[6]-Gingerol Facilitates CXCL8 Secretion And ROS Production in Primary Human Neutrophils By Targeting The TRPV1 Channel Part 2

2.6. Impact of [6]-Gingerol on CXCL8 Secretion upon fMLF Stimulation

The previous result prompted the question of whether the altered protein expression on the surface of neutrophils affects the general responses of these cells to well-known activating molecules like fMLF. 

When isolated neutrophils were pre-incubated with [6]- gingerol in a concentration of 50 nM for 2 h and stimulated with different concentrations of fMLF for additional 4 h, an increase in CXCL8 secretion after subsequent stimulation with fMLF at concentrations of 0.3 nM (22%, p = 0.08), 0.5 nM (32%, p = 0.05), and 1 nM (33%, p = 0.03) was demonstrated as compared to sole stimulation with fMLF (Figure 6). Also, pre-incubating the neutrophils for 2 h with 50 nM [6]-gingerol promoted a shift of the EC50 value from 0.66 to 0.49, indicating augmented responsiveness of the cells towards fMLF. In contrast, pre-incubating the neutrophils with 50 nM [6]-gingerol for 2 h alone did not induce CXCL8 secretion (CXCL8 not detectable, data not shown).

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2.7. Impact of [6]-Gingerol on ROS Production

Next, we analyzed whether incubation of neutrophils with [6]- gingerol affects their generation of reactive oxygen species after the 2 h incubation period with [6]-gingerol and after subsequent stimulation with 1 nM fMLF.

The analyses showed no statistically significant impact of a 2 h incubation of neutrophils with 50 nM [6]-gingerol (Figure 7A). However, pre-incubating neutrophils for 2 h with 50 nM of [6]- gingerol and subsequently stimulating the cells with 1 nM fMLF did increase the ROS production by 27.8% ± 2.2% (Figure 7B).

2.8. Pharmacological Inhibition of TRPV1 Reverses [6]-Gingerol-Induced Effects

To verify the involvement of TRPV1 in the [6]-gingerol-induced increases in CXCL8 secretion and ROS production after fMLF stimulation, the TRPV1 specific inhibitor trans-tertbutylcyclohexanol (BCH) was applied.[5a] Binding of BCH to TRPV1 interrupts its interaction with [6]-gingerol, and should thereby inhibit the [6]-gingerol-induced effects, which would allow the conclusion that the observed effects are TRPV1 mediated. 

For this approach, human neutrophils were either incubated with [6]-gingerol for 2 h or with [6]-gingerol and BCH for 2 h, and subsequently stimulated with 1 nM fMLF. We could show, that the increase in CXCL8 production induced by pre-incubating the neutrophils with 50 nM [6]-gingerol and subsequent stimulation with 1 nM fMLF could not be detected, when the cells were preincubated with a combination of [6]-gingerol and 100 μM BCH (Figure 8A), confirming the involvement of TRPV1 in the [6]- gingerol induced effects. In the case of ROS production, preincubating the neutrophils with a combination of [6]-gingerol and BCH led to even lower values than the solvent control (Figure 8B).

3. Discussion

While the chemosensory properties of some TRP channels have been well described, their function in non-sensory tissues is far less clear. Regarding their general expression profile in nonsensory tissue like blood leukocytes, only a few studies have been carried out in the past. In 2011, Wenning et al.[24] analyzed the RNA expression of members of the TRPC, TRPM, and TRPV families in primary human CD4+ T cells via RT-PCR. They found consistent expression of TRPC1, TRPC3, TRPV1, TRPM2, and TRPM7. 

Furthermore, TRPC3 was shown to modulate the Ca2+-dependent proliferation of primary CD4+ T cells. Regarding the expression profile, the reported results[24] are consistent with our data, except for TRPC3, which, in the present work, was detected only with a frequency of 80%. In human neutrophils, Heiner et al.[11] analyzed the expression pattern of TRP-specific transcripts via RT-PCR. They demonstrated specific transcripts for LTRPC2 (TRPM2), TRPV1, vanilloid receptor-like protein 1 (TRPV2), epithelial Ca2+ channel 1 (TRPV5), epithelial Ca2+ channel 2 (TRPV6), and TRPC6, which is completely in line with our results.

However, until now, according to our knowledge, no comprehensive quantitative data about the RNA expression of all 27 members of the human TRP superfamily have been analyzed for five of the most prominent blood leukocyte cell types. The results we obtained allow us to conclude that the TRP channel expression is not lineage-dependent. Hence, one might hypothesize that expression of TRP channels is regulated rather than individually. Which circumstances influence the expression of each TRP channel in each cell type at a certain point in time can only be speculated, but might, besides general physiological aspects, also include the individual’s dietary habits and nutritional status. 

For example, it was shown that high glucose levels increased the expression of TRPC1, TRPC3, TRPC5, TRPC6, TRPM6, and TRPM7 in human monocytes.[25] It is noteworthy that we could not detect TRPC3 and TRPC5 at all in monocytes, which could be explained by analyzing cells from different donors, supporting a highly individual regulation of TRP channel expression. 

On the other hand, stimulation of neutrophils with the TRPV1 ligand [6]-gingerol at a concentration of 50 nM for 2 h showed no significant impact on its RNA and surface protein expression. The latter result may hint at the conclusion that the incubation does not lead to an internalization of the receptor. This is to the finding that capsaicin provokes TRPV1 internalization only at higher concentrations (EC50: 500 nM) in TRPV1+ HEK293 cells.[26]

Incubating neutrophils with [6]-gingerol in a concentration of 50 nM for 2 h led to significant changes in protein surface expression other than TRPV1, i.e., an increased expression of the fMLF receptor FPR1, CD11b, and CD66b. This finding was interesting in that it has previously been shown that priming of neutrophils results in elevated surface expression of the fMLF receptor FPR1, CD11b, CD35, and CD66b.[27] Inversely, CD62L expression was reduced via enzymatic shedding in primed neutrophils.[28] Priming is a process by which neutrophils are transferred from a resting state to a “ready to go” state that is characterized by an ability to respond more strongly to activating stimuli, without features of full activation, e.g., ROS production.[16b] Whether the observed phenotypic changes can be attributed to neutrophil priming remains elusive at this point. 

However, increased surface expression of FPR1 may lead to increased neutrophil responsiveness to its ligand, fMLF, the most important and well-studied neutrophil activator. Stimulation of neutrophils with fMLF leads, amongst other responses, to the release of chemokines, with CXCL8 (IL-8) being crucial for this response. CXCL8 functions as a chemoattractant to other cells, thereby directing them to the site of infection. Indeed, pre-incubation of human neutrophils with [6]-gingerol and subsequent stimulation with fMLF led to increased secretion of the chemokine CXCL8. However, this effect could only be observed in a concentration range of 0.3– 1 nM fMLF. The reason for this could be that higher fMLF concentrations induce receptor desensitization or internalization,

which would abolish the [6]-gingerol-induced increase in FPR1 expression.[29] The increased response towards fMLF was also shown for ROS production after pre-incubation of the cells with [6]-gingerol and subsequent stimulation with 1 nM fMLF. Pharmacological inhibition of TRPV1 by trans-test-butyl cyclohexanol led to even lower ROS levels than in the control, which might indicate a general partial involvement of TRPV1 in ROS production in human neutrophils as it was shown for mouse dorsal root ganglion neurons.[30]

The 2 h pre-incubation itself did not increase ROS production, which also points to enhanced responsiveness of the cells, but no initiation of ROS production by [6]-gingerol. In addition, the cytokine and chemokine screening revealed only a quite low number of transcripts regulated by incubating neutrophils with [6]-gingerol, with even unchanged TRPV1 transcript and surface protein levels. This implies, that the observed functional and phenotypic changes occur in the absence of de novo synthesis.

About somewhat inconsistent previous findings, our results support a functional expression of TRPV1 in human neutrophils. However, Schepetkin et al.[22] showed a TRPV1-mediated inhibitory impact of geranyl acetone on neutrophil migration towards fMLF, and a CXCL8-induced intracellular Ca2+ mobilization, in contrast to our results which point to a stimulatory effect on TRPV1 upon subsequent activation. Besides being different compounds, one reason for this discrepancy might be the different compound concentrations used, since Schepetkin et al.[22] applied a concentration of 50 μM geranyl acetone, whereas in our work we applied only 50 nM [6]- gingerol. This concentration was chosen based on our previous work, which showed a maximum plasma concentration of 42.0 ± 16.3 nmol L−1 at 30 min after ginger tea consumption and decreasing concentrations over a subsequent time course of 2 h. However, the ginger tea was consumed within 20 min, which may not correspond to the usual consumption behavior. Therefore, we assumed that a plasma concentration of 50 nM [6]-gingerol is reachable via dietary intake, whereas higher concentrations are not very likely to occur.[9]

The inhibitory effect of geranyl acetone was attributed to cross desensitization, which would lead to decreased responses towards activating stimuli, and one might assume that this effect is only present at higher TRPV1 ligand concentrations in the μM range, leading to higher intracellular Ca2+ concentrations. In this context, it is worth mentioning that concentration-response analyses in our previous work regarding cytokine secretion in T cells revealed an IC50 value of 82.2 μM for [6]- gingerol.[9] This, together with the observed inhibitory effect of 50 μM geranyl acetone, would strengthen the assumption that high concentrations of TRPV1 ligands act rather intuitively, whereas lower concentrations augment immune cell functions. [6]-gingerol concentrations below 50 nM were not included in the study. Therefore, the question remains, whether lower concentrations would reveal significant, biologically relevant effects. Nonetheless, the study presented demonstrates, that a concentration of [6]-gingerol, reached in the blood plasma after habitual dietary intake of ginger tea, is sufficient to facilitate CXCL8 secretion as well as ROS production in human neutrophils.

Our findings show that at nutritionally relevant concentrations, a sensory active food ingredient can affect general cellular responses in non-sensory tissue such as blood leukocytes via the TRPV1 channel. The observed modifications of neutrophil function, together with the extensive-expression of TRP channels in blood leukocytes as potential target structures, underline the importance of assessing the bioactive potential of food ingredients with a particular focus on concentrations achievable through common dietary habits.

4. Experimental Section

Isolation of Leukocytes: Commercial blood samples from anonymized healthy donors were received from SonnenGesundheitszentrum/Transfusionsmedizin, (English translation: Sun Health Center/Transfusion Medicine) Munich (Germany). Neutrophils, NK cells, B cells, and T cells were isolated using the respective MACSxpress Whole Blood Isolation Kits (Miltenyi Biotec, Bergisch Gladbach, Germany) according to the manufacturer’s protocol. Afterward, the remaining erythrocytes were depleted using the MACSxpress Erythrocyte Depletion Kit (Miltenyi Biotec). For the isolation of monocytes, the StraightFrom Whole Blood CD14 Micro Beads, the Whole Blood Column Kit, and Red Blood Cell Lysis Solution (Miltenyi Biotec) were used.

Isolation of RNA, Reverse Transcription, and Quantitative Real-Time PCR: RNA from leukocytes was isolated using QIAzol Lysis Reagent (Qiagen, Hilden, Germany) following a DNase treatment using the RNase-free DNase set (Qiagen) according to the manufacturer’s protocol. RNA integrity was determined via the RNA 6000 Nano Kit (Agilent Technologies, Waldbronn, Germany) using the Bioanalyzer 2100 (Agilent Technologies). RNA was transcribed into cDNA using the iScript gDNA Clear cDNA Synthesis Kit (Bio-Rad Laboratories GmbH, Feldkirchen, Germany). For qPCR 50 ng of cDNA and SsoAdvanced Universal SYBR Green Supermix (BioRad Laboratories GmbH) were mixed with specific primer pairs. 

The respective primers were validated via sequencing PCR products derived from qRT-PCR experiments using RNA from the human brain or testis (from the FirstChoice Human Total RNA Survey Panel, Thermo Fisher Scientific) for TRPM4, TRPM5, and TRPV5, respectively. Primer and the respective product sequences were given in Table S1, Supporting Information. ACTB, B2M, HMBS, and HPRT1 were used as reference genes and the respective primers were purchased from Bio-Rad Laboratories. RT control, PCR control, gDNA control, and RNA quality control (Bio-Rad Laboratories) were used as general PCR controls. PCR reactions were performed on a CFX 96 Real-Time System (Bio-Rad Laboratories) using the following PCR conditions: 95 °C for 1 min, followed by 45 cycles of 95 °C for 15 s and 60 °C for 1 min. Frequencies, which indicated how many of the donors investigated the transcript could be detected, and Δct values were calculated using Microsoft Excel.

Incubation of Neutrophils: After isolation from the blood of healthy donors, neutrophils were suspended in RPMI 1640 (Gibco, Thermo Fisher Scientific, Schwerte, Germany) containing no further supplements with a cell density of 1 × 106 mL−1. [6]-gingerol (Sigma, ≥98% purity) or DMSO, respectively was added in a final concentration of 50 nM or 0.02%. Afterward, the cells were incubated at 37 °C and 5% CO2 for 2 h, and either used directly for further analyses or centrifuged at 300 × g for 10 min at room temperature, washed with RPMI 1640 (Gibco), and further incubated with fMLF (Alomone Labs, Jerusalem, Israel) for 4 h or DMSO (0.02%) as solvent control, respectively.

Immunostaining: For immunostaining, 105 neutrophils, either treated or non-treated, were centrifuged at 300 × g for 10 min. The supernatant was aspirated and the cells were resuspended in a PBS buffer. The antibodies were added according to the manufacturer, and the suspensions mixed and incubated at 4 °C for 10 min in the dark. Afterward, the cells were washed using 1 mL of PBS buffer and resuspended in 500 μL of PBS buffer for analysis using a MACSQuant Analyzer 16 (Miltenyi Biotec). Antibodies used were: CD15-VioBlue (final concentration: 2.5 μg mL−1), CD62LVioGreen (final concentration: 7.5 μg mL−1), CD11b-APC (final concentration: 2 μg mL−1), CD66b-APC/Vio770 (final concentration: 1 μg mL−1), fMLP receptor antibody-FITC (final concentration: 20 μg mL−1) (Miltenyi Biotec), anti-TRPV1-FITC (final concentration: 25 μg mL−1) (Alomone Labs), Rabbit IgG Isotype Control-FITC (final concentration: 25 μg mL−1) (Thermo Fisher Scientific). Cell viability was assessed using propidium iodide staining (Miltenyi Biotec). Mean fluorescence intensity (MFI) values and the corresponding figures were obtained by using the Flowlogic 7.3 software (minivan Technologies, Mentone, Victoria, Australia).

ROS Measurement: Reactive oxygen species were detected using the CellROX Green Reagent (Thermo Fisher Scientific) according to the manufacturer’s instructions. Briefly, neutrophils were treated with either 50 nM [6]-gingerol for 2 h or 50 nM [6]-gingerol for 2 h and subsequently with 1 nM fMLF for 4 h, or the respective solvent controls. The CellROX reagent was added to the cells at a final concentration of 5 μM 30 min before the end of the incubation period and the mixture was further incubated for 30 min at 37 °C and 5% CO2. Afterward, the medium was removed, and the cells were washed three times with PBS and analyzed using a MACSQuant Analyzer 16 (Miltenyi Biotec). Mean fluorescence intensity (MFI) values were obtained by using the Flowlogic 7.3 software (minivan Technologies).

Spectrofluorimetry: For measuring intracellular Ca2+ concentrations, neutrophils were suspended in RPMI 1640 (Gibco) with a cell density of 1 × 106 mL−1. The cells were loaded with Fura-2 AM (Promocell, Heidelberg, Germany) for 45 min at 37 °C and 5% CO2, washed, and resuspended in RPMI 1640 (Gibco). The fluorescence of the cells was assessed using a SAFAS Xenius XC Spectrofluorometer (SAFAS, Monaco) at the following wavelengths: 340 nm excitation/510 nm emission and 380 nm excitation/510 nm emission. Fura-2 M is a ratiometric dye that can be excited at 340 nm for its calcium-bound form and 380 nm for its unbound form. An increase in the intracellular calcium concentration would lead to an increased fluorescence for the calcium-bound form and a decreased fluorescence for the unbound form. The 340/380 ratio, which was used to account for unequal cellular loading of the dye, was calculated using Microsoft Excel. The 340/380 ratio for the cells treated with the ionophore ionomycin after 2 h was set to 100% and the ratio for the cells treated with 50 nM [6]-gingerol and the solvent control was referred to this.

Enzyme-Linked Immunosorbent Assay: The CXCL8 concentrations in the supernatants derived from the incubation experiments (Section 4.3) were determined via Sandwich ELISA (DuoSet, R&D Systems, bio-techne, Minneapolis, MN, USA) according to the manufacturer’s protocol.

Statistical Analyses: All experiments were performed using cells from at least three independent donors. Unless otherwise stated, data were shown as mean ± standard deviation (SD) calculated by GraphPad Prism 9.0. For the analyses of statistically significant differences, two sample of Student’s t-tests, or, in case more than two variables were compared, a one-way ANOVA followed by a Tukey’s multiple comparisons test as a posthoc test was performed. The respective test used was indicated in the corresponding figure legend. A p-value of ≤0.05 was considered statistically significant.

Supporting Information

Supporting Information is available from the Wiley Online Library or the author.

Acknowledgments

The Data Availability Statement was added on February 20, 2023.

Conflict of Interest

The authors declare no conflict of interest.

Author Contributions

G.A., D.K., and V.S. conceived and planned the experiments; G.A. and K.K. carried out the experiments; V.S. supervised the project; G.A. wrote the first draft of the manuscript. All authors reviewed the final manuscript.

Data Availability Statement

Data is available on request from the authors.

Keywords

food, ginger, immune system, immunomodulation, polymorphonuclear leukocytes (PMNs).

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