Lessons from dermatology about inflammatory responses in Covid‐19

Summary The SARS‐Cov‐2 is a single‐stranded RNA virus composed of 16 non‐structural proteins (NSP 1‐16) with specific roles in the replication of coronaviruses. NSP3 has the property to block host innate immune response and to promote cytokine expression. NSP5 can inhibit interferon (IFN) signalling and NSP16 prevents MAD5 recognition, depressing the innate immunity. Dendritic cells, monocytes, and macrophages are the first cell lineage against viruses' infections. The IFN type I is the danger signal for the human body during this clinical setting. Protective immune responses to viral infection are initiated by innate immune sensors that survey extracellular and intracellular space for foreign nucleic acids. In Covid‐19 the pathogenesis is not yet fully understood, but viral and host factors seem to play a key role. Important points in severe Covid‐19 are characterized by an upregulated innate immune response, hypercoagulopathy state, pulmonary tissue damage, neurological and/or gastrointestinal tract involvement, and fatal outcome in severe cases of macrophage activation syndrome, which produce a ‘cytokine storm’. These systemic conditions share polymorphous cutaneous lesions where innate immune system is involved in the histopathological findings with acute respiratory distress syndrome, hypercoagulability, hyperferritinemia, increased serum levels of D‐dimer, lactic dehydrogenase, reactive‐C‐protein and serum A amyloid. It is described that several polymorphous cutaneous lesions similar to erythema pernio, urticarial rashes, diffuse or disseminated erythema, livedo racemosa, blue toe syndrome, retiform purpura, vesicles lesions, and purpuric exanthema or exanthema with clinical aspects of symmetrical drug‐related intertriginous and flexural exanthema. This review describes the complexity of Covid‐19, its pathophysiological and clinical aspects.

gangrene in seven patients, four of them diagnosed as disseminated intravascular coagulation (DIC), 6 besides other two reports, in China and Italy resembling acral characteristics of DIC, and severe perniosis (acute across-ischemia in the child), respectively. 7,8 Probably due to the lack of adequate personal protective equipment (PPE) for frontline health care workers, dermatologists have not registered adequately the cutaneous findings in Covid-19 patients. 20 Viral infections can produce specific clinical and non-specific manifestations, due to the direct action in infected cells or as a phenomenon of immune system hyperactivity. Since some of the associations are considered to be causal, it is instructive to consider by specific cases what evidence is generally accepted to establish a causal relation and which factors may be dispensable. 21 We would like to highlight some interesting aspects of SARS-CoV-2 infections for allergists and dermatologists and their possible relation with the skin: 1 The SARS-CoV-2 is composed of 16 non-structural proteins (NSP [1][2][3][4][5][6][7][8][9][10][11][12][13][14][15][16] with specific roles in the replication of coronaviruses (CoVs). 21 NSP3 prevents host innate immune response and promotes cytokine expression, NSP5 can inhibit interferon (IFN) signalling and NSP16 prevents MDA5 recognition, depressing innate immunity. 21 Four proteins are structural and essential for viral assembly and infection: homotrimers of S proteins (spikes on the viral surface), M protein (three transmembrane domains), E protein (involved in viral pathogenesis) and N protein. 22 2 Aerosolized uptake of SARS-CoV-2 leads to infection of target cells expressing angiotensin-converting enzyme (ACE) type II (ACE2) such as alveolar type 2 (that produce lung surfactant) or other unknown target cells. 23 3 Dendritic cells, monocytes, and macrophages are the first line cell lineage against viral infections. Antiviral proteins that are at the front lines of immune defence are the main targets of virusencoded antagonists. Protein suppressors, or others involved in upregulation of the innate response, restore the antiviral response and provide an advantage to the host immune defence against a specific viral infection. 24 4 Immunopathogenesis is associated with an uncontrolled immune response, which may result in pulmonary tissue damage, functional impairment and reduced lung capacity. Chemotactic factors are essential to the immune responses in viral infections and spectral changes in such factors may lead to severely maladjusted immune responses, increased viral replication and tissue damage. 25 In a subset of patients, the disease can progress to pneumonia, respiratory failure and death related to an extreme rise in inflammatory cytokines including interleukin (IL)2, IL6, IL7, IL10, GCSF, IP10, MCP1, MIPI A and TNFα. 1 The increase in pro-inflammatory cytokines is associated with severe pneumonia and it can have deleterious effects on the adaptative immune system. 26,27 In a subset of patients, overactive immune responses may induce immunopathological conditions, termed cytokine storm, and in some individuals, may proceed to macrophage activation syndrome (MAS), often causing a fatal outcome. 25 5 Hammining et al 28 28 Soler et al studied ACE2 and ACE in acute and chronic rejection after human heart transplant and found both expressions in endothelial and inflammatory cells. 29 Angiotensin-converting enzyme (ACE)-2 is homologous to ACE. In addition, ACE2 protein levels are increased in failing human hearts, suggesting an important role in the negative modulation of the renin-angiotensin system (RAS), leading to the generation and degradation of angiotensin peptides after cardiac damage. 29 6 A transmembrane protease, serine 2 (TMPRSS2), a type II transmembrane serine protease (TTPS), plays a critical role in SARS and MERS coronavirus (COV) indicating that it could be a novel antiviral strategy to treat coronavirus and some low pathogenic influenza virus infections. 30 SARS-COV-2 and SARS-COV bind to ACE2 by the protein s (spike) and allow virus to enter and infect cells. 31 In order for the virus to complete entry into the cell, the spike protein has to be primed by TMPRSS2 to complete this process. 31 In order to attach virus receptor (SPIKE) to host cellular ligand (ACE2), activation by TMPRSS2 as a protease is needed. 31 TMPRSS2 gene is located on human chromosome 21, and a significant feature of the TMPRSS2 gene is that several androgen receptor elements (ARES) are located up-stream of the transcription start site and the first intron. 30 Lukassen et al 32 investigated ACE2 and TMPRSS2 expression levels and their distribution across cell types in lung tissue and in cells derived from subsegmental bronchial branches by single nuclei and single-cell RNA sequencing, respectively. TMPRSS2 is expressed in both tissues, in the subsegmental bronchial branches and ACE2 is predominantly expressed in a transient secretory cell type. Interestingly, these transiently differentiating cells show enrichment for pathways related to RHO GTPase function and viral processes suggesting increased vulnerability for SARS-CoV-2 infection. 32 Cleavage of the SARS-CoV S protein (SARS-S) by host cell proteases is essential for viral infectivity, and the responsible enzymes constitute potential targets for intervention. 33 The SARS-CoV can hijack two cellular proteolytic systems to ensure adequate processing of its S protein. 33 Cleavage of SARS-S can be facilitated by cathepsin L, a pH-dependent endo-/lysosomal host cell protease, upon uptake of virions into target cell endosomes. 33 Alternatively, TMPRSS2 and human airway trypsin-like protease (HAT) can activate SARS-S, presumably by cleavage of SARS-S at or close to the cell surface, and activation of SARS-S by TMPRSS2 allows for cathepsin L-independent cellular entry. 33 Both TMPRSS2 and HAT are expressed in ACE2-positive cells in the human lung and results obtained with surrogate cell culture systems suggest that TMPRSS2 might play a significant role in SARS-CoV spread in the human respiratory tract via two independent mechanisms: ACE2 cleavage by these proteases increases entry efficiency, while SARS-S cleavage by TMPRSS2 activates the S protein for cathepsin L-independent host cell entry. 33 Meanwhile, HAT and TMPRSS2 are known to cleave the glycoprotein haemagglutinin (HA) of influenza A viruses, a prerequisite for fusion between viral and host cell membranes and viral cell entry. 34 Hoffmann et al 35 suggested that the serine protease inhibitor Camostat mesylate, which blocks TMPRSS2 activity, has been approved in Japan for human use, but for an unrelated indication. This compound or related ones with potentially increased antiviral activity could thus be considered for controlled clinical trials of treatment for SARS-CoV-2-infected patients. 35 Interestingly, in lung cancer cell line, A549, and prostate cancer cell line, LnCap, TMPRSS2 exhibits an androgen-dependent pattern and activates protease-activated receptor 2 (PAR2), causing the upregulation of matrix metalloproteinase-2 (MMP-2) and MMP-9, both of which play key roles in the metastatic tumour cells. 30 SARS-CoV may enter human cells via two distinct pathways, dependent on the availability of human cell proteases required for virus activation. The first pathway is processed if no SARS-Sactivating proteases are expressed on the cell membrane. Upon binding of the virus envelope protein S to ACE2, viruses are taken up into endosomes (phagosomes), and in a second step SARS-S is cleaved and activated by pH-dependent cysteine protease cathepsin L (acid pH is necessary). The second pathway of activation can be pursued if the SARS-S activating protease TMPRSS2 is co-expressed with ACE2 on human membrane cells. Binding to ACE2 and processing by TMPRSSS2 are recognized to allow fusion at the human cell surface or upon uptake into cellular vesicles, but before transport of viruses into host cell endosomes.
7 Angiotensin II (Ang II) is the major biologically active molecule of the RAS, which is involved in the homeostasis of blood pressure through its effects on water and electrolyte balance, as well as peripheral vascular resistance. 30 Ang II is produced from Ang I. When bound to the angiotensin type 1 receptor (AT1R) on the vasculature, Ang II initiates vasoconstriction, whereas it elicits nitric oxide (NO)-mediated vasodilation via binding to the endotheliumlocated AT2R (cardioprotective effects). 30 Alternatively, cleavage of Ang II by ACE2 produces the heptapeptide angiotensin 1-7 (Ang 1-7), which binds to the MAS receptor (MASR) and initiates downstream vasodilator activity that can counteract the hypertensive effects of AT1R signaling. Ang 1-7, therefore, possesses cardioprotective properties through effects that can prevent and/or reverse heart failure and blunt hypertensive cardiac remodeling. 30 In addition to the systemic RAS, local RAS has been found in many tissues, including the heart, which functions both independently and in correlation with systemic RAS components. Importantly, in the heart, there is an alternate pathway for Ang II synthesis by the endopeptidase chymase, a serine protease that is predominantly found in mast cells located within the tissue interstitium. 30 Mast cells contain granules disposed into cytoplasm with cytokines (TNFα and VEGF) and proteases (including chymase, tryptase, carboxypeptidase) that are released during the inflammatory process, via a classically mediated ligand-dependent pathway. 30 Chymase acts in the same manner as ACE but with a 20-fold higher catalytic activity for the conversion of Ang I to Ang II. 30 In this context, chymase is believed to serve as a major Ang II-forming enzyme in the human heart and is reported to be involved in many pathological processes, such as hypertension, atherosclerosis, vascular proliferation, development of cardiomyopathies, myocardial infarction and heart failure, as well as cardiac fibrosis. 30 8 During Covid-19, by the end of the first week, the disease can progress to pneumonia, respiratory failure and death. 1 This progression is associated with extreme rise in inflammatory cytokines. 1 The median time from onset of symptoms to dyspnea is 5 days, hospitalization 7 days and acute respiratory distress syndrome (ARDS) 8 days. 1 Intensive care admission was needed in 25% to 30% of affected patients in published series. 1 Considering current knowledge, the SARS-CoV-2 may bind to ACE2 in human cell membranes and these receptors are internalized into the human cytoplasm, forming a phagosome. This exposes the ACE/Ang II/ATR1/ROS signalling pattern so contributing to amplification of acute and chronic inflammation, fibrogenesis and cellular proliferation. 30 However, up to now, the research medical literature was unable

| INNATE AND ADAPTATIVE IMMUNE RESPONSE TO VIRAL INFECTIONS
Interferons (IFN) contribute significantly to the host response to virus infections, and impaired interferon production from mononuclear cells has been associated both to asthma and allergic sensitization. 36 Inadequate production of IFN-λ1 by airway mononuclear cells has been linked to more severe human rhinovirus (HRV)-induced illness and obstructive patterns on lung function testing in allergic asthmatics compared with non-allergic controls. 36,37 Moreover, plasmacytoid dendritic cell (pDCs) from allergic patients produce lesser IFN-α upon toll-like receptor (TLR)-9 intracytoplasmic stimulation, 38 as observed during influenza virus infection where pDCs from asthmatic patients produced less IFN-α. 39 In the skin, 20% to 40% of mast cells are in close contact with macrophages in a conformation known as 'baseball glove and ball', as an immune sentinel in the dermis ( Figure 1). 40 Diffuse alveolar damage (DAD) is a histological pattern characterized by hyaline membranes, intra-alveolar edema, alveolar epithelial cell injury and neutrophilic inflammation. 41 DAD is a pathological pattern that can occur as a consequence of distinct forms of lung insult, such as bacterial and viral infections, connective tissue diseases and sepsis, and others. 41 41 Both DAD groups presented higher alveolar and airway densities of neutrophils and macrophages. However, there was a significantly higher expression of F I G U R E 1 Mast cell during degranulation process (anaphylactic type) in intimal contact with dermal dendrocyte. Mast cell resembles a ball in a baseball glove (dermal dendrocyte). Note that membrane flaps of dermal dendrocyte consistently shrouded mast cell membrane for 50% to 90% of their perimeter. This suggests the presence of functional interactions between these cells. Immunoelectron microscopy (ITM) technique (original magnification ×40 000) CD4+ and CD8+ T lymphocytes, dendritic cells, granzyme A+, and NK cell density in the parenchyma of patients who died as a consequence of influenza A (H1N1) pdm09 DAD, and there was down cell density of tryptase+ mast cells, dendritic cells+ and an increased number of IL17+ cells in the airways, compared with non-viral DAD cases and the controls. 41 Tang et al 42 47 In some patients, these aPL/anti-β 2 -GPI antibodies are transient and disappear within 2 or 3 months. 47 In some susceptible individuals, they persist and raise the question of whether infections may be a trigger for the development of aPL and anti-β 2 -GPI antibodies in autoimmune diseases. 47 Moreover, Smeeth et al 48  plasminogen activator inhibitor-1 (PAI-1), are risk factors for ischemic heart disease, and venous thromboembolism. 49 Then, as there is extensive crosstalk between inflammation and coagulation, it is likely the prothrombotic mechanisms in VARI could be further exacerbated in patients suffering from severe forms, as Covid-19. 50 Tan et al 50

T A B L E 3 Pathogenic distinct mechanisms between Macrophage Activation Syndrome and Catastrophic Antiphospholipid Syndrome
Macrophage Activation Syndrome Catastrophic Antiphospholipid Syndrome • The immune hyperactivation in which dysregulation of macrophages and lymphocytes leads to excessive cytokine production (cytokine storm) • These cytokines include but are not limited to interleukin-18 (IL- 18) and its downstream effector interferon-γ (IFN-γ) • Elevated levels of IFN-γ and its downstream effectors are well documented in patients with HLH and MAS • Chronic IL-18 elevation, potentially mediated by an epithelial inflammasome source, has been shown to play an important role in the pathogenesis of MAS • A total IL-18 level ≥ 24 000 pg/mL was shown to distinguish MAS from familial HLH with 83% sensitivity and 94% specificity • The immune dysregulation and severe inflammation that characterize MAS result in tissue infiltration by lymphocytes and histiocytes, leading to organ failure and potentially death • Antiphospholipid antibodies and complement activation are felt to play a central role • During infections, molecular mimicry may provide the trigger that unleashes the thrombotic storm characteristic of this condition • Regardless of the trigger, however, antiphospholipid antibodies have been postulated to mediate disease by activating platelets, inhibiting anticoagulants, inhibiting fibrinolysis, and activating the classical complement pathway • The alternative complement pathway can be activated • Complement activation is expected to contribute to a prothrombotic state through endothelial activation and apoptosis mediated by the release of tissue factor and other prothrombotic substances

| Hyperinflammation states
Systemic immune-mediated diseases (SIDs) include autoimmune and autoinflammatory diseases affecting at least two organ systems. 55 Autoinflammatory diseases refer to a growing family of conditions characterized by episodes of unprovoked inflammation in the absence of high autoantibody titres or autoreactive T lymphocytes, reflecting a primary innate immune system dysfunction. 55 Conversely, Therapy approach for Livedoid vasculopathy, their possible action mechanism and Covid-19 injury and obstruction, increased vascular permeability, perfusion failure, and organ dysfunction in sepsis and associated syndromes may be related to widespread endothelial apoptosis. HBO therapy attenuates: (a) inhibition of fibrinolysis ("tPA and #PAI-1), (b) activation of the coagulation system, and (c) thrombocytopenia and platelet hyper aggregation caused by zymosan 83

Hydroxychloroquine (+) References 84 and 85
Useful as immunomodulator, especially in cases with antiphospholipid antibodies. The antithrombotic effect of chloroquine analogues has been attributed to a range of mechanisms, including reduction in red blood cell aggregation, inhibition of platelet aggregation and adhesion, reduction in blood viscosity and enhancement of antiplatelet activity 86 Hydroxychloroquine and chloroquine were indicated for treat patients with COVID-19, under in vitro effects due to capacity as 87 : (a) an inhibitor of endocytic pathways through an elevation of endosomal pH, and (b) these drugs shown to interfere with the terminal glycosylation of angiotensin-converting enzyme-2 (ACE2), which acts as a plasma membrane receptor for both SARS-CoV and SARS-CoV-2. 87 (+/−) There is limited evidence of in vitro activity of CQ/HCQ against SARS-CoV-2. 88 A number of in vivo clinical trials are underway. 88 The empirical data available from two of these trials reveal conflicting results. Both trials are characterised by small numbers of participants (n = 30 and n = 36) and suffer methodological limitations. 88 No medium or long-term follow-up data is available 88

Folate (folic acid) (+) References 84 and 89
Reducing homocysteine serum levels 84,89 (−) Alteplase (+) Reference 84 Intravenous tissue plasminogen activator is recommended in a dose of 10 mg administered intravenously initially six hourly and subsequently once daily for 14 days. 84 Tissue plasminogen activator therapy may be considered especially in patients who have not responded to multiple conventional therapies. 84 After tissue plasminogen activator therapy, maintenance with anti-platelet or anticoagulant agents have to be continued 84 (−/+) There is evidence in both animals and humans that fibrinolytic therapy in Acute Lung Injury and ARDS improves survival, which also points to fibrin deposition in the pulmonary microvasculature as a contributory cause of ARDS and would be expected to be seen in patients with ARDS and concomitant diagnoses of DIC 90 Intravenous Immunoglobulin (+) 94,95 Then, the viruses spread through the bloodstream and mainly in the lungs, gastrointestinal tract, and heart, presumably concentrated in the tissues expressing ACE2, the receptor of SARS-CoV-2. This phase occurs around 7-14 after the onset of the symptoms when the virus starts a second attack, which is also the main cause of the aggravation of symptoms. 93 At this time, pulmonary lesions became worse, and chest CT scans show imaging changes consistent with COVID-19. 94  In AOSD, a systemic inflammatory disorder, with intermittent spiking fevers, arthralgias or arthritis, leukocytosis and hyperferritinemia, an evanescent salmon-pink macule often appears on the skin during the evening with mild or without pruritus. 59  Early diagnosis and treatment are essential for preventing a suboptimal outcome in MAS. 61 High dose of corticosteroids with or without cyclosporine A was reported as first-line therapy, followed by combinations with anakinra or tocilizumab. 61 IL-1β is generated through cleavage of pro-IL1β via the inflammasome, and plays a clear role in AOSD/MAS. 61 IL-18 is also generated via the inflammasome through the same mechanism and is typically also very high in AOSD/ MAS, reflecting inflammasome hyperactivity. 61 99 Procoagulant factors, such F I G U R E 5 Clinical outcomes in SARS-CoV-2 infected patients/Covid-19, immune system responses, systemic and possible cutaneous manifestations.① The outcome spectrum is probably related to intrinsic host factors. Other factors are adequate type I IFN response, blood group type, high levels of proteases as plasmin(ogen) in the serum, which may cleave a newly inserted furin site in the S (Spike) protein of SARS-CoV-2, extracellularly and increasing its infectivity and virulence. 101 The affinity to human ACE2 receptors and activity of TMPRSS2 protease transmembrane, which may also cleave angiotensin-converting enzyme 2 (ACE2) for augmented viral entry. 102 The capacity of the virus nonstructural proteins like ORF1ab, ORF7a and ORF8 and surface glycoprotein to bind with the human porphyrin, respectively, while ORF1ab, ORF10 and ORF3a proteins could co-ordinately attack β1-heme chain of the human red blood cells contribute to impair the normal oxygen and carbon dioxide changes between pulmonary alveoli and interstitial capillaries, producing hypoxia 103 inducing macrophages responses. ② Most subjects when infected by a down or moderate load of SARS-CoV-2 produce an adequate and early synthesis of IFNγ and type I IFNs, their distinct cells of innate and acquired immune system will respond with pro-inflammatory cytokines and oxidative metabolites causing symptoms and probably an adequate host response to conducted for a favorable clinical outcome. ③ In a selected group of patients, with moderate and severe Covid-19, some authors proposed that a genetic background in these subjects might determinate one new immune response as ④ 'second wave' of cytokines production, the 'CSS' in response to the SARS-CoV-2 infection, similar to Macrophage Activation Syndrome (MAS-like/sHLH). The CSS can be the result of rare homozygous genetic defects in perforin pathway proteins, as proposed by Cron and Chatham, 104 due to the similarities with infants with familial HLH. ⑤There is evidence of D-dimer level elevation in Covid-19 pneumonia which might represent an extension of this novel virally induced hyper-inflammatory pulmonary immunopathology to the adjacent microcirculation with extensive secondary fibrinolytic activation. 105 The MAS that supervenes Covid-19 pneumonia is probably related to 'virally-induced immunosuppression or Covid-19 immunodeficiency status', by the viral escape of the human immune pathways, playing a key role. 104 ⑥ In this setting of hypercoagulability state and MAS/sLHL-like milieu several dermatological conditions could be observed. CSS, cytokine storm syndrome; HLH, hemophagocytic lymphohistiocytosis as high levels of Lp(a), in some genetically predisposed subjects ( Figure 4) may trigger this manifestation of the disease, and must, therefore, be considered significant factors in the pathogenesis of viral-induced myocardial ischaemia. 100 The interplay among genetic factors, environmental aspects, host immune responses, viral evasive properties and systemic and cutaneous involvement on Covid-19 patients are illustrated in Figure 5.