key: cord-0994708-26erz3sl
authors: Gasparello, Jessica; D'Aversa, Elisabetta; Papi, Chiara; Gambari, Laura; Grigolo, Brunella; Borgatti, Monica; Finotti, Alessia; Gambari, Roberto
title: Sulforaphane inhibits the expression of interleukin-6 and interleukin-8 induced in bronchial epithelial IB3-1 cells by exposure to the SARS-CoV-2 Spike protein
date: 2021-05-04
journal: Phytomedicine
DOI: 10.1016/j.phymed.2021.153583
sha: 4a5cedb04c9e0582f4b5367f97e7c4226c4f8ee9
doc_id: 994708
cord_uid: 26erz3sl

BACKGROUND: A key clinical feature of COVID-19 is a deep inflammatory state known as “cytokine storm” and characterized by high expression of several cytokines, chemokines and growth factors, including IL-6 and IL-8. A direct consequence of this inflammatory state in the lungs is the Acute Respiratory Distress Syndrome (ARDS), frequently observed in severe COVID-19 patients. Cytokine storm is associated with severe forms of COVID-19 and poor prognosis for COVID-19 patients. Sulforaphane (SFN), one of the main components of Brassica oleraceae L. (Brassicaceae or Cruciferae), is known to possess anti-inflammatory effects in tissues from several organs, among which joints, kidneys and lungs. PURPOSE: The objective of the present study was to determine whether SFN is able to inhibit IL-6 and IL-8, two key molecules involved in the COVID-19 cytokine storm. METHODS: The effects of SFN were studied in vitro on bronchial epithelial IB3-1 cells exposed to the SARS-CoV-2 Spike protein (S-protein). The anti-inflammatory activity of SFN on IL-6 and IL-8 expression has been evaluated by RT-qPCR and Bio-Plex analysis. RESULTS: In our study SFN inhibits, in cultured IB3-1 bronchial cells, the gene expression of IL-6 and IL-8 induced by SARS-CoV-2. This represents the proof-of-principle that SFN may modulate the release of some key proteins of the COVID-19 cytokine storm. CONCLUSION: The control of the cytokine storm is one of the major issues in the management of COVID-19 patients. Our study demonstrates that SFN can be employed in protocols useful to control hyperinflammatory state associated with SARS-CoV-2 infection.

The pandemic coronavirus disease 2019 caused by the Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) is characterized by two major clinical phases: (a) a SARS-CoV-2 infection of target cells and tissues and (b) a deep inflammatory state, known as "cytokine storm" (Pascarella et al., 2020; Pelaia et al., 2020) . A key step for SARS-CoV-2 infection is the binding of the SARS-CoV-2 Spike protein (S-protein) to angiotensin-converting enzyme 2 (ACE2). This dictates the wide host range and tropism of the infection in human organs and tissues, leading to important clinical manifestations and complications, such as pulmonary failure (Gao et al., 2020; Pascarella et al., 2020) . The interaction between the SARS-CoV-2 S-protein and ACE2 is a key step also for inducing the "cytokine storm". The attachment of the SARS-CoV-2 Sprotein to ACE2 is followed by deep cellular changes, among which the "cytokine storm", including the hyperactivation of Nuclear Factor kappa-B (NF-B) by IL-6/STATs axis (Ratajczak et al., 2020) . This induces in lungs the Acute Respiratory Distress Syndrome (ARDS) that had been frequently observed in severe COVID-19 patients (Soumagne et al., 2020) and is clearly associated with the severity of the pathology (Grasselli et al., 2020; Matthay et al., 2020) . Patients with COVID-19-related ARDS have a form of injury that, in many aspects, is similar to the ARDS unrelated to COVID-19 (Grasselli et al., 2020) . Importantly, patients with COVID-19-related ARDS have high mortality rates compared to COVID-19 patients without any ARDS symptoms (Matthay et al., 2020) .

The impact of anti-inflammatory protocols for anti-SARS-CoV-2 pharmacological strategies is clear, as recently demonstrated by the effective treatments targeting IL-6 (Nasonov et al., 2020) and IL-8 (Andreakos et al., 2020) . Notably, the pharmacological approach for treating ARDS steadily needs novel anti-inflammatory reagents as different COVID-19 patients might respond in a different way to these treatments (de Simone et al., 2020) .

Several anti-inflammatory strategies to reduce COVID-19 "cytokine storm" and associated ARDS have been proposed using biomolecules derived from herbal medicinal extracts and reviewed by several authors (Khalifa et al., 2020; Matveeva et al., 2020) . This was judged to be a key strategy at the beginning of the pandemic, in consideration of the unknown nature of the disease and the lack of effective treatment protocols and approved vaccines (Adem et al., 2020 , Ngwa et al., 2020 . Repurposing of known plant-derived reagents for anti-inflammatory activity against the COVID-19 "cytokine storm" might be of great interest (Dzobo et al., 2020; Wang et al., 2020) . In this respect, sulforaphane (SFN: 1-isothiocyanate-4-methyl sulfonyl butane), one of the main components of Brassica oleraceae L. (Brassicaceae) (common name broccoli) and other spp from the Cruciferae family, deserves consideration (Lattè et al., 2011) . SFN has been reported to exhibit anti-inflammatory effects (Sturm et al., 2017; Ruhee et al., 2019) in tissues of several organs, such as joints, kidneys and lungs. In particular, SFN is a potent Nuclear factor erythroid 2-related factor 2 (Nrf2) activator.

Interestingly Nrf2 inhibits the recruitment of inflammatory cells and orchestrates the anti-inflammatory process by regulating gene expression through the Antioxidant Response Element (ARE) (Ahmed et al., 2016) .

In the context of lung tissue, Qi et al. (2016) reported that SFN dependent Nrf2/ARE activation exerts protective effects against lipopolysaccharide (LPS)-induced acute lung injury (ALI). Thus, SFN may be a potential candidate for use in the treatment of acute lung injury.

The objective of the present study was to determine whether SFN inhibits IL-6 and IL-8, two important molecules involved in the COVID-19 "cytokine storm". The effects of SFN were studied in an experimental in vitro study using bronchial epithelial IB3-1 cells (Zeitlin et al., 1991) exposed to the SARS-CoV-2 Spike protein (S-protein), following a protocol developed using the S-protein of SARS-CoV-1 (Wang et al., 2007) . We first verified whether this novel experimental model employing SARS-CoV-2 S-protein would also induce an increase of proteins characterizing the COVID-19 "cytokine storm". Then the effects of SFN on expression of pro-inflammatory genes were analyzed by RT-qPCR and Bio-Plex assay in order to determine mRNA accumulation and extracellular protein release. Both these issues (the development of a SARS-CoV-2 Spike-induced experimental screening system and the effects of SFN on Spike-induced IL-6 and IL-8) are novel and might be considered of some interest for the screnning and characterization of new agents to be proposed as inhibitors of molecules of the COVID-19 "cytokine storm". Cell culture conditions. The human bronchial epithelial IB3-1 cell line (Zeitlin et al., 1991) was cultured in humidified atmosphere of 5% CO 2 /air in LHC-8 medium (Gibco, Thermo Fischer Scientific, Waltham, MA, USA) supplemented with 5% fetal bovine serum (FBS, Biowest, Nuaillé, France) in the absence of gentamycin. To verify the effect on proliferation, cell growth was monitored by determining the cell number/ml using a Z2

Coulter Counter (Coulter Electronics, Hialeah, FL, USA).

KDa; stock concentration = 7.2 M in 9% urea, 0.32% Tris-HCl pH 7.2, 50% glycerol) was diluted in 200 µl of LHC-8 medium to achieve the final concentrations used to treat IB3-1 cells. Briefly, cells seeded at 50% of confluence, were treated with Spike protein (5-50 nM) and incubated for 30 min at 4°C, then for 30 min at 37°C, according with the protocol published by Wang et al. (2007) (this procedure is expected to maximize Sprotein interaction with the receptor and the S-protein cellular uptake). After this incubation, LHC-8 medium supplemented with 5% (final concentration) FBS was added to a final 500 l volume and the cultures were further incubated at 37°C and for 24h.

Unstimulated cells (treated with DMSO) were used as reference control.

RNA extraction. Cultured cells were trypsinized (0,05% trypsin and 0,02% EDTA; Sigma-Aldrich) and collected by centrifugation at 1,000 x g for 8 min at 4 °C, washed twice with DPBS 1X (Gibco, Thermo Fischer Scientific) and lysed with Tri-Reagent (Sigma Aldrich), according to manufacturer's instructions. The isolated RNA was washed once with cold 75% ethanol, dried and dissolved in nuclease-free pure water before use. Obtained RNA was stored at -80°C until use (Gasparello et al., 2019) . After an incubation of 15 minutes at room temperature in the dark, samples were acquired and data were analyzed using Annexin V and Dead Cell Software Module (Merck Millipore) (Gasparello et al., 2020) .

Statistics. Results were expressed as mean ± standard error of the mean (SEM) and comparison among groups was made by using analysis of variances (ANOVA). Statistical significance was defined with p<0.05 (*, significant) and p<0.01 (**; highly significant).

We first analyzed the effects of exposure of IB3-1 cells to SARS-CoV-2 Spike protein (S-protein). To this aim, IB3-1 cells were treated with 5, 15 and 50 nM Sprotein for 24h, as indicated in the Materials and methods section and in the experimental flow-chart shown in Fig. 1A . After the treatment, RNA was purified from the treated cells for RT-qPCR analysis, and cellular supernatants were isolated for the analysis of secreted cytokines, chemokines and growth factors in order to identify SARS-CoV-2 Spike protein-induced alteration of the secretome profile.

Concerning mRNA expression, we have compared the mRNA coding for IL-6 ( Fig. 1B) and IL-8 (Fig. 1C) . The results obtained show that, after S-protein exposure, IB3-1 cells accumulate larger amounts of IL-6 and IL-8 mRNAs with respect to control untreated IB3-1 cells. This information was derived from RT-qPCR analysis using actin as internal control. Similar results were obtained using as internal control GAPDH and RPL13A sequences (data not shown). Interestingly, exposure of IB3-1 cells to 5 nM S-protein was sufficient to have significant differences of mRNA expression (p = 0.0294 for IL-6 and p = 0.001 for IL-8).

The data concerning IL-6 and IL-8 protein release were obtained by Bio-Plex analysis (Fig. 1, panels D-F) . Exposure to SARS-CoV-2 Spike protein was associated with a sharp increase of the release of IL-6 ( Fig. 1D ) and IL-8 (Fig. 1E ). In agreement with the RT-qPCR data exposure to 5 nM SARS-CoV-2 S-protein was sufficient to obtain a significant increase (2-3-fold) of the release of IL-6 (p = 0.0008) and IL-8 (p = 0.0116) (see also the summary of 5 independent experiments shown in Fig. 1F ).

Data from these experiments showed that the IL-6 and IL-8 gene expression ( Fig.   1B and 1C ) and the release of their respective proteins ( Fig. 1D and Fig. 1F ) are operating following treatment of IB3-1 with SARS-CoV-2 Spike protein. As IL-6 and IL-8 gene expression is under transcriptional control of NF-B (Bezzerri et al., 2011) , we also analyzed NF-B expression by Western blotting and found that exposure of IB3-1 cell to SARS-CoV-2 Spike protein leads to an increase of NF-B (Supplementary Figure S1 ). Altogether, these data support the concept that our experimental system recapitulates some of the key step of SARS-CoV-2 Spike-mediated biological changes, including NF-B stimulation and upregulation of IL-6 and IL-8.

In the experiment shown in Fig. 2 , the effects of SFN on S-protein induced IL-6 and IL-8 gene expression induced by S-protein were analyzed. Fig. 2A shows that both 5 and 10 M of SNF are potent inhibitors of IL-6 and IL-8 mRNA accumulation. This finding was highly reproducible. We avoided the use of higher concentrations of SFN, since it is known that this molecule is able to induce also pro-apoptotic effects at high concentrations in other cell types of cells (Gasparello et al., 2020) . No major effects of SFN on IB3-1 cell growth were observed when 5 M concentration was used. Fig. 2 shows that SFN inhibits the release of IL-6 and IL-8 and that this effect is dose-dependent. In addition, the inhibitory effects on protein release ( Fig. 2B and 2C) are, as expected, accompanied by a sharp inhibition of mRNA accumulation ( Fig. 2A) .

The experiment shown in Fig. 3 (Fig. 3B) and did not induce their apoptosis ( Fig. 3C and 3D ).

In order to verify the selectivity of the effects of SFN on overall protein released, the Bio-Plex analysis was conducted on secreted proteins exceeding 1 pg/ml in the extracellular medium of untreated S-protein induced IB3-1 cells. Fig.4A shows that 24h of S-protein treatment induced an increased release of IL-6, IL-8, IL-9, FGF, G-CSF, GM-CSF, MCP-1 and MIP-1.

The effects of SFN treatment on the release of these 13 proteins are shown in panels B-N of Fig. 4 . These results suggest a differential effect of SFN on the release of cytokines, chemokines and growth factors. SFN-mediated inhibitory effects were evident (in addition to IL-6 and IL-8, as shown in Fig. 2) for PDGF, IL-9, G-CSF, GM-CSF, IFN-, MCP-1, MIP-1. No effect was evident for IL-10, FGF, RANTES and VEGF.

One of the clinical features of COVID-19 is the hyperinflammatory activity that is characterized by high expression of IL-6, IL-8 and several other cytokines, chemokines and growth factors (Pelaia et al., 2020) . This hyperinflammatory activity is associated with severe forms of COVID-19 and poor prognosis for COVID-19 patients (Zeng et al., 2020) . For instance, Del Valle et al. (2020) found that high serum IL-6, IL-8 and TNF-α levels at the time of hospitalization are strong and independent predictors of patient survival. In another study, Burke et al. (2020) found that inflammatory phenotyping (revealing upregulation of IL-6 and IL-8 gene expression) predicts clinical outcome in COVID-19 subjects. Therefore, anti-inflammatory compounds and specific clinical protocols are highly needed.

Concerning this issue,different approaches targeting IL-6 and IL-8 have been proposed in several studies as well as in clinical trials (Ruhee et al., 2019; Sturm et al., 2017) . For instance, among these: NCT04381052 ("Study for the Use of the IL-6

Inhibitor Clazakizumab in Patients With Life-threatening COVID-19 Infection"), The results presented in our study are a proof-of-principle that the release of two key proteins of the COVID-19 "cytokine storm" (Soy et al., 2020) can be strongly inhibited by SFN. Since the control of the "cytokine storm" is a major issue in the management of COVID-19 patients (Mustafa et al., 2020) , our study could stimulate research activity that can contribute to the development of protocols useful to control hyperinflammatory state associated with SARS-CoV-2 infection. Of particular interest, in addition to IL-6 and IL-8, is the inhibitory effects of SFN on SARS-CoV-2 induced increase of G-CSF, GM-CSF and MCP-1. This paper is expected to sustain research activity on plant extracts and food supplement containing SFN, in order to support the integration of 'phytopreparations' into conventional/official medicine focusing on COVID-19 treatment. SFN is indeed present within several phytomaterials, derived for instance from broccoli sprout, broccoli, cauliflower, kale, Brussels sprout, cabbage, bok choy, collard, rugula, turnips (Farag et al., 2010) .

One of the limits of our study is the lack of explanation about the SFN mechanism of action. This should be considered a major issue of the research on this topic in the future, since it could help in finding new targets of possible use in therapeutic protocols. Among the several possibilities (that in any case are not mutually exclusive), SFN can exert its anti-inflammatory activity by JNK/AP-1/NF-κB inhibition and Nrf2/HO-1 activation (Subedi et al., 2019) . The SFN-mediated targeting of NRF2 has been described in other studies (Qi et al., 2016) . On the other hand, SFN is well known as a potent inducer of endocellular production of hydrogen sulfide (Pei et al., 2011) . In consideration of the large variety of anti-inflammatory effects of hydrogen sulfide and hydrogen sulfide-releasing chimeras, this issue should be considered of great interest for future studies.

We have developed a simple experimental system and analytical protocol for the screening of molecules interfering with the expression of proteins known to be involved in the COVID-19 "cytokine storm". The results here presented demonstrate that exposure of epithelial IB3-1 cells to the SARS-CoV-2 spike protein induces increased expression of NF-B and increased release particularly of IL-6 and IL-8, but also of IL-9, FGF, G-CSF, GM-CSF, MCP-1 and MIP-1 This allows the screening of possible inhibitors of these biochemical targets and possible agents to be proposed for the experimental treatment of this clinical phase of the disease.

Treatment with sulforaphane reverses IL-6 and IL-8 upregulation induced by SARS-CoV-2 Spike protein in IB3-1 cells. Furthermore, sulforaphane-mediated inhibitory effects were observed also for PDGF, IL-9, G-CSF, GM-CSF, IFN-, MCP-1 and MIP-1. Therefore, sulforaphane and sulforaphane-containing phytoproducts should be further evaluated as potential inhibitors of the COVID-19 "cytokine storm".

Further experiments should be programmed to identify other agents able to inhibit changes in gene expression induced by SARS-CoV-2 Spike, not only to identify novel molecules to be considered as positive control in this experimental system, but also to verify whether combined therapy with sulforaphane is possible.

No competing financial interests exist. The authors declare the absence of other types of conflict of interest.

This work was funded by Fondazione Fibrosi Cistica (FFC), Project "Revealing the microRNAs-transcription factors network in cystic fibrosis: from microRNA therapeutics to precision medicine (CF-miRNA-THER)", FFC#7/2018.

Jessica Gasparello, investigation, data curation and formal analysis; Elisabetta D'Aversa, data curation; Chiara Papi, data curation; Laura Gambari, resources and methodology; Monica Borgatti, supervision; Alessia Finotti and Brunella Grigolo, supervisors; Roberto Gambari, writing the manuscript and funding acquisition. 

Caffeic acid derivatives (CAFDs) as inhibitors of SARS-CoV-2: CAFDs-based functional foods as a potential alternative approach to combat COVID-19

Nrf2 signaling pathway: Pivotal roles in inflammation

Dexamethasone, pro-resolving lipid mediators and resolution of inflammation in COVID-19

Mapping the transcriptional machinery of the IL-8 gene in human bronchial epithelial cells

Inflammatory phenotyping predicts clinical outcome in COVID-19

Finding the right time for anti-inflammatory therapy in COVID-19

An inflammatory cytokine signature predicts COVID-19 severity and survival

Coronavirus Disease-2019 treatment strategies targeting interleukin-6 signaling and herbal medicine

Sulforaphane composition, cytotoxic andantioxidant activity of crucifer vegetables

ACE2 partially dictates the host range and tropism of SARS-CoV-2

High levels of apoptosis are induced in the human colon cancer HT-29 cell line by co-administration of sulforaphane and a Peptide Nucleic Acid targeting miR-15b-5p. Nucleic Acid Ther

Efficient delivery of microRNA and antimiRNA molecules using an argininocalix[4]arene macrocycle

Pathophysiology of COVID-19-associated acute respiratory distress syndrome: a multicentre prospective observational study

Screening for natural and derived bio-active compounds in preclinical and clinical studies: One of the frontlines of fighting the coronaviruses pandemic

Health benefits and possible risks of broccoli an overview

Biological mechanisms of COVID-19

Acute Respiratory Distress Syndrome

In search of herbal anti-SARS-Cov2 compounds

Cytokine Storm in COVID-19 patients, its impact on organs and potential treatment by QTY Code-Designed Detergent-Free Chemokine Receptors

The role of Interleukin 6 inhibitors in therapy of severe COVID-19

Potential of flavonoid-inspired phytomedicines against COVID-19

COVID-19 diagnosis and management: a comprehensive review

Hydrogen sulfide mediates the antisurvival effect of sulforaphane on human prostate cancer cells

Lung under attack by COVID-19-induced cytokine storm: pathogenic mechanisms and therapeutic implications

Sulforaphane exerts anti-inflammatory effects against lipopolysaccharide-induced acute lung injury in mice through the Nrf2/ARE pathway

SARS-CoV-2 entry receptor ACE2 is expressed on very small CD45(-) precursors of hematopoietic and endothelial cells and in response to virus Spike protein activates the Nlrp3 inflammasome

Sulforaphane Protects Cells against Lipopolysaccharide-Stimulated Inflammation in Murine Macrophages

Pulmonary embolism among critically ill patients with ARDS due to COVID-19

Cytokine storm in COVID-19: pathogenesis and overview of anti-inflammatory agents used in treatment

Brassica-derived plant bioactives as modulators of chemopreventive and inflammatory signaling pathways

Anti-Inflammatory effect of sulforaphane on LPS-activated microglia potentially through JNK/AP-1/NF-κB inhibition and Nrf2/HO-1 Activation

Up-regulation of IL-6 and TNF-alpha induced by SARS-coronavirus spike protein in murine macrophages via NF-kappaB pathway

Small molecule therapeutics for COVID-19: repurposing of inhaled furosemide

A cystic fibrosis bronchial epithelial cell line: immortalization by adeno-12-SV40 infection

Longitudinal changes of inflammatory parameters and their correlation with disease severity and outcomes in patients with COVID-19 from Wuhan

A) Flow-chart of the experimental plan. (B-E) Effects of 24h exposure of IB3-1 cells to increasing amounts of SARS-CoV-2 spike protein (S-protein) on IL-6 (B) and IL-8 (C) mRNA

Secreted proteins exceeding the concentration of 1 pg/ml in the extracellular medium are reported in the histogram. Data are reported as Fold Change (FC, S-protein infected IB3-1 cells versus control cells). (B-N) Effects of SFN on the inflammation-associated proteins released by S-protein exposed IB3-1 cells