In vivo Effects of Acute Inflammatory Responses on Dopaminergic Receptor Expression in Leukocytes and Marginal Effects of Hypoxia Pretreatment

Abstract

Introduction

Lipopolysaccharide (LPS) is widely used to study the mechanisms underlying acute inflammation. Interestingly, several studies suggest that LPS also regulates central dopaminergic signaling. Despite these findings in the brain, the effects of LPS on the dopaminergic system in the periphery remain poorly understood. Notably, peripheral immune cells express dopamine receptors (DRs) and can respond to dopamine. Dysregulation of the dopaminergic system in immune cells has been reported in various chronic inflammatory conditions. Additionally, studies suggest that hypoxia may also modulate dopamine synthesis and potentially amplify LPS-induced effects.

Methods

The aim of this study was to investigate the effects of peripheral LPS administration on the dopaminergic system in male human peripheral blood mononuclear cells by measuring dopamine plasma levels and the expression of tyrosine hydroxylase and DRs. Additionally, we explored whether these effects are modulated by prior hypoxic exposure.

Results

Our results suggest that in vivo LPS modulates the expression of DRs on monocytes and natural killer cells, as reflected by an upregulation after 24 h. In contrast, the effects of LPS on T and B cells were weaker, with a predominantly inhibitory influence on DR expression, supporting the notion of a cell-specific effect of LPS on dopaminergic signaling within the immune system. Additionally, our results indicate that hypoxic pretreatment did not alter LPS-induced changes in the dopaminergic pathway.

Conclusion

Taken together, this study demonstrates for the first time that systemic LPS administration modulates DR expression in male peripheral immune cells. Further, our in vitro findings suggest that it is the LPS-induced immune response, rather than LPS itself, that drives changes in the dopaminergic pathway in specific immune cell subpopulations. However, further research is needed to elucidate the functional relevance of these findings in clinical contexts.

Introduction

Lipopolysaccharide (LPS), also known as bacterial endotoxin, is widely used in experimental research to study the mechanisms underlying acute inflammatory activation and sepsis [1–3]. Upon administration, LPS binds to Toll-like receptor (TLR)-4, a pattern recognition receptor expressed on various immune cells including monocytes, B cells, and natural killer (NK) cells. This interaction triggers intracellular signaling cascades such as the nuclear factor kappa B (NF-κB) signaling pathway [4, 5], leading to the secretion of several pro- and anti-inflammatory cytokines, including tumor necrosis factor (TNF)-α, interleukin (IL)-1β, IL-6, IL-10, as well as to leukopenia and subsequent leukocytosis [1, 5–7]. Clinically, LPS administration dose-dependently induces a range of mild (headache, fatigue, myalgia, nausea) to more severe (vomiting, fever, hypotensive shock) physical and psychological sickness symptoms [1, 4, 8].

Regarding the effects of LPS on the central nervous system, several studies suggest that, besides classical immune cell activation, LPS also regulates dopamine and its signaling in the brain [9–11]. Chronic LPS exposure in murine models induced dopaminergic neurodegeneration, including a reduction of tyrosine hydroxylase (TH)-expressing cells and dopamine metabolites in the substantia nigra, similar to key features of Parkinson’s disease (PD) [10, 12]. Additionally, the phenomenon of “sickness behavior,” a behavioral response to acute infection characterized by fatigue, anhedonia, and social withdrawal, is thought to be partially mediated by reduced dopaminergic activity in brain regions involved in motivation and reward. This observation supports the connection between inflammation and the dopaminergic system [13, 14].

Despite these findings in the central nervous system, the effects of LPS on the dopaminergic system in the periphery, particularly in immune cells, remain poorly understood. Notably, peripheral immune cells express dopamine receptors (DRs) and other components of the dopaminergic machinery and can respond to dopamine [15, 16]. Furthermore, due to expression of TH, they synthesize dopamine themselves, providing an additional cellular communication system [17, 18]. Thus, in this context, dopamine is called a “neuro-immunotransmitter” which modulates immune cell functions, such as cytokine release, cell proliferation, apoptosis, antigen presentation, and cell migration, in an autocrine and paracrine manner [19–21].

Notably, this interaction is bidirectional as inflammatory conditions have also been shown to modulate the dopamine machinery. Dysregulation of the dopaminergic system in immune cells has been reported in various chronic inflammatory conditions such as rheumatoid arthritis, multiple sclerosis, PD, and attention deficit hyperactivity disorder as well as in acute inflammatory models [22–24]. Therefore, it is plausible that dopaminergic signaling also plays a role in the interaction of immune cells during acute LPS-induced inflammation and sepsis. Investigating this interplay may help identify new therapeutic targets and pathways.

Another modulator of both inflammation and dopamine signaling is hypoxia [25–27]. Previous studies have shown that hypoxia exposure prior to LPS injection (hypoxia priming) and the consequent transcriptional activation of various interconnected genes amplifies the pro-inflammatory response in healthy men [7, 28]. Additionally, experimental animal studies suggest that the hypoxia-inducible factor (HIF) may also regulate central dopaminergic signaling by modulating TH and dopamine synthesis [25, 26, 29]. However, whether hypoxia also influences LPS-induced alterations in the dopaminergic pathway, particularly in peripheral immune cells, remains largely unexplored.

The aim of this study was to investigate the effects of peripheral LPS administration on the dopaminergic system in human peripheral blood mononuclear cells (PBMCs). Specifically, we examined whether LPS alters dopamine levels in serum as well as the expression of TH and DRs on various PBMC subsets obtained from healthy male volunteers. Additionally, previous results from the same cohorts showed that hypoxia exposure prior to LPS administration amplified the increase in body temperature, noradrenaline secretion, and pro-inflammatory cytokine expression (i.e., IL-6 and TNF-α) compared to LPS exposure under normoxic conditions [7, 28]. Thus, we also explored whether LPS-induced effects on the dopaminergic system are modulated by prior hypoxic exposure. Through this, we aimed to contribute to a better understanding of neuroimmune crosstalk under acute inflammatory conditions.

Materials and Methods

Study Participants

Healthy male volunteers (N = 36) with a mean age of 25.9 ± 2.9 years (range 21–33), a mean body mass index (BMI) of 24.2 ± 2.1 (range 21.2–29.5), and a C-reactive protein level <2 mg/L were recruited through public advertisement in the surrounding community. All volunteers underwent an extensive medical screening performed and evaluated by physicians of the Institute of Physiology at the University Medicine Essen. A detailed description of this medical screening and exclusion criteria is stated in an associated study [7].

Study Design

Blinded study participants were randomly assigned to three different groups. Groups (LPS, LPS-HOX, and HOX-LPS) did not differ in age, BMI, or school education [7]. In brief, the LPS group received a LPS injection only, the LPS-HOX group received a LPS injection for the first 4 h and was afterward exposed to hypoxia for 4 h, and the HOX-LPS group was first exposed to hypoxia for 4 h and afterward injected with LPS for 4 h (also see [7, 28]).

To analyze short-term effects of LPS on dopaminergic signaling, samples of the LPS group (n = 6; age 25.40 ± 0.8; BMI 24.1 ± 0.6) and the LPS-HOX group (n = 15; age 26.1 ± 0.9; BMI 24.6 ± 0.7) were pooled, as both groups only received an i.v. injection of 0.4 ng/kg bw LPS (Escherichia coli, serotype O113:H10, HOK354, US Pharmacopeia, Rockville, MD, USA) for the first 4 h. LPS had been dissolved in sterile water, filtered through a 0.2-µm membrane, and subjected to microbiological safety testing by a cGMP-certified laboratory (Labor LS, Bad Bocklet, Germany). In contrast, to examine long-term dopaminergic effects induced by LPS, only samples of the LPS group were analyzed, since they were not exposed to hypoxia. Additionally, participants belonging to the HOX-LPS group (n = 15; age 25.7 ± 0.8; BMI 23.8 ± 0.4) were first exposed to acute normobaric hypoxic conditions with a simulated altitude of approx. 4,500 m and an oxygen concentration of 10.5% for 4 h in a hypoxia chamber (−4 h to 0 h). Afterward, they received an i.v. injection of 0.4 ng/kg bw LPS to examine the effect of hypoxia priming on LPS-induced effects regarding the dopaminergic system. Blood samples were collected via an indwelling venous catheter at baseline (0 h), subsequently every second h (+2 h to +4 h) after LPS administration (Fig. 1a) and 24 h (+24 h) after the intervention (Fig. 4a).

Fig. 1.

Short-term effect of lipopolysaccharide administration on plasma dopamine concentration. a Protocol of the short-term LPS administration. Blood samples were collected immediately before LPS injection as well as 2 h and 4 h later. b Dopamine concentrations measured in plasma. Data are shown as mean ± SEM. n = 15.

Fig. 4.

Long-term effect of lipopolysaccharide administration on plasma dopamine concentrations. a Protocol of the long-term LPS administration. Blood samples were collected immediately before the LPS injection as well as 24 h later. b Dopamine concentrations measured in plasma. n = 3. Data are shown as mean ± SEM.

Plasma Dopamine Analysis

Blood was collected in EDTA-treated tubes (S-Monovette, Sarstedt, Nümbrecht, Germany), and plasma was obtained by centrifugation (2,000 g, 10 min, 4°C). Plasma samples were stored at −80°C until analysis. Plasma dopamine concentrations were measured by ELISA as described by the supplier (Dopamine ELISA, Tecan RE59161, IBL International, Hamburg, Germany). The sensitivity of the assay was 4 pg/mL and cross-reactivity of the antiserum with other catecholamines and their metabolites was <0.05%. Mean inter- and intra-assay coefficients of variation were 11% and 16%, respectively.

PBMC Isolation

PBMCs were isolated using density-gradient centrifugation (Ficoll-Paque Plus; GE Healthcare) and SepMate PBMC Isolation Tubes (STEMCELL Technologies) according to manufacturer’s instructions. Isolated PBMCs were then washed in protein-free phosphate-buffered saline (PBS) with 2% fetal bovine serum (FBS) and counted using a fully automatic 3-part cell counter (Sysmex). Following centrifugation (300 g, 8 min, 20°C), 5–10 × 10⁶ PBMCs were resuspended in freezing medium (FBS + 10% DMSO) and gently frozen using a Mr. Frosty (Thermo Fisher Scientific). Samples were stored in liquid nitrogen until analyses.

Flow Cytometry Analysis of DRs and TH

For flow cytometry analysis, frozen PBMCs were thawed and counted. A total of 0.5 × 10⁶ cells were used per staining condition. The antibody panels used to analyze DR and TH expression on PBMC subsets are listed in Table 1. To exclude dead cells, samples were stained with Zombie NIR^™ Fixable Viability dye (1:1,000, BioLegend, 423108) in PBS for 20 min at 4°C. Afterward, nonspecific binding sites were blocked by incubation with Albumin Fraction V (2%, Carl Roth, 0163.4) in PBS for 20 min at 4°C. Extracellular antigens (CD3, CD14, CD19, CD56, DRD₁, DRD₃, DRD₅) were stained in staining buffer (PBS containing 2% FBS) for 20 min at 4°C. For intracellular antigen staining, cells were first fixed with paraformaldehyde (2%, Aldrich, 16005) for 10 min at room temperature (RT) and then permeabilized using FACS^™ Permeabilizing Solution 2 (diluted 1:10 in water, BD, 340973) for 10 min at RT. To block nonspecific intracellular binding sites, cells were incubated with Albumin Fraction V (2%) for 20 min at RT. Finally, intracellular antigens (DRD₂, DRD₄, TH) were stained with primary antibodies for 20 min at 4°C. A secondary antibody staining was performed for 20 min at 4°C. Cells were washed twice, and flow cytometric analysis was performed on the same day using an AURORA flow cytometer.

Table 1.

Antibody panels used for flow cytometry staining

Panel	Antibody	Dilution	Company
Panels 1–4	CD3-PerCP	1:200	BioLegend, 300428
	CD14-BV650	1:400	BioLegend, 302825
	CD19-BV510	1:200	BioLegend, 302241
	CD56-FITC	1:100	BioLegend, 362546
Panel 1	DRD1- PE	1:50	Bioss, BS-10610R-PE
	DRD3-Cy5	1:100	Bioss, BS-1743R-Cy5
	DRD5-AF405	1:100	R&D, FAB82861P
Panel 2	DRD2	1:200	LS-Bio, LS-A1405
Panel 3	DRD4	1:200	Biorbyt, orb39453
Panel 4	TH-AF647	1:100	Bioss, BS-0016R-A647
Panels 2–3	Donkey anti-rabbit Ab-PE	1:400	BioLegend, 406421

Statistical Analysis

Data were analyzed using GraphPad Prism (Version 10). Outliers were identified and excluded using the ROUT method. To test normal distribution, the Shapiro-Wilk test was used. Accordingly, raw data were analyzed using parametric tests for normally distributed values and non-parametric tests in case of non-normal distribution. To investigate changes over time, paired t test or Wilcoxon test was used for two time points of measurement, whereas repeated measures ANOVA or Friedman-ANOVA were used for more than two time points. To analyze differences between groups (dim vs. bright), unpaired t test or Mann-Whitney U test was conducted. Whenever appropriate, post hoc multiple comparison tests, including p value adjustment, were performed (Dunnett’s after repeated measures ANOVA and Dunn’s after Friedman-ANOVA). Details about the tests used are specified in the figure legends. If values are missing, e.g., due to poor sample substance, the reduced n is specified. The level of significance was set at p < 0.05. For reasons of clarity, the graphs show data normalized to the baseline, but the calculations were always based on the raw data.

Results

Short-Term LPS Administration Modulates the Dopaminergic Pathway in PBMCs but Barely Alters the Dopaminergic Concentration in Plasma

As shown in Figure 1b, short-term LPS had no impact on plasma dopamine concentration. To investigate TH and DR expression in PBMC subpopulations, flow cytometry was used for gating strategy (see online suppl. Fig. 1; for all online suppl. material, see https://doi.org/10.1159/000550768). Short-term administration of LPS had a strong effect on the expression of DR especially in monocytes, with a significant increase of DRD3 after 4 h and a decrease of DRD2 and DRD4 after 2 h (Fig. 2). Interestingly, the expression of TH was also significantly reduced 2 h after LPS treatment (Fig. 2). In contrast, no effects were observed in NK cells within 4 h after LPS administration, but a slight increase of DRD4 after 2 h was observed (Fig. 2). These findings suggest a cell-specific effect of LPS on the dopaminergic pathway.

Fig. 2.

Short-term effect of lipopolysaccharide administration on tyrosine hydroxylase and DR expression in monocytes and natural killer cells. Expression of TH and DRs was measured via flow cytometry 0 h, 2 h, and 4 h post-LPS injection. a Monocytes. b NK cells. n = 15. Post hoc Dunnett’s multiple comparison test: *p < 0.05; **p < 0.01; ***p < 0.001; post hoc Dunn’s multiple comparison test: ⁺p < 0.05. Data are shown as mean ± SEM.

The effects of LPS on the dopaminergic pathway in T cells and B cells were slightly inhibitory, with an inhibition of TH 4 h after LPS in B cells and T cells (Fig. 3) and a slight decrease of DRD3 in B cells (Fig. 3). These results suggest a weaker but inhibitory effect of LPS on the dopaminergic pathway in T cells and B cells and a more complex and strong modulation in monocytes and NK cells.

Fig. 3.

Short-term effect of lipopolysaccharide administration on tyrosine hydroxylase and DR expression in T cells and B cells. Expression of TH and DRs was measured via flow cytometry 0 h, 2 h, and 4 h after LPS injection. a B cells. b T cells. n = 15. Post hoc Dunnett’s multiple comparison test: *p < 0.05; post hoc Dunn’s multiple comparison test: ⁺p < 0.05. Data are shown as mean ± SEM.

We then investigated if the pretreatment with hypoxia had any effect on the LPS-modulation of dopaminergic pathways in PBMCs. Our results show that hypoxia pretreatment did not alter the LPS effects observed (online suppl. Fig. 2).

Long-Term LPS Effects Are Similar but Partially Stronger Compared to the Short-Term LPS Effects

One day after LPS administration (Fig. 4a), no long-term effect of LPS on dopamine plasma concentration was observed (Fig. 4b). In addition, TH expression was unaltered in monocytes, NK, T, and B cells (Fig. 5a, 6). Therefore, LPS seems not to have any long-term effect on dopamine synthesis by PBMCs.

Fig. 5.

Long-term effects of lipopolysaccharide administration on tyrosine hydroxylase and DR expression in monocytes and natural killer cells. Expression of TH and DRs was measured via flow cytometry 0 h and 24 h after LPS injection. a Expression of TH and DR in monocytes (upper row) and NK cells (lower row). n = 6. Paired t test: *p < 0.05. b Gating strategy for CD56dim and CD56bright NK cells. c Comparison of TH and DR expression in CD56bright (blue) and CD56dim (green). n = 5. Paired t test: *p < 0.05; **p < 0.01; unpaired t test: ^#p < 0.05; Mann-Whitney U test: °p < 0.05. Data are shown as mean ± SEM.

Fig. 6.

Long-term effects of lipopolysaccharide administration on tyrosine hydroxylase and DR expression in T cells and B cells. Expression of TH and DRs was measured via flow cytometry 0 h and 24 h after LPS injection. a B cells. b T cells. n = 6. Paired t test: *p < 0.05. Data are shown as mean ± SEM.

However, a long-term effect of LPS was observed in the form of an upregulation of DRD1 and DRD3 in monocytes and NK cells (Fig. 5a). Of interest, LPS affects expression of two DR (DRD1 and DRD4) in CD56dim NK cells, the more mature and cytotoxic NK cell subpopulation, whereas DRD3 expression changes were observed in the less mature and more cytokine-producing CD56bright NK cells (Fig. 5b, c).

Similar to the short-term effects, long-term effects of LPS on B cells and T cells were not as strong as the effects observed on monocytes and NK cells, but there is an overall inhibition of the dopaminergic pathway. Indeed, only DRD4 in T cells was reduced (Fig. 6). Pretreatment with hypoxia did not change the long-term effects of LPS (online suppl. Fig. 3), as already observed for the short-term effects after hypoxia priming and LPS administration.

In vitro Effects of LPS Differ from in vivo Effects

To test if the observed effects of LPS on immune cells were the result of a direct stimulation or mediated by secondary factors such as cytokines or other cell-cell interactions, we isolated PBMCs from age-matched healthy male donors and treated them with an LPS concentration (5 ng/mL) comparable to the peripheral concentration reached in vivo after i.v. administration, and with LPS from the same company as for in vivo experiments was used for a better comparison (online suppl. Fig. 4). Our in vitro results did not match the results obtained in vivo (online suppl. Fig. 5). Therefore, it is plausible that the observed effects of LPS on the dopaminergic pathway in PBMCs in vivo were not due to the direct binding of LPS to PBMCs. However, one must also consider that the in vitro conditions do not fully capture the complexity of the in vivo system.

Discussion

In this study, we investigated whether in vivo LPS administration, as a model of acute inflammatory activation, affects the dopaminergic system in male human immune cells. While the effects of LPS on the immune system are well established [4, 30, 31], and the involvement of the dopaminergic pathway in immune responses has also been described [12, 15, 24, 32], a direct interaction between LPS and the dopaminergic pathway in immune cells has not been reported. Although disorders such as PD, schizophrenia, or attention deficit hyperactivity disorder are primarily characterized by disturbances of dopaminergic signaling within the central nervous system, accumulating evidence indicates that alterations in central dopamine pathways may also be associated with peripheral immune mechanisms [22–24]. Moreover, many patients with these conditions receive dopaminergic and antidopaminergic therapeutics, which can act not only on neuronal receptors but also on DRs expressed by immune cells, highlighting that the interactions investigated in this study may also be clinically relevant in these patients. Several studies indicate that dopaminergic stimulation of peripheral immune cells can modulate key pro-inflammatory functions, including cytokine release, cell proliferation, apoptosis, antigen presentation, and cell migration [19–21]. Accordingly, the LPS-induced modulation of the dopaminergic pathway in immune cells observed in our study advances our understanding of neuroimmune interactions and may reveal new therapeutic targets in infectious or inflammatory conditions.

Our results suggest that LPS modulates the expression of dopaminergic receptors on immune cells, particularly in monocytes and NK cells. LPS induced an upregulation of specific DRs in these cell types, especially after 24 h. Previous experiments in murine RAW264.7 cells have suggested an anti-inflammatory role of dopamine in macrophages in response to LPS [33]. Whether the same mechanisms apply in humans remains unclear, but this could potentially explain the activation of the dopaminergic pathway in monocytes observed 24 h after LPS exposure. In contrast, the effects of LPS on T and B cells were weaker, with a predominantly inhibitory influence on DR expression. These findings support the notion of a cell-specific effect of LPS on dopaminergic signaling within the immune system. Interestingly, although D2-, D3-, and D4-like receptors are all coupled to inhibitory signaling, their differential regulation in response to LPS – upregulation of D3 versus transient downregulation of D2 and D4 – likely reflects receptor- and cell type-specific sensitivity and temporal dynamics, highlighting that even receptors sharing similar intracellular pathways can exhibit distinct, context-dependent responses.

Consistent with our findings, a growing body of experimental evidences supports the existence of cell type-specific responses to LPS [34]. Monocytes, NK cells, and B cells express TLR4 and other associated receptors such as LBP-binding protein (LBP) and CD14 that enable direct recognition of LPS. In contrast, T cells do not express these receptors under physiological conditions and are thus affected only indirectly [34–37]. However, it is worth noting that NK cells express only low levels of TLR4, and LPS appears to act differently on NK cells compared to monocytes [36, 37].

Previous studies have also shown that activation of the dopaminergic pathway in immune cells may vary depending on cell subtype and activation state [18, 38–40]. Therefore, the observed cell type-specific differences in dopaminergic signaling are not unexpected.

In our in vitro experiments, we observed effects that differed from those seen after in vivo LPS administration. This discrepancy suggests that LPS may also exert indirect effects on immune cells in vivo, potentially through the activation of specific inflammatory pathways and/or the sympathetic nervous system [7, 28]. The even stronger long-term effects of LPS support the hypothesis of indirect mechanisms and underscore the importance of in vivo studies, particularly in humans, to better understand the complex interplay between immune signaling pathways and organ systems.

In addition to DR expression, we also analyzed dopamine release in the blood following LPS administration. However, no significant changes in dopaminergic concentration were observed in our in vivo human model. This suggests that LPS does not directly influence dopamine release via activation of the sympathetic nervous system and that the observed effects on DR expression are not driven by altered dopamine levels. Nevertheless, since dopamine is rapidly degraded, short-term changes occurring within the first 2 h after LPS administration cannot be ruled out.

It is well established that the transcriptional responses to inflammation and hypoxia, mediated by NF-κB and HIF signaling pathways, involve substantial crosstalk [41–45]. Consistent with this, previous results from the same cohorts showed that hypoxia exposure prior to LPS administration amplified the increase in body temperature, noradrenaline secretion, and pro-inflammatory cytokine expression (i.e., IL-6 and TNF-α) compared to LPS exposure under normoxic conditions [7, 28].

Notably, experimental animal studies suggest that HIF may also regulate central dopaminergic signaling by modulating TH and dopamine synthesis [25, 26, 29]. Based on this, we investigated whether hypoxia pretreatment could influence the peripheral effects of LPS observed in the current study. However, our results indicate that hypoxic pretreatment did not alter LPS-induced changes in the dopaminergic pathway in healthy men. Moreover, hypoxia alone did not affect DR expression in immune cells after 4 h (data not shown), further supporting the conclusion that hypoxia does not modulate the dopaminergic pathway in human peripheral immune cells.

However, due to the use of relatively mild hypoxic conditions in our experimental setup, we cannot rule out the possibility that more severe hypoxia may exert effects on the dopaminergic system of peripheral immune cells. Moreover, HIF-dependent effects on TH and dopamine expression reported by others were observed in neuronal cells, which are among the most sensitive to hypoxia and may be more responsive than peripheral immune cells [46].

To date, it remains unclear whether the upregulation of DR expression on NK cells and monocytes following LPS has a pro- or anti-inflammatory effect. Functional analysis will require additional samples to address this question. Furthermore, it would be highly interesting to compare the effects observed in male subjects with those in a female cohort. Indeed, we have previously demonstrated that the dopaminergic pathway exerts markedly different effects on immune cells from female and male individuals [22, 23], supporting the plausibility of sex-specific differences. However, our present results apply only to one sex, namely, males, since sex hormones and hormonal fluctuations can introduce additional variance and would require a sampling approach in a larger cohort [47, 48].

Taken together, this study demonstrates for the first time that systemic LPS administration modulates DR expression in peripheral immune cells. Our findings suggest that it is the LPS-induced immune response, rather than LPS itself, that drives changes in the dopaminergic pathway in specific immune cell subpopulations. Further research is needed to elucidate the functional relevance of these findings in clinical contexts, particularly in patients receiving dopaminergic treatments or those affected by disorders involving the dopaminergic system. These insights may ultimately contribute to the development of novel therapeutic strategies.

Acknowledgments

The authors want to thank Dr. Sina Trebing for her support in blood sampling. Additionally, we would like to thank Sebastian Wenzlaff, Alexandra Kornowski, Julia Bihorac, Jannis Möller, Jana Schäfer, and Dina Wüster for technical assistance. We also thank Marion Page and Dr. Jörg Reinders from the analytical chemistry unit of the IfaDo for analyzing plasma dopamine concentrations. Finally, we want to thank all blood donors who participated in this study.

Statement of Ethics

Participants gave written informed consent after being well informed about the study protocol, which was approved by the Local Ethics Committee for Human Investigations of the University Medicine Essen (18-8258-BO) and conducted according to the principles of the Declaration of Helsinki.

Conflict of Interest Statement

Prof. Dr. Harald Engler, Prof. Manfred Schedlowski, and Prof. Silvia Capellino were members of the journal’s Editorial Board at the time of submission. The remaining authors have no conflicts of interest to declare.

Funding Sources

This research was intramurally funded by the IfADo-Leibniz Research Centre for Working Environment and Human Factors. Additionally, this work was partly funded by center grants of the German Research Foundation (Deutsche Forschungsgemeinschaft [DFG]), Project No. 316803389-SFB 1280 (TP A18 to Manfred Schedlowski). The funder had no role in the design, data collection, data analysis, and reporting of this study.

Author Contributions

Conceptualization: S.C., M.S., J.F., B.T., L.F., and T.H.-G.; methodology: L.F., G.B., M.J., T.M.S., T.H.-G., H.E., S.C., and A.L.F.; formal analysis: L.F. and G.B.; data curation: L.F., G.B., and S.C.; writing – original draft preparation: S.C., L.F., G.B., M.J., and M.S.; and writing – review and editing: S.C., L.F., G.B., M.J., M.S., and J.F. All authors have read and agreed to the published version of the manuscript.

Funding Statement

This research was intramurally funded by the IfADo-Leibniz Research Centre for Working Environment and Human Factors. Additionally, this work was partly funded by center grants of the German Research Foundation (Deutsche Forschungsgemeinschaft [DFG]), Project No. 316803389-SFB 1280 (TP A18 to Manfred Schedlowski). The funder had no role in the design, data collection, data analysis, and reporting of this study.

Data Availability Statement

The datasets supporting the conclusions of this article are included within the article and its additional file(s). Upon reasonable request, the corresponding author will provide access to the data used and/or analyzed in this study. Further inquiries can be directed to the corresponding author.