Cytokine Storm: The Primary Determinant for the Pathophysiological Evolution of COVID-19 Deterioration

1SARS-CoV-2 is the pathogen responsible for the COVID-19 global pandemic. As it is a novel and strongly infectious coronavirus, most people lack immunity to it. Studies have shown that the cytokine storm is closely associated with disease exacerbation and even death in COVID-19 patients. When the virus invades the body, it can cause an imbalance in the immune system that may result in a cytokine storm. COVID-19 patients deteriorate over a short period, leading to ARDS and coagulation disorders, and eventually also multiple organ failure. Understanding the pathophysiological processes involved in COVID-19 progression due to cytokine storms is extremely important, as each key link is expected to be a potential target for immune intervention against COVID-19 and will also help in the development of therapeutic drugs to prevent disease deterioration.

2COVID-19 patients can develop a fever and dry cough, and some may also experience dyspnea, muscle and/or joint pain, headache, dizziness, diarrhea, nausea, and hemoptysis. In severe cases of COVID-19, organs can be damaged to varying degrees and even result in MODS. COVID-19 can be complicated by ARDS, acute heart injury, acute kidney injury, septic shock, liver injury, and pancreatic tissue injury, among others. The effect of COVID-19 on the cardiovascular system is more severe in patients with elevated levels of inflammatory factors such as interleukin (IL)-6. In particular, a burst of inflammatory factors can result in diffuse microvascular thrombosis, leading to myocarditis, heart failure, arrhythmia, acute coronary syndrome, rapid deterioration, and even sudden death.

3The lungs are the organ most commonly affected in COVID-19 patients and display pathological features very similar to those of SARS or Middle East respiratory syndrome (MERS) coronavirus infection. Under macroscopic observation, patients with severe COVID-19 exhibit a large number of sticky secretions emanating from the alveoli, as well as some fibrous cords. White foamy mucus can be seen in the airway cavity, and sticky gelatinous mucus is observed in the bronchial cavity. Pathological examination shows significant desquamation of lung cells and alveolar hyaline membrane formation, suggestive of ARDS. In patients who die from COVID-19-related or flu-related respiratory failure, the histological pattern of the lungs manifests as a diffuse alveolar injury with perivascular T-cell infiltration. Additionally, the incidence of microthrombus and the number of new blood vessels are both higher in COVID-19 patients than in influenza patients. On admission, the typical chest CT findings of patients with severe COVID-19 are bilateral, multiple, lobular, and subsegmental consolidation, and even whole-lung consolidation with “white lung” appearance. Laboratory tests show that the number of lymphocytes is significantly reduced in COVID-19 patients; in particular, the levels of CD4+ T and CD8+ T cells in patients with severe disease show a significant and progressive decline. Concurrently, the plasma levels of IL-1β, Interleukin-1 receptor antagonist (IL1RA), IL-2, IL-7/8/9/10, basic fibroblast growth factor (BFGF), granulocyte colony-stimulating factor (GCSF), granulocyte-macrophage colony-stimulating factor (GM-CSF), interferon gamma (IFN-γ), interferon gamma-induced protein 10 (IP-10), monocyte chemotactic protein 1 (MCP-1), macrophage inflammatory protein 1-alpha (MIP-1-α), macrophage inflammatory protein 1-beta (MIP-1-β), platelet-derived growth factor (PDGF), tumor necrosis factor (TNF), and vascular endothelial growth factor (VEGF) are significantly increased in patients with severe COVID-19, among which IL-6 was significantly increased. The main cell sources, target cells, and primary immunobiological effects of the above-mentioned cytokines are shown in Table 1.

4The above-mentioned clinical characteristics of COVID-19 patients suggest that the accumulation and exudation of inflammatory substances caused by the cytokine storm destroy tissues and organs throughout the body, leading to multi-organ failure and acute respiratory distress, which is an important cause of death in patients with severe and critical cases of COVID-19. The determinants of the severity of COVID-19 disease deterioration are closely related mainly to the cytokine storm and a decrease in lymphocytosis, which are key early warning factors for the transition to severe disease.

5Adaptive immunity is suppressed in severe COVID-19 patients, leading to delayed clearance of the virus and hyperactivation of the innate immune response. However, the innate immune response makes it difficult to fight against the virus. The lack of negative feedback, as well as positive feedback aggravation, promotes the overproduction of various inflammatory factors, leading to an increase in the number of active immune cells at the sites of inflammation and, consequently, a cytokine storm.

6Angiotensin-converting enzyme 2 (ACE2) is a key receptor for coronavirus invasion. Studies have shown that the binding of SARS-CoV-2 to host cell membrane receptors and subsequent fusion with the cell membrane are mainly mediated by the spike (S) protein of the virus, which is closely associated with its pathogenicity. Furthermore, transmembrane serine protease 2 (TMPRSS2) is required for the virus to enter host cells, while ACE2 is considered to be the limiting factor for the initial SARS-CoV-2 infection. ACE2 and TMPRSS2 are reported to be co-expressed mainly in type II alveolar epithelial cells, nasal epithelial cells, cornea, esophagus, ileum, colon, gallbladder, and common duct. However, because most COVID-19 patients display lung manifestations, while relatively fewer present with abdominal pain, abdominal distention, diarrhea, and other digestive system symptoms, this suggests that the main target organ of SARS-CoV-2 is likely to be the lung.

7SARS-CoV-2 infects alveolar epithelial cells by recognizing the ACE2 receptor. Following cell invasion, the virus replicates in large quantities, which activates immune cells and the release of a large number of inflammatory cytokines, leading to a cytokine storm ( Figure 1 ). The internalization and exfoliation of ACE2 reduced the expression of ACE2 on the cell membrane but did not affect the level of ACE, resulting in an imbalance of ACE2 and ACE in the lung. ACE2 has a lung-protective effect. Reduced levels of ACE2 weaken its lung-protective effect, which may result in alveolar epithelial cell necrosis and aggravation of lung inflammation. Increased ACE expression upregulates the level of angiotensin II (Ang II) and further hyperactivates the pulmonary angiotensin II type-1 receptor (AT1R), thereby increasing pulmonary capillary permeability and leading to pulmonary edema and aggravation of lung damage. Ang II is able to bind to the AT1R to stimulate the Janus Kinase (JAK)/Signal transducers and activators of transcription (STAT) pathway and facilitates the production of downstream IL-6, which in turn triggers the JAK/STAT pathway through positive feedback to release more cytokines such as IL-6 and IFN. Ang II can also combine with nuclear factor kappa-B (NF-κB) and promote the transcription and production of inflammatory cytokines such as IFN-γ, IL-6, GM-CSF, TNF-β, and TNF-α. The plasma level of Ang II was reported to be significantly increased in SARS-CoV-2-infected patients and shows a linear correlation with viral load and lung injury. Additionally, Ang-(1–7) which is produced by ACE2 degradation of Ang II decreases, and the anti-inflammatory effect of the PIP3/Akt and ERK signaling pathway regulated by the MAS receptor specifically binding with Ang-(1–7) should be weakened. This promotes the cytokine storm, and accelerates the deterioration of patients to ARDS and multiple organ dysfunction, eventually leading to the death of patients.

8Damage-associated molecular patterns (DAMPs) produced by virus-infected epithelial cells and pathogen-associated molecular patterns (PAMPs) of the virus itself can combine with pattern recognition receptors (PRRs) expressed in pulmonary epithelial cells, macrophages, and dendritic cells, and then activate NF-κB and several mitogen-activated protein kinases (MAPKs) such as ERK1/2, p38, JNK, ERK5, triggering a series of cascades and release of chemokines. Normally, NF-κB inhibited protein (IκBS) is transported in the cytoplasm. SARS-CoV-2 promotes nuclear translocation of NF-κB by inducing phosphorylation, ubiquitination, and degradation of IκB through proteasomes. In the nucleus, NF-κB induces the transcription of several genes, which encode TNF-α, IL-6, and other cytokines and enhance the occurrence of cytokine storm. MCP-1, MIP-1-α, MIP-1-β, IP-10 chemotactic lymphocytes, macrophages, neutrophils, natural killer (NK) cells, basophils, and other immune cells gather at the site of injury; the interleukins IL-1α, IL-1β, IL-2, IL-8, and IL-10, among others, activate and strengthen lymphocytes, macrophages, neutrophils, NK cells, basophils, and other immune cells; IFN-γ and TNF enhance cytotoxicity; the release of the colony-stimulating factors GCSF and GM-CSF stimulates granulocyte, monocyte, and macrophage maturation and release into the blood; growth factors such as PDGF, VEGF, and basic FGF stimulate the proliferation of endothelial cells, smooth muscle cells, and fibroblasts. The activated immune cells generate new cytokines, recruit and activate more inflammatory cells, and release more cytokines, and this cascade amplification will finally lead to a cytokine storm. IL-6 is a key inducer of the cytokine storm, a key factor in disease deterioration. Cell and animal experiments, as well as results from human patients, have all shown that interferon (IFN) expression is reduced following SARS-CoV-2 infection. The levels of type I and III interferons are exceedingly low, and interferon expression is only activated at a high multiplicity of infection. IFN and other downstream molecules (including proinflammatory cytokines) controlled by IFN have a variety of functions such as direct inhibition of viral replication and recruitment and activation of various immune cells and are the first line of defense against viral infection. In contrast, this virus can induce elevated expression levels of IL-6 and IL-1RA, as well as other proinflammatory factors. This indicates that the host innate antiviral immunity is dysregulated and is likely to lead to a cytokine storm outbreak.

9Activated CD4+ T cells proliferate and differentiate into T-helper 1 (Th1) cells and produce GM-CSF, IL-6, and other factors. GM-CSF further activates CD14+ and CD16+ inflammatory monocytes and activates and recruits macrophages, which produce more IL-6 and other inflammatory factors, thereby promoting cytokine storm development.

10The first autopsy performed on a COVID-19 patient showed that a large number of monocytes (possibly T cells) had accumulated in the lung, and that the activity of T cells in the peripheral blood was low. Furthermore, the lymphocyte count and peripheral blood T-cell level have been reported to be low in COVID-19 patients. These findings indicate that T cells are attracted from the blood to the infected site to control the viral infection.

11The numbers of CD4+ and CD8+ T cells in peripheral blood significantly reduce in severe COVID-19. T cells may be infected by SARS-CoV-2, which is similar to the occurrence in Middle East respiratory syndrome (MERS) cases ( Figure 2 ). The MERS coronavirus can directly infect human primary T lymphocytes and induce T-cell apoptosis through extrinsic and intrinsic apoptotic pathways, but it cannot replicate in T lymphocytes. The numbers of Th17 CD4+ T cells, which play a proinflammatory role, were increased and CD8+ T cells were highly cytotoxic. This suggests that a decrease in the levels of CD4+ and CD8+ T cells leads to dysregulated innate and adaptive immune responses. The impaired immune response delays virus clearance, which stimulates non-specific immune cells, and leads to the release of more proinflammatory factors. The high concentrations of TNF, IL-6, and IL-10 during the cytokine storm exert a negative regulatory effect on T-cell survival and proliferation. The number of T cells is negatively correlated with the concentrations of IL-6, IL-10, and TNF. Furthermore, the expression of PD-1 and Tim-3 in T cells is upregulated from initial infection to disease onset, further indicating T-cell failure and leading to a vicious cycle. B cell immune responses and follicular helper T-cell responses occur simultaneously in COVID-19 patients. In addition to producing antibodies, activated B cells also secrete IL-1, IL-6, IL-8, TNF, lymphotoxin-alpha (LT-α), G-CSF, GM-CSF, M-CSF, IL-7, and other cytokines, which can aggravate the cytokine storm. The number of B cells in patients with severe disease is significantly reduced, meaning that not enough antibodies will be produced. Consequently, viral replication will not be inhibited, and virus infection will spread throughout the body, which will lead to a further imbalance in the immune response and the release of more proinflammatory cytokines by non-specific immune cells. Furthermore, IFNs induce cells to express a variety of proteins. The genes that express these proteins, such as ACE2, are called interferon-stimulating genes (ISGs). SARS-CoV-2 can exploit species-specific interferon-driven upregulation of ACE2. The increased expression of ACE2 increases the number of receptors available to the virus for host invasion, which inhibits the lung-protective role of ACE2. Increased IFN levels during the cytokine storm have been proposed to lead to the deterioration of COVID-19 patients, further aggravating the cytokine storm.

12The cytokine storm is an important factor in the deterioration of some COVID-19 patients, and leads to abnormalities such as ARDS, MODS, and coagulation defects ( Figure 3 ).

13COVID-19-related ARDS is closely related to the cytokine storm. Respiratory failure due to ARDS is the leading cause of death from COVID-19. A large number of immune cells resulting from the cytokine storm aggregate and are activated in the lungs. These cells then release oxygen free radicals, proteases, and inflammatory factors through a “respiratory burst,” which damages target cells. In particular, the combined effect of TNF-α and IFN-γ that sensitize the cells to undergo PANoptosis, inflammatory cell death involving components of pyroptosis, apoptosis, and necroptosis plays a key role in lung tissue injury. As a result, lung capillary endothelial cells and lung epithelial cells are damaged, lung microvascular permeability and microthrombus formation are increased, and a large amount of protein and fibrin-rich fluids are exuded into the lung stroma and alveoli, forming non-cardiac pulmonary edemas and opacities. Lung compliance decreases, alveolar inactivity increases, and persistent hypoxia is present, which leads to decreased Na-K-ATPase activity in alveolar epithelial cells, cellular metabolism disorders, lymphatic decompensation, and further exacerbates fluid retention and hypoxia. Concurrently, oxygen-sensitive proline hydroxylases are activated, and NF-κB is released, further exacerbating inflammatory responses. Inflammatory mediators can also damage the pulmonary capillary endothelium and alveolar epithelium, leading to the contraction of vascular endothelial cells, cell connection rupture, and significantly increased vascular permeability. Capillary leak results from inflammation driven by key inflammatory cytokines such as TNF, IL-1, IL-6, IL-8, and especially VEGF, which in the past was also called “vascular permeability factor.” Capillary leak is a major component of deteriorating lung function in COVID-19, resulting in ARDS. Several proinflammatory cytokines (IL-6, L-1, and CSF), chemokines (CCL2, CCL-5, IP-10, and CCL3), and reactive oxygen species have been identified as causing ARDS.

14Besides severe lung damage, cytokine storms can also cause cardiovascular symptoms, hematologic symptoms, acute kidney injury, and multiple organ failure, and can even be life-threatening. As mentioned above, SARS-CoV-2 mainly infects cells via ACE2. The virus may first infect type II alveolar epithelial cells through ACE2, leading to air–blood barrier damage. The virus can then enter the blood and infect the lungs again through ACE2 that is expressed on the endothelial cells of pulmonary capillaries. On the one hand, this leads to an increased viral load in the lungs; on the other hand, the virus binds to ACE2 on the endothelial cells, which enables the endothelial cells to be pathogen-labeled, and become recognized as targets for attack by the host immune system. Additionally, as the virus circulates to all parts of the body, it can infect other organs as well that express ACE2, such as the heart, kidney, and liver, further triggering an impaired immune response, such as an imbalance of Th1 and Th2 cells. This results in a cascade amplification, whereby a great number of inflammatory factors are produced, eventually leading to MODS.

15Abnormal coagulation may be the main reason for organ failure and death in patients with severe COVID-19, and a sign of organ damage in sepsis, which is mainly caused by the cytokine storm. Microthrombi have been reported in the heart, liver, lungs, lower limbs, hands, brain, and kidneys of COVID-19 patients. Microthrombi occur because tissue factor (TF) is expressed both in monocytes and in vascular endothelial cells through the activity of cytokines (mainly IL-6), which is thought to promote the conversion of prothrombin to thrombin, which then converts circulating fibrinogen to fibrin and the formation of fibrin-based blood clots. Neutrophils are absorbed by activated endothelial cells and release neutrophil extracellular traps (NETs), which activate the contact activation pathway of coagulation, as well as platelets, thereby amplifying blood clot formation. During the cytokine storm, major natural anticoagulant effectors, such as antithrombin or TF pathway inhibitors, are always inhibited, further promoting coagulation and leading to an irreversible situation. Cytokines IL-1, IL-6, TNF, signal transducer and transcriptional activator 3 (STAT3), NF-κB, and lipopolysaccharides were the highest regulators of thrombotic markers in COVID-19 patients with severe-to-critical disease. In the absence of vascular injury, the initiation of coagulation is completely dependent on activated endothelial cells, which express TF, for the recruitment of proinflammatory monocytes. The cytokine storm leads to abnormal coagulation in the body, and microthrombi formation in the organs further leads to MODS.

16Several treatments are available to prevent and suppress cytokine storm development in COVID-19 patients, such as glucocorticoids, immunomodulators, cytokines antagonists, cytokine receptor antagonists, and others ( Table 2 ).

17Glucocorticoids, which have a good anti-inflammatory effect, may play a vital role in the treatment of COVID-19. Glucocorticoids with a genomic mechanism inhibit the synthesis of proinflammatory cytokines such as IL-1, IL-2, IL-6, IL-8, TNF, IFN-γ, COX-2, VEGF, and prostaglandins. Prednisolone, for example, inhibits the production of TNF, IFN-γ, IL-1β, IL-6, IL-17, IL-10, and TGF-β. Dexamethasone significantly reduces the level of IL-6. Corticosteroids are known to suppress inflammation by non-genomic mechanisms such as inbinding to the membrane-associated glucocorticoid receptors of T cells resulting in perturbation of receptor signaling and immune response and interacting with the exchange of calcium-sodium across the cell membrane, resulting in a quick downturn in inflammation. Although studies have revealed that glucocorticoids do not improve the survival of patients with severe COVID-19, dexamethasone was the first drug shown to reduce mortality in COVID-19 patients. Recent studies have shown that elderly patients with severe COVID-19 who received systemic corticosteroid therapy have lower in-hospital mortality than patients who did not receive corticosteroid therapy. The study also demonstrated that it is a safe treatment with few serious adverse events. This indicates that glucocorticoids may be used in combination with other intensive treatments that may have significant effects.

18Focusing on responses to the cytokine storm in the treatment of COVID-19, trials are currently underway for several cytokine antagonists and cytokine receptor antagonists, including IFN, IL-6, IL-6 receptor, IL-1 receptor, IL-1, GM-CSF, and GM-CSF receptor antagonists. A new adjuvant therapy, CytoSorb, reduces the circulating levels of cytokines, DAMPs, and PAMPs, and improves immunopathology by absorbing a wide range of these factors.

19Immunomodulators such as thalidomide that were reported to be effective in treating one COVID-19 patient can inhibit TNF, IL-1, IL-6, and other cytokines. However, this was an isolated case, and its efficacy requires further verification. Another immunosuppressant—hydroxychloroquine or chloroquine—has shown apparent efficacy in treatment of COVID-19-associated pneumonia in clinical studies, but its efficacy remains controversial. A combination of azithromycin and hydroxychloroquine has been reported to yield better results, especially in the early stages of the disease. Studies have shown that statin use is associated with a significant reduction in mortality among hospitalized COVID-19 patients. Statins can be effective in treating COVID-19 patients as they inhibit the MYD88/NF-κB proinflammatory pathway and promote the upregulation of ACE2.

20Based on the pathophysiology of SARS-CoV-2 and the life cycle of the virus, several antiviral drugs will serve as effective anti-COVID-19 targets. Drugs such as remdesivir, favipiravir, and lopinavir/ritonavir have been shown to be effective. However, at present, most of these therapeutic drugs are still under clinical trials, and their efficacy against SARS-CoV-2 has not been determined holistically.

21Additional treatments that inhibit inflammatory responses, such as TNF blockers, ulinastatin, sphingosine-1-phosphate receptor agonists, Toll-like receptor 4 (TLR4) antagonists, stem cell therapy (especially mesenchymal stem cells), exogenous surfactants, intravenous immunoglobulin (IVIG), Nafamostat mesylate, convalescent plasma therapy, and blood purification therapy, also remain to be further explored and have the potential to effectively treat patients with COVID-19. Traditional Chinese medicine has also elicited positive effects in the treatment of COVID-19. For instance, the Lianhua–Qingwen formula can alleviate symptoms of COVID-19 by activating key molecules such as antiviral and anti-inflammatory synergids.

22Vaccines are the most reliable and cost-effective approach to avoid and manage infectious diseases. They are designed to build an appropriate and effective immune response without creating any imbalance. There are a number of vaccines in the research phase, including inactivated, live attenuated, viral vector-based, subunit, DNA, and RNA vaccines. In phase I clinical trials of the recombinant adenovirus type-5 vectored COVID-19 vaccine, all 108 volunteers showed significant cellular immune responses. mRNA vaccines have rapidly advanced into clinical trials, but most candidate DNA vaccines are currently in the preclinical stage. Moreover, the mRNA-1273 vaccine induced an immune response against SARS-COV-2 in all participants and was relatively safe. Now, the mRNA-1273 vaccine is currently in the phase 3 clinical trial. Another mRNA vaccine, the BNT162 (BioNTech, Mainz, Germany), is in advanced clinical trials and has four variants, namely, a1, b1, b2, and c2. Currently, mutations in the virus pose a challenge in vaccine development and production. In a phase II study in South Africa, AstraZeneca/Oxford adenovirus vector vaccine (Chadox1) was only 10.4% effective against mild-to-moderate infection caused by B.1.351. The development and application of vaccines are still met with many challenges, and future challenges are also anticipated.

23The pathogenesis of COVID-19 involves multiple cytokine pathways. Among them, the cytokine storm caused by SARS-CoV-2 is the key reason for deterioration within a short time-span in COVID-19 patients and subsequent development into acute respiratory distress syndrome (ARDS), leading to respiratory failure and eventual death from multiple organ failure. Increasing evidence suggests that cytokines such as IL-6, IL-1β receptor, IFN-γ, and TNF-α play a key role in the pathogenesis of COVID-19. Therefore, it is of great significance to accurately identify COVID-19 inflammatory pathways and therapeutic targets. Although many current therapies are based on inhibiting the occurrence and development of cytokine storms, the efficacy of the current therapies is still unsatisfactory, and more research is needed to unlock more key inflammatory pathways triggered by SARS-CoV-2.

24PZ and YG conceived and designed the study. RC and ZL performed the literature search and drafted the manuscript. All authors contributed to the article and approved the submitted version.

25Guangdong Medical Science and Technology Research Foundation (A2020162, C2019119, B2021182), Higher Education Teaching Reform Project of Southern Medical University (JG2019126, 2018JG43) Scientific Research Project of Chaozhou Health Bureau (2019110).

26The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.