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. Author manuscript; available in PMC: 2026 Jun 22.
Published in final edited form as: Neuropharmacology. 2025 Jun 22;278:110571. doi: 10.1016/j.neuropharm.2025.110571

Blocking IL-17A inhibits methamphetamine-induced hyperlocomotion and conditioned place preference and prevents methamphetamine abstinence-induced depression-like behaviors in mice

Saadet Inan 1, Sonita Wiah 1, Alexandra Szmacinski 1, Scott Dunn 1, Joseph J Meissler 1, Ekaterina K Koltsova 3, Sergey Grivennikov 4, Scott M Rawls 1,2
PMCID: PMC12369639  NIHMSID: NIHMS2092666  PMID: 40555304

Abstract

We demonstrated previously that blocking IL-17A, a proinflammatory cytokine, prevents oxycodone-induced depression-like effects and anxiety-like effects during abstinence from MDPV (a psychostimulant) in rats. Here, we tested the hypothesis that eliminating IL-17A signaling (pharmacological antagonism using IL-17A Ab or genetic deletion of IL-17RC) would inhibit behavioral and neurochemical effects elicited by methamphetamine (METH) exposure and abstinence in adult mice. We investigated rewarding and locomotor-activating effects of METH and withdrawal-induced anxiety-and depression-like effects during METH abstinence. Mice received saline or METH (5 mg/kg, IP) once daily for 18 d. Locomotion was measured on days 1 and 15. Anxiety-and depression-like effects were investigated 72 and 96 h after the last METH injection using the elevated plus maze and forced swim test, respectively. IL-17A antibody (Ab, 60 μg/100 μl, IP) was injected every 3rd day of METH exposure. METH-induced hyperlocomotion was significantly reduced in IL-17RC knockout mice or by treatment with the IL-17A Ab (100 μg/100μl). Neutralization of IL-17A or genetic deletion of IL-17RC prevented development of depression-like effects during METH abstinence. Also, mRNA levels of IL-17RC, but not IL-17RA, in the NAC were enhanced during METH abstinence. Development of METH conditioned place preference (CPP) was prevented by IL-17A Ab but was not affected by IL-17RC deletion in mice conditioned with METH (3 mg/kg) for 4 d. Our data show that abolishing IL-17A signaling reduces METH-induced hyperlocomotion and CPP and attenuates depression-like effects during METH abstinence. These results highlight studying IL-17A blockade as a neuroimmune-based approach to mitigate METH adverse effects.

Keywords: methamphetamine, IL-17A, IL-17RC, methamphetamine use disorder, mice

Graphical Abstract

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1. Introduction

IL-17A is a cytokine that binds to IL-17RA/RC and IL-17RA/RD receptor complexes (Shalom-Barak et al., 1998; Kuestner et al., 2007; Gaffen, 2009; Țiburcă et al., 2022; Douglas et al., 2023). Involvement of IL-17A in the pathogenesis of various inflammatory and autoimmune disorders has led to the development of therapeutic strategies blocking IL-17A signaling (Onishi and Gaffen, 2010; Chenuet et al., 2017; Adams et a., 2020). A growing body of literature also indicates that IL-17A contributes to the pathogenesis of anxiety and depression (Vieira et al., 2010; Liu et al., 2012; Pallavi et al., 2015; Li et al., 2019; Alves de Lima et al., 2020; Kim et al., 2021). Both anxiety and depression are common in patients with methamphetamine (METH) use disorder, especially during the METH abstinence period (Blanchard and Blanchard, 1999; Vorspan et al., 2015). Recent evidence also suggests that chronic METH exposure dysregulates IL-17A signaling in the brain and periphery. In human METH users, IL-17A is increased in saliva samples (Deng et al., 2022). In mice, repeated METH administration increases IL-17A levels in the prefrontal cortex (PFC) (Potula et al., 2023). Given that anxiogenic-like effects are detected in rats 48–72 h after cessation of chronic administration of cocaine or MDPV (a cathinone psychostimulant) (Harris et al., 1993; Basso et al., 1999; Philogene-Khalid et al., 2017), we previously investigated a role for IL-17A in MDPV abstinence-induced anxiety-like effects in rats. We detected an increase in IL-17A levels in the ventral tegmental area (VTA) of rats during the abstinence period following binge administration of MDPV (Inan et al., 2023). We also showed that treatment with the flavonoid cyanidin, a non-specific IL-17RA antagonist, prevents MDPV withdrawal-induced anxiety-like effects and normalizes increased cytokine levels in the VTA. Furthermore, in rats treated with chronic oxycodone, blocking IL-17A signaling with a monoclonal Ab inhibits oxycodone-induced depression-like effects and normalizes oxycodone-induced enhancement of IL-17A and IL-6 in the VTA (Inan et al., 2024). Taken together, these studies suggest involvement of IL-17A signaling in anxiety- and depression-like effects that can accompany psychostimulant exposure and abstinence.

In the present study, we investigated a role for IL-17A in METH-induced behavioral effects in mice using endpoints of locomotor activity, conditioned place preference (CPP), and abstinence-induced anxiogenic- and/or depression-like effects. To block IL-17A, both genetic and pharmacological approaches were utilized. Since IL-17A binds to IL-17RA/RC heterodimer receptor complex, IL-17RC KO and age matched WT mice were used for genetic inhibition of IL-17A signaling. For pharmacological inhibition, a monoclonal IL-17A Ab was given systemically every 3rd day throughout the studies. IL-17RC and IL-17RA mRNA expression levels were also investigated in reward regions of the brain.

2. Material and Methods

2.1. Animals

Adult (3–4 months-old) male and female C57BL/6 (Harlan Laboratories, Indianapolis, IN) and male and female IL-17RC knock out (KO) and their wild type (WT) littermates (C57BL/6 background) were used. IL17RC “floxed” mice were generated by Grivennikov and Koltsova labs as described previously (Lee et al., 2025). Briefly, C57Bl6 Agouti ES cell line C57BL/6N-A/a JM8A3.N1; allele Il17rctm1a(KOMP) Wtsi was generated obtained from EUCOMM/EuMMCR (IKMC project 27077) and microinjected into C57Bl6 Albino blastocysts and germline transmission was achieved. Deletion of neo cassette and LacZ reporter was performed in vivo by crossing the germline transmitted mice with β-actin FLP deleter (Jackson Laboratories, stock#003800). IL17RC loxP (“floxed “) strain was crossed to ubiquitous Cre-deleter to delete exon 6 and produce IL17RC null mice. Mice were routinely genotyped for floxed, wild type or deleted alleles by PCR.

Animals were housed in a humidity-controlled vivarium, maintained on a 12-h light/dark cycle with lights turning on daily at 7:00 AM. Mice were provided with ad libitum food and water access. All procedures and animal care were conducted in accordance with the National Research Council, Guide for the Care and Use of Laboratory Animals (2011) and Use of Laboratory Animals and Temple University’s Institutional Animal Care and Use Committee guidelines. Mice were allowed to acclimate for a week before starting procedures.

2.2. Compounds

Methamphetamine hydrochloride (METH) was purchased from Sigma-Aldrich (St. Louis, MO) and dissolved in sterile saline. Anti-mouse IL-17A antibody (Ab, clone 17F3) was obtained from Bio X Cell (West Lebanon, NH, USA) and diluted in sterile saline. Since specificity of Ab was confirmed in previous reports (Abt et al., 2015; Sugimura et al., 2019; Delgado-Ramirez et al., 2021) and multiple dilution with saline was performed, we used saline vehicle as control. Dose and frequency of administration of IL-17A Ab were determined based on previous studies from our laboratory and others that used intraperitoneal (IP) delivery for IL-17A Ab (Liang et al., 2018; Sugimura et al., 2019; Jung et al., 2022; Inan et al., 2024).

2.3. Experimental design

Although the present study did not aim to study sex differences, we used both male and female IL-17RC KO mice and their WT littermates (based on availability). Since the numbers of male and female mice were not always equivalent, the strategy of pooling both sexes is more representative of IL-17A effects versus using only one sex. Since mixed sexes were used in IL- the 17RC KO studies, both male and female mice were also used in pharmacological antagonist studies.

Time schedules for treatment and behavioral studies for METH withdrawal are summarized for IL-17RC KO and WT (Panel A) and IL-17A Ab treatment in WT mice (Panel B) in Fig. 1. Behavioral testing was performed between 10:00 AM and 1:00 PM. At the end of the experiments, mice were euthanized (carbon dioxide inhalation followed by cervical dislocation), and brain samples were collected.

Fig. 1. Experimental design. (Panel A) IL-17 RC KO:

Fig. 1.

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Adult IL-17 RC KO and aged-matched C57BLJ-6 mice (female and male) were used. Mice were injected with saline or METH (5 mg/kg) once daily for 18 days. Seventy-two and 96 h after the last injection, elevated plus maze (EPM) for anxiety-like behavior measurement and forced-swim test (FST) for depression-like effects were performed, respectively. (Panel B) IL-17A Antibody (Ab): Mice were injected with saline or METH one daily for 18 days. Mice were injected every 3rd day with vehicle or IL-17A Ab (60 ug/100 μl, IP) throughout the 22-d study. EPM was performed on day 21, and FST experiments were conducted on day 22.

Design for IL-17RC KO mice and WT littermates:

KO and WT mice were injected with saline or METH (5 mg/kg, IP) once daily for 18 d. KO and WT mice were grouped randomly as: KO mice receive saline; KO mice receive METH; WT mice receive saline; and WT mice receive METH. On days 1 and 15, locomotor activity was measured. Seventy-two h and 96 h after the last injection of METH or saline, testing on the elevated plus maze (EPM) and forced swim test (FST) were performed, respectively. Separate groups of mice were used for conditioned place preference (CPP) experiments. For these experiments, groups were KO mice treated with saline; KO mice treated with METH (3 mg/kg); WT mice treated with saline; and WT mice treated with METH (3 mg/kg). The METH dose for CPP experiments was chosen based on our previous study conducted in mice (Potula et al., 2023).

Design for IL-17A Ab treatment in WT mice:

METH (5 mg/kg, IP) or saline was administered once a day for 18 d. IL-17A Ab (60 μg/100 μl, IP) or saline was given every 3rd d starting 24 h before the first METH or saline injections. Treatment with IL-17A Ab or saline was continued until the end of all behavioral studies. Mice were randomly divided into 4 groups: saline (every 3rd day)-saline (daily); IL-17A Ab (every 3rd day)-saline (daily); saline (every 3rd day)-METH (daily); and IL-17A Ab (every 3rd day)-METH (daily). The EPM and FST experiments were conducted at 72 and 96 h, respectively, after the last injection of METH or saline. Separate groups of animals were studied for CPP experiments. Groups for these studies were saline-saline; saline-METH (3 mg/kg); IL-17A Ab-saline; and IL-17A Ab-METH.

2.3.1. Measurement of locomotor activity

Locomotor activity was measured as previously described using a Digiscan DMicro system (Accuscan Inc., Columbus, OH) (Nayak et al., 2020; Inan et al., 2023; Potula et al., 2023). Activity was measured following one-time administration of saline or METH on day 1, as well as after chronic administration of saline or METH on day 15. On the day of the experiment, mice were placed individually in the activity chamber and acclimated for 60 min. Animals were then injected with either saline or METH (5 mg/kg, IP), and locomotion was measured for 120 min. For IL-17A Ab studies, the first dose of IL-17A Ab was administered 24 h before the first METH injection. Then, Ab was injected every 3rd d through the study.

2.3.2. Determination of METH withdrawal-induced anxiety-like effects

Anxiety-like effects were measured 72 h after the last METH or saline injection using the elevated plus maze (EPM) (Holmes et al., 2000; Komada et al., 2008). Mice were acclimated for 60 min. The illumination level was maintained at 100 lux at the open arms. During testing, a mouse was placed in the center area of the maze with its head directed toward an open arm and was allowed to move freely for 10 min. Testing was recorded using a video camera. The numbers of open and closed arm entries and time spent in open and closed arms were measured. An entry to open or closed arms was defined as entrance of 2 forepaws and head of the mouse.

2.3.3. Determination of METH withdrawal-induced depression-like effects

Depressive-like effects were measured 96 h after the last METH or saline injection using the forced swim test (Can et al., 2012; Yankelevitch-Yahav et al., 2015). Mice were transported to the behavioral room and acclimated for 60 min. Transparent cylindrical glass containers were filled with water at 23–25 °C. Water was changed for each animal. Testing was recorded using a video camera. Mice were left in the water for 6 min. The first 2 min was designated as acclimation and 4 min was designated as the testing period. The time each mouse spent mobile was measured and, to calculate immobility time, was subtracted from 240 s.

2.3.4. Conditioned place preference (CPP) studies

CPP experiments were conducted as described previously (Hicks et al., 2017; Potula et al., 2023). In an initial 30-min pre-test on day 1, the compartment in which a mouse spent less time was determined and designated as the METH-paired side. A 4-d (days 2–5) conditioning paradigm was then initiated. Two sessions of 4 h apart were performed each conditioning day. In the morning session, mice were injected with METH (3 mg/kg) and placed in the METH-paired compartment for 30 min. In the afternoon session, mice were injected with saline and placed in the opposite compartment for 30 min. Mice assigned for saline control received saline both in the morning and afternoon sessions for both KO and IL-17A Ab studies. One day after the conditioning phase, mice were placed into behavioral chambers and allowed access to both compartments for 30 min. A difference score was calculated as the time spent in the METH-paired compartment during the post-test minus the time spent in METH-paired compartment during the pre-test.

For studies on IL-17A Ab pretreatment, as described above, an initial 30-min pre-test was conducted, and the least preferred side was designated as the METH-paired side. Mice received IL-17A Ab or saline following pre-test (day 1). On the 3rd day of conditioning, another dose of IL-17A Ab or saline was administered 30 min before the injection of METH or saline during the morning session. On day 6, a post-test was conducted. Mice were placed into the chamber, and given access to both compartments for 30 min. The time spent in dark and light compartments was measured as in pretest, and a difference score was calculated as described above.

2.3.5. Measurement of mRNA expression levels

Gene expression experiments were conducted on brain tissue harvested from mice following FST experiments, 96 h after the last METH injection as previously described (Nayak et al., 2020; Walters et al., 2024). The NAC, dorsal striatum, PFC, VTA and amygdala were dissected from frozen slices using 1- or 2-mm round punches. RNA was isolated using the Quick-RNA Miniprep kit (Zymo Research, Irvine, CA, USA), and cDNA was synthesized using RT^2 First Strand Kit (Qiagen, Germantown, MD, USA). Quantitative real-time PCR was carried out with TaqMan Fast Advanced Master Mix and the TaqMan Gene Expression Assays for IL-17RC (Mm00506606_m1), IL-RA (Mm00439618_m1), IL-17A (Mm00439618_m1), Drd1( D1, dopamine receptor D1) (Mm01353211_m1), Drd2 (D2) (Mm00438545_m1), Slc6a3 (DAT, dopamine transporter) (Mm00438388_m1), and Th (TH, tyrosine hydroxylase) (Mm00447557_m1) using the Quant Studio 3 Real-Time PCR System (Applied Biosystems). The internal control gene was Rn18s (housekeeping gene, Mm03928990_g1). All primers were purchased from ThermoFisher Scientific (Philadelphia, PA). Fold-change values were calculated using the 2 ΔΔCTmethod as described (Nayak et al., 2020; Walters et al., 2024).

2.3.6. Statistical analysis

Data are presented as Mean ± Standard Error of Mean (S.E.M.). Two-way ANOVA or three-way ANOVA followed by Tukey’s or Sidak’s as well as Neuman Keuls multiple comparison tests were used to analyze data. For IL-17RC KO and WT groups, sex was not considered since only a small number of mice were female. Graph Pad Prism version 10.1.0 was used for statistical analysis and generating graphs. p <0.05 was considered statistically significant in all cases.

3. Results

3.1. Effects of IL-7RC genetic deletion or IL-17A Ab on METH-induced hyperlocomotion

3.1.1. IL-17RC KO vs WT

Results from the IL-17RC KO and WT mice are shown in Fig. 2A-F. Top and bottom panels show horizontal, ambulatory, and stereotypic activity following METH injection on days 1 (A,B,C) and 15 (D,E,F), respectively. For day 1 horizontal activity, three-way ANOVA (treatment x genotype x time) revealed significant effects of treatment [F(1,600) =1091, p <0.0001)], genotype [F(1,600) =27.21, p <0;0001)], and time [F(24,600) =4.42, p <0.0001)], as well as a significant interaction between treatment and genotype [F(1,600) =70.51, p <0.0001)]. For ambulatory activity, there were significant effects of treatment [F(1,600) =954.3, p <0.0001)], genotype [F(1,600) =23.37, p <0;0001)], and time [F(24,600) =4.38, p <0.0001)]. There was also a significant interaction between treatment and genotype [F(1,600) =71.06, p <0.0001)]. For stereotypic activity, there was no significant effect of time or genotype, but there was a significant effect of treatment [F(1,600) =1005, p <0.0001)] and a significant interaction between treatment and genotype [F(1,600) =21.79, p =0.009)]. Tukey’s post hoc test revealed a significant difference between IL-17 RC KO and WT mice in response to METH (significance in different time points are shown in Figs. 2A-C, **p<0.01,***p<0.001).

Fig. 2. IL-17 RC genetic deletion reduced METH hyperlocomotion.

Fig. 2.

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Mice (WT and IL-17 RC KO) were injected with saline or METH (5 mg/kg) every other day. Locomotor experiments were conducted on days 1 and 15. Data are presented as horizontal (A, D), ambulatory (B, E), and stereotypical (C, F) activities. N=8–10 mice/group. *p<0.05, **p<0.01, ***p<0.001, or ****p<0.0001 compared to METH (WT).

Day 15 locomotor results after METH administration are shown in Figs. 2D-F. For horizontal activity, three-way ANOVA (treatment x genotype x time) revealed significant effects of treatment [F(1,600) =843, p <0.0001)], genotype [F(1,600) =68.47, p <0;0001)], and time [F(24,600) =5.59, p <0.0001)]. There was also a significant interaction between treatment and genotype [F(1,600) =113.3, p <0.0001)] and a significant interaction among time, genotype, and treatment [F(24,600) =1.58, p =0.035)]. For ambulatory activity, three-way ANOVA (treatment x genotype x time) revealed significant effects of treatment [F(1,600) =743.7, p <0.0001)], genotype [F(1,600) =69.13, p <0;0001)], and time [F(24,600) =5.18, p <0.0001)]. There was also a significant interaction between treatment and genotype [F(1,600) =123.5, p <0.0001)]. For stereotypic activity, three-way ANOVA (treatment x genotype x time) revealed significant effects of treatment [F(1,600) =571.2, p <0.000.1)], genotype [F(1,600) =15.06, p <0.0001)], and time [F(24,600) =3.28, p <0.0001)]. There was also a significant interaction between treatment and genotype [F(1,600) =3.74, p =0.07)]. Tukey’s post hoc test revealed a significant difference between IL-17 RC KO and WT mice in response to METH (significance in different time points are shown in Figs. 2D-F, *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001). These results indicate that METH-elicited hyperlocomotion is reduced by genetic elimination of IL-17RC.

3.1.2. Vehicle vs IL-17A Ab

Blocking IL-17A signaling using IL-17A Ab at 60 μg every 3rd day did not attenuate METH-induced hyperlocomotion on day 1 (A,B,C) or day 15 (D,E,F) (Fig. 3). For day 1 horizontal activity, two-way ANOVA (time x treatment) revealed significant effects of treatment [F(3,700) =532, p <0.0001)] and time [F(24,700) =4.08, p <0.001)] and a significant interaction between time and treatment [F(72,700) =1.31, p =0.048)]. For ambulatory activity, two-way ANOVA (time x treatment) revealed significant effects of treatment [F(3,700) =447, p <0.0001)] and time [F(24,700) =3.67, p <0.001)] but no interaction between treatment and time. For stereotypic activity, two-way ANOVA (time x treatment) revealed significant effects of treatment [F(3,700) =493.9, p <0.0001)] and time [F(24,700) =2.37, p =0.0003)] and a significant interaction [F(72,700) =3.19, p <0.0001)]. Tukey’s post hoc test revealed that horizontal, ambulatory, and stereotypic activities in METH-treated mice that were pretreated with saline or IL-17A Ab were significantly increased compared to drug-naïve (saline-saline) mice (Figs. 3A-C, ****p<0.0001).

Fig. 3. IL-17A Ab (60 μg) did not reduce METH-induced hyperlocomotion.

Fig. 3.

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Mice were injected with saline or METH one daily for 18 days. On every 3rd day, mice were injected with vehicle or IL-17A Ab (60 μg/100 μl, IP). Data are presented as horizontal (A, D), ambulatory (B, E), and stereotypical (C, F) activities.

For day 15, for horizontal activity, two-way ANOVA (time x treatment) revealed significant effects of treatment [F(3,700) =620.8, p <0.0001)] and time [F(24,700) =3.93, p <0.001) and a significant interaction between treatment and time [F(72,700) =1.31, p =0.022)]. For ambulatory activity, two-way ANOVA (time x treatment) revealed significant effects of treatment [F(3,700) =535, p <0.0001)] and time [F(24,700) =3.38, p <0.001)] but no interaction between treatment and time. For stereotypic activity, two-way ANOVA (time x treatment) indicated significant effects of treatment [F(3,700) =491.1, p <0.0001)] and time [F(24,700) =3.43, p <0.0001)] and a significant interaction between treatment and time [F(72,700) =2.94, p <0.0001). Horizontal, ambulatory, and stereotypic activities were significantly increased in METH-treated mice that were pretreated with saline or IL-17A Ab compared to saline-saline treated mice (Figs. 3D-F, ****p<0.0001).

Using separate mice, we next tested whether a higher dose of IL-17A Ab affected METH-induced hyperlocomotion. We administered 100 μg/ 100 μl IL-17A Ab a day before METH (5 mg/kg, IP) administration. Following METH injection, locomotor activity was measured for 120 min. Time courses for horizontal, ambulatory, and stereotypic activities of saline-METH and IL-17A Ab-METH groups are shown in Figs. 4A-C. Area Under the Curve for reciprocal activities are also shown in Figs. 4D-F. An unpaired Student’s t-test revealed that IL-17A Ab pretreatment significantly reduced METH-induced horizontal, ambulatory, and stereotypic activities compared to pretreatment with saline (**p<0.01, **p<0.01, and ***p<0.001, respectively).

Fig. 4. IL-17A Ab (100 μg) reduced acute METH-induced hyperlocomotion.

Fig. 4.

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Mice were injected with saline or METH (5 mg/kg). Twenty-four h before METH or saline injections, mice were administered either saline or IL-17A Ab (100 μg/100 μl, IP). Locomotor activity was measured for 120 min following METH or saline administration. Time course of horizontal (A), ambulatory (B), and stereotypical (C) activities, as well as AUCs for horizontal (D), ambulatory (E), and stereotypical (F) are shown. Unpaired Student’s-t-test was used for the analysis of AUC (**p<0.01, ***p<0.001, N=7–8).

3.2. METH abstinence does not induce anxiety-like effects

Results from IL-17RC KO and WT mice studies are shown in Supp Fig. 1A and B. METH withdrawal did not induce anxiety-like behaviors in WT or IL-17RC KO mice. Time spent in open arms (Panel A) and the number of open arm entries (Panel B) were similar among the groups. For time spent in open arms and open arm entries, two-way ANOVA (treatment x genotype) revealed no significant effects of treatment or time.

METH abstinence also did not elicit any anxiety-like effects in mice studied for the effect of IL-17A Ab. As seen in Supp Fig. 1, time spent in open arms (Panel C) and the number of open arm entries (Panel D) were similar in all groups. For time spent in open arms and open entries, two-way ANOVA (pretreatment x treatment) revealed that there were no significant effects of treatment or time.

3.3. Effects of IL-17RC genetic deletion or IL-17A Ab on depression-like effects during METH abstinence

3.3.1. IL-17RC KO vs WT

In WT mice treated with METH, there was a trend of increased immobility time compared to WT mice treated with saline as seen in Fig. 5 (panel A) (p=0.07). In IL-17RC KO mice treated with METH, a significantly lower immobility time was detected compared to WT mice treated with METH (***p<0.001). There was no significant difference in immobility time between IL-17RC KO and WT littermates treated with saline, and no difference between saline and METH treatments in IL-17RC KO mice. Two-way ANOVA (treatment x genotype) revealed a significant effect of genotype [F(1,39) =15.02, p = 0.0004)], no effect of treatment, and a significant interaction between treatment and genotype [F(1,39) =6.46, p = 0.015)]. Tukey’s post hoc test showed that immobility time was significantly less in IL-17RC KO mice treated with METH than in WT mice treated with METH (***p<0.001).

Fig. 5. IL-17 RC genetic deletion or IL17A Ab reduced depression-like effects during METH abstinence.

Fig. 5.

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Panel A (IL-17 RC KO): Mice (WT and IL-17 RC KO) injected with saline or METH (5 mg/kg) once daily for 18 d were tested on the FST 96 h after last METH injection. Data are presented as mean immobility time. N=10–12 mice/group. ***p<0.001 compared to METH WT. Panel B (IL-17A Ab ): Mice injected with saline or METH (5 mg/kg) once daily for 18 d were also injected every 3rd d with vehicle or IL-17A Ab (60 μg/100 μl, IP). FST was conducted 96 h after last METH injection. N=7–8 mice/group. **p<0.01 compared or *p<0.05 compared to Vehicle Meth.

3.3.2. Vehicle vs IL-17A Ab

Immobility time in mice treated with saline-METH was significantly increased during METH abstinence, suggesting depression-like effects (Fig. 5B). Two way-ANOVA (treatment x pretreatment) did not reveal significant effects of either treatment or pretreatment but did indicate a significant interaction between treatment and pretreatment [F(1,26) =13.75, p = 0.001)]. Immobility time was significantly increased in mice that received saline-METH compared to mice that received saline-saline (*p<0.05) or IL-17A Ab-METH (**p<0.01), suggesting that IL-17A Ab prevented development of depression-like behavior during METH withdrawal.

3.4. Effects of IL-17RC genetic deletion or IL-17A Ab on METH-induced CPP

3.4.1. IL-17RC vs WT

METH produced CPP in both IL-17RC KO and WT mice (Fig. 6A). Two-way ANOVA (treatment x genotype) revealed a statistically significant effect of treatment [F(1,33) =13.75, p = 0.0008)] and no significant effect of genotype, and no significant interaction between treatment and genotype. Sidak’s test revealed that CPP was significantly greater in METH-treated mice compared to saline-treated mice in both IL-17RC KO and WT mice (*p<0.05).

Fig. 6. IL-17A Ab, but not IL-17 RC genetic deletion, prevented development of METH CPP.

Fig. 6.

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Panel A (IL-17 RC KO): Mice (WT or IL-17 RC KO) were conditioned with METH (3 mg/kg, IP) or saline for 4 d. CPP testing was conducted the next day. Data are presented as a difference score (difference in time spent on METH-paired side between post-test and pre-test). N=10–12/mice. *p<0.05 compared to Saline IL-17 RC KO or Saline WT. Panel B (IL-17A Ab): Mice conditioned with METH (3 mg/kg, IP) or saline for 4 day were injected with IL-17A Ab or saline 1 d before beginning METH conditioning and on the third day of METH conditioning. CPP was conducted the day after METH conditioning was completed. Data are presented as a difference score. N=10–12/mice. *p<0.05 compared to Saline METH.

3.4.2. Vehicle vs IL-17A Ab

METH by itself (saline-METH) produced CPP that was not observed in mice that received IL-17A Ab and METH (Fig. 6B). Two way-ANOVA (treatment x pretreatment) revealed a significant effect of METH treatment [F(1,26) =17.22, p = 0.0003)] and IL-17A Ab pretreatment [F(1,26) =6.03, p = 0.021)] but no significant interaction between treatment and pretreatment. Sidak’s test revealed that CPP was significantly greater in mice treated with METH by itself (saline-METH) than in drug-naïve mice (saline-saline) (*p<0.01). In mice treated with METH, CPP was significantly greater in mice pretreated with saline than in mice pretreated with IL-17A Ab (*p<0.05).

3.5. METH abstinence causes an increase in IL-17RC mRNA levels in the NAC of WT mice

3.5.1. IL-17RC KO vs WT

In WT mice, IL-17RC mRNA levels during abstinence from chronic METH exposure were significantly increased compared to METH-naïve mice treated with saline (***p<0.001, Fig. 7A). Two way-ANOVA (treatment x genotype) revealed a significant effect of METH treatment [F(1,36) =10.47, p = 0.0026)], genotype [F(1,36) =363.3, p< 0.0001)], and a significant interaction between treatment and genotype [F(1,36) =10.47, p = 0.0026)]. In mice treated chronically with METH, IL-17RC mRNA levels during METH abstinence were significantly greater compared to METH-naïve mice treated with saline (***p<0.001). IL-17RC mRNA was not detected in IL-17RC KO mice treated with saline or METH, confirming knockout of IL-17RC. In contrast, during abstinence from chronic METH, IL-17RA mRNA levels in WT and IL-17RC KO mice were similar to levels in METH-naïve mice injected with saline (Fig. 7B).

Fig. 7. IL-17RC mRNA expression in the NAC was increased during METH abstinence in WT mice.

Fig. 7.

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mRNA levels of IL-17RC (A) and IL-17RA (B) in the NAC were quantified 96 h after discontinuation of chronic METH exposure (5 mg/kg, IP x 18 d). Data are presented as fold-change in mRNA. N=10–12 mice/group. ***p<0.001 or ****p<0.0001 compared to Saline WT.

4. Discussion

Our results provide the first evidence that IL-17A signaling contributes to METH-induced behavioral effects. We showed that elimination of IL-17A signaling in mice inhibited three established preclinical effects of METH: (1) hyperlocomotion induced by acute and chronic exposure to METH, (2) development of METH place preference in CPP assays, and (3) depression-like effects in the FST during abstinence from chronic METH exposure. At the cellular level, during METH abstinence, mRNA levels of IL-17RC in the NAC were elevated. In contrast, mRNA levels of IL-17RA were not affected during METH abstinence.

The most innovative aspect of our study was the use of IL-17RC KO mice. As expected, both acute and chronic METH administration caused hyperactivity, including increased ambulation and stereotypy (Kitanaka et al., 2003; Ozawa et al., 2006; Shobo et al., 2010; Potula et al., 2023). In mice lacking IL-17RC, METH-induced hyperactivity was reduced, with the most pronounced reduction detected after chronic METH exposure. Both METH-induced ambulation and stereotypy were diminished in IL-17RC KO mice. In METH-naïve mice, no differences in spontaneous locomotor activity were detected between WT and IL-17RC KO mice, suggesting that normal baseline locomotor activity is not tonically regulated by IL-17RC. Compared to IL-17RC KO, the effects of IL-17A Ab on METH-induced hyperlocomotion were more complex. A lower dose of IL-17A Ab that reduced METH CPP and depression-like effects during METH abstinence did not affect hyperactivity evoked by acute or chronic METH administration. However, a higher dose of IL-17A Ab administered 24 h before acute METH exposure did reduce hyperactivity. Chronic studies using 100 ug IL-17A Ab were not performed for other behavioral effects of METH since 60 ug of IL-17A Ab was effective in reducing these METH-induced effects.

Locomotor sensitization following chronic METH administration was not observed in our studies. Achat-Mendes et al. (2012) showed an inverted U-shaped response on locomotor activity following acute and chronic administration of METH, but locomotor sensitization was not observed with a dose of 5 mg/kg. In a recent study, Busceti et al. (2024) observed locomotor sensitization in mice following once daily chronic administration of METH at 1 or 2 mg/kg but not at 5 mg/kg. It should be noted that the present study aimed to not only assess METH-induced hyperlocomotion and rewarding effects but also withdrawal-induced depression- and anxiety-like effects during METH abstinence. Thus, the METH dose was chosen based on previous reports showing that 5 mg/kg elicits withdrawal-induced behavioral effects (Ru et al., 2019; Yang et al., 2022). Future studies will investigate if blocking of IL-17A signaling prevents development of sensitization to hyperlocomotion induced by chronic administration of lower doses of METH, such as 1 or 2 mg/kg, and by administration of METH under a design in which METH is given intermittently (e.g., every other day) rather than continuously.

Neutralization of IL-17A signaling with IL-17A Ab also inhibited METH-induced CPP in our experiments. This suggests that blockade of IL-17A signaling reduces both rewarding and locomotor-stimulant effects of METH. Moreover, IL-17A Ab reduced METH-induced CPP at a lower dose than that required to reduce METH-evoked hyperlocomotion, suggesting that place preference was more sensitive to the inhibitory effects of IL-17A Ab. Mechanistically, it is not clear how a systemically injected monoclonal Ab with limited brain penetrability inhibited effects of METH considered to be primarily of central origin. One possibility is that METH exposure, or even increased levels of IL-17A during METH exposure, caused damage to the blood-brain barrier (BBB) that increased BBB leakiness, thereby enabling IL-17A Ab to pass into the CNS. Prior work using in vivo and in vitro models shows that METH can damage the BBB (Sharma and Ali, 2006; Qie et al., 2017; Huang et al., 2022). Growing evidence also suggests that IL-17A itself exerts damaging effects on the BBB (Kebir et al., 2007; Huppert et al., 2010; Rahman et al., 2018; Setiadi et al., 2019; Chen et al., 2022; Wang et al., 2024). In murine bEnd.3 monolayer cells and human brain microvascular endothelial cells, IL-17A treatment reduces trans endothelial electrical resistance (TEER) (Kebir et al., 2007; Huppert et al., 2010), and pretreatment with neutralizing Ab normalizes the reduction in TEER (Huppert et al., 2010). Setiadi et al. (2019) showed that IL-17A diminishes TJ gene expression levels in human brain microvascular endothelial cells. Setiadi et al. (2019) also showed that systemic administration of IL-17A Ab reduces development of experimental autoimmune encephalomyelitis in mice. IL-17A also contributes to BBB damage following epilepsy in WT and IL-17RA KO mice. The IL-17RA KO mice showed reduced IL-17A levels as well as less severe epilepsy, less albumin leakage, reduced brain water content, and upregulated expression of TJ proteins compared to WT mice, suggesting that elimination of IL-17A signaling reduces BBB damage (Wang et al., 2024).

Collectively, the evidence discussed above suggests that BBB integrity is worsened by METH exposure or enhancement IL-17A signaling and improved by reduction of IL-17A signaling. Thus, for our experiments, one explanation is that IL-17A Ab reduced METH-induced CPP and hyperlocomotion in part by preserving BBB integrity during chronic METH exposure. In this scenario, less METH reaches the brain in the presence of an IL-17A block, resulting in a diminished central effect of METH including reduced hyperlocomotion and CPP. A related possibility is that a leakier BBB, damaged by METH exposure, allowed greater concentrations of IL-17A Ab to enter the brain and directly block central IL-17A signaling that contributes to METH-evoked behaviors. A limitation of the study that precludes more extensive speculation about the BBB interpretation, and how it synchronizes with behavioral outcomes, is that levels of IL-17A in blood or CSF were not measured. Future studies will determine if chronic METH exposure increases levels of IL-17A in plasma or brain, or in both compartments, and if METH-induced changes in IL-17A levels are normalized by IL-17A Ab.

Unlike neutralization of IL-17A signaling with IL-17A Ab, which reduced both METH induced hyperlocomotion and CPP, genetic deletion of IL-17RC only reduced locomotor stimulant effects of METH. Somewhat surprisingly, genetic elimination of IL-17RC did not affect METH-induced CPP. Given that IL-17A can bind to and activate multiple receptors (IL-17RA, IL-17RC and IL-17RD), it is conceivable that each of these IL-17 receptors has different biological functions. If so, approaches that neutralize the ligand, IL-17A, such as administration of IL-17A Ab, would reduce activity at all the IL-17 receptors and have broader effects compared to a more specific strategy that eliminates a specific IL-17 receptor. For our results, this might explain why IL-17A Ab inhibited a broader range of METH-induced effects than genetic deletion of IL-17RC. In IL-17RC KO mice, IL-17A signaling is still intact at other IL-17 receptors such as IL-17RA or IL-17RD. Thus, it is possible that IL-17A signaling at these receptors may have exerted a greater influence on METH-induced CPP behaviors. A recently published study shows brain mapping of IL-17 receptors in mice (Lee et al., 2025). Results of the study show that while IL-17RA is strongly expressed in deeper layers of the cortex, hippocampus, and amygdala, IL-17RC is expressed extensively in the striatum and thalamus but sparsely expressed in the cortex. Since CPP tests context associations based on rewarding properties of an unconditioned stimulus and is a complex behavior that incorporates memory and motivated behaviors (Cunningham et al., 2019), the scarcity of IL17-RC in the cortex, hippocampus and amygdala may explain why knocking out only IL-17RC had no effect on METH-associated rewarding effects. Also, in the IL-17RC KO mice, compensation by IL-17RC-independent mechanisms may have contributed to the lack of effect of IL-17RC genetic deletion on METH-induced CPP.

We also investigated impacts of IL-17A Ab and genetic deletion of IL-17RC on behavioral effects caused by abstinence from chronic METH exposure. Human users of psychostimulants report heightened anxiety and depression during abstinence that increases the risk of relapse (Gawin and Kleber, 1986; Elman et al., 2002), and both anxiety and depression can be studied in animal models (Erb et al., 2006; Perrine et al., 2008). We found that concurrent administration of IL-17A Ab during chronic METH exposure prevented depressive-like behaviors 96 h after discontinuation of METH exposure. In mice that entirely lacked IL-17RC, which was confirmed in our mRNA experiments, the depressive-like effects during METH abstinence were also prevented. Our outcomes are supported by emerging preclinical and clinical evidence suggesting that IL-17A contributes to the pathogenesis of depression. For example, in mice with depression-like symptoms caused by chronic stress or psoriatic inflammation, IL-17A levels are increased in the hippocampus and PFC (Nadeem et al., 2017; Kim et al., 2021). In both studies, systemic administration of IL-17A Ab (100 ug, 10 d) prevented development of the depression-like effects and normalized cortical and hippocampal IL-17A levels. At the clinical level, Ixezumab, an IL-17A inhibitor used to treat psoriasis, decreases (30–40%) depression symptoms associated with psoriasis (Griffiths et al., 2017). In patients with first time diagnosed depression, blood levels of IL-17A levels are elevated but normalized after treatment of the depression (Mao et al., 2022). Blood levels of IL-17A, and the TH-17 cell population, are elevated in patients with post-partum depression (Min et al., 2022). Previously, we have shown that systemic administration of IL-17A Ab prevents development of chronic oxycodone treatment-induced depression-like effects in rats (Inan et al., 2024). These collective data suggest that active IL-17A signaling contributes to depression-like symptoms precipitated by multiple factors, including abstinence from different classes of addictive drugs, and that neutralization of IL-17A signaling reduces these symptoms.

Clues about which IL-17 receptor contributed to depressive-like behavior during METH abstinence are offered by our gene expression results. IL-17RC mRNA levels in the NAC of WT mice were upregulated 96 h after discontinuation of chronic METH exposure. In contrast, IL-17RA mRNA levels in the NAC of WT mice were not changed during METH abstinence. Given that METH abstinence was associated with both upregulation of IL-17RC mRNA and depressive-like behavior, and genetic deletion of IL-17RC abolished the depressive behavior, a functional role of IL-17RC in METH withdrawal is plausible.

Some limitations of the present study should be discussed. One was that brain tissue was collected only for the METH abstinence period. In future experiments, we plan to quantify IL-17RC and IL-17RA mRNA levels at different stages of METH exposure and abstinence to better correlate neurochemical and behavioral outcomes. Protein levels of these IL-17 receptors will also be quantified to compare with changes in mRNA. Another limitation was that plasma samples were not collected. In future experiments, blood and CSF samples will be obtained at different time points of METH exposure and abstinence, and we will quantify IL-17A levels to synchronize with behavioral outcomes and provide insight into the source of the IL-17A (peripheral, central, or both). Finally, the experiments included a mix of male and female mice, but limited numbers of KO mice precluded identification of sex differences. Since female rats are typically more sensitive to locomotor-stimulant effects of METH, self-administer more METH, and are more likely to reinstate METH seeking behavior following periods of abstinence (Reichel et al., 2012), it will be important to compare the effectiveness of blocking IL-17A signaling on METH-induced effects in male and female rats.

In conclusion, we found that elimination of IL-17A signaling with a neutralizing Ab reduced locomotor stimulant and rewarding effects of METH and prevented depressive-like behaviors during METH abstinence. We also identified a role for IL-17RC - one of the five established IL-17 receptors - in the behavioral effects of METH by showing that genetic deletion of IL-17RC reduced METH-induced hyperactivity and prevented METH abstinence-induced depressive like behaviors. We also showed that IL-17RC in the NAC is upregulated during abstinence from chronic METH exposure. Neither IL-7A Ab or genetic knockdown of IL-17RC affected spontaneous locomotor activity or caused aversive effects. These findings suggest that inhibitors of IL-17A signaling, including those that are clinically available, warrant further investigation as potential cytokine-based therapeutics for managing adverse effects of METH.

Supplementary Material

1

Fig. Suppl. 1. IL-17 RC genetic deletion or IL-17A Ab did not affect elevated plus maze (EPM) behaviors in METH-abstinent or METH-naïve mice. (A, B) IL-17RC KO mice: Mice (WT and IL-17 RC KO) were injected with saline or METH (5 mg/kg) once daily for 18 d. (C, D) IL-17A Ab: Mice injected with saline or METH (5 mg/kg) once daily for 18 d were also injected every 3rd d with vehicle or IL-17A Ab (60 μg/100 μl, IP). In all cases, EPM experiments were conducted 72 h after the last METH injection. Data are again presented as time spent in the open arm (A, C) or number of open arm entries (B, D). (N=7–8 mice/group).

Highlights:

  • METH-induced hyperlocomotion was reduced in IL-17RC KO mice vs WT mice.

  • Treatment with IL-17A Ab reduced METH-induced hyperlocomotion.

  • Treatment with IL-17A Ab prevented METH-elicited conditioned place preference.

  • IL-17A Ab treatment prevented METH abstinence-induced depression-like behavior.

  • Depression-like behavior during METH abstinence was not observed in IL-17RC KO mice.

Funding

This study was supported by the National Institute on Drug Abuse P- 30 DA013429 (EMU) Core of Excellence Grant by NIDA to our Center for Substance Abuse Research, R01 DA045499 (SMR) and The National Heart, Lung, and Blood Institute R01 HL 173975 (EKK).

Footnotes

Declaration of Generative AI and AI assisted technologies in the writing process

The authors declare that AI and AI assisted technologies were not used in the writing process.

CRediT authorship contribution statement

Saadet Inan: Conceptualization, Methodology, Investigation, Formal analysis, Data curation, Writing – original draft. Sonita Wiah: Methodology, Data curation, Formal analysis. Alexandra Szmacinski: Methodology, Data curation, Formal analysis. Scott Dunn: Methodology, Data curation, Final analysis. Joseph J Meissler: Methodology, Data curation, Final analysis. Ekaterina K Koltsova: Conceptualization, Methodology, Funding acquisition. Sergey Grivennikov: Conceptualization, Methodology, Writing –original draft. Scott Rawls: Conceptualization, Writing – review & editing, Supervision, Funding acquisition.

Declaration of competing interest

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

Publisher's Disclaimer: This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

Data availability

Data will be made available on request.

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Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

1

Fig. Suppl. 1. IL-17 RC genetic deletion or IL-17A Ab did not affect elevated plus maze (EPM) behaviors in METH-abstinent or METH-naïve mice. (A, B) IL-17RC KO mice: Mice (WT and IL-17 RC KO) were injected with saline or METH (5 mg/kg) once daily for 18 d. (C, D) IL-17A Ab: Mice injected with saline or METH (5 mg/kg) once daily for 18 d were also injected every 3rd d with vehicle or IL-17A Ab (60 μg/100 μl, IP). In all cases, EPM experiments were conducted 72 h after the last METH injection. Data are again presented as time spent in the open arm (A, C) or number of open arm entries (B, D). (N=7–8 mice/group).

NIHMS2092666-supplement-1.jpg (101KB, jpg)

Data Availability Statement

Data will be made available on request.

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