Storms in COVID-19

Introduction

Coronavirus disease 2019 (COVID-19) is caused by severe acute respiratory syndrome coronavirus 2 (SARSCoV2).¹ The disease was first identified in December 2019 in Wuhan, China. The World Health Organization declared the outbreak a Public Health Emergency of International Concern on 30th January 2020 and a pandemic on 11th March 2020. As of 13th June 2021, 175,333,154 cases and 3,793,230 deaths have been documented around the world. The current evidence shows that high concentration of proinflammatory cytokines is implicated in severely ill patients compared to those who are moderately ill, indicating a poor prognosis in COVID-19.² Besides, excessive infiltration of pro-inflammatory cells, mainly involving macrophages, T-helper 17 cells, have been found in lung tissues of patients with COVID-19 on postmortem pathological examination. Increasing studies indicate that the “cytokine storm” may contribute to the mortality in COVID-19. Recent studies have shown that bradykinin storm also contributes to severe mortality in COVID-19 patients.³

Cytokines

Cytokines are proteins released by the immune system to help fight against infection. Cytokine in Greek means ‘cell signaling’. They are an important part of the inflammatory process.⁴ Cytokines are produced by innate macrophages, dendritic cells, natural killer cells and also the adaptive T and B lymphocytes. Interferons, Chemokines, TNF (tumor necrosis factor), and Interleukins are the cytokines released by immune cells.⁵ Interferons (IFN) are a group of signaling proteins made and released by host cells in response to virusinfection causing nearby cells to increase their anti-viral defenses. IFN-alpha is produced by leukocytes, IFNbeta by fibroblasts, IFN-gamma by lymphocytes on contact with viruses.⁶ Chemokines are a family of small cytokines. Their name is derived from their ability to induce directed chemotaxis in nearby responsive cells. Tumor Necrosis Factor (TNF) is a multifunctional, proinflammatory cytokine that plays an important role in cell survival, proliferation, differentiation, and death. It may also be involved in inflammation-associated carcinogenesis. TNF-alpha and TNF-beta are the two important cytokines in this category. Interleukins (ILs) are a group of cytokine secreted proteins that were first seen to be expressed by leukocytes. ILs can be divided into four major groups based on distinguishing structural features. These groups include the genes encoding the IL-1-like cytokines, the class I helical cytokines (IL4- like, g-chain and IL6/12-like), the class II helical cytokines (IL-10-like and IL-28-like) and the IL-17- like cytokines. Many interleukins are considered to be lymphokines.

a) IL-1: Indirectly stimulates immune responses via various effector proteins and other cytokines.

b) IL- 2: Regulation of T cells growth.

c) IL-3: Stimulates hematopoietic stem cells to produce myeloid progenitor cells. However, an interaction between IL-3 and IL-7 results in the production of lymphoid progenitor cells from hematopoietic stem cells.

d) IL-4: Proliferation of T cells and B cell stimulation to humoral and adaptive immunity. Production of a number of cells including dendritic cells, Th1 cells, as well as Interferon Gamma cells.

e) IL-5: Similar to IL-4 and IL-13. It is involved in stimulating the growth of B-cells, increased secretion of immunoglobin and activation of eosinophils.

f) IL-6: Primary mediator in illness such as fever, activating the expression of Prostaglandin E2 in the hypothalamus, which results in body temperature change.

g) IL-8: Induction of chemotaxis

h) IL-10: Inflammation as well as regulation of the immune responses

i) IL-18: Immune responses in the body (innate and adaptive responses)

j) IL-33: Involved in immune responses of Th28

Origin and spread of COVID-19  

COVID-19 is caused by the SARS-CoV-2 that belongs to the beta-coronavirus subfamily. Corona viruses are enveloped, positive single stranded large RNA viruses. The primary data available about COVID-19 indicates possible animal-to-human transmission via wild animals in seafood market in Wuhan. Epidemiological data and studies, after that, have increasingly demonstrated that the virus transmits human-to-human, through droplets or direct contact.⁹ The virus was confirmed to spread through respiratory droplets from cough or sneeze with the power of the host to shed the infection while asymptomatic. Studies now are also proposing the possible feco-oral transmission of the virus.¹⁰

SARS-CoV-2 is thought to enter the body through Angiotensin converting enzyme 2 (ACE2) receptors present on the surface of cells that line the respiratory tract in the nose and throat. Once in the lungs, the virus appears to move from the alveoli, the air sacs in the lung, into the blood vessels, which are also rich in ACE2 receptors.¹¹

Cytokine storm

Cytokine storm, named after the substances called cytokines rampage through the bloodstream. These small proteins are the immune army’s messengers, transiting between cells with a spread of effects; some raise more immune activity, some raise less.¹²

Normally, when human body encounters a microorganism, the system attacks the invader. Pattern recognition receptors (PRRs) activate the transcription factors - nuclear factor kappa B (NF-kB), activation protein 1, interferon response factors three and seven. These transcription factors induce the expression of genes encoding inflammatory cytokines, chemokines and adhesion molecules. This cytokines and chemokines help in recruitment of leukocytes and plasma proteins to the site of infection, where they perform various effector functions that serve to combat the triggering infection.¹³ Although generally a positive step towards recovery, variants on this hyperactive response occur in an array of conditions, triggered by infection, faulty genes or autoimmune disorders, during which the body thinks its own tissues are invaders, resulting in an unbridled immune response leading to continuous activation and expansion of immune cells. This results in sudden acute increase in massive amounts of cytokines, resulting in a cytokine storm.¹⁴

The cytokine storm clinical findings are allocated to the action of the pro-inflammatory cytokines like IL-1, IL6, IL-18, IFN-γ, and TNF-a. A severity of reaction is due to release of too many cytokines into the blood too quickly.¹⁵ This increase in cytokines results in influx of various immune cells such as macrophages, neutrophils and T cells from the circulation into the site of infection with destructive effects on human tissues resulting in disruption of endothelial cell to cell interactions, damage of vascular barrier, capillary damage, diffuse alveolar damage, multiorgan failure, and ultimately death.¹⁶ Levels of acute-response cytokines - TNF Alpha, IL6, ferritin, C reactive protein (CRP), High Sensitive C reactive protein (hsCRP) and pro-coagulant factors D-Dimer, Prothrombin time (PT) and activated Partial thromboplastin time (aPTT), many of them measurable are elevated.¹⁷

Cytokine storm leads to a critical life-threatening condition requiring intensive care admission and having a quite high mortality in many conditions including viral infections, cancer, sepsis, rheumatic diseases and systemic juvenile idiopathic arthritis.¹⁸

Lung injury is also one of the consequences of the cytokine storm that can progress into acute lung injury or it is a more severe form Acute Respiratory Distress Syndrome (ARDS).17 Cytokine storm has been reported in several viral infections including influenza H5N1 virus, influenza H1N1 virus, and the two corona viruses highly related to COVID-19; severe acute respiratory syndrome coronavirus (SARS-CoV) and Middle East Respiratory Syndrome coronavirus (MERS-CoV). Both pro-inflammatory cytokines (e.g., IL-1, IL-6, and TNF-α) and anti-inflammatory cytokines (e.g., IL-10 and IL-1 receptor antagonist) are increased in the serum of cytokine storm patients. The main contributors to the interplay of the cytokine storm are IL-6 and TNF-α.¹⁹

Cytokine storm in COVID-19

Cellular entry of SARS-CoV-2 depends on the binding of S proteins covering the surface of the virion to the cellular ACE2 receptor and on S protein priming by transmembrane protease, serine 2 (TMPRSS2), a host membrane serine protease. In SARS1, the protein that is required to cleave the virus is likely present only in the lung environment, so that is where it can replicate. According to Lee et al., “SARS-CoV-2 is cleaved by a protein called furin and due to its ubiquitous nature, it helps in systemic spread of the virus.²⁰

After entering respiratory epithelial cells, SARS-CoV-2 provokes an immune response with inflammatory cytokine production accompanied by a weak interferon (IFN) response. The pro-inflammatory immune responses of pathogenic Type 1 T helper cells (Th1) and intermediate Cluster differentiation (CD) 14+, CD16+ monocytes are mediated by membrane-bound immune receptors and downstream signaling pathways. This is followed by the infiltration of macrophages and neutrophils into the lung tissue, which results in a cytokine storm. Particularly, neutrophilic extracellular traps released by neutrophils, may contribute to cytokine release.²¹

SARS-CoV-2 can rapidly activate pathogenic Th1 cells to secrete pro-inflammatory cytokines, such as granulocyte-macrophage colony-stimulating factor (GM-CSF) and interleukin-6 (IL-6). GM-CSF further activates CD14+ and CD16+ inflammatory monocytes to produce large quantities of IL-6, tumor necrosis factor-α (TNF-α), and other cytokines. Membrane-bound immune receptors (e.g., Fragment crystallizable (Fc) and Toll-like receptors) may contribute to an imbalanced inflammatory response, and weak IFN-γ induction may be an important amplifier of cytokine production.²²

The cytokine storm in COVID-19 is characterized by high expression of IL-6 and TNF-α. Hirano et al, proposed a potential mechanism of the cytokine storm caused by the angiotensin 2 (AngII) pathway. SARSCoV-2 activates Nuclear factor Kappa B (NF-κB) via pattern recognition receptors (PPRs). It occupies ACE2 on the cell surface, resulting in a reduction in ACE2 expression, followed by an increase in AngII.²³ In addition to activating NF-κB, the AngII-angiotensin receptor type 1 (AngII-AT1R) axis can also induce TNF-α and the soluble interleukin 6 receptor alpha (sIL6Ra) via disintegrin and metalloprotease 17 (ADAM17). IL-6 binds to sIL-6R through gp130 to form the IL-6- sIL-6R complex, which can activate signal transducer and activator of transcription 3 (STAT 3) in non-immune cells. Both NF-κB and STAT 3 are capable of activating the IL-6 amplifier (IL-6 Amp) to induce various proinflammatory cytokines and chemokines, including vascular endothelial growth factor (VEGF), monocyte chemoattractant protein-1 (MCP-1), IL-8, and IL-6. IL-6 not only binds to sIL-6R to act in cis signaling, but can also bind to membrane-bound IL-6 receptor (mIL-6R) through gp130 to act in trans-signaling. The latter can lead to pleiotropic effects on acquired and innate immune cells, resulting in cytokine storms. Collectively, the impaired acquired immune responses and uncontrolled inflammatory innate responses to SARS-CoV-2 may cause cytokine storm.²⁴

Patients with COVID-19 who required ICU admission have the cytokine storm. The virus-mediated downregulation of ACE2 causes a burst of inflammatory cytokine release through dysregulation of the reninangiotensin-aldosterone system (ACE/angiotensin II/ AT1R axis), attenuation of Mas G protein-coupled receptor (ACE2/MasRaxis), increased activation of [des-Arg9]-bradykinin (ACE2/bradykinin B1R/DABK axis) and activation of the complement system including C5a and C5b-9 components.²⁵

Analysis of cytokine levels in plasma of 41 COVID-19 confirmed cases in China revealed elevated levels of IL-1β, IL-7, IL-8, IL-9, IL-10, Fibroblast growth factor (FGF), G-CSF, GM-CSF, IFN-γ induced protein (IP) 10, monocyte chemoattractant protein 1 (MCP-1), Macrophage Inflammatory Proteins (MIP) 1α, MIP1-ß, Platelet derived growth factor (PDGF), TNF-α, and Vascular endothelial growth factor (VEGF) in both patients admitted to the intensive care unit (ICU) and non-ICU patients compared to healthy adults. All the patients included in the study had pneumonia and 2/3rd of the patients were admitted to ICU.²⁶

Lung injury is one of the consequences / major consequence of the cytokine storm that can progress into acute lung injury or its more severe form ARDS. ARDS leading to low oxygen saturation levels is a major cause of mortality in COVID-19. Although the exact mechanism of ARDS in COVID-19 patients is not fully understood, the excessive production of pro-inflammatory cytokines is considered to be one of the major contributing factors (Figure 1).^27-28

When the cytokines that raise the immune activity become too abundant, the system might not be ready to stop itself. Immune cells spread beyond infected body parts and begin attacking healthy tissues, gobbling up red and white blood cells and damaging the liver. Vascular walls open to immune cells into surrounding tissues, but the vessels get so leaky that the lungs may fill with fluid and vital signs drops. Blood clots throughout the body, further obstructing the blood flow.²⁹ Decreased blood supply leads to shock and permanent organ damage or death. The phenomenon has been implicated in critically ill patients infected with SARS-CoV-2, the novel coronavirus implicated in COVID-19. An impairment in blood circulation and in 40% of deaths from COVID-19 are related to cardiovascular complications, and the disease starts to look like a vascular infection instead of a purely respiratory one.³⁰

Higher levels of the cytokines, Interleukin 2 receptor and IL-6 were found in more severe COVID-19 infections. Molecular indicators for a cytokine storm includes IL6, CRP and ferritin which are increased and required in intensive care of patients with severe cases and lung symptoms. IL-6 was an early indicator of a cytokine storm-like condition, blood-clotting rates seem to go beyond those often seen in other storm conditions. In COVID-19, doctors may observe immune cells attacking the lungs so early and so harshly, that a sort of scar tissue called fibrosis forms.³¹

AngII- Angiotensin II, ACE2- Angiotensin Converting Enzyme -2, TNF- Tumor Necrosis Factor, S1, S2 – Spike 1,2, PPRs- pattern recognition receptors, AT1RAngiotensin II type 1 receptor, TMPRSS2- transmembrane protease serine 2, ADAM17- Adisintegrin and Ametalloprotease, domain, IL6 AMP -IL6 Amplifier, STAT3- signal transducer and activator of transcription.

Bradykinin storms in COVID-19

Bradykinin is a biologically active, short – lived peptide and is a vasoactive substance. Bradykinin is formed by the interaction of factor XII, prekallikrein, and highmolecular-weight kininogen on negatively charged inorganic surfaces (silicates, urate, and pyrophosphate) or macromolecular organic surfaces (heparin, other mucopolysaccharides, and sulfatides) or on assembly along the surface of cells.³³ Catalysis along the cell surface requires zinc dependent binding of factor XII and high-molecular-weight kininogen to proteins, such as the receptor for the globular heads of the C1q subcomponent of complement, cytokeratin 1, and urokinase plasminogen activator receptor. These three proteins complex together within the cell membrane and initiation depends on autoactivation of factor XII on binding to gC1qR (the receptor for the globular heads of the C1q subcomponent of complement). There is also a factor XII–independent bypass mechanism requiring a cell-derived cofactor or protease that activates prekallikrein. Kallikreins are produced by the inactive precursor prekallikreins after activation in plasma mediated by Factor XII (Hageman factor), which is an enzyme part of the clotting mechanism.³⁴

Kallidin is a product of the enzymatic action of kallikrein to kininogens. Kallidin then is transformed into bradykinin after enzymatic action of plasma aminopeptidase. Separate studies measuring bradykinin and kallidin peptides demonstrated the differential regulation of the plasma and tissue kallikrein – kinin systems, providing different insights on understanding this complex system.³⁵

Bradykinin is degraded by carboxypeptidase N and angiotensin-converting enzyme. The renin–angiotensin system (RAS) controls many aspects of the circulatory system, including the body’s levels of a chemical called bradykinin, which normally helps to regulate blood pressure. Bradykinin storm could be a potent part of the vasoconstrictor system that induces cardiovascular disease and dilatation and is degraded by ACE and increased by the hypertension created by ACE2.

Angioedema that is bradykinin dependent results from hereditary or acquired C1 inhibitor deficiencies or use of angiotensin-converting enzyme inhibitors to treat hypertension, heart failure, diabetes, or scleroderma.36 Kinins are low-molecular-weight peptides that participate in inflammatory processes by virtue of their ability to activate endothelial cells and as a consequence, leads to vasodilatation, increased vascular permeability, production of nitric oxide and mobilization of arachidonic acid. Kinins also stimulate sensory nerve endings to cause a burning dysaesthesia. Thus, the classical parameters of inflammation (i.e., redness, heat, swelling, and pain) can all result from kinin formation.³⁷

The role for bradykinin in allergic rhinitis, asthma, and anaphylaxis is to contribute to tissue hyper responsiveness, local inflammation and hypotension. Activation of the plasma cascade occurs as a result of heparin release and endothelial-cell activation and as a secondary event caused by other pathways of inflammation.

Comparison with bronchoalveolar lavage fluid (BALF) between COVID-19 and controls showed a crucial imbalance in RAS, pictured by slashed expression of ACE together with increase in ACE2, rennin, hypertension, key RAS receptors, kininogen and lot of kallikrein enzymes that activate it and each bradykinin receptors in COVID-19 patients.³⁸

According to the team’s analysis, when the virus tweaks the RAS, it causes the body’s mechanisms for regulating bradykinin to go haywire. Bradykinin receptors are resensitized, and the body also stops effectively breaking down bradykinin. ACE normally degrades bradykinin, but when the virus downregulates it, it cannot do this as effectively. The end result, the researchers say, is to release a bradykinin storm - a massive, runaway buildup of bradykinin in the body. According to the bradykinin hypothesis, it is this storm that is ultimately responsible for many of COVID-19’s deadly effects.³⁹

These bradykinin-driven consequences explain several of the symptoms being discovered in COVID-19. Bradykinin B1 and B2 receptors are constitutively expressed within the airways on many domestic and/or immune cells.

Bradykinin, especially at high doses cause the bloodbrain barrier to break down which could allow harmful cells and compounds into the brain, leading to inflammation, potential brain damage, and many of the neurological symptoms COVID-19 patients experience.

It is a reasonable hypothesis that many of the neurological symptoms in COVID-19 could be due to an excess of bradykinin. In addition, similar neurological symptoms have been observed in other diseases that result from an excess of bradykinin. Increased bradykinin levels could also account for other common COVID-19 symptoms.⁴⁰

As shown in the figure 2, after the activation of RAS, angiotensinogen is converted to angiotensin I and further to angiotensin 1-9 by ACE II, as conversion of angiotensin I to angiotensin II by ACE I is inhibited by COVID -19 (as there is inhibition of ACE I by COVID-19) leading to accumulation of angiotensin I and angiotensin 1-9 which brings about vasodilatation and stimulatory effect on bradykinin receptors, which also brings about vasodilation. Once bradykinin is formed by the activation of bradykinin pathway, bradykinin acts on bradykinin receptors and brings about vasodilatation. This is further augmented by inhibition of bradykinin degradation by COVID-19.⁴¹

RAS – Renin angiotensin system, COVID–19 – corona virus disease 19, ACE – angiotensin converting enzyme, LMWK – low molecular weight kininogen.⁴²

Conclusion

Renin angiotensin system and bradykinin may act together in causing damage to the biological system. Bradykinin can explain few symptoms of COVID-19, which was unexplainable by cytokine storm. This helps the researcher to further narrow the drugs which are currently available (tocilizumab, anakinra, and baricitinib) for the treatment, that can bring down the bradykinin storm and cytokine storm, and immediately used for patient care without any delay. The effectiveness of these findings and theory will only be evident after the patient care providers starts using the drugs to prevent bradykinin storm and document the improvement.

Conflict of Interest

None.

Financial Support

Nil.

Authorship credit

• Dr Wilma Delphine Silvia CR – Conception, design & final approval of the version to be published.

• Mr Venkatesha - Acquisition of review articles, data & drafting the article

• Dr Girish KS - Drafting the article & revising it critically for important intellectual content