 gene neighbor (Figure 1). These genes could determine a different susceptibility to Covid–19 infection.
In this network we detected several pathway connected with COVID–19 that are affected in DS that will be discussed in the following sections (Figure 2).
Mechanisms of viral entry
Trisomy of TMPRSS2 might enhance virus activation
TMPRSS2 was upregulated across the different DS studies, with a median fold change of 1.59, in cortical tissue(28, 29), white blood cells(30), lymphoblastoid cell lines(28), iPSCs(31), mouse fetal liver and placental tissue(32) (Figure 3A). This suggests that the proteolytic activation of the viral spike protein for its interaction with the receptor ACE2 (angiotensin I converting enzyme 2), would be favored in DS, allowing increased entrance of the SARS-CoV–2 virus in the host cell. Conversely, we found downregulation of the endosomal protease cathepsin B that, similarly to cathepsin L, could also prime S-protein, suggesting that the preferential virus entry in DS is through TMPRSS2. In fact, only TMPRSS2 activity is essential for viral spread and pathogenesis in the infected host whereas CatB/L activity is dispensable(33–35). However, ACE2 itself is not consistently differentially expressed, and it is not a HSA21 neighbor. We found ACE2 upregulated in DS induced pluripotent stem cells (iPSCs) (homo sapiens) (31), downregulated in peripheral blood from DS individuals(36); slightly upregulated in the hippocampus of the DS model Ts1Cje (6–7 months old)(37), covering most of the region orthologous to human chromosome 21q22, DS human postnatal inferior temporal cortex(29) and adult dorsolateral prefrontal cortex(29), and slightly downregulated in post-mortem dorsolateral DS prefrontal cortex(38) (Figure 3B).
Other ACE2-related mechanisms
Interestingly, in DS we detected an upregulation of the bradykinin receptor B1 (BDKRB1)(29, 39), one of the HSA21 neighbors revealed in our analysis, and of the metalloprotease CPA3, that is normally upregulated in asthma patients. BDKRB1 upregulation, as part of the kallikrein-kinin system could determine a higher susceptibility in DS individuals to ARDS. Following viral binding, ACE2 expression and activity is eventually downregulated by the virus through multiple mechanisms, preventing it from performing its usual function in states of health(40). The downregulation of ACE2 signaling induces the kallikrein-kinin system which is activated during inflammatory conditions with vascular-alveolar fluid extravasation, leukocyte extravasation and recruitment to the lung and acute respiratory distress syndrome (ARDS), lung injury and pneumonia(41).
The interferon signaling is activated in DS
We detected 21 proteins in our network belonging to the IFN-I signaling pathway, and several of them were found upregulated in DS transcriptomic datasets: IFNAR1(12, 29, 42, 43), IFNAR2(12, 29, 32, 42, 44–47), IFNA2(29), IFNB1(29), STAT1(42), STAT2(48),
OAS1(12, 49), OAS2(12, 50), NF- KBIA(36, 38, 49). Of those, two genes present in three copies in trisomic cells are the Interferon Alpha and Beta Receptor Subunit 1 and 2 (IFNAR1 and IFNAR2), that form a heterodimeric interferon receptor. IFNAR1 and IFNAR2 initiate the innate antiviral immune response, that leads to the phosphorylation of STAT1-STAT2, that dimerize and activate transcription of inflammatory genes in the nucleus. OAS1 and OAS2 are two HSA21 neighbor proteins, activated by the interferon signaling pathway, leading to activation of the RNAseL that, in turn, leads to the degradation of the viral genome(51). On the other hand, Nuclear Factor kappa B (NF- kB) inhibitor alpha, is activated by the inflammatory NF- kB signaling and inhibits the translocation of the same NF- kB into the nucleus. With the exception of IFNA1 expression, type I interferons (IFNA2 and IFNB1) were all upregulated, indicating that the axis IFNAR-STAT-OAS is upregulated in Down syndrome.
A recent paper shows that SARS-CoV–2 receptor ACE2 is an interferon-stimulated gene in human airway epithelial cells and is detected in specific cell subsets across tissues(52). Therefore, we predict that virus entry might be significantly increased in DS patients that have both an increased interferon signaling and triplication of the protein S-priming through TMPRSS2.
Tightly connected to this pathway, the MAPK signaling acts as an integration point of several biological processes (53). In this pathway, we found 7 genes downregulated (BCL2(28, 30, 36, 54), FOS(29, 39, 46, 54–56), IFITM2(36, 54, 56), MAPK3(36, 39, 56), MAPK10(29, 54), MAPK13(30, 46, 57), MAPK14(29, 30)) and 5 upregulated (MAPK1(42, 58), MAPK11(12, 59), IFITM1(29, 38, 42, 49), IFI27(29, 36, 48, 49, 60) and BST2(12, 29, 38, 46, 48, 54)). Interestingly, some of these proteins, such as Interferon Induced Transmembrane Protein 1 (IFITM1), Interferon Induced Protein 27 (IFI27) and Bone marrow stromal antigen 2 precursor (BST2), are all interferon-induced proteins with antiviral properties. Specifically, IFITM1 is active against multiple viruses, including SARS-CoVs(61), preventing the viral fusion after endocytosis and the release of viral contents into the cytosol.
The viral Orf3a protein from SARS-CoV can bind TRAF3 and activate the NLRP3 inflammasome(62), leading to the cytokine storm. Given the higher basal inflammation in DS we would have expected the inflammasome to be upregulated. Instead, we detected a strong downregulation of the NLRP3 gene(36), critical for maintenance of homeostasis against pathogenic infections, as previously identified(63), along with lower levels of the gene for the NF- kB subunit RELA. Actually, even if the IFN-I signaling in the beginning induces an antiviral response, it eventually exerts an anti-inflammatory action inhibiting the NLRP3 inflammasome through STAT1. Although this could potentially be beneficial in later stage to shut inflammation down, it could also be one of the reasons why DS patients with influenza often manifest bacterial infection complications (64).
Moreover, DS individuals could be more susceptible to late onset complication such as lung fibrosis upon COVID19 infection, because they upregulate some of the cytokines responsible for the so-called “cytokine storm”. Specifically, we detected upregulation of the chemokine CXCL10(42) that induces chemotaxis and stimulation of monocytes, and of Interleukin 10(29). IL10 is an anti-inflammatory cytokine necessary for regulated resolution of inflammation. However, IL10 recruits fibrocytes and activates M2 macrophages in a CCL2/CCR2 axis and mice overexpressing IL10 show lung fibrosis(65).
Finally, we found upregulated ZAP70 (29), a tyrosine kinase that regulates motility, adhesion and cytokine expression of mature T-cells, as well as thymocyte development. A recent study found the SARS-CoV–2 nsp9 and nsp10 interact with NKRF(66), that inhibits IL–8 and IL–6 induction by competing with NF-КB for promoter binding. This interaction would lead to IL8/IL6 induction, and, among other, inhibition of ZAP70.
Apoptosis is inhibited in DS
As regards apoptosis, it is known that the apoptotic effect of SARS-CoV is mediated by its M protein through inhibition of the proto-oncogene AKT1(67). Interestingly, AKT1 is a HSA21 neighbor and is consistently upregulated(58). We therefore speculate that trisomic cells might be less sensitive to the apoptotic effect of coronaviruses.
Apoptosis can also be induced through endoplasmic reticulum (ER) stress. As a matter of fact, coronavirus replication is associated with the endoplasmic reticulum and this often induces ER stress, with subsequent activation of the unfolded protein response (UPR). UPR leads to the blockade of protein synthesis, and subsequent apoptosis through EIF2AK2(68). EIF2AK2 is an interferon-induced serine-threonine protein kinase (PKR), that, once activated by the viral RNA, phosphorylates and inhibits the initiation factor eIF2α, preventing viral replication. The coronavirus’ non-structural related protein 15 is an endoribonuclease that can inhibit EIF2AK2 preventing a premature block of protein synthesis upon viral infection(69). However, in DS, EIF2AK2 levels are elevated(29, 37- 39), and therefore, once again, trisomic cells might be more resistant to this inhibition. Supporting this, the serine/threonine-protein phosphatase PP1-alpha catalytic subunit PPP1CA, one of the three catalytic subunits of protein phosphatase 1 (PP1), is also downregulated in DS(29, 39), leading again to the translation shutoff mediated by phosphorylated eIF2α.
Host proteins interacting with viral proteins are enriched in viral life cycle and tight junction
When we analyzed the portion of DS-SARS-CoV–2 network of host proteins interacting with viral proteins (squared nodes) we found that they were enriched in two main categories: the biological process “viral life cycle” and the KEGG pathway “tight junction”. Among the first are worth mentioning the nucleoporin NUP62(30, 36) and NUP210(36, 39), that are downregulated in DS. Upon viral infection, nucleoporins are normally degraded to suppress innate immune responses, and improve viral replication and transmission(70). This may indicate altered virus-host interactions in DS leading to more efficient viral innate immune evasion(71). As regards the tight junction pathway, we detected 4 genes upregulated and 2 downregulated in DS. Even though the net effect of these deregulations cannot be predicted, interestingly, many viruses, including coronaviruses, disrupt epithelial tight junctions of the respiratory tract that serve as a barrier to invaders(72).
Another interesting protein is a disintegrin and metalloproteinase with thrombospondin motifs 1 (ADAMTS1) that interacts with the viral helicase/triphosphatase nsp13. While the significance of this interaction is unknown, it is known that ADAMTS1 is triplicated and upregulated in DS, both in blood datasets and in DS lung(12, 27, 36, 39, 73), where it contributes to the global anti-angiogenic milieu leading to higher risk for developing pulmonary arterial hypertension (PAH) in infants with DS. Moreover, it induces fibrosis in myocardial viral infection(74, 75).
Antiviral properties of EGCG
Compared to SARS-CoV, SARS-CoV2 seems to be much more infectious. This might be due to the ability of the virus to be primed not only by TMPRSS2 but also by FURIN.
EGCG, the main polyphenol of green tea, showed some beneficial effects on cognition in a phase II clinical trial with DS individuals(76). Interestingly, EGCG perfectly fits in the FURIN pocket and is therefore predicted to be a FURIN inhibitor(77–79). Concordantly, lipophilic EGCG derivatives showed antioxidant and antiviral properties(80). Therefore, the use of EGCG in individuals with DS, might be considered in the context of this world pandemic.