key: cord-0801808-iojt8oxg
authors: Erol, Adnan
title: Importance of Efferocytosis in COVID-19 Mortality
date: 2022-03-10
journal: Infect Drug Resist
DOI: 10.2147/idr.s348639
sha: 950cc266bbc50c88eddfefe8551959a611bc2ac4
doc_id: 801808
cord_uid: iojt8oxg

COVID-19 is a generally benign coronavirus disease that can spread rapidly, except for those with a group of risk factors. Since the pathogenesis responsible for the severity of the disease has not been clearly revealed, effective treatment alternatives has not been developed. The hallmark of the SARS-CoV-2-infected cells is apoptosis. Apoptotic cells are cleared through a sterile process defined as efferocytosis by professional and nonprofessional phagocytic cells. The disease would be rapidly brought under control in the organism that can achieve effective efferocytosis, which is also a kind of innate immune response. In the risk group, the efferocytic process is defective. With the addition of the apoptotic cell load associated with SARS-COV-2 infection, failure to achieve efferocytosis of dying cells can initiate secondary necrosis, which is a highly destructive process. Uncontrolled inflammation and coagulation abnormalities caused by secondary necrosis reason in various organ failures, lung in particular, which are responsible for the poor prognosis. Following the short and simplified information, this opinion paper aims to present possible treatment options that can control the severity of COVID-19 by detailing the mechanisms that can cause defective efferocytosis.

pathogenesis of SARS-CoV-2, suggesting that the furin cleavage site has a critical role in the transmission of SARS-CoV-2 infection. 9 SARS-CoV-2 genome contains 29 open reading frames (ORFs), which encode 29 proteins. Among these, the ORF3a protein is an accessory protein specific to SARS-CoVs. ORF3a is involved in critical steps of the viral infection cycle and determines viral replication and virulence of SARS-CoV-2. 10 In cells infected with SARS-CoV-2, the virus enforces the apoptotic process through ORF3a. 11 ORF3a-mediated caspase-3 activation initiates both the apoptotic process and a series of changes for the rapid elimination of the apoptotic cell by the phagocytic system: 1. Phospholipase A2 activation increases the level of lysophosphatidylcholine on the outer membrane surface. 12 2. The opening of membrane Pannexin channels causes an increase in extracellular ATP, UTP, and spermidine levels. 12 3. An increase in oxidized PS and phosphatidylcholine levels occurs. 13 All of these three pathways act as "find me" signals for the phagocytic cells. 4. Irreversible activation of Xkr8, a membrane scramblase, causes PS to be expressed in the membrane outer leaflet, which is the essential "eat me" signal for cells of the phagocytic system. 14, 15 In a healthy organism, professional and non-professional phagocytic cells detect "find me" and "eat me" signals and rapidly clear apoptotic cells without damaging the environment through a process known as efferocytosis. [16] [17] [18] While macrophages, neutrophils and dendritic cells originating from the bloodstream are professional; local epithelial, endothelial, and fibrotic cells are the elements of the non-professional phagocytic system. 19 A large number of different PSRs that recognize PS directly and indirectly in phagocytic cells facilitate the functioning of the efferocytosis process. 5, 6, 14 While TIM receptors (T cell immunoglobulin and mucin domain-containing molecules) provide direct binding, TAM (Tyro3, Axl, and MerTK) receptors indirectly bind to apoptotic cells via mediator molecules, such as Gas6 and protein S (ProS). 5, 18 Among several other PSRs, the importance of the phagocytic cell scavenger receptor CD36 in engulfing the apoptotic cell should be particularly emphasized. 20 Cells exposed to apoptotic environments undergo conformational and biochemical changes in their membranes. The hydrolysis of phosphatidylcholine to lysophosphatidylcholine renders the phosphatidylcholine group accessible for the binding of C-reactive protein (CRP). CRP irreversibly marks apoptotic cells for the activation of the classical complement pathway, ultimately those opsonized cells are disposed of by phagocytosis. 21, 22 Apoptotic cells tagged with CRP may also interact with the scavenger CD36 receptors, which are expressed by the macrophages and thus may facilitate efferocytosis. 23 In other words, CRP can promote efferocytosis by binding to the apoptotic cell surface, leading to recognition and phagocytosis by macrophages. 24 Consistently, CRP apheresis has been reported to be an effective treatment option for patients severely challenged by SARS-CoV-2 infection, most likely by regulating efferocytosis. 25, 26 Efficient phagocytosis of apoptotic cells, efferocytosis, is one of the most essential components of maintaining tissue homeostasis. After engulfment of the apoptotic cell, phagocytic cells actively control inflammation ("tolerate me") by releasing anti-inflammatory cytokines such as transforming growth factor-beta (TGF-β) and IL-10. 14 This specialized phagocytosis of apoptotic cells also accelerates the presentation of viral antigens to lymphocytes and T cells, enhancing the activation of antigen-specific B and T cells. 27, 28 If the efferocytosis-mediated clearance process is disrupted, the apoptotic cells undergo secondary necrosis. This is a highly detrimental process that results in leakage of toxic intracellular antigens, tissue destruction, and intensification of the existing inflammation. 14, 29 In secondary necrosis, increased neutrophil pro-inflammatory activity and excessive degranulation may cause acute respiratory distress syndrome (ARDS) or multiple organ failure as a result of neutrophil-mediated systemic inflammatory response syndrome. 30, 31 Membrane rupture due to secondary necrosis triggers the development of a different type of autoimmune response to auto-antigen release with auto-reactive CD4 + cell activation. 32 Thus, efferocytosis, which means rapid and effective non-inflammatory and non-immunogenic apoptotic cell clearance through the interrelated "find me", "eat me", and "tolerate me" pathways, performs a unique function in the maintenance of homeostasis at the tissue and system level.

Collectively, COVID-19 patients with a relatively healthy premorbid history recover following a mild-to-moderate course through the functioning of the following mechanisms ( Figure 1 ).

1. SARS-CoV-2 replication may lead to the pro-apoptotic changes that would trigger the exposure of normally intracellular anionic phospholipids, PS in particular, on the outer leaflet of the membrane surface of virus-infected cells. These virus-induced apoptotic cells need to be rapidly and efficiently removed otherwise they may produce secondary necrotic debris that promotes the release and hyperactivation of pro-inflammatory molecules from the immune effector cells. 2. Efficient efferocytosis is an essential component of tissue homeostasis and the resolution of inflammation. SARS-CoV-2-infected apoptotic cells expose PS as an "eat me" signal, which distinguishes them from live cells, initiates efferocytosis by professional and non-professional phagocytic cells in a non-inflammatory manner. After engulfing an apoptotic cell, macrophages actively dampen inflammation by releasing anti-inflammatory cytokines, such as (TGF-β) and interleukin-10, as well as pro-resolving lipid mediators for the resolution of inflammation. 3. SARS-CoV-2-infected, apoptotic cells are phagocytosed and digested together with the virus itself through the actions of degrading enzymes that exist in lysosomes of phagocytes, leading to the inhibition of viral growth. This mode of phagocytic elimination of invading pathogens may be considered as a cellular innate immune response. 33 Consistently, binding and preventing PS-exposing cells from being phagocytosed by annexin V, which binds specifically to PS, has been reported to augment the rate of mortality among virus-infected mice. 34 

antigen presentation to induce regulatory T-cell differentiation from naïve CD4 + T-cell precursors and to tolerize effector CD8 + T cells. 35 Furthermore, efferocytosis of infected apoptotic cells can help to mount an adaptive immune response against the virus through the process of antigen cross-presentation. 36 Thus, while COVID-19 is highly contagious due to the multibasic furin cleavage site in the S protein, 37 it follows a self-limiting course through the operation of the mechanisms briefly described. However, the disease may progress to a more severe and even fatal stage in those with chronic degenerative and cardiovascular diseases, especially in the elderly. If the underlying pathogenesis of this adverse progression could be demonstrated, it may also provide possible target(s) for the treatment.

Despite the death of billions of cells every day, effectively functioning efferocytosis maintains normal physiology in the majority of the population; however, it becomes defective in unresolved chronic inflammatory diseases, causing apoptotic cell accumulation. 38 Diseases with increased morbidity and mortality, such as aging, obesity, heart diseases, and diabetes, cause an immuno-lipidomic imbalance with lipidome disorder and chronic inflammatory pathologies. 39, 40 The characteristic determinants of these disorders are low-level chronic inflammation and increased apoptotic cell load. 41 Altogether, defective efferocytosis is an essential pathology responsible for the development and progression of chronic inflammatory diseases, including atherosclerosis, obesity, diabetes, heart failure, chronic lung disease, and neurodegenerative disease, as well as cancer. 42 The common feature of most of these degenerative chronic disorders is increased ADAM-17 (a disintegrin and metalloproteinase 17) activity in most of the cells, including bronchial epithelial cells, endothelial cells, and phagocytic cells. 43, 44 ADAM-17, also known as TNFα-converting enzyme (TACE), is a sheddase, capable of proteolytic cleavage and release of membrane-bound molecules. 45, 46 Studies revealing the relationship between ADAM-17 and a group of inflammatory degenerative diseases, including cancer, are reported with increasing frequency. Interestingly, transcriptional ADAM-17 protein expression has been observed to increase in diabetic subjects through the hyperglycemia-induced HIF-1 activity. 47, 48 In addition, the euglycemic effect of insulin restored ACE2 and ADAM-17 expression to the physiological levels, 47 suggesting that insulin may work as an ADAM17 inhibitor. 49 Furthermore, evidence is provided that ADAM-17 plays a role in metabolic syndrome-related pathologies. 50, 51 On the other hand, some clear data also show the possible relationship between the presence of metabolic syndrome and the severity of COVID-19. 52 Accordingly, several of the features of the metabolic syndrome seem to be associated with a worse prognosis in patients with COVID-19. 53 ADAM-17 is the main secretase responsible for ectodomain shedding of most of the cell surface and the transmembrane proteins. At least 90 substrates are processed by ADAM-17. Its activity towards TNFα and IL-6Rα could potentiate the proinflammatory effects of the cytokines. 54 In addition, ADAM-17 activity promotes lymphocyte and neutrophil translocation to the inflammatory sites. 55 Taken together, premorbid patients with increased ADAM17 and thus proinflammatory activity, if contracted SARS-CoV-2 infection, would have the inflammatory process more severe. 56 PS translocation to the membrane surface, an exclusive marker of apoptosis, is also required for the sheddase function of ADAM-17. The externalized PS in the membrane interacts electrostatically with ADAM-17 and then enables substrate processing to take place. 57 Signaling pathways destined to activate ADAM-17-sheddase function converge at the final step leading to scramblase activation with a breakdown of phospholipid asymmetry and exposure of PS at the outer leaflet. 58 On the other hand, PS surface exposure is required for the regulation of cholesterol metabolism, inflammation, and hemostasis. 59 Recalling that anti-inflammatory cytokines are secreted during PS-induced efferocytosis, it is possible to consider this as an effort to control the existing inflammatory pathology. 60 Therefore, exposure to PS on the outer plasma membrane is increased in chronic inflammatory disorders, including atherosclerotic diseases and diseases associated with coagulation disorders, 59,61 which may potentiate the activity of ADAM-17.

In principle, all-natural membranes may function as a procoagulant phospholipid surface, if sufficient PS is present in the exofacial leaflet. 62 Microparticles and microvesicles refer to large extracellular vesicles, which are small membranous structures. 63 Microparticles are present in the blood of healthy individuals, but their number is considerably increased in diseased states, such as inflammation, cardiovascular events, and cancer, where they may contribute to the increased risk of thrombotic events in these patients. 62 Circulating microparticles are shed from the plasma membrane of a variety of cells, including platelets, erythrocytes, leukocytes, endothelial cells, and tumor cells as a result of activation and/or apoptosis. 62, 64 These microparticles have lost lipid asymmetry and expose PS, thus providing a procoagulant surface. 62 The prothrombotic state mediated by PS-exposing microparticles is implicated in the adverse progression of chronic inflammatory diseases, including atherosclerosis. 59 In addition, ADAM-17 was shown to present in microparticles released from activated cells; thus, ADAM-17 activity can be distributed to more distant cells. 65 Remarkably, increased levels of microparticles in the circulation of severe COVID-19 patients that trigger the risk of thromboembolic complications were reported. 66 Furthermore, the level of PS-exposing microparticles correlated more strongly with the severity of COVID-19 than indices, such as lymphopenia, IL-6, D-Dimer, fibrinogen, and other established laboratory parameters. 67 ACE2, a metallopeptidase and a key component of the renin-angiotensin system (RAS), is the main receptor for the cellular entry of SARS-CoV and SARS-CoV-2. The binding of SARS viruses to ACE2 triggers the viral conformational changes, which may increase the proteolytic digestion between the S1 and S2 subunits. 68, 69 Indeed, ACE2 cleavage by ADAM-17 is required for efficient SARS-S-driven entry. Furthermore, ADAM-17-mediated shedding of ACE2 results in the release of the ectodomain into the circulation, which preserves its catalytic and bioactive potency, and the binding site for SARS-CoV-2. 70 Recent studies demonstrated that ADAM-17-driven soluble ACE2 (sACE2) levels are elevated in COVID-19 patients. The binding of SARS-CoV-2 by sACE2 may enable cell entry of tissues where membrane-bound ACE2 (mACE2) is poorly expressed. 71, 72 Low expression levels of mACE2 and associated higher ADAM-17 activity have been related to cardiovascular diseases. Consequently, the activity of the sACE2 is increased in patients with cardiovascular disease and hypertension, which correlates with the severity of the disease. 73 Thus, chronic diseases with increased membrane PS exposure and associated ADAM-17 activity may adversely affect the prognosis of the COVID-19 through the facilitated cellular virus entry as a result of the increased sACE2 levels. 56 PSRs, including TIM1, TAM, and CD36, can also be proteolytically cleaved by the transmembrane protease ADAM-17. 20,74 This shedding can prevent receptor functions in macrophage-mediated apoptotic cell engulfment. 75 The soluble cleavage products may function as sequester ligands and decoy receptors, which can act as antagonists for those PSRs. 75, 76 Accordingly, the deletion of ADAM-17 has been shown to increase macrophage-mediated efferocytosis in vivo, resulting in an enhanced anti-inflammatory response. 77 Programmed cell death 1 (PD-1), a surface molecule and a member of the immunoglobulin superfamily, functions as coinhibitory receptors during immune responses against pathogens and cancer. Hence, PD-1 is important in the regulation of the magnitude and quality of T cell response. 78 PD-1 interacts with the ligands PD-L1 and PD-L2. PD-L1 is expressed in all hematopoietic cells and also by many non-hematopoietic cell types, such as endothelial and epithelial cells. PD-L2 expression, on the other hand, is limited to hematopoietic cells, such as dendritic cells, B cells, and monocytes/macrophages. 78, 79 During the acute phase of virus infection, stimulation of the PD-1/PD-L1 signaling pathway modulates the strength and quality of cytotoxic CD8 + T cell attack, maintaining a balance between virus elimination and tissue damage. 78 ADAM17 can cleave PD-L1 from the surface of vesicles and cells. 80 Although circulating soluble PD-L1 (sPD-L1) has been largely known as a prognostic biomarker for cancers, 81 sPD-L1 has also been detected in various pathologies, often associated with markers of inflammation. 82 Of note, a recent study suggests that an increase in sPD-L1 may be a sign of poor prognosis as a result of dysregulation of the PD-1/PD-L1 axis in COVID-19 patients. 83 To summarize, the following mechanisms due to the increased PS exposure and ADAM-17 activity adversely affect the prognosis of the disease in those who have comorbidities along with the COVID-19 disease (Figure 2 ).

1. The proinflammatory burden that will determine the severity of COVID-19 is already at high levels before the onset of the disease. 2. ACE2 cleavage and sACE2 levels are increased. The facilitated extracellular association between sACE2 and SARS-CoV-2 may reinforce the infection more widespread and devastating. 3. Circulating microparticles have been implicated in cellular signaling processes in immune responses, inflammation, and coagulation. In addition to PS exposure on their membrane, they may contain ADAM17, which can contribute to substrate shedding on more distant cells. 65 

cause an excessive immunopathology.

Collectively, potentiated dysregulation of ADAM-17 activity may lead to the chronic operation of defective efferocytosis. In turn, defective efferocytosis accompanying several non-resolving, chronic inflammatory diseases may lead to the accumulation of dead cells. The dead cells can become secondarily necrotic, which can lead to tissue necrosis, autoimmunity, and pathological inflammation. Thus, defective efferocytosis is emerging as a key mechanism driving the development and progression of chronic inflammatory diseases, including atherosclerosis, obesity, diabetes, heart failure, chronic lung disease, neurodegenerative disease, and cancer. 42

Efferocytosis is a mechanism for eliminating pathogens and pathogen-infected cells in an immunologically silent manner. The quality of efferocytic clearance of virus and virus-infected apoptotic cells depends on the virus load and the operation of efferocytic machinery. 17 Therefore, increased viral load and defect in efferocytic cells seem to be the crucially important factors that will determine the prognosis of the disease. Patients in the risk group who already have problems in apoptotic cell clearance due to defective efferocytosis cannot cope with the additional acute apoptotic burden that occurs with COVID-19 disease. Consequently, the intractable inflammation that increases with secondary necrosis causes destruction and inevitable organ failure. Support for this approach came from a recent study showing that dysfunctional efferocytosis is the leading cause of the increased inflammatory response and widespread tissue damage seen in COVID- 19. 84 Older age, obesity, diabetes, cardiovascular diseases, and active cancer have each been found to be independently associated with a higher mortality risk for COVID-19. 85 Furthermore, COVID-19 does not spare young people as well. The majority of comorbidities that play a role in the prognosis of the elderly have also been shown to be risk factors that may be responsible for the severity of COVID-19 for the young adult population aged 16-28 years. 86

The clinical trial research has occurred at an unprecedented pace during the COVID-19 pandemic. Among the many potential treatment options for COVID-19, only a few are effective and predictable for the clinical treatment of patients. Some immunomodulatory drugs such as anti-TNF, anti-IL-1, anti-IL-6 used in COVID-19 patients have been shown to support recovery, possibly by neutralizing the increased cytokine levels by the augmented ADAM17 activity. 87 In a study showing the effect of glucocorticoids, another group of immune modulators, dexamethasone treatment in hospitalized COVID-19 patients provided lower mortality among those requiring respiratory support. 88 Glucocorticoids are powerful anti-inflammatory agents by their ability to inhibit the recruitment of inflammatory cells and to downregulate proinflammatory cytokine expression. 89 Glucocorticoids have been shown to modulate the expression of over 100 genes, including those known to be associated with efferocytosis, such as Mer tyrosine kinase (MerTK), a member of TAM family PS receptors. 90 Dexamethasone was also shown to promote the expression of two PS-binding opsonins, protein S and MFG-E8, which facilitate MerTK-dependent efferocytosis of apoptotic cells. 89, 91 Thus, corticosterones, which provide limited support for defective efferocytosis, find wide use in clinical treatment as an effective drug that can control the severity of cases.

Peroxisome proliferator-activated receptor γ (PPARγ) agonists such as pioglitazone alter cellular metabolism of the liver, adipose tissue, and muscle cells, as well as inflammatory macrophages. In addition, they have many antiinflammatory effects including the suppression of Th1 and Th17-induced inflammation, inhibition of NFκB, and upregulation of IL-10. 92 macrophages, similar to glucocorticoids. 94 Furthermore, treatment with pioglitazone, either prophylactically or during inflammation, while significantly enhancing PPARγ-mediated programming of efferocytes, reduces the accumulation of apoptotic cells. 92, 95 Therefore, low-dose pioglitazone alone or combination with glucocorticoids seems to be a readily available inexpensive, and effective treatment option for the correction of defective efferocytosis. 93, 96 While ADAM-17 plays a crucial role in the pathogenesis of defective efferocytosis, it was shown that ADAM-17 activity is also important in the development and worst prognosis of COVID-19. 97 Therefore, inhibiting ADAM-17 activity seems to be a rational option in severe cases of COVID-19. Experimental evidence has been shown that the inhibition of ADAM-17 protects against lung inflammation by reducing cytokine storm and excessive neutrophil recruitment to the lung in a mouse model associated with COVID-19. 98 However, ADAM-17 has a broad regulatory role in human biological processes. Consistently, dramatic disorders affecting the intestines, lungs, eyes, and hair of ADAM-17-deficient mice and humans have prompted caution regarding therapeutic interventions for ADAM-17 blockade in humans. 99, 100 Nevertheless, researchers are nowadays focusing on developing strategies for selective inhibition of ADAM-17. Several compounds have recently been entered into clinical trials but have been withdrawn due to emerging adverse events. 97 Therefore, despite the central importance of ADAM-17 in the pathogenesis of the disease, the option of inhibiting ADAM-17 in severe cases of COVID-19 should be viewed with caution.

It has been emphasized above that the level of sACE2 in the circulation increases due to defective efferocytosis in diseases that constitute a risk group for COVID-19. However, another therapeutic approach proposes using sACE2 to trap and neutralize SARS-CoV-2. From this perspective, while mACE2 may mediate cell entry of SARS-CoV-2, a genetically modified soluble form of ACE2, called human recombinant soluble angiotensin-converting enzyme-2 (hrsACE2), may decrease cell entry of SARS-CoV-2 competing for mACE2. 101 However, the plasma RAS balance in COVID-19 patients was already characterized by a strong transient increase in circulating plasma sACE2. 72 Therefore, in COVID-19 patients with comorbid diseases, both Angiotensin II and virus adversely affect the prognosis by affecting the RAS balance in a way that down-regulates ACE2. 102 Interestingly, a recent study reports encouraging results in the first severe COVID-19 patient treated with hrsACE2. 103 However, sACE2 concentrations used in experimental studies are much higher (μg/mL) than the physiological range of sACE2 concentrations (ηg/mL). While a high range of sACE2 concentrations can compete with the SARS-CoV-2-ACE2 complex for cell entry, the physiological range of sACE2 may facilitate virus entry and thus SARS-CoV-2 infectivity. Therefore, a treatment that may alter sACE2 levels in the physiological range in patients with COVID-19 should be carefully considered. 71 In severe cases of COVID-19 where defective efferocytosis is essential in the pathogenesis, the characteristic feature of infected-apoptotic cells waiting to be cleared is that they express PS on the outer surface of the membrane. Therefore, triggering antibody-dependent cellular phagocytosis (ADCP) with PS-specific antibodies can be considered as an important alternative for the treatment of such pathologies in which PS receptors are deficient. The possibility of such a specific monoclonal antibody already exists. Bavituximab (PGN401) is a monoclonal human-mouse chimeric antibody directed against the membrane phospholipid PS. Bavituximab and other antibodies that target PS were developed by Philip Thorpe's laboratory to specifically target tumor vasculature. 15, 104 Bavituximab does not bind PS directly. It binds to PS-expressing membranes by crosslinking two molecules of β2-glycoprotein 1 (β2GP1) bound to PS on the membrane. 14,105 Bavituximab could be expected to act by at least two mechanisms in severe COVID-19 patients: i) it can reduce the amount of virus to infect other tissues as a result of opsonization-mediated clearance of SARS-CoV2; ii) it induces ADCP of virus-infected apoptotic cells; thus, in pathologies where efferocytosis is defective, an opportunity arises for clearing apoptotic cells. 106 β2GP1 is an evolutionarily conserved single-chain anionic phospholipid-binding glycoprotein. It has gained much attention since the discovery of its role in antiphospholipid syndrome (APS). 107 Accordingly, β2GP1 binds to the PS expressed on apoptotic cells. When the amount of β2GP1 bound to PS reaches a certain threshold, antibodies dimerize the adjacent β2GP1 molecules. As a consequence, these high-affinity anti-β2GP1-β2GP1 complexes may activate targeted cells, causing anti-phospholipid antibodies-related manifestations. 108 However, during the clinical trials, bavituximab therapy appeared safe and well tolerated, and reductions were observed in virus load in the blood. Furthermore, coagulation parameters remained within the normal range. 106, 109 In the light of those data and considering the FDA approval of the drug, I had planned a clinical trial to investigate the efficacy of bavituximab treatment in patients with severe COVID-19 in October 2020. Unfortunately, the opinion remained in theory, as OncXerna, the company that owns the drug, did not welcome this offer. Since the hallmark autoantibodies of APS are anti-beta2 glycoproteins, a series of fully human PS-targeting antibodies were generated by phage display technology. 110 To further explore the antiviral activity of PS-targeting antibodies, my efforts to develop a specific antibody in my country, such as PGN632, that could bind directly to PS and would not need the cofactor β2GP1 protein, 109 were also failed.

The majority of those who are infected with COVID-19 have a self-limiting infection and do recover. However, a minority with comorbidities proceeds to more severe disease, requiring intensive care unit admission. 111 Therefore, the disease may cease to be a threat following the treatment of patients who are in the risk group or whose condition deteriorates with the monoclonal anti-PS antibody to be developed. Moreover, such a treatment would also have the potential to control the morbidity and mortality caused by other enveloped viruses such as influenza, Ebola, dengue, and even HIV.

Vaccine and therapeutic development are predominantly focused on the essential virus-encoded Spike protein. However, the variation of the S gene between SARS-like CoV viruses and SARS-CoV-2 variants may affect the efficacy and particularly for cross-protection of a vaccine. Nowadays when mutations against vaccines are confusing, other proteins of SARS-CoV-2 seem to be important targets to develop alternative treatments against potential resistance or emerging new viruses in the future. 112 SARS-CoV-2 ORF3a is a putative viral ion channel implicated in autophagy inhibition, inflammasome activation, and apoptosis. As explained above, it is a specific viral protein that may be responsible for the development of the virus-induced apoptotic load and ultimately the adverse course of the disease in cases of defective efferocytosis.

SARS-CoV-2 enters the host cell by both membrane fusion and by clathrin/caveolin-mediated endocytosis after binding to the ACE2 cell-surface receptors. 113 Caveolin-1 is a major component of caveolae membrane microdomains, which are involved in non-clathrin-mediated virus uptake into cells. 114 Viral ORF3a protein binding to host caveolin-1 was shown to be essential for entry and endomembrane trafficking of SARS-CoV-2. In addition to viral entry, caveolin-1 was also associated with all stages of the viral life cycle. 113, 114 Even if ORF3a is expressed on viral particles, anti-ORF3a antibodies will not neutralize the virus by preventing attachment or fusion. 115 In other words, antibodies that bind to ORF3a while disrupting the functions of the protein, the antibody-bound virus would be able to enter the cell. In turn, entry of the antibody-bound virus into the cell may present an opportunity for an unusual cytosolic antibody receptor, called TRIM21. Critically, TRIM21 is also a ubiquitin ligase and can efficiently target antibody-bound substrates for proteasomal degradation. 116 Therefore, the development of an antibody or vaccine, targeting ORF3a, may provide more effective results in controlling severe cases of COVID-19 with a high risk of mortality.

Every virus infection and even mRNA vaccines have pro-apoptotic properties. 117 Therefore, effective removal of this burden in cell death is inevitable for the maintenance of cellular homeostasis. Since defective efferocytosis is a potential threat to immune system dysregulation, a drug is urgently needed to support efferocytosis, "as it becomes more and more obvious that vaccination alone might not sufficient to curb the pandemic caused by this rapidly mutating virus". 118 Therefore, we will be awaiting further research in the hope that this work will be evaluated to respond to this urgent need.

The author reports no conflicts of interest in this work.

Outbreak of SARS-CoV-2 infections, including COVID-19 vaccine breakthrough infections

Shedding of infectious SARS-CoV-2 despite vaccination when the delta variant is prevalent -Wisconsin

Delta variants of SARS-CoV-2 cause significantly increased vaccine breakthrough COVID-19 cases in

SARS-CoV-2 Lambda variant exhibits higher infectivity and immune resistance. Prepr bioRxiv

Role of host factors in SARS-CoV-2 entry

Phosphatidylserine receptors enhance SARS-CoV-2 infection: AXL as a therapeutic target for COVID-19

A multibasic cleavage site in the spike protein of SARS-CoV-2 is essential for infection of human lung cells

Cell entry mechanisms of SARS-CoV-2

Loss of furin cleavage site attenuates SARS-CoV-2 pathogenesis

Variations in Orf3a protein of SARS-CoV-2 alter its structure and function

The ORF3a protein of SARS-CoV-2 induces apoptosis in cells

ATP and large signaling metabolites flux through caspase-activated Pannexin 1 channels

Mechanisms underlying production and externalization of oxidized phosphatidylserine in apoptosis: involvement of mitochondria

Phosphatidylserine is a global immunosuppressive signal in efferocytosis, infectious disease, and cancer

TAMs, and PS-antibody targeting: implications for cancer immunotherapy

Efferocytosis of dying cells differentially modulate immunological outcomes in tumor microenvironment

Efferocytosis of pathogen-infected cells

Biology of phosphatidylserine (PS): basic physiology and implications in immunology, infectious disease, and cancer

Scrambled eggs: apoptotic cell clearance by non-professional phagocytes in the drosophila ovary

Phosphatidylserine receptors: enhancers of enveloped virus entry and infection

CRP and the disposal of dying cells: consequences for systemic lupus erythematosus and rheumatoid arthritis

C-reactive protein triggers cell death in ischemic cells

C-reactive protein-mediated low density lipoprotein uptake by macrophages

Efferocytosis and outside-in signaling by cardiac phagocytes. links to repair, cellular programming, and intercellular crosstalk in heart

Successful treatment of a 39-year-old COVID-19 patient with respiratory failure by selective C-reactive protein apheresis

Case report: c-reactive protein apheresis in a patient with COVID-19 and fulminant CRP increase. Front Immunol

Dendritic cells acquire antigen from apoptotic cells and induce class I-restricted CTLs

Beyond antigens and adjuvants: formulating future vaccines

Secondary necrosis: the natural outcome of the complete apoptotic program

Tissue damage from neutrophil-induced oxidative stress in COVID-19

Excessive neutrophils and neutrophil extracellular traps in COVID-19

The immune response to secondary necrotic cells

Evidence for phagocytosis of influenza virus-infected, apoptotic cells by neutrophils and macrophages in mice

Augmentation of fatality of influenza in mice by inhibition of phagocytosis

Enhanced efferocytosis by dendritic cells underlies memory T-cell expansion and susceptibility to autoimmune disease in CD300f-deficient mice

Activation of dendritic cells alters the mechanism of MHC class II antigen presentation to CD4 T cells

SARS-CoV-2 and bat RaTG13 spike glycoprotein structures inform on virus evolution and furin-cleavage effects

Mechanisms and consequences of defective efferocytosis in atherosclerosis

Cutting edge: severe SARS-CoV-2 infection in humans is defined by a shift in the serum lipidome, resulting in dysregulation of eicosanoid immune mediators

Regulation of apoptotic cell clearance during resolution of inflammation

The effect of ageing on the resolution of inflammation

Efferocytosis in health and disease

ADAM-17: the enzyme that does it all

Role of endothelial ADAM17 in early vascular changes associated with diabetic retinopathy

A metalloproteinase disintegrin that releases tumour-necrosis factor-α from cells

Angiotensin II induced proteolytic cleavage of myocardial ACE2 is mediated by TACE/ADAM-17: a positive feedback mechanism in the RAS

Insulin treatment attenuates renal ADAM17 and ACE2 shedding in diabetic Akita mice

High glucose up-regulates ADAM17 through HIF-1α in mesangial cells

Biologia futura: is ADAM 17 the reason for COVID-19 susceptibility in hyperglycemic and diabetic patients?

The role of ADAM17 in metabolic inflammation

Implications of ADAM17 activation for hyperglycaemia, obesity and type 2 diabetes

COVID-19 alters thinking and management in metabolic diseases

Coronavirus and obesity: could insulin resistance mediate the severity of Covid-19 infection? Front Public Health

Molecular switch in human diseases-disintegrin and metalloproteinases

Regulation of mature ADAM17 by redox agents for L-selectin shedding

ACE2/ADAM17/TMPRSS2 interplay may be the main risk factor for COVID-19

How membrane asymmetry regulates ADAM17 sheddase function

The plasma membrane: penultimate regulator of ADAM sheddase function

Phosphatidylserine in atherosclerosis

Modulation of cardiac macrophages by phosphatidylserine-presenting liposomes improves infarct repair

Contribution of ADAM17 and related ADAMs in cardiovascular diseases

Getting to the outer leaflet: physiology of phosphatidylserine exposure at the plasma membrane

Cellular mechanisms underlying the formation of circulating microparticles

Microparticles in hemostasis and thrombosis

Stimulated release and functional activity of surface expressed metalloproteinase ADAM17 in exosomes

Circulating platelet-derived extracellular vesicles are a hallmark of Sars-Cov-2 infection

Binding of phosphatidylserine-positive microparticles by PBMCs classifies disease severity in COVID-19 patients

TMPRSS2 and ADAM17 cleave ACE2 differentially and only proteolysis by TMPRSS2 augments entry driven by the severe acute respiratory syndrome coronavirus spike protein

SARS-CoV-2 cell entry depends on ACE2 and TMPRSS2 and is blocked by a clinically proven protease inhibitor

Priming time: how cellular proteases arm coronavirus spike proteins

Soluble ACE2-mediated cell entry of SARS-CoV-2 via interaction with proteins related to the reninangiotensin system

Soluble angiotensin-converting enzyme 2 is transiently elevated in COVID-19 and correlates with specific inflammatory and endothelial markers

Detection of soluble angiotensin-converting enzyme 2 in heart failure

Macrophage ADAM17 deficiency augments CD36-dependent apoptotic cell uptake and the linked anti-inflammatory phenotype

Soluble T cell immunoglobulin and mucin domain (TIM)-1 and −4 generated by A Disintegrin And Metalloprotease (ADAM)-10 and −17 bind to phosphatidylserine

ADAM17-mediated shedding of inflammatory cytokines in hypertension

Effect of soluble cleavage products of important receptors/ligands on efferocytosis: their role in inflammatory, autoimmune and cardiovascular disease

The PD-1/PD-L1 axis and virus infections: a delicate balance

The diverse functions of the PD1 inhibitory pathway

ADAM10 and ADAM17 cleave PD-L1 to mediate PD-(L)1 inhibitor resistance

Soluble PD-1 and PD-L1: predictive and prognostic significance in cancer

Soluble programmed death ligand-1 (sPD-L1): a pool of circulating proteins implicated in health and diseases

Efferocytosis of SARS-CoV-2-infected dying cells impairs macrophage anti-inflammatory programming and continual clearance of apoptotic cells

Factors associated with death in critically ill patients with coronavirus disease 2019 in the US

Risk factors for severity of COVID-19 in hospital patients age 18-29 years

Immunomodulatory drugs in the management of SARS-CoV-2. Front Immunol

Dexamethasone in hospitalized patients with Covid-19

Glucocorticoids induce protein S-dependent phagocytosis of apoptotic neutrophils by human macrophages

Glucocorticoids induce differentiation of a specifically activated, anti-inflammatory subtype of human monocytes

Milk fat globule-EGF factor 8 mediates the enhancement of apoptotic cell clearance by glucocorticoids

Impaired efferocytosis in human chronic granulomatous disease is reversed by pioglitazone treatment

Role of oxidized LDL-induced "trained macrophages" in the pathogenesis of COVID-19 and benefits of pioglitazone: a hypothesis

The PPAR-γ antagonist GW9662 elicits differentiation of M2c-like cells and upregulation of the MerTK/Gas6 axis: a key role for PPAR-γ in human macrophage polarization

PPARγ activation normalizes resolution of acute sterile inflammation in murine chronic granulomatous disease

Pioglitazone enhances the beneficial effects of glucocorticoids in experimental nephrotic syndrome

Strategies to target ADAM17 in disease: from its discovery to the iRhom revolution

ADAM17 inhibition prevents neutrophilia and lung injury in a mouse model of Covid-19. bioRxiv Prepr

Inflammatory skin and bowel disease linked to ADAM17 deletion

Loss of ADAM17 is associated with severe multiorgan dysfunction

Inhibition of SARS-CoV-2 infections in engineered human tissues using clinical-grade soluble human ACE2

Potential detrimental role of soluble ACE2 in severe COVID-19 comorbid patients

Human recombinant soluble ACE2 in severe COVID-19

Antibody targeting of phosphatidylserine for the detection and immunotherapy of cancer

Effective binding of a phosphatidylserine-targeting antibody to Ebola virus infected cells and purified virions

Targeting inside-out phosphatidylserine as a therapeutic strategy for viral diseases

Quantitative determination of the binding of β2-glycoprotein I and prothrombin to phosphatidylserine-exposing blood platelets

β2GP1, anti-β2GP1 antibodies and platelets: key players in the antiphospholipid syndrome

Protective effect of anti-phosphatidylserine antibody in a guinea pig model of advanced hemorrhagic arenavirus infection

Targeting phosphatidylserine on apoptotic cells with phages and peptides selected from a bacteriophage display library

Evaluation, and Treatment of Coronavirus (COVID-19). StatPearls; 2021

Cryo-EM structure of SARS-CoV-2 ORF3a in lipid nanodiscs

Partha basuchowdhuri SSJ. D155Y substitution of SARS-CoV-2 ORF3a weakens binding with caveolin-1: an in silico study. bioRxiv Prepr

Severe acute respiratory syndrome coronavirus Orf3a protein interacts with caveolin

SARS-CoV-2 vaccines in advanced clinical trials: where do we stand?

Viral nucleoprotein antibodies activate TRIM21 and induce T cell immunity

mRNA vaccines for infectious diseases: principles, delivery and clinical translation

Getting hands on a drug for Covid-19: inhaled and intranasal niclosamide

Defective efferocytosis as a predictor of COVID-19 mortality

Infection and Drug Resistance is an international, peer-reviewed open-access journal that focuses on the optimal treatment of infection (bacterial, fungal and viral) and the development and institution of preventive strategies to minimize the development and spread of resistance. The journal is specifically concerned with the epidemiology of antibiotic resistance and the mechanisms of resistance development and diffusion in both hospitals and the community. The manuscript management system is completely online and includes a very quick and fair peer-review system, which is all easy to use