Methamphetamine induces a low dopamine transporter expressing immunophenotype without altering total number of peripheral immune cells

Abstract

Methamphetamine is a widely abused psychostimulant and one of the main targets of dopamine transporter (DAT). Methamphetamine reduces DAT-mediated dopamine uptake and stimulates dopamine efflux leading to increased synaptic dopamine levels many folds above baseline. Methamphetamine also targets DAT-expressing peripheral immune cells, reduces wound healing, and increases infection susceptibility. Peripheral immune cells such as myeloid cells, B-cells and T-cells express DAT. DAT activity on monocytes and macrophages exhibits immune suppressive properties via an autocrine paracrine mechanism, where deletion or inhibition of DAT activity increases inflammatory responses. In this study utilizing a mouse model of daily single dose of methamphetamine administration, we investigated the impact of the drug on DAT expression in peripheral immune cells. We found in methamphetamine-treated mice, DAT expression was downregulated in most of the innate and adaptive immune cells. Methamphetamine did not increase or decrease total number of innate and adaptive immune cells but changed their immunophenotype to low-DAT expressing phenotype. Moreover, serum cytokine distributions were altered in methamphetamine-treated mice. Therefore, resembling its effect in the CNS, in the periphery, methamphetamine regulates DAT expression on peripheral immune cell subsets, potentially describing methamphetamine regulation of peripheral immunity.

1. INTRODUCTION

Amphetamines, such as methamphetamine (METH), are dopamine transporter (DAT) substrates [1–3]. METH competes with dopamine uptake and induces DAT-mediated dopamine efflux increasing synaptic dopamine levels more than many folds above baseline [1, 4]. While there is a wealth of evidence addressing the behavioral and cognitive impairments following METH abuse, METH also affects peripheral immunity, wound healing, and increases susceptibility to acquisition of infectious diseases [5–7]. METH users frequently experience formication, a sensation that feels like insects crawling on or beneath the skin. As a result of formication, METH users engage in persistent skin picking, resulting in the creation of ulcers that are associated with reduced immune responses, and highly susceptible to infections [4, 8, 9].

Dopamine signaling in the brain impacts peripheral immune responses, linking reward processing to peripheral immunity [10, 11]. The activation of ventral tegmental area dopamine neurons regulates innate and adaptive immunity [10, 12]. While it is unclear which dopaminergic projections are crucial for the CNS-to-periphery circuit, we have shown that altered CNS dopamine levels affect DAT expressing peripheral immunity [11]. Importantly, functional DAT molecules are expressed at the plasma membrane of peripheral monocytes, T-cell, B-cells, and macrophages. DAT activity on human macrophages, via an autocrine/paracrine signaling loop, regulates macrophages’ response to immune stimulation [13]. Lymphocytes and monocytes express catecholamine biosynthetic enzyme tyrosine hydroxylase, but not dopamine beta hydroxylase [14–16], indicating peripheral immune cells produce dopamine [15, 17–19], store the synthesized dopamine in VMAT2 positive vesicles [20], release and uptake dopamine via DAT [11, 13]. Thus, the activity of DAT expressing peripheral immune cells and their phenotype are regulated by drugs targeting dopamine transmission. Therefore, METH modulation of peripheral immunity may be due to 1) METH regulation of dopamine signaling in the brain which has shown to impact peripheral immune responses[10, 12] and/or 2) the direct effects of METH on peripheral immune cells.

METH not only regulates DAT activity, it also downregulates DAT expression [4, 21–25]. DAT activity on monocytes and macrophages exhibits immune suppressive properties, where deletion or inhibition of DAT activity on peripheral immune cells increases inflammatory responses [11, 13]. In the current study, we used a mouse model of a daily single dose of METH administration and investigated the impact of the drug on DAT expression on peripheral immune cells. We also determined the effect of METH on pro- and anti-inflammatory cytokine production. We hypothesized that METH does not alter total innate and adaptive cell populations but changes their immune phenotype in blood circulation. We found that except for Ly6C+ cells or inflammatory monocytes, METH does not affect CD11b+ (myeloid), Ly6G+ (neutrophils) or T and B lymphocyte total populations. Whereas DAT expression was downregulated in many of the innate and adaptive immune cells in METH-treated mice. Moreover, serum cytokine distributions were altered in METH-treated mice. Thus, investigation of DAT activity on peripheral immune cells may help to explain METH reduction of wound healing, susceptibility to microbial infections, behavioral responses, and the immunomodulatory role for DAT in the peripheral immune system.

2. MATERIALS AND METHODS

The study was conducted in accordance with the Basic & Clinical Pharmacology & Toxicology policy for experimental and clinical studies[26].

2.1. Animals

All animal studies were conducted according to the experimental practices and standards approved by the Institutional Animal Care and Use Committee (IACUC) at the University of Florida. A single dose of 2 mg/kg of METH (Sigma Aldrige) were intraperitoneally (i.p.) administered daily to male and female C57BL/6 mice (age, 12-16 weeks; Envigo) over 7 days. Sterile saline-treated animals were used as controls. Food and water were available ad libitum for the mice in their home cage. The room was maintained under standard 12 h light/dark cycles, at 22-24°C with 50-60% humidity.

2.2. Blood collection and peripheral blood mononuclear cell (PBMC) isolation

Whole blood was obtained from isoflurane anesthetized mice via cardiac puncture. Up to 1 mL of whole blood was collected using a 1 mL syringe with a 25-gauge needle, transferred to K2EDTA vacutainer tubes and kept on ice for 30 min prior to PBMC isolation. Whole blood was transferred into a 5 mL flow cytometry tube containing 1 mL of PBS (1:1 dilution in PBS) then overlaid atop 1 mL of Ficoll-Paque Plus (GE) in 5 mL flow cytometry tubes. Overlaid blood samples were centrifuged for 20 min at 400g with brakes off and acceleration set to minimum. PBMCs collected from the interphase of Ficoll and PBS were transferred to a fresh 5 mL flow cytometry tube (Table 1), resuspended with 4 mL PBS, and centrifuged for 10 min at 100g twice. Then, cells were resuspended in 200 μL of PBS and used in the experiment.

Table 1.

Reagents and materials used in this study.

Reagent	Supplier	Catalog #	Purpose	Concentration
Ficoll-Paque Plus	GE	45-001-750	PBMC isolation	N/A
PBS	In house	N/A	PBMC isolation, FC	1x
K2EDTA Vacutainer	BD	366643	Blood collection	N/A
FACS tubes	Fisher		FC, mouse PBMC isolation	N/A
Fix/Perm Kit	eBioscience	88-8824-00	FC	Stock
Syringe	Exel	26016	IP injection, cardiac puncture blood draw	N/A
Isoflurane	Patterson	07-893-8441	Anesthesia	1-5%
Legendplex	Biolegend	740150	Cytokine Analysis	N/A

2.3. Flow cytometry

Antibody concentrations, vendor, catalog numbers, and fluorochromes are shown in Table 1. Reagent details are shown in Table 2. For flow cytometry staining, PBMCs were isolated from blood from six mice treated with saline or METH as described above; the cells were washed and then stained with fluorescence-labeled antibodies against CD11b, CD45, CD19, CD27, CD3, CD4, CD8, Ly6C, and Ly6G for 30 min on ice and protected from light. All samples included NIR viability dye to allow exclusion of dead cells from analysis.

Table 2.

Antibodies used in this study.

Specificity	Clone/Species	Conjugate	Vendor	Catalog Number	Purpose	Dilution
CD11b	M170/Rat	PerCP-Cy5.5	Biolegend	101228	FC	1:100
CD45	30-F11/Mouse	FITC	Biolegend	334824	FC	1:200
CD19	6D5/Rat	BV605	Biolegend		FC	1:100
CD27	LG.3A10/Mouse	APC	Biolegend	124212	FC	1:100
CD3	17A2/Mouse	PacBlue	Biolegend	100214	FC	1:50
CD4	GK1.5/Mouse	AF700	Biolegend	100429	FC	1:50
CD8a	QA17A07/Mouse	SV538	Biolegend	155020	FC	1:50
Ly6C	HK1.4/Rat	BV785	Biolegend	128041	FC	1:200
Ly6G	1A8/Rat	PE	Biolegend	127607	FC	1:100
CD11b	M1-70/Rat	FITC	Biolegend	101206	FC	1:100
F4/80	BM8/Rat	AF700	Biolegend	123129	FC	1:100
MHC-II	M5-114.15.2/Rat	APC-Cy7	Biolegend	107602	FC	1:100
Zombie NIR	N/A	N/A	Biolegend	423105	FC	1:1000
TH	poly/chicken	AF647	Encor	CPCA-TH	FC	1:50
DAT	poly/rabbit	N/A	Sigma	AB5802	FC	1:200
Rabbit	poly/goat	BV421	BD	565014	FC	1:25

Cells were washed, stained with viability dye, washed with FACS buffer, and fixed for 20 min (eBioscience) at room temperature, protected from light. Immediately following fixation and series of washes with permeabilization buffer, and primary and secondary staining against DAT were performed. After final washes, samples were resuspended in 300μL of phosphate buffered saline (PBS). Data were immediately acquired on Cytek Aurora 5L Spectral Analyzer. Each experiment included single color compensation, followed by automatic compensation calculation. Compensation matrices were not altered thereafter. Data were analyzed using Flowjo (Treestar Software).

Samples were analyzed using gates set by fluorescence minus one. For each panel used, a separate set of samples was prepared in which a single fluorochrome was omitted per sample. After compensation, negative space created by omission of the fluorochrome was used to set positive gates. Set gates were verified using a fully stained sample.

2.4. Serum cytokine analysis

200uL of whole blood was diluted in PBS containing Li-Heparin anticoagulant and centrifuged for 5 min at 2,700g to separate serum from other blood components. Serum was collected, aliquoted, and stored with protease inhibitors a-80°C until tested. Sera were tested for IL-1 alpha, IL-1 beta, IFN-beta, IFN-gamma, CCL2, IL-6, IL-10, GM-CSF, IL-12p70, IL-23, and IL-17A by 13-plex mouse inflammation panel according to the manufacturer’s protocol (BioLegend). All samples were analyzed in duplicate and two technical replicates were performed. Data were acquired on a Beckman Coulter Cytoflex LX and analyzed using BioLegend LEGENDPlex Analysis software.

2.5. Statistical analysis

All data were subjected to statistical analysis using Prism 8.0 (Graph Pad). Analyses were conducted under blinded conditions. P-values for individual comparisons were calculated using unpaired two-tailed Studenťs t-test analysis. p values of < 0.05 were considered significant.

3. RESULTS

3.1. METH does not change total number of CD11b+ myeloid cells but reduces DAT expression and the number of inflammatory monocytes.

There is an unmet need to understand whether METH regulates DAT expression on peripheral immune cells. We tested the hypothesis that systemic METH (2 mg/kg/day for 7 days) administration induces a low DAT-expressing phenotype without altering the total number of immune cells (Fig. 1). We used flow cytometry to immune profile blood PBMCs from mice receiving systemic saline or METH for 7-days. After gating single, live cells (Fig. 1A), we first analyzed the myeloid cells in the PBMC fraction, including CD11b+ total myeloid cells, and Ly6C+ and Ly6G+ inflammatory monocytes and reactive neutrophils (Fig. 1B), respectively [27–29]. We found no change in total numbers of CD11b+ myeloid cells (Fig. 1B–C), but we found a significant decrease in inflammatory Ly6C+ monocytes in METH-treated mice (p<0.05). Although no difference was observed in Ly6G+ cells in saline or METH-treated animals, there was a reduction trend in METH-treated mice (Fig. 1B–C). These results are consistent with the interpretation that systemic METH downregulates DAT expression in CD11b+ peripheral myeloid cells but not in Ly6C+ monocytes or Ly6G+ neutrophils, suggesting that unlike its effect in the CNS, in the periphery, METH differentially regulates DAT expression on different immune cell subsets.

Fig. 1. Methamphetamine (METH) does not change total number of CD11b+ myeloid cells, but induces an immunophenotype shift to low-dopamine transporter (DAT) expression, and reduces the number of inflammatory monocytes.

C57BL/6 mice were injected daily with 2 mg/kg of METH for 7 days. Blood samples were collected 24 h after the last treatment. A) Single live cells were isolated from blood and gated for expression of the pan-leukocyte marker CD45, B) then analyzed for the expression of the myeloid cell markers CD11b, and myeloid subsets Ly6C and Ly6G in mice treated with saline (top row) or METH (bottom row). C) Percentage of CD11b+, Ly6C+, and Ly6G+ cells in blood of saline- and METH-injected mice. In METH-treated mice, inflammatory Ly6C+ myeloid cells are significantly reduced, while total myeloid cells identified by CD11b, and reactive neutrophils identified by Ly6G are not significantly altered. By contrast, D) when assessed for DAT expression, CD11b+/DAT+ total myeloid cells are significantly reduced in METH-vs saline-treated mice, while Ly6C+/DAT+ inflammatory monocytes nor Ly6G+/DAT+ reactive neutrophils are not affected. E) Percentage of CD11b+, Ly6C+, and Ly6G+ and DAT+ myeloid cells. For C and E, bars represent the means for saline- (grey; n = 9) and METH-treated (pink; n = 4) mice, and error bars indicate standard error (SEM). Asterisks denote p value significance (*, p< 0.05) calculated using two-tailed student’s t-test.

3.2. Systemic METH induces an immunophenotype shift to low-DAT expressing CD4+ and CD8+ T-cells but no change in total numbers of T-cells.

Multiple reports have shown T-cells in various tissues are adversely impacted following METH exposure [30, 31], by dysregulating T-cells’ classical functions such as proliferation, cytokine secretion, tissue infiltration and cytotoxicity [31–35]. In addition, in individuals with HIV, METH increases viral adhesion, replication and disease progression, leading to persistent reductions in T-cell populations [31, 36]. Hence, we tested whether DAT expression is reduced in T-cell subsets following methamphetamine treatment focusing on DAT-expressing CD3+, CD4+ and CD8+ T-cells (Fig. 2). Single, live cells were gated for pan-leukocyte marker CD45 (Fig. 2A), then analyzed for expression of pan-T-cell marker CD3. Cells expressing CD3 were gated and analyzed for CD4 and CD8 expression (Fig. 2B). METH- and saline-treated mice show similar percentage of CD3+, CD4+, and CD8+ cells (Fig. 2C), suggesting that METH does not alter total numbers of T-cells in blood circulation. We then, documented the impact of METH on DAT-expressing CD3+, CD4+, and CD8+ cells (Fig. 2D). CD4+ (p<0.05) and CD8+ (p<0.05) T cells, in blood collected from METH-treated animals, demonstrated a significant decrease in DAT expression (Fig. 2E), whereas CD3+ T cells remained unchanged. These findings suggest that METH reduces DAT expression on a subset of T-cells in the periphery, potentially affecting the adaptive immunity’s responses to infection and wound healing.

Fig. 2. Systemic METH induces an immunophenotype shift to low-DAT expressing CD4+ and CD8+ T-cells without altering the total numbers of T-cells.

C57BL/6 mice were injected daily with 2 mg/kg of METH for 7 days. Blood samples were collected 24 h after the last treatment. A) Single, blood live cells were gated for CD45 expression, then B) analyzed for expression of T-cell markers CD3, CD4 and CD8 in mice treated with saline (top) or METH (bottom). C) Percentage of CD3+, CD4+, and CD8+ cells in blood of saline- and METH-injected mice. While no differences are evident in saline vs METH in terms of total CD3, CD4 or CD8 expressing T-cells, D) analysis for expression of DAT in these subsets indicates E) that both CD4+ and CD8+ T-cells expressing DAT are significantly reduced in METH-treated mice, with no change in DAT+ CD3+ T-cells, suggesting that T-lymphocytes shifted into a low-DAT-expressing immunophenotype after METH treatment. For C and E, bars represent the means for saline- (grey; n = 9) and METH-treated (pink; n = 4) mice, and error bars indicate SEM. Asterisks denote p value significance (*, p< 0.05) calculated using two-tailed student’s t-test.

3.3. METH reduces B cell DAT expression that may impact humoral responses.

B cells are important for adaptive immunity especially for humoral or antibody-based responses and antigen presentation. METH alters B cell tissue infiltration and antibody production suggesting a possible detrimental impact on humoral immunity to infection or autoimmunity [36–38]. Thus, we evaluated whether METH compromises B cell and B memory cell populations as well as their DAT expression (Fig. 3). We first gated for CD45+ cells (Fig. 3A), followed by CD19+ cells (naïve B cells) and CD19+/CD27+ (memory B cells) (Fig. 3B). Both, saline and METH-treated mice had similar number of CD19+ and CD19+/CD27+ cells in the blood stream (Fig. 3C). However, the expression of DAT in naïve (p<0.05) and memory (p<0.01) B cells from METH-treated mice was significantly higher than in the saline control group (Fig. 3D–E). These data show that B cells are not altered by METH, although DAT expression on B cells is significantly reduced, suggesting notable implications for host defense against pathogens following METH exposure.

Fig. 3. B-cells show an immunophenotype shift to low-DAT expression after METH treatment for 7 days.

A) Live, single blood cells were gated for CD45 expression, then B) assessed for expression of CD19, a marker of naïve total B-cells, and CD27, a marker of memory B-cells, in saline- and METH-treated mice. C) Percentage of CD19+ and CD19+/CD27+ cells in blood of saline- and METH-injected mice. METH treatment did not affect total numbers of naïve CD19+ B-cells nor CD19+/CD27+ memory B-cells. D) However, when assessed for expression of DAT, we observed a significant reduction in DAT+ B-cells of both subsets (naïve and memory), again indicating that METH treatment induces an immunophenotype shift of B-cells towards low-DAT expression, without affecting total numbers of B-cells. E) Percentage of CD19+/DAT+ and CD19+/CD27+/DAT+ lymphocytes. For C and E, bars represent the means for saline- (grey; n = 9) and METH-treated (pink; n = 4) mice, and error bars indicate SEM. Asterisks denote p value significance (*, p< 0.05) calculated using two-tailed student’s t-test.

3.4. METH differentially regulates cytokine levels in blood.

Cytokines are involved in regulation of innate and adaptive immune cell communications in blood and tissue [39–41]. Since we found METH reduces inflammatory monocytes (Ly6C+) without altering other myeloid cells (CD11b+), neutrophils (Ly6G+), or lymphocytes (T and B cells), we assessed the role of METH on pro- and anti-inflammatory cytokine expression in blood of saline- or METH-treated mice (Fig. 4). We found, IL-1 beta (p<0.01), IFN-gamma (p<0.001), and IL-23 (p<0.05) were significantly reduced in serum of METH-treated mice relative to saline-treated mice. In contrast, CCL2, IL-6, IL-10, and IL-12p70 were significantly higher in METH-treated mice than in the saline-treated mice (p<0.05 for each mediator). The levels of IL-1 alpha, IFN-beta, GM-CSF, and IL-17A were not different in METH vs. saline treated mice. These results suggest that METH modifies pro- and anti-inflammatory cytokines and reduced immune cells’ DAT expression may modulate immunity against infection, wound healing, or autoimmunity. Since METH increases blood brain barrier permeability [42], METH-regulation of DAT expression on peripheral immune cells may increase the CNS penetrance of immune cells. Therefore, the CNS landscape, peripheral immunity, and intervening factors such as blood-brain-barrier integrity should be considered when investigating METH-induced behavioral and neurochemical responses.

Fig. 4. METH differentially regulates serum cytokine levels, impairing both inflammatory immune signals and innate-to-adaptive immune crosstalk.

The serum from saline (n = 6)- and METH (n = 5)-treated mice for 7 days were processed and analyzed for (A) IL-1 alpha, (B) IL-1 beta, (C) IFN-beta, (D) IFN-gamma, (E) CCL2, (F) IL-6, (G) IL-10, (H) GM-CSF, (I) IL-12p70, (J) IL-23, and (K) IL-17A levels with the BioLegend 13-plex mouse inflammation panel. Bars represent the mean values; error bars indicate SEM. Asterisks indicate p value significance (*, p<0.05; **, p<0.01; ***, p<0.001) calculated using two-tailed student’s t-test.

4. DISCUSSION

METH is a DAT substrate and a potent psychostimulant that increases CNS dopamine levels many folds above baseline, induces erratic and risky behaviors, increasing the susceptibility to acquiring infectious diseases such as AIDS, hepatitis B or C, and/or skin infections. METH has formidable effects on peripheral immunity [34, 35, 43]. For instance, METH facilitates HIV replication and induces transcriptional changes in monocytes, increases the susceptibility to recurrent skin infections, and alters the antimicrobial efficacy of phagocytic cells in murine cutaneous wounds [9, 44–46]. In DAT expressing cells (dopamine neurons or immune cells) METH reduces dopamine uptake, induces dopamine efflux [1–4] and downregulates DAT expression [4, 21–25, 47–49]. Although peripheral immune cells express DAT [11, 13], limited data is available on METH-regulation of DAT expression on peripheral immune cells. Recently, we have shown that DAT activity on immune cells regulate the activity of immune cells via a paracrine mechanism, supporting the interpretation that immunological synapse between two different immune cells can be regulated by DAT[13]. In addition, we found that DAT-mediated dopamine efflux regulates the host cell’s dopamine receptors to influence its function, i.e., autocrine mechanism[13]. Hence, it is important to investigate the impact of METH on circulating immune cells, which are modulated by dopamine tone [16] and are required to control disseminated infections or tissue migration to combat infection. Importantly, multiple peripheral immune cell subtypes express DAT, including myeloid cells (monocytes and macrophages) and lymphocytes (B-cells and T-cells) [50]. Consistent with the literature regarding METH-mediated downregulation of CNS DAT, we found DAT expression is almost universally reduced among peripheral immune cells of METH-treated mice. In addition, METH regulates fundamental immune functions, such as impairing baseline leukocyte proliferation and cytokine production in different tissues [5]. We found that METH decreases inflammatory monocytes (Ly6C+) in blood but not myeloid cells (CD11b+) or neutrophils (Ly6G+), that are consistent with the literature supporting METH-mediated dysregulation of peripheral immunity.

As shown in Fig. 2 and 3, METH did not alter the total number of T and B cells in blood but reduced DAT expression in these immune cells, which is in contrast with studies showing that METH is detrimental to lymphocytes [5, 32, 37, 51]. This could be due to the different model and duration of METH exposure [52]. It is conceivable that METH downregulates the expression of DAT on the surface of immune cells, thus, compromising the inflammatory or adaptive immune responses. Also, it is plausible that altered dopamine peripherally stimulates inflammatory cells to migrate to the brain upon antigenic stimulation resulting in a detrimental effect on the CNS. We previously showed that combination of METH and LPS stimulate the infiltration of macrophages (F4/80+cells) and neutrophils (Ly6G+cells) into the brainstem [53]. High CCL2, IL-6, and IL-12p70 levels found in our current studies indicate that METH can orchestrate phagocytic cell chemotaxis [5, 54, 55]. As show in Fig. 4, METH decreases some pro-inflammatory cytokines such as IL1-beta and IFN-gamma but elevates other immunosuppressive cytokines. Recent data suggest that the endotoxin, LPS, does not change surface DAT expression, but it reduces dopamine uptake and increase dopamine efflux [13]. Future studies should investigate how these pro-inflammatory and immunosuppressive cytokines regulate DAT expression at the surface membrane, DAT activity in the context of METH regulation of peripheral immunity. In addition, the elevated IL-10 are consistent with the suppressive effects of METH on peripheral immune function and the inhibitory effects of METH on transcription factors critical for the expression of pro-inflammatory cytokines [7, 8, 51, 52, 56]. Importantly, IL-10 can be induced by IL-6 expression [57, 58]. IL-10 can also contribute to bone marrow retention of monocytes or reduce differentiation and generation of Ly6C+ inflammatory monocytes [59, 60]. This observation may explain the reduction of Ly6C+ in circulation in METH-treated mice. Moreover, dopamine receptor 5 in monocytes obtained from patients with multiple sclerosis showed reduction of STAT3 activation, a transcription factor that limits the production of IL-23, therefore, impairing IL-17A expression [61]. METH stimulates HIV replication and macrophage infection [44, 62]. Hence, a reduction in DAT expression in T cells, the main producers of IFN-gamma during viral infections, may explain the low levels of this cytokine in circulation, an important immunomodulator to combat viral infections [63].

Limitations of this study:

In this study, we treated the animals with a concentration of METH that has shown to produce conditioned place preferences and increased psychomotor responses. Lower or higher concertation of METH may or may not yield similar results. We also selected a repeated non-contingent METH exposure model (7 days), therefore it is possible that acute, shorter, or longer noncontingent METH exposure or a contingent METH exposure model, i.e., METH self-administration differentially regulates DAT expression on peripheral immune cells, and blood cytokine levels. The Rolls’ laboratory has shown that activation of reward pathway regulates immune responses [10, 12], thus data shown in the current study represent a combination of METH regulation of peripheral immunity and METH regulation of CNS-to-periphery communication. Lastly, although we did not detect sex differences in the measured responses, this could be due to the limited number of metrics studied in this study. It is possible that different METH exposure paradigms or employing a more comprehensive immunophenotyping approach reveal sex differences in DAT expressing peripheral immune cells following METH exposure.

Summary and broader impact:

In conclusion, the routine clinical use and extensive illicit abuse of psychostimulants underscores the broad impacts of this study. METH affects both CNS reward pathway and peripheral immunity, contributing to systemic effects of METH including wound healing and HIV infection. In addition, dysregulated peripheral immunity may contribute to a feed-forward loop that can exacerbate the neurotoxic and neurochemical effects of METH. Our data suggest that the CNS landscape, peripheral immunity, and intervening factors such as blood-brain-barrier integrity and CNS-to-peripheral immune crosstalk should be considered when investigating METH-induced behavioral and neurochemical responses. The contribution of DAT activity on peripheral immune cells in drug addiction, neurological, neuropsychiatric, and immunological diseases remains incomplete, but it is an exciting research area and a focus of future studies.

Table 3.

Equipment used in this study.

Equipment	Supplier	Part Number	Purpose
Centrifuge	Sorvall	ST8	PBMC isolation
Cytometer	BD	Canto II	FC
Spectral Analyzer	Sony	SP6800	FC
Spectral Analyzer	Cytek	Aurora 5L	FC
Flow cytometer	Beckman	Cytoflex LX	FC, 13-plex ELISA
Microcentrifuge	Fisher	59A	FC

DATA AVAILABILITY STATEMENT:

Data will be made available upon reasonable request.