Functional characterization of the biogenic amine transporters on human macrophages
Phillip M. Mackie, Adithya Gopinath, Dominic M. Montas, Alyssa Nielsen, Aidan Smith, Rachel A. Nolan, Kaitlyn Runner, Stephanie M. Matt, John McNamee, Joshua E. Riklan, Kengo Adachi, Andria Doty, Adolfo Ramirez-Zamora, Long Yan, Peter J. Gaskill, Wolfgang J. Streit, Michael S. Okun, Habibeh Khoshbouei
Phillip M. Mackie, Adithya Gopinath, Dominic M. Montas, Alyssa Nielsen, Aidan Smith, Rachel A. Nolan, Kaitlyn Runner, Stephanie M. Matt, John McNamee, Joshua E. Riklan, Kengo Adachi, Andria Doty, Adolfo Ramirez-Zamora, Long Yan, Peter J. Gaskill, Wolfgang J. Streit, Michael S. Okun, Habibeh Khoshbouei
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Research Article Inflammation Neuroscience

Functional characterization of the biogenic amine transporters on human macrophages

Abstract

Monocyte-derived macrophages (MDMs) are key players in tissue homeostasis and diseases regulated by a variety of signaling molecules. Recent literature has highlighted the ability for biogenic amines to regulate macrophage functions, but the mechanisms governing biogenic amine signaling in and around immune cells remain nebulous. In the CNS, biogenic amine transporters are regarded as the master regulators of neurotransmitter signaling. While we and others have shown that macrophages express these transporters, relatively little is known of their function in these cells. To address these knowledge gaps, we investigated the function of norepinephrine transporter (NET) and dopamine transporter (DAT) on human MDMs. We found that both NET and DAT are present and can uptake substrate from the extracellular space at baseline. Not only was DAT expressed in cultured MDMs, but it was also detected in a subset of intestinal macrophages in situ. Surprisingly, we discovered a NET-independent, DAT-mediated immunomodulatory mechanism in response to LPS. LPS induced reverse transport of dopamine through DAT, engaging an autocrine/paracrine signaling loop that regulated the macrophage response. Removing this signaling loop enhanced the proinflammatory response to LPS. Our data introduce a potential role for DAT in the regulation of innate immunity.

Authors

Phillip M. Mackie, Adithya Gopinath, Dominic M. Montas, Alyssa Nielsen, Aidan Smith, Rachel A. Nolan, Kaitlyn Runner, Stephanie M. Matt, John McNamee, Joshua E. Riklan, Kengo Adachi, Andria Doty, Adolfo Ramirez-Zamora, Long Yan, Peter J. Gaskill, Wolfgang J. Streit, Michael S. Okun, Habibeh Khoshbouei

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Figure 7

LPS-stimulation increased DAT-mediated dopamine efflux and decreased DAT–JHC1-064 binding.

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LPS-stimulation increased DAT-mediated dopamine efflux and decreased DAT...
(A) Schematic representation of experimental design using simultaneous whole-cell patch-clamp and amperometry technique to measure DAT-mediated dopamine efflux. (B) Representative amperometric traces from unstimulated and LPS-stimulated human macrophages. (C) Bar graphs show basal or amphetamine-induced DAT-mediated dopamine efflux. While unstimulated macrophages did not exhibit measurable DAT-mediated dopamine efflux at baseline, LPS-stimulation significantly increased basal DAT-mediated dopamine efflux (top, P = 0.05, Welch’s 2-tailed t test) but did not further increase or decrease the amphetamine-induced dopamine efflux (bottom, P = 0.9, Mann Whitney U test). Data are from n = 5–6 experiments/group. (D) Representative 40× images of JHC1-064 binding to unstimulated and LPS-stimulated macrophages. (E) Quantification of fluorescence signal showed that LPS significantly decreased the magnitude of JHC1-064–DAT binding to levels similar to those seen with DAT blockade (nomifensine-treated, *P < 0.05, 2-way ANOVA with Tukey’s post hoc test for multiple comparisons). (F) The average rate of JHC1-064–DAT binding (average slope from E) was similarly decreased by LPS-stimulation (P = 0.02) and by presence of DAT-specific antagonist, nomifensine (P = 0.02). Data in D–F are from n = 3–6 independent experiments/group, and statistical analysis was performed by Brown-Forsythe ANOVA with Tukey’s post hoc test for multiple comparisons. (G) Representative images of unstimulated and LPS-stimulated macrophages labeled with CTxB-555 and DAT. (H) Quantifying colocalization between GM-1 and DAT using Pearson’s correlation coefficient showed LPS-stimulated macrophages has significantly increased DAT-CTxB colocalization compared with unstimulated macrophages (P = 0.04, unpaired 2-tailed t test). Images and data in G and H are from n = 5 and 8 experiments for unstimulated and LPS-stimulated groups, respectively.