Hydroxytyrosol Recovers SARS-CoV-2-PLpro-Dependent Impairment of Interferon Related Genes in Polarized Human Airway, Intestinal and Liver Epithelial Cells

The SARS-CoV-2 pandemic has caused approximately 6.3 million deaths, mainly due to the acute respiratory distress syndrome or multi-organ failure that characterizes COVID-19 acute disease. Post-acute COVID-19 syndrome, also known as long-COVID, is a condition characterized by a complex of symptoms that affects 10–20% of the individuals who have recovered from the infection. Scientific and clinical evidence demonstrates that long-COVID can develop in both adults and children. It has been hypothesized that multi-organ effects of long-COVID could be associated with the persistence of virus RNA/proteins in host cells, but the real mechanism remains to be elucidated. Therefore, we sought to determine the effects of the exogenous expression of the papain-like protease (PLpro) domain of the non-structural protein (NSP3) of SARS-CoV-2 in polarized human airway (Calu-3), intestinal (Caco-2), and liver (HepG2) epithelial cells, and to evaluate the ability of the natural antioxidant hydroxytyrosol (HXT) in neutralizing these effects. Our results demonstrated that PLpro was able to induce a cascade of inflammatory genes and proteins (mainly associated with the interferon pathway) and increase the apoptotic rate and expression of several oxidative stress markers in all evaluated epithelial cells. Noteably, the treatment with 10 μM HXT reverted PL-pro-dependent effects almost completely. This study provides the first evidence that SARS-CoV-2 PLpro remaining in host cells after viral clearance may contribute to the pathogenetic mechanisms of long-COVID. These effects may be counteracted by natural antioxidants. Further clinical and experimental studies are necessary to confirm this hypothesis.


Introduction
Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) was discovered in January 2020 in Wuhan, China, during the recent epidemic of pneumonia. The virus has spread rapidly throughout the world causing the COVID-19 pandemic [1,2]. tion KIT (Minerva Biolabs GmbH, Berlin, Germany). All the experiments were performed only in Mycoplasma-free cells.

Transfection and Treatment
Plasmid transfections in Calu-3, Caco-2 and HepG2 cells were performed by using Lipofectamine2000 (Thermo Fisher Scientific-Invitrogen, Waltham, MA, USA) following the manufacturer's instructions. In detail, cells were seeded at a density of 300,000 cells/well in a 6-wells plate and were transfected with 2 µg of a plasmid containing SARS-CoV-2 (2019-nCoV) Papain-like proteinase PLpro/NSP3 Gene ORF cDNA clone expression plasmid (Catalog Number: VG40593-UT; Sino Biological, Eschborn, Germany) or with pCMV3untagged Negative Control Vector (Catalog Number: CV011; Sino Biological, Eschborn, Germany). The day after transfection, cells were cultured for 24 h in a medium containing HXT (Catalog No. S3826; Selleck Chemicals, Houston, TX, USA) at a concentration of 10 µM. All transfection experiments were performed with 80% confluent cells.

Total Protein Extraction and JESS Capillary Western Blotting
Total protein extraction was performed by homogenizing cells in Ripa lysis buffer (Merck-Sigma-Aldrich) and Halt Protease and Phosphate Inhibitor Cocktail (100X) (Thermo Fisher Scientific-Thermo Scientific), and incubated on ice for 30 min. The homogenates were then centrifuged at 13,000 rpm for 10 min and the resulting supernatant was taken as a protein sample and quantified using the BCA™ Protein Assay (Thermo Fisher Scientific-Thermo Scientific). Capillary western analyses were performed using the Jess Simple Western System (Bio-Techne, Minneapolis, MN, USA). Samples were diluted with 0.1X Sample Buffer. Then four parts of the diluted sample were combined with 1 part 5× Fluorescent Master Mix (containing 10× sample buffer and 400 mM DTT) and heated at 95 • C for 5 min. After this denaturation step, prepared samples, antibody diluent, primary antibodies, streptavidin-HRP, secondary conjugate, luminol-peroxide mix and wash buffer were dispensed into designated wells in an assay plate. A biotinylated ladder provided molecular weight standards for each assay. After plate loading, the separation electrophoresis and immunodetection steps took place in the fully automated capillary system. The following antibodies were used: (i) rabbit anti-SARS NSP3 (dilution 1:1000; Code: ab181620 Abcam, Cambridge, MA, USA); (ii) mouse anti-αTubulin (dilution 1:5000; code: nb100-690; Novus Biologicals, Littleton, CO, USA).

Human IFN Pathway
96-well plates are pre-configured with the most appropriate TaqMan ® Gene Expression Assays for a specific human IFN pathway (TaqMan Array96 well Human Interferon Pathway, Number Catalog 4414285; Thermo Fisher Scientific-Thermo Fisher). The contains 93 assays associated with interferon pathway genes and 3 assays to candidate endogenous control genes.
Pathway analysis was conducted by querying Reactome annotations using the R/Bioconductorlibraryreactome.db (accessed on 20 July 2022) [22]. For Reactome analysis, only pathways with an FDR lower than 0.05 and p < 0.05 were considered.

Cell Viability Assays
Cell viability was evaluated using the Cell Proliferation Kit II-XTT (Merck-Roche Molecular Biochemicals, Basel, Switzerland), according to the manufacturer's protocol. Two independent experiments were conducted. Into each well of a 96-well cell culture plate, 20,000 Calu-3, Caco-2 and HepG2 were seeded, and 24 h after plating were treated with or without HXT at different concentrations (5 µM, 10 µM, 20 µM and 30 µM). Assays were performed in quintuple. At the end of the treatment, 50 µL of a mixture of XTT labeling reagent and electron-coupling reagent were added and incubated at +37 • C for 4 h. The absorbance of the water-soluble formazan formed was measured at 492 and 620 nm using an ELISA microplate spectrophotometer.

Apoptosis
Apoptosis was assessed by Fluorescein-5-isothiocyanate (FITC) Annexin V Apoptosis Detection Kit I (code: 556547; Becton Dickinson-BD, Franklin Lakes, NJ, USA). Cells were seeded at a density of 100,000 cells/well in a 12-wells plate and were transfected with 800 nanograms of a plasmid containing SARS-CoV-2 (2019-nCoV) Papain-like proteinase/NSP3 Gene ORF cDNA clone expression plasmid or with pCMV3-untagged Negative Control Vector (all by Sino Biological Inc., Beijing, China). Briefly, cells were washed twice with cold PBS and resuspended in 1X Annexin Binding Buffer. Cells were then stained with 5 µL of FITC Annexin V and with 5 µM of Propidium Iodide (PI) for 15 min before analyzing. Acquisition and analysis were carried out on a FACSCanto II flow cytometer, using DiVa Software, version 6.3 (Becton, Dickinson and Company, Franklin Lakes, NJ, USA).

SDS-PAGE and Western Blotting
Total protein extraction was performed by homogenizing cells in Ripa lysis buffer (Merck-Sigma-Aldrich) and Halt Protease and Phosphatase Inhibitor Cocktail (100×) (Thermo Fisher Scientific-Thermo Scientific), and incubated on ice for 30 min. The homogenates were then centrifuged at 13,000 rpm for 15 min and the resulting supernatant was taken as a protein sample and quantified using the BCA™ Protein Assay (Thermo Fisher Scientific-Thermo Scientific). The samples were then diluted in the sample buffer 4× and resolved in 12% SDS-PAGE, then transferred and immobilized onto the nitrocellulose membranes (GE Healthcare, Munich, Germany). The membranes were blocked using 5% non-fat dry milk for 30 min and incubated with the appropriate primary and secondary antibodies. Protein expression was quantified by densitometric analysis using the open source Image J v3.91 software (https://imagej.nih.gov/ij/download.html, accessed on 20 July 2022). As antibodies were used: (i) rabbit anti-active caspase-8 anti-body (dilution 1:1000; code: NB100-56116; Novus Biological LLC, Centennial, CO, USA); (ii) rabbit anti-GAPDH (dilution 1:1000; code 5174; Cell Signaling, Danvers, MA, USA).

HPLC to Analyze Various Forms of Glutathione
Reduced and oxidized glutathione (GSH and GSSG, respectively) were analyzed by HPLC as previously described [23]. In detail, for glutathione forms determination, cells were mixed with 100 µL of 10 mmol/L phosphate buffer, pH 7.2 (for GSH), or with 100 µL of the same buffer containing 5 mmol/L N-ethylmaleimide (for GSSG, and protein-bound glutathione, GS-Pro). Cells are then lysed by sonication three times for 2 s. After sonication, 50 µL of 12% sulfosalicylic acid was added; the protein pellet is dissolved in 150 µL of 0.1 M NaOH, and the glutathione content in the acid-soluble fraction is determined. Proteins were measured using BCA™ Protein Assay (Thermo Scientific, Rockford, IL, USA). For GS-Pro determinations, the protein pellet was re-suspended in 150 µL of NaOH 0.1 M and derivatized. The levels of the different forms of glutathione were determined by using the derivatization and chromatography procedures. Total glutathione (TotGSH) amounts were calculated by the sum of free GSH, GSSG and GS-Pro. GSH was calculated by subtracting GSSG to free GSH.

Determination of the Oxidative Stress Parameters
Determination of the oxidative stress parameters was carried out on 2 × 10 7 cells for each treatment collected by centrifugation at 1500× g for 10 min at 4 • C. Cell lysates were obtained by treating the cell pellets with 1 mL of cold phosphate buffer saline (PBS, pH 6.7) containing 1 mM EDTA, sonicating in an ice-cold bath for 5 min using a 3 mm titanium probe set to 200 W and 30% amplitude (Vibra Cell™ Sonics Materials, Inc., Danbury, CT, USA). PBS was used as a blank in each assay. The intracellular Reactive Oxygen Species (ROS) levels, expressed as percentage (%), were assessed according to Smeriglio et al. [24] by recording the fluorescence resulting from the intracellular oxidation of 2 ,7dichlorofluorescin diacetate (DCF-DA). The probe, diluted in PBS (10 µM) was added, and the cell was incubated for 30 min to label intracellular ROS. The medium was then removed, and the cells were washed five times with 1× PBS. Fluorescence of the labeled intracellular ROS was recorded by a plate reader (FLUOstar Omega, BMG LABTECH, Ortenberg, Germany) at the following excitation and emission wavelengths: λ ex 485 nm; λ em 535 nm. The release of Nitric Oxide (NO) was evaluated according to Smeriglio et al. [24] by Griess's reagent. The absorbance was measured at 550 nm using a UV-Vis plate reader (Multiskan GO, Thermo Scientific, Waltham, MA, USA). NO was quantified using sodium nitrite as a reference standard ( was measured colorimetrically at 540 nm by using the UV-Vis plate reader reported above, and using MDA as the reference standard (0.625-50 µM).
The protein carbonyl colorimetric assay kit Item No. 10005020 (Cayman Chemical, Ann Arbor, MI, USA) based on 2,4-dinitrophenylhydrazine reaction, was used to quantify the protein carbonyl content according to the manufacturer's instructions. The amount of protein-hydrazone produced (nM) was quantified spectrophotometrically at 370 nm by using the UV-Vis plate reader reported above. Both kits provide a simple, reproducible, standardized and validated (precision, intra-assay and inter-assay coefficient of variation, and recovery) tool for assaying lipid peroxidation and protein carbonyl content in cell lysate, respectively. Furthermore, several reagents were tested by the manufacturer for interference such as buffers, detergents, protease inhibitors and chelators, highlighting that no interferences can occur in the experimental condition adopted in the present study. All data were normalized for protein concentration.

Statistical Analysis
The data are presented as mean ± standard deviation (SD). Comparisons were made between means from at least two independent experiments repeated in duplicate. Statistical differences were analyzed using the Student's t-test. Values of p < 0.05 were considered to be statistically significant. Data analysis was performed with GraphPad Prism 9.0 (GraphPad Software, San Diego, CA, USA).

Expression of SARS-CoV-2-PLpro Induced an Up-Regulation of Genes of the IFN Pathway and of Pro-Inflammatory Cytokines
Polarized human Calu-3, Caco-2, and HepG2 epithelial cells were transfected with the SARS-CoV-2-PLpro (PLpro) plasmid or with an empty vector (pCMV3) as confirmed by the PLpro gene ( Figure 1A) and protein (Supplementary Figure S1) expression. Since PLpro plays a key role in triggering IFN-related signaling and pro-inflammatory molecules in host cells, the expression of 93 genes related to the IFN pathway was evaluated in all epithelial cell lines. Gene array analysis was performed in Calu-3 cells expressing PLpro, compared to cells expressing pCMV3 (Supplementary Figure S2A). As shown in Supplementary Figure S2B, results in PLpro Calu-3 cells showed an up-regulation of nine interferon-related genes, including IFN1A, IFNA7, ATF4, CREB3, CREB3L4, HIST1H3F, HIST1H3H, HIST3H3 and MCF2L, respect to pCMV3; whereas no IFN-related genes were significantly down-regulated. Reactome enrichment analysis for the up-regulated genes showed that these genes belong to the following top-ten pathways: Cellular responses to stress; Cellular responses to stimuli; Factors involved in megakaryocyte development and platelet production; Hemostasis; TRAF6-mediated IRF7 activation; DDX58/IFIH1mediated induction of interferon-α/β; Unfolded Protein Response; Cytokine Signaling in Immune system; Innate Immune System; and Infectious disease ( Figure 1B). These results were confirmed by a single SYBR Green qRT-PCR assay in Calu-3, Caco-2, and HepG2 epithelial cells ( Figure 1C-E). Moreover, the exogenous expression of PLpro also caused the transcriptional increase in genes encoding for pro-inflammatory cytokines such as IL-1β, IL-6 and TNF-α ( Figure 1F-H).
ated induction of interferon-α/β; Unfolded Protein Response; Cytokine Signaling in Immune system; Innate Immune System; and Infectious disease ( Figure 1B). These results were confirmed by a single SYBR Green qRT-PCR assay in Calu-3, Caco-2, and HepG2 epithelial cells ( Figure 1C-E). Moreover, the exogenous expression of PLpro also caused the transcriptional increase in genes encoding for pro-inflammatory cytokines such as IL-1β, IL-6 and TNF-α ( Figure 1F-H).

The Treatment with HXT Reduced the PLpro mRNA Expression and the Apoptosis
HXT, one of the main phenolic compounds in olive oil, has several biological activities, including anti-inflammatory, anti-microbial and antioxidant properties [20].
In order to evaluate if the treatment with HXT was able to reduce the effects of exogenous expression of PLpro, Calu-3, Caco-2 and HepG2 cells were treated with different concentrations of HXT (5 µM, 10 µM, 20 µM and 30 µM). Cell viability was unaffected by PLpro expression in all cell lines, while it already significantly increased after the treatment with 5 µM HXT for 24 h and reached a plateau at 10 µM HXT (Supplementary Figure S3A). Therefore, this latter concentration of HXT was chosen for the next experiments.

The Treatment with HXT Reduced the PLpro mRNA Expression and the Apoptosis
HXT, one of the main phenolic compounds in olive oil, has several biological activities, including anti-inflammatory, anti-microbial and antioxidant properties [20].
In order to evaluate if the treatment with HXT was able to reduce the effects of exogenous expression of PLpro, Calu-3, Caco-2 and HepG2 cells were treated with different concentrations of HXT (5 μM, 10 μM, 20 μM and 30 μM). Cell viability was unaffected by PLpro expression in all cell lines, while it already significantly increased after the treatment with 5 μM HXT for 24 h and reached a plateau at 10 μM HXT (Supplementary Figure  S3A). Therefore, this latter concentration of HXT was chosen for the next experiments.
A recent study reported that SARS-CoV-2 infection could activate caspase-8 to induce apoptosis [25]. Therefore, we evaluated apoptosis by Annexin V assay and the expression of active caspase-8. Exogenous expression of PLpro increased the apoptotic rate in all cell lines compared to cells with empty vector, whereas the treatment with 10 μM HXT reduced apoptosis when compared to their untreated counterpart ( Figures 2B and S3B,C). Values are plotted as mean ± SD of three independent experiments repeated at least in duplicate. Data were analyzed by 2-tailed t-tests, * p < 0.05, ** p < 0.01, ** p < 0.001 vs. pCMV3; § p < 0.05, § § p < 0.01 vs. PLpro.

The Treatment with HXT Reduced PLpro-Dependent Inflammatory Response
Next, we tested the ability of HXT into reducing the PLpro-dependent inflammatory response in polarized human epithelial cells. The inflammatory profile was evaluated in terms of both gene and protein expression levels. As shown in Figure 3A-C, the exposure to 10 μM HXT caused a statistically significant reduction of mRNA levels of several IFNrelated genes and cytokines in the treated cells (PLpro + HXT) compared to untreated cells Values are plotted as mean ± SD of three independent experiments repeated at least in duplicate. Data were analyzed by 2-tailed t-tests, * p < 0.05, ** p < 0.01, *** p < 0.001 vs. pCMV3; § p < 0.05, § § p < 0.01 vs. PLpro.
A recent study reported that SARS-CoV-2 infection could activate caspase-8 to induce apoptosis [25]. Therefore, we evaluated apoptosis by Annexin V assay and the expression of active caspase-8. Exogenous expression of PLpro increased the apoptotic rate in all cell lines compared to cells with empty vector, whereas the treatment with 10 µM HXT reduced apoptosis when compared to their untreated counterpart ( Figures 2B and S3B,C).

The Treatment with HXT Reduced PLpro-Dependent Inflammatory Response
Next, we tested the ability of HXT into reducing the PLpro-dependent inflammatory response in polarized human epithelial cells. The inflammatory profile was evaluated in terms of both gene and protein expression levels. As shown in Figure 3A-C, the exposure to 10 µM HXT caused a statistically significant reduction of mRNA levels of several IFNrelated genes and cytokines in the treated cells (PLpro + HXT) compared to untreated cells expressing viral peptide (PLpro). In particular, the data highlights that HXT significantly down-regulated: IFN7A, IL-1β, IL-6 and TNF-α gene expression in all cell lines; IFN1A and CREB3 gene expression in Calu-3 and Caco-2; CREB3L4 and MCF2L gene transcription  Figure S4A,B). As reported in Figure 4A-C and Supplementary Table S2 exogenous expression of PLpro caused changes in the protein expression levels of several cytokines and chemokines. Among the significantly up-regulated and down-regulated pro-inflammatory molecules, seven were common to all polarized human epithelial cells ( Figure 4D,E).

man inflammation array (Supplementary
Finally, as shown in Figure 5A-C, the treatment with 10 μM HXT significantly reduced the expression levels of EOTAXIN, EOTAXIN-2, IL-1β, IL-2, IL-6, and TNF-α in all cell lines, while the natural antioxidant significantly decreased the expression levels of IL-3 in Caco-2 cells only. These effects of HTX were not ascribable to a direct anti-inflammatory action on cells but rather to an indirect action on PLpro expression (Supplementary Figure S4C).   Figure S4A,B). As reported in Figure 4A-C and Supplementary Table S2 exogenous expression of PLpro caused changes in the protein expression levels of several cytokines and chemokines. Among the significantly up-regulated and down-regulated pro-inflammatory molecules, seven were common to all polarized human epithelial cells (Figure 4D,E).
Antioxidants 2022, 11, x FOR PEER REVIEW 10 of 18 mean ± SD of two independent experiments repeated at least in duplicate. Data were analyzed by 2-tailed t-tests, * p < 0.05, ** p < 0.01, *** p < 0.001 vs. PLpro.  Finally, as shown in Figure 5A-C, the treatment with 10 µM HXT significantly reduced the expression levels of EOTAXIN, EOTAXIN-2, IL-1β, IL-2, IL-6, and TNF-α in all cell lines, while the natural antioxidant significantly decreased the expression levels of IL-3 in Caco-2 cells only. These effects of HTX were not ascribable to a direct anti-inflammatory action on cells but rather to an indirect action on PLpro expression (Supplementary Figure S4C). Values are plotted as mean ± SD of two independent experi ments repeated in duplicate. Data were analyzed by 2-tailed t-tests, * p < 0.05, ** p < 0.01, *** p < 0.001 vs. PLpro.

Expression of SARS-CoV-2-PLpro Induced Oxidative Stress That was Counteracted by the Treatment with HXT
Unbalanced redox homeostasis and accumulation of ROS seem to be crucial to pro moting most of the inflammatory conditions linked to COVID-19 [26,27]. These effects have been associated with the activity of specific viral proteins, such as the binding o SARS-CoV-2-Spike protein to ACE-2, but to date, the possible contribution of PLpro remains to be explored. Therefore, here we analyzed the effect of PLpro in polarized human epithelial cells, and then we evaluated whether HXT was able to counteract these effects.
As shown in Figure 6A-E, the exogenous expression of PLpro caused a significan decrease in GSH/GSSG ratio, a significant increase in ROS release percentage, NO levels TBARS levels and protein carbonyl levels in Calu-3, Caco-2 and HepG2 cells.
Finally, the treatment of PLpro expressing cells with 10 μM HXT for 24 h caused a significant increase in GSH/GSSG ratio, and a significant decrease in all oxidative stress markers ( Figure 7A-E). Values are plotted as mean ± SD of two independent experiments repeated in duplicate. Data were analyzed by 2-tailed t-tests, * p < 0.05, ** p < 0.01, *** p < 0.001 vs. PLpro.

Expression of SARS-CoV-2-PLpro Induced Oxidative Stress That Was Counteracted by the Treatment with HXT
Unbalanced redox homeostasis and accumulation of ROS seem to be crucial to promoting most of the inflammatory conditions linked to COVID-19 [26,27]. These effects have been associated with the activity of specific viral proteins, such as the binding of SARS-CoV-2-Spike protein to ACE-2, but to date, the possible contribution of PLpro remains to be explored. Therefore, here we analyzed the effect of PLpro in polarized human epithelial cells, and then we evaluated whether HXT was able to counteract these effects.
As shown in Figure 6A-E, the exogenous expression of PLpro caused a significant decrease in GSH/GSSG ratio, a significant increase in ROS release percentage, NO levels, TBARS levels and protein carbonyl levels in Calu-3, Caco-2 and HepG2 cells.
Finally, the treatment of PLpro expressing cells with 10 µM HXT for 24 h caused a significant increase in GSH/GSSG ratio, and a significant decrease in all oxidative stress markers ( Figure 7A-E). Antioxidants 2022, 11, x FOR PEER REVIEW 12 of 18

Discussion
Here, we reported for the first time the effects of the exogenous expression of the catalytic subunit PLpro SARS-CoV-2 NSP3 protein in the polarized human airway, intestinal, and liver epithelial cells. Our results demonstrated that PLpro was able to induce a cascade of inflammatory genes and proteins, apoptosis and a strong increase in several oxidative stress markers. Of note, we demonstrated that HXT reversed the effects on most of these altered pathways.
SARS-CoV-2 infection was initially described as causing severe respiratory disease, but several clinical and experimental studies have demonstrated that infected individuals may exhibit a severe inflammatory response, which in turn results in a severe multi-organ dysfunction and finally death [28]. In addition, even patients with mild or asymptomatic disease may present a variety of symptoms including fatigue, intermittent fever, shortness of breath, cough, joint, chest and muscle pain. These clinical manifestations persisting for more than 12 weeks after acute infection have been called long-COVID syndrome, named also PASC (post-acute sequelae of SARS-CoV-2). Patients with long-COVID are often defined as long haulers [12]. The clinical manifestations may derive from the involvement of tissues and cells for which SARS-CoV-2 has an extended tropism, including lung, intestinal and liver epithelia and could be linked to the interplay between chronic inflammation and persistence of viral proteins [8,17,29]. Regarding mechanisms explaining long-COVID syndrome, there are various hypotheses, such as the presence of persistent viral reservoirs in the body and harmful immune response. Recently, SARS-CoV-2 RNA persistence has been reported in monocytes and the gut [30,31]. Even though the persistence NSPs has not yet been reported, NSP3, NSP4 and NSP6, may contribute to forming double-membrane vesicles where these proteins could be retained and hidden to host cell defense [32]. Therefore, we hypothesize that SARS-CoV-2 PLpro/NSP3 of SARS-CoV-2 could persist for several months after acute infection, thus playing a central role in the pathogenesis of long-COVID syndrome. In this scenario, the catalytically active PLpro may play a dual role in COVID-19. On the one hand, PLpro may enzymatically reduce the ISGylation of MDA5 and IRF3 genes, thus inhibiting the host response to viral infection [33]; on the other PLpro may induce the extracellular release of ISG15, thus amplifying the production and release of pro-inflammatory cytokines and chemokines in host infected cells [34]. A similar phenomenon could be caused by the presence of virally inert SARS-CoV-2 RNA and peptides that could remain in host cells also after virus eradication and trigger the onset of long-COVID syndrome [35]. However, further studies are required to support our hypothesis.
Here, we show that polarized human airway, intestinal and liver epithelial cells expressing exogenous PLpro up-regulate the expression of IFN-related genes (i.e., IFNA1, IFNA7, ATF4, CREB3, CREB3L4, HIST3H3, HIST1H3F, HIST1H3H, MCF2L) and proinflammatory cytokine genes/proteins (mainly IL-1β, IL-6 and TNF-α). These results are in line with previous studies that highlighted the role of PLpro expression in human cells and its ability to establish a functional interactome with host factors that elicit anti-viral signaling and inflammatory response [33,36,37]. The activation of inflammatory signals in response to SARS-CoV-2 infection response may induce five kinds of regulated cell death, including apoptosis, necroptosis, pyroptosis, autophagy and PANoptosis. In the proposed model, SARS-CoV-2-induced apoptosis was mediated by caspase-8 activation and mitochondrial pathways [38]. SARS-CoV-2 ORF3 has been reported as a pivotal inductor of apoptosis in several cell lines, even though more moderately than its SARS-CoV homolog [39]. Accordingly, we demonstrated for the first time that SARS-CoV-2 PLpro/NSP3 induced increased expression of active caspase-8 and consequent apoptosis. Evidence in cells expressing SARS-CoV 3CLpro reported a significant increase in apoptotic rate associated with ROS production [40]. This phenomenon was also confirmed in our polarized human epithelial cells, where exogenous expression of SARS-CoV-2 PLpro/NSP3 caused a decrease in GSH/GSSG and induction of several markers of oxidative stress (i.e., ROS, NO, TBARS and protein carbonyl).
Our data and other reported evidence suggest that SARS-CoV-2 PLpro/NSP3 may be a highly attractive and druggable target not only in acute COVID-19 but also in long-COVID [41,42]. Moreover, several lines of evidence demonstrated that inflammation and oxidative stress mutually reinforce each other in COVID-19 and presumably also in long-COVID, highlighting the major role of oxidative unbalance as a trigger of both acute and chronic inflammation [27,43]. Therefore, it is plausible that natural antioxidant molecules may counteract SARS-CoV-2 redox status derangement and consequent inflammation representing a possible therapy for improving signs and symptoms of long-COVID syndrome [26,27,44]. Accordingly, recently, Bartolini et al. demonstrated that SARS-CoV-2 infection may cause a marked decrease in cellular thiols, mainly GSH, and that the antioxidant N-acetyl-cysteine may cooperate with Nelfinavir restoring protective levels of GSH in VERO E6 cells [45].
HXT is a phenolic compound found in the leaves and fruits of olive with antioxidant, anti-inflammatory and antimicrobial activities [20,21]. The antiviral activity of olive leaf metabolites against SARS-CoV-2 was reported in several in silico computational studies [43,44]. In particular, Yu et al. tested several viral targets, such as viral proteases (Mpro/3CLpro, PLpro), TLRs, ACE2, RBD, NSP15, HSPA5 SBDβ, TMPRSS2, S protein and furin [46]. Furthermore, Takeda et al. showed the antiviral activities of HXT against SARS-CoV-2. In particular, an HXT-rich cream showed virucidal activity through the induction of structural changes in SARS-CoV-2, by modifying the molecular weight of the spike proteins and disrupting the viral genome [47]. In addition, HXT attenuated the pro-inflammatory agents in both in vitro and in vivo disease models [48][49][50][51].
Hence, here we investigated whether the treatment with HXT may improve inflammation, apoptosis and oxidative stress induced by SARS-CoV-2 PLpro/NSP3 in the polarized human airway, intestinal and liver epithelial cells. Confirming our hypothesis, our results demonstrated that the treatment of PLpro expressing cells with HXT restored the expression of pro-inflammatory genes/proteins at levels similar to those expressed in controls, reduced apoptotic rate and pro-oxidant state. It is plausible that the HXT-dependent reduction of inflammation could be mainly due to its capacity in reducing the expression of PLpro in infected cells. These findings increase the chances that HXT could be used as a preventive treatment to avoid long-COVID development.

Conclusions
Many researchers have focused on designing drugs, which can affect the replication or protease activity of SARS-CoV-2 to reduce severe/mild COVID-19 disease, but currently, there is an urgent need for the requirement of safe and effective treatments to alleviate long-COVID syndrome. Natural antioxidants, such as HXT, may have health benefits in this scenario.
Here, we provide the first evidence that SARS-CoV-2 PLpro promotes pro-inflammatory, pro-apoptotic and pro-oxidants effects in epithelial cells, thus suggesting that if this portion of NSP3 remains in host cells after viral clearance it could participate in long-COVID pathogenesis. Further clinical and experimental studies are needed to confirm this hypothesis. Moreover, we demonstrate that PLpro-dependent adverse effects are reversed by the treatment with HXT, thus indicating the possible effectiveness of this molecule as a longer-term and safe approach to reduce the symptoms of long-COVID, in adults, but also in children and adolescents where this condition is a relevant, unrecognized health problem [52].
Supplementary Materials: The following supporting information can be downloaded at: https://www.mdpi.com/article/10.3390/antiox11081466/s1, Figure S1: Expression of PLpro protein in epithelial cells; Figure S2: PLpro expression up-regulates genes of IFN pathways; Figure S3: Analysis of apoptosis; Figure S4: Inflammatory pattern by antibody array; Table S1: Primers sequences for SYBR Green qRT-PCR; Table S2: Means of raw data for pro-inflammatory protein array.