Discussion
SARS-coV-2 infection causes surge in a number of pathways related to inflammation, cytokine signaling, leukocyte and lymphocyte activation, innate and adaptive immune response marking the phenomenon of “cytokine storm”. As the whole immune system is affected during the SARS-coV-2 infection, immunomodulators would be highly beneficial in treating the symptoms. A COVID-19 patient with pneumonia was treated successfully with thalidomide and low dose glucocorticoid. There was a significant decrease in the inflammatory cytokines including IL1-, IL-6 and IFN-ϒ and increase in the CD4+ and CD8+ T cells and NK cells. Thalidomide reduced the severity of many COVID-19 symptoms such as lung lesions, exudation due to its pleiotropic effects on the human system . Hemophagocytic syndrome, a hyperinflammatory disorder is also another condition in which cytokine storm occurs. It is frequently present with extranodal natural killer/T cell lymphoma (ENKTL). Thalidomide was effective in suppressing the cytokine storm through inhibition of NF-κB based transcription of IFN-ϒ and TNF genes and thalidomide along with P-Gemox was highly effective in treating ENKTL patients in a Phase II clinical trial . Comparison of SARS-coV-2 expression profiles with drug signatures through enrichment analysis revealed striking actions of thalidomide and lenalidomide in A549 and endothelial cells. The results suggest that thalidomide and lenalidomide could reverse the devastating effects of SARS-coV-2 infections on immune system. We selected A549, an adenocarcinomic human alveolar basal epithelial cell line to test our hypothesis that thalidomide would be effective against the cytokine storms. The A549 cell line is an appropriate model for testing cytokine storm targeting drugs since a previous study established this model by infecting the cells with influenza A/H1N1 virus (PR-8) or nonstructural protein 1 (NS1) plasmid to test the mechanisms behind inflammatory cytokines/chemokines mediated “cytokine storm” Studies have utilized A549 cells to show the effects of thalidomide on lung fibrosis . A limitation of this study is that only 978 genes called “landmark genes” are profiled in the iLINCS drug signatures. However, the profiles are highly reproducible and represent the whole transcriptome . Our models of A549 and HUVEC effectively capture the effects of thalidomide in lungs as well as endothelium.
It is also emerging that SARS-coV-2 infections perturb vascular plexus significantly and there is a substantial increase in the growth of new blood vessels and evidence of intussusceptive angiogenesis with overexpression of angiogenesis and hypoxia genes in the lungs of COVID-19 patients . Cytokine storm and atherosclerosis are tightly connected in SARS-coV-2 which is consistent with our analysis revealing the enrichment of atherosclerosis in the SARS-coV-2 signatures (Figure 6). Thalidomide is a renowned modulator of vascular system, and it is known to transcriptionally or functionally target various genes (Table S1) up-regulated genes in the lungs of COVID-19 patients . As SARS-coV-2 infection has a huge impact on the hematopoietic system affecting the myeloid cell maturation, we meta-analyzed the effects of thalidomide and its derivatives on PBMC, bone marrow cells as well as lymphoma cells. Thalidomide and lenalidomide exhibited attenuation of cytokine signaling and inflammation in addition to its anti-angiogenic action (Figure 3A). The drugs affected most of the pathways up-regulated in SARS-coV-2 affected lungs and PBMC (Figure 1A, 3A) in A549 cells, mandating direct investigations in SARS-coV-2 infected models.
COVID-19 coincides with a strong neuro-endocrine modulation because the disease devastates functions of the organs, and naturally the reciprocal communication between the organs of the endocrine stress system gets a set-back . ACE2 is expressed along the hypothalamus, pituitary and adrenal (HPA) axis which is implicated in the stress response and adrenal glands has the highest concentration of virus particles next to lung . A high expression of ACE2 in brain is believed to be the reason for the possible infection of the central nervous system in SARS patients . Chronic elevated stress levels has been reported in SARS and SARS-coV-2 patients even long after the outbreak . Notably, thalidomide is also known for its neuro-endocrine modulation properties. Thalidomide modulates CNS by reducing the generation of pro-inflammatory cytokines such as IL-1, IL-6, IL-8 and TNF-α through NF-κB inhibition . There was a down-regulation of genes involved in circadian wake cycle (Figure 1B, S2) including PER3 in the PBMC of COVID-19 patients hinting on the possible sleep disturbances in SARS-coV-2 patients. Thalidomide being a well-known antiemetic and sedative action on the neuroendocrine axis would relax the patients which is supported by the report that thalidomide was effective in treating the anxiety and digestive symptoms in the COVID-19 patient .
The anti-inflammatory properties of thalidomide and its analogs through reduction of IL-1β, TNF-α expression and NF-κB inhibition are well established . SARS-coV-2 infections showing elevated NF-κB signaling and rampage activation of immune response. Unlike other RNA viruses, SARS-coV-2 suppresses TNF receptor-associated factors 3 (TRAF3) activation, inhibiting NF-κB and IRFs, leading to suppression of early pro-inflammatory and antiviral responses. Whereas later stages of the infection show an enhanced expression of IRF targets in the lungs with an activation of IL-1, IL-6 and TNF-α expression and inhibition of type I interferon signaling . Activation of IRF and ISRE transcriptional targets in SARS-coV-2 affected lungs is in agreement with previous studies reporting the SARS biology . Thalidomide inhibited LCK activity affecting STAT1 phosphorylation, cytokine mediated signaling, NF- κB signaling, osteoclast differentiation and MAPK signaling through modulation of various upstream activators and downstream effectors. Lenalidomide, in addition, suppressed leukocyte differentiation, TLR signaling along with IRF activation in A549 and lymphoma cells. The effects of thalidomide and lenalidomide observed in our study are consistent with the previous studies where thalidomide and lenalidomide has been shown to inhibit IRF and STAT1 phosphorylation resulting in the downregulation of interferon expression and TLR signaling .
The expression profile of SARS-coV-2 infected lungs, PBMC as well as A549 cells show resemblance with profiles of lymphoma, multiple myeloma and SLE. Therefore, drugs which are effective in treating SLE, lymphoma and multiple myeloma might be effective against SARS-coV-2 infection. Thalidomide and its derivatives show impressive efficacy in treating multiple myeloma and certain forms of lymphoma . Remarkably, hydroxychloroquine, an FDA approved SLE drug is currently being used in the management of critically ill SARS-coV-2 patients . CC-220, another thalidomide analog shows very promising results in phase I/II clinical trials against SLE . CC-220 through suppression of Ikaros and Aiolos expression , transcription factors which are essential for differentiation of leukocyte and NK cells thus modulating the innate immune system. As innate immune system pathways are deregulated in SARS-coV-2 infected lung and PBMC, further studies are warranted to investigate the efficacy and safety of CC-220 in treating COVID-19
Any treatment strategy with thalidomide and its analogs including repurposing thalidomide for COVID-19, should consider thalidomide-induced adverse effects including neuropathy and venous thromboembolism (VTE) . There have been many reports on COVID-19 patients develop blood clots , a dangerous issue which might be aggravated with the use of thalidomide and lenalidomide. In addition, lenalidomide might cause cytokine release syndrome in chronic lymphocytic leukemia patient . Therefore, a very careful dosage regimen has to be followed with all these drugs as serious adverse effects have been observed during dose escalation earlier.