SARS-CoV-2 infection as a model to study the effect of cinnamaldehyde as adjuvant therapy for viral pneumonia.

doi:10.21203/rs.3.rs-3072092/v1

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Research Article

SARS-CoV-2 infection as a model to study the effect of cinnamaldehyde as adjuvant therapy for viral pneumonia.

https://doi.org/10.21203/rs.3.rs-3072092/v1

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Introduction:

The recent pandemic outbursts, due to SARS-CoV-2, has highlighted once more the central role of the inflammatory process in the propagation of viral infection. In fact, the main consequence of COVID-19 is the induction of a diffuse pro-inflammatory state, also defined as cytokine storm, that affects different organs, but mostly the lungs. Therefore, despite the progress in the vaccination campaign, the high mutability of this virus also requires effective pharmaceutical approaches aimed to reduce the inflammatory spread.

Methods

The effect of SARS-CoV-2 infection on IL-1β and IL-6 levels was assessed by performing ELISA on patients' serum and stimulated-PBMCs. The therapeutic potential of cinnamaldehyde on COVID-19 was evaluated in vitro, testing the effects on THP-1 macrophages' inflammatory state, and on the viral replication in Calu-3 and Vero6 cells. Moreover, we assessed the effect of cinnamaldehyde also in vivo generating a lung-inflammatory model by intranasal administration of LPS.

Results

In this study we highlight that COVID-19 patients have higher IL-1β and IL-6 plasma levels compared to non-COVID-19 pneumonia patients, showing that human mononuclear cells (PBMCs) isolated from SARS-CoV-2 infected patients are more prone to release pro-inflammatory cytokines upon stimuli. Furthermore, we demonstrated how cinnamaldehyde, a natural compound easily tolerated by the majority of human beings, significantly reduces SARS-CoV-2-related inflammation by inhibiting IL-1β release by macrophages, and viral replication in epithelial cells. In addition, we confirmed the ability of aerosol-administered cinnamaldehyde to in vivo reduce IL-1β release in a lung-inflammatory model.

Conclusion

The obtained results suggest the possible use of cinnamaldehyde suitable as a co-adjuvant preventive treatment for COVID-19 disease together with vaccination, but also as a promising dietary supplement to reduce, more broadly, viral induced inflammation.

COVID-19

SARS-CoV-2

Cinnamaldehyde

IL-1β

IL-6

viral replication

inflammation

COVID-19 pandemic, induced by SARS-CoV-2 infection, has highlighted once more the need for understanding the immune-inflammatory pathways involved in the regulation of the advancement of pulmonary infections. In fact, as for COVID-19, we lack specific pharmacological approaches targeting virus-induced inflammation, mainly because the underlying molecular mechanisms are still to be investigated. Different researchers have reported that COVID-19 patients show increased levels of pro-inflammatory cytokines, such as IL-6 and IL-1β [1, 2], indicating that their modulation might affect patients’ outcomes [3]. Despite the high number of reviews aiming to prove how the modulation of the NLRP3 inflammasome, the main producer of IL-1β, might be beneficial in COVID-19 patients [3–5], only a few studies have been conducted to verify that hypothesis [6–8], and they provided limited data to support the use of NLRP3 inhibitor compounds in COVID-19 patients.

The NLRP3 inflammasome is involved in the regulation of a variety of diseases, from autoimmune disorders to cancer, representing a promising transversal target. During viral infection, NLRP3 inflammasome activation involves multiple cellular and molecular signaling resulting in the development of a consistent inflammatory state, which in turns promotes fibroblast proliferation, matrix deposition, and aberrant epithelial-mesenchymal function [9]. Accordingly, different NLRP3 inflammasome inhibitors have been reported to date, including the ones that directly inhibit the NLRP3 and those that act on its related signaling pathways [10]. Indeed, recently we identified a series of aryl sulfonamide derivatives (ASDs) and natural compounds with high potency and selectivity in inhibiting NLRP3 activation in vitro and in vivo [11, 12]. Among all the cited inhibitors, nutraceutical compounds might be of high impact for their low side effects in prolonged treatments. Notably, cinnamaldehyde (CINNA), the active compound of cinnamon (Cinnamomum cassia and Cinnamomum verum) which is often used as a flavoring condiment, is widely used in herbal medicine in China and Southeast Asia to treat inflammatory autoimmune diseases. Interestingly, it has been shown that CINNA acts on IL-1β release by modulating NLRP3 in both renal inflammation and rheumatoid arthritis in vivo [13, 14]. Moreover, it has been speculated that CINNA may also regulate oxidative stress [15], even if there are no effective studies to support this theory. Based on these concepts, CINNA treatment represents a promising therapeutic approach to modulate viral-induced lung inflammation. In this study, we aimed to assess CINNA efficacy by using SARS-CoV-2 infection as a viral activator of inflammation. In order to achieve our goal we: i) assessed the levels of pro-inflammatory cytokines as IL-1β and IL-6, in the plasma samples of severe COVID-19 patients compared to controls in order to prove the involvement of NLRP3 activation in the development of the disease; ii) evaluated the LPS-induced production of IL-1β and IL-6 from PBMCs of severe COVID-19 patients compared to healthy controls, in order to prove the effect of COVID-19 induced pro-inflammatory state, on the further activation of the NLRP3 axis; iii) the effects of CINNA in vitro in inhibiting IL-1β and IL-6 release as well as modulating viral replication; iv) the effects of CINNA in vivo in a lung-inflammatory model.

All the reagents used have been purchased from Merck Millipore (Burlington, USA) if not otherwise specified.

2.1. Subjects’ enrollment

The present analysis included blood samples of patients from the “Pro-thrombotic status in patients with SARS-CoV-2 infection” (ATTAC-Co) study that was approved by the local Ethic Committee CE-AVEC (Comitato Etico di Area Vasta Emilia Centro, Bologna, Italy) [16]. The ATTAC-Co study is an investigator-initiated, prospective, single-center study recruiting consecutive patients admitted to the hospital (University Hospital of Ferrara, Italy) because of respiratory failure due to COVID-19 between April and May 2020. Inclusion criteria are detailed elsewhere [17]. Briefly, patients were (i) age > 18 year; (ii) confirmed SARS-CoV-2 infection; (iii) hospitalized for respiratory failure; (iv) need for invasive or non-invasive mechanical ventilation or only oxygen support. SARS-CoV-2 infection was confirmed by RT-PCR assay (Liaison MDX; Diasorin) from a nasopharyngeal swab specimen. Respiratory failure was defined as a PaO2/FiO2 (P/F) ratio ≤ 200 mmHg. Clinical management was in accordance with current guidelines and specific recommendations for the COVID-19 pandemic by Health Authorities and Scientific Societies. A control group of patients, hospitalized in the same period and hospital settings with acute respiratory failure due to respiratory/cardiovascular acute conditions not related to SARS-CoV-2, was also included. This analysis was performed on blood samples collected just after admission. Ethics approval has been obtained. All patients gave their written informed consent. In case of unconsciousness, the informed consent was signed by the next of kin or legally authorized representative. The study is registered at www.clinicaltrials.gov with the identifier NCT04343053.

2.2. Plasma collection and PBMCs isolation

Plasma was collected by centrifuging patients’ blood at 1000 × g for 30 min, the obtained aliquots were stored at -80°C.

Peripheral blood mononuclear cells (PBMCs) were isolated from blood samples of a subgroup of COVID-19 patients and healthy donors. Ficoll-Paque density gradient centrifugation was used for PBMC separation. Blood was diluted with an equal volume of PBS. Diluted blood was layered over 4 mL of the Ficoll-Paque PLUS. Gradients were centrifuged at 400 × g for 30 min at room temperature in a swinging-bucket rotor without the brake applied. The PBMCs interface was carefully removed by pipetting and washed with PBS by centrifugation at 250 × g for 10 min then a total of 5x10⁵ cells/well were seeded in 24-well tissue culture plates in RPMI medium supplemented with 10% FBS, 100 U/mL penicillin, and 100 mg/mL streptomycin. PBMCs were maintained in a 5% CO₂ incubator at 37°C

2.3. Cell cultures

The human leukemia monocytic cell line THP-1 was grown in RPMI medium supplemented with 10% FBS, 100 U/mL penicillin, and 100 mg/mL streptomycin. THP-1 cells were stimulated by 100 ng/mL PMA overnight to differentiate into macrophages. The human lung cancer (CaLu-3) and human airway epithelial (VeroE6) cell lines were grown in Eagle’s minimal essential medium (MEM) with nonessential amino acids supplemented with 10% FBS, 100 U/mL penicillin, 100 mg/mL streptomycin and 2mM L-glutamine. All cells were grown in a 5% CO₂ incubator at 37°C.

2.4. Inflammasome activation assays in vitro

Human PBMCs and THP-1 cells were seeded at 5x10⁵ cells/well and 3 × 10⁵ cells/well respectively in 24 well plates. The following day cells were stimulated with LPS (1 µg/mL) for 3 hours and then with 2′(3′)-O-(4-Benzoylbenzoyl) adenosine 5′-triphosphate (100 µM) for 30 minutes.

2.5. SARS-CoV-2 Propagation and Infection

SARS-CoV-2 was isolated from a nasopharyngeal swab retrieved from a patient with COVID-19 (Caucasian man of Italian origin, genome sequences available at GenBank (SARS-CoV-2-UNIBS-AP66: ERR4145453). This SARS-CoV-2 isolate clustered in the B1 clade, which includes most of the Italian sequences, together with sequences derived from other European countries and the United States. As previously described, the viral titer was determined by plaque assay in Vero E6 cells [18]. SARS-CoV-2 manipulation was performed in the BSL-3 laboratory of the University of Ferrara, following the biosafety requirements and accordingly with the Institutional Biosafety Committee. THP-1, CaLu3 and VeroE6 cell lines were exposed to SARS-COV-2 as previously described [19] in the presence or absence of modulatory interventions as detailed below. Cells supernatant was collected 8, 24, or 48 hours post-infection and after the different pharmacological treatments.

2.6. Viral RNA Detection

RNA extraction was performed 8 and 24 h post-infection (hpi) with MagMAX viral/pathogen nucleic acid isolation kit (Thermo Fisher, Milan, Italy), a kit for the recovery of RNA and DNA from the virus. SARS-CoV-2 titration was obtained by TaqMan 2019nCoV assay kit v1 real-time qPCR (Thermo Fisher, Milan, Italy).

2.7. In vivo lung-inflammatory model

Procedures involving animals and their care were in conformity with institutional guidelines, and the Animal Ethics Committee approved all experimental protocols (authorization nos. 625/2018-PR and CBCC2.N. BH4 approved by Italian Ministry of Health). 12-weeks old C57BL/6J mice were injected intranasally with LPS: 10 µg of LPS diluted in 25µl of sterile PBS for each mouse. After 4 hours the mice were sacrificed by cervical dislocation and their lungs were collected and stored at -20°C until further processing. To control mice were administered 25µl of sterile PBS by intranasal injection.

2.8. ELISA

Plasma, PBMCs, THP-1, and CaLu-3 cell cultures supernatants were assayed for human IL-1β or IL-6 using either ELISA kits or Ella™ arrays following the manufacturer’s instructions (R&D Systems, Minneapolis, USA). Mice lungs were homogenized using TissueRuptor™ (Qiagen, Hilden, Germany) with an optimized lysis buffer (300 mM sucrose, 1 mM K2HPO4, 1mM MgSO4, 5.5 mM D-glucose, 20 mM HEPES, 0.5% IGEPAL and supplemented with protease inhibitor cocktail) following the manufacturer’s instructions. Following a centrifugation of 12,000 × g at 4°C for 15 min, the supernatant was collected and filtered with a 0,45 µm filter. The obtained specimens were assayed for mouse IL-1β using Ella™ arrays following the manufacturer’s instructions (R&D Systems, Minneapolis, USA).

2.9. Pharmacological treatments.

Cells were pre-treated before viral infection with the following compounds: MCC950 1mM for 30 min, cinnamaldehyde (CINNA) 50 µM for 30 min, and dexamethasone (DEXA) 100nM for 1 h. For the in vivo model, mice were pre-treated 30 min before LPS administration with the following compounds by aerosol administration: Cinna-Sol® (CinnaPharm, Italia) 1 vial each mouse, and dexamethasone (DEXA) 25mg/kg diluted in 5 ml of PBS.

2.10. Statistical analysis.

The data were analyzed by Prism 6 (GraphPad). Normal distributions were assessed using the Shapiro-Wilk test. Student’s t-test for unpaired data with Welch’s correction was used to compare two datasets with normal distribution, while the Mann-Whitney test was used for non-normal datasets. Multiple comparisons of ≥ 3 experimental groups were performed using Brown-Forsythe and Welch ANOVA one-way test by comparing all the groups with the untreated or with the control ones (Dunnett’s T3 multiple comparison test). P < 0.05 indicates statistical significance.

3.1. Increased blood plasma levels and increased PBMCs release of IL-1β and IL-6 were found in severe COVID-19 patients compared to controls.

In line with previous publications [20], we found significantly increased plasma levels of IL-1β and IL-6 compared to control hospitalized subjects with similar clinical severity but with non-COVID conditions (Fig. 1). Numerically higher levels of IL-1β and IL-6 were found in patients who died compared to those who survived during the study period (Fig. 1c, d). Interestingly, following LPS and Bz-ATP stimulation, we observed that PBMCs of COVID-19 patients significantly release more IL-1β and IL-6 compared to the control group (Fig. 1e, f). These results show that SARS-CoV-2 infection can trigger a specific systemic inflammation and suggest that the pro-inflammatory cytokines IL-1β and IL-6 play a central role in this pro-inflammatory systemic response.

3.2. Macrophages mediate pro-inflammatory cytokine storm while lung cells support SARS-CoV-2 replication

To explore the relative contribution of immune and lung cells in the release of pro-inflammatory cytokines that follows SARS-COV2 infection, we evaluated the production of IL-1β in macrophages (THP-1) and lung (CaLu-3) cell lines. We highlight that only THP-1 cells showed a significant increase in IL-1β production after viral infection, while CaLu-3 cells were completely unaffected (Fig. 2a, b). Remarkably, we found that IL-1β production occurred in both cell lines when stimulated with an NLRP3 activator (LPS + BzATP), thus indicating a selective inability of SARS-CoV-2 virus in activating the same effect in CaLu-3 cells (Fig. 2a, b). Nevertheless, the main consequence of viral infection on CaLu-3 cells is the replication of the virus, instead of the promotion of a pro-inflammatory response, as shown by the progressive viral genome increase detected in SARS-CoV-2 infected CaLu-3 (Fig. 2c). Accordingly, THP-1 cells also showed an increased release of IL-6 upon SARS-CoV-2 infection (Fig. 2d), further supporting their potential role in modulating the pro-inflammatory cytokine storm observed in COVID-19 patients. These data suggest that while lung cells are important for virus infection and replication, activation of immune-inflammatory cells is key in the pro-inflammatory cascade that follows the infection.

3.3. Cinnamaldehyde treatment strongly reduces IL-1β and IL-6 secretion together with SARS-CoV-2 replication

Besides the effects of vaccines and monoclonal therapies, there is still a lack of drugs that allow an efficient pharmacological approach to COVID-19 patients. Therefore, we aimed to identify a compound that might counteract the previously observed effects of SARS-CoV-2 infection on pro-inflammatory cytokines release. We identified the natural compound cinnamaldehyde (CINNA) as a promising candidate since it has been shown to act on NLRP-3 inflammasome reducing IL-1β release [13, 14, 21]. At the same time, we tested the effects of dexamethasone on these pathways, which nowadays represents one of the standard treatments for severe COVID-19. Also, the known NLRP3 inhibitor MCC950 was used as a control. Notably, CINNA pretreatment strongly decreased LPS + BzATP-induced inflammation in COVID-19 patients’ PBMCs, by inhibiting SARS-CoV-2 enhanced proinflammatory state (Fig. 3a, b). The effects of CINNA were stronger even compared with dexamethasone and MCC950, which were both ineffective in diminishing IL-6 release (Fig. 3b). We then tested the direct effect of CINNA pretreatment on LPS-induced inflammation and SARS-CoV-2 infection on THP-1 cells. Interestingly, in this scenario, CINNA strongly reduced IL-1β and IL-6 secretion induced by LPS as well as dexamethasone and MCC950 (Fig. 3c, d) as well known from literature [22, 23]. However, after viral infection, CINNA could reduce IL-1β levels but not significantly counteract the IL-6 one (Fig. 3e, f). Similarly, dexamethasone and MCC950 only showed their efficacy in counteracting NLRP3 activation (Fig. 3e, f). Lastly, we investigated the effects of CINNA in affecting SARS-CoV-2 viral replication by using two different in vitro cell models, the aforementioned CaLu-3 and the VeroE6, the gold standard cell line to study viral replication for SARS-CoV-2. As shown in Fig. 3g, CINNA pretreatment lessens SARS-CoV-2 viral replication 3-fold both 8- and 24-hours post-infection in CaLu-3 cells. An equal effect was observed also on VeroE6 cells after 48 hours from infection (Fig. 3h).

3.4. Aerosol administration of CINNA reduces IL-1β secretion in an in vivo lung-inflammatory model

Next, we aimed to assess the effects of CINNA pretreatment on the release of IL-1β in an in vivo model of LPS-induced lung inflammation. In order to induce direct inflammation into lungs, we performed intranasal administration of LPS into C57BL/6J mice, and in the control group we administered PBS (Fig. 4a). We tested the anti-inflammatory capacity of CINNA, giving it by aerosol as pretreatment before LPS intranasal injection (CINNA + LPS). As a control, we performed aerosol administration of DEXA (DEXA + LPS). As expected, LPS treatment (LPS) was able to increase IL-1β release compared to the control group (CTRL), while both treatments with CINNA and DEXA exhibited a large inhibitory effect on the levels of IL-1β release induced by intranasal LPS administration (Fig. 4b). These findings suggest that CINNA has a pronounced ability to mitigate the release of IL-1β in the context of LPS-induced lung inflammation not only in vitro but also in vivo.

Chronic inflammation is one of the main hallmarks in several pulmonary diseases and pathogens infectious. Over the years, research has made significant strides in the treatment of respiratory diseases, due to bacterial and viral infections. However, a treatment for the reduction of pro-inflammatory cytokine storms induced by pathogens, with minimal or no side effects, is still an important goal to be reached. Here, we developed a model of pathogen-induced pro-inflammatory condition by using SARS-CoV-2 infection, and we evaluated the anti-inflammatory properties of the natural compound CINNA.

First, we confirm previously reported data showing that COVID-19 patients show increased plasma levels of IL-1β and IL-6 compared to pneumonia patients non-SARS-CoV-2 related [24, 25]. Interestingly, our data highlight that PBMCs of COVID-19 patients are more prone to release proinflammatory cytokines, such as IL-1β and IL-6, upon stimulation, thus indicating that virus infection might prime and boost the systemic inflammatory responses exposing patients to inflammation-related clinical impairment. Furthermore, we have shown that SARS-CoV-2 infection elicits IL-1β and IL-6 release only in macrophages, while lung cells are the site for viral replication. These results suggest that the preventive use of anti-inflammatory compounds might result in a beneficial effect on disease resolution, by reducing the diffuse COVID-19 pathology.

Several studies have shown the promising anti-inflammatory effects of some phyto-compounds, used to reduce inflammation in different conditions [26]. Phyto-compounds, extracted from plants, are generally characterized by reduced side effects in long-term treatments and low production costs. In addition, complementary approaches based on natural compounds are gaining increasing attention due to superior patient compliance compared to traditional synthetic drugs [27–29]. Our data have shown that CINNA administration is able to reduce LPS-induced IL-1β and IL-6 release in human PBMCs, and consistently also in THP-1 macrophages. However, CINNA treatment, as well as dexamethasone and MCC950, were able to reduce only SARS-CoV-2-induced IL-1β release in THP-1 macrophages, leaving SARS-CoV-2-induced IL-6 secretion unaffected. However, CINNA treatment, as well as dexamethasone and MCC950, were able to significantly reduce the release of IL-1β induced by SARS-CoV-2 in THP-1 macrophages, while having only a moderate effect on SARS-CoV-2-induced IL-6 secretion.

It is well-known that IL-1β possesses a broad spectrum of pro-inflammatory properties, including the ability to induce the synthesis of other pro-inflammatory cytokines and chemokines (such as TNF-α, IL-6, and IL-8) [30, 31]. Nevertheless, in the cited experiment performed on THP-1 macrophages, CINNA, dexamethasone, or MCC950 pre-treatment only moderately affect IL-6 release, suggesting that in SARS-CoV-2 infection, IL-6 release might be independent of IL-1β activation. Therefore, considering that pre-treatment with the employed compounds only had a modest impact on IL-6 release in THP-1 macrophages infected with SARS-CoV-2, it is plausible to suggest that in this condition, the release of IL-6 may occur independently of IL-1β activation.

Several studies have reported that the use of anakinra, a dual blocker of IL-1α and IL-1β, in COVID-19 patients reduced the mortality risk, especially in the presence of signs of hyperinflammation [6, 32–34]. However, facing the severity of COVID-19, it is imperative to identify treatments that are easily accessible to the population who do not need to be hospitalized [35]. In this scenario, CINNA, as an easy-to-assume nutraceutical, represents a promising preventive approach to be used to prevent the appearance or aggravation of COVID-19 respiratory symptoms. Indeed, our in vivo lung inflammatory model demonstrates a significant reduction in IL-1β release following pre-treatment with CINNA. In addition, our data proved that CINNA pre-treatment reduces the viral replication rate in lung cells, suggesting that CINNA might provide beneficial effects by down-regulating the synthesis of IL-1β also by reducing viral spreading.

Inflammation is not an exclusive characteristic of COVID-19 but is also present in many other forms of pneumonia caused by various pathogens. Therefore, its application could also be relevant in the treatment of different types of pneumonia. Hence, CINNA represents a promising dietary supplement to reduce the pro-inflammatory effects due to pathogens-induced pulmonary infections.

Author Contributions: Conceptualization, C.G. and P.P.; methodology, M.Ca., B.V., M.P., P.C., V.G. and R.R.; formal analysis, B.V., M.P.; investigation, G.C.; resources, M.Co., L.M., S.S. and A.P.; data curation, B.V., M.P.; writing—original draft preparation, B.V. and M.Co.; writing—review and editing, M.Ca., B.V., C.G., P.P. and M.P.; visualization, B.V.; supervision, C.G.; funding acquisition, C.D., A.T. and D.T.. All authors have read and agreed to the published version of the manuscript.

Disclosures: The authors declare no conflict of interest. The CinnaPHARM had no role in the design of the study; in the collection, analysis, or interpretation of data; in the writing of the manuscript; or in the decision to publish the results.

Compliance with Ethics Guidelines: The present study was conducted following ethical approval from CE-AVEC (Comitato Etico di Area Vasta Emilia Centro, Bologna, Italy). Informed consent was obtained from all subjects involved in the study. The study was performed in accordance with the Helsinki Declaration of 1964, and its later amendments. The study is registered at www. clinicaltrials.gov with the identifier NCT04343053.

Acknowledgments: All the authors are grateful to Cristina Mantovani for the support received during the final revisions and editing of the manuscript and to the association A-rose ODV.

Funding: B.V. is supported by the Italian Ministry of Health (SG-2019-12369531). M.P. is supported by the Italian Association for Cancer Research (ID-26665). R.R. is supported by a COVID-19 grant from the University of Ferrara. P.P. is supported by local funds from the University of Ferrara, Italian Association for Cancer Research (AIRC, IG-23670), Italian Ministry of Education, University and Research (PRIN, 2017 E5L5P3). C.G. is supported by local funds from the University of Ferrara, Italian Association for Cancer Research (AIRC, IG-19803), the European Research Council (ERC; 853057-InflaPML), and Italian Ministry of Education, University and Research (PRIN, 2017 7E9EPY).

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No competing interests reported.

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SARS-CoV-2 infection as a model to study the effect of cinnamaldehyde as adjuvant therapy for viral pneumonia.

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Abstract

Introduction:

Methods

Results

Conclusion

Figures

1. Introduction

2. Materials and Methods

2.1. Subjects’ enrollment

2.2. Plasma collection and PBMCs isolation

2.3. Cell cultures

2.4. Inflammasome activation assays in vitro

2.5. SARS-CoV-2 Propagation and Infection

2.6. Viral RNA Detection

2.7. In vivo lung-inflammatory model

2.8. ELISA

2.9. Pharmacological treatments.

2.10. Statistical analysis.

3. Results

3.2. Macrophages mediate pro-inflammatory cytokine storm while lung cells support SARS-CoV-2 replication

3.3. Cinnamaldehyde treatment strongly reduces IL-1β and IL-6 secretion together with SARS-CoV-2 replication

3.4. Aerosol administration of CINNA reduces IL-1β secretion in an in vivo lung-inflammatory model

4. Discussion

Declarations

References

Additional Declarations

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