Fibrin Deposition, Lung Failure, Fibrosis , Effects in Many Tissues and COVID-19: Proposal That Excessive Inflammasome Activation Is the Basic Disease Mechanism

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Fibrin deposition, lung failure, fibrosis , effects in many tissues and COVID-19: Proposal that excessive inflammasome activation is the basic disease mechanism. Good Biomarker Sciences, Leiden, The Netherlands, [email protected] Correspondence C. Kluft, [email protected] Good Biomarker Sciences, Zernikedreef 8, 2333 CK, Leiden, The Netherlands. Declaration: No conflict of interest Key words: COVID-19, inflammasome, fibrin, fibrosis, bradykinin Running title: Inflammasome and COVID-19. Abstract The activation of the innate immune system during COVID-19 infection, in particular the inflammasome, and the overrepresentation of severe symptoms in patients presenting with overweight and diabetes raises the possibility that excessive inflammasome activation occurs in these patients and that this is the pivotal mechanism that causes the severity of the disease. The observed effects on fluid homeostasis, respiratory resistance, fibrosis and fibrin deposition in multiple tissues, can be brought together under the umbrella of excessive inflammasome activation in COVID-19 infection. The proposal is to use specific biochemical measurements or biomarkers in order to (a) identify patients that have excessive inflammasome activation and cells loaded with pro-cytokines and (b) follow the disease progression and assessing the effect of experimental treatments using these biomarkers. It is expected that these biomarkers also provide proof of concept for the proposed mechanisms. They may also replace clinical determinants to define the patients at risk more precisely. In our opinion the best approach for treatment is to partially inhibit the excessive inflammasome activation and keep the activation intact at a lower level to maintain the antiviral effect. Specific inhibition of the multiple downstream effects of inflammasome activation, most notably interleukin 1 beta and 6 effects, bradykinin action and coagulation activation, may require treatment with multiple agents to be sufficiently effective.

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1.1 Introduction A small proportion of patients infected with COVID-19 have severe lung problems and display fibrin formation, and problems in multiple tissues. Patients presenting with overweight and diabetes [1,2] are especially at risk. Inflammasome activation, identified by, for instance increased IL-18, is known to occur in obesity and diabetes [3-6]. Inflammasome activation is part of the innate immune system and at the maximum of its activation it contributes to counteract infection by pyroptosis (cell death) of the white blood cell. This aims at recruiting more cells to cope with the infection [7]. In addition, this process can also take place in tissue cells and is held responsible for reduction in pancreatic beta cells that produce insulin (Diabetes) and in neurons to reduce brain function (Alzheimer, Parkinson). In the case of COVID-19 infection, cells with the receptor ACE2 are more sensitive for uptake of virus (since it acts as a docking station for the virus) and inflammasome activation. The ACE2 receptor is present on lung cells (endothelial and epithelial cells of the air-blood barrier) and for instance also on skeletal muscle cells, neural cells and on 33 other cell types in tissues (www.genecards .org for ACE2) The main question is why the symptoms are so severe in the mentioned high risk groups. It appears that these patients have activation of the inflammasome already in tissue cells. Further activation of the inflammasome by the virus has severe effects and can result in several consequences

• Fluid homeostasis. Inflammasome activation may disrupt the lung fluid balance and induce increased fluid extravasation in the lung. We suggest that the following three mechanisms are involved: (a) inflammasome-mediated pyroptosis of air-blood barrier cells, i.e. lung epithelial and endothelial cells, which carry the ACE2 receptor that acts as the docking station for the virus entry [8]. (b) reduced ACE2 activity (due to internalisation with the virus) and consequent reduction of inactivation capacity for the vasodilation hormone, bradykinin. (c) release of HMBG1, reducing the air-blood barrier function [9]. • Respiratory system resistance. The inflammasome activation can generate increased amounts of interleukin 6 via interleukin 1 beta. Interleukin 6 can induce respiratory system resistance causing an increase in the mechanical work of breathing during exertion [10]. High Il-6 was related to fatality in Wuhan [11]. • Fibrin formation. The inflammasome activation can result in cell death (pyroptosis) which creates microvesicle-bound tissue factor and causes the procoagulant phosphatidylserine to appear in the membrane [12]. This results in activation of the coagulation system that may cause the observed fibrin deposits / (hyaline) thrombosis at multiple locations in most of the severe cases. The remarkably high D-dimer also indicates the occurrence of fibrin formation and dissolution [13]. In combination with the metabolic syndrome where reduced lysis capacity is due to an increase in the inhibitor of fibrinolysis, plasminogen activator inhibitor 1 [14], the effects can be more severe.

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• Fibrosis. The interleukin 1 beta is capable of stimulating fibrosis. That is elegantly shown in rat lungs where adenoviral gene transfer to transiently over express interleukin 1 beta led to severe tissue fibrosis [15]. The increase in interleukin 1 beta due to strong inflammasome activation thus can contribute to fibrotic effects. This appears to happen during COVID-19 infection in the lung where fibrosis has been observed but should also be considered for other tissues with expression of ACE2 such as skeletal muscle. Neutralisation of interleukin 1 beta with interleukin1 RAA has been shown to attenuate fibrosis [16]. The activation of the inflammasome requires two signals [7]

• Signal one: activation via NFkB resulting in the production of pro-cytokines of IL- 1beta and Il-18. • Signal two: activation via, as the case with COVID-19, the formation of caspase-1 which generates mature interleukin-1 beta and 18 from the pro-cytokines stock in the cell. Caspase-1 also activates gasdermin D which is the pathway to pyroptosis. HMGB1 is generated and affects the barrier function of epithelial cells. Interleukin 1 beta also stimulates fibrotic reactions. It is proposed that signal one is increased in subgroups with the consequence that in these patient groups the cytokine and pyroptosis effects are strong (a loaded gun), when a second hit takes place The subgroups contain those with the metabolic syndrome which occur in overweight and diabetic individuals. This increased signal one also occurs in other situations such as periodontitis [17], chronic stress [18], autoimmune diseases [19, 20] and lifestyle factors including the effects of chronic excessive alcohol use on the lung [21]. Also, genetic variability in the inflammasome may contribute [22].

1.2. Dilemma’s posed by treatment options Reducing inflammasome activation with medication produces a dilemma since the inflammasome is also active as an antiviral mechanism. This requires inhibiting inflammasome activation partially (only to reduce the excessive reaction), while monitoring the effects is also required. In the case of COVID-19 infection in susceptible individuals the excessive activation requires strong inhibition, which may not be desired for all cell types. 1.2.1 Inhibition of consequences from inflammasome activation There is a second option where the consequences from inflammasome activation such as coagulation, unrestricted bradykinin action (to compensate for loss of ACE2) and interleukin-1 beta effects may be targeted. These specific inhibitions of consequences are: • For fluid homeostasis, the inhibition of bradykinin formation (kallikrein inhibition, monoclonal, DX2930, Ianadelumab, recombinant c1-inactivator) [23] or blocking the bradykinin receptor B2 (Decapeptide Icatibant) to compensate for the reduced ACE2 [see: 24]. • For coagulation applying anticoagulant treatment with standard methods such as heparinoids (high dose) (see review of [25]). Monitoring microvesicle TF

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might be a rational option to follow the effects. More direct inhibition of tissue factor expression by Wogonin might be an opportunity [26]. Another therapeutic option to combat fibrin deposits/thrombi is to use thrombolysis. Here caution is required since thrombolysis with t-PA has been suspected of increasing bradykinin formation [27], which is clearly undesirable in COVID-19 infection. To combine thrombolysis with a treatment blocking bradykinin formation or bradykinin effects seems a logical option. • In the downstream chain of interleukin-1 beta to interleukin 6 there are also possibilities. It has been previously shown in infections that breaking this chain with an antibody to interleukin-1 beta action (Anakinra) in sepsis was beneficial (28). Other antibodies directed at interleukin-1 beta action (Arcalyst, Canakinumab) are at present under investigation in humans [29]. COVID-19 infection trials involving the use of antibodies against interleukin-6 receptor (Sarilumab) have been started, with high expectations of benefit. The use of natural inhibitory cytokines (Interleukins 37 and 38) has also been proposed [30]. In the downstream chain of interleukin-1 beta to fibrosis also the use of Anakinra is an option. Proof of concept in animals was obtained with the receptor antagonist for interleukin-1 beta [16].

• HMBG1 effects can be inhibited by using its natural antagonist IFN-gamma and its release using nicotine [31] and green tea [32].

In all these cases follow-up with biomarkers (notably interleukin-1 beta, interleukin-18, ASC, interleukin-6, interleukin-37 and -38, HMBG1 and the further consequences on e.g. CRP and ferritin and fibrosis markers) is important in order to identify the mechanisms and effects of treatment. To counteract the activation of the inflammasome in this manner by acting on the consequences may have the need to combine targeting the various mechanisms together with multiple medications. 1.2.2. Partial inhibition of inflammasome activation A most rational option is to partially inhibit inflammasome activation. The aim is to reduce the excessive activation to a lower level. The clue is to identify the patients with excessive activation for treatment. Currently these appear to be subgroups of overweight and diabetic patients. It is expected that in healthy, young individuals the inflammasome activation is less and this can be supported by analysing biomarkers such as interleukin-18 as reporter of inflammasome activation To identify patients with strong inflammasome activation, it is also desirable to develop an algorithm at multiple levels of action. This algorithm may include interleukin-18, d- dimer, micro-vesicle tissue factor, interleukin-6, HMBG1, CRP, fibrosis markers and urinary bradykinin. A longitudinal study is also desirable since the infection shows a strong progressive course.

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The tests are mostly performed on blood. Blood collection is not an appropriate option when patients become very ill. It may be more suitable to adapt less invasive technologies using finger-prick drops and dry spot technology for this situation (33). The selection of biomarkers can be adapted to technical feasibility. Partial inhibition may be achieved (and monitored with biomarkers) with currently known modalities from regular as well as complementary medicine. Evidence is currently experimental and preclinical. Medications reported are (the list is not exhaustive and does not include anticoagulants): caffeine [34], MCC950 [35], C1-inactivator, bradykinin antibodies, bradykinin receptor blocker, EPO [36], atorvastatin [37], gamma-tocotrienol [38], curcumin [39], glibenclamide [40], atractylodin [41], beta-hydroxybutyrate, Kanglexin [42], parthenolides [43], flavonoids [44,45], resveratrol [46], isoliquiritigenon [47], JC-124 [48], dexmedetomidine [49], nicotine [31], green tea (epigallocatechin-3 gallate)[32], colchicine [50], chloroquine [51], Chrysanthemum indicium [52] and by activating cannabinoid receptor 2 [53]. The inhibition can be documented in a cell system with THP-1 cells (similar to blood cells [54]). The experiments with MCC950 and glibenclamide are shown in figure 2. For the model, with LPS as signal 1 (5 ng/ml, 3 hr incubation) and alum (3 hr incubation) as signal 2, the inhibitors added first, show a dose dependent reduction in production of interleukin-1 beta. The IC-50 for glibenclamide is in the therapeutic range 12.5-50 M, 5-20mg/day.

It shows how in vitro the IC50 can be determined to give guidanceμ for in vivo application and further evaluation of other effects and toxicity The list of options gives the impression that there are enough inhibitor candidates to explore, but the additional and negative effects attached to all these possibilities need to be evaluated further before considering them for in vivo use. 1.3. Discussion / Conclusion Partial inhibition of inflammasome activation is feasible with various experimental compounds. Our opinion is that this is a way to go, using biomarkers to monitor the biological magnitude of the effects. Monitoring is required to titrate the inhibitory effect to achieve partial inhibition of the excessive activation and leaving intact the antiviral effect of the inflammasome The same biomarkers may be used to identify the patients for treatment, more specifically using the biological determinants and not selecting broad clinical groups with general characteristics such as obesity, diabetes, and age. Alternatively, treatments for the various consequences or downstream effects of inflammasome activation can be aimed at but may require to be combined.

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25. Bikdeli, B., Madhavan, M. V.,Jimenez, D., Chuich, T., Dreyfus, I., Driggin, E., et al. "COVID-19 and Thrombotic or Thromboembolic Disease: Implications for Prevention, Antithrombotic Therapy, and Follow- up." J Am Coll Cardiol. 2020, [In press]. 26. Wu, Y. H., Chuang, L. P., Yu, C. L., Wang, S. W., Chen, H. Y., Chang, Y. L. "Anticoagulant effect of wogonin against tissue factor expression." Eur J Pharmacol 2019, 859: 172517. 27. Gauberti, M., Potzeha, F., Vivien, D., Martinez de Lizarrondo, S. "Impact of Bradykinin Generation During Thrombolysis in Ischemic Stroke." Front Med (Lausanne) 2018, 5: 195. 28. Shakoory, B., Carcillo, J. A., Chatham, W. W., Amdur, R. L., Zhao, H., Dinarello, C. A., et al. "Interleukin- 1 Receptor Blockade Is Associated With Reduced Mortality in Sepsis Patients With Features of Macrophage Activation Syndrome: Reanalysis of a Prior Phase III Trial." Crit Care Med 2016, 44: 275-81. 29. Goh, A. X., Bertin-Maghit, S., Ping Yeo, S., Ho, A. W., Derks, H., Mortellaro, A., et al. "A novel human anti-interleukin-1beta neutralizing monoclonal antibody showing in vivo efficacy." MAbs 2014, 6: 765-73. 30. Conti, P., Ronconi, G., Caraffa, A., Gallenga, C. E., Ross, R., Frydas, I.., et al. (2020). "Induction of pro- inflammatory cytokines (IL-1 and IL-6) and lung inflammation by Coronavirus-19 (COVI-19 or SARS- CoV-2): anti-inflammatory strategies." J Biol Regul Homeost Agents 34(2). 31. Wang, H., Liao, H., Ochani, M., Justiniani, M., Lin, X., Yang, L.,et al. (2004). "Cholinergic agonists inhibit HMGB1 release and improve survival in experimental sepsis." Nat Med 10(11): 1216-21. 32. Li, W., Ashok, M., Li, J., Yang, H., Sama, A. E., Wang, H. "A major ingredient of green tea rescues mice from lethal sepsis partly by inhibiting HMGB1." PLoS One 2007, 2: e1153. 33. Freeman, J. D., Rosman, L. M., Ratcliff, J. D., Strickland, P. T., Graham, D. R., Silbergeld, E. K. State of the Science in Dried Blood Spots. Clin Chem. 2017, 64: 656-79. 34. Zhao, W., Ma, L., Cai, C., Gong, X. "Caffeine Inhibits NLRP3 Inflammasome Activation by Suppressing MAPK/NF-kappaB and A2aR Signaling in LPS-Induced THP-1 Macrophages." Int J Biol Sci 2019, 15: 1571-81. 35. Coll, R. C., Robertson, A. A., Chae, J. J., Higgins, S. C., Munoz-Planillo, R., Inserra, M. C., et al. "A small-molecule inhibitor of the NLRP3 inflammasome for the treatment of inflammatory diseases." Nat Med 2015, 21: 248-56. 36. Cao, F., Tian, X., Li, Z., Lv, Y., Han, J., Zhuang, R., et al. "Suppression of NLRP3 Inflammasome by Erythropoietin via the EPOR/JAK2/STAT3 Pathway Contributes to Attenuation of Acute Lung Injury in Mice." Front Pharmacol 2020, 11: 306. 37. u, L. M., Wu, S. G., Chen, F., Wu, Q., Wu, C. M., Kang, C. M., et al. Atorvastatin inhibits pyroptosis through the lncRNA NEXN-AS1/NEXN pathway in human vascular endothelial cells. Atherosclerosis 2020, 293: 26-34. 38. Kim, Y., Wang, W., Okla, M., Kang, I., Moreau, R., Chung, S. "Suppression of NLRP3 inflammasome by gamma-tocotrienol ameliorates type 2 diabetes." J Lipid Res 2016, 57: 66-76. 39. Yin, H., Guo, Q., Li, X., Tang, T., Li, C., Wang, H., et al. "Curcumin Suppresses IL-1beta Secretion and Prevents Inflammation through Inhibition of the NLRP3 Inflammasome." J Immunol 2018, 200: 2835-46. 40. Tamura, K., Ishikawa, G., Yoshie, M., Ohneda, W., Nakai, A., Takeshita, T., et al. "Glibenclamide inhibits NLRP3 inflammasome-mediated IL-1beta secretion in human trophoblasts." J Pharmacol Sci 2017, 135: 89- 95. 41. Tang, F., Fan, K., Wang, K., Bian, C. "Atractylodin attenuates lipopolysaccharide-induced acute lung injury by inhibiting NLRP3 inflammasome and TLR4 pathways." J Pharmacol Sci 2018, 136: 20311. 42. Zheng, Z., Li, G. Mechanisms and Therapeutic Regulation of Pyroptosis in Inflammatory Diseases and Cancer. Int J Mol Sci. 2020, 21: DOI 10.3390/ijms21041456. 43. Juliana, C., Fernandes-Alnemri, T., Wu, J., Datta, P., Solorzano, L., Yu, J. W., et al. "Anti-inflammatory compounds parthenolide and Bay 11-7082 are direct inhibitors of the inflammasome." J Biol Chem 2010, 285: 9792-802. 44. Lim, H., Min, D. S., Park, H., Kim, H. P. "Flavonoids interfere with NLRP3 inflammasome activation." Toxicol Appl Pharmacol 2018, 355: 93102. 45. Yi, Y. S. "Regulatory Roles of Flavonoids on Inflammasome Activation during Inflammatory Responses." Mol Nutr Food Res 2018, 62: e1800147. 46. Chang, Y. P., Ka, S. M., Hsu, W. H., Chen, A., Chao, L. K., Lin, C. C., et al. "Resveratrol inhibits NLRP3 inflammasome activation by preserving mitochondrial integrity and augmenting autophagy." J Cell Physiol 2015, 230: 156779. 47. Honda, H., Nagai, Y., Matsunaga, T., Okamoto, N., Watanabe, Y., Tsuneyama, K., et al. "Isoliquiritigenin is a potent inhibitor of NLRP3 inflammasome activation and diet-induced adipose tissue inflammation." J Leukoc Biol 2014, 96: 1087-100.

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48. Yin, J., Zhao, F., Chojnacki, J. E., Fulp, J., Klein, W. L., Zhang, S., et al. "NLRP3 Inflammasome Inhibitor Ameliorates Amyloid Pathology in a Mouse Model of Alzheimer's Disease." Mol Neurobiol 2018, 55: 1977- 89. 49. Yin, D., Zhou, S., Xu, X., Gao, W., Li, F., Ma, Y., et al. "Dexmedetomidine attenuated early brain injury in rats with subarachnoid haemorrhage by suppressing the inflammatory response: The TLR4/NF-kappaB pathway and the NLRP3 inflammasome may be involved in the mechanism." Brain Res 2018, 1698: 1-10. 50. Martinez, G. J., Celermajer, D. S., Patel, S. The NLRP3 inflammasome and the emerging role of colchicine to inhibit atherosclerosis-associated inflammation. Atherosclerosis, 2018, 269: 262-71. 51. Fujita, Y., Matsuoka, N., Temmoku, J., Furuya, M. Y., Asano, T., Sato, S., et al. "Hydroxychloroquine inhibits IL-1beta production from amyloid-stimulated human neutrophils." Arthritis Res Ther 2019, 21: 250. 52. Yu, S. H., Sun, X., Kim, M. K., Akther, M., Han, J. H., Kim, T. Y., et al. Chrysanthemum indicum extract inhibits NLRP3 and AIM2 inflammasome activation via regulating ASC phosphorylation." J Ethnopharmacol 2019, 239: 111917. 53. Yu, W., Jin, G., Zhang, J., Wei, W. "Selective Activation of Cannabinoid Receptor 2 Attenuates Myocardial Infarction via Suppressing NLRP3 Inflammasome." Inflammation 2019, 42: 904-14. 54. Tran, T. A. T., Grievink, H. W., Lipinska, K., Kluft, C., Burggraaf, J., Moerland, M., et al. Whole blood assay as a model for in vitro evaluation of inflammasome activation and subsequent caspase-mediated interleukin-1 beta release. PLoS One. 2019, 14: e0214999.

Figure 1: Simplified biochemical scheme of inflammasome activation and consequences. The virus is signal 2. The virus can enter by endocytosis, but more effectively with the help of binding to ACE2, at the same time creating a deficiency in ACE2 action outside the cell.

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Figure 2: Dose dependent effect of glibenclamide and MCC950 on stimulation of the inflammasome in THP-1 cells (ATCC) with LPS + Alum (InVitrogen). The data are normalized to the maximum IL-1β release of the positive control (LPS + Alum), all data is depicted as mean value ± standard deviation.