Enteroviruses are responsible for emerging diseases which cause diverse symptoms and may result in neurological complications. An antiviral with multiple mechanisms of action can help prevent enterovirus mediated disease despite differences in the pathogenesis between enteroviruses, including the recently identified enterovirus 69 (EV-69) for which pathogenesis is not well understood. This study investigated the efficacy of epigallocatechin-3-gallate stearate (EGCG-S), a modified form of the antioxidant green tea catechin epigallocatechin-3-gallate (EGCG), in inhibiting EV-69 infection of lung fibroblast cells
in vitro
. Treatment with EGCG-S resulted in moderate protection from EV-69 mediated cytotoxicity as demonstrated by increased metabolic activity as well as maintenance of cell morphology and mitochondrial function. These effects were correlated with reduced hydrogen peroxide production in infected cells following EGCG-S treatment with concentrations less than 100 μM, suggesting a role for inhibition of EV-69 mediated oxidative stress. This study provides insight into characteristics of EV-69 infection as well as the efficacy of EGCG-S mediated inhibition of EV-69 infection.
Enteroviruses comprise a large and diverse genus of non-enveloped RNA viruses in the Picornaviridae family. Members of this genus are grouped into fifteen species all of which are associated with a wide variety of diseases due to their tropism encompassing gastrointestinal, respiratory, and neuronal cells [1]. This allows enterovirus infections affecting the gastrointestinal or respiratory tract to also cause neuropathogenesis and fatal conditions such as meningitis [2] [3].
The rapid mutation rate of enteroviruses allows for emergence of strains capable of causing severe disease and even within the same species of enteroviruses pathogenesis can greatly vary. The Human enterovirus D species, for example, includes enterovirus 68 (EV-68) which causes respiratory disease and acute flaccid paralysis while another serotype, EV-70, is primarily associated with acute hemorrhagic conjunctivitis [4] [5]. These serotypes are two of four enteroviruses most recently isolated, along with EV-69 and EV-71. EV-71 causes hand, foot, and mouth disease [6]. Both EV-68 and EV-70 along with EV-71, have been associated with severe neurological disease [7] [8] [9]. Much about the pathogenesis of EV-69, however, is still not known since its first reported isolation in the U.S. in 1959 [10]. This type was originally associated with respiratory illness similar to EV-68 [11] but in recent years has been isolated worldwide from patients presenting with acute flaccid paralysis and encephalitis [12] - [18]. While some common features of the enterovirus infectious cycle likely apply to EV-69, there is a lack of knowledge on the tissue tropism of this enterovirus serotype. This and other emerging enteroviruses may continue to cause outbreaks and disease associated mortality, necessitating novel antivirals that can alleviate infection regardless of the enterovirus type. Such antivirals would thus ideally be able to simultaneously target multiple stages of the infectious cycle to account for differences, such as different host receptor utilization, between enteroviruses.
Here, we investigate the modulation of oxidative stress and the therapeutic properties of a compound derived from the Camellia sinensis plant, known as epigallocatechin-3-gallate (EGCG). EGCG is a green tea polyphenol which has been applied to numerous infectious disease models for the inhibition of microbial growth and viral infectivity [19] [20] [21]. The antiviral effect of EGCG has been reported for both enveloped and non-enveloped viruses including the enteroviruses Coxsackie B3 and EV-71 [21] - [26]. Additionally, it has been demonstrated to be well tolerated in vivo and even utilized for alleviating neurotoxicity associated viral proteins in mice [20] [27]. While the mechanism behind EGCG mediated antiviral effects has been often proposed to be due to interfering with virus attachment to host cells [28], some studies have found that it is the antioxidant property and ability of EGCG to modulate cellular redox that is associated with inhibition of infection [26] [29] [30] [31]. The ability of EGCG to inhibit viral infection through various ways shows promise for the development of an antiviral agent that can be applicable to emerging viruses for which pathogenesis is still unknown. Furthermore, modifications have been made to the structure of EGCG to enhance its stability and bioavailability [23] [32]. Such include palmitoylation of EGCG (pEGCG), which resulted in more effective inhibition of HSV-1 adsorption and infection in Vero cells than did EGCG [23]. Furthermore, addition of stearic acid to EGCG to make epigallocatechin-3-gallate-stearate (EGCG-S) is another modification that enhanced stability and was also shown to inhibit HSV-1 infection in A549 cells without causing cytotoxicity at up to 75 μM, similar to EGCG [33]. In this study, we investigate the cytoprotective effects of EGCG-S on infected cells and evaluate the efficacy of EGCG-S in inhibiting infection of EV-69 in MRC-5 cells and A549 cells. MRC-5 and A549 lung fibroblast cells are appropriate models for in vitro study because EV-69 is associated with atypical respiratory illness.
2. Materials and Methods
Cell Culture Maintenance
The adherent human lung epithelial A549 cells (CCL-185) and the fetal lung fibroblast IRR-MRC-5 cells (ATCC 55-X) (American Type Culture Collection (ATCC) Manassas, VA) were maintained in T25 flasks at 37˚C in a 5% CO2 incubator. The A549 cell line and MRC-5 cell line were propagated in F12K and MEM media (Gibco, ThermoFisher Scientific), respectively, both of which were supplemented with 10% Fetal Bovine Serum and 1% gentamicin.
Virus Propagation
Cells were sub-cultured into a T25 flask and incubated 24 hrs to reach 70% - 80% confluency. 100 μL of Enterovirus type 69 [American Type Culture Collection (ATCC® VR-1077TM) Manassas, VA] was used to infect the confluent cell monolayer for 1 hr after which 5mL of media was supplemented. MRC-5 cells were checked after 24 hrs and A549 cells were checked after 48 - 72 hrs for cytopathic effect (CPE). Virus was harvested when at least 90% of cells showed CPE, through removal of the media and centrifugation for 5 min at 1500 RPM to eliminate cellular debris. The supernatant containing virus was stored in cryogenic tubes at −80˚C and used for the following experiments.
EGCG-S Preparation
EGCG-stearate (EGCG-S) from green tea extract in powder form (US Patent 20120172423) was obtained from Camellix, LLC (Georgia Health Sciences University Center of Innovation for Life Sciences, August, GA). It was dissolved in DMSO to a final stock concentration of 5 mM and stored at 4˚C. Treatment concentrations were prepared from this stock by diluting in F12K or DMEM to make 25, 50, 75, and 100 μM concentrations.
MTS cell viability assay
The CellTiter 96Aqueous One Solution Cell Proliferation Assay (MTS) kit (Cat#G3580, Promega Corp., Madison, WI) was used to quantify cell viability. A549 and MRC-5 cells were seeded from a 100% confluent flask into a 96-well plate and incubated for 24 hrs at 37˚C under 5% CO2 prior to performing the assay. Cells at 70% - 80% confluency were then treated with 100 μL of 25, 50, 75, or 100 μM of EGCG-S for 1 hr. The unadsorbed EGCG-S was then aspirated and cells were supplemented with medium and incubated for 24 hrs at 37˚C under 5% CO2. After incubation, 20 μL of MTS reagent was added to each well and the plate was incubated for 1 hr. Absorbance was read at 490 nm using an Infinite Pro 200 microplate reader (Tecan Life Sciences). The percent viability was determined for each sample by first normalizing absorbance values according to the untreated cells using Microsoft Excel (Microsoft Office, New York, NY, USA), following the formula below. The Mean and Standard error of mean (SEM) of three to five replicates were then calculated using Prism 9 software (GraphPad, San Diego, CA, USA).
MRC-5 or A549 cells were seeded in a 96-well plate and incubated for 24 hrs at 37˚C in a humidified CO2 atmosphere until 80% - 90% confluent. Virus was treated with 25, 50, 75, and 100 μM EGCG-S for 1 hr prior to infection. Cells were then infected with 100 μL EGCG-S treated and untreated virus, respectively, for 1 hr after which unadsorbed virus was aspirated and cells were replenished with media. The plate was incubated for 24 hrs at 37˚C in a humidified CO2 atmosphere, at which point 20 μL of the MTS reagent was added and absorbance was read at 490 nm using a 96-well plate reader spectrophotometer following 1 hr of incubation with the reagent. The % Inhibition was calculated with Microsoft Excel using the formula below for each replicate absorbance value of each condition. Mean and Standard error of mean (SEM) for three to five replicates per experiment were then calculated using Prism 9 Software (GraphPad, San Diego, CA, USA).
A 96-well plate was plated with MRC-5 cells and incubated for 24 hrs at 37˚C in a humidified CO2 atmosphere until 80% - 90% confluent. For infection assays, virus was treated with 25, 50, 75, and 100 μM EGCG-S for 1 hr prior to infection. Media was aspirated from wells and cells were then infected with 100 μL EGCG-S treated and untreated virus for 1 hr. After infection, unadsorbed virus was aspirated and cells were replenished with media. The Viral ToxGloTM assay kit (Cat#G8941, Promega Corp., Madison, WI) was used to detect viable cells with normal mitochondrial function by measuring ATP levels in test conditions. The plate was incubated for 24 hrs at 37˚C in a humidified CO2 atmosphere, at which point 10 μL of the ATP detection reagent was added to each well and the plate was incubated for 1 hr. Relative luminescence units (RLU) were then read as a correlate of ATP levels using a Biotek SynergyTM 2 luminometer.
Antiviral ROS-Glo H2O2 assay
The ROS-GloTM H2O2 assay kit (Promega Corp., Madison, WI) was used to assess oxidative stress induced by viral infection by measuring the levels of H2O2, a reactive oxygen species (ROS). MRC-5 cells were seeded in a 96-well plate and allowed to reach 80% confluency, then infected with EGCG-S treated or untreated virus for 1 hr as described in the previous infection assays. The lytic assay protocol for product Cat#G8820 was followed as per manufacturer’s directions. Cells were then incubated at 37˚C in a humidified CO2 atmosphere for 7 hrs to allow infection, after which 20 µL of H2O2 substrate solution was added to each well and cells were incubated for an additional 5 hrs, for a total of 12 hrs of infection. After incubation, 100 μL of the ROS-GloTM detection solution reaction was added to each well and the plate was incubated for 20 mins. ROS levels were measured in RLU and read using a Biotek SynergyTM 2 luminometer.
Determination of cytopathic effects in cells with EGCG-S treated virus
MRC-5 and A549 cells were seeded in a 6-well plate and incubated for 24 hrs until 70% - 80% confluency. Virus was treated with 25, 50, 75, or 100 μM EGCG-S for 1 hr prior to infection. Media was aspirated from adherent cells in each well and cells were then infected with 100 μL EGCG-S treated and untreated virus, respectively, for 1 hr after which unadsorbed virus was aspirated and cells were replenished with media. The plate was incubated at 37˚C under 5% CO2. Cells were examined for CPE at 400× using an inverted microscope and images were taken at 48 and 72 hrs post infection (hpi).
Statistical Analysis
Graphing and statistical analysis was done using Prism 9 Software (GraphPad, San Diego, CA, USA). Data was analyzed using a one-way Anova with Dunnett’s post-hoc test unless otherwise mentioned.
3. Results3.1. Cytotoxicity Study of Treatment of A549 and MRC-5 Cells with EGCG-S
Enteroviruses are able to infect a variety of cells which include cells of the respiratory tract and can be found in respiratory secretions [34] [35]. To understand the potential effect of EGCG-S treatment on EV-69 infection in cells of the respiratory tract, A549 lung epithelial cells and MRC-5 lung fibroblast cells which were previously demonstrated to be highly susceptible to enterovirus infection were used [36]. Cytotoxicity to EGCG-S was assayed at a concentration range of 25 - 100 μM. Since EGCG-S was dissolved in DMSO, the effect of DMSO alone on cells was evaluated. Microscopy analysis showed that cells were not affected by the DMSO vehicle control even when treated at concentrations greater than 0.5% (The highest final concentration of DMSO used in the study). This correlated well with a previously reported study [37]. Cell viability of A549 cells and MRC-5 cells treated with 25 - 100 µM EGCG-S was determined using an MTS assay, which quantifies viable cells through detection of cellular respiration using the insoluble formazan product as an indicator. This formazan product is derived from the MTS substrate (3-(4, 5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium, inner salt) which becomes converted by the mitochondrial dehydrogenase enzymes present in viable cells. Cytotoxicity did not occur with EGCG-S treatment at up to 75 µM (Figure 1(a)) for A549 cells and no cytotoxicity was observed for MRC-5 cells at each concentration used (25, 50, 75, and 100 µM) (Figure 1(b)). Treatment with these concentrations even resulted in a slightly higher cell viability relative to the untreated control for MRC-5 cells, possibly as a result of antioxidant or protective effects of EGCG-S against regular cell
damage pathways. The cytotoxicity of EGCG-S on cells is dose dependent. EGCG-S was safely applied to cultured A549 cells [33] and cultured Vero cells (unpublished data) up to 75 μM. The results indicated that EGCG-S is non-cytotoxic to A549 and MRC-5 cells.
3.2. Proliferation Assay of A549 and MRC-5 Cells Infected with EV-69 and EGCG-S Treated EV-69
The ability of EGCG-S to protect against EV-69 mediated cytotoxicity was further investigated by measuring viability with respect to metabolically active cells as well as ATP levels as an indicator of normal mitochondrial function. The percent inhibition of infection was determined based on these viabilities as described in the Methods section. An increased viability of cells with treated EV-69 as compared to the untreated EV-69 was observed (Figure 2(a)). The highest inhibition of infection observed was 47% after 75 µM treatment for A549 cells (Figure 2(b)). The percent inhibition of infection was overall lower for MRC-5 cells at the same EGCG-S concentrations tested for A549 cells, at most being 24.5% after 50 µM treatment (Figure 2(b)). Altogether, this demonstrates that EGCG-S treatment has some efficacy in inhibiting EV-69 infection but highlights the differences in efficacy of EGCG-S in vitro that can be expected between different cells of the respiratory tract infected with EV-69. Infection with EGCG-S treated EV-69 results in increased cell viability of both A549 and MRC-5 cells compared with untreated virus infected cells. Since the A549 cells failed to support productive EV-69 infections, the extent of EGCG-S mediated protection against the cytotoxic effects of EV-69 infection of the MRC-5 cells was further investigated.
3.3. Viral ToxGlo ATP Detection Assay
To gain insight into the metabolic activity of the MRC-5 cells for which EGCG-S mediated inhibition of infection was not prominent, we also assayed for ATP levels as an indicator of mitochondrial function and cell viability. Treatment with EGCG-S alone did not negatively impact ATP production in these cells, as measured through the ToxGlo ATP detection assay (Figure 3(a)). Treatment of MRC-5 with EGCG-S did not influence ATP production, therefore treatment with EGCG-S had no negative impact on cell viability. There is no statistical difference between untreated MRC-5 cells and cells treated with EGCG-S up to 100 µM
(Figure 3(a)). However, the limited efficacy of EGCG-S in inhibiting infection was further observed when examining the ATP levels of infected MRC-5 cells. Intracellular ATP for these cells was increased with EGCG-S treatment but was still substantially lower than the uninfected control (Figure 3(b)). This suggests that EV-69 infection still greatly compromised MRC-5 cells, and EGCG-S treatment only moderately protects MRC-5 cells from mitochondrial dysfunction as well loss of metabolic activity induced by EV-69 infection.
3.4. ROS Detection Assay
Treatment with EGCG-S has thus far been demonstrated to reduce mitochondrial dysfunction, and overall cytotoxicity caused by EV-69 infection. These effects are known to occur in enterovirus infection as a result of oxidative stress [38] [39] [40]. While MRC-5 cells are highly susceptible to EV-69 mediated infection, EGCG-S treatment moderately increased cell viability in comparison to untreated controls (Figure 2(a) and Figure 2(b)). EGCG has been reported to modulate cellular redox that is associated with inhibition of infection [26] [29] [30] [31]. Therefore, this study investigated the antioxidant potential of EGCG-S in inhibiting EV-69 infection. To assess whether the protective effects of EGCG-S observed may be due to its antioxidant potential, the total intracellular level of a major reactive oxygen species (ROS), hydrogen peroxide (H2O2) was further examined. EGCG-S treatment resulted in a statistically significant decrease in the total H2O2 levels following 25 - 75 µM treatments in comparison to the untreated EV-69 infected control (Figure 4). Conversely, treatment at 100
µM resulted in greater H2O2 levels than in uninfected cells. This may be a result of EGCG-S mediated cytotoxicity in addition to EV-69 infection, as EGCG may produce H2O2 in culture at concentrations above 50 µM [41] [42] [43]. This suggests the protective effects of EGCG-S may be due to its antioxidant activity, at concentrations below 100 µM. While a difference in H2O2 levels were observed in this infection assay, the effect of EGCG-S on total intracellular ROS needs to be investigated to further understand if the antiviral effect of EGCG-S may be mediated by its ability to scavenge radicals that result in cell damage caused by total ROS collectively.
3.5. Microscopic Observation of EV-69 Infected MRC-5 Cells and EGCG-S Treated Infected MRC-5 Cells
Infection with EV-69 causes a decrease in viability, characterized as more than 90% of cells exhibiting rounding cytopathic effects or by the presence of cellular debris as a result of prominent cell lysis at 48 hrs post infection (hpi). These cytopathic effects (CPE) were investigated in MRC-5 cells infected with untreated EV-69 or EV-69 that was pretreated for 1 hr with EGCG-S (25 to 100 µM) before infecting cells. As a result of EGCG-S treatment of EV-69, more cell proliferation was observed for MRC-5 cells, particularly at 50 and 75 µM, than for the untreated EV-69 infected control (Figure 5). Cytotoxicity became apparent at 100 µM for MRC-5 cells, however, as demonstrated by increased rounding and debris relative to cells infected with untreated EV-69 (Figure 5). These morphological changes may be a result of both viral infection and toxicity of EGCG-S.
Thus, microscopic observation indicated that EGCG-S reduces EV-69 induced cytopathic effects. Altogether, this supports that EGCG-S can prevent EV-69 mediated damage to cells but at concentrations lower than 100 μM.
4. Discussion
The benefits of EGCG-S treatment in viral infections with greatly differing clinical manifestations have been extensively investigated. In this study, we assess the antiviral effect of EGCG-S on EV-69, an enterovirus that was observed to cause productive infection in respiratory epithelial and fibroblast cells in our study. Following treatment with EGCG-S, MRC-5 cells had reduced EV-69 mediated cytotoxicity in comparison to the untreated virus control, albeit with low percent of inhibition of infection. In contrast, other studies reported EGCG-S mediated inhibition of infection to be much higher for HSV-1 infected Vero cells and A549 cells [23] [33]. This difference in efficacy of EGCG-S observed in our study may be attributed to the structural differences in viruses treated, as this modified form of EGCG is postulated to be more efficacious than unmodified EGCG due to having better affinity for the viral envelope and EV-69 is a non-enveloped virus [23]. Furthermore, it is possible that the modes of action of EGCG-S against cells infected with RNA versus DNA viruses also differ. Moreover, we observed that levels of hydrogen peroxide were reduced in MRC-5 cells infected with EGCG-S treated virus at concentrations lower than 100 µM, suggesting EV-69 mediated oxidative stress was inhibited. Determining the causes of the moderate antiviral activity observed in this study requires understanding of both EV-69 pathogenesis as well as the full range of mechanisms in which EGCG-S inhibits viral infections.
The proposed antiviral mechanism(s) of EGCG has differed among studies. Researchers demonstrated that it is likely due to EGCG competing with sialic acid or heparin sulfate containing host cell receptors for viral attachment. This finding is supported by reduced antiviral efficacy against poliovirus. This reduction was associated with poliovirus not being dependent on sialic acid or heparin sulfate containing receptors for entry [21]. However, a different study attributed EGCG-mediated inhibition of EV-71 replication to a reduction of EV-71 induced oxidative stress [26]. Our study demonstrated that hydrogen peroxide was reduced with EGCG-S treatment. EV-69 was treated with EGCG-S prior to infection, which would allow attachment of EGCG-S to the virus. Thus, it is possible that inhibition of EV-69 infection may have occurred through both modulation of cellular redox and prevention of virus attachment to cells. Subsequent research in our laboratory found that EGCG-S inhibited attachment and penetration of HSV-2 in cultured Vero cells (unpublished data). These reports suggest that there may be multiple mechanisms whereby EGCG-S inhibits infection, depending on different virus specific factors that cause cytotoxicity.
While EGCG-S has been formulated as a topical cream for treatment of HSV-1 [23] [44] its clinical application for treatment of other viral diseases is at the preliminary stages. Murine models of Coxsackie B3 and influenza A virus infection supported the bioavailability of EGCG in vivo with oral administration, as treatment of mice resulted in improved survival correlated with reduced viral replication [25] [45] [46] [47]. Our study suggests that EGCG-S reduced the oxidative damage caused by EV-69 infection and increased the viability of infected cells, thus potentially reducing viral replication. While EGCG was shown to reduce viral replication in previous studies, contrasting effects on the inflammatory response associated with viral infections were reported. For influenza A infection, antiviral effects were associated with reduced lung inflammation, in agreement with studies reporting the overall anti-inflammatory effect of EGCG [48] [49] [50]. On the other hand for Coxsackie B3 infection, EGCG treatment was found to greatly inhibit viral replication in heart tissues of mice and associated myocarditis but did not decrease pro-inflammatory cytokine levels [25]. Dampening of the pro-inflammatory cytokine response may be beneficial for viral diseases that cause severe symptoms in result of a cytokine storm, such as influenza A virus or the newly emerged SARS-COV-2 [51], but inhibition of inflammation in other viral infections may lead to prolonged disease or persistent infection due to the suboptimal immune response necessary for clearance of infection. The role of EGCG-S in inhibiting infection will thus require closer examination of the antiviral immune response, including the production of type I interferons (IFNs), in untreated versus treated infected cells. Examination of the cytokine milieu as well as antigen presenting cell chemotaxis and targeting of EGCG-S treated infected cells would help elucidate whether EGCG-S treatment facilitates clearance of enterovirus infection or enables persistent infection due to interference with the antiviral response.
Furthermore, as enterovirus infections may cause severe disease progression to neurological complications, the ability of EGCG-S to serve as an antiviral in early versus later stages of infection will need to be assessed. Future studies investigating the application of EGCG-S 24 or 48 hrs following infection can help characterize the efficacy of EGCG-S in inhibiting EV-69 infection at different stages, as well as further distinguish the role of EGCG-S in virus attachment. Thus far, our data suggest that EV-69 mediated cytotoxicity can be reduced with EGCG-S application in the early stages of infection and merit further investigation into maximization of efficacy alone or in combinatorial strategies. If applicable to enteroviral infections in vivo, EGCG-S can contribute to the prevention of disease progression and spread in areas in which enterovirus outbreaks occur.
Acknowledgements
We would like to thank Dr. A. DiLorenzo in the Biology Department at Montclair State University for providing us with MRC-5 cells and an ROS-Glo assay kit for H2O2 measurement. We would also like to acknowledge Dr. J. Siekierka in the Chemistry and Biochemistry Department at Montclair State University for providing us his facility to conduct luminescence-based experiments. This research was funded in part by the Faculty Scholarship Program at Montclair State University.
Authors’ Contributions
HM, LHL, and SDA designed the study. SDA supervised HM in the laboratory. HM and SDA conducted the experiments. HM performed all statistical analyses. HM, LHL, and SDA drafted the manuscript. All authors read and approved the final manuscript.
Conflicts of Interest
The authors declare no conflicts of interest.
Cite this paper
Mohamed, H., Lee, L.H. and Adams, S.D. (2021) EGCG-S Impacts Oxidative Stress and Infection of Enterovirus 69 in Lung Cells. Advances in Bioscience and Biotechnology, 12, 109-124. https://doi.org/10.4236/abb.2021.125008
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