https://orcid.org/0000-0003-2641-1759
Figures
Abstract
Background
Chronic infection of Toxoplasma gondii (T. gondii) induces the anxiety-like behavior in hosts, which is closely linked to neuroinflammatory processes. Cis-aconite decarboxylase 1 (Acod1) is an enzyme that is responsible for itaconate production in Krebs Cycle. Emerging evidence highlights the Acod1/itaconate axis as a key regulatory node in macrophage immune-metabolic reprogramming. However, its role in infection-induced neurobehavioral alterations remains unclear. Here, we investigated the role of Acod1/itaconate axis in the anxiety induced by T. gondii chronic infection in mice.
Methods
To assess anxiety-like behaviors, we performed open field test and elevated plus maze test. Transcriptomic alterations and neuroinflammatory responses in the mouse amygdala were profiled via RNA sequencing, immunofluorescence staining, quantitative PCR (qPCR), and western blot. The functional role of the Acod1/itaconate axis was further investigated using Acod1-/- mice. Additionally, the therapeutic potential of dimethyl itaconate (DI), a cell-permeable itaconate derivative, was evaluated in chronically T. gondii-infected mice. The levels of indoleamine 2,3-dioxygenase (IDO), and serotonin (5-hydroxytryptamine, 5-HT) in serum were measured by enzyme-linked immunosorbent assay. Finally, DI’s anti-inflammatory mechanism was identified in the microglial cell line BV-2 cells.
Results
Chronic T. gondii infection induced anxiety-like behaviors in mice and triggered the activation of Acod1/itaconate axis in the amygdala. Transcriptomic and histological analyses revealed upregulation of neuroinflammation-related genes, along with microglia activation. Genetic knockout of Acod1 induced the anxiety-like phenotypes, which were rescued by DI administration. Notably, DI treatment conferred both prophylactic and therapeutic benefits, effectively mitigating anxiety induced by infection. Mechanistically, DI suppressed T. gondii-induced M1 polarization in microglia to mitigate neuroinflammation via activating Nrf2 signaling. These events further reduced indoleamine IDO expression, leading to increased 5-HT levels and subsequent amelioration of anxiety-like behavior.
Conclusions
Our findings demonstrate that the Acod1/itaconate axis plays an important role in regulating anxiety-like behavior by modulating neuroinflammation during chronic T. gondii infection. These results reveal a promising immune-metabolic drug target for treating T. gondii-associated neuropsychiatric conditions.
Author summary
Anxiety is closely linked to neuroimmune dysfunction. Chronic infection with the neurotropic parasite T. gondii triggers significant neuroinflammation in the amygdala, a key brain region for emotional processing, leading to anxiety-like behaviors. This study identifies the immune-metabolic Acod1/itaconate axis as a central regulator in this process. Using Acod1 knockout mice and pharmacological intervention with DI, a cell-permeable itaconate derivative, we demonstrate that DI effectively alleviates infection-induced anxiety. Mechanistically, DI activates the protective Nrf2 signaling pathway, which in turn dampens microglial-driven neuroinflammation. Furthermore, DI treatment reduces the expression of IDO, thereby restoring the balance of serotonin (5-HT), a crucial neuromodulator involved in mood regulation. Our findings not only provide a novel molecular perspective on how chronic infection promotes anxiety but also highlight the Acod1/itaconate axis as a promising therapeutic target for treating neuropsychiatric sequelae associated with pathogenic infection.
Citation: Yan Z, Zhou Y, Huang Y, Lv H, Li S, Zhang Y, et al. (2026) Acod1/itaconate axis controls anxiety-like behaviors induced by chronic infection of Toxoplasma gondii in mice. PLoS Negl Trop Dis 20(3): e0014077. https://doi.org/10.1371/journal.pntd.0014077
Editor: Luisa Magalhães, Universidade Federal de Minas Gerais, BRAZIL
Received: October 2, 2025; Accepted: February 23, 2026; Published: March 13, 2026
Copyright: © 2026 Yan et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Data Availability: The datasets of RNA-seq presented in this study can be found in online repositories. The names of the repository/repositories and accession number(s) can be found below: https://www.ncbi.nlm.nih.gov/; PRJNA1158573.
Funding: Project support was provided in part by National Nature Science Foundation of China (No. 82372283 to YS), the 333 High-level Talent Training Project of Jiangsu Province to WP, the Training Programs of Innovation and Entrepreneurship for College Students in Jiangsu Province (No. 202210313056Z to ZY; 202410313014Z to YH), and the XZHMU-QL Joint Research Fund (No. QL-YB022 to WP). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
Competing interests: The authors have declared that no competing interests exist.
Introduction
Neuropsychiatric disorders including anxiety, represent a leading cause of global health burden [1–3]. Nevertheless, it is frustrating that an effective treatment for those health issues is still unavailable due to the low cure rate and high recurrence rate. Pathogen infection has emerged as a significant etiological factor in mental disorders [4,5]. Toxoplasma gondii (T. gondii) is a neurotropic protozoan parasite, affecting approximately a third of the world’s population [6]. Growing evidence suggests a strong association between T. gondii infection and various neuropsychiatric conditions [7–9]. In particular, individuals with elevated anti-T. gondii antibody levels or seropositivity show increased risk of anxiety disorders [10]. Consistent with human data, recent studies, including our, have reported that chronic T. gondii infection induces anxiety-like behavior in mice [11–13]. Nevertheless, the mechanistic basis through which T. gondii influences anxiety-like behavior remains poorly understood.
Neuroinflammation, as a primary immune response to cerebral infections, is closely linked to the development of anxiety-like behavior [14–16]. Microglia is a key regulator of neuroinflammation [17,18] and latent T. gondii infection has been shown to activate microglia and promote neuroinflammation [19,20]. Interestingly, TgCtWh6, a predominant strain of T. gondii in China [21], can enhance TNF-α production in bone marrow-derived macrophages (BMDM) in vitro [22]. TNF-α is implicated in anxiety pathogenesis through its modulation of serotonin (5-hydroxytryptamine, 5-HT) metabolism [23–26]. Specifically, TNF-α stimulates indoleamine 2,3-dioxygenase (IDO), which depletes tryptophan (the precursor of 5-HT) and further leads to reduced 5-HT availability. Therefore, targeting neuroinflammatory pathways may represent a promising therapeutic strategy for alleviating T. gondii-induced anxiety-like behavior.
The emerging field of immunometabolism highlights the intricate integration and mutual regulation between metabolic processes and inflammatory responses [27]. Aconitate decarboxylase 1 (Acod1), also known as Immune-responsive gene 1 (Irg1), is a strongly upregulated gene in macrophages under inflammatory conditions [28]. Acod1 catalyzes cis-aconitate in Krebs Cycle to generate itaconate, a metabolite renowned for its potent anti-inflammatory properties [29]. For instance, itaconate has been reported to suppress inflammation through the activation of nuclear factor erythroid2-related factor 2 (Nrf2)/Kelch-like ECH-Associating protein 1 (Keap1) pathway via alkylation [30]. We recently reported that dimethyl itaconate (DI), a cell-permeable derivative of itaconate, alleviates neuroinflammation and cognitive impairment induced by T. gondii infection [31]. Also, the protective effect of DI on cognition has been observed in a mouse model of Alzheimer’s disease [32]. However, the role of Acod1/itaconate axis in T. gondii infection-induced neuropsychiatric disorder remains elusive.
The present study demonstrates that chronic T. gondii infection induces anxiety-like behavior in mice, concomitant with upregulation of Acod1 expression and elevated neuroinflammation in the amygdala, a key region that controls anxiety. Intriguingly, Acod1-/- mice exhibited obvious anxiety-like behavior, which were ameliorated following DI treatment. Notably, DI administration markedly attenuated both behavioral deficits and pathological changes associated with chronic T. gondii infection. Collectively, our findings identify Acod1/itaconate as a novel regulatory target for developing the drugs against T. gondii-related mental disorders.
Methods
Ethics statement
Mice were used for experiments in accordance with the guidelines of the Chinese Animal Care Committee and approved by the Institutional Animal Care Committee of Xuzhou Medical University (Xuzhou, China, SCXK (Su) 2020–0048).
Animals and parasites
C57BL/6J (7 weeks old) mice were purchased from Jiangsu Jicui Pharmaceutical Technology Corporation (Jiangsu Province, China). Acod1-/- mice (No: 029340) were purchased from the Jackson Laboratory (Maine, USA). Animals were housed in the room of university institute with specific temperature (24℃) and specific light (a cycle of 12 h dark/light). All mice were accessible to regular food and water freely. After a 1-week acclimation period, T. gondii cysts (Wh6 isolate, a low virulent strain and usually causes chronic infection) [21] were used to established the infection model. T. gondii tachyzoites of Wh6 isolate were used for cell experiment.
Cysts burden counting
After establishing the chronic infection model, brain cyst burden evaluated the cysts in the brain using a classical protocol [33]. Briefly, fresh brain tissue was homogenized in 1 mL PBS. Then, 10 μL of the brain suspension was examined under a light microscope (×20 magnification) to estimate the total cysts burden. All counts were performed in quadruplicate for each mouse in a blinded manner.
In-vivo experimental design
There were 4 animal experiments in the present study. Experiment 1 aimed to evaluate the effect of T. gondii chronic infection on anxiety. Mice were randomly separated into 2 groups (10 animals for each group): (a) Mice received phosphate buffer saline (PBS) by gavage as control (Con) group; (b) Mice received T. gondii cysts by the gavage (10 cysts for each mouse) as Tg group.
Experiment 2 aimed to explore the role of Acod1/itaconate axis in the anxiety-like behavior. Acod1-/- mice and wild type mice were randomly divided into 3 groups (10 animals for each group) as follows: (1) wild-type mice received PBS as WT group; (2) Acod1-/- mice received PBS as Acod1-/- group; (3) Acod1-/- mice supplemented with DI (Acod1-/- + DI). DI (Lot. 617527, Sigma-Aldrich, USA) was administrated by peritoneally injecting DI solution (40 mg per kilogram body weight) twice a week. After 4 weeks, the behavioral tests were performed.
Experiment 3 aimed to determine the preventive effect of DI on T. gondii-induced anxiety-like behavior. Mice were randomly separated into 4 groups (10 animals for each group): Con and Tg groups were treated as mentioned in Experiment 1. In the Con + Veh group, mice received PBS as vehicle control. In the Con + DI group, mice received 40 mg/kg DI. In the Tg + Veh group, the T. gondii-infected mice were received with PBS. In the Tg + DI group, each T. gondii-infected mouse received 40 mg DI per kilogram body weight. DI administration (intraperitoneal injection, twice per week) started one week before T. gondii infection and lasted until the end of the experiment.
Experiment 4 aimed to evaluate the therapeutic effect of DI on T. gondii-induced anxiety-like behavior deficits and neuropathologic lesion in the amygdala. Mice were randomly separated into 4 groups (10 animals for each group): Con + Veh, Con + DI, Tg + Veh, and Tg + DI groups. Con and Tg groups were treated as mentioned in Experiment 1. Four weeks after infection, the infected mice were intraperitoneally injected with DI (40 mg/kg) as the Tg + DI group, while the infected mice received PBS as the Tg + Veh group. DI administration (twice per week) lasted until the end of the experiment.
All mice were sacrificed 4 days after behavioral tests with CO2. The amygdala tissues were collected for further analyses.
In-vitro experimental design
Two independent in vitro experiments were performed using the mouse microglial cell line BV2 and T. gondii tachyzoites.
- Experiment 1 was designed to examine the effects of DI on the inflamamtory response and Nrf2-Keap1 pathway in microglia. BV2 cells were seeded in 6-well plates at a density of 5 × 105 cells per well and randomly assigned to four groups: (1) control cells treated with PBS (Con + Vehicle, Con + Veh); (2) cells treated with DI (100 μM, Con + DI); (3) cells infected with T. gondii tachyzoites at a multiplicity of infection (MOI) of 1 (Tg + Veh); (4) cells pretreated with DI (100 μM) for 3 h before T. gondii infection (MOI = 1, Tg + DI).
- Experiment 2 aimed to further investigate the role of Nrf2 pathway in DI’s anti-inflammatory function by using the pharmacological Nrf2 agonist sulforaphane (SFN, Cat. No. 142825-10-3, MeilunBio, China). BV2 cells were prepared as above and divided into 4 groups: (1) PBS-treated control (Con + Veh); (2) cells treated with SFN (2.5 μM; Con + SFN); (3) PBS-treated cells infected with T. gondii tachyzoites (MOI = 1; Tg + Veh); (4) cells pretreated with SFN (2.5 μM) for 3 h prior to infection (MOI = 1, Tg + SFN).
In both experiments, the cell pellets were harvested 24 h after T. gondii infection for subsequent analyses.
Open field test (OFT)
OFT is one of the most used behaviors to evaluate the autonomous and inquiry behaviors of experimental animals in a novel environment [34]. The open field was divided into two different areas: a central area (36 cm diameter) and a peripheral area. Twenty-four hours before the test, mice were acclimatized to the room for 1-1.5 h. During the test, each mouse was allowed explore freely in the open field for 10 minutes and its behavior track was recorded and analyzed.
Elevated plus maze test (EPMT)
EPMT is also a widely used behavior test to evaluate the anxiety level [35]. The elevated plus labyrinth is designed with two open arms (35 × 5 cm) and two closed arms of the same size and are raised to a height of 50 cm above the ground. During the test, the mouse was allowed to move freely in the elevated plus maze for 5 minutes and its behavior track was recorded and analyzed.
RNA-sequencing (RNA-seq)
Total RNA was first extracted from fresh amygdala tissue. The concentration, purity, integrity, and RNA Integrity Number (RIN) of the extracted RNA were then assessed to ensure its suitability for subsequent experiments. Messenger RNA (mRNA) was isolated from the total RNA using Oligo(dT) magnetic beads, based on the principle of base pairing. The enriched mRNA was fragmented by adding a fragmentation buffer. These fragmented mRNA sections were then reverse transcribed into complementary DNA (cDNA) using random hexamers and reverse transcriptase (RT). Subsequently, the cDNA underwent end repair and was ligated to adapters. Finally, the adapter-ligated cDNA libraries were sequenced on the Illumina NovaSeq 6000 platform. Based on the sequencing data, differentially expressed genes (DEGs) between the control group and the infected group were identified. DEGs were defined as genes with a P value < 0.05 and |log1.5fold change| ≥ 1. Enrichment analysis of Gene ontology and Kyoto Encyclopedia of Genes and Genomes was performed on the identified DEGs to investigate the associated biological changes. Data analysis was performed using the online platform of Majorbio Cloud Platform (http://www.majorbio.com).
Western blotting
The freshly isolated amygdala tissue and precipitated BV2 cells were firstly homogenized in ice-cold RIPA lysis buffer containing protease inhibitor cocktail (Beyotime, Jiangsu, China) and Phosphatase inhibitor cocktail (Beyotime, Jiangsu, China). The homogenate was sonicated on ice for 4 s (total six times and at 6 s intervals) and centrifuged for 20 min at 4°C and 12000 rpm. The supernatant was collected, and the protein concentration was determined by the BCA assay. Then proteins were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred onto polyvinylidene difluoride (PVDF) membranes. Membranes were blocked with 5% non-fat milk for 1 h at room temperature. For protein detection, the membranes were incubated overnight at 4°C with primary antibodies. The primary antibodies used were anti-IRG1 (1:1000, 17805S, CST), anti-KEAP1 (1:2000, 80744–1-RR, Proteintech), anti-NRF2 (1:1000, 12721T, CST), and anti-β-actin (1:10000, 81115–1-RR Proteintech). After 3 washes in TBST, the membrane was incubated with HRP-inked anti-rabbit IgG secondary antibody (1:3000, GB23303, Servicebio) for 1 h at room temperature. After the last 3 washes in TBST, the Clarity ECL western blot substrate (Bio-Rad, 1,705,060) and ChemiDoc Touch imaging system (Bio-Rad) were respectively used to detect and visualize the protein bands.
Immunofluorescence
After fixation, brain tissue was placed in 30% sucrose water for two days to dehydrate. The frozen brain was sectioned to 20 μm sections at −18°C by cryostat and the slices were further blocked with 1% BSA for 40 min at 37℃ and then incubated with the primary antibodies at 4℃ overnight. The primary antibody was shown as follows: rabbit anti-calcium-binding adapter molecule 1 (Iba1, 019–19741, 1:500, Wako). The secondary antibody was shown as follows: goat-anti-rabbit Cy3 conjugated antibody (gb21303, 1:400, Servicebio). After washing with PBS, the second primary antibody was added to the dealt sections for incubation at room temperature for 2 h. Following the incubation, DAPI solution (P0131, Beyotime) was used to counterstain sections. Finally, the images were photographed by OLYMPUS IX51 microscope (Tokyo, Japan) and analyzed by the ImageJ software (https://imagej.nih.gov/ij/).
Quantitative (q) real-time polymerase chain reaction (qPCR)
Total RNA isolated from the tissue of amygdala and precipitated BV2 cells was extracted homogenized in RNA isolator total RNA extraction reagent (Vazyme Biotech Co.,Ltd, Nanjing, China). The concentration of total RNA was determined at 260 nm and 280 nm by the spectrophotometer (DU800, Beckman Coulter Inc., Brea, CA, USA). The cDNA was generated from the purified RNA with HiScript II Q RT SuperMix for qPCR (+g DNA wiper) (Vazyme Biotech Co.,Ltd, Nanjing, China). qPCR was conducted under the ChamQ SYBR qPCR Master Mix (Vazyme Biotech Co.,Ltd, Nanjing, China) and determined on the real-time PCR detection system (Roche). Using β-actin as the internal reference control, the data of the relative mRNA expression level was analyzed by the 2-ΔΔCT method. Primer sequences were shown in the S1 Table.
Enzyme linked immunosorbent assay (ELISA)
After collecting the blood samples from mice, the determination of IDO and 5-HT was performed via enzyme linked immunosorbent assay (ELISA). IDO level was respectively determined by commercial ELISA kits (RXM203494M, Ruixin Biotechnology, Quanzhou, China). 5-HT level was determined by ELISA universal kit (RXJ203223M, Ruixin Biotechnology, Quanzhou, China). All procedures were conducted according to the provided instructions.
Statistical analysis
The data was presented as mean ± standard error of the mean (SEM) and processed by the software GraphPad Prism (Version 8.0.2). Difference between two groups was performed by the unpaired tailed Student’s t-test. Additionally, the difference among multiple groups was conducted by One-way analysis of variance. P < 0.05 was considered as significant.
Results
T. gondii chronic infection induces anxiety-like behavior in mice
Chronic T. gondii infection has been implicated in the development of anxiety-like disorders [10,36,37]. Before conducting the behavioral tests, we evaluated the cysts burden in infected mice to confirm successful establishment of chronic T. gondii infection model. A large number of T. gondii cysts were detected in the brain tissues of the infected mice (S1A Fig). In addition, the mRNA expression level of chronic infection marker bradyzoite antigen 1 (BAG1) in the amygdala tissues was also analyzed [38]. The result showed that compared with the uninfected control group, the expression level of BAG1 in Tg group was significantly upregulated (P < 0.01; S1C Fig). These findings provided a reliable basis for subsequent behavioral experiments. In the present study, we performed the open field test (OFT) and elevated plus maze test (EPMT) to evaluate whether chronic T. gondii infection induces anxiety-like behavior in mice. In OFT, infected mice displayed significantly reduced exploratory behavior in the central zone, as indicated by decreased time and distance travelled in the center (all P < 0.05; Fig 1A-1E). The number of entries into the center was also markedly lower in infected mice compared with controls (P < 0.05; Fig 1F). In EPMT, the infected group exhibited a pronouncedly decreased tendency to explore the open arms, along with lower frequency and time of head entries in the open arms (P < 0.05, P < 0.01; Fig 1H and 1I). Moreover, these mice spent less time to explore the open arms compared with the control group. (both P < 0.001; Fig 1J and 1K). Additionally, serum levels of serotonin (5-HT), a key neuromodulator involved in anxiety, were significantly reduced in infected mice (P < 0.05; Fig 1L). Correlation analysis further revealed that 5-HT levels were positively correlated with the time spent in central zone and open arms of mice (Pearson’s r = 0.73 and 0.64, both P < 0.05; Fig 1M). Together, these results demonstrate that chronic T. gondii infection induces anxiety-like behavior in mice.
(A-F) showed the results of OFT. (A) Representative track plots of OFT. (B) Time in the central zone. (C) Percentage of time in the central zone to total time. (D) Distance in the central zone. (E) Percentage of distance in the central zone to total distance. (F) Entries into the central zone. (G-K) showed the results of EMPT. (G). Representative track plots of EPMT. (H) Frequencies of head entries in open arms. (I) Time of head entries in open arms. (J) Time in open arms. (K) Percentage of time in open arms to total time. (L) 5-HT level in serum of mice. Values are mean ± SEM. * P < 0.05, ** P < 0.01, *** P < 0.001 compared with the Con group. (M) Pearson Correlation of 5-HT level with the time spent in the central zone (TCZ) and open arms (TOZ). * P < 0.05, ** P < 0.01. n = 10 for each group.
T. gondii chronic infection upregulates neuroinflammation in the amygdala of mice
Neuroinflammation is widely recognized as a critical factor in the pathogenesis of anxiety [39]. In order to investigate the impact of chronic T. gondii infection on neuroinflammatory pathways in the amygdala (a significant brain region regulating anxiety) [40], we performed RNA sequencing and the results revealed 649 upregulated and 143 downregulated DEGs post infection (Fig 2A). Gene Ontology enrichment analysis indicated that these DEGs were significantly associated with terms such as “response to stress” and “regulation of response to stress” (Fig 2B), which are closely linked to anxiety-related mechanisms. Furthermore, the expression of several anxiety-associated genes was markedly altered induced by infection (Fig 2C). In addition, several immune cell activation-related pathways including “microglial cell activation”, “macrophage activation”, and “astrocyte activation” were observed in the Gene Ontology terms (Fig 2D), alongside significant enrichment of DEGs in inflammatory pathways (Fig 2E). Notably, genes associated with M1 microglia polarization were significantly upregulated in infected mice (Fig 2F).
(A) The numbers of differentially expressed genes (DEGs) in the amygdala. (B) The biological processes associated with behavior change. (C) The relative change of DEGs associated with anxiety and neurotransmitters. (D) The biological processes of DEGs enriched in the activation of astrocytes and microglia. (E) The pathways of DEGs associated with pro-inflammatory cytokine production. (F) The expression of M1 microglia related markers. n =3 for each group.
To further validate neuroinflammatory changes, we assessed microglial activation and cytokine expression in the amygdala. Immunofluorescence staining for ionized calcium-binding adaptor molecule-1 (Iba-1) revealed a significant increase in microglial density in infected mice compared to controls (P < 0.001; Fig 3A and 3B). Morphological analysis indicated an activated microglial phenotype, characterized by increased solidity and circularity (both P < 0.01; Fig 3C and 3D). Consistent with these findings, mRNA expression levels of pro-inflammatory cytokines (TNF-α, IL-6 and IL-1β) were significantly elevated in the amygdala of infected mice (all P < 0.01; Fig 3E–3G). Overall, these results indicate that chronic T. gondii infection contributed pronounced neuroinflammation and microglial activation in the amygdala.
(A) The immunofluorescent staining of Iba-1 in the amygdala (n = 3, 10 images per mouse, scale bar: 50 μm). The image captured from the box was marked with a dotted line (scale bar 10 μm). (B) The quantification of Iba-1+ cells number in amygdala. (C, D) The solidity and circularity of Iba-1+ cells in the amygdala. (E-G) The mRNA expression levels of TNF-α, IL-6 and IL-1β in amygdala. (H) The Acod1expression of amygdala in the transcriptome. (I) The protein expression level of Acod1 in the amygdala (n=3). (J) The protein expression level of Nrf2 in the amygdala (n = 3). (K) The protein expression level of Keap1 in the amygdala (n = 3) Values are mean ± SEM. *P < 0.05, **P < 0.01, ***P < 0.001 compared with the Con group.
T. gondii chronic infection activates Acod1 expression in the amygdala of mice
Acod1/itaconate axis serves as a critical immune-metabolic node regulating inflammatory responses in macrophages [30,41]. We therefore investigated whether this pathway is involved in the amygdala following chronic T. gondii infection. Our results showed that T. gondii infection caused significantly upregulated expression of Acod1 in both the mRNA and protein levels (both P < 0.05; Fig 3H and 3I). Consistently, protein expression of Nrf2 was increased, while Keap1 was decreased (both P < 0.01; Fig 3J and 3K). Given its well-established role in mitigating inflammation, these findings suggest that Acod1/itaconate axis may be implicated in anxiety-like behavior by modulating T. gondii-induced neuroinflammation.
Acod1 deficiency causes anxiety-like behavior in mice
To further elucidate the role of the Acod1/itaconate axis in anxiety-like behavior, we evaluated Acod1-/- mice in behavioral paradigms. In OFT, Acod1 deficiency significantly reduced exploratory activity in the center, manifested as decreased time spent (both P < 0.001; Fig 4A-4C) and reduced distance travelled (P < 0.01 and P < 0.001; Fig 4D and 4E) in the central zone compared with wild-type controls. Furthermore, Acod1-/- mice exhibited a significant reduction in the number of entries into the center (P < 0.05; Fig 4F). In EPMT, Acod1-/- mice displayed reduced open-arm exploration, lower frequency and time of head entries in the open arm (P < 0.05, P < 0.01; Fig 4H and 4I). Moreover, these mice spent significantly less time exploring the open arms overall (both P < 0.001; Fig 4J and 4K).
(B) Time in the central zone. (C) Percentage of time in the central zone to total time. (D) Distance in the central zone. (E) Percentage of distance in the central zone to total distance. (F) Entries into the central zone. (G). Representative track plots of EPMT. (H) Frequencies of head entries in open arms. (I) Time of head entries in open arms. (J) Time in open arms. (K) Percentage of time spent in open arms to total time. Values are mean ± SEM. vs WT, * P < 0.05, ** P < 0.01, *** P < 0.001. vs Acod1-/-, # P < 0.05, ## P < 0.01, ### P < 0.001.
Interestingly, the administration of dimethyl itaconate (DI), a cell-permeable derivative of itaconate, significantly alleviated anxiety-like behaviors in Acod1-/- mice (Fig 4A–4K). In OFT, DI treatment enhanced the exploratory behavior of Acod1-/- mice, as reflected by increased time spent (both P < 0.01; Fig 4B and 4C), distance (P < 0.001 and P < 0.05; Fig 4D and 4E), and entries (P < 0.001; Fig 4F) in the central zone. In EPMT, DI supplementation increased the preference for exploring the open arms in Acod1-/- mice, with significant increases in the frequency and duration of head dips into the open arms (all P < 0.05; Fig 4H–4K). Taken together, these findings demonstrate that Acod1/itaconate axis plays a critical role in regulating anxiety-like behavior during chronic T. gondii infection.
DI pre-treatment ameliorates anxiety induced by T. gondii chronic infection
Given the pivotal role of Acod1/itaconate axis in anxiety-like behaviors, we next evaluated whether DI could prevent such behaviors induced by chronic T. gondii infection (Fig 5A). In the OFT, DI administration significantly enhanced the preference of infected mice to explore the central zone, with the Tg + DI group exhibiting increased time spent, distance traveled, and number of entries into the center (all P < 0.05; Fig 5B-5F). Similarly, in the EPMT, DI-treated infected mice displayed a marked increase in the frequency and duration of head dips into the open arms compared to the untreated infected group (all P < 0.05; Fig 5G–5J). At the molecular level, DI supplementation also attenuated the elevated mRNA expression of proinflammatory cytokines (TNF-α, IL-6, and IL-1β) in the amygdala of T. gondii-infected mice (all P < 0.001; Fig 5K–5M). Together, these results demonstrate that DI treatment effectively prevents T. gondii-induced anxiety-like behavior, likely through modulation of neuroinflammatory pathways.
(A) The schematic timeline of experiment 3, in which DI administration started one week before T. gondii infection and lasted until the end of the experiment. The aim of experiment 3 was to investigate the preventive effect of DI on T. gondii-induced anxiety-like behavior. (B) Time in the central zone. (C) Percentage of time in the central zone to total time. (D) Distance in the central zone. (E) Percentage of distance in the central zone to total distance. (F) Entries into the central zone. (G) Frequencies of head entries in open arms. (H) Time of head entries in open arms. (I) Time in open arms. (J) Percentage of time in open arms to total time. (K-M) The mRNA expression levels of TNF-α, IL-6 and IL-1β in the amygdala. Values are mean ± SEM. vs Con + Veh, * P < 0.05, ** P < 0.01, *** P < 0.001. vs Tg + Veh, # P < 0.05, ## P < 0.01, ### P < 0.001.
DI therapeutically reverses anxiety induced by T. gondii chronic infection
To determine whether DI could reverse pre-existing anxiety-like phenotypes, we administered DI to mice at 4 weeks post-infection (Fig 6A). Strikingly, DI treatment effectively normalized the behavioral deficits in infected mice. In the OFT (Fig 6B), DI supplementation significantly restored the willingness to explore the aversive center zone, as evidenced by increased center time (both P < 0.001; Fig 6C and 6D), distance moved (both P < 0.05; Fig 6E and 6F), and number of entries (P < 0.01; Fig 6G). Similarly, in the EPMT (Fig 6H), DI treatment robustly increased open arm entries (both P < 0.001; Fig 6I and 6J) and open arm time (both P < 0.001; Fig 6K and 6L), indicating a strong anxiolytic effect. In addition, we evaluated the cyst burden in the brains of infected mice to investigate whether DI exerts anti-T. gondii effects. The results showed that brain cyst counting in Tg + DI group did not show differ significantly from Tg + Veh group (S1B Fig). In parrel, the expression of BAG1 showed no significant change after DI treatment (S1D Fig). These findings demonstrate the therapeutic efficacy of DI in alleviating anxiety-like behaviors induced by chronic T. gondii infection, which appears to be independent of cyst reduction.
(A) The schematic timeline of experiment 4, in which DI administration started four weeks after infection and lasted until the end of the experiment. The aim of experiment 4 was to evaluate the therapeutic effect of DI on T. gondii-induced anxiety. (B) Representative track plots of OFT. (C) Time in the central zone. (D) Percentage of time in the central zone to total time. (E) Distance in the central zone. (F)Percentage of distance in the central zone to total distance. (G) Entries in the central zone. (H) Representative track plots of EPMT. (I) Frequencies of head entries in open arms. (J) Time of head entries in open arms. (I) Time in open arms. (J) Percentage of time in open arms to total time. Values are mean ± SEM. vs Con + Veh, * P < 0.05, ** P < 0.01, *** P < 0.001. vs Tg + Veh, # P < 0.05, ## P < 0.01, ### P < 0.001.
DI attenuates anxiety-related neuropathological alterations induced by T. gondii chronic infection
To elucidate the mechanism underlying the protective effect of DI against T. gondii-induced anxiety, we examined its impact on neuroinflammation and related signaling pathways in the amygdala. DI treatment significantly reduced the infection-induced increase in Iba1 ⁺ microglia density (P < 0.01; Fig 7B) and suppressed microglial activation (Fig 7A), as indicated by decreased solidity and circularity (both P < 0.05; Fig 7C and 7D). Consistent with these morphological changes, DI downregulated the mRNA expression of pro-inflammatory cytokines (TNF-α, IL-6, and IL-1β) in infected mice (all P < 0.05; Fig 7E–7G).
(A) The immunofluorescent staining of Iba-1 in the amygdala (n = 3, 10 images per mouse, scale bar: 50 μm). The image captured from the box was marked with a dotted line (scale bar 10 μm). (B) The quantification of Iba-1+ cells number in amygdala. (C, D) The solidity and circularity of Iba-1+ cells in amygdala. (E-G) The mRNA expression levels of TNF-α, IL-6 and IL-1β in amygdala. (H, I) The mRNA expression levels of IDO and 5-HT1AR in the amygdala. (J, K) The IDO and 5-HT levels in the serum. Values are mean ± SEM. Tg + Veh vs Con + Veh, * P < 0.05, ** P < 0.01, *** P < 0.001. Tg + DI vs Tg + Veh, # P < 0.05, ## P < 0.01, ### P < 0.001.
Furthermore, the depletion of 5-HT induced by IDO, which is a critical event for anxiety [25] controlled by neuroinflammation [42–44] were also evaluated. In the amygdala, T. gondii infection respectively upregulated IDO and downregulated 5-HT1A receptor (5-HT1AR) mRNA expression (both P < 0.05; Fig 7H and 7I). Correspondingly, serum levels of IDO were elevated and 5-HT was reduced in infected mice (both P < 0.05; Fig 7J and 7K). Importantly, DI administration revised these alterations, suppressing IDO overproduction and restoring 5-HT levels (both P < 0.05; Fig 7H-7K). These results collectively indicate that DI alleviates T. gondii-triggered anxiety by dampening microglia-driven neuroinflammation and subsequently rectifying the IDO/5-HT pathway.
DI activates the Nrf2-Keap1 pathway to suppress pro‑inflammatory responses in microglia
To establish a more direct mechanistic link for the anti-inflammatory function of DI, we performed a series of in vitro experiments using the BV2 microglial cell line (Fig 8A). Cells were pretreated with DI before stimulation with T. gondii tachyzoites. The results showed that DI pretreatment activated the Nrf2 signaling pathway, as evidenced by increased Nrf2 protein and decreased Keap1 protein levels (Fig 8B and 8C). In parallel, DI pretreatment significantly suppressed the infection-induced upregulation of key pro-inflammatory cytokine mRNAs, including TNF-α, IL-6 and IL-1β (Fig 8D-8F). These results suggest that DI suppresses microglial inflammatory responses at least in part through Nrf2 pathway activation.
(A) The schematic timeline of in-vitro experiment 1 and 2, in which BV2 cells were pretreated with DI or SFN for 3h prior to T. gondii infection. The aim of experiments was to evaluate the mechanistic link underlying the anti-inflammatory function of DI. (B) The protein expression level of Nrf2 in DI pretreated group. (C) The protein expression level of Keap1 in DI pretreated group. (D-F) The mRNA expression levels of TNF-α, IL-6, and IL-1β in DI pretreated group. (G, H) The mRNA expression levels of CD86 and iNOS in DI pretreated group. (I-K) The mRNA expression levels of TNF-α, IL-6, and IL-1β in SFN pretreated group. (L, M) The mRNA expression levels of CD86 and iNOS in SFN pretreated group. Values are mean ± SEM. Tg + Veh vs Con + Veh, * P < 0.05, ** P < 0.01, *** P < 0.001. Tg + DI or Tg + SFN vs Tg + Veh, # P < 0.05, ## P < 0.01, ### P < 0.001.
Having established that DI activates the Nrf2-Keap1 pathway in microglia, we next asked whether this signaling axis mediates the suppression of microglial M1 polarization. To evaluate the polarization state accurately, we first measured the mRNA expression of canonical M1 markers (CD86 and iNOS) [45,46]. Stimulation with T. gondii tachyzoites robustly induced the expression of CD86 and iNOS, and DI pretreatment significantly inhibited this induction (Fig 8G and 8H), indicating that DI attenuates the M1-polarized state. Consistent results were observed in vivo: T. gondii infection significantly upregulated CD86 and iNOS levels in the tissue of amygdala compared to uninfected control (S2A and S2B Fig) and DI treatment potently reversed this upregulation (S2A and S2B Fig).
To causally link Nrf2 activation to this anti-inflammatory effect, we further used sulforaphane (SFN), a pharmacological Nrf2 agonist [47,48]. SFN pretreatment successfully recapitulated the inhibitory effect of DI on cytokine expression following tachyzoite stimulation. Specifically, SFN activated the Nrf2 pathway and thereby reduced the expression of inflammatory cytokines (Fig 8I–8K). Moreover, a similar suppression of polarization was observed in cells pretreated with the SFN in vitro (Fig 8L and 8M). Together, these findings demonstrate that DI targets microglia to suppress M1 polarization through activation of the Nrf2 pathway, providing a mechanistic basis for its anti‑inflammatory and anxiolytic effects.
Discussion
Our study demonstrates that chronic T. gondii infection triggers anxiety-like behaviors in mice, concomitant with significant neuroinflammation and activation of the Acod1/itaconate axis in the amygdala. We reported the critical role for Acod1/itaconate, in which the global Acod1 deficiency induced the anxiety-like behavior and this phenotype could be rescued by supplementation with DI. Importantly, DI administration demonstrated both prophylactic and therapeutic efficacy in mitigating infection-induced anxiety. Mechanistically, DI exerted its anxiolytic effects by suppressing neuroinflammation, reducing IDO activity, and consequently restoring 5-HT levels (Fig 9). Collectively, these findings position the Acod1/itaconate axis as a novel and promising therapeutic target for managing anxiety associated with chronic T. gondii infection.
(A) In mice, T. gondii infection upregulates the expression of pro-inflammatory cytokines in microglia, which increases IDO and decreases 5-HT levels, and consequently, leading to anxiety. Dimethyl itaconate (DI) ameliorates T. gondii- induced anxiety via suppressing neuroinflammation. (B) Acod1 loss induces anxiety in mice, which can be restored by supplementing DI. IDO, indoleamine 2,3-dioxygenase; 5-HT, 5-hydroxytryptamine; DI, dimethyl itaconate. Created in BioRender. Zhou, Y. (2026) https://BioRender.com/7xh71r1.
Pathogen infection is increasingly recognized as a significant etiology of neuropsychiatric disorders, including anxiety. T. gondii, one of the world’s most widespread infectious agents with an exceptionally broad host range, has been reported to induce anxiety [10,36]. The Wh6 isolate is a predominant strain prevalent in China [21] and demonstrates a strong propensity to invade the brain and establish chronic infection [6]. In the present study, using two classic behavioral tests (OFT and EPMT), we report that Wh6 infection induces anxiety-like behaviors in mice. Furthermore, we observed that infection increases IDO levels and decreases 5-HT levels in mouse serum. Actually, 5-HT plays a pivotal role in mood regulation and significantly influences anxiety [49] and IDO which is an enzyme in the tryptophan catabolism pathway, has the capability to degrade tryptophan (the essential precursor of 5-HT) into kynurenine [50], thereby limiting 5-HT availability and influencing mood and anxiety-related behaviors [51]. Thus, the alterations in 5-HT and IDO observed in our study further support the anxiety-like behavior induced by T. gondii infection. Epidemiological studies have indicated an association between seroprevalence of anti–T. gondii IgG and anxiety [10,52–54]. However, biomarkers underlying this relationship remain poorly defined. Our findings provide evidence that 5-HT and IDO levels may serve as potential biomarkers of anxiety related to T. gondii infection.
Neuroinflammation represents a hallmark pathological feature in the brains of mice chronically infected with T. gondii. The Wh6 strain has previously been reported to induce M1-type polarization in macrophages in vitro [22]. In the present study, we focused on the amygdala, a key brain region governing emotional processing and implicated in neuropsychiatric disorders, which is also a known site for T. gondii persistence [55]. Here, we found that chronic Wh6 infection triggered significant microglial accumulation and promoted their activation toward an M1-type polarization in the amygdala. Transcriptomic profiling of the amygdala revealed that infection-upregulated DEGs were significantly enriched in pathways related to microglial activation and neuroinflammatory responses. Consistent with this, the expression of several classic M1 marker genes was elevated post-infection. Furthermore, immunohistochemical and molecular analyses confirmed an increase in microglial density alongside upregulated expression of the pro-inflammatory cytokines TNF-α, IL-6, and IL-1β. These specific cytokines have been established as key contributors to anxiety pathogenesis in various classical models, whether induced by acquired or hereditary factors [15,56–58]. Importantly, T. gondii cyst burden in the brain remained unchanged upon DI treatment (S1B and S1D Fig), further indicating that the anxiolytic effects of DI are mediated primarily through modulation of the host neuroinflammatory response, rather than through pathogen clearance. Therefore, our data strongly suggest that microglia-driven neuroinflammation is a critical mechanism underlying anxiety-like behaviors induced by the Wh6 strain of T. gondii.
In addition, it should be emphasized that although our study employed a chronic T. gondii infection model, the microglia-driven neuroinflammatory mechanism revealed here is not limited to infectious etiology. Substantial evidence indicates that such neuroinflammation represents a common pathological feature across various anxiety disorders, including those induced by psychological stress, genetic predisposition, or environmental factors [59–62]. Based on the sustained neuroinflammatory phenotype induced by the Wh6 strain, this study demonstrates that the Acod1/itaconate axis can modulate anxiety-like behaviors by regulating microglial activation and cytokine production. Therefore, this mechanism is likely applicable to other anxiety disorders involving neuroinflammation, suggesting that targeting neuroinflammatory pathways such as the Acod1/itaconate axis may represent a broad-spectrum therapeutic strategy for anxiety.
Metabolic reprogramming is increasingly recognized as a critical regulator of microglial activation and function, thereby influencing anxiety-related behaviors [63]. Acod1 encodes the enzyme that catalyzes the conversion of cis-aconitate to itaconate within the Kreb’s cycle [64]. Notably, Acod1/itaconate axis has emerged as a key metabolic checkpoint that integrates immune-metabolic signaling in macrophages [65]. Of particular relevance, previous work has shown that Acod1 is highly upregulated in microglia during T. gondii infection [66]. In line with this, we found that chronic Wh6 infection activates the Acod1/itaconate axis in the mouse amygdala. Notably, global deletion of Acod1 induced anxiety-like behaviors in mice, an effect that was rescued by supplementation with DI, suggesting the functional importance of this pathway in anxiety regulation. Furthermore, we demonstrated that DI administration confers not only prophylactic but also therapeutic benefits against anxiety induced by chronic Wh6 infection. At the mechanistic level, DI treatment attenuated infection-driven neuroinflammation and microglial activation. This anti-inflammatory effect of itaconate signaling is consistent with findings in other neuroinflammatory disease models, such as experimental autoimmune encephalomyelitis (EAE) [67], reinforcing its broad therapeutic potential.
As the current report, 5-HT/IDO is well-established indicators of anxiety [25] and is closely modulated by neuroinflammatory signals [42–44]. Excess production of proinflammatory cytokines can enhance IDO activity, which diverts tryptophan metabolism away from 5-HT synthesis, ultimately reducing 5-HT availability in the brain [23–26]. In the present study, we observed that T. gondii infection induced neuroinflammation and microglial activation, accompanied by increased IDO expression and decreased 5-HT levels in mice. In recent years, DI has garnered considerable attention for its potent anti-inflammatory properties. For instance, DI has been shown to suppress TNF-α and IL-6 production in LPS-stimulated BMDMs [68]. Consistent with these reports, our findings demonstrate that DI alleviated T. gondii-induced neuroinflammation and microglial activation—effects potentially mediated through the Nrf2/Keap1 pathway. Furthermore, DI treatment restored 5-HT levels and suppressed IDO upregulation in infected mice. Taken together, these results suggest that the protective effect of DI against T. gondii-induced anxiety may operate through neuroinflammation/IDO/5-HT pathway.
Conclusion
In summary, this study demonstrates that chronic T. gondii infection induces anxiety-like behaviors in mice, driven by microglia-mediated neuroinflammation in the amygdala. We identify the immune-metabolic regulator Acod1/itaconate as a critical node in this process and show that its robust neural protection. DI alleviates the anxiety phenotype by suppressing neuroinflammation, normalizing the IDO/5-HT level via modulating the Nrf2/Keap1 pathway. Our findings thus establish the Acod1/itaconate axis as a novel and promising therapeutic target for mitigating neuropsychiatric sequelae associated with the chronic infection of T. gondii.
Supporting information
S1 Fig. Quantitative assessment of cysts loading in brains of mice.
The cysts burden in mice’s brains was evaluated respectively at the stages of Experiment 1 and Experiment 4. (A) Cysts burden counting in Tg + Veh before behavior tests in Experiment 1. (B) Cysts burden counting in Tg + Veh and Tg + DI groups after DI treatment in Experiment 4. (C) The mRNA expression level of BAG1 before behavior tests in Experiment 1. (D) The mRNA expression level of BAG1 after DItreatment in Experiment 4. Values are mean ± SEM. * P < 0.05, ** P < 0.01, *** P < 0.001. ns: no significance.
https://doi.org/10.1371/journal.pntd.0014077.s001
(DOCX)
S2 Fig. The mRNA expression levels of M1 polarization markers of microglia in the amygdala tissue before and after DI treatment.
(A) The mRNA expression level of CD86 in the amygdala. (B) The mRNA expression level of iNOS in the amygdala. Values are mean ± SEM. * P < 0.05, ** P < 0.01, *** P < 0.001.
https://doi.org/10.1371/journal.pntd.0014077.s002
(DOCX)
S1 Table. The qPCR primer sequences used in the study.
The qPCR primer sequences used in the study (β-actin, IL-1β, IL-6, TNF-α, IDO, 5-HT1AR, iNOS, CD86, and BAG1) are shown in the table.
https://doi.org/10.1371/journal.pntd.0014077.s003
(DOCX)
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