Single-nucleus lung transcriptomics and inammatory responses in lethal COVID-19 reveal potential drugs in advanced-stage clinical trials

There is pressing urgency to identify drugs that allow treating COVID-19 patients effectively. Respiratory failure is the leading cause of death in patients with severe COVID-19, and the host inammatory response at the lungs remains poorly understood. Therefore, we retrieved data from postmortem lungs from COVID-19 patients and performed in-depth in silico analyses of single-nucleus RNA sequencing data, inammatory protein interactome network, functional enrichment, and shortest pathways to cancer hallmark phenotypes to reveal potential therapeutic targets and drugs in advanced-stage COVID-19 clinical trials. Herein, we analyzed transcriptomics data of 719 inammatory response genes across 19 cell types (116,313 nuclei) from lung autopsies. The functional enrichment analysis of the 233 signicantly expressed genes showed that the most relevant biological annotations were: inammatory response, innate immune response, cytokine production, interferon production, macrophage activation, thymic stromal lymphopoietin, blood coagulation, IL-1 and megakaryocytes in obesity, NLRP3 inammasome complex, and the TLR, JAK-STAT, NF-κB, TNF, oncostatin M, AGE-RAGE signaling pathways. Subsequently, we identied 34 essential inammatory proteins with both high-condence protein interactions and shortest pathways to inammation, cell death, glycolysis, and angiogenesis. Lastly, we propose ve small molecules involved in advanced-stage COVID-19 clinical trials: baricitinib, pacritinib, and ruxolitinib are tyrosine-protein kinase JAK2 inhibitors, losmapimod is a MAP kinase p38 alpha inhibitor, and eritoran is a TLR4/MD-2 antagonist. After being thoroughly analyzed in COVID-19 clinical trials, these drugs can be considered for treating severe COVID-19 patients. activation, Toll-like receptor signaling pathway, interferon production, JAK-STAT signaling pathway, NF-κB signaling pathway, thymic stromal lymphopoietin, TNF signaling pathway, blood coagulation, oncostatin M signaling pathway, AGE-RAGE signaling pathway, IL-1 and megakaryocytes in obesity, and NLRP3 inammasome complex. UMAP: uniform manifold approximation and projection for dimension reduction.


Introduction
The rst zoonotic transmission of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) occurred in China in late December 2019 1 , and it is the etiological agent of the coronavirus disease 2019 (COVID-19) 2 . Since the World Health Organization (WHO) declared the outbreak of COVID-19 as a pandemic on March 11, 2020, the SARS-CoV-2 infection has led to more than 200 million cases and more than 4 million deaths globally 3 .
At the molecular level, amino-acid changes that result in reduced tness are generally removed by negative selection, whereas changes that increase virus tness are maintained by positive selection 12 . The most signi cant mutation observed in SARS-CoV-2 is probably the D614G substitution in the S1 subunit of the S protein. This mutation confers a 20% increase in infectivity and is associated with a higher ACE2-binding a nity 13 . Additionally, SARS-CoV-2 tness is also enhanced in presence of the E484K mutation, which increases resistance to antibodies 14 .
This transmissibility advantage was further increased to approximately 50% by the emergence of the B.1.1.7 variant 15 . Subsequently more variants have emerged, some of them capable of escaping monoclonal antibodies, partially eluding the polyclonal immune responses induced by previous infection or even allowing re-infections. It should be noted that recent improvements in immune escape are linked to mutations that alter the N-terminal domain (NTD) rather than the receptor-binding domain (RBD) of the S protein, where early and functionally important alterations prevailed 16 . However, improved transmissibility, rather than immunoevasion or increased lethality, are considered as the main route for the virus to become tter and more viable 17 .
The variants that are being carefully monitored include: A) Variants of concern (VOCs): characterized by increased transmissibility, cause a more severe manifestation of the disease or signi cant reduction in neutralizing antibodies generated during a previous infection or after vaccination, reduced effectiveness of treatments, or diagnostic detection failures. This group includes the B. VOCs, however precise information is still lacking. D) High consequence variants: those that have signi cantly reduced the e cacy of vaccines in relation to previously circulating variants, and additionally cause failures in their diagnosis; however, there are not SARS-CoV-2 variants that rise to the level of high consequence yet 18, 19 . It is expected that more variants will emerge over time that will need to be closely monitored, since they are a potential threat to public health. Nevertheless, this will not happen inde nitely because -over time-the virus will reach its maximum transmission point, therefore, new variants will not acquire more advantages in terms of infectivity. Thereafter, virus infectivity will stabilize and experience occasional and minimal variations 20,21 .
SARS-CoV-2, enriched by the previously mentioned genomic variants, has the ability to infect human body cells -especially in the lung microenvironment-through the angiotensin-converting enzyme 2 (ACE2) protein receptor 4 . Lung homeostasis necessitates a ne balance between tolerance mechanisms against non-pathogenic agents, pro-in ammatory immune system activation to ght off respiratory tract infections and anti-in ammatory and pro-brotic processes that will minimize the immune-mediated tissular lesion and promote tissue remodeling and repair. These complex mechanisms are mediated by a variety of tissue-resident and recruited cell types. The pulmonary alveolar epithelium is mainly composed of alveolar type I cells (AT1), which are essential for the gas-exchange function of the lungs, and alveolar type II cells (AT2), which are best known for their functions in synthesizing and secreting pulmonary surfactant factors 22 . Airway epithelial cells are central players in mucociliary clearance of the lungs. They produce a variety of antimicrobial substances, cytokines, and growth factors that mediate leukocyte recruitment, modulation of innate and adaptive immunity, and tissue repair 23,24 . They also constitute the rst cells that contact invading pathogens and are responsible for early pathogen recognition and induction of the antiviral state through pro-in ammatory cytokines and type I interferon secretion 25 .
Pulmonary endothelial cells are localized in the interface between the pulmonary tissue and the bloodstream. Their strategic location is also re ected in their pleiotropic functions that range from gas interchange to regulating vascular tone and facilitating immune cell recruitment and diapedesis upon receiving pro-in ammatory stimuli 26 . Mast cells are innate immune cells, involved in immune defense and surveillance. They are lled with secretory granules, which upon activation, release bioactive mediators to ght pathogens or induce allergic reactions 27 . Macrophages are key sentinel cells residing in peripheral tissues that detect pathogen invasion or tissue damage and initiate acute in ammatory processes triggering recruitment and activation of innate and -in second step-adaptive immune responses. Macrophages are also key inducers of the respiratory burst, professional antigen-presenting cells and tissue repair and remodeling mediators 28 . Dendritic cells form a heterogeneous population of the immune system that have a wide array of immune functions. Conventional dendritic cells bridge the innate and adaptive immune responses as they are constantly sampling antigens from the airways and/or the infected lung tissue, migrate to T-cell areas of secondary lymphoid organs and present it to T lymphocytes thereby activating them 29 . Monocytes, subsets of leukocytes mostly originated from myeloid progenitors in the bone marrow, are able to differentiate into macrophages or dendritic cells in peripheral tissues. They seed tissues with enough macrophages to replace their loss through infection and tissue damage and can adopt speci c macrophage or dendritic cell phenotypes depending on the cytokine milieu they encounter upon arrival to the in amed tissue 30 . Natural killer (NK) cells are lymphocytes of the innate immune system, that play a main role in anti-viral and anti-tumor responses [31][32][33] . They can identify and kill infected or stressed cells by releasing perforin and granzymes or by death receptor signaling (FasL/Fas interactions and the subsequent induction of apoptosis). NK cells can also release IFNγ upon activation, thereby contributing to naive T helper cell activation and differentiation and classical activation of macrophages 34 . The CD4 + T helper cell population orchestrates the innate and adaptive immune responses in acute and chronic viral infections by secreting a panel of immunomodulatory cytokines. These cells also play a key role for the establishment of long-term cellular and humoral antigen-speci c immunity, which is the basis of long-term protection induced by a plethora of viral infections and vaccines 35 . The cytotoxic CD8 + T cells play a pivotal role in controlling infections caused by intracellular pathogens. These cells can be considered the adaptive immunity counterparts of NK cells, but unlike their innate immunity counterparts CD8 + T cells are activated by speci c pathogen or tumor-derived antigen presented on class I major histocompatibility complex molecules (MHC I). The three major mechanisms of action of these cells are quite similar to NK cell functions: a) direct killing of infected or tumor cells by release of perforin and granzymes, b) indirect destruction of cells via death receptor signaling (Fas/FasL interactions), and c) secretion of cytokines that can direct and potentiate immune responses of nearby cells 36 . Treg cells are potent immunosuppressive cells that play a vital role in maintaining immune homeostasis and in the prevention of autoimmune responses by suppressing the activation of conventional T-cells 37,38 . B cells have a key role in the humoral adaptive immune response and -once activated-are responsible for the production of antigen-speci c immunoglobulins 39 . Plasma blasts are terminally differentiated populations of effector B cells cells, which produce antibodies, providing immunity during initial exposure to a pathogen and mediating the protective effects of vaccination 40 . Fibroblasts are key cells in the wound repair process and tissue scarring. They participate in the immune response by producing cytokines and chemokines that initiate the recruitment and retention of bone marrow-derived immune effector cells 41  Previous studies have reported profound SARS-CoV-2-induced transcriptional and immunological changes in animal models 44 , as well as in bronchoalveolar lavage uid (BALV) 45 , nasopharyngeal 46 , and human blood samples 47 . However, the respiratory failure is the leading cause of death in patients with severe COVID-19 disease and the host in ammatory response at the lung tissue level remains poorly understood 48, 49 . To shed light on this physiological response, we retrieved data from COVID-19 autopsies and performed in-depth in silico analyses of single-nucleus RNA sequencing (snRNA-seq) data, in ammatory protein-protein interactome (iPPI) network, miRNome enrichment, gene ontology (GO), and the shortest paths to cancer hallmark phenotypes to reveal potential therapeutic targets and drugs in advanced stage COVID-19 clinical trials.
The criteria of the analysis of the lung transcriptomics data was the following: 'uniform manifold approximation and projection (UMAP)' as load cluster, 'cell type intermediate' as selected annotation, and 'all cells' as subsampling threshold. Additionally, we adjusted the mRNA expression taking into account the Z-scores, that is, overexpressed mRNAs with Z-scores ≥ 2 and underexpressed mRNAs with Z-scores ≤ -2. Regarding visualization of transcriptomics data, we designed dot plots to visualize the percentage of cells expressing a certain gene, box plots to compare the mean Z-score across cell types, and scatter plots of 2D UMAPs to visualize signi cantly expressed multiple genes per subpopulation cell, and biological annotations across cell types.
Functional enrichment analysis. The functional enrichment analysis gives curated signatures of gene sets generated from omics-scale experiments 11,53,54 . We performed the enrichment analysis to validate the correlation between signi cantly expressed genes and biological annotations related to lethal COVID-19. The enrichment was calculated using g:Pro ler version e101_eg48_p14_baf17f0 (https://biit.cs.ut.ee/gpro ler/gost) to obtain signi cant annotations (Benjamini-Hochberg FDR q-value < 0.001) related to gene ontology: biological processes, the Kyoto Encyclopedia of Genes and Genomes (KEGG) signaling pathways, Reactome signaling pathways, and Wikipathways [53][54][55][56][57] . Lastly, the expression of genes involved in signi cant annotations was visualized in scatter plots, and the signi cant terms related to lethal COVID-19 pathology were manually curated. miRNome enrichment analysis. The Gene Set Enrichment Analysis (GSEA) (https://www.gseamsigdb.org/gsea/index.jsp) is a powerful analytical method for interpreting gene expression data that share common biological functions or regulations 58 . Therefore, we performed a miRNome enrichment analysis using the 'microRNA targets' option to compute overlaps between miRNAs and signi cantly expressed mRNAs in > 50% of lung cells from lethal COVID-19 patients. Lastly, we proposed the most signi cant miRNAs with a false discovery rate (FDR) q-value < 0.01.
In ammatory protein-protein interactome network. The iPPI network with zero node addition and a highest con dence cutoff of 0.9 was created between the human proteins physically associated with SARS-CoV-2 and human proteins involved in the pulmonary in ammatory response. This network was generated using the human proteome from the Cytoscape StringAPP 59,60 , which imports protein interactions from the STRING database 60 . The degree centrality represents the number of edges the node has in a network 11,61,62 , and it was calculated using the CytoNCA app 63 . The network elements were organized through the organic layout producing a clear representation of complex networks, and the iPPI network was visualized through the Cytoscape software v.3.7.1 64 . Finally, we ranked the in ammatory response proteins with the highest protein-protein interactions to the human-SARS-CoV-2 proteins.
Shortest paths from in ammatory response proteins to cancer hallmark phenotypes. CancerGenNet (https://signor.uniroma2.it/CancerGeneNet/) is a resource that links frequently altered proteins to cancer hallmark phenotypes 65 . This bioinformatic tool, curated by SIGNOR 66 , is based on experimental information that allows to infer likely paths of causal interactions linking proteins to oncogenic phenotypes. The shortest distance scores or paths from proteins to cancer phenotypes were programmatically implemented using the shortest path function of igraph R package 65 . Hence, we calculated the shortest distance scores of positive regulation from the in ammatory response proteins with the highest con dence interactions to the human-SARS-CoV-2 proteins to the in ammation, cell death, angiogenesis, and glycolysis hallmark phenotypes.
Drugs involved in current COVID-19 clinical trials. The Open Targets Platform version 21.06 (https://www.targetvalidation.org) is comprehensive and robust data integration for access to and visualization of potential drug targets associated with several diseases including COVID-19 67 . This platform has developed the COVID-19 Target Prioritization Tool (https://covid19.opentargets.org/) that integrates molecular data from the ChEMBL database 68 to provide an evidence-based framework to support decision-making on potential drug targets for COVID-19. Lastly, this platform shows all drugs in clinical trials associated with target proteins, detailing its modality, mechanism of action, phase, status, type of drug, target class, and clinical trial number 67 .
Functional enrichment analysis. The functional enrichment analysis was performed using g:Pro ler to obtain signi cant GO: biological processes, KEGG signaling pathways, Reactome signaling pathways, and Wikipathways related to lethal COVID-19 (Benjamini-Hochberg FDR q < 0.001) 11,53,54 . Figure 3 shows scatter plots of signi cantly expressed genes (n = 233) in lung cells of lethal COVID-19 autopsies. After a manual curation of biological annotations, the most signi cant GO terms were in ammatory response  Table 2). miRNome enrichment analysis. After identifying the signi cantly expressed genes in lung cells of lethal COVID-19 autopsies, we performed the GSEA analysis to compute overlaps between miRNAs and mRNAs 58 . In ammatory protein-protein interactome network. We generated the iPPI network encompassing 265 nodes and 2052 edges (Fig. 5). Of them, 159 pulmonary in ammatory response proteins had a mean of degree centrality of 8 and 108 human-SARS-CoV-2 proteins had a mean of degree centrality of 7.2. The top ten in ammatory response proteins with the highest degree centrality were APP (38), NFKB1 (36),  Table 3).
Shortest pathways from in ammatory response proteins to cancer hallmark phenotypes. We analyzed the 111 pulmonary in ammatory response proteins with the highest condifence interactions (cutoff = 0.9) to human-SARS-CoV-2 proteins in order to nd the shortest pathways toward in ammation, cell death, angiogenesis, and glycolysis according to Iannuccelli et al 65 . Figure 6A shows box plots encompassing proteins with the shortest distance scores to cancer hallmark phenotypes related to COVID-19 pathology.
Cell death was the phenotype with the shortest mean of distance score (2.82), followed by in ammation (3.06), glycolysis (3.12), and angiogenesis (3.79). Figure 6B shows a Venn diagram integrating in ammatory proteins with shortest pathways to biological phenotypes related to COVID-19. We found 34 essential in ammatory response proteins with shortest pathways simultaneously to in ammation, glycolysis, cell death, and angiogenesis (Supplementary Table 4). Figure 6C Table 5). Lastly, Fig. 7 details all shortest pathways and distance scores of positive regulation from the 34 essential proteins to the in ammation phenotype.
Drugs involved in current COVID-19 clinical trials. Figure 8 details the current status of COVID-19 clinical trials regarding to essential in ammatory proteins, according to the Open Targets Platform 67 . There are 5 drugs (small molecules) that are being analyzed in 8 clinical trials in advanced stages (phases III and IV) for 3 essential in ammatory proteins. Baricitinib is a tyrosine-protein kinase JAK1/2 inhibitor that acts on the JAK proteins and it is being studied in 4 clinical trials in phase III (NCT04640168, NCT04693026, NCT04421027, and NCT04401579). Similarly, pacritinib and ruxolitinib are tyrosine-protein kinase JAK1/2 inhibitors. They are currently been evaluated in a phase III clinical trial NCT04404361 and NCT04362137, respectively. Losmapimod is a MAP kinase p38 alpha inhibitor that acts on the MAPK14 protein and it is being studied in a phase III clinical trial (NCT04511819). Lastly, eritoran is a toll-like receptor 4/MD-2 antagonist that acts on the TLR4 protein and it is being studied in one clinical trial in phase IV (NCT02735707).

Discussion
Since the identi cation of patient zero in China, a wide spectrum of clinical features have been discovered in severe COVID-19. For instance, dyspnea, acute respiratory distress syndrome (ARDS) 71 , respiratory failure, lung edema, severe hypoxemia, cardiac arrhythmias, lymphopenia 72 , hyperferritinemia, rhabdomyolysis, intravascular coagulopathy 73,74 , and pulmonary thromboembolism 75 . Nowadays, it is known that SARS-CoV-2 not only causes respiratory tract infection, but also skin, kidneys, blood, and central neural system pathologies 76 . Therefore, it is imperative to continuously review the clinical manifestations and physiopathological mechanisms of the SARS-CoV-2 infection, especially with the appearance of new genomic variants.
Single-cell biology provides unprecedented resolution to the cellular underpinnings of biological processes in order to nd therapeutically actionable targets for complex diseases 69, 70  Macrophages are cells that perform crucial functions in the immune system, from the phagocytosis of the viruses and bacteria to maintaining homeostasis 88 . Precisely, macrophages produce high amounts of pro-in ammatory cytokines in patients with ARDS, who present an activated state known as cytokine storm or macrophage activation syndrome 89 . The overexpression of cytokines (i.e., TNF-α, IL-2, IL-10, IL-1, and IL-6) leads to a hyperin ammatory response, which has been reported as a remarkable feature of SARS-CoV-2 infection [90][91][92] . IL-6 plays a main role in the severity of COVID-19, while TNF-α and IL-1β trigger the NF-κB signaling pathway 93,94 . The excessive production of cytokines leads to development of pathological symptoms, such as lung damage, cell death, severe pneumonia, ARDS, lung brosis, and multiple organ failure 93,95 . Hence, this cytokine storm plays a crucial role in the progression of SARS-CoV-2 infection and is considered as one of the main causes of lethal COVID-19 92,93 .
TNF is considered as one of the most important pro-in ammatory cytokines, affecting different parts of the immune system and regulating various pathological and physiological processes 96 . Therefore, the TNFα-NF-κB axis is considered as a potential therapeutic target in COVID-19 97 . Initially, NF-κB is present within the cytoplasm, after activation of IκB through phosphorylation of IκB kinase, NF-κB is activated and translocated to the nucleus where it regulates the transcription of various target genes 98,99 . To date, SARS-CoV-2-mediated NF-κB activation has been observed in several cells such as macrophages of liver, kidney, lung, central nervous system, cardiovascular system, and gastrointestinal system. This causes a chronic production of IL-1, IL-2, IL-6, IL-12, TNF-α, LT-α, LT-β, GM-CSF, and several chemokines, leading to the aforementioned pathological symptoms 100 . Catanzaro et al have recently published a report analyzing the role of the TNFα-NF-κB pathway in COVID-19. In their report, it was suggested that inhibiting this axis may prevent pulmonary complications in COVID-19 patients 97 . This was also observed in SARS-CoV infection. NF-κB expression was elevated in the lungs of recombinant SARS-CoV-1-infected mice, while NF-κB inhibitors reduced SARS-CoV-related expanding survival of these mice 101 .
The cytokine signaling depends on the JAK and STAT which are phosphorylated and activated upon cytokines binding to their receptors. The STAT homodimers translocate into the nucleus, where they upregulate the transcription of several genes that participate not only in cytokine production but also in apoptosis, immune regulation, and cell cycle differentiation 102 . In the context of SARS-CoV-2 infection, inhibition of the JAK-STAT pathway seems as promising approach to prevent cytokines storm in fatal cases or in patients with comorbidities that express high levels of in ammatory markers such as of IL-6, TNFα, IL-17a, GM-CSF, and G-CSF 103 . In fact, the GenOMICC GWAS study suggests that individuals with a variant on chromosome 19: 10,466,123 that affects expression of tyrosine kinase 2 (TYK2), member of the JAK family, could be associated with a host-driven in ammatory response that leads to severe lung injury 104 . Thus, several clinical trials have shown that baricitinib, a JAK inhibitors possesses a good safety and e cacy pro les in reducing cytokine levels of severe COVD-19 patients without side effects 105 .
Nevertheless, the JAK/SAT pathway is also necessary to mediate the immune response to clear viral infections and prolonged inhibition of the pathway could lead to immunosuppression and prolonged infections 106 . For instance, SARS-CoV-2 is able to hijack the JAK/STAT pathway in order to increase its proliferation by evading the immune response. Li et al showed that SARS-CoV-2 infected cell had a decreased expression of JAK1, JAK2, TYK2, and STAT2 proteins. This is explained by action of viral nsp1, ORF6, and ORF8 that prevent the phosphorylation of STAT1 and STAT3 to inhibit IFN production 107,108 . Therefore, the timeline for administration of JAK/STAT inhibitors should be carefully analyzed since reducing the hyperin ammation could affect viral clearance. Due to the narrow therapeutic window of JAK/STAT inhibitors, dosage should aim to restore the immune response homeostasis.
The incidence of thrombotic events in COVID-19 patients responsible for strokes and heart attacks raises the concern about the abnormal coagulation patterns and poor prognosis in the actual pandemic. Tang et al reported that 71.4% of non-surviving COVID-19 patients met the criteria for disseminated intravascular coagulation and presented high levels of coagulation-related biomarkers such as D-dimer and brin degradation products 109 . The mechanisms of the coagulopathy are not clear; however, some reports indicate that dysregulated immune responses are involved in such processes. Exacerbation of in ammatory cytokines promoting proliferation of megakaryocytes, lymphocyte cell-death, hypoxia, endothelial damage contributing to ischemia and organ dysfunction, and the association between autoantibodies and neutrophil extracellular traps seem to be involved in the abnormal thrombotic events in COVID-19 patients [110][111][112] .
Oncostatin M is a cytokine involved in homeostasis and chronic in ammation that has pleiotropic functions such as cell differentiation and proliferation, and it is present in hematopoietic, immunological, and in ammatory networks 113 . One of the most important functions of oncostatin M is the stimulation of the chemokines CCL1, CCL7 and CCL8 in primary human dermal broblasts at a faster kinetics than IL-1β or TNF-α 114 . In 2020, it was proposed as a new mortality biomarker in patients with acute respiratory failure supported by venous-venous extracorporeal membrane oxygenation 115 . In the case of COVID-19, an increase of OSM plasma levels and other in ammatory mediators was detected; this nding was correlated with the severity of disease and the increase of bacterial products in plasma 116 . Finally, OSM is curiously elevated in obese patients and upon recognition by its speci c receptor (OSMRβ) induces obesity and insulin resistance conditions 117 .
Obesity is one of the main risk factors associated with lethal COVID-19, and levels of pro-in ammatory cytokines increase under this pathology 118 . Low NAD + levels in obese individuals decrease the activity of SIRT1, a molecule that modulates cytokine production 119 . However, the excess of amino acid availability hyperactivates the mTOR signaling pathway increasing viral replication and in ammatory response 120 . Additionally, because adipose tissue has a considerable level of ACE2 expression, viral shedding increases, as well as the production of pro-in ammatory factors 121 . This in ammatory process contributes to thrombotic problems, a probable cause of multiorgan failure, which has been evidenced by the presence of elevated levels of megakaryocytes in COVID-19 autopsies 122,123 .
Thymic stromal lymphopoietin is an epithelial cytokine normally produced by airway epithelial cells. It has been associated with T-helper type 2 (Th2) responses in allergic diseases, highlighting its role in in ammatory disease pathogenesis. It has been discovered that TSLP can be triggered by respiratory viral infections, bacteria, allergens and injuries 124 . TSLP acts upon cells with TSLP receptor such as hematopoietic progenitor cells, eosinophils, basophils, mast cells, airway smooth muscle cells, group 2 innate lymphoid cells, lymphocytes, dendritic cells and monocytes/macrophages. When several immune mediators were measured in patient's plasma suffering from in uenza A (H1N1) and COVID-19, TSLP levels were signi cantly upregulated in COVID-19 patients. This fact suggests a possible contribution of TSLP in COVID-19 pathogenesis and perhaps aids differential diagnosis 125 . Besides, since TSLP concentration was reported to be higher in severely affected than in mild and moderated COVID-19 cases, it may be potentially used as a biomarker for disease severity 126 .
Optimal NLRP3 in ammasome activation is crucial for host immune defense against several pathogenic infections 127 . SARS-CoV-2 activates in ammasomes, which are large multiprotein assemblies that are broadly responsive to pathogen-associated cellular insults, leading to secretion of cytokines and an in ammatory form of cell death 128 . However, excessive activation can lead to systemic in ammation and tissue damage which are detrimental to the host 129 . Patients with severe COVID-19 have been found to have higher serum concentrations of pro-in ammatory cytokines and chemokines such as granulocyte-colony stimulating factor (GCSF), monocyte chemoattractant protein 1 (MCP1), TNF, IL-6, and IL-1β compared with healthy individuals. A uni ed mechanism for NLRP3 in ammasome activation has not been proposed yet; however, some researchers have found that SARS-CoV-2 ORF-8b interacts with the LRR domain of NLRP3 in ammasome activating IL-1β secretion in THP-1 macrophages 130 .
Findings suggest that SARS-Cov-2 infection leads to NLRP3 in ammasome activation, caspase-1 cleavage, and the release of IL-1β stimulating pyroptosis in peripheral blood mononuclear cells from severe COVID-19 patients 131 .
In a biological system approach, SARS-CoV-2 employs a suite of virulent proteins that interacts with host targets to extensively rewire the ow of information and cause COVID-19 11,132−134 . The human proteins physically associated with SARS-CoV-2 are the rst line of host proteins 10 , which also interact with proteins involved in a wide spectrum of signaling pathways and biological processes within lung cells. In this study, we identi ed 111 pulmonary in ammatory response proteins with the highest con dence interactions to human-SARS-CoV-2 proteins, being the top ten: C3, FN1, NFKB1, RPS19, CTSC, HSPD1, APP, ITGAM, SNAP23, and MAPK14.
Subsequently, we analyzed these 111 in ammatory response proteins to identify those with the shortest pathways to four cancer hallmark phenotypes. In ammation is a hallmark of cancer observed in patients with SARS-CoV-2 infection 135 . The chronic in ammatory process causes cell death 136,137 , angiogenesis 138 , and during the peak of in ammation, immune cells preferentially use glycolysis as a source of energy 139 . These facts provide a biological rationale to analyze and prioritize the in ammatory response proteins with the shortest distance scores to these biological phenotypes. Consequently, we identi ed 34 essential in ammatory response proteins highly associated with cell death, glycolysis, and angiogenesis. Recent studies showed that SARS-CoV-2 rewires human monocytes in a high glucose culture medium.
This induces viral replication and cytokine production, and might be the reason why people suffering from diabetes, obesity and other related metabolic diseases are more susceptible to developing severe COVID-19 139 . For instance, people with type 2 diabetes show an increased glucose metabolism due to hyperglycemia, which may boost SARS-CoV-2 pathogenesis 139 . Codo et al proved that glycolytic ux is essential for SARS-CoV-2 impact 141 . Through several assays, they inhibited glycolysis by blocking 2deoxy-D-glucose (2-DG) and glycolytic enzymes 6-phospho-fructo-2-kinase/fructose-2,6-biphosphatase-3 (PFKFB3) and lactate dehydrogenase A (LDH-A), as a consequence, they observed that both viral replication and cytokine response stopped 141 . The metabolic transcription factor HIF-1α activity and related genes are strongly stimulated in SARS-CoV-2 infected blood monocytes isolated from severe COVID-19 patients 141 . HIF-1α is also a major glycolysis regulator, when inhibited, viral replication and cytokine expression were also blocked. Overall, these experiments showed that high glucose concentration and glycolysis are essential for SARS-CoV-2 replication, in ammatory response, and upregulation of ACE2 141 .
Angiogenesis occurs in response to the activation of acute in ammation or chronic systemic hypoxia pathways that increase the expression of proteins and factors (HIF-1α, VEGF, NO) associated with its development 142 . During the SARS-CoV-2 infection, local endothelial damage, known as endotheliitis, is associated with acute in ammation of the outermost endovascular layers, triggering a cascade of reactions that result in endothelial in ammation, platelet aggregation, and impaired laminar ow 138,143 .
In the context of COVID-19 disease, the reported vasoconstriction and subsequent hypoxia, stimulate the formation of new blood vessels by promoting branching of pre-existing blood vessels (intussusception) and de novo angiogenesis that contributes to the already established systemic hypoxia 144 . This process together with the systemic hypoxia observed in severe COVID-19 patients cause a structural and functional reorganization of the pullmonary tissue, which ultimate function is to allow an adequate gas exchange between the tissue and the cells 142 .
Regarding drugs against COVID-19 disease, in this study we propose ve small molecules (ruxolitinib, baricitinib, pacritinib, losmapimod, and eritoran) that after being thoroughly analyzed in COVID-19 clinical trials, these drugs can be considered for treating severe COVID-19 patients.
A systematic review and meta-analysis published by Walz et al concluded that Janus kinase-inhibitor treatment is signi cantly associated with positive clinical outcomes in terms of mortality, intensive care unit admission, and discharge 145 . Ruxolitinib is a tyrosine-protein kinase JAK1/2 inhibitor 146 that is currently used for myelo brosis and polycythemia vera, both hematologic malignancies. The use of ruxolitinib in these diseases is based on its ability of being a kinase inhibitor, which mediates the  150 . The use of baricitinib is indicated in COVID-19 critically ill patients with high oxygen needs despite the use of dexamethasone (the only approved corticosteroid), however it should not be used when IL-6 inhibitors such as tocilizumab have been started, given that its combined use has not yet been tested as well as its safety. The known e cacy of Baricitinib is from emerging data from an unpublished article where the 27.8% of participants receiving baricitinib vs 30.5% receiving placebo progressed (primary endpoint, odds ratio 0.85, 95% CI 0.67-1.08; p = 0.18), and the all-cause mortality was 8.1% for baricitinib and 13.1% for placebo, corresponding to a 38.2% reduction in mortality (hazard ratio [HR] 0.57, 95% CI 0.41-0.78; nominal p = 0.002) 151 . Pacritinib is also a protein-kinase inhibitor mainly focused on JAK2 and FLT3 protein targets.
This small molecule has been developed for the treatment of myelo brosis 152 . On the other hand, losmapimod is a MAP kinase p38 alpha inhibitor that has been investigated for the prevention of chronic obstructive pulmonary disease and cardiovascular disease 153 . The therapeutic hypothesis for the use of losmapimod in COVID-19 is that increased mortality is caused by p38 MAPK-mediated exaggerated acute in ammatory response resulting in SARS-CoV-2 infection. Lastly, eritoran is a Toll-like receptor 4 / MD-2 antagonist that downregulates the intracellular generation of pro-in ammatory cytokines IL-6 and TNFalpha in human monocytes, and has been developed for the treatment of severe sepsis. Shirey et al examined how antagonizing TLR4 signaling has been effective experimentally in ameliorating acute lung injury and lethal infection in challenge models triggered by acute lung injury-inducing viruses 154 .
Considering the enormous pressure that health systems are facing due to the COVID-19 pandemic and the continuous need to present and implement comprehensive health strategies that can address the global situation; mainly after the emergence of different variants, it is imperative to recognize the urgent need to diminish the gaps between research and the implementation of public health measures. In fact, it may be unprecedented in the history of science to know how many research articles related to  have been submitted and published. However, according to Park et al, the research community has emphasized on "the new norm of publishing: quantity over quality" and this is also related to the well known problems that clinical trials faced even before the pandemic 155 . This is of particular interest to our research given that we acknowledge that clinical trials are essential in evidence-based medicine, and consequently, in the decision making process of public health policies and strategies. Relevantly, the need to smartly invest not only in randomized clinical trials but also in large-scale clinical trials with master protocols and conducted by coordinated and collaborative structures, as also supported by Park et al 155 .
These clinical trials networks are essential to coordinate actions between clinical researchers and health practitioners, also promoting knowledge sharing, leadership, and cost-time reductions. In addition, it is critical to decentralize, improve and increase clinical trials in low and middle-income countries, as current evidence shows large inequalities and concentrations of funds and information in high-income countries 156 . This holds true especially for Latin America, one of the most affected regions in the world by the pandemic 157 .
The role of health research is fundamental in the response to COVID-19, considering the importance of data sharing and assuring e ciency, equity, and effectiveness in the diverse processes. Contradictorily, a large number of clinical trials might never be completed and others are done with doubtful methodologies 155,158 . Thus, analyzing potential drugs targets for COVID-19, especially the ones which can serve for severe cases, need an urgent and e cient development of well designed and managed clinical trials, which can provide potential interventions that help people to live longer, diminish long-term effects, manage pain and/or possible disabilities; not to mention the possible positive effects on the reduction of hospitalization costs, both at the individual level and in terms of possible savings for the national health system. As another study also mentioned, the potential and bene ts of repositioning clinical trials are directed to use the already available information of safe and affordable generic drugs and propose "potential, prompt, cost-effective, and safe solutions for the public and global health problems, with a human-centered approach" 11 . This is also conveyed by the Pan American Health Organization (PAHO), which adds to the bene ts, the idea of having already pharmaceutical formed supply chains 159 .
Finally, as other authors have contributed, the current global research situation must be guided towards a collaborative and synergetic approach instead of being conceived as a competitive and isolated process. The COVID-19 pandemic assures the need to eliminate structural barriers that increase health inequalities, and in this perspective, bene ts, knowledge, and of course potential treatments must be available for all, in order to achieve universal health coverage and equity. Figure 1 Signi cantly expressed genes across lung cell types. Box plots show the number of signi cantly expressed genes, their Z-scores, and p-values per each lung cell type. Neural cells were the cell type with the highest mean Z-score, followed by B cells, mast cells, broblast cells, alveolar type II cells, cycling natural killer / T cells, endothelial cells, macrophages, airway epithelial cells, alveolar type I cells, natural killer cells, dendritic cells, smooth cells, Treg cells, plasma cells, monocytes, other epithelial cells, CD4+ T cells, and CD8+ T cells.  Functional enrichment analysis. UMAPs show the most signi cant genes per lung cell type involved in gene ontology biological processes and signaling pathways. The most signi cant biological term was in ammatory response, followed by cytokine production, innate immune response, macrophage activation, Toll-like receptor signaling pathway, interferon production, JAK-STAT signaling pathway, NF-κB signaling pathway, thymic stromal lymphopoietin, TNF signaling pathway, blood coagulation, oncostatin M signaling pathway, AGE-RAGE signaling pathway, IL-1 and megakaryocytes in obesity, and NLRP3 in ammasome complex. UMAP: uniform manifold approximation and projection for dimension reduction.   Shortest paths to cancer hallmark phenotypes. A) Box plots encompassing in ammatory response proteins with the shortest mean of distance score per phenotype. Cell death was the phenotype with the shortest paths, followed by in ammation, glycolysis, and angiogenesis. B) Venn diagram of in ammatory response proteins with shortest paths to hallmarks of cancer related to COVID-19. C) Ranking of the most essential proteins with shortest paths to cell death, in ammation, glycolysis, and angiogenesis.

Supplementary Files
This is a list of supplementary les associated with this preprint. Click to download. SupplementaryDataset.xlsx