Associations between immune-suppressive and stimulating drugs and novel COVID-19—a systematic review of current evidence

Background Cancer and transplant patients with COVID-19 have a higher risk of developing severe and even fatal respiratory diseases, especially as they may be treated with immune-suppressive or immune-stimulating drugs. This review focuses on the effects of these drugs on host immunity against COVID-19. Methods Using Ovid MEDLINE, we reviewed current evidence for immune-suppressing or -stimulating drugs: cytotoxic chemotherapy, low-dose steroids, tumour necrosis factorα (TNFα) blockers, interlukin-6 (IL-6) blockade, Janus kinase (JAK) inhibitors, IL-1 blockade, mycophenolate, tacrolimus, anti-CD20 and CTLA4-Ig. Results 89 studies were included. Cytotoxic chemotherapy has been shown to be a specific inhibitor for severe acute respiratory syndrome coronavirus in in vitro studies, but no specific studies exist as of yet for COVID-19. No conclusive evidence for or against the use of non-steroidal anti-inflammatory drugs (NSAIDs) in the treatment of COVID-19 patients is available, nor is there evidence indicating that TNFα blockade is harmful to patients in the context of COVID-19. COVID-19 has been observed to induce a pro-inflammatory cytokine generation and secretion of cytokines, such as IL-6, but there is no evidence of the beneficial impact of IL-6 inhibitors on the modulation of COVID-19. Although there are potential targets in the JAK-STAT pathway that can be manipulated in treatment for coronaviruses and it is evident that IL-1 is elevated in patients with a coronavirus, there is currently no evidence for a role of these drugs in treatment of COVID-19. Conclusion The COVID-19 pandemic has led to challenging decision-making about treatment of critically unwell patients. Low-dose prednisolone and tacrolimus may have beneficial impacts on COVID-19. The mycophenolate mofetil picture is less clear, with conflicting data from pre-clinical studies. There is no definitive evidence that specific cytotoxic drugs, low-dose methotrexate for auto-immune disease, NSAIDs, JAK kinase inhibitors or anti-TNFα agents are contraindicated. There is clear evidence that IL-6 peak levels are associated with severity of pulmonary complications.


Introduction
Since the outbreak of severe acute respiratory syndrome (SARS)-CoV-2 or COVID-19 coronavirus started in China in December 2019, there is increasing evidence that those with existing comorbidities, older age or a compromised immune system are at higher risk of developing severe and even fatal respiratory diseases [1]. Cancer and transplant patients are also considered to be in this risk group [2,3], especially as they may be treated with immune-suppressive or immune-stimulating drugs.
The current review focuses on the effects of immune-suppressive or immune-stimulating drugs on the host immunity against COVID-19. Here, we report a short introduction to each drug, followed by a summary of the results from the identified studies for each relative drug.
We hypothesise that the drugs selected will likely be categorised into one of two groups: 1) those that may be harmful for patients and put them at risk of increased morbidity/mortality associated with COVID-19 infection and 2) those that may be used to treat the immunopathology associated with severe persistent viral infection. The clinical impact of this review is, thus, twofold. It aims to identify which drugs clinicians should be thinking about taking patients off to protect them from increased harm from COVID-19 and also which drugs could be potentially beneficial in the fight against the disease.
This review covers the information available today. As the COVID-19 pandemic progresses, there is an opportunity and responsibility to collect prospective data using established randomised controlled trial involving drugs of interest and cohort-based translational studies.

Methods
A search was conducted using Ovid MEDLINE and a total of 89 studies were included in this review for 10 different types of immunesuppressing or immune-stimulating drug groups. Table 1 shows the search terms and the number of studies included in the review. Low-dose steroids/ NSAIDs (SARS-CoV-2 or coronavirus or COVID or betacoronavirus or MERS-CoV or SARS-CoV) AND (anti-inflammatory or ibuprofen or isobutylphenylpropionic acid or cortisone or non-steroidal anti-inflammatory)

Table 1. Search terms and the number of studies included for each investigated drug group. (continued)
IL-6 blockade Two different search strategies were explored due to the number of agents that block the IL-6: 1. ((SARS-CoV-2 or coronavirus or COVID or betacoronavirus or MERS-CoV or SARS-CoV) AND (Anti-IL-6 therapy or anti-interleukin-6 receptor antibody or anti-interlukin-6 therapy or interlukin 6 blockage or interlukin-6 blockage or IL 6 blockage or IL-6 blockage or tocilizumab or siltuximab or Sylvant or sarilumab or olokizumab or CDP6038 or elsilimomab or BMS-945429 or ALD518 or sirukumab or CNTO 136 or CPSI-2364 or ALX-0061 or clazakizumab or olokizumab or sarilumab or sirukumab or ARGX-109 or FE301 or FM101)) 2. ((SARS-CoV-2 or coronavirus or COVID or betacoronavirus or MERS-CoV or SARS-CoV) and (IL-6 or interlukin 6 or Anti-IL-6 therapy or IL-6 blockage or tocilizumab or siltuximab)) Anti-CD20 ((SARS-CoV-2 or coronavirus or COVID or betacoronavirus or MERS-CoV or SARS-CoV) AND (anti-cd20 monoclonal antibodies or anti-cd20 or rituximab or truxima or zevalin or ruxience or rituxan or arzerra or gazyva) The only other study to investigate a non-steroidal anti-inflammatory drug was one which looked at indomethacin [13]. This study suggested that indomethacin exhibited potent antiviral activity against canine coronavirus (dramatically inhibiting virus replication and protecting the host cell from virus-induced damage). This activity was also observed against human SARS-CoV at a concentration dose of 1 mg/kg.
In general, there appeared to be a few positive results for the use of corticosteroids in viral infections such as SARS-CoV [14][15][16][17][18][19]. Corticosteroids were widely used during the SARS-CoV outbreak due to their known ability to modulate a variety of involved cytokines (including IL-1, IL-6, IL-8, IL-12 and TNFα) [14,16]. Various studies in humans noted that corticosteroids appeared effective in reducing immunopathological damage but concerns centred around the promotion of viral rebound and association with adverse events (including acute respiratory distress syndrome) [14]. One laboratory study which treated porcine respiratory coronavirus infected pigs with dexamethasone suggested that one or two doses of the corticosteroid in the acute phase of infection may effectively alleviate early pro-inflammatory response, but prolonged administration may play a role in enhancing viral replication [18]. A separate Chinese study, which separated SARS-CoV patients into 4 treatment groups, identified early high-dose steroids in combination with a quinolone as producing the best patient outcomes [19]. Nevertheless, one review stated that the WHO does not currently recommend corticosteroids in other viral diseases such as Dengue as the 'glucocorticoid-mediated stimulation of the hypothalamic-pituitaryadrenal axis can also drive lymphocytopenia, or it may promote exaggerated pro-inflammatory responses that eventually cause a worsening of the pathogenic condition' [20].

Conclusion
The current literature does not give conclusive evidence for or against the use of NSAIDs in the treatment of COVID-19 patients, though there appears to be some evidence that corticosteroids may be beneficial in the treatment of SARS-CoV. However, it is important to note this is not specific to COVID-19.

TNFa blocker
TNF family of receptors and cytokines is very large and are often the targets for drugs. One example are TNFa inhibitors, which act by supressing the physiologic response to TNFa. TNFa is a pro-inflammatory cytokine involved in autoimmune and immune-mediated disorders such as rheumatoid arthritis, ankylosing spondylitis, inflammatory bowel disease, psoriasis, hidradenitis suppurativa and refractory asthma. Inhibitors of TNFa may be used in their treatment.

Results
A total of three studies were identified, of which two were deemed suitable for inclusion (Supplementary Table 3). The first study was a research letter that suggested that TNFa has been implicated in the severe immune-based pulmonary injury caused by SARS coronavirus, suggesting that TNFa inhibitors could be a potential treatment for the acute respiratory disease syndrome caused by coronavirus [21] The second study utilised 22 piglets to assess the efficacy of an anti-TNFa)therapy for endotoxin respiratory diseases and observed that TNFa blockade was not associated with decrease in disease severity [22].

Conclusion
Currently, there is no evidence indicating that TNFa blockade is harmful to patients in the context of COVID-19.

Results
A total of 108 studies were identified from the second search strategy, 23 were suitable for inclusion (Supplementary Table 4). The first search strategy found no hits.

IL6 an actor in the pathogenetic mechanisms of the coronavirus infection
• COVID-19 induces a pro-inflammatory generation and secretion of cytokines including IL-1b and IL-6 via the toll like receptors (TLR) that causes the production of active mature IL-1b which is a mediator of lung inflammation, fever and fibrosis. Anti-inflammatory cytokines, such as IL1-Ra, IL-37 or IL-38 could potentially provide relief in both systemic inflammation and fever occurring after infection [24]. • Cytokine profiles in patients diagnosed with SARS showed marked elevation of T-helper lymphocyte type 1 (Th1) cytokine interferongamma (IFN-γ), inflammatory cytokines IL-1β, IL-6 and IL-12 for at least two weeks after disease onset. Children however presented a much milder cytokine and chemokine storm [16]. • The high levels of IL-6 in the acute stage associated with lung lesions found in SARS patients are activated by the viral nucleocapsid SARS-CoV N protein [25]. • Over induction of inflammatory cytokine and dysregulation of cytokine signalling has been observed in patients with SARS in comparison with other respiratory viruses including respiratory syncytial virus (RSV), influenza A virus (FluAV), and human parainfluenza virus type 2 (hPIV2). SARS-CoV and RSV induced high levels of IL-6 and RANTES compared with FluAV and hPIV2 [26]. • The N-protein of SARS-CoV induces pulmonary inflammatory reaction and acute lung injury, which were related to the increase and imbalance of pro-inflammatory and anti-inflammatory cytokines. Glucocorticoids could effectively alleviate the pulmonary inflammatory reaction induced by N-protein of SARS-CoV [15]. • SARS-CoV does not productively infect human macrophages (Mphi) or dentritic cells (DCs), however it modulates a massive release of IL-6 and IL-12 and compromises the endocytic capacity (e.g., antigen capture capture) of Mphi was significantly compromised [27]. • Changes in plasma T helper (Th) cell cytokines, inflammatory cytokines and chemokines in 20 patients diagnosed with SARS were assessed. The elevation of Th1 cytokine IFN-γ, inflammatory cytokines IL-1, IL-6 and IL-12 and chemokines IL-8, MCP-1 and IP-10 confirmed the activation of Th1 cell-mediated immunity and hyper-innate inflammatory response in SARS through the accumulation of monocytes/macrophages and neutrophils [17].

IL-6 as a potential marker of disease severity in coronavirus infected patients
• IL-6 blood measurements seem useful to diagnose severe COVID-19 cases. The findings suggest that IL-6 and D-Dimer level can be used to estimate the severity of COVID-19. The optimum critical point of IL-6 in the group was 24.3 pg/ml, which was the upper limit of no severe pneumonia [28]. • The increased expression of IL-2R and IL-6 in serum is expected to predict the severity of the 2019-nCoV pneumonia and the prognosis of patients [29]. • The serum levels of IL-6 and CXCL-10 were significantly elevated in MERS-CoV patients who developed severe diseases [30]. • A new lethal animal model was characterised for SARS-CoV. Strain v2163 had nine mutations that increased levels of IL-1alpha and IL-6 in mice. The high IL-6 expression was correlated with mortality [31]. • SARS vaccination was tested in a murine SARS model. A high level of IL-6 and on days 2 and 3 after SARS-CoV infection was closely linked to the virus replication and disease severity [32] • Interleukin-6 (IL-6) and IL-8 are key SARS-CoV-induced epithelial cytokines capable of inhibiting the T-cell-priming ability of dendritic cells, a cellular element of the host innate defenses against respiratory infections, leading to an exacerbated inflammatory cascades and severe tissue damage in SARS patients [33].
• In patients with a diagnosis of SARS-associated coronavirus infection, there were no significant differences in peak levels of IL-6, IL-8 and TNFα between patients with and without acute respiratory distress syndrome. However , CRP and TNFα were associated with worse outcomes and might be used as prognostic markers of SARS [34]. • IFN-gamma, IL-18, TGF-beta, IL-6, IP-10, MCP-1, MIG were highly elevated in the acute phase sera of Taiwan SARS patients, being IL-18, IP-10, MIG, and MCP-1 were significantly higher in the death group than in the survival group. It suggests that an interferongamma-related cytokine storm was induced post SARS coronavirus infection [35]. • Eight patients with SARS [36] were treated with ribavirin, which was not effective in reducing the SARS coronavirus load in three of eight. Elevated levels of interleukin (IL)-6 and IL-8 subsequent to the peak viral load were found in eight and six cases [37]. • A cytokine profiling was performed for 110 serum from healthy donors, patients with SARS, patients with severe SARS, and patients with SARS in convalescence. IL6 concentrations were significantly elevated in severe SARS patients, but the IL-6 concentrations were similar in convalescent patients and control subjects which suggested that IL6 is associated with SARS severity [38]. • The authors of this study set out to study the inflammatory cytokine profile in children with SARS. They found that the plasma concentrations' key proinflammatory cytokines, including IL-6, were not substantially increased in any of the patients throughout the course of illness. From this, the authors stated that the cytokine results cast doubt on the liberal use of corticosteroids in paediatric SARS patients [39].

Potential targets for therapeutic intervention related to IL6 regulation during infection
• ADAM-17 regulates IL-6 class switching as a mediator between pro-and anti-inflammatory responses to viral antigenic stimuli in Ebola, SARS-CoV and dengue infections in humans. Therefore, ADAM-17 should be considered as a potential target molecule for novel antiviral drug discovery for infections, such as SARS-CoV [20] • SARS-CoV ssRNA is a new therapeutic target given its capacity to cause acute lung injury in mice with a high mortality rate in vivo experiment suggesting that SARS-CoV specific GU-rich ssRNA plays a very important role in the cytokine storm associated with a dysregulation of the innate immunity [40]. • DUSP1 and p38 MAPK are potential therapeutic targets for coronavirus infectious bronchitis virus, given their capability to reduce the production of an excessive amount of IL-6 and IL-8 in the infected cells [41].

Potential therapeutic agents that inhibit the infection-induced production of IL6
• Pretreatment of primary cultures of human nasal and tracheal epithelial cells with glycopyrronium or formoterol decreased viral RNA levels and/or titers, the expression of the HCoV-229E and the infection-induced production of cytokines, including IL-6, IL-8 and IFNbeta. Treatment of the cells with the CD13 inhibitor 2'2'-dipyridyl decreased viral titers. Pretreatment of the cells with a combination of three drugs (glycopyrronium, formoterol and budesonide) exerted additive inhibitory effects on viral titers and cytokine production [42].

Conclusion
It has been suggested that the pathogenesis of SARS-CoV is mediated by disproportional immune responses and the ability of the virus to circumvent innate immunity. The COVID-19 infection has also been observed to induce pro-inflammatory cytokine generation and secretion of cytokines, such as IL-6, which dysregulates the local inflammatory responses that have been suggested as partially responsible for the devastating acute respiratory distress syndrome.
Moreover, it has been observed that COVID-19 induces high levels of IL-6 for at least 2 weeks after disease onset. Children presented lower levels of cytokine production. IL-6 has been suggested as a potential prognostic marker of COVID-19 disease severity. Different molecules related with the IL6 pathway have been suggested as potential therapeutic targets such as ADAM-17, SARS-CoV ssRNA, DUSP1 and p38 MAPK.
Currently, there is no scientific evidence of the beneficial impact of IL-6 inhibitors in the modulation of the COVID-19 infection. Further understanding of the role of IL-6 reduction will be forthcoming as the pandemic progresses and further clinical data becomes available. In vitro treatment with glycopyrronium, formoterol and budesonide exerted additive inhibitory effects on viral titers and cytokine production human nasal and tracheal epithelial cells.

JAK inhibitors
JAK inhibitors work by inhibiting the activity of one or more of the JAK family of enzymes, including, JAK1, JAK2, JAK3 and TYK3. JAKs interact with signal transducer and activator of transcription proteins (STATs) and the JAK-STAT pathway is central to cellular response to exogenous signals in the immune system. The JAK family of enzymes are responsible for signal transduction and JAK inhibitors play a major role in inhibiting and blocking cytokine release that can contribute to growth of malignant cells in cancer. JAK inhibitors are used in the treatment of cancer and inflammatory diseases such as rheumatoid arthritis [43].
This review focuses on how the JAK-STAT pathway can be manipulated to reduce viral entry and inflammation in patients with coronavirus. The main targets that the review highlighted were baricitinib (JAK inhibitor), IRE1α (an endoplasmic reticulum stress sensor, leading to an increased expression of negative regulators of JAK-STAT SOCS1 and SOCS3) and combination therapies using tylophorine-based compounds with JAK2 inhibitors.

Results
Fifteen studies were yielded from the search of which 4 were deemed suitable for inclusion (Supplementary Table 5).

Baricitinib
Baricitinib (Olumiant) is a JAK inhibitor that was approved by the European Medicines Agency in February 2017 for the treatment of moderate-to-severe active rheumatoid arthritis in adults with an inadequate response to one or more disease-modifying anti-rheumatic drugs [44]. One study suggested the use of Baricitinib to reduce the viral entry and inflammation caused by 2019-nCoV. Most viruses enter cells through receptor-mediated endocytosis. ACE2, a cell-surface protein expressed on cells in the kidney, blood vessels, heart, and alveola type 2 (AT2) cells in the pulmonary epithelia, may be the receptor that 2019-nCoV uses to infect lung cells. The authors of Richardson et al [45] suggest that by inhibiting adaptor associated protein kinase 1 (AAK1) receptor that promotes endocytosis involved in ACE2, Baricitinib may reduce both the viral entry and the inflammation in 2019-nCoV patients.

IRE1α
Inositol-requiring transmembrane kinase/endoribonuclease 1α (IRE1α) is an endoplasmic reticulum stress sensor that leads to increased expression of negative regulators of JAK-STAT, suppressor of cytokine signalling (SOCS)-1 and SOCS-3 [46]. Therefore, IRE1α may be a novel target against coronavirus infection requiring further exploration.

Tylophorine-based compounds
Tylophorine-based compounds are isolated from plants and exert potent anti-coronaviral activities against SARS-CoV and MERS-CoV [47]. Nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) activation is a common pro-inflammatory response of host cells to viral infection. Following in vitro analysis, Yang et al [48] suggests the use of a combination therapy for SARS-CoV or MERS-CoV, wherein a tylophorine compound known to target transmissible gastroenteritis virus and a JAK2 inhibitor synergise to block the alternative dominant NF-κB activation mediated by JAK2. Therefore, the combination treatment for the inhibition of coronavirus per se, e.g. viral genome replication, and blocking cellular NF-κB activation by coronaviruses, is a promising approach for the development of anti-coronavirals.

Conclusion
Current studies suggest that although there are potential targets in the JAK-STAT pathway that can be manipulated in the treatment for coronaviruses, they are all in early stages and require further in vitro and in vivo studies to confirm their therapeutic effects.

IL-1 blockade
IL-1 is a pro-inflammatory cytokine and an important mediator of local and systemic inflammation. Excessive IL-1 release during viral infections can cause lung and tissue inflammation, fever and fibrosis. IL-1 suppression has found to be effective in many inflammatory diseases including rheumatoid arthritis [24].
It is well established that an over-expression of interleukin-1 is a hallmark of SARS-CoV infection, probably through activation of transcription factor nuclear factor, activator protein 1 and activating factor 2. In COVID-19 specifically, the virus is thought to bind to TLRs which activate the formation of pro-IL-1 and activation of the inflammasome [24]. This inflammasome activation is important for the regulation of cells of both the innate and adaptive immune system paving the way for specific immune responses. As part of the inflammasome activation, IL1-b is subsequently produced which mediates the inflammation of the lungs, fever and fibrosis thus causing respiratory complications in the infected host.

Results
A total of 37 studies were identified from the search of which 9 were deemed suitable for inclusion (Supplementary Table 6). Many of the studies were in vitro studies and repeatedly demonstrated increased IL-1 levels in patients infected with a coronavirus. One study investigated the levels of various inflammatory cytokines in 29, COVID-19 patients in China and compared the levels between general, severe and critically ill groups [49]. The authors reported no significant differences in IL-1b levels between the three groups of patients. Another study involving 20 consecutive SARS patients admitted to a Hong Kong hospital identified significantly elevated levels of IL-1β within the first 12, 7 and 5 days following onset of infection [17]. Those patients with more severe disease were treated with pulsed methylprednisolone and IL-1β levels returned to normal after 7 days. The seven patients with less severe disease did not receive any dosage of corticosteroids and their cytokine levels returned to normal range levels over the same 7-day time period.
A further Chinese study also identified reduced levels of IL-1β following administration of corticosteroids-suggesting inhibition of proinflammatory cytokines such as IL-1 may be a beneficial treatment strategy for treatment of SARS [50]. A third study, which measured serum cytokine levels in four patient groups including controls, patients with SARS, patients with severe SARS and convalescent SARS patients suggested that longer term treatment (over a period of 7-10 days) with low-dose steroids can alter serum cytokine levels, including IL-1α [38].
One rat model showed promising results for an IL-1 receptor antagonist which reduced the chemokine expression in infected animals [51]. However, this result cannot be generalised for humans. Unfortunately, one study, in which the authors state the 'demonstrate for the first time that inflammation by coronavirus may be inhibited by anti-inflammatory cytokines belonging to the IL-1 family', was only available as an abstract [52]. Therefore, further evidence or information to back this claim up is not available.

Conclusion
Overall, this review demonstrated that although it is evident that IL-1 is elevated in patients infected with a coronavirus, there is not at present evidence for an established role for IL-1 blockers in the treatment of COVID-19 in humans. The literature did, however, suggest a potential role for low-dose corticosteroids to reduce levels of pro-inflammatory markers, such as IL-1, which are elevated as part of the immune response and may have a role in the severe lung damage associated with human coronaviruses.

Mycophenolate
Mycophenolate mofetil (which is a derivative of mycophenolic acid (MPA)) is an immune suppressant, antineoplastic and antiviral mediation. According to the British National Formulary, mycophenolate mofetil is used for the prophylaxis of acute rejection in renal transplantation and is usually used in combination with a corticosteroid and ciclosporin.

Results
Almost all of the studies investigated MPA as a potential therapy for MERS-CoV due to its anti-viral properties. Six of the 13 selected studies were in vitro studies, two were in vivo, one was a clinical example and four were reviews (therefore there was some overlapping of results) (Supplementary Table 7).

In-vitro studies
In general, the in vitro studies looked positive with MPA targeting the papain like proteases of both MERS-CoV and SARS-CoV [53,54]. The studies found that MPA showed strong inhibition of the virus with a very low IC50 [55][56][57].

In vivo study
MPA can been used in combination with interferon-beta (IFN-b). One study, which applied this regime in marmosets exhibiting a severe disease resembling human MERS, reported high viral loads with more severe or even fatal disease [28]. The authors of this study state that MPA is likely to cause more harm than benefit to MERS patients.

Clinical studies
According to the review by Mo and Fisher [58], MPA monotherapy had not been tested in a clinical setting for the treatment of MERS-CoV. Al Ghamdi et al [59], presented an example where eight patients were treated with MPA for MERS-CoV, seven in combination with IFN-β. All eight of these patients survived, however the review by Mo and Fisher stated that this group of patients had lower Acute Physiology and Chronic Health Evaluation II scores compared with others in the cohort who received a variety of antiviral agents including ribavirin and IFNa, steroids and antibiotics. Therefore, the results must be interpreted with caution.

Conclusion
Whilst the in vitro studies showed promising results for MPA against MERS, the in vivo studies suggest that its use is likely to cause more harm than benefit and hence is not likely to be useful against coronavirus infections. The clinical studies are too small to confirm or deny any beneficial use for MERS-CoV patients.

Tacrolimus
Tacroliumus, also known as fujimycin, envarsus or FK506, is an immunosuppressive drug which is mainly administered after allogeneic organ transplant to lower risk of organ rejection. It's mechanism of action focusses on inhibition of calcineurin which is involved in the production of IL-2. IL-2 is a cytokine which promotes the development and proliferation of T cells which form a vital component of the human adaptive immune response.

Results
A total of 18 studies were identified from the search terms, of which three were deemed suitable for inclusion (Supplementary Table 8).
Overall, the literature appeared to suggest a potential role for tacrolimus in the treatment of human coronaviruses. In a case study of two renal transplant recipients who tested positive for MERS CoV, a patient who was being treated with an immunosuppressive regimen of tacrolimus underwent full recovery whilst the other patient (who was not on this treatment regimen) succumbed to the infection [60]. The patient who eventually made a full recovery was also treated with antibacterial therapy and a reduced dose of mycophenolate mofetil and it is therefore not possible to conclude that patient recovery due to tacrolimus.
The two other included studies were both laboratory studies involving cell line culture. The first investigated pathways of coronavirus viral replication as potential antiviral therapeutic targets [61]. Genome-wide SARS-CoV yeast-two-hybrid interaction screen with human cDNA libraries identified FK506-binding proteins as interaction partners of SARS-CoV non-structural protein 1. Subsequently, the authors investigated whether tacrolimus inhibits viral replication of human coronaviruses. VeroFM cells infected with SARS-CoV and other human coronaviruses were treated with FK506. Results showed that FK506 effectively inhibited viral replication of SARS-CoV, HCoV-NL63 and HCoV-229E at non-toxic, low-micromolar concentrations with a reduction in viral titers to undetectable levels. The second study further confirmed this inhibition using novel non-immunosuppressive derivatives of FK506 in the context of HCoV-NL63 at low-micromolar, non-cytotoxic concentrations in cell culture [62].

Conclusion
Overall the small amount of literature available suggests a potential role of FK506 (tacrolimus) as a potent antiviral in the treatment of human coronaviruses. It is important to note, however, that COVID-19 is a novel disease and may have different aetiology and mechanistic action compared to existing strains and to date, this immunosuppressive drug and its derivatives has not been tested in humans. Further study is warranted, both in the clinical setting and laboratory.

Anti-CD20
No studies were identified for inclusion.

CTLA-4 Ig
No studies were identified for inclusion.

Overall conclusion
The rapidly progressing SARS-CoV-2 pandemic has led to challenging decision-making about the treatment of critically unwell patients with the novel viral infection. In parallel, doctors across multiple specialties are making clinical decisions about the appropriate continuation of treatments for patients with chronic illnesses requiring immune-suppressive medication. This systematic review looks to provide guidance from the current available literature.
As the COVID-19 pandemic progresses, collective effort to capture data from prospective trials is required. Sponsors of randomized controlled trials recruiting patients randomized to receive immune modulatory drugs that may be affected by COVID-19 should collect data about the disease outcomes and consider interim analysis of potential advantages and disadvantages associated with using one of these medications.
Low-dose prednisolone and tacrolimus therapy may have beneficial impacts on the course of SARS-CoV-2. This observation requires further validation. The mycophenolate mofetil picture is less clear, with conflicting data from pre-clinical studies. There is no definitive evidence that specific cytotoxic drugs, low-dose methotrexate for auto-immune disease, NSAIDs, JAK kinase inhibitors or anti-TNFa biological agents are contraindicated. There is evidence that IL-6 peak levels are associated with severity of pulmonary complications. Ongoing studies of blockade of the IL-6 pathway are rational and will hopefully inform practice as the pandemic progresses.

Funding declaration
We would like to thank our various funders: Guy's and St Thomas' Charity, Cancer Research UK (C45074/A26553), and the CRUK King's Health Partner Centre.

Conflicts of interest
The authors have no conflicts of interest to declare.  • Loss of pulmonary ACE2 function has been suggested to be associated with acute lung injury; the reduction in ACE2 function can cause dysfunction of the reninangiotensin system [23] and enhance inflammation and vascular permeability. • ACE2-associated lung injury has been suggested in SARS-CoV infection • From reading this article the following article was then discovered… [24] Are patients with hypertension and diabetes mellitus at increased risk for COVID-19 infection?

Fang, Karakiulakis and Roth
Greece 2020 The expression of ACE2 is substantially increased in patients with type 1 or type 2 diabetes, who are treated with ACE inhibitors and angiotensin II type-I receptor blockers (ARBs). Hypertension is also treated with ACE inhibitors and ARBs, which results in an upregulation of ACE2 ACE2 can be increased by thiazolidinediones and ibuprofen. These data suggest that ACE2 expression is increased in diabetes and treatment with ACE inhibitors and ARBs increases ACE2 expression. This review article aimed to look at multiple diseases caused by various viruses including Dengue, Ebola and SARS-CoV. They stated that ribavirin (an antiviral) is the most frequently used drug to combat SARS-CoV, and is administered together with corticosteroids. Although immunosuppressive corticosteroid drugs are commonly used in an attempt to reduce fatality rates, the results of clinical trials are not sufficiently satisfactory to approve as an effective class of therapeutic Glucocorticoid-mediated stimulation of the hypothalamic-pituitaryadrenal axis can also drive lymphocytopenia, or it may promote exaggerated pro-inflammatory responses that eventually cause a worsening of the pathogenic condition. (Please note this is not specific for SARS-CoV-19 and is referring to the 3 viral diseases mentioned). [28] The management of coronavirus infections with particular reference to SARS. Results also suggest that dexamethasone induced reduction of IL-6 might play a role in delayed onset of lung lesions but increases of IL-6 at later infection stage may contribute to severity of lung lesions (mimicking delayed onset of disease severity as seen in SARS patients treated with corticosteroids).

Overall findings suggest that 1 or 2 doses of dexamethasone in acute phase of infection may effectively alleviate early pro-inflammatory response, but prolonged DEX administration may play role in enhancing viral replication.
[30] Indomethacin has a potent antiviral activity against SARS coronavirus. Indomethacin is common NSAID. Report describes how indomethacin was unexpectedly found to possess potent antiviral activity against canine coronavirus (dramatically inhibit virus replication and protect the host cell from virus-induced damage). Activity also observed in vivo and against human SARS-CoV. Oral administration of concentration 1mg/kg found to be effective. [31] A study of pulmonary inflammatory reaction induced by N-protein of SARS-CoV in rat models and effects of glucocorticoids on it been reported to be beneficial for treatment of the non-infectious idiopathic pneumonia syndrome which can follow stem-cell transplantation, a pulmonary syndrome that resembles SARS pneumonia in some respect. • If the SARS coronavirus does indeed lead to massive release of TNF-αf rom alveolar macrophages, then early inhibition of TNF-α might be able to prevent TNF-αmediated immune activation and therefore reduce pulmonary injury in these patients. • Compared with the use of corticosteroids, the use of biologic TNF inhibitors, including etanercept, has the potential to be a more specific and more effective method of ameliorating the severe alveolar damage which can occur following infection with these agents. [37] Anti-TNF alpha therapy does not ameliorate disease in a model of acute virus-endotoxin mediated respiratory disease in pigs. COPD and bronchial asthma exacerbation induced by viral infection. The decreased production of IL-6 and IL-8 in cells pretreated with glycopyrronium, formoterol, and budesonide, as well as the increased inhibitory effects of GFB observed in the present study, may be associated with the inhibitory effects of these drugs on COPD and bronchial asthma exacerbation. China 2020 • 29 patients with 2019-ncov admitted to the isolation ward of Tongji hospital ( mild (15 cases), severe (9 cases) and critical (5 cases). • The expression levels of inflammatory cytokines and other markers in the serum of each group were detected. • There were statistically significant differences in the expression levels of interleukin-2 receptor (IL-2R) and IL-6 in the serum of the three groups (p < 0.05), among which the critical group was higher than the severe group and the severe group was higher than the mild group. • However, there were no statistically significant differences in serum levels of tumor necrosis factor-alpha (TNF-alpha), IL-1, IL-8, IL-10, hs-CRP, lymphocyte count and LDH among the three groups (P>0.05). [26] Severe acute respiratory syndrome: clinical and laboratory manifestations.  (12) years, range 21-58] for 19 consecutive days upon hospital admission (from ≤ 2 days after disease onset). • The cytokine profile showed marked elevation of T-helper lymphocyte type 1 (Th1) cytokine IFN-γ, inflammatory cytokines IL-1β, IL-6 and IL-12 for at least two weeks after disease onset, but there was no significant increase in inflammatory cytokine TNF-α, anti-inflammatory cytokine IL-10, Th1 cytokine IL-2, and T-helper lymphocyte type 2 (Th2) cytokine IL-4. • We have also performed serial studies of plasma cytokine and chemokine profiles of 8 children with SARS (5 boys and 3 girls, age 0.3 -6.2 years) and found that these patients had a much milder cytokine and chemokine storm, rendering the use of corticosteroids more controversial if not unjustified. • IL-6 Interleukin acts as a mediator between pro-and anti-inflammatory reactivity by initiating trans-and classical-signalling, which might relate to the cytokine storm that is triggered by excessive pro-inflammatory responses to Ebola, SARS-CoV and dengue infections in humans. • Future antivirals should thus aim to target the mechanism that regulates switching between IL-6 trans-and classical-signaling • The tumour necrosis factor-α converting enzyme ADAM-17 could be the master molecule involved in regulating IL-6 class switching and through this in controlling pro-and anti-inflammatory responses to viral antigenic stimuli. • ADAM-17 should be considered as a potential target molecule for novel antiviral drug discovery that would regulate host reactivity to infection and thereby limit or prevent fatal outcomes. [43] Extraordinary GU-rich singlestrand RNA identified from SARS coronavirus contributes an excessive innate immune response. • They identified a set of SARS-CoV specific GU-rich ssRNA fragments with a high-density distribution in the genome. • In vitro experiments, the result showed the representative SARS-CoV ssRNAs had powerful immunostimulatory activities to induce considerable level of pro-inflammatory cytokine TNF-a, IL-6 and IL-12 release via the TLR7 and TLR8, almost 2-fold higher than the strong stimulatory ssRNA40 that was found previously from other virus. • Moreover, SARS-CoV ssRNA was able to cause acute lung injury in mice with a high mortality rate in vivo experiment. It suggests that SARS-CoV specific GU-rich ssRNA plays a very important role in the cytokine storm associated with a dysregulation of the innate immunity and could open a new therapeutic strategy.

US 2009
• A new strain of SARS-CoV (strain v2163) was characterised and it was highly lethal in 5-to 6-week-old BALB/c mice. It had nine mutations affecting 10 amino acid residues. • Strain v2163 increased IL-1alpha, IL-6, MIP-1alpha, MCP-1, and RANTES in mice, and high IL-6 expression correlated with mortality. [46] Neutralising antibody against severe acute respiratory syndrome -coronavirus spike is highly effective for the protection of mice in the murine SARS model. • The efficacy of three SARS vaccine candidates was tested in a murine SARS model utilising low-virulence Pp and SARS-CoV coinfection. • Vaccinated mice were protected from severe respiratory disease. • A high level of IL-6 and on days 2 and 3 after SARS-CoV infection was closely linked to the virus replication and disease severity, suggesting the importance of these cytokines in the lung pathogenicity of SARS-CoV infection. [47] Severe acute respiratory syndrome (18) coronavirusinduced lung epithelial cytokines exacerbate SARS pathogenesis by modulating intrinsic functions of monocyte-derived macrophages and dendritic cells.   [18], were included to study the link between viral load, ribavirin, proinflammatory cytokines, and clinical progression. • ribavirin was not effective in reducing the SARS coronavirus load in 3 of 8 probable cases studied • elevated levels of interleukin (IL)-6 and IL-8 subsequent to the peak viral load were found in 8 and 6 cases, respectively.

Supplementary Table 4. Current studies investigating COVID-19/ other coronavirus strains and IL-6. (continued)
[54] Analysis of serum cytokines in patients with severe acute respiratory syndrome. • The authors of this study set out to study the inflammatory cytokine profile in children with SARS. They found that the plasma concentrations key proinflammatory cytokines, including IL-6 were not substantially increased in any of the patients throughout the course of illness. From this the authors stated that the cytokine results cast doubt on the liberal use of corticosteroids in paediatric SARS patients, as the host immunologic response did not seem to be as severe as initially anticipated. The authors provide evidence that SARS-CoV 3a protein activates the NLRP3 inflammasome in lipopolysaccharide-primed macrophages and that the ion channel activity of the 3a protein was essential for 3a-mediated IL-1β secretion. The macrophages obtained in this study were from 6-week-old female mice. Note. (PLpros)= Papain-like proteases MERS-CoV and SARS-CoV -act as a potential anti-viral target for drugs Multiple inhibition assays also support a kinetic mechanism by which disulfiram together with 6TG and/or MPA can synergistically inhibit MERS-CoV PLpro, but not, due to its competitive mode of inhibition, SARS-CoV PLpro. Synergistic inhibition of MERS-CoV PLpro by disulfiram and 6-thioguanine or mycophenolic acid implies the potential for combination treatments using these three clinically available drugs. The combination of mycophenolic acid and interferon beta 1b shows synergistic activity against MERS-CoV in Vero cells. The desirable pharmacokinetics of mycophenolic acid compared to ribavirin warrants further evaluation, although the potential inhibitory effect on the immune system and therefore neutralising antibody production should be fully assessed in animal models before use in humans. A fatal case of MERS was reported in a renal transplant recipient who was receiving antirejection therapy consisting of prednisone, mycophenolate mofetil, and cyclosporine, but the dosage, serum drug level of mycophenolate mofetil, and resulting lymphocyte count were not reported [80] A review of candidate therapies for Middle East respiratory syndrome from a molecular perspective.

Germany/ Switzerland 2012
Coronaviruses represent the group of RNA viruses with the largest RNA genome to date therefore the development of resistant mutants to targeted drugs remains a concern. Viral replication represents potential antiviral targets. Study performed genome-wide SARS-CoV yeast-two-hybrid interaction screen with human cDNA libraries identifying FK506-binding proteins as interaction partners of SARS-CoV non-structural protein 1 (Nsp1). FK506-binding proteins bind the immunosuppressive drug FK506 (tacrolimus). Since Nsp1 interacts with FK506-binding proteins, investigated whether FK506 inhibits replication of human coronaviruses. VeroFM cells infected with SARS-CoV and other human coronaviruses and treated with FK506. Study found that FK506 inhibits the replication of SARS-CoV, HCoV-NL63 and HCoV-229E at non-toxic, lowmicromolar concentrations with reduction in viral titers to undetectable levels. All human coronaviruses sensitive to FK506 indicating the involvement of FK506-binding proteins in viral replication. PPIase-independent activities of CsA and FK506 exerted by gain-of-function, result from binary complexes formed by binding of the drugs to FKBPs. Based on inhibition of the protein phosphatase activity of calcineurin, these complexes block the cellular calcineurin/NFAT pathway thereby interfering with T-cell activation and IL-2 production. FK506 analogues (altered by side chain modification)