Abstract
Influenza viruses, corona viruses and related pneumotropic viruses cause sickness and death partly by inducing a hyper-proinflammatory response by immune cells and cytokines in the host airway. Here we show that the cardiac glycoside digitoxin suppresses this response induced by influenza virus strain A/Wuhan/H3N2/359/95 in the cotton rat lung. The cytokines TNFα, GRO/KC, MIP2, MCP1, TGFβ, and IFNγ. are significantly reduced. Since the hyper-proinflammatory overproduction of cytokines is a host response, we suggest that digitoxin may have therapeutic potential for not only influenza and but also for coronovirus infections.
Introduction
Influenza viruses, corona viruses and related pneumotropic viruses cause sickness and death partly by inducing a hyper-proinflammatory immune response in the host airway. This immune overreaction, called a cytokine storm, can lead to multiorgan failure and death 1. For example, Influenza A (H5N1) has been shown to activate the TNFα-driven NFκB signaling pathway in a mouse host during viral infection generating a cytokine storm 2. Similarly, inhibitors of NFκB acutely suppress cytokine storm and increase survival in a mouse model of SARS CoV infection 3. Recent data show that COVID-19 also activates NFκB 4. A recent description of COVID-19 infection in humans has stressed that cytokine storm marks the airways of infected patients that were admitted to the Intensive Care Unit (ICU) with more severe disease 5.
The clinical problem is that there are limited options for treating respiratory cytokine storm, most of which are predicated on inhibiting NFκB-activated cytokine expression 6-8. The absence of NFκB inhibitory drugs from the human formulary is due to most candidate drugs being either neurotoxic or nephrotoxic when administered chronically 9. One drug that lacks these toxicities is the cardiac glycoside digitoxin. We have previously shown digitoxin to be among the most potent inhibitors of the proinflammatory TNFα/NFκB pathway in the human airway and in other epithelial cells, both in vitro 9, and in vivo 11-13. Corroborating this is a screen of 2800 drugs and bioactive compounds which found digitoxin to be the 2nd most potent inhibitor of TNFα/NFκB activity 14. Digitoxin has been a drug to treat heart failure for decades, and is safe for children and adults with normal hearts 15. In a clinical trial of digitoxin administered to young adults with the proinflammatory lung disease cystic fibrosis, digitoxin was safe 13. The study showed “the mRNAs encoding chemokine/cytokine or cell surface receptors in immune cells were decreased in nasal epithelial cells…leading to pathway-mediated reductions in IL-8, IL-6, lung epithelial inflammation, neutrophil recruitment and mucus hypersecretion.” 13.
To test the ability of digitoxin to inhibit cytokine storm, we used the cotton rat to investigate the effects of digitoxin on influenza virus strain A/Wuhan/H3N2/359/95. The cotton rat model has the important advantage of susceptibility to influenza infection without engineered adaptation. In addition, it has been shown that the response of the cotton rat to this virus strain evokes a pattern of pulmonary cytokine changes that parallel the human response 16
Results
Digitoxin blocks cytokine storm
Figure 1 shows the changes in cytokine protein in the lung due to digitoxin administration in the cotton rat after intranasally instilling 107TCID50/100 gm of influenza strain A/Wuhan/H3N2/359/95 virus. Animals were given four different doses of digitoxin, starting one day prior to virus administration and continuing until sacrifice on day 7. The maximum dose of digitoxin, 30 μg/kg, was calculated to be similar to the human dose routinely used to treat heart failure. As shown in Figure 1, protein data were collected for IFNγ (Interferon gamma); GRO/KC (rodent equivalent of human IL8); MIP2 (Chemokine (C-X-C motif) ligand 2, CXCL2, Macrophage inflammatory protein 2-alpha); TNFα (Tumor Necrosis Factor alpha); IL-1β (Interleukin one beta); MCP1 (Monocyte chemoattractant Protein 1, CCL2); and TGFβ (Transforming Growth Factor beta). As summarized in Table 1, digitoxin-dependent changes in protein were found to be significant for 6 of the 7 cytokines. The digitoxin-dependent reductions are specific and saturating for each cytokine, but do not reduce any of them to zero.
Digitoxin differentially affects cytokine expression
Table 1 shows that the greatest significant digitoxin-dependent reductions in cytokine proteins were found for IFNγ (68.9%), GRO/KC (46.6%), and MCP1 (54.9%). Smaller but still significant reductions in cytokine proteins were found for MIP2 (32.2%) and TNFα (38.4%). As also shown in Table 1, a significant reduction of only 15.3% was found for TGFβ cytokine protein at a concentration of 3 μg/kg, while only trending significance was noted at higher digitoxin concentrations. In the case of IL-1β only the highest digitoxin concentration trended towards significance. Thus digitoxin independently, dose-dependently and significantly lowers the individual concentrations in the lung of at least these six cytokines which have been induced by viral exposure.
Discussion
Administration of digitoxin to the cotton rat inhibits expression of many cytokines in the lung that are induced by influenza strain A/Wuhan/H3N2/359/95, including TNFα, the key activator of the TNFα/NFκB inflammation pathway. With the exception of IFNγ, which is secreted only from activated T lymphocytes and NK cells of the immune system 17, the rest of the cytokines are secreted by epithelial cells in the airway, as well as by endothelial cells, immune cells and others 18,19. GRO/KC (CXCL1, the rodent equivalent of human IL8), a key target of NFκB signaling, is the most powerful known chemoattractant for drawing neutrophils into the lung. MIP2 and MCP1 induce entry and accumulation of monocytes and macrophages into the lung, and are targets of NFκB. TGFβ drives, and is driven by, NFκB-signaling for inflammation and fibrosis. IL-1β also drives NFκB and is driven by NFκB. It appears that digitoxin-dependent reduction in TNFα/NFκB signaling is sufficient to suppress influenza A-driven cytokine storm.
Digitoxin also blocks IFNγ, which is secreted by NK cells and activated T lymphocytes from the innate and adaptive immune systems 17. In the cotton rat lung, IFNγ mRNA expression in response to infection is biphasic 16. There is an early phase, from 6 hours after infection on day 1 to day 6, which may reflect the presence of activated NK cells. The late phase, from day 6 to day 28, may be the product of incoming antigen-specific T cells. Importantly, simply neutralizing INFγ in a mouse model of infection with influenza A virus strain A/California/07/2009 (H1N1v; “Swine Flu”) is sufficient to not only alleviate acute lung injury but also to increase weight and survival rate 20. Reduced IFNγ is associated with reduced TNFα and NFκB activation. The data also show that digitoxin treatment causes profound reduction in INFγ expression. It is further known that IFNγ expression is driven by a combination of both NFκB and NFAT 21. As summarized in Figure 2, digitoxin not only reduces NFκB expression, but also reduces NFAT through digitoxin-dependent activation of Caspase 3 22.
Finally, since antiviral properties have been reported for digitoxin and other cardiac glycosides, it is limitation of the study that we cannot exclude other antiviral effects by digitoxin from contributing to the reduction in influenza-dependent cytokine concentration 23-25.
In conclusion, these data show that digitoxin blocks the host cytokine storm induced by influenza strain A/Wuhan/H3N2/359/95 in the cotton rat lung. Since digitoxin has already been shown in people to improve respiratory cytokines in human disease, this drug may be a good candidate for further investigation into therapy for influenza and potentially for COVID-19.
Methods
Drugs and protocol for preparation
Digitoxin (μg/kg) was obtained from Sigma-Aldrich (> 95% pure). The drug was prepared as a stock solution in 95% ethanol, and further diluted in PBS before administration.
Animals and protocol for drug administration
Cotton rat experiments were managed as previously described 16. Digitoxin was administered to cotton rats one day before intranasal administration of 107TCID50/100 gm cotton rat with influenza strain A/Wuhan/H3N2/359/95. Daily digitoxin treatment continued until harvest on day 7 of the experiment. Lungs were dissected with the lower one-third of the trachea left attached. The left lung was first tied off and reserved for cytokine analysis. Lung samples were then immediately frozen on dry ice, and then kept at −80°C until further processed.
Biochemical analysis
Frozen lung samples were weighed, thawed and then minced with scissors in 10% (v/v) ice cold PBS, homogenized in 10 strokes in a Ten Broeck homogenizer, and centrifuged at 20,000 X g for 30 minutes. The supernatant solution was kept at −80°C until assayed by ELISA. The supernatant solutions were first tested by ELISA at a now-defunct company in Ijamsville, MD. The data were corroborated by the Searchlight® ELISA platform from Pierce-Thermo to assay for cytokines and chemokines. Rat reagents were used in both instances.
Acknowledgements
The authors thank Val Hemming, M.D. for advice, Jorge C Blanco, Ph.D. for managing the experiment, and Max Tran, MBA, for providing statistical analysis.
The opinions, interpretations, conclusions and recommendations are those of the authors and are not necessarily endorsed by the Department of Defense, the U.S. Government or the Uniformed Services University of the Health Sciences. The use of trade names does not constitute an official endorsement or approval of the use of such reagents or commercial hardware or software. This document may not be cited for purposes of advertisement.