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T Beyond the anti-viral therapy: A comprehensive review Jaume Alijotas-Reiga,b,c,⁎, Enrique Esteve-Valverded,e, Cristina Beliznaf,g,h, Albert Selva-O'Callaghana,c, Josep Pardos-Geaa,i, Angela Quintanaj, Arsene Mekiniank, Ariadna Anunciacion-Llunellj, Francesc Miró-Murj a Systemic Autoimmune Diseases Unit, Department of Internal Medicine-1, Vall d’Hebron University Hospital, Barcelona, Spain b Systemic Autoimmune Research Unit, Vall d’Hebron Reseacrh Institute, Spain c Department of Medicine, Universitat Autònoma de Barcelona, Barcelona, Spain d Department of Internal Medicine, Althaia Network Health, Manresa, Barcelona, Spain e Universitat Central de Catalunya, Spain f Vascular and Coagulation Department, University Hospital Angers, Angers, France g UMR CNRS 6015, Angers, France h INSERM U1083, Angers, France i Universitat Autònoma de Barcelona, Barcelona, Spain j Systemic Autoimmune Research Unit, Vall d’Hebron Research Institute, Barcelona, Spain k Service de Médecine Interne, Centre de référence AO Bradykiniques et compétence Maladies Auto-immunes FAI2R, Hôpital Saint Antoine Hôpitaux Universitaires de l'Est Parisien, Professeur des Universités-Praticien Hospitalier Sorbonne Université, France ARTICLE INFO ABSTRACT Keywords: Severe Acute Respiratory Syndrome related to Coronavirus-2 (SARS-CoV-2), coronavirus disease-2019 (COVID- Acute respiratory distress syndrome 19) may cause severe illness in 20% of patients. This may be in part due to an uncontrolled immune-response to COVID-19 SARS-CoV-2 infection triggering a systemic hyperinflammatory response, the so-called “cytokine storm”. The Cytokine storm reduction of this inflammatory immune-response could be considered as a potential therapeutic target against Immunosuppressive severe COVID-19. The relationship between inflammation and clot activation must also be considered. SARS-CoV-2 Treatment Furthermore, we must keep in mind that currently, no specific antiviral treatment is available for SARS-CoV-2. While moderate-severe forms need in-hospital surveillance plus antivirals and/or hydroxychloroquine; in severe and life-threating subsets a high intensity anti-inflammatory and immunomodulatory therapy could be a ther- apeutic option. However, right data on the effectiveness of different immunomodulating drugs are scarce. Herein, we discuss the pathogenesis and the possible role played by drugs such as: antimalarials, anti-IL6, anti- IL-1, calcineurin and JAK inhibitors, corticosteroids, immunoglobulins, heparins, angiotensin-converting en- zyme agonists and statins in severe COVID-19. Abbreviations: ACE-2, Angiotensin-converting enzyme-2; AD, Autoimmune diseases; ADE, Antibody dependent enhancement; ADRS, Acute distress respiratory syndrome; APC, Antigen-presenting cells; aPL, Antiphospholipid antibodies; CD, Cluster of differentiation or cluster of designation or classification determinant; CDC, Centres for disease control; COVID-19, Coronavirus disease 2019; CQ, Chloroquine; CyA, Cyclosporine A; FDA, Food and Drugs Administration; GCS, Glucocorticoids; HCQ, Hydroxychloroquine; HPS, Haemophagocytic syndrome; IFNγ, Interferon gamma; IL, Interleukin; JAK, Janus-Kinase family of enzymes (JAK1, JAK2, JAK3, TYK2); IVIG, Intravenous immunoglobulins; MDA5, Melanoma differentiation-associated gene 5; MHC-II, Major histocompatibility type-II; LMWH, Low-molecular weight heparin; MAS, Macrophage activation syndrome; MERS-CoV, Middle East Respiratory Syndrome Coronavirus; mTOR, Mammalian target of Rapamycin; NHC, National Health Council; NK, natural killer cells; NF-kβ, Nuclear Factor-Kβ; PIC, Pulmonary intravascular coagulation; PTE, Pulmonary thromboembolism; RA, Rheumatoid arthritis; SARS-CoV-2, Severe Acute Respiratory Syndrome Coronavirus-2; SLE, Systemic Lupus Erythematosus; TCZ, Tocilizumab; TLR, Toll-Like Receptor; TNF-α, Tumour necrosis factor-alpha; TRAASVIR, Thrombotic Risk Associated with Antiphospholipid Syndrome after Viral infection; TRALI, Transfusion- related acute lung injury; TGF-β, Transforming growth factor-beta; Tregs, Regulatory T-cells; WHO, World Health Organization ⁎ Corresponding author at: Systemic Autoimmune Diseases Unit, Department of Internal Medicine-1, Vall d'Hebron University Hospital, Passeig Vall d'Hebron 119- 129, 08035 Barcelona, Spain. E-mail address: jalijotas@vhebron.net (J. Alijotas-Reig). https://doi.org/10.1016/j.autrev.2020.102569 Received 14 April 2020; Accepted 15 April 2020 Available online 03 May 2020 1568-9972/ © 2020 Published by Elsevier B.V. J. Alijotas-Reig, et al. Autoimmunity Reviews 19 (2020) 102569 Table 1 neurologic dysfunction, coagulopathy, liver dysfunction, cytopenias, World Health Organization. Clinical management of severe acute respiratory pulmonary involvement, hypertriglyceridemia, hyperferritinemia, he- infection [SARI] when COVID-19 disease is suspected Interim guidance 13 mophagocytosis, and diminished NK cell activity [6]. According to Prof. March 2020 Shoenfeld [7], two more syndromes may present with similar severe Mild ARDS: 200 mmHg < PaO2/FiO2 a ≤ 300 mmHg [with PEEP or CPAP ≥5 clinical pictures and hyperferritinaemia: the catastrophic antipho- cmH2O, or non-ventilated] • spholipid syndrome and the adverse reaction to the biological com- Moderate ARDS: 100 mmHg < PaO2/FiO2 ≤ 200 mmHg [with PEEP ≥5 pound anti-CD-28 [7–9]. Interestingly, these syndromes share similar cmH2O, or non-ventilated] • clinical picture that severe COVID-19. Additionally, it has also been Severe ARDS: PaO2/FiO2 ≤ 100 mmHg [with PEEP ≥5 cmH2O, or non- ventilated] • When PaO2 is not available, SpO2/FiO2 ≤ 315 suggests ARDS suggested to test for soluble CD-163 (sCD-163), which represents [including in non-ventilated patients]. macrophage activation, the level of which was found to increase in MAS Oxygenation impairment in children: note OI = Oxygenation Index and and to parallel the ferritin level [10]. Whether the hyperferritinaemia is OSI = Oxygenation Index using SpO2. Use PaO2-based metric when available. If just an epiphenomenon that can help us for diagnostic, or the ferritin is PaO2 not available, wean FiO2 to maintain SpO2 ≤ 97% to calculate OSI or SpO2/FiO2 ratio: • Bilevel [NIV or CPAP] ≥ 5 cmH2O via full face mask: PaO2/ “per se” able to perpetuate the inflammatory loop is speculative. FiO2 ≤ 300 mmHg or SpO2/FiO2 ≤ 264 • Mild ARDS [invasively ventilated]: However, Ruscitti and Shoenfeld [11] have found that the H-chain of 4 ≤ OI < 8 or 5 ≤ OSI < 7.5 • Moderate ARDS [invasively ventilated]: the ferritin is able to activate macrophages to increase the secretion of 8 ≤ OI < 16 or 7.5 ≤ OSI < 12.3 Severe ARDS [invasively ventilated]: OI ≥ 16 inflammatory cytokines and eventually, ferritin. This pathway would be or OSI ≥ 12.3. Sepsis: Adults: life-threatening organ dysfunction caused by a common to all hyperferritinaemic syndromes, COVID-19 included [11]. dysregulated host response HPS and MAS could be triggered by an excessive immune response to a viral infection [6,12,13]. Different virus has been related to HPS, mainly Epstein-Barr, cytomegalovirus, other herpes viruses, and human 1. Introduction immunodeficiency virus [6,14]. Pathology similar to HPS was reported in both SARS-CoV and Middle East Respiratory Syndrome (MERS)-CoV Severe Acute Respiratory Syndrome related to Coronavirus-2 (SARS- infections [15,16]., and has been suggested in COVID-19 [5]. However, CoV-2), is the infectious agent causing coronavirus disease 2019 McGonagle et al [17] hypothesised that MAS related to severe SARS- (COVID-19) may cause systemic inflammatory response with severe CoV-2 infection has distinctive characteristics than classical MAS/HPS. acute respiratory syndrome [1,2] (Table 1). It emerged as an outbreak They argue that, at the beginning, the hyper-inflammatory reaction in early December 2019 in Wuhan, China [3] and from March 13 is a appears to be more confined to the lungs based on histopathological pandemic according to WHO criterion, carrying a mortality of ap- findings (see below). In addition, COVID-19 patients usually do not proximately 3,7% [4]. Some previous pathological conditions of the present lymphadenopathy or splenomegaly. Furthermore, pulmonary patients may have a major impact on survival [Table 2]. We must bear hemophagocytosis has not yet been reported in COVID-19 associated in mind that currently, no specific antiviral treatment is available for pneumonia [18]. COVID-19, and therefore further research into the pathogenesis of Since HPS and MAS are a hyperinflammatory syndrome, an extreme human coronavirus infection is imperative for identifying appropriate elevation in pro-inflammatory cytokine and acute phase reactants levels therapeutic targets. Right from the first time that the outbreak appeared are observed (the exception to this is the erythrocyte sedimentation in China, the importance of immunomodulation in the clinical pro- rate, which may be low due to hypofibrinogenaemia), among them: IL- gression of the infection was described [5]. Given the severity of the 1β, IL-2, IL-6, IL-7, G-CSF1, IP-10, MCP-1α, MIP-1α, TNF-α, and sIL2R immunological picture it has been suggested a series of etiopathogeneic [6,19,20]. A hyperinflammatory profile that resembles HPS is described mechanisms that are close to the ones involved in other im- in clinical features of hospitalised COVID-19 patients, [7,18]. In addi- munomediated disorders. tion, the binding of SARS-CoV-19 to the Toll Like Receptor (TLR) will be followed by the release of pro-IL-1b cleaved by caspase-1, will in- 2. SARS-CoV-2, cytokine storm and hyperinflammatory syndrome duce inflammasome and IL-1b activation, which is a mediator of lung inflammation. Clinical observations indicate that few patients' health One of the principal candidates to explain the catastrophic evolution worst at 6–8 days after infection. According to theory of disease toler- of some people infected with SARS-CoV-2 is the haemophagocytic ance the most severe cases of COVID-19 could be in part due to an syndrome (HPS) also named haemophagocytic lymphohistiocytosis [6] uncontrolled immune response to SARS-CoV-2 infection [21]. The re- and similarly, the macrophage activation syndrome (MAS), a subtype of duction of this inflammatory immune response could be considered as a HPS in which the syndrome develops in the background of autoimmune potential therapeutic target against severe COVID-19. Consequently, disease, particularly in adults, Still's disease, systemic lupus er- the proposal to treat severe COVID-19 with immunomodulatory ythematosus (SLE) and vasculitis [6,7]. HPS is a life-threatening dis- therapy as it is done in HPS is likely to be beneficial to tackle hyper- order characterized by unbridled activation of cytotoxic T lymphocytes, inflammation and eventually to ameliorate the severe clinical syndrome natural killer (NK) cells, and macrophages resulting in hypercytoki- [5,22]. Theoretically, corticosteroid treatment could have a role to nemia and immunomediated injury of multiple organs or systems [6]. suppress systemic and lung inflammation related to COVID-19. Never- Clinical and laboratory manifestations include fever, splenomegaly, theless, the therapeutic use of corticosteroids that has excellent phar- macological effects to suppress exuberant and dysfunctional systematic Table 2 inflammation is still controversial [22–25]. Thus, the current National Variables related to high risk to develop severe COVID⁎ Health Council (NHC) guideline emphasizes that the routine use of systematic corticosteroids is not recommended unless indicated for 1. Older age (> 65 years old) another reason. In this line, there was no available data showing that 2. High Sequential Organ Failure Assessment [SOFA] score 2. Comorbidities: the patients benefited from corticosteroid treatment in SARS-CoV or Hypertension MERS infection, which might be attributed to the suppression of im- Cardiovascular (mainly, coronary heart disease) mune response and virus clearance [23]. In addition, the use of the sera Diabetes Mellitus of immunised people for treating severe case of COVID-19 has also been 3. Laboratory test D-dimer levels >1 μg/mL on admission suggested. However, some concerns arise on this proposal. In relation with the discrepancy on the severity of cases observed with current ⁎ The role played by obesity, smoking, asthma, hyperferritinaemia and high COVID-19, one possible answer could be the role played by antibody IL-6 levels as risk factors for severe COVID are not clearly stablished to date. dependent enhancement (ADE) of SARS-CoV-2 [26]. Immunization 2 J. Alijotas-Reig, et al. Autoimmunity Reviews 19 (2020) 102569 with sera of patients that contains non-neutralising antibodies could were included for this review. enhance disease by promoting virus infection in monocytes/macro- phages or by inducing complement activation leading to vascular injury 4. Results and discussion such as systemic necrotising vasculitis and disseminated intravascular coagulation. Furthermore, ADE up-modulates the immune response and 4.1. Antimalarials can elicit sustained inflammation, lymphopenia, and cytokine storm, as documented in COVID-19 severe cases [26]. It could happen in relap- Old anti-infectious drugs, such as chloroquine (CQ) and hydroxy- sing COVID-19 patients possessing naturally acquired or passively ad- chloroquine (HCQ) firstly used as anti-malarial drugs and later as im- ministered antibodies against SARS-CoV-2, or in patients previously munomodulatory treatment for autoimmune and rheumatic diseases, exposed to other coronaviruses. A mechanism for ADE in SARS-CoV-2 mainly SLE and rheumatoid arthritis, have also shown a potential an- could be the evasion of neutralising antibodies against Spike protein tiviral effect against SARS and avian influenza H5N1. Their effects are produced in previous virus infection [27]. ADE in SARS-CoV-2 may related to the change of cell membrane pH necessary for viral fusion account for the geographic discrepancy in the pathogenesis. Thus, an and the interference with glycosylation of viral proteins. Furthermore, individualised monitoring of the harshest cases in order to identify CQ/HCQ appear to have a summatory anti SARS-CoV-2 effects when coagulopathies associated with hyperinflammatory syndrome and, administered with antivirals [29] and with azithromycin [30] We consequently, to treat them with immunosuppressing drugs could be a would stress on other possible pleiotropic effects of CQ/HCQ other than good approach. anti-infectious. Antimalarials have many anti-inflammatory, anti-ag- The advent of biologic therapies that target cytokines has expanded gregant and immune-regulatory properties: they inhibit phospholipase the armamentarium of treatments available for HPS/MAS and for si- activity, stabilize lysosomal membranes, block the production of several milarity for COVID-19, to include drugs that block IL-1, IL-2, IL-6, IL- pro-inflammatory cytokines and, in addition, impair complement-de- 18, and IFNγ [28]. In addition, other drugs capable to block other in- pendent antigen-antibody reactions [31,32]. Currently, at least twenty flammatory pathways such as anti-malarials, baricitinib or ruxolitinib clinical trials have already been registered to test the usefulness of CQ [Janus-kinase inhibitors] should also be considered. (Table 3). and HCQ for the treatment of COVID-19 [33]. The just finished Chinese clinical trial - ChiCTR2000029559 – have shown the potential of HCQ 3. Review of the literature in the treatment of COVID-19 [Chen Z, et al.: submitted]. In vivo re- sults, although promising, are limited to date. Thus, considering the 3.1. Search strategy and selection criteria antiviral and immunomodulatory properties of the anti-malarials and the pre-clinical evidence of effectiveness and safety from long-time Data for this review were obtained through a comprehensive lit- clinical use for other indications, clinical research on CQ/HCQ in pa- erature search using the keywords: “immunosuppressives”, “anti- tients with COVID-19 is warranted [34]. So, on the basis of the weak malarials”,” hydroxychloroquine” “chloroquine”, “anakinra”, “tocili- evidence available, treatment guidelines have already incorporated the zumab”,” corticosteroids”, “heparin” “low-molecular-weight heparin” use of CQ/HCQ for treating patients with COVID-19. Thus, HCQ asso- “immunoglobulins”, “sarilumab”, “JAK inhibitors”, “cyclosporine” ciated with other drugs could play a role in the treatment of SARS-2- “ACE inhibitors” “statins” “haemophagocytic syndrome” “acute re- CoV-19 infection [35]. spiratory distress syndrome”. The search was restricted to: “SARS-CoV- 2″, “COVID-19″, “treatment”, to identify articles published in English 4.2. Interleukin-6 blockade [anti-IL-6] from MEDLINE, PubMed and The Cochrane Library (January 2020–March 30th, 2016). Some interesting papers related to SARS-CoV, Tocilizumab (TCZ), is an anti-human IL-6 receptor monoclonal an- MERS, thrombosis-related viruses, cytokines and inflammation from tibody that inhibits signal transduction by binding sIL-6R and mIL-6R. 2003 were also reviewed and included according to their relevance. The main indication of the TCZ use is rheumatoid arthritis [36] and Clinical trials, case-control or cohort studies, brief reports, commu- giant-cell arteritis [37]. In 2017, the U.S. Food and Drug Administration nications, reviews and systematic reviews were included. Current na- (FDA) approved TCZ for the treatment of cytokine release syndrome tional guidelines on management of COVID-19 were also retrieved and (CRS) consisting of a systemic inflammatory response caused by the included (CDC, Australian, WHO, Spanish and Italian). The authors massive release of pro-inflammatory cytokines in response to iatrogenic reviewed the selected manuscripts and finally the most appropriated (e.g. CAR-T therapies) or infective stimuli [38]. In COVID-19 patients, IL-6 plasmatic levels were especially high in Table 3 List of drugs potentially useful for treating severe “cytokine storm” associated severe cases. Histological examination of lung tissue showed diffuse with COVID-19⁎ alveolar damage with cellular fibromyxoid exudates and interstitial mononuclear inflammatory infiltrates suggesting severe immune injury Antimalarials: hydroxychloroquine and chloroquine in a biopsy sample from a severe COVID-19 patient [39]. A case series IL-6 blockers: Tocilizumab and Sarilumab Calcineurin inhibitors: Cyclosporine A and Tacrolimus in 20 Chinese patients reported that TCZ allowed the lung lesion opa- IL-1 blocker: Anakinra, Canakinumab city to be erased in 19 patients (90.5%), oxygen intake lowered in 15 Heparins: low-molecular-weight and unfractionated heparin (75%) and oxygen stopped in one case. Moreover, elevated C-reactive Intravenous immunoglobulins (IVIG) protein decreased significantly in 84.2% of the patients, lymphocytes Hiperimmune immunoglobulins (neutralising antibodies) JAK inhibitors: Ruloxitinib, Bariticinib count normalised in 52.6% patients [40]. In China TCZ was recently Corticosteroids (methylprednisolone) approved for patients affected by severe SARS-CoV-2 pulmonary com- Statins plications by the National Health Commission of the People's Republic Recombinant human angiotensin-converting enzyme 2 (rhACE2) of China. Further, a randomised controlled clinical trial is ongoing in China (ChiCTR2000029765) [41]. In this way, sarilumab [Sanofi / ⁎ Other procedures such as CytoSorb® adsorber, that can lead to a reduction Regeneron] is an anti-human IL-6 receptor monoclonal antibody laun- of the circulating pro-inflammatory (and anti-inflammatory) cytokines could improve the course of the disease and the outcome of the patients when used ched for the treatment of rheumatoid arthritis. Sarilumab is able to together with other therapy. Currently there is a trial to study the efficacy of block the IL-6 as the form that TCZ does and could exert positive effects this procedure (University Hospital Freiburg, Germany NCT 04324528). in cases of COVID-19 with severe manifestations and high IL6 levels. Currently, a phase II-III clinical trial has started in United States of America and five European countries [42]. 3 J. Alijotas-Reig, et al. Autoimmunity Reviews 19 (2020) 102569 4.3. Interleukin-1 blockade (anti-IL-1) results of an off-the-shelf, currently available agent are encouraging because clinical trials could readily be undertaken in humans to treat Anakinra, an anti-IL-1, is another therapeutic option for treating autoimmune or inflammatory-based disorders [53]. Thus, a clinical patients suffering of severe COVID-19. It is a recombinant and slightly trial with ruxolitinib in patients with SARS-CoV-2/COVID-19 has been modified version of the human interleukin 1 receptor antagonist protein started with encouraging preliminary results. (Capoachiani E, et al.: (IL-1Ra). It is naturally secreted by monocytes and tissue macrophages unpublished results). that selectively binds to IL-R and modulates its activity. The blockade of IL-1 leads to the inhibition of inflammatory responses [43]. In a phase 3 4.7. Intravenous immunoglobulins / Hyperimmune immunoglobulin randomised controlled trial the IL-1 blockade (anakinra) in sepsis has been shown as beneficial with increased survival without increased High doses of intravenous immunoglobulins (IVIG) exert anti-in- adverse events [44]. Thus, considering the similar “cytokine storm” flammatory and immunomodulating effects. Applications involving between severe sepsis and severe COVID-19, anakinra may play a role immunoglobulin have expanded to include treatment for im- in the treatment of some severe or refractary cases. munodeficiency diseases, immune thrombocytopenia (ITP), Kawasaki disease, neurologic disorders, SLE and other severe or refractory auto- 4.4. Interleukin-2 inhibition immune diseases [54]. Among the multiple effects on the innate and adaptive immune pathways related to IVIG, doses >0,5 g per kg The cyclosporine-cyclophilin A complex inhibits a calcium/calmo- weight/day can interrupt the storm of inflammatory cytokines caused dulin-dependent phosphatase, calcineurin, the inhibition of which is by different stimuli. Although preliminary studies have shown efficacy thought to suppress organ rejection by halting the production of the of the IVIG in the treatment of patients with severe inflammatory pro-inflammatory molecules TNF-α and interleukin 2 (IL-2). Due to complaints related to influenza [52] and SARS-CoV [55] infections, we these effects, cyclosporine A (CyA) has been proven very useful in the need more clinical data of COVID-19 patients as evidence [56]. Cur- management of autoimmune diseases. Cyclophilin is also known to be rently, a randomised controlled clinical trial of IVIG in patients with recruited by the Gag polyprotein during HIV-1 virus infection, and its severe SARS-CoV-2 infection has been initiated (Clinical Trial.gov: NCT incorporation into new virus particles is essential for HIV-1 infectivity 04261426). We want to alert on the two potential and severe IVIG [45]. Experimental studies showed that cyclophilin inhibitors v.g. CyA adverse effects which could have a negative survival impact on patients exert and inhibitory effect on SARS-CoV through the calcineurin with severe COVID-19: a/ the transfusion (immunoglobulin)-related pathway inhibition that, at the same time, plays an important role in acute lung injury (TRALI), that may be a serious immunoglobulin the SARS-CoV virus replication. In addition, CyA and tacrolimus down- transfusion-related adverse effect with high mortality, manifests with modulated the calcineurin/Nuclear Factor of activated-T cells in- acute respiratory distress syndrome within 6 h of perfusion [57]. TRALI flammatory pathway induced by SARS- [46,47]. In this way, pathogenic is an immune-mediated process and the neutrophil-priming hypothesis similarities between severe pulmonary COVID-19 and the anti-mela- have been proposed as possible mechanisms [58,59]; b/ thrombotic noma differentiation-associated gene 5-positive (anti-MDA-5) amyo- events related to IVIG treatment with an estimated incidence of trophic dermatomyositis-associated rapidly progressive interstitial lung 1–16.9% [57]. According to the doubtful effectiveness of IVIG treat- disease could be taken in account. Interestingly, MDA5 cell sensor may ment in patients with SARS-CoV and the risk of severe lung injury and be activated by viruses [48]. CyA or tacrolimus with or without in- thrombosis [60], we think the IVIG option should be carefully analyzed travenous immunoglobulins (IVIG) is a mainstay of pharmacologic before its use in severe COVID-19 patients. In addition, the con- treatment of the anti-MDA5 syndrome [49]. In addition, cases of anti- valescent plasma (CP) and hyperimmune immunoglobulin (HIVIG) – MDA5 syndrome complicated by haemophagocytic syndrome have neutralising antibodies - have been tried for the treatment of severe been described [50]. Thus, according to the antiviral and anti-in- acute respiratory syndrome of viral etiology, including MERS-CoV and flammatory properties of calcineurin inhibitors, CyA and tacrolimus SARS-CoV-2 infection. Although preliminary reports have shown that could be potential effective drugs for treating the severe forms of CP/HIVIG are able to reduce the mortality, especially when adminis- COVID-19. tered early after symptoms onset, the real effectiveness is controversial, and this therapy should be evaluated within the context of a well-de- 4.5. Other cytokine-targeted therapy signed clinical trials [61–64]. In the same line, other targets could be therapeutic options such as 4.8. Anticoagulants: heparin and fondaparinux IL-37 and IL-38 [51]. IL-37 inhibits inflammation by acting on IL-18Ra receptor, on mTOR and increasing the adenosine monophosphate Severe SARS2-CoV-2 as well as other infections are associated with (AMP) kinase. IL-38 is also a suppressor cytokine which inhibits IL-1b clot pathway hyperactivity, probably related to pro-inflammatory state. and other pro-inflammatory IL-family members [51]. Treatment of WT High levels of D-dimer, which is indicative of the activation of the mice with recombinant human IL-37 has been shown to be protective in coagulation and/or thrombosis pathways as well as the risk of suffering several models of inflammation and injury. These cytokines might re- venous or pulmonary thromboembolism (PTE), are found high in present novel therapeutic targets in patients with systemic in- COVID-19 hospitalised patients. In addition, high levels of D-dimer are flammatory syndromes. related with poor-outcomes and high mortality rate [65–67]. Although D-dimer levels are elevated in most patients with blood clots, D-dimer 4.6. Janus kinase pathway [JAK] inhibition levels also are elevated in many other disorders including infection. In any event, multiple pulmonary embolus has also been observed in these JAK inhibition could affect both inflammation and cellular viral patients. [68,69]. Moreover, it has been observed that COVID-19 pa- entry in COVID-19. Therefore, Richardson et al. reported that bar- tients with severe type may develop disseminated intravascular coa- icitinib could be a potential treatment for acute respiratory disease gulation [DIC], PTE and arterial thrombosis [Esteve-Valverde E, et al.: related to SARS-CoV-2 infection [52]. The Janus kinase 1/2 inhibitor unpublished data], in similar way that occurred is SARS-CoV [67]. ruxolitinib, currently FDA approved in the USA for the treatment of Furthermore, different diagnostic approaches have been proposed in primary myelofibrosis, polycythemia vera and rheumatoid arthritis, has COVID patients with clinical suspicion of PTE [70]. Finally, data ob- been examined in a murine model of HPS. Mice with the manifestations tained from autopsies of 50 COVID-19 patients showed microthombosis of HPS were treated with ruxolitinib, with improvement in manifesta- and sometimes thrombus affecting large pulmonary arteries even in the tions and rapid decrease in serum IL-6 and TNF-α levels. Such positive superior cava vein and right auricula. [Gianatti&Sonzogni: unpublished 4 J. Alijotas-Reig, et al. Autoimmunity Reviews 19 (2020) 102569 results]. Interestingly histologic data such as alveolar and interstitial Prudent use with low-to-moderate doses and short courses of treatment inflammation extends to the closely juxtaposed pulmonary vasculature could be advised in selected cases [87,92,93]. According to Cochrane and the normal circulatory fibrinogen levels and regional fibrinolysis review [88], the WHO recommendations [91] and expert consensus with elevated D-dimer formation seen in early COVID-19 pneumonia statement from Chinese Thoracic Society [90], some basic principles are not a features of typical acute onset MAS/HPS [17]. This hyper- should be followed when using corticosteroids in severe COVID-19 inflammatory intra-pulmonary inflammation might influence a pro- patients: [a] benefits and risks should be carefully weighed before using pensity toward severe local vascular dysfunction including micro- GCS [b] GCS should be used carefully in critically ill patients with thrombosis and haemorrhage resulting in a lung centric pulmonary SARS-CoV-2 pneumonia; [c] for patients with hypoxemia due to un- intravascular coagulopathy (PIC) presentation rather than a DIC pre- derlying diseases or who regularly use corticosteroids for chronic dis- sentation [7]. eases, further use of corticosteroids should be cautious and [d] dosage Anticoagulation therapy is recommended for COVID-19 patients should be low to moderate (≤ 0.5–1 mg/ kg daily of methylpredniso- when high D-Dimer levels are detected, except for patients in whom lone or equivalent) and duration should be short (≤7 days). Thus, high anticoagulants are contraindicated [71]. doses or pulses of GCS are not recommended. Furthermore, WHO [82] Tang et al. [72] reported a major improvement of clot activation and CDC [93] recommend that corticosteroids not to be routinely used markers and a reduction of 28-days mortality when COVID-19 patients in patients with COVID-19 for treatment of viral pneumonia or ARDS were treated with heparin. The recommended dose of low-molecular- unless indicated for another reason. Finally, in cases of life-threatening weight heparin (LMWH) is 100 U per kg weight per 12 h by sub- septic COVID-19 patients, clinicians who consider to add corticoster- cutaneous injection at least 5 days. Clinicians should closely monitor oids to “goal standard” treatment should balance the potential small the laboratory values of patients to be alert for side effects after an- reduction in mortality with potential effects of prolonged coronavirus ticoagulant treatment. In addition, heparin exerts other pharmacolo- shedding. gical effects beyond its antithrombotic properties. Furthermore, a large body of evidence supports the concept that heparin has anti-in- 4.10. Other possible complementary nonviral drugs to be used: ACE agonists flammatory and immune-modulating properties. LMWH promotes sur- and statins vival of human endothelial cells undergoing apoptosis in response to TNF-α. Heparins are able to bind anionic molecules, such as aPL, and Patients with severe COVID-19 infection are at risk of acute re- block complement pathway activation [73–75]. Other anti-in- spiratory distress syndrome and death. Angiotensin-converting enzyme-2 flammatory effects of LMWH have been postulated to be specifically (ACE2) is a homologue of ACE, and functions as a negative regulator of TNF-α- mediated [76]. In patients with heparin allergy, fondaparinux is the renin–angiotensin system and it is expressed in the human lungs a good option. Fondaparinux is a synthetic pentasaccharide whose an- [94]. ACE-2 is also present in heart tissue. In addition, angiotensin- tithrombotic activity is the result of anti-thrombin-mediated selective converting enzyme II is a key molecule involved in the development and inhibition of Factor Xa [FXa]. Neutralization of FXa interrupts the blood progression of acute lung injury [95]. It is known that ACE-2 is a func- coagulation cascade and thus inhibits thrombin formation and further tional receptor for the SARS-coronavirus, SARS-CoV-2 included [96]. thrombus development [77]. In animal models, fondaparinux is able to SARS-CoV-2 infects ACE2+ cells in the oral mucosa and lungs, including prevent endothelial damage, and to bind anionic molecules such as ACE-2 cells in the alveoli [97]. Thus, SARS-CoV-2 induces direct lung β2GP-I and β2GP-I/anti-β2GP-I complexes [78]. Furthermore, Amara injury by involving ACE enzyme, which contributes to diffuse alveolar et al. [79] reported the capability of fondaparinux to block FXa-C3 damage, and high levels of ACE2 can protect against ARDS [98]. It is cleavage, and probably further delivery of C3a and C5a. In addition, worthy of mention that higher levels of ACE-2 and ACE2+ cells, higher diverse virus infections, SARS-CoV and SARS-CoV-2 included, appear to regenerative capacity and a strong immune response lead to an effective be associated with an antiphospholipid antibody positivity with po- viral clearance [99]. With age or when certain comorbidities are present, tential pathogenic effects [ [80], [81] and Esteve-Valverde et al.: such as hypertension or diabetes, the ACE-2 levels decrease with subse- TRAASVIR study: unpublished results]. quently slower in viral clearance, sustained ACE cell injury, lung in- Thus with the anticoagulant therapy we could kill two birds with flammation and risk of precipitating into ARDS [97,100]. Numerous one stone: to prevent thrombosis and to down-regulate the pro-in- agents have been shown to modulate and upregulated ACE2 expression, flammatory pathways. Nevertheless, only with clinical trials we will be including angiotensin peptides and some other peptide and steroid hor- able to solve this conundrum. mones [98]. Interestingly, ACE inhibitors and angiotensin II receptor blockers increase ACE2 levels. This fact could partially explain the re- 4.9. Glucocorticoids lationship between increased fatality rate of COVID-19 in patients with cardiovascular diseases, including hypertension [101]. Currently, a Glucocorticoids (GCS) exert inhibitory effects on a broad range of clinical trial (Clinical Trials.gov: NCT04287686) using recombinant innate and adaptive immune responses. Because of their inhibitory ef- human angiotensin-converting enzyme 2 (rhACE2) as a treatment for fects on multiple types of immune cells, GCS are remarkably efficacious patients with COVID-19 is ongoing. in managing many of the acute disease manifestations of inflammatory Statins induced potent inhibition via protein geranyl geranylation of and autoimmune disorders [82]. The anti-inflammatory and im- pro-inflammatory cytokine production (TNF-α, IL-10, IL-6 and IL-8) in munosuppressive effects of GCS rely on three main mechanisms that mononuclear, synovial and endothelial cells. Statins also inhibit T-cell include, a/ direct effects on gene expression by the binding of gluco- activation and proliferation, leading to the immunomodulatory effects. corticoid receptors to glucocorticoid-responsive elements, b/ indirect Furthermore, statins inhibit MHC-II expression on endothelial cells and effects on gene expression through the interactions of glucocorticoid monocyte-macrophages via inhibition of the promoter IV of the transac- receptors with other transcription factors i.e., NF-kβ and activator tivator and thereby repress MHC-II mediated T-cell activation [102,103]. protein 1, and c/ glucocorticoid receptor–mediated effects on second- In 2014, it was suggested that statins might be used to treat patients with messenger cascades [83]. GCS have been used with different success in Ebola virus disease [104]. A supply of a generic statin and a generic an- life-threatening conditions such as CID, sepsis and acute distress re- giotensin receptor blocker [ARB] was sent to Sierra Leone. Experimental spiratory syndrome (ADRS) [84–86], By its clinical similarity, GCS are studies had shown that both drugs improved outcomes in experimental currently empirically used in some hospitals in the treatment severe acute lung injury/ARDS models [105,106]. A far as we know no data on COVID −19 patients, and different therapeutic local guidelines have use of these drugs on SARS-2-CoV-19 patients have been published so far. included them [87]. However, existing evidence is inconclusive or does Clinical trials are needed to determine whether this drug combination not support for GCS treatment of COVID-19 patients to date [88–92]. might be used to treat patients with severe COVID-19 [107]. 5 J. Alijotas-Reig, et al. Autoimmunity Reviews 19 (2020) 102569 5. Infectious agents, cytokines and induction of autoimmunity autoimmune dysfunction and eventually AD. It is already known, even in individuals with appropriate genetic background, that environmental factors participate to trigger auto- 6. Conclusions immune disease (AD). It is also possible that infections accelerate an already stablished subclinical autoimmune disorder [108]. Diverse COVID-19 is a primarily respiratory tract infection with different mechanisms have been proposed i.e. molecular mimicry (cross-re- forms of clinical manifestations. While most infected people only de- activity between the microorganisms and host tissue), the production of velop mild illness, approximately 15–20% develop severe disease that superantigens, deviation of the balance between T-helper subsets to- requires hospitalization and 5% require admission to an intensive care ward Th1 or increase Th1/Th2/ regulatory T cells (Treg) ratios (loss of unit. In severe cases, COVID-19 with MAS occurs in patients with ARDS, active cytokine regulation) and the apparition of self-neoantigens sepsis and septic shock, and ultimately, multiorgan failure and death, [108,109]. Thus, a variety of hypotheses have been put forward to linked to sustained IL-6 and IL-1 elevation. explain the onset of AD, but all of them are dependent of the auto- While mild clinical forms only require symptomatic management, in reactivity of T and/or B lymphocytes that have escaped of the reg- moderate-severe forms in-hospital surveillance with general measures ulatory controls. The increased levels of Th1-derived cytokines, mainly plus antivirus and/or HCQ administration is necessary. However, in IL-1, IL-6, TNF-α and IFN-γ may be the effects of autoreactivity but, in more severe and life-threating cases, a high intensity pharmacological turn, may facilitate the loss of immune control and eventually the ap- treatment is recommended. The pathogenesis of the acute pulmonary parition of AD [109]. As an example, IL-6, a proinflammatory cytokine, injury related to COVID-19 is very similar that occur in other disorders also affects T cells. IL-6 is one of the factors that determine how naïve that induce high hyperinflammatory state with a release of high CD4+ T cells differentiate into particular effector T cell subsets. IL-6 in amounts of pro-inflammatory cytokine mainly, IL-1, IL-2, IL-6 and TNF- combination with transforming growth factor-beta (TGF)-β pre- α. A pro-thrombotic status appears later. Thus, drugs that usually serve ferentially induces differentiation into Th17 cells, whereas IL-6 inhibits to treat rheumatic or autoimmune syndromes may play a major role in TGFβ-induced Treg development. The resultant predominance of Th17 this setting. To date, only HCQ has proved to be useful for the treatment cells over Treg cells may be responsible for the breakdown of im- of severe cases of pneumonia related to COVID-19. Attention should be munological tolerance, and may therefore be pathologically involved in paid with cardiac side effects when high HCQ doses are administered in the development of AD [110]. Deregulated excessive IL-6 synthesis COVID patients. However, pre-clinical and few clinical made in patients during this protective process or persistent IL-6 production leads to the with severe COVID-19 show that intense immunosuppressive drugs development of a severe acute life-threatening complication, the so- improve clinical severity and reduce the mortality rate. Thus, antivirals called cytokine storm or AD respectively [111]. HLA-class II and par- and supportive measures apart, the combination of high HCQ doses plus ticularly HLA-DR are expressed constitutively mainly by antigen pre- immunomodulatory agents such as tocilizumab, cyclosporine or others senting cells (APC), and B cells and by some activated T cells. Their are warranted mainly in the context of clinical trials, in order to de- expression is essential for starting the adaptive immune response and monstrate a possible benefit in those severe COVID-19 patients. help to clear infections through it. Furthermore, HLA-DR over- If this schema fails, IVIG or short course of GCS can be tried. High expression on these cells or, the apparition “de novo” class-II-DR prophylactic or full heparin dose should be administered according to complex in other cells that usually lack HLA-DR, may facilitate its re- D-dimer levels. The role played by JAK-inhibitors, statins, or ACE-2- cognition as non-self cells and eventually, cause AD. IFN-γ leads to agonist is currently unknown. In addition, the effectiveness of the HLA-DR gene expression concomitant with inflammatory cytokine transfusion of hyperimmune plasma – neutralising antibodies -obtained genes such as IL-1 beta TNF-α, and IL-6 in vitro [112]. This MHC class II of cured COVID-19 patients is speculative. Attention should be paid upregulation increases MHC-restricted antigen presentation and adap- when neutralising antibodies are used, since the effectiveness or dele- tive immune response. In addition, IFN-γ can inhibit the differentiation terious effect can be time-dependent. Only randomised clinical trials and proliferation of Th2 cells, and the sustained response of Th1 cells is although difficult to perform in this context, would be the pathway to involved in the occurrence and development of AD. Many infectious exit from this labyrinth and allow the scientific community to affront agents like Epstein-Barr virus, CMV and parvovirus, among others, have been implicated in the pathogenesis of certain AD v.g. rheumatoid ar- Table 4 thritis (RA), SLE and psoriasis. Although the exact mechanism by which Recommended doses of drugs potentially useful for treating severe “cytokine pathogens causes pathology is unknown, presence of class II molecules storm” associated with COVID-19⁎. is mandatory [108,113]. Thus, certain viral infections, coronavirus Hydroxychloroquine phosphate: 400 mg tablets: 1 tablet q12 as loading dose, among them, are able to induce a high amount of proinflammatory followed by 200 mg tablets, 1 tablet q12, during 10 days, or 1 and half tablet q12 cytokines in predisposed host, that eventually may cause hyperferriti- during 7–10 days. Alternatively: Chloroquine phosphate 250 mg tablets, 2 tablet q12, during naemic syndrome (haemophagocityc/MAS-like syndrome) [7,108]. In 10 days. addition, the increase of Th1 and Th17-derived cytokines will induce, Heparin: LMWH at high prophylactic dose, i.e. enoxaparin 1 mg q24. Consider proinflammatory milieu apart, a decrease in IL-4, IL-10 and TGF-β with full anticoagulant dose if D-dimer >1500–3000 a subsequent decrease in total and functional Tregs (CD4 + CD25 and Tocilizumab#: 8 mg/kg (maximum 800 mg/dose), single dose intravenously (1-h CD4 + CD25 + FoxP3+) that limits the major arm to control self- infusion); in absence or with poor clinical improvement a second dose should be administered after 8–12 h (maximum recommended doses: 3) tolerance. Another issue that deserves some consideration is the role of IVIG§: 0.5–1.0 g/Kg (maxium doses: 2 g/kg) Treg cells in the severity of the COVID-19. It has been reported that Methtyl-prednisolone¶: 1 g/Kg q24 (IV) x 3 days, followed by 0.5 mg/kg q24 x severe cases present a lower proportion of naïve Tregs but a higher of 3 days. Alternatively: 250 mg q 24 × 3 d (IV) memory Tregs which in turn, although not demonstrated, probably may play a role leading to a high intensity autoimmune response [114]. ɸ Although lopinavir/ritonavir appears not to be effective, preliminary results with Remdesivir showed positive effect in 68% of cases [121]. Furthermore, IFN-γ is able to increase the expression of the class-II-DR #: In cases with plasmatic IL-6 leves ≥40 pg/mL. + molecules in immune cells and to induce their expression in non- §: Some authors recommended doses of 0.5–0.5 g/Kg q24 h per 3 days [122]. immune cells, thereby facilitating the apparition of auto-neoantigens, ¶: The is no agreement in its usual use. autoimmune response and eventually AD [115]. In conclusion, combi- Cyclosporin A, Anakinra and Canakinumab could empirically be administered if nation of the possible molecular mimicry (viral particles) plus cytokine tocilizumab fail or it cannot be used. imbalance [116] plus Treg cells decrease and class-II-DR molecules ⁎ See references: [82, 83, 90, 93, 117, 118, 119, 120]. Standard of care in- overexpression, are the “perfect storm” for a loss of self-tolerance, cludes: antiviralsɸ plus azithromycin plus hydroxychloroquine. 6 J. Alijotas-Reig, et al. Autoimmunity Reviews 19 (2020) 102569 this colossal challenge. In these lines, different trials involving hydro- of the iceberg. Eur J Rheumatol 2019 Dec 3:1–8. doi:.5152/eur- xychloroquine, tocilizumab, sarilumab, anakinra, immunoglobulins, jrheum.2019.19150. [14] Telles JP, de Andrade Perez M, Marcusso R, Correa K, Teixeira RFA, Tobias WM. plasma hyperimmune, cyclosporine A and ruloxitinib are ongoing or Hemophagocytic syndrome in patients living with HIV: a retrospective study. Ann just started. A possible therapeutic approach can be seen at Table 4. Hematol 2019 Jan;98(1):67–72. https://doi.org/10.1007/s00277-018-3500-9. Thus, we face a double edge sword when considering treatment with [15] Nicholls JM, Poon LL, Lee KC, Ng WF, Lai ST, Leung CY, et al. Lung pathology of fatal severe acute respiratory syndrome. Lancet. 2003;361:1773–8. immunosuppressive drugs in those patients. One the one hand it may be [16] Peiris JS, Lai ST, Poon LL, Guan Y, Yam LY, Lim W, et al. SARS study group. useful to control the inflammatory response that certainly may be Coronavirus as a possible cause of severe acute respiratory syndrome. Lancet. harmful for the patient, and on the other side, it could favour the virus 2003;361:1319–25. [17] McGonagle D, Sharif K, O’Regan A, Bridgewood C. The role of cytokines including shedding. However, taking in account the poor outcomes of these pa- interleukin-6 in COVID-19 induced pneumonia and macrophage activation syn- tients, and meanwhile we are waiting for more results based on clinical drome-like disease. Autoimmun Rev 2020. https://doi.org/10.1016/j.autrev. trials, our feeling is that immunosuppressors play a major role and that 2020.102537. [18] Xu Z, Shi L, Wang Y, Zhang J, Huang L, Zhang C, et al. Pathological findings of as earlier the immunosuppressive treatment is started the less compli- COVID-19 associated with acute respiratory distress syndrome. Lancet Respir Med cations and deaths there will be. The future will show us the correct 2020;8. https://doi.org/10.1016/S2213-2600(20)30076-X. 420–122. answer. [19] Nikiforow S, Berliner N. The unique aspects of presentation and diagnosis of he- mophagocytic lymphohistiocytosis in adults. Am Soc Hematol Educ Program 2015:183–9. Funding [20] Cron RQ, Davi S, Minoia F, Ravelli A. Clinical features and correct diagnosis of macrophage activation syndrome. Expert Rev Clin Immunol 2015;11:1043–53. There is no funding source. https://doi.org/10.1586/1744666X.2015.1058159. [21] Medzhitov R, Schneider DS, Soares MP. Disease tolerance as a defense strategy. Science. 2012;335(6071):936–41. https://doi.org/10.1126/science.1214935. Ethical approval [22] Li H, Yunjiao Zhou Y, Meng Zhang M, Haizhou Wang H, Qiu Zhao Q, Liu J. Updated approaches against SARS -CoV – 2. Antimicrob Agents Chemother 2020. https://doi.org/10.1128/AAC.00483-20. This article does not contain any studies with human participants or [23] Russell CD, Millar JE, Baillie JK. Clinical evidence does not support corticosteroid animals performed by any of the authors. treatment for 2019-nCoV lung injury. Lancet. 2020 Feb 15;395(10223):473–5. https://doi.org/10.1016/S0140-6736[20]30317-2. [24] Martinez MA. Compounds with therapeutic potential against novel 336 respiratory Declaration of Competing Interest 2019 coronavirus. Antimicrob Agents Chemother 2020;337. https://doi.org/10. 1128/AAC.00399 -20. [25] Huang C, Wang Y, Li X, Ren L, Zhao J, Hu Y, et al. Clinical features of patients The author also state that they do not have any commercial or any infected with 2019 novel coronavirus in Wuhan, China. Lancet 2020;395:497–506. other type of interest that may have influenced the drawing up and the [26] Tetro JA. Is COVID-19 receiving ADE from other coronaviruses? Microbes Infect results of this paper. 2020 Mar;22(2):72–3. https://doi.org/10.1016/j.micinf.2020.02.006. [Epub 2020 Feb 22]. [27] Yang ZY, Werner HC, Kong WP, Leung K, Traggiai E, Lanzavecchia A, et al. Evasion Acknowledgements of antibody neutralization in emerging severe acute respiratory syndrome cor- onaviruses. Proc Natl Acad Sci U S A 2005 Jan 18;102(3):797–801. The authors thank Ms. Christine O'Hara for reviewing and cor- [28] Halyabar O, Chang Margaret H, Schoettler Michelle L. Calm in the midst of cy- tokine storm: a collaborative approach to the diagnosis and treatment of hemo- recting the style and grammar of the manuscript. phagocytic lymphohistiocytosis and macrophage activation syndrome. Pediatric Rheumatol 2019;17:7–13. References [29] Zhou D, Dai SM, Tong Q. COVID-19: a recommendation to examine the effect of hydroxychloroquine in preventing infection and progression. J Antimicrob Chemother 2020 Mar 20. https://doi.org/10.1093/jac/dkaa114. pii: dkaa114. [1] Chan JF, Yuan S, Kok KH, To KK, Chu H, Yang J, et al. A familial cluster of [Epub ahead of print]. pneumonia associated with the 2019 novel coronavirus indicating person-to- [30] Gautret P, Lagier JC, Parola P, Hoang VT, Meddeb L, Mailhe M, et al. person transmission: a study of a family cluster. Lancet 2020;395:514–23. Hydroxychloroquine and azithromycin as a treatment of COVID-19: results of an [2] Chen N, Zhou M, Dong X, Qu J, Gong F, Han Y, et al. Epidemiological and clinical open-label non-randomized clinical trial. Int J Antimicrob Agents 2020 characteristics of 99 cases of 2019 novel coronavirus pneumonia in Wuhan, China: Mar;20:105949. https://doi.org/10.1016/j.ijantimicag.2020.105949. [Epub a descriptive study. Lancet 2020;395:507–13. ahead of print]. [3] Huang Y, Tu M, Wang S, Chen S, Zhou W, Chen D, et al. Clinical characteristics of [31] Wozniacka A, Lesiak A, Narbutt JM, DP McCauliffe, Sysa-Jedrzejowska A. laboratory confirmed positive cases of SARS-CoV-2 infection in Wuhan, China: A Chloroquine treatment influences proinflammatory cytokine level in systemic retrospective single center analysis Travel. Med Infect Dis 2020 Feb;27:101606. lupus erythematosus patients. Lupus 2006;15:268–75. https://doi.org/10.1016/j.tmaid.2020.101606. [32] Rynes RI. Antimalarial drugs. In: Kelley WN, Harris ED, Ruddyn S, Sledge CB, [4] WHO Coronavirus Disease. [COVID-19] situation report-52. March 12, 2020. editors. Textbook of Rheumatology. 4th ed.Philapelphia: W.B. Saunders; 1993. p. http://www.who.int/docs/default-source/coronaviruse/20200312; 2019 (ac- 731–42. cessed March 13, 2020). [33] Qi Zhang Q, Yakun Wang Y, Changsong Qi C, Shen L, Li J. Clinical trial analysis of [5] Mehta P, McAuley DF, Brown M, Sanchez E, Tattersall RS. COVID-19: consider 2019-nCoV therapy registered in China. J Med Virol 2020. https://doi.org/10. cytokine storm syndromes and immunosuppression. Lancet 2020. https://doi.org/ 1002/jmv.25733 Available at:. Acccessed on April 6. 10.1016/S0140-6736[20]30628-0. published Online March 13, 2020. [34] Cortegiani A, Ingoglia G, Ippolito M, Giarratano A, Einav S. A systematic review on [6] Hanny Al-Samkari H, Berliner N. Hemophagocytic lymphohistiocytosis. Annu Rev the efficacy and safety of chloroquine for the treatment of COVID-19. J Crit Care Pathol Mech Dis 2018;13:27–49. 2020 Mar 10. https://doi.org/10.1016/j.jcrc.2020.03.005. pii: S0883–9441[20] [7] Shoenfeld Y. Corona (COVID-19) time musings: our involvement in COVID-19 30390–7. [Epub ahead of print]. pathogenesis, diagnosis, treatment and vaccine planning. Autoimmun Rev 2020. [35] Colson P, Rolain JM, Lagier JC, Brouqui P, Raoult D. Chloroquine and hydroxy- https://doi.org/10.1016/j.autrev.2020.102538. chloroquine as available weapons to fight COVID-19. Int J Antimicrob Agents 2020 [8] Agmon-Levin N, Rosario C, Porat-Katz B-S, Zandman-Goddard G, Meroni P, Mar;4:105932. https://doi.org/10.1016/j.ijantimicag.2020.105932. Cervera R, et al. Ferritin in the antiphospholipid syndrome and its catastrophic [36] Scott LJ. Tocilizumab: a review in rheumatoid arthritis. Drugs 2017 Nov. https:// variant (cAPS). Lupus 2013;22:1327–35. doi.org/10.1007/s40265-017-0829-7. 7717]1865–1879. [9] Suntharalingam G, Perry MR, Ward S, Brett SJ, Castello-Cortes A, Brunner MD, [37] Serling-Boyd N, Stone JH. Recent advances in the diagnosis and management of et al. Cytokine storm in a phase 1 trial of the anti-CD28 monoclonal antibody giant cell arteritis. Curr Opin Rheumatol 2020 Mar 9;32:201–7. https://doi.org/ TGN1412. N Engl J Med 2006;355(10):1018–28. 10.1097/BOR.0000000000000700. [10] Colafrancesco S, Priori R, Alessandri C, Astorri E, Perricone C, Blank M, et al. [38] Drug Approval Package: ACTEMRA tocilizumab–FDA. Available on: fda.gov/ sCD163 in AOSD: a biomarker for macrophage activation related to hyperferriti- drugsatfda_docs/label/2017/125276s114lbl.pdf; 2020 Accessed March 28, 2020. nemia. Immunol Res 2014;60:177–83. [39] Zhang H, Zhou P, Wei Y, Yue H, Wang Y, Hu MD, et al. Histopathologic Changes [11] Ruscitti P, Berardicurti O, Cipriani P, Iagnocco A, Shoenfeld Y. Severe hyper-in- and SARS–CoV-2 Immunostaining in the lung of a patient with COVID-19. Ann flammatory COVID-19, another piece in the puzzle of the “htperferritinemic syn- Intern Med 2020. https://doi.org/10.7326/M20-0533. drome”. The rheumatologist’s point of view. 2020. (Submitted for publication). [40] Xu Xiaoling, Han Mingfeng, Li Tiantian, Sun Wei, Wang Dongsheng, Fu Binqing, [12] Crayne CB, Albeituni S, Nichols KE, Cron RQ. The immunology of macrophage et al. Effective treatment of severe COVID-19 patients with tocilizumab. chinaXiv activation syndrome. Front Immunol 2019 Feb 1;10:119. https://doi.org/10. 2020 Apr 29. https://doi.org/10.1073/pnas.2005615117. 202003.00026v1, pii: 3389/fimmu.2019.00119. [eCollection 2019]. 202005615. [13] Crayne C, Cron RQ. Pediatric macrophage activation syndrome, recognizing the tip [41] Chinese Clinical Trial Registry. A multicenter, randomized controlled trial for the 7 J. Alijotas-Reig, et al. Autoimmunity Reviews 19 (2020) 102569 efficacy and safety of tocilizumab in the treatment of new coronavirus pneumonia [68] Ng KHL, Wu AKL, Cheng VCC, Tang BSF, Chan CY, Yung CY, et al. Pulmonary [COVID-19]. http://www.chictr.org.cn/showprojen.aspx?proj=49409; 2020 artery thrombosis in a patient with severe acute respiratory syndrome. Postgrad Date: Feb 13, Date accessed: March 6, 2020. Med J 2005;81:e3https://doi.org/10.1136/pgmj.2004.030049. [42] European Pharmaceutical Review Report. Global trial to evaluate Kevzara® [sar- [69] Danzi GB, Loffi M, Galeazzi G, Gherbesi E. Acute pulmonary embolism and COVID- ilumab] as COVID-19 therapy initiated Available at: https://www. 19 pneumonia: a random association? Eur Heart J 2020 Mar 30. https://doi.org/ europeanpharmaceuticalreview.com/news/116003/global-trial-to- 10.1093/eurheartj/ehaa254. pii: ehaa254. evaluatekevzara-sarilumab-as-covid-19-therapy-initiated/; 2020 [accessed on [70] Zuckier LS, Moadel RM, Haramati LB, Freeman LM. Diagnostic evaluation of April, 4, 2020]. pulmonary embolism during the COVID-19 pandemic. J Nucl Med 2020. https:// [43] Calabrese LH. Molecular differences in anticytokine therapies. Clin Exp Rheumatol doi.org/10.2967/jnmed.120.245571. 2003;21:241–8. [71] Li T, Lu H, Zhang W. Clinical observation and management of COVID-19 patients. [44] Shakoory B, Carcillo JA, Chatham WW, Ambdur RC, Zhao H, Dinarello CA, et al. Emerg Microbes Infect 2020 Dec;9(1):687–90. https://doi.org/10.1080/ Interleukin-1 receptor blockade is associated with reduced mortality in sepsis 22221751.2020.1741327. patients with features of macrophage activation syndrome: reanalysis of a prior [72] Tang N, Bai H, Chen X, Gong J, Li D, Sun Z. Anticoagulant treatment is associated phase iii trial. Crit Care Med 2016;44:275–81. with decreased mortality in severe coronavirus disease 2019 patients with coa- [45] Thali M, Bukovsky A, Kondo E, et al. Functional association of cyclophilin a with gulopathy. J Thromb Haemost 2020 Mar 27. https://doi.org/10.1111/jth.14817. HIV-1 virions. Nature. 1994;372:363–5. https://doi.org/10.1038/372363a. [73] Oberkersch R, Attoresi A, Calabrese GC. Low-molecular-weight heparin inhibition [46] Pfefferle S, Schöpf J, Kögl M, Friedel CC, Müller MA, Carbajo-Lozoya J, et al. The in classical complement activation pathway during pregnancy. Thromb Res SARS-coronavirus-host interactome: identification of cyclophilins as target for 2010;125:e240–5. pan-coronavirus inhibitors. PLoS Pathog 2011 Oct;7(10):e1002331. https://doi. [74] Lim W. Complement and the antiphospholipid syndrome. Curr Opin Hematol org/10.1371/journal.ppat.1002331. 2011;18:361–5. [47] Channappanavar R, Perlman S. Pathogenic human coronavirus infections: causes [75] Girardi G, Redecha P, Salmon JE. Heparin prevents antiphospholipid-induced fetal and consequences of cytokine storm and immunopathology. Semin Immunopathol loss by inhibiting complement activation. Nat Med 2004;10:1222–6. 2017;39:529–39. [76] Carr JA, Cho JS. Low molecular weight heparin suppresses tumour necrosis factor [48] Romero-Bueno F, Diaz del Campo P, Trallero-Araguás E, Ruiz-Rodríguez JC, expression from deep vein thrombosis. Ann Vasc Surg 2007;21:50–5. Castellvi I, Rodriguez-Nieto MJ, et al. On behalf of the MEDRA5 [Spanish MDA5 [77] Samama MM, Gerotziafas GT. Evaluation of the pharmacological properties and Register] group. Recommendations for the treatment of anti-melanoma differ- clinical results of the synthetic pentasaccharide [fondaparinux]. Thromb Res entiation-associated gene 5-positive dermatomyositis-associated rapidly pro- 2003;109:1–11. gressive interstitial lung disease. Semin Arthritis Rheum 2020. [in press]. [78] Banzato A, Frasson R, Acqusaliente L, et al. Circulating B2glycoprotein I-IgG anti- [49] Griger Z, Nagy-Vincze M, Dankó K. Pharmacological management of dermato- B2glycoprotein I immunocomplexes in patients with definite antiphospholipid myositis. Expert Rev Clin Pharmacol 2017 Oct;10(10):1109–18. https://doi.org/ syndrome. Lupus 2012;21:784–6. 10.1080/17512433.2017.1353910. [Epub 2017 Jul 17]. [79] Amara U, Fliert MA, Rittirsch D, Klos A, Chen H, Acker B, et al. Molecular inter- [50] Fujita Y, Fukui S, Suzuki T, Ishida M, Endo Y, Tsuji S, et al. Anti-MDA5 antibody- communications between the complement and coagulation systems. J Immunol positive dermatomyositis complicated by autoimmune-associated 2010;185. 5268–36. Hemophagocytic syndrome that was successfully treated with immunosuppressive [80] Sun W, Wang BL, Liu BL, Zhao FC, Shi ZC, Guo WS, et al. Osteonecrosis in patients therapy and plasmapheresis. Intern Med 2018;57:3473–8. https://doi.org/10. after severe acute respiratory syndrome [SARS]: possible role of anticardiolipin 2169/internalmedicine.1121-18. [Epub 2018 Jul 6]. antibodies. J Clin Rheumatol 2010 Mar;16(2):61–3. https://doi.org/10.1097/ [51] Conti P, Ronconi G, Caraffa A, Gallenga CE, Ross R, Frydas I, et al. Induction of RHU.0b013e3181cf3464. pro-inflammatory cytokines [IL-1 and IL-6] and lung inflammation by COVID-19: [81] Zhang Y, Xiao M, Zhang S, Xia P, Cao W, Jiang W, et al. Coagulopathy and anti- anti-inflammatory strategies. J Biol Regul Homeost Agents 2020;34:1–42. phospholipid antibodies in patients with Covid-19. N Engl J Med 2020. https:// [52] Richardson P, Griffin I, Tucker C, Smith D, Oechsle O, Phelan A, et al. Baricitinib as doi.org/10.1056/NEJMc2007575. potential treatment for 2019-nCoV acute respiratory disease. Lancet. [82] Vandewalle J, Luypaert A, De Bosscher K, Libert C. Therapeutic mechanisms of 2020;395(10223):e30–1. glucocorticoids. Trends Endocrinol Metab 2012;9. https://doi.org/10.1016/j.tem. [53] Elli EM, Baratè C, Mendicino F, Palandri F, Palumbo GA. Mechanisms underlying 2017.10.010. 42–. the anti-inflammatory and immunosuppressive activity of ruxolitinib. Front Oncol [83] Rhen T, Cidlowski JA. Antiinflammatory action of glucocorticoids— new me- 2019;9:1186. https://doi.org/10.3389/fonc.2019.01186. chanisms for old drugs. N Engl J Med 2005;353:1711–23. https://doi.org/10. [54] Galeotti C, Kaveri SV, Bayry J. IVIG-mediated effector functions in autoimmune 1056/nejmra050541. and inflammatory diseases. Int Immunol 2017;29:491–8. [84] Matthay MA, Zemans RL, Zimmerman GA, Arabi YM, Beitler JR, Mercat A, et al. [55] Hung IFN, To KKW, Lee C-K, et al. Hyperimmune iv immunoglobulin treatment: a Acute respiratory distress syndrome. Nat Rev Dis Primers 2019;5(1):18. https:// multicenter double-blind randomized controlled trial for patients with severe 2009 doi.org/10.1038/s41572-019-0069-0. Mar 14. influenza a [H1N1] infection. Chest 2013;144:464–73. [85] Lamontagne F, Rochwerg B, Lytvyn L, Guyatt G, Moller MH, Annane D, et al. [56] Lai ST. Treatment of severe acute respiratory syndrome. Eur J Clin Microbiol Infect Corticosteroid therapy for sepsis: a clinical practice guideline. BMJ. 2018;362:1–8. Dis 2005;24:583–91. https://doi.org/10.1136/bmj.k328430097460. [57] Li T, Lu H, Zhang W. Clinical observation and management of COVID-19 patients. [86] Nicastri E, Petrosillo N, Ippolito G, D’Offizi G, Marchioni L, Ascoli Bartoli T, et al. Emerg Microbes Infect 2020 Dec;9(1):687–90. https://doi.org/10.1080/ National Institute for the Infectious Diseases “L. Spallanzani”, IRCCS 22221751.2020.1741327. Recommendations for COVID-19 Clinical Management. Infectious disease report. [58] Reddy DR, Guru PK, Blessing MM, Stubbs JR, Rabinstein AA, Wijdicks EF. 2020. p. 1–28. Transfusion-related acute lung injury after IVIG for myasthenic crisis. Neurocrit [87] Russell CD, Millar JE, Baillie JK. Clinical evidence does not support corticosteroid Care 2015;23:259–61. https://doi.org/10.1007/s12028-015-0115-z. treatment for 2019-CoV lung injury. Lancet 2020;395:473–5. https://doi.org/10. [59] Vlaar AP, Juffermans NP. Transfusion-related acute lung injury: a clinical review. 1016/S0140-6736[20]30317-2. PMID: 32043983. Lancet 2013;382(9896):984–94. https://doi.org/10.1016/S0140-6736[12] [88] Cochrane Database Syst Rev. Pharmacological agents for adults with acute re- 62197-7. spiratory distress syndrome. 2019 Jul 23. https://doi.org/10.1002/14651858. [60] Woodruff RK, Grigg AP, Firkin FC, Smith IL. Fatal thrombotic events during CD004477.pub3. PMID: 31334568. treatment of autoimmune thrombocytopenia with intravenous immunoglobulin in [89] Shang L, Zhao J, Hu Y, Du R, Cao B. On the use of corticosteroids for 2019-nCoV elderly patients. Lancet 1986;2(8500):217–8. https://doi.org/10.1016/S0140- pneumonia. Lancet. 2020;395:683–4. https://doi.org/10.1016/S0140-6736[20] 6736[86]92511-048. 30361-5. [Epub 2020 Feb 12]. [61] Cunningham AC, Goh HP, Koh D. Treatment of COVID-19: old tricks for new [90] National Health Commission & State Administration of traditional Chinese medi- challenges. Crit Care 2020;24:91–2. cine. Diagnosis and treatment protocol for novel coronavirus pneumonia. From [62] Mair-Jenkins J, Saavedra-Campos M, Baillie JK, et al. The effectiveness of con- China consulate website. Accessed 2020 Mar 20 http://busan.china-consulate.org/ valescent plasma and hyperimmune immunoglobulin for the treatment of severe chn/zt/4/P020200310548447287942.pdf; 2020. acute respiratory infections of viral etiology: a systematic review and exploratory [91] Brody B. Prednisone and coronavirus: Do corticosteroids Make You im- meta-analysis. J Infect Dis 2015;211:80–90. munosuppressed and Higher Risk for COVID-19? Available at. creacyjoints.org; [63] Arabi YM, Hajeer AH, Luke T, Raviprakash K, Balkhy H, Johani S, et al. Feasibility 2020 Accessed on March 30, 2020. of using convalescent plasma immunotherapy for MERS-CoV infection. Saudi [92] World Health Organization. Clinical management of severe acute respiratory in- Arabia Emerg Infect Dis 2016;9:1554–61. fection [SARI] when COVID-19 disease is suspected Interim guidance. 13 March [64] Shen C, Wang Z, Zhao F, Yang Y, Li J, Yuan J, et al. Treatment of 5 critically ill 2020. patients with COVID-19 with convalescent plasma. JAMA. 2020. https://doi.org/ [93] Interim clinical guidance for management of patients with confirmed coronavirus 10.1001/jama.2020.4783. disease [COVID-19]. https://www.cdc.gov/coronavirus/2019-ncov/hcp/clinical- [65] Heng M, Yu H. Characteristics, causes, diagnosis and treatment of coagulation guidance-management-patients.html; 2020. dysfunction in patients with COVID-19. Chin J Hematol 2020;41. https://doi.org/ [94] Tipnis S. R. Hooper NM, Hyde R, Karran E, Christie G, Turner AJ. A human 10.3760/cma.j.issn.0253-2727.2020.0002. homolog of angiotensin-converting enzyme. Cloning and functional expression as a [66] Zhou F, Yu T, Du R, Fan G, Liu Y, Liu Z, et al. Clinical course and risk factors for captopril-insensitive carboxypeptidase. J Biol Chem 2000;275:33238–43. https:// mortality of adult inpatients with COVID-19 in Wuhan, China: a retrospective doi.org/10.1074/jbc.M002615200. cohort study. Lancet 2020;95:1054–62. https://doi.org/10.1016/S0140-6736[20] [95] Imay Y, Kuba K, Rao S, Huan Y, Guo F, Guan B. Angiotensin-converting enzyme 2 30566-3. protects from severe acute lung failure. Nature 2005;436:112–6. https://doi.org/ [67] Stockman LJ, Bellamy R, Garner P. SARS: system rev treat effects. PLoS Med 10.1038/nature03712. 2006;9. e343. [96] Li W, Moore MJ, Vasilieva N, Sui J, Wong SK, Berne MA. Angiotensin-converting 8 J. Alijotas-Reig, et al. Autoimmunity Reviews 19 (2020) 102569 enzyme 2 is a functional receptor for the SARS coronavirus. Nature. [110] Kishimoto T, Kang S, Tanaka T. IL-6: a new era for the treatment of autoimmune 2003;426(6965):450–4. inflammatory diseases. In: Nakao K, Minato N, Uemoto S, editors. Innovative [97] Xudong X, Junzhu C, Xingxiang W, Furong Z, Yanrong L. Age- and gender-related medicine Tokyo: Springer; 2015. p. 131–47. https://doi.org/10.1007/978-4-431. difference of ACE2 expression in rat lung. Life Sci 2006;78:2166–71. https://doi. [111] Kimura A, Kishimoto T. IL-6: regulator of Treg/Th17 balance. Eur J Immunol org/10.1016/j.lfs.2005.09.038. 2010;40:1830–5. [98] Clarke NE, Turner AJ. Angiotensin-converting enzyme 2: the first decade. Int J [112] Hamano H, Haneji N, Yanagi K, Ishimaru N, Hayashi Y. Expression of HLA-DR and Hypertens 2012;2012:307315. cytokine genes on interferon-γ-stimulated human salivary gland cell line. [99] Felice Rivellese F, Prediletto E. ACE2 at the Centre of COVID-19 from pauci- Pathobiology 1996;64:255–61. symptomatic infections to severe pneumonia. Autoimmun Rev 2020. https://doi. [113] Taneja V. Cytokines pre-determined by genetic factors are involved in pathogen- org/10.1016/j.autrev.2020.102536. esis of rheumatoid arthritis. Cytokine. 2015;75:216–21. https://doi.org/10.1016/ [100] Xudong X, Junzhu C, Xingxiang W, Furong Z, Yanrong L. Age- and gender-related j.cyto.2014.11.028. difference of ACE2 expression in rat lung. Life Sci 2006;78:2166–71. https://doi. [114] Ritchie AI, Singanayagam A. Immunosuppression for hyperinflammation in org/10.1016/j.lfs.2005.09.038. COVID-19: a double-edged sword? Lancet. 2020;395(10230):1111. https://doi. [101] Cao Y, Li L, Feng Z, et al. Comparative genetic analysis of the novel coronavirus org/10.1016/S0140-6736(20)30691-7. [2019-nCoV/SARS-CoV-2] receptor ACE2 in different populations. Cell Discov [115] Rosa FM, Fellous M. Regulationof HLA-DRgene by IFN-gamma. Transcriptional 2020;6:11. https://doi.org/10.1038/s41421-020-0147-1. and pos-transcriptional control. J Immunol 1988;140:1660–4. [102] Satoh M, Takahashi Y, Tabuchi T, et al. Cellular and molecular mechanisms of [116] O’Shea J, Ma A, Lipsky P. Cytokines and autoimmunity. Nat Rev Immunol statins: an update on pleiotropic effects. Clin Sci (Lond) 2015;129:93–105. 2002;2:37–45. https://doi.org/10.1038/nri702. [103] Esteve-Valverde E, Ferrer-Oliveras R, Gil-Aliberas N, Baraldès-Farré A, Llurba E, [117] World Health Organization. Clinical management of severe acute respiratory in- Alijotas-Reig J. Pravastatin for preventing and treating preeclampsia: a systematic fection [SARI] when COVID-19 disease is suspected Interim guidance 13 March. review. Obstet Gynecol Surv 2018 Jan;73(1):40–55. 0.1097/ 2020. OGX.0000000000000522. [118] Inpatient guidance for treatment of covid-19 in adults and children. University of [104] Fedson DS. A practical treatment for patients with Ebola virus disease. J Infect Dis Michigan; 2020http://www.med.umich.edu/asp/pdf/adult_guidelines/COVID- 2015;21:661–2. https://doi.org/10.1093/infdis/jiu474. 19-treatment.pdf Accessed on March 31th, 2020. [105] Fedson DS, Opal SM, Rordam OM. Hiding in plain sight: an approach to treating [119] The Australian and New Zealand Intensive Care Society [ANZICS]. COVID-19 patients with severe COVID-19 infection. mBio 2020;11. https://doi.org/10.1128/ Guidelines. Version 1. 16 March Available at: www.anzics.com.au/wp-content/ mBio.00398-20. e00398-20. uploads/2020/03/ANZICS-COVID-19-Guidelines-Version-1.pdf; 2020 Accessed on [106] Shyamsundar M, McKeown STW, O’Kane CM, Craig TR, Brown V, Thickett DR, March 30, 2020. et al. Simvastatin decreases lipopoly-saccharide-induced pulmonary inflammation [120] Alhazzani W, Møller MH, Yaseen M, Arabi YM, Loeb M, Gong MN, et al. Surviving in healthy volunteers. Am J Respir Crit Care Med 2009;179:1107–14. https://doi. sepsis campaign: Guidelines on the management of critically ill adults with cor- org/10.1164/rccm.200810-1584OC. onavirus disease 2019 [COVID-19] Intensive Care Med. 2020. https://doi.org/10. [107] Shen L, Mo H, Cai L, Kong T, Zheng W, Ye J, et al. Losartan prevents sepsis- 1007/s00134-020-06022. induced acute lung injury and decreases activation of nuclear factor kappa/β and [121] Grein J, Ohmagari N, Shin D, Diaz G, Asperges E, Castagna A, et al. Compasionate mitogen-activated protein kinases. Shock 2009;3:500–6. https://doi.org/10.1097/ use of remdesivir for patients with severe Covid-19. N Engl J Med 2020(April 10). SHK.0b013e318189017a. https://doi.org/10.1056/NEJMoa2007016. [108] Shepshelovich D, Shoenfeld Y. Prediction and prevention of autoimmune diseases: [122] Cao W, Liu X, Bai T, Fan H, Hong K, Song H, et al. High-dose intravenous im- additional aspects of the mosaic of autoimmunity. Lupus 2006;15:183–90. munoglobulin as a therapeutic option for deteriorating patients with coronavirus [109] Volpe R. Autoimmune diseases. Encyclopedia of life sciences. London: Nature disease 2019. Open Forum Infect Dis 2020 Mar 21;7(3). https://doi.org/10.1093/ Publishing Group; 2001. ofid/ofaa102. ofaa102. eCollection 2020. 9