Since the identification 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 find therapeutically actionable targets for complex diseases 69,70. Melms et al have previously published the molecular single-cell lung atlas of lethal COVID-19 51. Motivated by this study, we performed an in-depth in silico analysis comparing the transcriptional data of 719 inflammatory response genes across 19 lung cell types belonging to COVID-19 autopsies. The functional enrichment analysis of the 233 significantly expressed inflammatory genes showed that the most significant biological annotations were inflammatory response (5.9 x 10− 241), cytokine production (9.5 x 10− 62), innate immune response (1.0 x 10− 30), macrophage activation (1.1 x 10− 29), TLR signaling pathway (3.8 x 10− 15), type I and II interferon production (1.8 x 10− 13), JAK-STAT signaling pathway (9.0 x 10− 8), NF-κB signaling pathway (2.0 x 10− 6), thymic stromal lymphopoietin (4.5 x 10− 6), TNF signaling pathway (4.9 x 10− 6), blood coagulation (5.6 x 10− 6), oncostatin M signaling pathway (5.9 x 10− 6), AGE-RAGE signaling pathway (5.9 x 10− 6), IL-1 and megakaryocytes in obesity (6.8 x 10− 6), and the NLRP3 inflammasome complex (2.5 x 10− 4).
The innate immune response is the first line of defense against new invading pathogens 77. Pattern recognition receptors (PRRs) are capable of recognizing molecules with conserved motifs commonly shared by pathogen groups 78. Recognition by these receptors triggers innate immune responses and induces multiple IFN and pro-inflammatory cytokine secretion in COVID-19 patients 79. Upon ligand recognition PRRs initiate a signaling pathway that activate key transcription factors, such as NF-κB, AP-1, and interferon regulatory factors (IRF3 and IRF7) that induce pro-inflammatory cytokines and type I interferon. Type I IFNs are responsible for inducing the JAK-STAT signaling pathway to activate IFN-stimulated genes and develop the “anti-viral state” in the infected organism 80,81.
Interferon is a cytoplasmic glycoprotein with antiviral activity. This cytokine is another contributing factor in the humoral immunological response against respiratory viruses 82,83. Through a bronchoalveolar lavage in severely ill patients, evidence of local induction of interferon and the stimulation of interferon genes was found. In contrast, minimal levels of interferon were found in peripheral blood of severely ill patients 84. This increased cytokine production with limited interferon levels might be due to an antagonist mechanism of the nsp1 protein against interferon signaling 85. Regarding the genetics underlying severe COVID-19, Zhang et al concluded that genetics may determine the clinical course of SARS-CoV-2 infection identifying mutations in genes involved in the regulation of type I and III IFN immunity 86, and Bastard et al identified high titers of neutralizing autoantibodies against type I IFN-α2 and IFN-ω in 10% of patients with severe COVID-19 87.
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-inflammatory 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 hyperinflammatory response, which has been reported as a remarkable feature of SARS-CoV-2 infection 90–92. 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 fibrosis, 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-inflammatory 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 inflammatory 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 inflammatory response that leads to severe lung injury 104. Thus, several clinical trials have shown that baricitinib, a JAK inhibitors possesses a good safety and efficacy profiles 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 hyperinflammation 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 fibrin 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 inflammatory 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 110–112.
Oncostatin M is a cytokine involved in homeostasis and chronic inflammation that has pleiotropic functions such as cell differentiation and proliferation, and it is present in hematopoietic, immunological, and inflammatory 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 fibroblasts 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 inflammatory mediators was detected; this finding 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 specific 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-inflammatory 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 inflammatory response 120. Additionally, because adipose tissue has a considerable level of ACE2 expression, viral shedding increases, as well as the production of pro-inflammatory factors 121. This inflammatory 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 inflammatory 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 influenza A (H1N1) and COVID-19, TSLP levels were significantly 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 inflammasome activation is crucial for host immune defense against several pathogenic infections 127. SARS-CoV-2 activates inflammasomes, which are large multiprotein assemblies that are broadly responsive to pathogen-associated cellular insults, leading to secretion of cytokines and an inflammatory form of cell death 128. However, excessive activation can lead to systemic inflammation 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-inflammatory 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 unified mechanism for NLRP3 inflammasome activation has not been proposed yet; however, some researchers have found that SARS-CoV-2 ORF-8b interacts with the LRR domain of NLRP3 inflammasome activating IL-1β secretion in THP-1 macrophages 130. Findings suggest that SARS-Cov-2 infection leads to NLRP3 inflammasome 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 flow of information and cause COVID-19 11,132−134. The human proteins physically associated with SARS-CoV-2 are the first 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 identified 111 pulmonary inflammatory response proteins with the highest confidence 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 inflammatory response proteins to identify those with the shortest pathways to four cancer hallmark phenotypes. Inflammation is a hallmark of cancer observed in patients with SARS-CoV-2 infection 135. The chronic inflammatory process causes cell death 136,137, angiogenesis 138, and during the peak of inflammation, immune cells preferentially use glycolysis as a source of energy 139. These facts provide a biological rationale to analyze and prioritize the inflammatory response proteins with the shortest distance scores to these biological phenotypes. Consequently, we identified 34 essential inflammatory response proteins highly associated with cell death, glycolysis, and angiogenesis. These proteins were: PTGS2, PRKCZ, NFKBIA, MAPK14, TNFRSF1B, TLR4, ATM, MECOM, PIK3CG, EGFR, JAK2, LYN, CYLD, PRKCQ, STAT3, TGFB1, RBPJ, TNFAIP3, NOTCH1, IGF1, CD28, CCL5, PTAFR, FPR1, EDNRA, EDNRB, CYSLTR1, CNR2, HGF, EPHA2, FN1, CSF1, PTGFR, and APP.
The SARS-CoV-2 infection of lung epithelial cells activates caspase-8 to trigger the three major cell death pathways, including apoptosis, pyroptosis, and necroptosis. Cell death and inflammatory responses are intimately linked during SARS-CoV-2 infection 140. Lastly, analysis of postmortem lung sections of lethal COVID-19 patients has revealed that inflammatory responses from lung epithelial cells may induce infiltration of inflammatory cells that trigger strong immune pathogenesis 137.
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 flux is essential for SARS-CoV-2 impact 141. Through several assays, they inhibited glycolysis by blocking 2-deoxy-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, inflammatory response, and upregulation of ACE2 141.
Angiogenesis occurs in response to the activation of acute inflammation 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 inflammation of the outermost endovascular layers, triggering a cascade of reactions that result in endothelial inflammation, platelet aggregation, and impaired laminar flow 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 five 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 significantly 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 myelofibrosis 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 signaling of a number of cytokines and growth factors that are important in hematopoiesis and immune function. Based on this principle, it is reasonable then, from a clinical point of view, to use this drug to specifically manage cytokine storm in COVID-19 147. According to Yan et al, ruxolitinib normalized interferon signature genes and all complement gene transcripts induced by SARS-CoV-2 in lung epithelial cell lines. They proposed that combination therapy with JAK inhibitors and drugs that normalize NF-κB-signaling could potentially have clinical application for severe COVID-19 146. Baricitinib is a tyrosine-protein kinase JAK1/2 inhibitor 148 mainly used for rheumatoid arthritis, and among its pharmacological properties it has an antiviral effect on the entry of a virus 149. At the moment, baricitinib is approved by the WHO, the Food and Drug Administration (FDA) of the United States, and the National Institutes of Health (NIH) for emergent use in severe pneumonia due to COVID-19 150. 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 efficacy 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 myelofibrosis 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 inflammatory 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-inflammatory cytokines IL-6 and TNF-alpha 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 COVID-19 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 al155. 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 efficiency, 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 efficient 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 benefits 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 benefits, 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, benefits, knowledge, and of course potential treatments must be available for all, in order to achieve universal health coverage and equity.