Neutrophil Immunomodulatory Activity of Natural Organosulfur Compounds

Organosulfur compounds are bioactive components of garlic essential oil (EO), mustard oil, Ferula EOs, asafoetida, and other plant and food extracts. Traditionally, garlic (Allium sativum) is used to boost the immune system; however, the mechanisms involved in the putative immunomodulatory effects of garlic are unknown. We investigated the effects of garlic EO and 22 organosulfur compounds on human neutrophil responses. Garlic EO, allyl propyl disulfide, dipropyl disulfide, diallyl disulfide, and allyl isothiocyanate (AITC) directly activated Ca2+ flux in neutrophils, with the most potent being AITC. Although 1,3-dithiane did not activate neutrophil Ca2+ flux, this minor constituent of garlic EO stimulated neutrophil reactive oxygen species (ROS) production. In contrast, a close analog (1,4-dithiane) was unable to activate neutrophil ROS production. Although 1,3-dithiane-1-oxide also stimulated neutrophil ROS production, only traces of this oxidation product were generated after a 5 h treatment of HL60 cells with 1,3-dithiane. Evaluation of several phosphatidylinositol-3 kinase (PI3K) inhibitors with different subtype specificities (A-66, TGX 221, AS605240, and PI 3065) showed that the PI3K p110δ inhibitor PI 3065 was the most potent inhibitor of 1,3-dithiane-induced neutrophil ROS production. Furthermore, 1,3-dithiane enhanced the phosphorylation of extracellular signal-regulated kinase 1/2 (ERK1/2), glycogen synthase kinase 3 α/β (GSK-3α/β), and cAMP response element binding (CREB) protein in differentiated neutrophil-like HL60 cells. Density functional theory (DFT) calculations confirmed the reactivity of 1,3-dithiane vs. 1,4-dithiane, based on the frontier molecular orbital analysis. Our results demonstrate that certain organosulfur compounds can activate neutrophil functional activity and may serve as biological response modifiers by augmenting phagocyte functions.

The compounds were first evaluated for their effects on Ca 2+ flux in human neutrophils, and we found that garlic EO activated neutrophil Ca 2+ flux with an EC50 of 34.9 µg/mL. A representative kinetic curve is shown in Figure 1. We found that four of the 22 tested compounds (allyl propyl disulfide, dipropyl disulfide, diallyl disulfide, and allyl isothiocyanate (AITC)) directly activated neutrophil Ca 2+ flux, with the most potent being AITC, a major component of mustard oil (Table 2). Thus, diallyl disulfide, a major component of garlic EO, may be a principal molecule responsible for activating neutrophil Ca 2+ mobilization.
The compounds were first evaluated for their effects on Ca 2+ flux in human neutrophils, and we found that garlic EO activated neutrophil Ca 2+ flux with an EC50 of 34.9 µg/mL. A representative kinetic curve is shown in Figure 1. We found that four of the 22 tested compounds (allyl propyl disulfide, dipropyl disulfide, diallyl disulfide, and allyl isothiocyanate (AITC)) directly activated neutrophil Ca 2+ flux, with the most potent being AITC, a major component of mustard oil (Table 2). Thus, diallyl disulfide, a major component of garlic EO, may be a principal molecule responsible for activating neutrophil Ca 2+ mobilization.
The compounds were first evaluated for their effects on Ca 2+ flux in human neutrophils, and we found that garlic EO activated neutrophil Ca 2+ flux with an EC50 of 34.9 µg/mL. A representative kinetic curve is shown in Figure 1. We found that four of the 22 tested compounds (allyl propyl disulfide, dipropyl disulfide, diallyl disulfide, and allyl isothiocyanate (AITC)) directly activated neutrophil Ca 2+ flux, with the most potent being AITC, a major component of mustard oil (Table 2). Thus, diallyl disulfide, a major component of garlic EO, may be a principal molecule responsible for activating neutrophil Ca 2+ mobilization.

Allium sativum
1.6-9.5 [38][39][40] Allium ursinum 0.1-0.3 [20] Dimethyl disulfide  The compounds were first evaluated for their effects on Ca 2+ flux in human neutrophils, and we found that garlic EO activated neutrophil Ca 2+ flux with an EC50 of 34.9 µg/mL. A representative kinetic curve is shown in Figure 1. We found that four of the 22 tested compounds (allyl propyl disulfide, dipropyl disulfide, diallyl disulfide, and allyl isothiocyanate (AITC)) directly activated neutrophil Ca 2+ flux, with the most potent being AITC, a major component of mustard oil (Table 2). Thus, diallyl disulfide, a major component of garlic EO, may be a principal molecule responsible for activating neutrophil Ca 2+ mobilization.

Allium sativum
0.4-1.4 [38] Allium ursinum 0.7-2.3 [20] Methyl propyl disulfide  The compounds were first evaluated for their effects on Ca 2+ flux in human neutrophils, and we found that garlic EO activated neutrophil Ca 2+ flux with an EC50 of 34.9 µg/mL. A representative kinetic curve is shown in Figure 1. We found that four of the 22 tested compounds (allyl propyl disulfide, dipropyl disulfide, diallyl disulfide, and allyl isothiocyanate (AITC)) directly activated neutrophil Ca 2+ flux, with the most potent being AITC, a major component of mustard oil (Table 2). Thus, diallyl disulfide, a major component of garlic EO, may be a principal molecule responsible for activating neutrophil Ca 2+ mobilization.

Allium sativum
tr [13] Dipropyl disulfide  The compounds were first evaluated for their effects on Ca 2+ flux in human neutrophils, and we found that garlic EO activated neutrophil Ca 2+ flux with an EC50 of 34.9 µg/mL. A representative kinetic curve is shown in Figure 1. We found that four of the 22 tested compounds (allyl propyl disulfide, dipropyl disulfide, diallyl disulfide, and allyl isothiocyanate (AITC)) directly activated neutrophil Ca 2+ flux, with the most potent being AITC, a major component of mustard oil (Table 2). Thus, diallyl disulfide, a major component of garlic EO, may be a principal molecule responsible for activating neutrophil Ca 2+ mobilization.

Allium porrum
29.8 [40] Allium ursinum 0.0-0.3 [20] Allyl methyl disulfide  The compounds were first evaluated for their effects on Ca 2+ flux in human neutrophils, and we found that garlic EO activated neutrophil Ca 2+ flux with an EC50 of 34.9 µg/mL. A representative kinetic curve is shown in Figure 1. We found that four of the 22 tested compounds (allyl propyl disulfide, dipropyl disulfide, diallyl disulfide, and allyl isothiocyanate (AITC)) directly activated neutrophil Ca 2+ flux, with the most potent being AITC, a major component of mustard oil (Table 2). Thus, diallyl disulfide, a major component of garlic EO, may be a principal molecule responsible for activating neutrophil Ca 2+ mobilization.

Allium sativum 4.4-8.3 [38]
Allium ursinum 1.1-18.9 [20] Allyl propyl disulfide  The compounds were first evaluated for their effects on Ca 2+ flux in human neutrophils, and we found that garlic EO activated neutrophil Ca 2+ flux with an EC50 of 34.9 µg/mL. A representative kinetic curve is shown in Figure 1. We found that four of the 22 tested compounds (allyl propyl disulfide, dipropyl disulfide, diallyl disulfide, and allyl isothiocyanate (AITC)) directly activated neutrophil Ca 2+ flux, with the most potent being AITC, a major component of mustard oil (Table 2). Thus, diallyl disulfide, a major component of garlic EO, may be a principal molecule responsible for activating neutrophil Ca 2+ mobilization.

Allium sativum
3.1 [39] Diallyl disulfide  The compounds were first evaluated for their effects on Ca 2+ flux in human neutrophils, and we found that garlic EO activated neutrophil Ca 2+ flux with an EC50 of 34.9 µg/mL. A representative kinetic curve is shown in Figure 1. We found that four of the 22 tested compounds (allyl propyl disulfide, dipropyl disulfide, diallyl disulfide, and allyl isothiocyanate (AITC)) directly activated neutrophil Ca 2+ flux, with the most potent being AITC, a major component of mustard oil (Table 2). Thus, diallyl disulfide, a major component of garlic EO, may be a principal molecule responsible for activating neutrophil Ca 2+ mobilization.

Allium ursinum
9.9-20.7 [20] Allium sativum 20.8-29.1 [38][39][40] Dimethyl trisulfide  The compounds were first evaluated for their effects on Ca 2+ flux in human neutrophils, and we found that garlic EO activated neutrophil Ca 2+ flux with an EC50 of 34.9 µg/mL. A representative kinetic curve is shown in Figure 1. We found that four of the 22 tested compounds (allyl propyl disulfide, dipropyl disulfide, diallyl disulfide, and allyl isothiocyanate (AITC)) directly activated neutrophil Ca 2+ flux, with the most potent being AITC, a major component of mustard oil (Table 2). Thus, diallyl disulfide, a major component of garlic EO, may be a principal molecule responsible for activating neutrophil Ca 2+ mobilization.  The compounds were first evaluated for their effects on Ca 2+ flux in human neutrophils, and we found that garlic EO activated neutrophil Ca 2+ flux with an EC50 of 34.9 µg/mL. A representative kinetic curve is shown in Figure 1. We found that four of the 22 tested compounds (allyl propyl disulfide, dipropyl disulfide, diallyl disulfide, and allyl isothiocyanate (AITC)) directly activated neutrophil Ca 2+ flux, with the most potent being AITC, a major component of mustard oil (Table 2). Thus, diallyl disulfide, a major component of garlic EO, may be a principal molecule responsible for activating neutrophil Ca 2+ mobilization.

Allium ursinum
tr [20] Diallyl trisulfide  The compounds were first evaluated for their effects on Ca 2+ flux in human neutrophils, and we found that garlic EO activated neutrophil Ca 2+ flux with an EC50 of 34.9 µg/mL. A representative kinetic curve is shown in Figure 1. We found that four of the 22 tested compounds (allyl propyl disulfide, dipropyl disulfide, diallyl disulfide, and allyl isothiocyanate (AITC)) directly activated neutrophil Ca 2+ flux, with the most potent being AITC, a major component of mustard oil (Table 2). Thus, diallyl disulfide, a major component of garlic EO, may be a principal molecule responsible for activating neutrophil Ca 2+ mobilization. The compounds were first evaluated for their effects on Ca 2+ flux in human neutrophils, and we found that garlic EO activated neutrophil Ca 2+ flux with an EC50 of 34.9 µg/mL. A representative kinetic curve is shown in Figure 1. We found that four of the 22 tested compounds (allyl propyl disulfide, dipropyl disulfide, diallyl disulfide, and allyl isothiocyanate (AITC)) directly activated neutrophil Ca 2+ flux, with the most potent being AITC, a major component of mustard oil (Table 2) The compounds were first evaluated for their effects on Ca 2+ flux in human neutrophils, and we found that garlic EO activated neutrophil Ca 2+ flux with an EC50 of 34.9 µg/mL. A representative kinetic curve is shown in Figure 1. We found that four of the 22 tested compounds (allyl propyl disulfide, dipropyl disulfide, diallyl disulfide, and allyl isothiocyanate (AITC)) directly activated neutrophil Ca 2+ flux, with the most potent being AITC, a major component of mustard oil (Table 2). Thus, diallyl disulfide, a major component of garlic EO, may be a principal molecule responsible for activating neutrophil Ca 2+ mobilization.

Allium sativum
(3 mg/g) b [42] 2,5-Dimethylthiophene The compounds were first evaluated for their effects on Ca 2+ flux in human neutrophils, and we found that garlic EO activated neutrophil Ca 2+ flux with an EC50 of 34.9 µg/mL. A representative kinetic curve is shown in Figure 1. We found that four of the 22 tested compounds (allyl propyl disulfide, dipropyl disulfide, diallyl disulfide, and allyl isothiocyanate (AITC)) directly activated neutrophil Ca 2+ flux, with the most potent being AITC, a major component of mustard oil (Table 2) The compounds were first evaluated for their effects on Ca 2+ flux in human neutrophils, and we found that garlic EO activated neutrophil Ca 2+ flux with an EC50 of 34.9 µg/mL. A representative kinetic curve is shown in Figure 1. We found that four of the 22 tested compounds (allyl propyl disulfide, dipropyl disulfide, diallyl disulfide, and allyl isothiocyanate (AITC)) directly activated neutrophil Ca 2+ flux, with the most potent being AITC, a major component of mustard oil (Table 2). Thus, diallyl disulfide, a major component of garlic EO, may be a principal molecule responsible for activating neutrophil Ca 2+ mobilization.

Allium sativum
2.1 [13,39] a Percentage composition is indicated for EOs. b Based on the weight of dry plant material.
The compounds were first evaluated for their effects on Ca 2+ flux in human neutrophils, and we found that garlic EO activated neutrophil Ca 2+ flux with an EC 50 of 34.9 µg/mL. A representative kinetic curve is shown in Figure 1. We found that four of the 22 tested compounds (allyl propyl disulfide, dipropyl disulfide, diallyl disulfide, and allyl isothiocyanate (AITC)) directly activated neutrophil Ca 2+ flux, with the most potent being AITC, a major component of mustard oil (Table 2). Thus, diallyl disulfide, a major component of garlic EO, may be a principal molecule responsible for activating neutrophil Ca 2+ mobilization. The compounds were also evaluated for their ability to inhibit fMLF-induced Ca 2+ flux in neutrophils. The cells were pretreated with the organosulfur compounds for 30 min and then treated with 5 nM fMLF. Six compounds, including the four compounds shown above to have agonist activity (i.e., allyl propyl disulfide, dipropyl disulfide, diallyl disulfide, and AITC), as well as methyl propyl disulfide and allicin were found to be modest inhibitors of fMLF-induced Ca 2+ mobilization, suggesting that pre-exposure of neutrophils to these compounds may desensitize them to activation by other agonists (Table 2). For example, garlic EO inhibited fMLF-induced Ca 2+ flux in neutrophils with an IC50 of 14.7 µg/mL.

Effect of Garlic EO and Organosulfur Compounds on Neutrophil ROS Production
The organosulfur compounds and garlic EO were evaluated for their ability to modulate neutrophil ROS production. We found that garlic EO and three of the individual compounds (diallyl trisulfide, ajoene, and allicin) inhibited spontaneous ROS production by neutrophils. These compounds, as well as alliin, N-acetyl-S-allyl-L-cysteine, and S-allyl-L-cysteine, also inhibited phorbol-12-myristate-13-acetate (PMA)-stimulated ROS production by these cells (Table 2). Ajoene and allicin were the most potent inhibitors of neutrophil ROS production. It should be noted that during the process of steam distillation, allicin, which is a precursor of ajoene, is completely eliminated from garlic EO [44]. Thus, diallyl trisulfide, a major component compound of garlic EO, could be one of the main components responsible for ROS inhibition by garlic EO. The compounds were also evaluated for their ability to inhibit f MLF-induced Ca 2+ flux in neutrophils. The cells were pretreated with the organosulfur compounds for 30 min and then treated with 5 nM f MLF. Six compounds, including the four compounds shown above to have agonist activity (i.e., allyl propyl disulfide, dipropyl disulfide, diallyl disulfide, and AITC), as well as methyl propyl disulfide and allicin were found to be modest inhibitors of f MLF-induced Ca 2+ mobilization, suggesting that pre-exposure of neutrophils to these compounds may desensitize them to activation by other agonists (Table 2). For example, garlic EO inhibited f MLF-induced Ca 2+ flux in neutrophils with an IC 50 of 14.7 µg/mL.

Effect of Garlic EO and Organosulfur Compounds on Neutrophil ROS Production
The organosulfur compounds and garlic EO were evaluated for their ability to modulate neutrophil ROS production. We found that garlic EO and three of the individual compounds (diallyl trisulfide, ajoene, and allicin) inhibited spontaneous ROS production by neutrophils. These compounds, as well as alliin, N-acetyl-S-allyl-L-cysteine, and S-allyl-L-cysteine, also inhibited phorbol-12-myristate-13-acetate (PMA)-stimulated ROS production by these cells (Table 2). Ajoene and allicin were the most potent inhibitors of neutrophil ROS production. It should be noted that during the process of steam distillation, allicin, which is a precursor of ajoene, is completely eliminated from garlic EO [44]. Thus, diallyl trisulfide, a major component compound of garlic EO, could be one of the main components responsible for ROS inhibition by garlic EO.
Reactive oxygen species (ROS) production was monitored as L-012-dependent chemiluminescence (CL). EC 50 and IC 50 values were determined by nonlinear regression analysis of the dose-response curves, as described under Section 3.5, and are presented as the mean ±S.D. of three independent experiments. For the stimulation of ROS production, neutrophils were pretreated with phorbol-12-myristate-13-acetate (PMA) (100 nM). N.A., no inhibitory activity was found at concentrations up to 50 µM. * Instead of inhibition, 1,3-dithiane directly activated neutrophil ROS production (see below). In contrast to the ROS inhibition exhibited by several compounds (Table 2), 1,3-dithiane actually activated neutrophil ROS production. This compound stimulated ROS production with unimodal kinetics, which is similar to that observed for other phagocyte activating agents [45] (Figure 2A). Note that 1,3-dithiane-stimulated ROS production was completely inhibited by superoxide dismutase (SOD, 100 U/mL), and 1,3-dithiane had no effect on the ROS assay system in the absence of cells, indicating neutrophil NADPH oxidase activation rather than an artifact (data not shown). Although 1,4-dithiane (an analog of 1,3-dithiane) has not been reported to be present in plant extracts/EOs, it has been reported to be present in products of bacterial fermentation and boiled beef extract [46,47]. Thus, we also tested 1,4-dithiane under the same conditions and found that it had no effect on ROS production, demonstrating that the effects of 1,3-dithiane were specific for this compound ( Figure 2B). Since 1,3-dithiane is efficiently oxidized by flavin monooxygenases into the corresponding sulfoxide enantiomer (R)-1,3-dithiane-1-oxide [48], we could not exclude that the biotransformation products of 1,3-dithiane might also activate neutrophils. Thus, we tested 1,3-dithiane-1-oxide and found that its activity was similar to 1,3-dithiane itself ( Figure 2B), indicating that either 1,3-dithiane or its oxide could be responsible for the observed activation of neutrophil ROS production. To further investigate this issue, we incubated HL60 cells with 1,3-dithiane for 0, 1, and 5 h and evaluated the culture medium for oxidation products. Analysis of the cell lysates by gas chromatography-mass spectrometry (GC-MS) showed the m/z of the 1,3-dithiane-1-oxide (M + ) ion to be 136.00. The electron impact (EI) mass spectrum also indicated the presence of trace amounts of 1,3-dithiane-1-oxide, but only after the 5 h incubation, and the identity of this compound was confirmed using a reference compound and the NIST 14 MS library embedded in the Agilent data analysis software (data not shown). Thus, neutrophil activation is primarily due to 1,3-dithiane, especially during the earlier treatment times evaluated in this study (0-60 min), whereas trace amounts of the oxidation product 1,3-dithiane-1-oxide could contribute to cell activation at much later times.
investigate this issue, we incubated HL60 cells with 1,3 -dithiane for 0, 1, and 5 h and evaluated the culture medium for oxidation products. Analysis of the cell lysates by gas chromatography -mass spectrometry (GC-MS) showed the m/z of the 1,3-dithiane-1-oxide (M + ) ion to be 136.00. The electron impact (EI) mass spectrum also indicated the presence of trace amounts of 1,3-dithiane-1-oxide, but only after the 5 h incubation, and the identity of this compound was confirmed using a reference compound and the NIST 14 MS library embedded in the Agilent data analysis software (data not shown). Thus, neutrophil activation is primarily due to 1,3-dithiane, especially during the earlier treatment times evaluated in this study (0-60 min), whereas trace amounts of the oxidation product 1,3-dithiane-1-oxide could contribute to cell activation at much later times. shown (% of control). The data in Pane ls B and C are pre se nte d as me an ±S.D. of triplicate sample s from one e xpe riment that is re presentative of thre e inde pendent e xperiments.
Although various EOs and their major compounds have been reported to modulate ROS production and Ca 2+ mobilization in human neutrophils, including oils with a high content of organosulfur compounds [11,30,31], the current study is the first to report the biological effects of 1,3-dithiane, a small molecule organosulfur compound identified in A. sativum and boiled beef extracts [13,39,47]. Here, we found that 1,3-dithiane stimulates ROS production by human neutrophils, and this response was inhibited by specific inhibitors of PI3Kβ, γ, and δ. Activation of PI3K signaling usually occurs following the stimulation of receptor tyrosine kinases or G-protein-coupled receptors (GPCRs) [56]. We can exclude a stimulatory effect of 1,3-dithiane on several GPCRs, including N-formyl peptide receptors (FPRs) 1 and 2, C5a receptor, and chemokine receptors CXCR1/2, as this compound did not stimulate neutrophil Ca 2+ mobilization ( Table 2). PI3Ks phosphorylate phosphatidylinositol-(4,5) bisphosphate to form phosphatidylinositol (3,4,5)-trisphosphate (PIP3), and the accumulation of PIP3 facilitates the localization of Akt to the plasma membrane and its subsequent activation following phosphorylation. PI3K/Akt signaling regulates many of the molecular mechanisms contributing to increased ROS production through the phosphorylation and activation of NADPH oxidase subunits [56]. In this study, we also established that 1,3-dithiane activates ERK signaling, which in turn phosphorylates CREB, which has been reported for other bioactive molecules, such as lysophosphatidic acid [57]. Indeed, we found that 1,3-dithiane stimulated the phosphorylation of CREB and GSK-3α/β in differentiated HL60 cells. It should be noted that PI3K generation of PIP3 could directly or indirectly affect CREB phosphorylation [58].

2D Structure
Molecules 2019, 24, x FOR PEER REVIEW 8 of 17 Although various EOs and their major compounds have been reported to modulate ROS production and Ca 2+ mobilization in human neutrophils, including oils with a high content of organosulfur compounds [11,30,31], the current study is the first to r eport the biological effects of 1,3dithiane, a small molecule organosulfur compound identified in A. sativum and boiled beef extracts [13,39,47]. Here, we found that 1,3-dithiane stimulates ROS production by human neutrophils, and this response was inhibited by specific inhibitors of PI3Kβ, γ, and δ. Activation of PI3K signaling usually occurs following the stimulation of receptor tyrosine kinases or G-protein-coupled receptors (GPCRs) [56]. We can exclude a stimulatory effect of 1,3-dithiane on several GPCRs, including Nformyl peptide receptors (FPRs) 1 and 2, C5a receptor, and chemokine receptors CXCR1/2, as this compound did not stimulate neutrophil Ca 2+ mobilization ( Table 2). PI3Ks phosphorylate phosphatidylinositol-(4,5) bisphosphate to form phosphat idylinositol (3,4,5)-trisphosphate (PIP3), and the accumulation of PIP3 facilitates the localization of Akt to the plasma membrane and its subsequent activation following phosphorylation. PI3K/Akt signaling regulates many of the molecular mechanisms contributing to increased ROS production through the phosphorylation and activation of NADPH oxidase subunits [56]. In this study, we also established that 1,3-dithiane activates ERK signaling, which in turn phosphorylates CREB, which has been reported for other bioactive molecules, such as lysophosphatidic acid [57]. Indeed, we found that 1,3-dithiane stimulated the phosphorylation of CREB and GSK-3α/β in differentiated HL60 cells. It should be noted that PI3K generation of PIP3 could directly or indirectly affect CREB phosphorylation [58].
Using the SwissADME online tool, we found that 1,3 -and 1,4-dithianes have similar bioavailabilities (not shown) and physicochemical properties (Table 3). Numerous bioavailability parameters for these compounds are very close to each other, except lipophilicities (consensus Log Po/w values of 1.86 and 1.70, respectively). Table 3. Physicoche mical prope rtie s of 1,3-and 1,4-dithiane s. To estimate the reactivity of 1,3-and 1,4-dithiane in biological environment s, we performed a theoretical study of the molecules using the density functional theory (DFT) method. For geometry optimization and electronic structure calculations, the BP86 functional [59] and def2-TZVPP basis set [60] were used. At this level of theory, high-quality results can be obtained for conformations, energies, and electronic properties of organic compounds [61]. The values of Gibbs energies indicate that the two dithianes are thermodynamically most s table in the "chair" conformation, for which w e have analyzed their calculated properties. We found that 1,3 -and 1,4-dithiane differ markedly in their electronic structures. In contrast to the zero polarity of 1,4-dithiane, the calculated dipole moment equals 2.11 D for 1,3-dithiane, which is very close to the experimental values of 2.13 and 2.09 D measured in tetrachloromethane and benzene solutions, respectively [62].

Properties 1,3-Dithiane 1,4-Dithiane
The lowest unoccupied molecular orbital (LUMO) energies were equal to -0.702 eV (1,3dithiane) and -0.280 eV (1,4-dithiane). The difference in LUMO energies of >0.4 eV is substantial and indicates that 1,3-dithiane is a better electron acceptor and can relatively easily form an anion -radical Although various EOs and their major compounds have been reported to modulate ROS production and Ca 2+ mobilization in human neutrophils, including oils with a high content of organosulfur compounds [11,30,31], the current study is the first to r eport the biological effects of 1,3dithiane, a small molecule organosulfur compound identified in A. sativum and boiled beef extracts [13,39,47]. Here, we found that 1,3-dithiane stimulates ROS production by human neutrophils, and this response was inhibited by specific inhibitors of PI3Kβ, γ, and δ. Activation of PI3K signaling usually occurs following the stimulation of receptor tyrosine kinases or G-protein-coupled receptors (GPCRs) [56]. We can exclude a stimulatory effect of 1,3-dithiane on several GPCRs, including Nformyl peptide receptors (FPRs) 1 and 2, C5a receptor, and chemokine receptors CXCR1/2, as this compound did not stimulate neutrophil Ca 2+ mobilization ( Table 2). PI3Ks phosphorylate phosphatidylinositol- (4,5) bisphosphate to form phosphat idylinositol (3,4,5)-trisphosphate (PIP3), and the accumulation of PIP3 facilitates the localization of Akt to the plasma membrane and its subsequent activation following phosphorylation. PI3K/Akt signaling regulates many of the molecular mechanisms contributing to increased ROS production through the phosphorylation and activation of NADPH oxidase subunits [56]. In this study, we also established that 1,3-dithiane activates ERK signaling, which in turn phosphorylates CREB, which has been reported for other bioactive molecules, such as lysophosphatidic acid [57]. Indeed, we found that 1,3-dithiane stimulated the phosphorylation of CREB and GSK-3α/β in differentiated HL60 cells. It should be noted that PI3K generation of PIP3 could directly or indirectly affect CREB phosphorylation [58].
Using the SwissADME online tool, we found that 1,3 -and 1,4-dithianes have similar bioavailabilities (not shown) and physicochemical properties (Table 3). Numerous bioavailability parameters for these compounds are very close to each other, except lipophilicities (consensus Log Po/w values of 1.86 and 1.70, respectively). To estimate the reactivity of 1,3-and 1,4-dithiane in biological environment s, we performed a theoretical study of the molecules using the density functional theory (DFT) method. For geometry optimization and electronic structure calculations, the BP86 functional [59] and def2-TZVPP basis set [60] were used. At this level of theory, high-quality results can be obtained for conformations, energies, and electronic properties of organic compounds [61]. The values of Gibbs energies indicate that the two dithianes are thermodynamically most s table in the "chair" conformation, for which w e have analyzed their calculated properties. We found that 1,3 -and 1,4-dithiane differ markedly in their electronic structures. In contrast to the zero polarity of 1,4-dithiane, the calculated dipole moment equals 2.11 D for 1,3-dithiane, which is very close to the experimental values of 2.13 and 2.09 D measured in tetrachloromethane and benzene solutions, respectively [62].
The lowest unoccupied molecular orbital (LUMO) energies were equal to -0.702 eV (1,3dithiane) and -0.280 eV (1,4-dithiane). The difference in LUMO energies of >0.4 eV is substantial and indicates that 1,3-dithiane is a better electron acceptor and can relatively easily form an anion -radical To estimate the reactivity of 1,3-and 1,4-dithiane in biological environments, we performed a theoretical study of the molecules using the density functional theory (DFT) method. For geometry optimization and electronic structure calculations, the BP86 functional [59] and def2-TZVPP basis set [60] were used. At this level of theory, high-quality results can be obtained for conformations, energies, and electronic properties of organic compounds [61]. The values of Gibbs energies indicate that the two dithianes are thermodynamically most stable in the "chair" conformation, for which we have analyzed their calculated properties. We found that 1,3-and 1,4-dithiane differ markedly in their electronic structures. In contrast to the zero polarity of 1,4-dithiane, the calculated dipole moment equals 2.11 D for 1,3-dithiane, which is very close to the experimental values of 2.13 and 2.09 D measured in tetrachloromethane and benzene solutions, respectively [62].
The lowest unoccupied molecular orbital (LUMO) energies were equal to −0.702 eV (1,3-dithiane) and −0.280 eV (1,4-dithiane). The difference in LUMO energies of >0.4 eV is substantial and indicates that 1,3-dithiane is a better electron acceptor and can relatively easily form an anion-radical via one-electron reduction. The highest occupied molecular orbitals (HOMOs) have a smaller energy difference (−5.162 and −5.252 eV for 1,3-dithiane and 1,4-dithiane, respectively). Nevertheless, the HOMO of 1,3-dithiane is higher in energy (less negative value) than that of 1,4-dithiane. This reflects a higher tendency to oxidation, in accordance with previous results [48], and the greater ability of 1,3-dithiane to form complexes with transition metals. It should also be noted that HOMOs in both dithianes are localized mainly on sulfur atoms participating in complexation with metal ions (see the isosurfaces of frontier orbitals in Figure 4).
Molecules 2019, 24, x FOR PEER REVIEW 9 of 17 via one-electron reduction. The highest occupied molecular orbitals (HOMOs) have a smaller energy difference (-5.162 and -5.252 eV for 1,3-dithiane and 1,4-dithiane, respectively). Nevertheless, the HOMO of 1,3-dithiane is higher in energy (less negative value) than that of 1,4 -dithiane. This reflects a higher tendency to oxidation, in accordance with previous results [48], and the greater ability of 1,3-dithiane to form complexes with transition metals. It should also be noted that HOMOs in both dithianes are localized mainly on sulfur atoms participating in complexation with metal ions (see the isosurfaces of frontier orbitals in Figure 4). Chemical hardness, calculated as a HOMO-LUMO energy gap [63], is equal to 4.460 eV for 1,3dithiane and 4.972 eV for 1,4-dithiane, also indicating the higher chemical reactivity of 1,3 -dithiane, including reactivity to possible biotargets. Thus, the characteristics of the electronic structure obtained from the DFT calculations differ noticeably between the two dithianes, which may explain their differences in biological activity. Further studies are needed to examine the molecular targets of 1,3-dithiane or its biotransformation products that could be involved in pha gocyte stimulatory effects.
Various disulfides and trisulfides derived from garlic and onion have been shown to modulate immune functions and inflammation [32,64]. Previous studies demonstrated that some allylcontaining garlic-derived organosulfur compounds, including diallyl sulfide, diallyl disulfide, and diallyl trisulfide, can induce Ca 2+ flux in several cell types, such as human glioblastoma cells, human colon cancer cells, and Madin-Darby canine kidney renal tubular cells [65][66][67]. Moreover, diallyl disulfide activated transient receptor potential (TRP) A1 channels [68]. However, there are no reports on the neutrophil modulatory effects of pure organosulfur compounds. Here, we show that several Chemical hardness, calculated as a HOMO-LUMO energy gap [63], is equal to 4.460 eV for 1,3-dithiane and 4.972 eV for 1,4-dithiane, also indicating the higher chemical reactivity of 1,3-dithiane, including reactivity to possible biotargets. Thus, the characteristics of the electronic structure obtained from the DFT calculations differ noticeably between the two dithianes, which may explain their differences in biological activity. Further studies are needed to examine the molecular targets of 1,3-dithiane or its biotransformation products that could be involved in phagocyte stimulatory effects.
Various disulfides and trisulfides derived from garlic and onion have been shown to modulate immune functions and inflammation [32,64]. Previous studies demonstrated that some allyl-containing garlic-derived organosulfur compounds, including diallyl sulfide, diallyl disulfide, and diallyl trisulfide, can induce Ca 2+ flux in several cell types, such as human glioblastoma cells, human colon cancer cells, and Madin-Darby canine kidney renal tubular cells [65][66][67]. Moreover, diallyl disulfide activated transient receptor potential (TRP) A1 channels [68]. However, there are no reports on the neutrophil modulatory effects of pure organosulfur compounds. Here, we show that several pure organosulfur compounds, including allyl propyl disulfide, dipropyl disulfide, diallyl disulfide, and AITC activate neutrophils, resulting in increased intracellular Ca 2+ , with the most potent being AITC. Previously, it was reported that some organosulfur compounds with an allyl group (diallyl sulfide, diallyl disulfide, diallyl trisulfide, and AITC) and ajoene were able to activate TRPA1 and/or TRPV1 channels [68][69][70][71][72][73]. TRPA1 is also activated by allicin from garlic and S-alkyl-S-alkenyldisulfides from asafoetida [68,69,71]. Various TRP channels are functionally expressed in neutrophils, and a transient elevation in intracellular [Ca 2+ ] i through activation of these channels can regulate various aspects of inflammatory and immune responses [74]. Human neutrophils only express members of the TRPC, TRPM, and TRPV channels [75]. However, SB 366791, a specific TRPV1 antagonist, did not inhibit Ca 2+ flux in neutrophils induced by our active organosulfur compounds (diallyl sulfide, diallyl disulfide, diallyl trisulfide, and AITC), indicating that TRPV1 is likely not involved in this response (data not shown). Thus, further studies are needed to examine the specific mechanisms and targets involved.
In conclusion, our data provide a molecular basis to explain at least part of the beneficial therapeutic effects of mustard oil, garlic EO, and garlic extracts and suggest that neutrophil stimulation by organosulfur components from Allium spp. might enhance resistance to infection. Future studies are planned to investigate the therapeutic potential of organosulfur compounds for various disorders with immune and/or inflammatory mechanisms.

Isolation of Human Neutrophils
For the 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 Ficoll-Paque 1077 gradient separation and hypotonic lysis of red blood cells, as described previously [31]. 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 donors (n = 8); however, the cells from different donors were never pooled together during experiments.

Cell Culture
Human promyelocytic leukemia HL60 cells were cultured in RPMI 1640 medium supplemented with 10% heat-inactivated fetal calf serum, 10 mM HEPES, 100 µg/mL streptomycin, and 100 U/mL penicillin. For the differentiation of HL60 into neutrophil-like cells, DMSO was added to a final concentration of 1.2%, and the cells were cultured for 6 days. Differentiation was monitored by a gain in responsiveness of the cells to f MLF by measuring f MLF-induced Ca 2+ mobilization (data not shown).

Ca 2+ Mobilization Assay
Changes in neutrophil 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 aliquoted into the wells of flat-bottom, half-area well black microtiter plates (2 × 10 5 cells/well). Test compounds diluted in DMSO were added to the wells (final concentration of DMSO was 1%), and the changes in fluorescence were monitored (λ ex = 485 nm, λ em = 538 nm) every 5 s for 240 s at room temperature. The maximum change in fluorescence observed after subtracting the background signal from DMSO-treated cells was used to determine the agonist response. Antagonist activity was evaluated after a 30 min pretreatment with test compounds at room temperature, followed by the addition of the peptide agonist (5 nM f MLF). Maximum change in fluorescence during the first 3 min, expressed in arbitrary units over baseline, was used to determine a response. Responses for test compounds were normalized to the response induced by 5 nM f MLF, which was assigned a value of 100%. Curve fitting (5-6 points) and calculation of median effective inhibitory concentrations (EC 50 or IC 50 ) were performed by nonlinear regression analysis of the dose-response curves generated using Prism 8 (GraphPad Software, Inc., San Diego, CA, USA), as described previously [30].

ROS Production Assay
ROS production was determined by monitoring L-012-enhanced CL, which represents a sensitive and reliable method for detecting ROS production [76]. Human neutrophils were resuspended at 10 6 cells/mL in HBSS + supplemented with 40 µM L-012. Cells (100 µL) were aliquoted into wells of 96-well flat-bottomed white microtiter plates containing test compounds (final DMSO concentration of 1%). Neutrophils were stimulated by the application of 100 nM PMA. Luminescence was monitored for 60 min (2-min intervals) at 37 • C using a Fluroscan Ascent FL microtiter plate reader (Thermo Electron, Waltham, MA, USA). To calculate IC 50 values, individual ROS responses were normalized to the maximal response in a given experiment, which was assigned a value of 100%. Curve fitting (at least five or six points) and calculation of IC 50 values were performed by nonlinear regression analysis of the dose-response curves generated using Prism 8 (GraphPad Software, Inc., San Diego, CA, USA).

Protein Kinase Array
Analysis of the phosphorylation profiles of MAPKs and related kinases and their substrates was performed using a human phospho-kinase array kit, Proteome Profiler (R&D Systems, Minneapolis, MN, USA). The array simultaneously detects 24 kinase phosphorylation sites, including MAPKs (ERK1/2, c-Jun N-terminal kinases (JNK 1-3), p38 isoforms α, β, γ, σ), MSK2, MKK3, MKK6, hsp27, p53, mTOR, Akt 1-3, GSK-3α/β, RSK 1/2, p70S6K, and CREB. For the analysis, differentiated neutrophil-like HL60 cells were incubated for 15 min with 500 µM of 1,3-dithiane or negative control (1% DMSO) at 37 • C. The cells were then lysed, and the arrays were incubated overnight at 4 • C with lysates obtained from 10 7 cells for each sample. The arrays were washed three times with 20 mL of the wash buffer, followed by incubation for 2 h with the detection antibody cocktail containing phospho-site-specific biotinylated antibodies. The wash steps were repeated, the arrays were exposed to chemiluminescent reagents, and the signal was captured with an Alpha Innotech FluorChem FC2 imaging system.

Analysis of 1,3-Dithiane Biotransformation
Biotransformation of 1,3-dithiane in culture medium from HL60 cells was evaluated by GC-MS analysis. HL60 cells were cultured in RPMI 1640 medium supplemented with 10 mM HEPES and 4 mM 1,3-dithiane for 0, 1, and 5 h at 37 • C. The samples were extracted with methanol for 4 h and then centrifugated. GC-MS analysis was performed on an Agilent 7890A GC with a 5890A mass-selective detector (MSD) system. A Restek Rtx-VMS column (30 m × 0.25 mm, 1.40 µm film thickness) was used with He as the carrier gas (2.0 mL/min). GC oven temperature was kept at 30 • C for 1 min, increased to 250 • C at a rate of 11 • C/min, and kept constant at 250 • C for 4 min. The split ratio was adjusted to 10:1, and the injector temperature was 250 • C. Commercial 1,3-dithiane-1-oxide was used as a reference. The MS parameters were as follows: transfer line temperature was set at 250 • C, source temperature at 230 • C and quad temperature at 150 • C. Acquisition range was set between 30 and 550 amu at a scan rate of 5.2 scans/sec. Standard 1,3-dithiane and 1,3-dithiane-1-oxide were injected to identify the retention time. Under these conditions, 1,3-dithane eluted at 15.1 min and 1,3-dithiane-1-oxide eluted at 21.6 min. The corresponding M + ions of 1,3-dithiane and 1,3-dithiane-1-oxide were seen at 120.00 and 136.00 m/z, respectively.
DFT calculations were performed with the ORCA 4.  [59], def2-TZVPP orbital basis set [60], resolution of identity (RI) approximation with def2/J auxiliary basis set [61], and D3BJ dispersion correction [77] were applied. The attainment of real energy minima on geometry optimizations in vacuo was confirmed by frequency calculations. The obtained results were visualized and analyzed with the use of Chemcraft graphical software for the visualization of quantum chemistry computations (https://www.chemcraftprog.com).