key: cord-0750182-sgqucz6x authors: Brandao-Rangel, M. A. R.; Melamed, D.; Silva-Reis, A.; Brill, B.; Zamarioli, L. d. S.; Oliveira, C. R.; Vieira, R. P. title: Virlaza Inhibits Sars-COV-2-induced Inflammatory Response of Bronchial Epithelial Cells and Pulmonary Fibroblast date: 2021-07-22 journal: nan DOI: 10.1101/2021.07.17.21260123 sha: 606add6c276dfeb31e77832e975e8fad4a66b02e doc_id: 750182 cord_uid: sgqucz6x Coronavirus disease 2019 (COVID-19), which is currently a global public health emergency and beyond vaccines as a prophylactic treatment, no specific and effective therapeutical treatments are available. COVID-19 induces a massive release of proinflammatory cytokines, which drives COVID-19 progression, severity, and mortality. In addition, bronchial epithelial cells are the first pulmonary cells activated by coronavirus-2 (SARS-Cov-2) leading to massive cytokine release, which can hyperactivate lung fibroblasts, resulting in pulmonary fibrosis, a phenomenon observed even in moderate COVID-19 survivors. This in vitro study tested the hypothesis that Virlaza, a herbal medicine, could inhibit the hyperactivation of human bronchial epithelial cells (BEAS-2B) and pulmonary fibroblasts (MRC-5) induced by SARS-Cov-2. BEAS-2B (5x104/mL/well) and MRC-5 (5x104/mL/well) cells were co-cultivated with 1ml of blood of a Sars-Cov-2 infected patient for 4 hours and Virlaza (1ug/mL) was added in the first minute of the co-culture. After 4 hours, the cells were recovered and used for analysis of cytotoxicity by MTT and for mRNA expression of P2X7 receptor E iNOS. The supernatant was used to measure ATP and cytokines. Sars-Cov-2 incubation resulted in increased release of ATP, IL-1beta, IL-6, IL-8, and TNF-alpha by BEAS-2B and MRC-5 cells (p<0.001). Treatment with Virlaza resulted in reduction of ATP, IL-1beta, IL-6, IL-8, and TNF-alpha release (p<0.001). In addition, Sars-Cov-2 incubation resulted in increased expression of P2X7 receptor and iNOS (p<0.001), which has been reversed by Virlaza (p<0.001). In conclusion, Virlaza presents important anti-inflammatory effects in the context of Sars-Cov-2 infection. The quick spread of Coronavirus disease resulted in a pandemic in the whole world, reaching more than 100 million cases and 2 million deaths due to the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) [1] . The progression, severity, and mortality of COVID-19 differ from no, mild, or moderate symptoms in most of the patients, while up to 15% develop the severe and critical form of the disease with high mortality rates [2] . Among the factors underlying the development of the severe and critical forms of COVID-19, advanced age [3] , comorbidities [3] , hyperactivation of the immune system [3, 4] are proven to have a central role, hyperactivation of the immune system results in the cytokine storm, which is characterized by the synthesis and release of high levels of proinflammatory cytokines [3, 4] . In this way, cytokine storm plays a major role in Covid-19 pathogenesis [3, 4] . In this context, bronchial epithelial cells have been described not only as a mechanical barrier for the respiratory tract, but also as an important cell layer centrally involved in the innate immune response, which actively synthetize and release a plethora of inflammatory and fibrotic mediators [5] , capable to activate fibroblasts inducing fibrosis. In addition, activation of the purinergic receptor P2X7 in the airway epithelium has been described as a key receptor involved in the inflammatory [6] and fibrotic [7] responses of the lungs to a variety of stimulus, including SARS-CoV-2 [8] . Furthermore, P2X7 is also involved in pulmonary fibroblast proliferation and activation, driven the pulmonary fibrotic response as well [7] . Activation of P2X7 receptor may be a trigger by increased levels of nitric oxide (NO) mediated by increased expression and activation of inducible nitric All rights reserved. No reuse allowed without permission. (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. The copyright holder for this preprint this version posted July 22, 2021. ; https://doi.org/10.1101/2021.07.17.21260123 doi: medRxiv preprint oxide synthase (iNOS) [9] . Indeed, increased iNOS expression drives an increased inflammatory and fibrotic responses of the lungs, such as in asthma [10] , pulmonary fibrosis [11] , and bacterial infections [12, 13] . Thus, this study tested the hypothesis that Virlaza™ could inhibit bronchial epithelial cells and lung fibroblast activation in vitro, involving iNOS and P2X7 signaling. This study and all experimental procedures were analyzed and approved by ethical committee of School of Medicine of Anhembi Morumbi University were co-cultured with whole blood from an infected patient with Sars-Cov-2. All rights reserved. No reuse allowed without permission. (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. 2), presenting an estimated high viral load based on the cycle threshold (CT = 15). Both BEAS-2B and MRC-5 cells were co-cultured at a concentration of 5x10 4 /mL/well in 48 wells plate, at a humidified atmosphere in a CO2 incubator (5% CO2, 37ºC) [14] . The blood was collected immediately after hospitalization, prior any antibiotic and corticosteroid administration. The written consent inform was obtained from the patient volunteer for this study. The cells (BEAS-2B or MRC-5) were incubated at the same time with Virlaza™ (1ug/mL) and with 500ul of whole blood of Sars-Cov-2 infected patients and the cells and supernatant was obtained 4 hours after stimulation. The cells and supernatant were recovered, centrifuged at 900g for 5 minutes at 4ºC, and then the supernatant immediately used for ATP measurement and later for cytokine measurement. The cells were washed using phosphate-buffered saline (PBS) and then subjected to RT-PCR protocol. To assess the Virlaza™ cytotoxicity through cell viability, MTT assay was carried out. Briefly, 5x10 4 viable BEAS-2B and MRC-5 cells were placed into clear 96-well flat-bottom plates (Corning USA) in DMEM high glucose medium supplemented with 10% fetal calf serum and immediately after different concentrations (0.1ug/mL; 1ug/mL; 10ug/mL; 100ug/mL; and 1000ug/mL) of Virlaza™. Following 24h after incubation in a humidified atmosphere of a CO2 incubator (5% CO2, 37ºC), 10μL/well of MTT (5mg/mL) was added to the cells All rights reserved. No reuse allowed without permission. (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. The copyright holder for this preprint this version posted July 22, 2021. ; https://doi.org/10.1101/2021.07.17.21260123 doi: medRxiv preprint (both in the control and Virlaza™), which was incubated for 4h. After this time, 100μL of 10% sodium dodecyl sulfate (SDS) solution in deionized water was added to the cells and incubated overnight [15, 16] . The absorbance was measured at 595nm in a benchtop multimode reader SpectraMax i3 (Molecular Devices, San Jose, CA, USA). The ATP concentration was determined in the cell culture supernatant were expressed as pg/mL [14] [15] [16] [17] . Total RNA isolation from cell pellets was used RNeasy mini kits (Qiagen, Hilden, Germany). Reverse transcription was used Stratascript reverse transcriptase (Stratagene, CA, USA) and random primers (Invitrogen, Germany). Quantitative PCR used Taqman Universal PCR Mastermix (Applied Biosystems, USA) and preformulated primers and probe mixes (Assay on Demand, Applied Biosystems, USA). PCR conditions were 2 min at 50°C, 10 All rights reserved. No reuse allowed without permission. (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. GAGCACGCTGAGTACCTCATT-3′ primers [16] . The software Graph Pad Prism 5.0 was used to perform the statistical analysis and build de graphs. The results were expressed as mean ± standard error of the mean (SEM) from at least three independent experiments. One-way analysis of variance (ANOVA) was used for multiple comparisons, followed by the Bonferroni post hoc test for comparison among groups. A p-value <0.05 was considered significant. All rights reserved. No reuse allowed without permission. (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. while Virlaza™ resulted in a significant reduction of the synthesis and release of All rights reserved. No reuse allowed without permission. (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. The copyright holder for this preprint this version posted July 22, 2021. ; https://doi.org/10.1101/2021.07.17.21260123 doi: medRxiv preprint SarsCov2 = Stimulated with blood infected with Sars-Cov-2; SarsCov2+Vir = Stimulated with blood infected with Sars-Cov-2 + 10ug/mL of Virlaza™. All rights reserved. No reuse allowed without permission. (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. All rights reserved. No reuse allowed without permission. (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. show that 10 μ g/mL was reached, was the dose corresponding to IC50, and was chosen was the study dose. All rights reserved. No reuse allowed without permission. (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. The copyright holder for this preprint this version posted July 22, 2021. ; https://doi.org/10.1101/2021.07.17.21260123 doi: medRxiv preprint Figure 6 -Cell viability (%) measured by MMT assay in BEAS-2B and MRC-5 cells stimulated with growing doses of Virlaza™ (0.1ug/mL, 1ug/mL, 10ug/mL, 100ug/mL, 1000ug/mL). This study shows for the first time that Virlaza™ was able to block the cytokine storm caused by Sars-Cov-2-induced epithelial (BEAS-2B) and lung fibroblast (MRC-5) activation, involving dampening of exacerbated purinergic signaling and iNOS expression. In addition, the study demonstrated the safety of an active therapeutic dose Virlaza™ in the context of cellular toxicity. Considering the lack of effective treatment for COVID-19, and the evidence that severely ill COVID-19 patients develop a cytokine storm, some studies are testing the hypothesis that the blockade of some proinflammatory cytokines, such as IL-6 [18] and IL-1beta [19] could be beneficial for severely ill COVID-19 patients. In addition, beyond massive infiltration by neutrophils and macrophages into the lungs, increased blood levels of IL-1β, IL-6, and TNFalpha have been linked to higher severity and mortality in COVID-19 patients [18, 19] . In this context, bronchial epithelial cells are the main entrance door and the main target of different types of respiratory bacteria and viruses, harming the immune system by exacerbating release of pro-inflammatory cytokines and growth factors [20, 21] . In the present study, we demonstrated that bronchial epithelial cells (BEAS-2B) when stimulated with Sars-Cov-2, responded to increased release of IL-1beta, IL-6, IL-8 and TNF-alpha, which were abolished All rights reserved. No reuse allowed without permission. (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. Importantly, the literature demonstrates the importance of increased expression of P2X7 receptor and consequent activation triggering proinflammatory and pro-fibrotic response in the context of respiratory diseases [4] [5] [6] [7] . Such importance has been demonstrated in several cells of the hematopoietic system [6] , but also in lung structural cells, such as in epithelial cells [4, 5] and lung fibroblasts [7] . , the activation of P2X7 receptors is classically related to the initiation, maintenance, progression, and severity of inflammatory states triggering IL-1beta release [7] [8] [9] . Herein, this study demonstrated that Virlaza™ inhibited IL-1beta release not only by epithelial cells (BEAS-2B) but also by lung fibroblasts . Particularly about fibroblasts, the inhibitory effect of Virlaza™ inhibiting the release of IL-1beta is of particular importance, since IL-1beta directly stimulates collagen synthesis and proliferation in fibroblasts [22, 23] . Therefore, it is plausible to hypothesize that Virlaza™ could also have a potential effect to inhibit lung fibrosis, which is commonly observed in COVID-19 survivors [24] . Such observed effects of Virlaza™ on IL-1beta release can be supported by the additional results of the present study, which demonstrated that Virlaza™ inhibited ATP release and accumulation, resulting in decreased expression of P2X7 receptor in BEAS-2B and in MRC-5 cells. Therefore, further clinical trial investigating the effects of Virlaza™ in COVID-19 survivors were guaranteed. Beyond P2X7 receptor, inducible nitric oxide synthase (iNOS), is though as a key enzyme involved in all aspects of the pathophysiological process of pulmonary infections with diverse etiologies, including for the Sars-Cov-2 [25, All rights reserved. No reuse allowed without permission. (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. 26 ]. In the context of COVID-19, intense and diffuse pulmonary inflammation results in endothelial dysfunction of the lung vasculature, uncoupling eNOS activity, lowering nitric oxide (NO) production, resulting in different pulmonary alterations and coagulopathy [25, 26] . On the contrary, viral infections trigger an increasing in iNOS activity, which may be acutely advantageous for host defense, once NO plays antiviral effects. However, sustained overproduction of NO mediates deleterious proinflammatory effects [25, 26] . In the present study, it was observed that Sars-Cov-2 increased iNOS expression in epithelial cells (BEAS-2B) and in lung fibroblasts (MRC-5 cells), which was dampened by Virlaza™. Interestingly, the literature demonstrates that NO synthesized specifically by the airway epithelium is vital to antiviral, inflammatory, and immune defense of the lungs [26] . However, again, the increased NO mediated by iNOS is related to an unresolved inflammatory process [27] as well as profibrotic response [28] . iNOS overexpression is linked to the release of proinflammatory cytokines, such as IL-1beta, IL-6, IL-8, and TNF-alpha, particularly in the context of acute respiratory distress syndrome (ARDS), phenomena present in cases of severe COVID-19 [27] . In the present study, Sars-Cov-2 stimulation resulted in an increase expression of iNOS by epithelial cells (BEAS-2B) and in lung fibroblasts (MRC-5 cells) and increased release of IL-1beta, IL-6, IL-8, and TNF-alpha, Virlaza™ significantly reduced iNOS expression and pro-inflammatory cytokines release, reinforcing the antiinflammatory and anti-fibrotic effects of Virlaza™. These findings concerning the iNOS inhibition by Virlaza™ can be strengthened once the literature already points out the possible therapeutic role of iNOS blockade for acute and long COVID-19 [29] . All rights reserved. No reuse allowed without permission. (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. The copyright holder for this preprint this version posted July 22, 2021. ; https://doi.org/10.1101/2021.07.17.21260123 doi: medRxiv preprint Beyond to inhibit the release of proinflammatory cytokines (IL-1beta, IL-6, IL-8, and TNF-alpha), Virlaza™ also induced the release of anti-inflammatory cytokine IL-1RA by epithelial cells (BEAS-2B) and by lung fibroblasts (MRC-5 cells). Functionally, interleukin-1 receptor antagonist (IL-1RA) inhibits the activities of interleukin 1 alpha (IL-1alpha) and interleukin 1 beta (IL-1beta), modulating a wide range of immune and inflammatory responses driven by interleukin 1 related cytokines [30] . Anakinra is a recombinant IL-1RA that has been tested for the treatment of COVID-19 patients, resulting in better clinical outcomes [31] [32] [33] . Therefore, the present study concludes that Virlaza™ was capable not only to inhibit the release of proinflammatory cytokines but also to stimulate the release of the anti-inflammatory cytokine IL-1RA, which has already been demonstrated to be a promising therapeutic option to prevent the development of severe forms of COVID-19 including respiratory failure, as well to treat the severe forms of COVID-19, improving the clinical outcomes and survival [31] [32] [33] . Thus, based on the findings of the present study, further clinical trials with Virlaza™ for the prevention and treatment of COVID-19 are guaranteed. Pulmonary Thrombosis and Thromboembolism in COVID-19 Epub ahead of print All rights reserved. No reuse allowed without permission. (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. The copyright holder for this preprint this version posted Cytokine Overproduction and Immune System Dysregulation in alloHSCT and COVID-19 Patients. Front Immunol COVID-19 severity correlates with airway epitheliumimmune cell interactions identified by single-cell analysis More Than Just a Barrier: The Immune Functions of the Airway Epithelium in Asthma Pathogenesis. Front Immunol Contribution of the Purinergic Receptor P2X7 to All rights reserved. No reuse allowed without permission. (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. The copyright holder for this preprint this version posted Extracellular ATP is a danger signal activating P2X7 receptor in lung inflammation and fibrosis A rationale for targeting the P2X7 receptor in Coronavirus disease 19 P2X7 receptor activation amplifies lipopolysaccharide-induced vascular hyporeactivity via interleukin-1 beta release Comparison of inducible nitric oxide synthase mRNA expression in different airway portions and association with nitric oxide parameters from patients with asthma Myeloid cell dynamics in bleomycin-induced pulmonary injury in mice; effects of anti-TNFalpha antibody No reuse allowed without permission. (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. The copyright holder for this preprint this version posted Porphyromonas gingivalis induced inflammatory responses and promoted apoptosis in lung epithelial cells infected with H1N1 via the Bcl-2/Bax/Caspase-3 signaling pathway Purinergic receptor type 6 contributes to airway inflammation and remodeling in experimental allergic airway inflammation Extracellular adenosine 5'-triphosphate modulates interleukin-6 production by human thyrocytes through functional purinergic P2 receptors Anti-Inflammatory Activity of Miodesin™: Modulation of Inflammatory Markers and Epigenetic Evidence No reuse allowed without permission. (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. The copyright holder for this preprint this version posted COVID-19: consider cytokine storm syndromes and immunosuppression Continuous Intravenous Anakinra Infusion to Calm the Cytokine Storm in Macrophage Activation Syndrome. ACR Open Rheumatol DP1 prostanoid receptor activation increases the severity of an acute lower respiratory viral infection in mice via TNF-α-induced immunopathology Aerobic All rights reserved. No reuse allowed without permission. (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. The copyright holder for this preprint this version posted Modulation of fibroblast functions by interleukin 1: increased steady-state accumulation of type I procollagen messenger RNAs and stimulation of other functions but not chemotaxis by human recombinant interleukin 1 alpha and beta Interleukin-1 increases collagen production and mRNA levels in cultured skin fibroblasts Post-COVID-19 Pulmonary Fibrosis: Novel Sequelae of the Current Pandemic Implications of SARS-Cov-2 infection on eNOS and iNOS activity: Consequences for the respiratory and vascular systems Role of epithelial nitric oxide in airway viral infection Expression of inducible nitric oxide synthase and inflammatory All rights reserved. No reuse allowed without permission. (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. The copyright holder for this preprint this version posted Abd El-Hack ME. Thymoquinone-PLGA-PVA Nanoparticles Ameliorate Bleomycin-Induced Pulmonary Fibrosis in Rats via Regulation of Inflammatory Cytokines and iNOS Signaling. Animals (Basel) Dual inhibition of CB(1) receptors and iNOS, as a potential novel approach to the pharmacological management of acute and long COVID-19 Acute Respiratory Distress Syndrome: A Retrospective, Observational Study Interleukin-1 receptor antagonist anakinra in association with remdesivir in severe COVID-19: A case All rights reserved. No reuse allowed without permission.(which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. All rights reserved. No reuse allowed without permission.(which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity.The copyright holder for this preprint this version posted July 22, 2021. ; https://doi.org/10.1101/2021.07.17.21260123 doi: medRxiv preprint