Neutrophil Immunomodulatory Activity of Nerolidol, a Major Component of Essential Oils from Populus balsamifera Buds and Propolis

Propolis is a resinous mixture of substances collected and processed from various botanical sources by honeybees. Black poplar (Populus balsamifera L.) buds are one of the primary sources of propolis. Despite their reported therapeutic properties, little is known about the innate immunomodulatory activity of essential oils from P. balsamifera and propolis. In the present studies, essential oils were isolated from the buds of P. balsamifera and propolis collected in Montana. The main components of the essential oil from P. balsamifera were E-nerolidol (64.0%), 1,8-cineole (10.8%), benzyl benzoate (3.7%), α-terpinyl acetate (2.7%), α-pinene (1.8%), o-methyl anisol (1.8%), salicylaldehyde (1.8%), and benzyl salicylate (1.6%). Likewise, the essential oil from propolis was enriched with E-nerolidol (14.4%), cabreuva oxide-VI (7.9%), α-bisabolol (7.1%), benzyl benzoate (6.1%), β-eudesmol (3.6%), T-cadinol (3.1%), 2-methyl-3-buten-2-ol (3.1%), α-eudesmol (3.0%), fokienol (2.2%), nerolidol oxide derivative (1.9%), decanal (1.8%), 3-butenyl benzene (1.5%), 1,4-dihydronaphthalene (1.5%), selina-4,11-diene (1.5%), α-cadinol (1.5%), linalool (1.4%), γ-cadinene (1.4%), 2-phenylethyl-2-methyl butyrate (1.4%), 2-methyl-2-butenol (1.3%), octanal (1.1%), benzylacetone (1.1%), and eremoligenol (1.1%). A comparison between P. balsamifera and propolis essential oils demonstrated that 22 compounds were found in both essential oil samples. Both were enriched in E-nerolidol and its derivatives, including cabreuva oxide VI and nerolidol oxides. P. balsamifera and propolis essential oils and pure nerolidol activated Ca2+ influx in human neutrophils. Since these treatments activated neutrophils, the essential oil samples were also evaluated for their ability to down-regulate the neutrophil responses to subsequent agonist activation. Indeed, treatment with P. balsamifera and propolis essential oils inhibited subsequent activation of these cells by the N-formyl peptide receptor 1 (FPR1) agonist fMLF and the FPR2 agonist WKYMVM. Likewise, nerolidol inhibited human neutrophil activation induced by fMLF (IC50 = 4.0 μM) and WKYMVM (IC50 = 3.7 μM). Pretreatment with the essential oils and nerolidol also inhibited human neutrophil chemotaxis induced by fMLF, again suggesting that these treatments down-regulated human neutrophil responses to inflammatory chemoattractants. Finally, reverse pharmacophore mapping predicted several potential kinase targets for nerolidol. Thus, our studies have identified nerolidol as a potential anti-inflammatory modulator of human neutrophils.


Effect of PBO, PRO, and Nerolidol on Neutrophil Ca 2+ Influx
PBO and PRO were evaluated for their immunomodulatory effects on human neutrophils. Specifically, their effects on intracellular Ca 2+ flux [Ca 2+ ] i were evaluated, which is a key component of neutrophil activation and function [78]. Treatment of neutrophils with either PBO or PRO activated human neutrophils, resulting in increased [Ca 2+ ] i (EC 50 = 10.5 µg/mL and 18.3 µg/mL, respectively) ( Table 2). Analysis of the direct effect of nerolidol on human neutrophils showed that nerolidol (mixture of E/Z isomers) activated [Ca 2+ ] i with an EC 50 = 0.8 µM, and a representative kinetic curve for neutrophil [Ca 2+ ] i induced by nerolidol is shown in Figure 1.
Analysis of the direct effect of nerolidol on human neutrophils showed that nerolidol (mixture of E/Z isomers) activated [Ca 2+ ]i with an EC50 = 0.8 µM, and a representative kinetic curve for neutrophil [Ca 2+ ]i induced by nerolidol is shown in Figure 1.

Ca 2+
Flux (Relative Fluorescence) It is well recognized that agonists can down-regulate neutrophil responses to subsequent treatment with heterologous or homologous agonists [79]. Thus, whether PBO, PRO, or nerolidol could alter agonist-induced [Ca 2+ ]i in human neutrophils stimulated with N-formyl chemotactic peptide was evaluated. As shown in Table 2 and Figure 2A, It is well recognized that agonists can down-regulate neutrophil responses to subsequent treatment with heterologous or homologous agonists [79]. Thus, whether PBO, PRO, or nerolidol could alter agonist-induced [Ca 2+ ] i in human neutrophils stimulated with N-formyl chemotactic peptide was evaluated. As shown in Table 2 and Figure (Table 2). A representative, dose-dependent response for the inhibition of f MLF-induced neutrophil [Ca 2+ ] i by nerolidol is shown in Figure 2B.

Effect of PBO, PRO, and Nerolidol on Neutrophil Chemotaxis
Various essential oils and their components were reported previously to inhibit neutrophil chemotaxis, including farnesene and germacrene D, which are minor components of PBO and PRO (Table 1). In the present study, the effects of PBO and PRO on human neutrophil chemotaxis were evaluated. Pretreatment with these essential oils dose-dependently inhibited f MLF-induced neutrophil chemotaxis (IC 50 = 1.5 and 2.9 µg/mL, respectively) ( Figure 3A). Likewise, pretreatment with nerolidol also inhibited f MLF-induced human neutrophil chemotaxis (IC 50 = 3.9 µM) ( Figure 3B). pretreatment with either PBO or PRO inhibited [Ca 2+ ]i in fMLF-and WKYMVM-stimulated neutrophils, with IC50 values in the micromolar range. Likewise, nerolidol pretreatment also potently inhibited fMLF-and WKYMVM-stimulated neutrophil [Ca 2+ ]i ( Table  2). A representative, dose-dependent response for the inhibition of fMLF-induced neutrophil [Ca 2+ ]i by nerolidol is shown in Figure 2B. Previously, several of the compounds that are also present in PBO and PRO (i.e., αpinene, β-pinene, limonene, 1,8-cineole, myrcene, sabinene, and terpinolene; see Table 1) were shown to have no inhibitory effect on human neutrophil Ca 2+ influx [34,35], whereas To ensure that the effects of the essential oils and nerolidol were not influenced by possible toxicity, we evaluated the cytotoxicity of the PBO and PRO (up to 25 µg/mL) and pure nerolidol at various concentrations (up to 25 µM) in human neutrophils. We found that the PRO had little to no cytotoxicity at concentrations up to 25 µg/mL during 30and 90-min incubation periods, which covers the times used to measure Ca 2+ influx (up to 30 min) and cell migration (up to 90 min), although PBO had some cytotoxicity at the highest concentration (25 µg/mL) ( Figure 4A). Note that the inhibitory effects of PBO on neutrophil functional activity were found at much lower concentrations (1-10 µg/mL) (see Figures 2 and 3). Consistent with these results, nerolidol had no cytotoxicity for neutrophils at all concentrations and times tested ( Figure 4B).

Effect of PBO, PRO, and Nerolidol on Neutrophil Chemotaxis
Various essential oils and their components were reported previously to inhibit neutrophil chemotaxis, including farnesene and germacrene D, which are minor components of PBO and PRO (Table 1). In the present study, the effects of PBO and PRO on human neutrophil chemotaxis were evaluated. Pretreatment with these essential oils dose-dependently inhibited fMLF-induced neutrophil chemotaxis (IC50 = 1.5 and 2.9 µg/mL, respectively) ( Figure 3A). Likewise, pretreatment with nerolidol also inhibited fMLF-induced human neutrophil chemotaxis (IC50 = 3.9 µM) ( Figure 3B). min) and cell migration (up to 90 min), although PBO had some cytotoxicity at the highest concentration (25 µg/mL) ( Figure 4A). Note that the inhibitory effects of PBO on neutrophil functional activity were found at much lower concentrations (1-10 µg/mL) (see Figures 2 and 3). Consistent with these results, nerolidol had no cytotoxicity for neutrophils at all concentrations and times tested ( Figure 4B).

Identification of Potential Protein Targets for Nerolidol
The application of nerolidol is widespread across different industries, and it has been used in cosmetics and non-cosmetic products [80,81]. In fact, the U.S. Food and Drug Administration (FDA) has also permitted the use of nerolidol as a food flavoring agent [82]. The fact that nerolidol is a common ingredient in many products has attracted researchers to explore more medicinal properties of nerolidol that may exert beneficial effects on human health (reviewed in [80]).
The most important physicochemical parameters of nerolidol were calculated using SwissADME [92]. These parameters were identical for E/Z isomers, with the exception of a slight difference in iLogP values (Table 3). SwissADME also predicted that nerolidol can permeate the blood-brain barrier (BBB). In addition, nanotechnology using nerolidolloaded nanospheres may also improve passage through the BBB [93]. Table 3. Chemical structures and physicochemical properties of nerolidol isomers according to SwissADME results.
The most important physicochemical parameters of nerolidol were SwissADME [92]. These parameters were identical for E/Z isomers, with a slight difference in iLogP values (Table 3). SwissADME also predicted t permeate the blood-brain barrier (BBB). In addition, nanotechnology loaded nanospheres may also improve passage through the BBB [93].  [80]). In addition to the immunomodulatory activity reported here, nerolidol was repor to exhibit a number of biological activities. For example, nerolidol has demonstrated ticholinesterase, antioxidant, antinociceptive, antibacterial, anti-parasite, anti-inflam tory, and anxiolytic activities, suggesting it may be a promising phytochemical for development of therapeutic drugs (reviewed in [63,80,83]). Likewise, nerolidol was ef tive against schistosomiasis [84] and babesiosis [85]. Nerolidol was also reported to h neuroprotective properties, presumably through its anti-inflammatory and antioxid activities [86,87]. Likewise, nerolidol reduced adjuvant arthritis by down-regulating p inflammatory cytokines and up-regulating anti-inflammatory cytokines [88]. It also at uated hypertension-induced hypertrophy in spontaneously hypertensive rats thro modulation of insulin-like growth factor receptor II (IGF-IIR) signaling [89]. This acy sesquiterpene induced apoptosis via phosphatidylinositol-3-kinase (PI3K) and c-Jun terminal kinase (JNK) regulation through cell cycle arrest in MG-63 osteosarcoma c [90]. Finally, changes in tissue myeloperoxidase concentrations, neutrophil and ma phage mRNA expression of monocyte chemoattractant protein-1 (MCP-1), and proinfl matory cytokine content (interleukin (IL)-1β, IL-6, and tumor necrosis factor (TNF)) p tein and mRNA levels were significantly reduced by nerolidol [91].
The most important physicochemical parameters of nerolidol were calculated us SwissADME [92]. These parameters were identical for E/Z isomers, with the exceptio a slight difference in iLogP values (Table 3). SwissADME also predicted that nerolidol permeate the blood-brain barrier (BBB). In addition, nanotechnology using neroli loaded nanospheres may also improve passage through the BBB [93].
Nerolidol is a lipophilic molecule (Table 3). Thus, the neutrophil signaling inhibitory mechanisms may be based on allosteric interactions of the nerolidol chain with the membrane portion of a receptor, and this issue mechanism is currently being investigated. Indeed, other lipophilic compounds, such as the bile acids deoxycholic acid and chenodeoxycholic acid, were reported to antagonize FPR1 at high concentrations (>100 µM) [94][95][96]. Moreover, lipoxin A 4 was also reported as an allosteric modulator of the CB1 cannabinoid receptor and FPR2 [97,98]. To further investigate this issue, lipoxin A 4 was aligned with the Eand Z-nerolidol enantiomers using FieldTemplater software ( Figure 5). The results demonstrated that the alignments were governed mainly by the hydrophobic hydrocarbon skeletons of the compounds. The combined similarity measure of the superimpositions were relatively high (S = 0.690 and 0.678 for the or Z and E isomers, respectively; each of the superimpositions was obtained with two enantiomers of nerolidol aligned onto lipoxin A 4 ), suggesting that nerolidol might mimic lipoxin A 4 and maybe other related specialized pro-resolving mediators, including resolvins, maresins, and protectins [99]. Indeed, many of these molecules were demonstrated to act allosterically on a number of GPCRs (reviewed in [100]). Interestingly, we found previously that other compounds structurally similar to nerolidol (i.e., 6-methyl-3,5-heptadien-2-one (MHDO), geranylacetone, and farnesene) also inhibited agonist-induced neutrophil activity [34,35,48]. Although molecular targets for MHDO and farnesene were not identified, we showed that geranylacetone is a TRPV1 agonist [34].
receptor and FPR2 [97,98]. To further investigate this issue, lipoxin A4 was aligned with the E-and Z-nerolidol enantiomers using FieldTemplater software ( Figure 5). The results demonstrated that the alignments were governed mainly by the hydrophobic hydrocarbon skeletons of the compounds. The combined similarity measure of the superimpositions were relatively high (S = 0.690 and 0.678 for the or Z and E isomers, respectively; each of the superimpositions was obtained with two enantiomers of nerolidol aligned onto lipoxin A4), suggesting that nerolidol might mimic lipoxin A4 and maybe other related specialized pro-resolving mediators, including resolvins, maresins, and protectins [99]. Indeed, many of these molecules were demonstrated to act allosterically on a number of GPCRs (reviewed in [100]). Interestingly, we found previously that other compounds structurally similar to nerolidol (i.e., 6-methyl-3,5-heptadien-2-one (MHDO), geranylacetone, and farnesene) also inhibited agonist-induced neutrophil activity [34,35,48]. Although molecular targets for MHDO and farnesene were not identified, we showed that geranylacetone is a TRPV1 agonist [34].  Reverse-pharmacophore mapping [101] of the molecular structures of E/Z-nerolidol in the R and S enantiomer forms of each geometric isomer was performed to identify potential biological targets. PharmMapper compared a database of pharmacophore patterns with these compounds and generated target information, such as pharmacophoric characteristics and normalized fitness scores. Note, however, that PharmMapper depends on the availability of structures for pharmacophore mapping, and most GPCRs are not represented in the database.
MAPK signaling plays an important role in neutrophil signal transduction cascades [102], and studies have shown that members of the MAPK, JNK, and the ERK families of proteins are activated in response to neutrophil priming/activation (reviewed in [103]). It is also clear from previous studies that one or more of these MAPK pathways is induced by FPR [104][105][106]. Thus, nerolidol may be a general inhibitor of neutrophil activation through GPCRs, and further studies are in progress to evaluate this idea and identify the specific molecular targets.

Essential Oil Extraction
Essential oil was obtained by hydrodistillation of P. balsamifera buds using a Clevengertype apparatus, as previously described [35]. Crude propolis (100 g) was divided into small pieces and extracted by the same procedure. Conditions accepted by the European Pharmacopoeia (European Directorate for the Quality of Medicines, Council of Europe, Strasbourg, France, 2014) were used to avoid artifacts. Yields of the essential oils were calculated based on the amount of air-dried plant material used. Stock solutions of the essential oils were prepared in DMSO (10 mg/mL) for biological evaluation and in n-hexane (10% w/v) for gas chromatographic analysis.

Gas Chromatography-Flame Ionization Detector (GC-FID) and Gas Chromatography-Mass Spectrometry (GC-MS) Analysis
GC-MS analysis was performed with an Agilent 5975 GC-MSD system (Agilent Technologies, Santa Clara, CA, USA), as reported previously [107]. An Agilent Innowax FSC column (60 m × 0.25 mm, 0.25 µm film thickness) was used with He as the carrier gas (0.8 mL/min). The GC oven temperature was kept at 60 • C for 10 min, increased to 220 • C at a rate of 4 • C/min, kept constant at 220 • C for 10 min, and then increased to 240 • C at a rate of 1 • C/min. The split ratio was adjusted to 40:1, and the injector temperature was 250 • C. MS spectra were monitored at 70 eV with a mass range of 35 to 450 m/z. GC analysis was performed on an Agilent 6890N GC system. To obtain the same elution order as with GC-MS, the line was split for FID and MS detectors, and a single injection was performed using the same column and appropriate operational conditions. FID temperature was 300 • C. The essential oil components were identified by co-injection with standards (whenever possible), which were purchased commercially or isolated from natural sources. In addition, compound identities were confirmed by comparison of their mass spectra with those in the Wiley GC/MS Library (Wiley, NY, USA), MassFinder software 4.0 (Dr. Hochmuth Scientific Consulting, Hamburg, Germany), Adams Library, and NIST Library. Confirmation was also achieved using the in-house "Başer Library of Essential Oil Constituents" database obtained from chromatographic runs of pure compounds performed with the same equipment and conditions. A C 8 -C 40 n-alkane standard solution (Fluka, Buchs, Switzerland) was used to spike the samples for the determination of relative retention indices (RRI). Relative percentage amounts of the separated compounds were calculated from the FID chromatograms.

Isolation of Human Neutrophils
For isolation of human neutrophils, blood was collected from healthy donors in accordance with a protocol approved by the Institutional Review Board at Montana State University (protocol #MQ041017). Neutrophils were purified from the blood using dextran sedimentation, followed by Histopaque 1077 gradient separation and hypotonic lysis of red blood cells, as described previously [108]. Isolated neutrophils were washed twice and resuspended in HBSS -. Neutrophil preparations were routinely >95% pure, as determined by light microscopy, and >98% viable, as determined by trypan blue exclusion. Neutrophils were obtained from multiple different donors; however, the cells from different donors were never pooled during experiments.

Ca 2+ Mobilization Assay
Changes in intracellular Ca 2+ concentrations ([Ca 2+ ] i ) were measured with a FlexStation 3 scanning fluorometer (Molecular Devices, Sunnyvale, CA, USA). Briefly, human neutrophils were suspended in HBSS − , loaded with Fluo-4AM at a final concentration of 1.25 µg/mL, and incubated for 30 min in the dark at 37 • C. After dye loading, the cells were washed with HBSS − , resuspended in HBSS + , separated into aliquots, and loaded into the wells of flat-bottom, half-area-well black microtiter plates (2 × 10 5 cells/well). To assess the direct effects of test compounds or pure essential oils on Ca 2+ influx, the compound/oil was added to the wells (final concentration of DMSO was 1%), and changes in fluorescence were monitored (λ ex = 485 nm, λ em = 538 nm) every 5 s for 240 s at room temperature after addition of the test compound. To evaluate the inhibitory effects of the compounds on N-formyl peptide receptor 1/2 (FPR1/FPR2) dependent Ca 2+ influx, the compound or essential oil was added to the wells (final concentration of DMSO was 1%) with human neutrophils. The samples were preincubated for 10 min, followed by the addition of 5 nM f MLF or 5 nM WKYMVM. The maximum change in fluorescence, expressed in arbitrary units over baseline, was used to determine the agonist response. Responses were normalized to the response induced by 5 nM f MLF or 5 nM WKYMVM, which were assigned as 100%. Curve fitting (at least five or six points) and calculation of median effective concentration values (EC 50 or IC 50 ) were performed by nonlinear regression analysis of the dose-response curves generated using Prism 9 (GraphPad Software, Inc., San Diego, CA, USA).

Chemotaxis Assay
Human neutrophils were resuspended in HBSS + containing 2% (v/v) heat-inactivated fetal bovine serum (2 × 10 6 cells/mL), and chemotaxis was analyzed in 96-well ChemoTx chemotaxis chambers (Neuroprobe, Gaithersburg, MD, USA). In brief, neutrophils were preincubated with the indicated concentrations of the test sample (essential oil or pure compound) or DMSO (1% final concentration) for 30 min at room temperature and added to the upper wells of the ChemoTx chemotaxis chambers. The lower wells were loaded with 30 µL of HBSS + containing 2% (v/v) fetal bovine serum and the indicated concentrations of test sample, DMSO (negative control), or 1 nM f MLF as a positive control. Neutrophils were added to the upper wells and allowed to migrate through the 5.0 µm pore polycarbonate membrane filter for 60 min at 37 • C and 5% CO 2 . The number of migrated cells was determined by measuring ATP in lysates of transmigrated cells using a luminescence-based assay (CellTiter-Glo; Promega, Madison, WI, USA), and chemiluminescence measurements were converted to absolute cell numbers by comparison of the values with standard curves obtained with known numbers of neutrophils. Curve fitting (at least eight to nine points) and calculation of effective concentration values (IC 50 ) were performed by nonlinear regression analysis of the dose-response curves generated using GraphPad Prism 9 (San Diego, CA, USA).

Cytotoxicity Assay
Cytotoxicity of essential oils and pure nerolidol in human neutrophils was analyzed with a CellTiter-Glo Luminescent Cell Viability Assay Kit (Promega) according to the manufacturer's protocol. Briefly, human neutrophils were cultured at a density of 10 4 cells/well with different concentrations of essential oil or nerolidol (final concentration of DMSO was 1%) for 30 and 90 min at 37 • C and 5% CO 2 . Following treatment, substrate was added to the cells, and the samples were analyzed with a Fluoroscan Ascent FL microplate reader.

Molecular Modeling
PharmMapper [109] was used to identify potential protein targets for four possible geometric and optical isomers of nerolidol. PharmMapper recognizes potential targets based on "invert" pharmacophore mapping. The protein biotargets are represented by sets of pharmacophore points in reference databases incorporated in the software. PubChem (https://pubchem.ncbi.nlm.nih.gov) and ChemSpider (https://www.chemspider.com/) databases (accessed on 25 August 2022) were used as sources of initial 3D structures of our compounds: (3R,6E)-nerolidol (PubChem CID: 11241545), (3S,6E)-nerolidol (PubChem CID: 5281525), (3S,6Z)-nerolidol (ChemSpider ID: 21427544). The 3D structure of (3R,6Z)nerolidol was obtained by a mirror reflection of the corresponding S-enantiomer using ChemOffice 2016 software. The 3D structures were saved in SDF format and uploaded into PharmMapper. The system automatically generated up to 300 conformers of each compound based on the software option. Pharmacophore mapping was performed using the "Human Protein Targets Only" database, which contained 2241 targets. The top 250 potential targets were retrieved for each compound evaluated, and the potential targets were sorted by normalized fit score.
Alignments of lipoxin-A 4 and enantiomers of Zand E-nerolidol were performed using FieldTemplater software (Cresset Group, Cambridgeshire, UK).

Statistical Analysis
One-way analysis of variance (ANOVA) was performed on the data sets, followed by Tukey's pair-wise comparisons. Pair-wise comparisons with differences at p < 0.05 were considered statistically significant.

Conclusions
Analysis of the composition of essential oils extracted from P. balsamifera buds and propolis collected in Montana showed that they were enriched with nerolidol and its derivatives. Further analysis of the immunomodulatory activity of these essential oils and nerolidol showed that they activated human neutrophils and were able to inhibit FPR1/FPR2 agonistinduced neutrophil activation and chemotaxis, which might contribute to the reported antiinflammatory activity and other pharmacological properties of these extracts. The biological effects of PBO and PRO might be attributable primarily to nerolidol or to the synergistic effects of nerolidol with other active constituents. However, to verify the key targets responsible for the immunomodulatory effects of nerolidol, further experimental investigation is needed.

Conflicts of Interest:
The authors declare no conflict of interest.
Sample Availability: Samples of essential oils are available from the authors.