Relationship of COVID-19 pathogenesis for periodontal medicine research. Part I: Pathogenesis of COVID-19

Cell invasion mediated by angiotensin-converting enzyme 2 (ACE2) ectoenzyme and cellular proteases, such as trypsinlike proteases, cathepsins, transmembrane serine protease 2 and furin, target different tissues and organs as lung, gut, colon, ileum, kidney, gallbladder, heart muscle, epididymis, breast, ovary, stomach, bile duct, liver, oral cavity, lung, thyroid, esophagus, bladder, breast, uterus, prostate, pancreas, cerebellum, as well as calyx secreting cells in the nasal and sinus tissue. Loss of homeostasis of the renin-angiotensin system deregulates different axes compromising metabolic, cardiorespiratory, renal and hepatic control. SARS-CoV-2 infected cell undergoes pyroptosis and releases molecular patterns associated with damage: pro-inflammatory interleukin (IL) -1, IL-6, IL-8, IL-10, IL-17, induced protein-10, interferon gamma, interferon gamma-induced protein-10, granulocyte colony-stimulating factor, granulocyte-macrophage colony-stimulating factor, macrophage inflammatory protein 1α and 1β, monocyte chemotherapy activating protein 1, inflammatory macrophage protein 1, tumor necrosis-α, and mediators of immunemediated inflammatory diseases. Cytokine storm and non-neutralizing antibodies produced by B cells circulate, cause/exacerbate damage to various organs. During viral replication and low oxygen saturation, loss of HIF-mediated cell homeostasis can lead to cell death/lysis and tissue damage, related to the hyperinflammatory response. The SARSCoV-2-ACE2 can increase permeability, inflammation and microbial transmission by bacteremia or endotoxemia, in addition to dysbiosis. Thrombotic potential and the immunoinflammatory imbalance compromise function or lead to injuries and multiple organ failure. Infection by SARS-CoV-2 has the potential to modify the natural history of diseases, the relationships or interactions between the different systems and pathologies and the effects of their treatments, as in periodontal medicine approach.


Introduction
SARS-CoV-2 infection appears to directly affect tissues and organs by exposure and presence of the angiotensinconverting enzyme 2 (ACE2) ectoenzyme and cellular proteases (Bertram et al., 2011, Glowacka et al., 2011, Raj et al., 2013, Wang et al., 2013, Gheblawi et al., 2020, Gralinski & Menachery, 2020, Hoffmann et al., 2020, Wan, Shang, Graham, Baric & Li, 2020. The lungs are the most affected organs and the clinical evolution of severe forms of  leads to abnormalities in the blood hematological and biochemical index, and systemic conditions/diseases on kidney, liver and coagulation biomarkers (Tay, Poh, Rénia, MacAry & Ng, 2020, Pedersen & Ho, 2020, Schett, Sticherling & Neurath, 2020. The pathogenesis of COVID-19 and its systemic impacts are associated with intense pro-inflammatory events and loss of homeostasis, associated with a hyperinflammatory state, secondary bacterial infections, bacteremia, endotoxemia, loss of function and multiple organ failure (Cao & Li, 2020, Hadjadj et al., 2020, Henry, de Oliveira, Benoit, Plebani & Lippi, 2020, Mehta, McAuley, Brown, Sanchez, Tattersall & Manson, 2020, Merad & Martin, 2020, Qin et al., 2020, Wang, Jiang, Chen & Montaner, 2020, Ye, Wang & Mao, 2020, García-Sastre, 2017, Schulert & Grom, 2015, Mayer-Barber et al., 2014. The systemic impacts of the COVID-19 have the potential to influence the relationships and interactions between periodontal diseases and systemic conditions/diseases, previously reported in the literature. In addition, the periodontal medicine research, the natural history of periodontal disease and the response to periodontal therapy during and after the COVID-19 pandemic may be affected by the disease. Therefore, the aim of this study was to review the literature and propose a conceptual hypothesis on the subject, based on the interception between the pathogenesis of COVID-19 and its main systemic repercussions, and periodontal medicine.

Methodology
Theoretical essay based on studies on the pathogenesis of COVID-19, potentially related to systemic interactions of periodontal diseases. Searches were performed in the MEDLINE|PubMed, Scopus, Embase, Web of Science, Cochrane Library, and BIREME|bvs databases for articles published up to 2020 December 20, using MeSH terms, Emtree terms and DeCS/MeSH terms related to 'COVID-19', 'SARS-CoV-2', and 'pathogenesis', combined by the Boolean operators "OR" and "AND". The studies, mostly experimental and review, published in the main journals, were qualitatively summarized. The comparison of these findings with the main systemic interactions of periodontal diseases previously described resulted in conceptual hypotheses based on the literature about the potential impacts of the COVID-19 pandemic on the scientific investigation of these interactions.

New coronavirus SARS-CoV-2 and host cell infection
The first cases of COVID-19 (coronavirus disease 2019) were reported to the World Health Organization (WHO) on December 31, 2019, where 27 individuals suffered pneumonia with no known cause and all were related to a wholesale market for wet animals in the city of Wuhan, China. All available evidence for COVID-19 suggests that SARS-CoV-2 has a zoonotic source. The clinical signs and symptoms and the genetic similarity of the pathogen to the SARS-CoV virus resulted in the taxonomic characterization of a new coronavirus identified as SARS-CoV-2 (severe acute respiratory syndrome coronavirus 2) (Peiris, Guan & Yuen, 2004, Cui, Li & Shi, 2019, Committee on Taxonomy of Viruses, 2020, Lai, Shih, Ko, Tang & Hsueh, 2020, World Health Organization, 2020a. In January 2020, the outbreak of COVID-19 was declared by WHO as a Public Health Emergency of International Importance, and on March 11 the pandemic of COVID-19 in the world was declared (World Health Organization, 2020b).
Structural proteins are encoded by the structural genes spike (S), envelope (E), membrane (M) and nucleocapsid (N) (Chen, Liu & Guo, 2020, Hui et al., 2020. SARS-CoV-2 differs from SARS-CoV in the absence of protein 8a and the number of amino acids in protein 8b and 3c varies . Spike is a transmembrane trimetric glycoprotein protruding from the viral surface. It is related to the diversity of coronaviruses and host tropism. SARS-CoV-2 Spike glycoprotein was modified by homologous recombination as a mixture of bat SARS-CoV and an unknown Beta-CoV;  the N501T mutation in the SARS-CoV-2 Spike may have significantly increased its binding affinity for ACE2 (Wan, Shang, Graham, Baric & Li, 2020) SARS-CoV-2 also expresses other polyproteins, nucleoproteins and membrane proteins, such as RNA polymerase, protease type 3-chymotrypsin, protease type papain, helicase, glycoprotein and accessory proteins .

Viral replication
The life cycle of SARS-CoV-2 in host cells begins when the cleavage at the S1/S2 cleavage site of the Spike protein for binding to the ectoenzyme ACE2 (step 1, attachmentstabilization of the membrane-anchored S2 subunit), followed by changes in conformation through protease cleavage for activation at the S'2 of the S2 subunit, resulting in fusion of the viral envelope with the host cell membrane and endocytosis (step 2, penetration via endosomal). Among coronaviruses, only SARS-CoV-2 presents a furin cleavage site ("RPPA" sequence) at the S1/S2 site. Although the S1/S2 site was also subjected to cleavage by TMPRSS2 and cathepsin B and L, the ubiquitous expression of furin likely makes this virus very pathogenic (Gheblawi et al., 2020, Hoffmann et al., 2020, Caly, Druce, Catton, Jans & Wagstaff, 2020, Moore, 2001. The virus's positive single-stranded RNA (RNA + ) is released into the host cell and its genome is translated into viral polyproteins replicase pp1a and 1ab, cleaved into small products by viral proteinases (step 3, biosynthesis). The viral replication of SARS-CoV-2 depends on the cytoplasmic mechanism of the infected cell and occurs through series of subgenomic mRNA production by discontinuous transcription. Viral replication of SARS-CoV-2 can occur in the cytoplasm of the host cell, with the viral RNA + acting as mRNA.
After a polymerase action, the mRNAs are translated into relevant viral proteins that are assembled into virions with RNA from the genome in the endoplasmic reticulum (ER), intermediate compartment ER-Golgi and Golgi complex (step 4, maturation).
Gene expressions by STAT1 are related to cell viability and survival, and responses to pathogens (Baris et al., 2016). Most interferon stimulated genes possess binding sites for STAT-or IFN-regulatory factor transcription factor-mediated expression (Kent et al., 2002, Read, Obeid, Ahlenstiel & Ahlenstiel, 2019.
PAMPs (pathogen associated molecular patterns) virus such as glycoproteins from the viral envelope (13 MHC-I and 3 MHC-II epitopes in SARS-CoV-2 Spike) and nucleic acids are recognized by cell surface and cytosolic pattern recognition receptors (TLR5/7/8 → innate immune), and endosomal toll-like receptors 7 and 8 (TLR7/8) that primarily bind viral nucleic acids (RNA + ) (Pande, Kawai & Akira, 2014, Moreno-Eutimio, López-Macías & Pastelin-Palacios, 2020, Noorimotlagh, Karami, Mirzaee, Kaffashian, Mami & Azizi, 2020. Thus, the downstream signaling intermediates activate both inflammatory and innate immune transcription factors and induce expression of IFN-α and IFN-β, IFN-γ, and/or IFN-λs, by a signaling cascade resulting in STAT1 and STAT2 heterodimerizing and binding interferon regulatory factor 9 (IRF9). Its translocation into the nucleus is related to gene promoter transcription, followed by immune cell chemotaxis and activation, and antiviral mechanisms to inhibit viral replication in the host cells (Read, Obeid, Ahlenstiel & Ahlenstiel, 2019). An association has been reported between ACE2 expression and IFN-stimulated canonical genes (ISGs) or components of the IFN signaling pathway, comparing ACE2 + and ACE2cells; both type I and type II IFNs induced ACE2 expression in human epithelial cells and keratinocytes. SARS-CoV-2 does not appear to induce type I, II or III interferons in infected human lung tissues, which suggests that ACE2 may not be increased in this organ in COVID-19 (Su & Jiang, 2020, Ziegler et al., 2020. The coronavirus infections induced expression of IFN, activating canonical IFN-induced genes (ISGs) and ACE2 expression, and contributing to the viral infection and acute lung injury (Su & Jiang, 2020).
The systemic interactions of COVID-19 with obesity, diabetes mellitus, metabolic syndrome, hypertension, cerebrovascular and cardiovascular diseases, acute kidney injury, chronic liver diseases, chronic kidney and liver diseases, maternal and perinatal results can be confirmed in blood biomarkers (Ali et al., 2020, Huang, Lim & Pranata, 2020, Malik, Ravindra, Attri, Bhadada & Singh, 2020, Mantovani, Beatrice & Dalbeni, 2020, Pranata, Huang & Lim, 2020, Zaigham & Andersson, 2020. Patients with COVID-19 and acute respiratory distress syndrome may have lower levels of serum D-dimer and activated partial thomboplastin time, and higher levels of fibrinogen, antithrombin, prothrombin time and platelet count than non-COVID-19 patients with acute respiratory distress syndrome (Helms et al., 2020). Viral pulmonary sepsis can lead to vasoconstriction and thrombotic events potentially related to myocardial infarction and necrosis of the renal and hepatic tissues .
PAMPs recognition receptors such as TLR4 and TLR7, DAMPs and cytokines (IL-6 and CCL2) activate blood monocytes that express tissue factor (TF) in the cell membrane. Cytokines (IL-6 and TNF) and viral particles stimulate endothelial cells to produce chemo-attractants of monocytes and adhesion molecules (P-selectin) and expose TF in the lumen, which recruits monocytes activated endothelial cells, TF and microvesicles derived from activated monocytes stimulate fibrin deposition and blood clotting (extrinsic coagulation pathway). Extracellular neutrophil traps (NETs) activate the coagulation contact pathway and bind and activate platelets (amplification of coagulation). In COVID-19, the inhibitor of the tissue factor pathway (TFPI), antithrombin and protein C (endogenous anticoagulant pathways) are reduced. The von Willebrand factor and the exposure of collagen from endothelial injury lead to the accumulation of platelets and fibrin and stimulate the intrinsic/contact coagulation pathway (Merad & Martin, 2020).
Thrombosis and pulmonary embolism in severe forms of COVID-19 are correlated with elevated levels of D-dimer and fibrinogen and abnormalities in the endothelium related to vasodilation, fibrinolysis and anti-aggregation. Significant endothelial lesions compromise thrombotic regulation leading to a hypercoagulable condition (Wang, Hao, Leeper & Zhu, 2018).
Endothelial cells express the ACE2 ectoenzyme and correspond to one third of lung cells. The increase in ACE2-mediated microvascular permeability, inflammatory events and microbial challenge (viral infections and/or bacterial superinfections) can potentiate coronavirus infection, bacteremia, endotoxemia, hyperinflammatory status and systemic complications in multiple tissues and organs (Yuki, Fujiogi & Koutsogiannaki, 2020, Yuki K, Fujiogi M, Koutsogiannaki et al., 2008, Sluimer et al., 2008, Zeng et al., 2012. Pulmonary injury lesions in SARS-CoV-2 infection occur, in part, because ACE2 is highly expressed on the apical side of pulmonary epithelial cells in the alveolar space. The main cellular components of innate immunity in the lungs are epithelial cells, alveolar macrophages located on the apical side of the epithelium and subepithelial dendritic cells (Yoshikawa, Hill, Li, Peters & Tseng, 2009). These cells present the antigen (coronavirus) to T cells in the lymph nodes by phagocytosis of apoptotic cells infected by the virus, through PAMPs (Fujimoto, Pan, Takizawa & Nakanishi, 2000, Channappanavar, Zhao & Perlman, 2014. ACE2 expression in dendritic (splenic) cells and alveolar macrophages is limited, however, the SARS-CoV virus can bind to dendritic-cell specific intercellular adhesion molecule-3-grabbing nonintegrin (DC-SIGN) and DC-SIGN-related protein (DC-SIGNR, L-SIGN), highly expressed in dendritic cells and macrophages (Jeffers et al., 2004, Marzi et al., 2004, Yang et al., 2004. Therefore, it is possible that these antigen-presenting cells may be infected with coronary viruses, in addition to phagocyting them. CD4 + cells activate B cells and promote the production of antibodies specific to the virus (IgM antibodies (recent exposure or acute events) and IgG (delayed immunity)). CD8 + T cells can kill infected viral cells, especially in the early stages of the disease (Yuki, Fujiogi & Koutsogiannaki, 2020).
SARS-CoV-infected lung epithelial cells produce IL-6 and IL-8. IL-8-mediated neutrophil chemotaxis increases the number of innate and adaptive immune inflammatory cells in the lungs of critically ill patients with COVID-19 (Tian et al., 2020. The innate neutrophil-mediated immune response is correlated with lung injury, , Koutsogiannaki, Shimaoka & Yuki, 2019, Fang et al., 2012 while the adaptive immune response mediated by CD8 + T cells (primary cytotoxic) and pathological cytotoxic T cells derived from CD4 + T cells kills the virus, cause lung injury, and recruit monocytes (CD14 + CD16 + ) by GM-CSF. These inflammatory monocytes showed high expression of IL-6 and increased the systemic inflammatory response (Yuki, Fujiogi & Koutsogiannaki, 2020, Fang et al., 2012, Small et al., 2001. ACE2 is also expressed in internal lymphoid cells (ILC) 2 and ILC3, responsible for mucosal homeostasis.
Approximately 95 % of lung ILCs are Natural Killer (NK) cells, type ILC1. The relationship of coronaviruses infection with ILC2 and ILC3 cells has not yet been defined (Yuki, Fujiogi & Koutsogiannaki, 2020).
These signaling molecules attract monocytes and T lymphocytes to the site of infection, but not neutrophils (Tian et al., 2020. The recruitment of immune blood cells and the lymphocytic infiltration in the lungs result in lymphopenia and an increase in the neutrophil-lymphocyte ratio in patients with COVID-19 (Hamming et al., 2004, Qin et al., 2020. Critical COVID-19 patients admitted to the intensive care unit show dysfunctional immune responses and elevated plasma levels of IL-2, IL-6, IL-7, IL-7, IL-10, G -CSF, IP-10, MCP1, MIP1α and TNF. IL-6 levels increase according to the severity of the disease and are correlated with cases of death . In addition, these patients have a population of FCN1 macrophages derived from highly inflammatory monocytes in the bronchoalveolar region (Chua et al., 2020) and a significantly higher percentage of CD14 + CD16 + inflammatory monocytes in the peripheral blood (Wilk et al., 2020). Cytokines secreted by these monocytes like MCP-1, IP-10 and MIP1α contribute to the cytokine storm (Tay, Poh, Rénia, MacAry & Ng, 2020).
As in SARS-CoV infection, it is possible that SARS-CoV-2 influences the stages of the interferon signaling pathway and prevents the recognition of viral RNA for PRR, (Wilk et al., 2020, Siu, (Frieman et al., 2007) and inhibiting the host protein translation by mRNA degradation (Narayanan et al., 2008).
This condition helps in viral replication and leads to aberrant inflammatory responses from pyroptosis. The species of virus, protease and reactive oxygen are associated with local tissue damage [diffuse alveolar damage (desquamation of alveolar cells), formation of hyaline membrane and pulmonary edema] (Tian et al., 2020. As a result, in addition to reducing oxygen saturation, secondary infections can occur (Tay, Poh, Rénia, MacAry & Ng, 2020).
The cytokine storm is related to local tissue damage and negative effects in the body, reaching septic shock and multiple organ failure associated with elevated levels of TNF. The dysfunctional immune response that causes pathology and also fails to successfully eradicate pathogens is more evident in people with comorbidities. It is still controversial whether the persistence of the virus is necessary to cause damage and loss of function in tissues and organs (Tay, Poh, Rénia, MacAry & Ng, 2020. Viral infection of immune cells by SARS-CoV, even if it is not productive, leads to a hyperinflammatory response of monocytes and macrophages, for example; , Law et al., 2005, Tseng, Perrone, Zhu, Makino & Peters, 2005, Yilla et al., 2005 the same should occur with exposure to SARS-CoV-2 (Tay, Poh, Rénia, MacAry & Ng, 2020).
The acute respiratory distress syndrome associated with impaired lung function in COVID-19 was attributed to a nonadaptive immune response (Guan et al., 2020). Vascular and cellular events of inflammation mediated by pro-inflammatory cytokines produced by immune cells resident in the lung lead to the leakage of neutrophils and monocytes from the blood into the bronchi, breaking through the air-blood barrier and causing damage to epithelial cells and local vascular endothelial cells (express ACE2); vascular endothelial injury may result in thrombotic microangiopathies. In the innate immune response to viruses, the complement system appears to induce pro-inflammatory responses via activation of the C3 component (observed in SARS-CoV-2 infections), associated with the acute respiratory distress syndrome (Mastellos, Ricklin & Lambris, 2019. These findings did not occur in mice with C3 deficiency infected with SARS-CoV, where the neutrophilic infiltrate and the levels of IL-6 in the lung were significantly reduced (Gralinski et al., 2018). The immunoinflammatory events previously described for other coronavirus infections can be repeated in the pathogenesis of COVID-19; however, clinical data on the role of complement activation associated with SARS-CoV-2 are still limited. Despite this, lung biopsies from patients with COVID-19 confirmed complement activation with a generation of C3a and deposition of C3 fragments, in addition to a significant increase in serum C5a levels. Treatment of COVID-19 with anti-C5a antibody (well-established axis C5a-C5aR in the Research, Society andDevelopment, v. 10, n. 5, e1910513729, 2021 (CC BY 4.0) | ISSN 2525-3409 | DOI: http://dx.doi.org/10.33448/rsd-v10i5.13729 pathophysiology of acute respiratory distress syndrome) resulted in an increase in pulmonary oxygenation and a decrease in systemic inflammation and in the clinical improvement of patients (Campbell CM, Kahwash, 2020, Risitano et al., 2020.

Final Considerations
The affinity of SARS-CoV-2 for ACE2 and the complications of loss of homeostasis of the renin-angiotensin system result in decompensations or deregulations of different axes compromising metabolic, cardiorespiratory, renal and hepatic control. The thrombotic potential and the hyperinflammatory cell response potentiate this imbalance and can compromise function or lead to injuries and multiple organ failure. Based on the pathogenesis of coronavirus infections, this theoretical essay proposes the conceptual hypothesis that infection by SARS-CoV-2, especially in cases of severe COVID-19, is able to modify the natural history of diseases, the relationships or interactions between the different systems and pathologies, and the consequences of their treatments.
New experimental studies, epidemiological studies, epigenetic, immunoinflammatory and microbiological characterization, and the comparison of the results of clinical trials during or after the COVID-19 pandemic with the results of the pre-pandemic period, will contribute to establish the impacts of SARS-CoV-2 infection in periodontal medicine.