key: cord-0333331-07171pev
authors: Shao, Rui; Zhou, Xiangda; Zhao, Renping; Yanamandra, Archana K.; Xing, Zhimei; Dai, Xiangdong; Zhang, Han; Wang, Yi; Wang, Yu; Qu, Bin
title: Bioactive constituents of Verbena officinalis regulate killing efficiency of primary human natural killer cells by accelerating killing processes
date: 2021-11-08
journal: bioRxiv
DOI: 10.1101/2021.10.22.465498
sha: acd457fc649a7f99d8d9dc290d7a00e7b9cf72a9
doc_id: 333331
cord_uid: 07171pev

Background Natural killer (NK) cells play a key role in eliminating tumorigenic and pathogen-infected cells. Verbena officinalis (V. officinalis) has been used as a medical plant in traditional and modern medicine, exhibiting anti-tumor and anti-inflammation activity. Purpose The impact of bioactive constituents of V. officinalis on immune responses still remains largely elusive. In this work we investigated the potential targets of V. officinalis and focused on killing efficiency and related functions of NK cells regulated by bioactive constituents of V. officinalis. Study design/methods We used primary human NK cells from peripheral blood mononuclear cells. Potential regulatory roles of selected compounds were analyzed by network pharmacology approaches. Killing efficiency was determined with real-time killing assay and live-cell imaging in 3D. Proliferation was examined by CFSE staining. Expression of cytotoxic proteins was analyzed using flow cytometry. Lytic granule release was quantified by CD107a degranulation assay. Contact time required for killing and determination of serial killers were analyzed using live cell imaging results. Results: Using network pharmacology approaches, we analyzed potential regulatory roles of five compounds (Acteoside, Apigenin, Kaempferol, Verbenalin and Hastatoside) from V. officinalis on immune cell functions and revealed NK cells as a major target. The effect of these compounds on NK killing efficiency was examined with real-time killing assay, and Verbenalin enhanced NK killing efficiency significantly. Further investigation showed that Verbenalin did not affect proliferation, expression of cytotoxic proteins, or lytic granule degranulation, but rather reduced contact time required for killing therefore enhancing total killing events per NK cell, suggestively via inhibition of inhibitory receptors as determined by docking assay. Conclusions Our findings reveal the underlying mechanisms how V. officinalis regulates functions of immune cells, especially NK cells, suggesting Verbenalin from V. officinalis as a promising therapeutic reagent to fight cancer and infection.

Verbena officinalis L. (V. officinalis), also known as common vervain, is a medicinal herb, widespread throughout the globe, mainly in the temperate climate zone (Kubica, Szopa et al., 2020b) . In China, V. officinalis is widely distributed in the southern part of the Yellow River and has been used as traditional Chinese medicine for the treatment of rheumatism, bronchitis, depression, insomnia, anxiety, liver and gallbladder diseases (Khan, Khan et al., 2016 , Kubica, Szopa et al., 2020a . Recent reports suggest that V.

officinalis has a number of scientifically proven activities, such as antioxidant, antibacterial, antifungal, anti-inflammatory, analgesic, anticonvulsant, anxiolytic, antidepressant, sedative, hypnotic, wound healing, gastro-protective, anti-cancer and insecticidal properties (Casanova, Garcia-Mina et al., 2008 , Kubica et al., 2020b , Lopez-Jornet, Camacho-Alonso et al., 2014 , Speroni, Cervellati et al., 2007 . In addition to being used as an antimicrobial, secretolytic, expectorant and diuretic agent, V. officinalis is also widely used in food and cosmetics, especially due to its antioxidant, antibacterial, and anti-inflammatory properties (Kubica et al., 2020a) .

V. officinalis, blooming overground parts of the plant, contains flavonoids, terpenoids, phenolic acids, phenylpropanoids and iridoids (Khan et al., 2016 , Kubica et al., 2020a , Kubica, Szopa et al., 2017 , Shu, Chou et al., 2014 . Verbenalin and hastatoside, belonging to iridoids, are characteristic constituents of V. officinalis, and they exhibit various biological activities including sleep-promoting, antioxidant, and hepatoprotective activity (Shu et al., 2014) . Moreover, the relative content of Verbenalin and hastatoside are higher compared to that of other characteristic iridoids in V.

officinalis, serving as promising target constituents for quality control of V. officinalis.

Kaempferol and apigenin represent the most encountered aglycone flavonoids isolated from V. officinalis Kaempferol has been shown to be cardioprotective, neuroprotective, anti-inflammatory, anti-diabetic, anti-oxidant, anti-microbial, and have anticancer activities (Imran, Salehi et al., 2019) . Acteoside, belonging to phenylpropanoid glycosides, is water-soluble and exhibits a wide range of biological activities, such as anti-tumor, anti-microbial, anti-inflammatory, anti-thrombotic and wound healing properties (Funes, Laporta et al., 2010) .

Natural killer (NK) cells are specialized immune killer cells belonging to innate immune system, which play a key role in eliminating tumorigenic and pathogeninfected cells. To successfully execute their killing function, NK cells need to infiltrate into the target tissue to search for their target cells. Once the target cells are identified, a tight junction termed the immunological synapse (IS) will be formed between the NK cell and the target cells. Consequently, lytic granules (LG) in NK cells, which contain cytotoxic protein such as pore-forming protein perforin and serine proteases granzymes, are enriched and released at the IS to induce destruction of target cells.

NK cells express activating receptors and inhibitory receptors to govern NK activation. Inhibitory receptors engage with major histocompatibility complex (MHC) Class I molecules, which are expressed on all healthy self-cells. In order to bypass immunosurveillance of cytotoxic T lymphocytes, which recognize antigens presented by MHC-I molecules, some tumorigenic cells or pathogen-infected cells down-regulate the expression of their surface MHC-I molecules. Loss or down-regulation of MHC-I molecules results in activation of NK cells to initiate the corresponding killing processes.

In this work, we aimed to investigate regulation of NK killing efficiency by active constituents of V. officinalis and the underlying mechanisms. We were focused on five compounds (Acteoside, Apigenin, Kaempferol, Verbenalin and Hastatoside) from V. officinalis. We employed network pharmacology approaches to predict possible targets and various functional assays to determine the effects of the compounds on NK killing functions.

Prediction of targets of five representative components of V. officinalis was performed by TCMSP, TCM-MESH, Symmap, and TCMID database. The Kyoto Encyclopedia of Genes and Genomics (KEGG) pathway analysis and gene ontology (GO) analysis of biological processes (BP), molecular function (MF) and cellular component (CC) using the DAVID online database were used for exploring gene function. We constructed a PPI (protein-protein interaction) network to elucidate the molecular mechanisms of effects of V. officinalis by using the Cytoscape software (version 3.7.2; http://www.cytoscape.org) and the STRING database (version 11.0, http://www.stringdb.org/) with a required confidence >0.4. Next, the degree of connectivity in the PPI network was analyzed by Cytoscape software (version 3.7.2) and the top 20 hub genes were obtained. We used online tools such as Gene Cards (http://www.genecards.org/) to find potential targets for NK cell-mediated cytotoxicity. Venn Diagram (http://bioinformatics.psb.ugent.be/webtools/Venn/) was used to find overlapping genes between potential targets of V. officinalis and NK cell-mediated cytotoxicity related targets. The further analyses of theses overlapping genes for functional prediction were using CluGO in Cytoscape software (version 3.7.2).

To investigate the interactions between small molecules and receptor proteins, the CDOCKER module in Discovery Studio software was used for molecular docking. The three-dimensional structures of five small molecules were download from TCMSP (https://tcmsp-e.com/), and then perform hydrogenation through the Prepare Ligand module, optimize the energy with CHARMM force field. The three dimensional structure of target protein was downloaded from the PDB database (https://www.rcsb.org/). Then run the Prepare Protein module to optimize the protein structure: delete redundant protein conformations, delete water molecules, complete incomplete residues, hydrogenation and distribution of related charges. The prepared target proteins and small molecules were introduced into Discovery Studio and docked using CDOCKER module. The semi flexible docking method and simulated annealing algorithm are used to find the optimal conformation of ligand and receptor. And according to the level of CDOCKER Interaction Energy to evaluate the degree of docking, the lower the scoring function value, the stronger the affinity of the small molecule with the receptor.

Human primary NK cells were isolated from peripheral blood mononuclear cells (PBMCs) of healthy donors using Human NK Cell Isolation Kit (Miltenyi). The isolated NK cells were cultured in AIM V medium (ThermoFischer Scientific) with 10% FCS and 100 U/ml of recombinant human IL-2 (Miltenyi). K562 and K562-pCasper cells were cultured in RPMI-1640 medium (ThermoFischer Scientific) with 10% FCS.

For K562-pCasper cells, 1.25mg/ml G418 was added. All cells were kept at 37 °C with 5% CO2.

Real-time killing assay was conducted as reported previously (Kummerow, Schwarz et al., 2014) . Briefly, target cells (K562 cells) were loaded with Calcein-AM (500 nM, ThermoFisher Scientific) and settled into a 96-well plate (2.5×10 4 target cells per well).

NK cells were subsequently added with an effector to target (E:T) ratio of 2.5:1 if not otherwise mentioned. Fluorescence intensity was determined by GENios Pro microplate reader (TECAN) using the bottom-reading mode at 37°C every 10 min for 4 hours.

Target lysis (t) % = 100 × (Flive(t)-Fexp(t))/(Flive(t)-Flysed(t)). (F: fluorescence intensity)

Briefly, target cells (K562-pCasper cells) were embedded into 2 mg/ml of pre-chilled neutralized Bovine type I collagen solution (Advanced Biomatrix) in a 96 well plate.

The collagen was solidified at 37℃ with 5% CO2 for 40 min. NK cells were subsequently put on top of collagen as effector cells. The cells were visualized using ImageXpress Micro XLS Widefield High-Content Analysis System (Molecular Devices) at 37 ℃ with 5% CO2. For 3D killing assay, as described previously (Zhao, Zhou et al., 2021) , the killing events were visualized every 20 min for 36 hours, and live target cell numbers were normalized to hour 0 based on area. For live cell imaging to determine time required for killing and the average kills per NK cell, the cells were visualized every 70 sec for 14 hours and tracked manually. Image J software was used to process and analyze the images.

To examine proliferation, freshly isolated primary human NK cells were labelled with CFSE (1 μM, ThermoFischer Scientific) and then stimulated with recombinant human IL-2 in presence of Verbenalin at indicated concentrations for 3 days. Fluorescence was determined with a FACSVerse™ flow cytometer (BD Biosciences) and analyzed with

To test perforin and granzyme B expression, NK cells were fixed in pre-chilled 4% paraformaldehyde. Permeabilization was carried out using 0.1% saponin in PBS containing 0.5% BSA and 5% FCS. FACSVerse™ flow cytometer (BD Biosciences) was used to acquire data. FlowJo v10 (FLOWJO, LLC) was used for analysis.

For degranulation assay, K562 cells were settled with vehicle-treated or Verbenalintreated NK cells in the presence of Brilliant Violet 421™ anti-human CD107a (LAMP1) antibody (Biolegend) and GolgiStop TM (BD Biosciences). The incubation were carried out at 37°C with 5% CO2 for 4 hours. The cells were then stained with PerCP antihuman CD16 antibody (Biolegend) and APC mouse anti-human CD56 antibody (BD Biosciences) to define NK cells. Data was obtained with a FACSVerse™ flow cytometer (BD Biosciences) and was analyzed with FlowJo v10 (FLOWJO, LLC).

To explore the pharmacological mechanism of V. officinalis on immune regulation, five compounds (Acteoside, Apigenin, Kaempferol, Verbenalin and Hastatoside) were selected as bioactive ingredients based on the TCMSP, TCM-MESH, Symmap, and TCMID database analyses ( Figure 1A) . Then, potential targets of these five ingredients were predicted via the SwissTargetPrediction database based on their structure, and a total of 117 targets were obtained. The corresponding ingredient-target network is shown in Figure 1B . Since the protein-protein interaction (PPI) networks are relevant to visualize the role of various key proteins in disease, a visual PPI network of the 117 potential targets was subsequently constructed using the Cytoscape software. And the 20 potential therapeutic targets with the highest degree of nodes were considered as hub genes ( Figure 1C ). Among those genes, several targets with the highest degree of nodes are directly involved in killing functions of NK cells, for example AKT1 for activation (Ali, Nandagopal et al., 2015) , TP53 for NK maturation (Collin, St-Pierre et al., 2017) , JUN for NK cell-mediated target cell killing (Nausch, Florin et al., 2006) , and TNF for NK cell interferon-γ production (Almishri, Santodomingo-Garzon et al., 2016) . These data indicate that bioactive constituents of V. officinalis can regulate NK functions, especially the killing-relevant processes.

The 117 targets of the 5 bioactive ingredients from V. officinalis were further Figure 2B . In the BP category, complex biological processes are described, such as response to drug, negative regulation of apoptotic processes and response to hypoxia. In the MF category, molecular-level activities performed by target proteins include enzyme binding, Heme binding, and identical protein binding. In the CC category, locations on cellular structures in which a target protein performs a function are described, such as the cytosol, Cyclin-dependent protein kinase holoenzyme complex and GABA-A receptor complex. These findings indicate that the five bioactive ingredients in V. officinalis may act on multiple pathways to dampen inflammation and on the biological processes, which are closely related to NK killing functions.

To further identify the genes regulated by V. officinalis for NK cell-mediated cytotoxicity, we retrieved a total of 2489 targets from the Gene Cards databases in this aspect. We used Venn Diagram (http://bioinformatics.psb.ugent.be/webtools/Venn/) to find overlapping genes between potential targets of V. officinalis and NK related targets.

A total of 71 potential targets of V. officinalis on NK mediated cytotoxicity were obtained ( Figure 3A) . The corresponding PPI network of V. officinalis ingredient-NK target is shown in Figure 3B , where the size of the nodes positively correlates with their degree. We noticed that the largest nodes such as AKT1, TP53, JUN, and TNF are the same as identified in Figure 1C , strongly suggesting that these five bioactive ingredients of V. officinalis have a great potential to directly regulate NK killing functions. Furthermore, the common target genes between Verbena officinalis L and NK cells were further analyzed for functional prediction using CluGO ( Figure 3C ). The primary enriched GO biological process (BP) terms were mainly related to apoptosis, cell cycle and reactive oxygen species metabolism, suggesting that V. officinalis may influence viability of NK cells, especially under conditions with excessive reactive oxygen species.

In addition, we analyzed whether active compounds of V. officinalis could bind to key proteins of NK cells. To this end, we tested the interaction between the five active compounds of V. officinalis (Acteoside, Apigenin, Kaempferol, Verbenalin and Hastatoside) with inhibitory receptors of NK cells including NKG2A and KIR2DL1.

Interestingly, we found that NKG2A can engage with three bioactive compounds (Apigenin, Kaempferol, and Verbenalin) whereas KIR2DL1 can interact with all five (Figure 4 ). Of note, among these bioactive compounds, with NKG2A Kaempferol showed the strongest interaction, to comparable extent of which, three compounds (Acteonid, Verbenalin, and Hastaboside) can engage with KIR2DL1 (Table 1) .

In order to verify the impact of bioactive constituents of V. officinalis on NK killing efficiency, we cultured primary human NK cells with the corresponding compound (10 µM and 30 µM) for three days in presence with IL-2. Killing kinetics of NK cells was determined using the plate-reader based real-time killing assay (Kummerow et al., 2014) . We found that Acteoside, Apigenin, and Kaempferol slightly reduced NK killing efficiency, whereas Verbenalin and Hastatoside enhanced NK killing efficiency ( Figure   5A ). Verbenalin exhibited the highest potency for elevation of NK killing efficiency ( Figure 5A ). We further verified the impact of Verbenalin on NK killing efficiency in 3D. Target cells expressing FRET-based apoptosis reporter pCasper were embedded in collagen matrix and NK cells were added from the top. Target cells were yellow when alive and turned green when undergoing apoptosis (Backes, Friedmann et al., 2018) .

Results show that Verbenalin-treated NK cells exhibited significantly faster killing kinetics compared to their counterparts treated with vehicle. Thus, we conclude that among the bioactive constituents of V. officinalis, Verbenalin is able to increase NK killing efficiency under a physiologically relevant condition.

Next, we sought for the underlying mechanisms of increase in NK killing efficiency by Verbenalin. We examined proliferation, expression of cytotoxic proteins (perforin and granzyme B), and degranulation of lytic granules. None of those processes were affected by Verbenalin ( Figure 6A -C). Since our molecular docking data suggest that Verbenalin can bind to inhibitory receptors of NK cells (Figure 4) , we analyzed the times required for killing. Killing events were visualized with high-content imaging setup every 70 sec for 12 hours ( Figure 7A , Movie 1), and the time between the contact and the induced target cell apoptosis for randomly chose NK cells was analyzed. The quantification shows that the time required for killing was reduced by Verbenalintreatment ( Figure 7B ). Concomitantly, on average, the numbers of target cells killed per NK cell was almost doubled for Verbenalin-treated NK cells relative to their counterparts ( Figure 7C) . Notably, it is reported that a portion of NK cells can kill several target cells in a row (Bhat & Watzl, 2007 , Prager, Liesche et al., 2019 . We thus also analyzed fractions of serial killers (one NK killed more than one target cell), single killers (one NK killed only one target cell) and non-killers (NK cells did not kill any target cells). We found that Verbenalin-treatment substantially enhanced the portion of serial killers and concomitantly reduced the portion of non-killers ( Figure 7D ). Taken together, our results suggest that Verbenalin potentiates NK cell activation upon target recognition, shortening the time required to initiate destruction of target cells, therefore enhancing killing efficiency of NK cells.

In our work, molecular docking predicted interaction between the bioactive components of V. officinalis with two prominent inhibitory receptors of NK cells:

NKG2A and KIR2DL1. NKG2A (natural killer group 2A) form heterodimers with CD94 (Lazetic, Chang et al., 1996) , serving as one of the most prominent inhibitory receptors in NK cells to avoid killing healthy self-cells (Khan, Arooj et al., 2020) .

NKG2A contains a cytoplasmic immunoreceptor tyrosine-based inhibition motif, which recruits tyrosine phosphatases (SHP1 and SHP2) to initiate inhibition processes involving PI3K, MAPK, and NF-kappaB signaling (Khan et al., 2020) . One major ligand of NKG2A is human leukocyte antigen (HLA)-E, non-classical MHC class I molecules in humans (Lee, Llano et al., 1998) . Notably, blockage of NKG2A functionality substantially enhances NK killing capacity against HLA-E-expressing tumor cells (Kamiya, Seow et al., 2019) . In the context of COVID-19, elevation of NKG2A expression on NK cells from patients is positively correlated with functional exhaustion (Antonioli, Fornai et al., 2020) . Thus, blocking interaction between NKG2A with its ligand by bioactive components of V. officinalis could dampen the inhibitory signaling, thus leading to enhancement in NK-mediated cytotoxicity.

Killer cell immunoglobulin-like receptors (KIRs) are transmembrane receptors expressing on NK cells and also on a minority of T cells (Pende, Falco et al., 2019) . At least 15 different KIRs have been identified in humans (Wende, Colonna et al., 1999) .

Concerning the nomenclature for KIRs, the first digit following KIR corresponds to the number of the extracellular domains (2 or 3), L or S at the third digit indicates the length of the cytoplasmic domain (L for long and S for short), P at the third digit refers to pseudogenes (Khan et al., 2016 , Pende et al., 2019 . Similar as the other inhibitory KIRs, KIR2DL1 is expressed on the plasma membrane and recognize HLA class I ligands (Pende et al., 2019) . Remarkably, several interactive residues in KIR2DL1 with bioactive compounds identified by docking methods (HIS 138, TRP188, and TYR186) are very close to the interaction sites with HLA (ASP135, SER184, and GLU187) (Fan, Long et al., 2001) . These results implies that these bioactive compounds of V. officinalis, especially Verbenalin, can potentially inhibit interaction between KIR2DL1 and its ligand, leading to dampened inhibitory signals, therefore activate NK cells. We postulate that our observation that Verbenalin-treatment shortened required time to initiate apoptosis and enhances average kills per NK cells is very likely due to blockage of inhibitory receptors (including NKG2A and KIR2DL1) by Verbenalin, which eventually enhances NK killing efficiency.

In this work, we identified several promising target genes that are regulated by the five bioactive components of V. officinalis, including TP53, JUN, HIF1A, and VEGFA.

TP53, as one of the most intensively investigated tumor suppressor genes, plays an essential role in initiation and progression of malignant tumors (Zhu, Pan et al., 2020) .

A recent study has reported that pharmacological activation of wild-type p53 increases granzyme B-dependent NK killing capacity against tumor cells via elevation of autophagy (Chollat-Namy, Ben Safta-Saadoun et al., 2019). JUN is a key component of the transcription factor AP-1 (activator protein-1), which are involved in killingrelated processes in NK cells such as mobilization of lytic granules and production of cytokines (Abel, Yang et al., 2018) . Interestingly, on the target cell side, activation of AP-1 upregulates RAE-1, ligands of the activating receptor NKG2D on NK cells, leading to increased target cell killing mediated by NK cells (Nausch et al., 2006) . HIF1A (hypoxia inducible factor 1 subunit alpha) is a key player to regulate cellular functions in response to hypoxia, which often takes place in tumor microenvironments (Lee, Ko et al., 2019) . Activity of HIF1A regulates killing activity in tumor-infiltrating NK cells (Ni, Wang et al., 2020) . In addition, VEGF (vascular endothelial growth factor) can be induced under hypoxia condition to improve oxygen supply (Lee et al., 2019) .

In combination with our findings, it suggests that bioactive constituents of V. officinalis can regulate NK killing efficiency from the NK side as well as the tumor cell side.

V. officinalis has a long tradition being used as a medical herb throughout the world, e.g. in Europe, Asia, American and Australia (Kubica et al., 2020a , Vogl, Picker et al., 2013 . Anti-tumor activity of V. officinalis has been reported (Kou, Yang et al., 2013) .

Recently, a newly developed formula XuanFeiBaiDu composed of thirteen medical herbs including V. officinalis has shown very positive clinical outcome treating COVID-19 patients (Xiong, Wang et al., 2020) . However, the underlying mechanism is not known. In this work, we have established a direct link between Verbenalin, a bioactive constituent of V. officinalis, and killing efficiency of NK cells. It indicates that as a compound, Verbenalin has a promising potential for therapeutical applications fighting against cancer and/or infection. (D) Fraction of serial killers is elevated by Verbenalin treatment. Fraction of serial killer (one NK killed more than one target cell), single killer (one NK killed only one target cell) and non-killer (NK cells did not kill any target cells) for each donor was analyzed.

Results are from two donors. 21 NK cells were randomly chosen from each condition.

Mann-Whitney test was used for statistical analysis. Table 1 . Scores of docking and the interactive residues. Lower binding energies correspond to stronger interaction between the compounds and the target proteins. 

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A Review on the Investigations of This Medicinally Important Plant Species

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Killer Ig-Like Receptors (KIRs): Their Role in NK Cell Modulation and Developments Leading to Their Clinical Exploitation

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We thank the Institute for Clinical Hemostaseology and Transfusion Medicine for providing donor blood; Carmen Hässig, Cora Hoxha, and Gertrud Schäfer for excellent 

Research carried out for this study with material from healthy donors (leukocyte reduction system chambers from human blood donors) is authorized by the local ethic committee (declaration from 16.4.2015 (84/15, Prof. Dr. Rettig-Stürmer).

The authors have no financial conflicts of interest.