Toxoplasma gondii: Effects on Serum Serotonin Concentration and Indoleamine 2, 3-Dioxygenase Expression

doi:10.21203/rs.3.rs-7390460/v1

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Toxoplasma gondii: Effects on Serum Serotonin Concentration and Indoleamine 2, 3-Dioxygenase Expression

https://doi.org/10.21203/rs.3.rs-7390460/v1

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Background

Toxoplasma gondii is an obligate intracellular protozoan that infects approximately one-third of the global population. Research has increasingly suggested a connection between toxoplasmosis and alterations in behavior. This study aims to investigate the effects of T. gondii infection on serum serotonin levels and the expression of the indoleamine 2, 3-dioxygenase (IDO) gene in the brain cells of Balb/c mice.

Methods

A total of 72 female Balb/c mice were utilized in this study, with 36 assigned to the experimental group and 36 to the control group. The mice were further divided into six subgroups, each containing six mice. Serum serotonin levels were quantified using the ELISA method, while the expression of the IDO1 gene was assessed through quantitative real-time PCR.

Results

It is observed that the serotonin serum concentration in the infected Balb/c mice was substantially higher than the non-infected groups on the day 10th (371.17 ± 53.391 vs. 233.50 ± 1.225, p < 0.0001), 20th (283.33 ± 41.707 vs. 233.33 ± 1.033, p < 0.05 ), 30th (269.17 ± 36.766 vs. 233.67 ± 0.516, p < 0.05) and 40th (291.50 ± 62.956 vs. 233.67 ± 1.033, p < 0.05) post-infection, while the serotonin serum was dramatically diminished in the infected groups rather than the control mice on day 60th (197.50 ± 23.998 vs. 233.17 ± 1.472, p < 0.01). Notably, the expression of the IDO1 gene in brain cells increased by 5.65-fold on day 10 post-infection, followed by downward trends by day 40th (1.91-fold) was observed. Moreover, sharp fluctuations also took place on the 50th and 60th .

Conclusion

It is conclusively revealed that the serum level of serotonin and IDO1 mRNA expression were significantly higher in T.gondii infected Balb/c mice than normal control group. However, future investigations could explore therapeutic interventions targeting IDO1 or serotonin pathways to mitigate neurological impacts in chronic T.gondii infections.

Serum

Serotonin

Indoleamine 2

3-dioxygenase

Brain

Toxoplasma gondii

Toxoplasma gondii (T. gondii) is a highly successful intracellular protozoan parasite of the Apicomplexa phylum [1]. Approximately one-third of the human population worldwide is estimated to be infected with T. gondii [2]. Felidaes, either wild or domestic ones, are considered as the ultimate hosts, while all warm-blooded, in particular, human are intermediate hosts, in which the asexual life cycle occurs [3]. Humans can contract toxoplasmosis infection by consuming oocysts contaminated food, water, fruits, and vegetables; by ingesting tissue cysts in undercooked or raw meat; and by congenital transmission from mother to fetus during pregnancy [4]. Following the infection of T. gondii in an intermediate host, sporozoites released from oocysts or bradyzoites inside tissue cysts infect enterocytes, transforming into rapidly reproducing tachyzoites that spread to many organs and tissues, resulting in an acute phase of infection. Tachyzoites transform into slowly replicating bradyzoites in reaction to the host immune system, forming cysts especially muscle and brain to develop a chronic infection. These bradyzoites develop cysts encased in a thick membrane that conceals them from the host's immune response. In final Felidae host, Bradyzoites within tissue cysts to invade enterocytes and differentiate into merozoites. Merozoites differentiate into male and female gametes, facilitating sexual reproduction and the subsequent excretion of many oocysts [5]. T.gondii infection is related to neurological and neurobehavioral disorders including Alzheimer’s disease, Parkinson's disease, depression, bipolar disorder, schizophrenia, and obsessive-compulsive disorder [6].

In the central nervous system (CNS), T. gondii can breach the blood-brain barrier (BBB) and invade several parenchymal cells, such microglia, astrocytes, and neurons, which can contribute to the inflammatory response. T. gondii infection may trigger alterations in the numbers of CD4⁺ T cells and cytokines like Interferon-γ (IFN-γ). IFN-γ in CNS can stimulate astrocytes to generate chemokines that facilitate the entry of CD4⁺ T cells into the CNS [7]. This clarifies how T. gondii interacts with the BBB and immune response, leading to a complicated infection in the CNS.

IFNγ is most important cytokine in the host's anti-parasitic defense against T. gondii [8]. One of the ways that IFNγ exerts its anti-Toxoplasma effects is by stimulating the expression and activity of indoleamine 2,3-dioxygenase (IDO), which depletes L-tryptophan, an essential amino acid for the parasite's growth [9]. IDO, an intracellular heme-containing enzyme, catalyzes the first and rate-limiting step in the breakdown of _L-tryptophan in the kynurenine pathway [10]. IDO exists in two forms: IDO1 and IDO2. All vertebrates possess both genes, with IDO1 found in all mammals, exhibiting a high affinity for _L-tryptophan. IDO2 is present in both mammals and lower vertebrates but has a lower affinity for _L-tryptophan, resulting in a reduced catalytic effect [11]. In vitro experiments revealed that IDO inhibits T. gondii growth in various mice and human cell lines [12, 13]. A study showed that the administration of 1-methyltryptophan (1-MT), an IDO inhibitor, to mice in the chronic phases of T. gondii infection led to increased death rates associated with rising parasite loads in the brain [14]. Given that tryptophan is the precursor of serotonin, elevated IDO levels during T. gondii infection might reduce production of serotonin [15].

Serotonin (5-hydroxytryptamine, 5-HT) is both a neurotransmitter and a peripheral hormone (16). In CNS it serves as a neurotransmitter that regulates important brain functions, including mood, sleep, cognition, memory, body temperature, and stress response [16, 17]. Peripherally, 5-HT plays a significant role in major organ systems, regulating various processes such as hemostasis, organ development, cellular regeneration, gastrointestinal motility, immunology, cardiovascular and pulmonary physiology, and among others [17, 18]. Almost 95% of 5-HT is created by enterochromaffin cells in the gut, while the other 5% is mostly synthesized by serotonergic neurons in the central nervous system, with a smaller amount generated by adipose tissue, mammary glands, and pancreatic islets [19]. Most 5-HT in the body is circulated in the bloodstream, stored in platelets, and released upon platelet activation [18]. 5-HT has significant effects on the immune system, although research on this topic has yielded conflicting results. Some studies suggest that serotonin has pro-inflammatory properties [20], while others indicate anti-inflammatory effects [21]. It impacts various immune cells, stimulating the activation and proliferation of T lymphocytes, dendritic cells, neutrophils, B lymphocytes, and natural killer (NK) cells [18].

This study aims to evaluate IDO gene expression in brain cells and assess variations in serotonin levels in the blood of mice infected with T. gondii at various time points post-infection. Previous studies have independently explored the impact of Toxoplasma gondii on IDO expression in the brain and serotonin levels in the blood. However, research simultaneously examining the effect of Toxoplasma gondii on IDO expression in brain cells and serotonin levels remains limited. This study was therefore designed to investigate the dual impact of Toxoplasma gondii on IDO gene expression in brain cells and serum serotonin levels over a 60-day period with measurements taken at 10-day intervals.

2.1. Animals

The study involved 84 female Balb/c mice, each weighing approximately 200 grams and aged 5 weeks. Mice were bought from the animal house of Jondishapour Medical Sciences University of Ahvaz (Iran, Ahvaz) and care in specific pathogen-free conditions. The mice were divided into two groups: 42 in the experimental group and 42 in the control group. Each group was further subdivided into six subgroups of seven mice each. The animals were housed in polypropylene cages, with 4 to 5 mice per cage, and had unrestricted access to food and water. The experimental group received an intraperitoneal injection of T.gondii bradyzoites, while the control group was injected with phosphate-buffered saline (PBS). All procedures performed on animals in this study was approved by the Animal Care and Use Committee of Ahvaz Jundishapur University of Medical Sciences (IR.AJUMS.ABHC.REC.1401.021) and adhered to National Institutes of Health guidelines.

2.2. Inoculation of Cyst-free T. gondii Parasites

T.gondii Type II was isolated from young C57BL/6 mice infected via oral gavage with 10 cysts and sacrificed 4 to 5 weeks post-infection. Cyst-free parasites were prepared following established protocols. Briefly, fresh brain tissue was homogenized in 2 ml of sterile PBS using a Dounce homogenizer for one minute on ice. The homogenate was centrifuged at 2000 x g for 5 minutes at 4°C, and the supernatant was discarded. The pellet was resuspended in 20 ml of sterile 30% Percoll and centrifuged for 20 minutes at 2000 x g at 15°C with no brake. After discarding the supernatant, the pellet was treated with 100 µl of 0.25% (v/v) trypsin for 30 seconds, then diluted with 900 µl of PBS. This mixture was added to 4 ml of PBS and filtered through a 3 µm filter. Parasite concentration was determined using a hemocytometer, and each mouse received 100 parasites (0.2 ml) via intraperitoneal (IP) injection.

2.3. Sample Collection

Mice from each group were anesthetized with ketamine (30–50 mg/kg) administered IP injection every 10 days, and blood and brain samples were collected. Blood samples were obtained from tail vein and put to clot activator tubes. Plasma samples were collected after centrifugation at 3000 rpm for 15 minutes at 4°C and stored at -80°C. Brains were collected and washed in sterile ice-cold PBS. The tissue samples were immediately frozen in liquid nitrogen using a tissue-freezing medium (Leica Biosystems, USA) for further analysis.

2.4. Determination of Anti-T. gondii Antibodies

The presence of anti-T. gondii antibodies was assessed 10 days post-inoculation using a Mouse Toxoplasmosis (TOXO) Antibody (IgG) ELISA Kit (Novus Biologicals, USA). Optical densities (ODs) were measured at λ = 450 nm on two wells per sample using a BioTek SP2 microplate reader (USA).

2.5. Histopathological Studies

Brain tissues were fixed in 10% formalin solution, then embedded in paraffin, and sectioned to a thickness of 5–7 µm. Serial sagittal sections were stained with hematoxylin and eosin (H&E).

2.6. Analysis of Serum 5-HT Concentration

Serum 5-HT levels were quantified using BioVision's Serotonin ELISA Kit (Milpitas Blvd., Milpitas, CA 95035, USA), a competitive ELISA assay. Standards were processed according to the reference protocol for each assay. OD values for the standards were used to create a semi-logarithmic reference curve, allowing the 5-HT content of each serum sample to be extrapolated from its corresponding OD value.

2.7. Gene expression analysis

The mRNA levels of IDO were assessed by quantitative real-time polymerase chain reaction (qRT–PCR) (Fig. 1). Total RNA was isolated from the cerebral cortex and striatum using an RNeasy Mini Kit (Qiagen, Germany) according to mentioned protocol. The concentration of extracted RNAs was quantified utilizing a NanoDrop spectrophotometer (Thermo Scientific NanoDrop). Reverse transcription of 1 µg of total RNA to complementary DNA (cDNA) was carried out employing the ExcelRT™ Reverse Transcription Kit (SMOBIO, Taiwan) in accordance with the manufacturer's instructions. The qRT-PCR was run with the TaKaRa PCR Amplification Kit (Takara Bio Inc., Japan) and primers and carried out on a StepOne^Plus Real-Time PCR system (Applied Biosystems, Branchburg, New Jersey). Each PCR reaction (20 µl) contained 1 µl of cDNA, 6.25 µl of QPCR Master Mix SYBR Green, 0.25 µl of each primer, and 4.75 µl of distilled water. The PCR cycles consisted of an initial 5 minutes at 95°C, followed by 40 cycles of 15 seconds at 95°C, 20 seconds at 60°C, and 20 seconds at 72°C. Gene expression was normalized to the GAPDH, a housekeeping gene. Gene expression levels were calculated using the 2^−ΔΔCt method. The specific primer sequences were as follows:

- IDO1

Forward: 5’-CCTTCTGGGAATAAAACACGAGG-3’

Reverse: 5’-CTAAGAAGA AAAGGAAGTTCCGG-3’

- IDO2

Forward: 5’-ATCTCCACGTAGCTCCTTCT-3’

Reverse: 5’-GCCTCCACACACTGGTTATAG-3’

- GAPDH

Forward: 5’-GGTCACCAGGGCTGCTTTTA-3’

Reverse: 3’ -CCGTTCTCAGCCATGTAGT- 5’

2.8. Statistical Analysis

The levels of serotonin and IDO1 mRNA gene expression folding changes were reported as mean ± standard deviation (SD) and Data were measured in triplicate. The 2^–ΔΔCt method was also implemented to calculate the IDO1 mRNA gene expression folding changes Student t-test and two-way ANOVA tests were employed for comparison between unpaired and within the study groups, respectively. P-values less than 0.05 were considered as significant level. P-values < 0.05 (*), P-values < 0.01 (**), P-values < 0.001 (***) and P-values < 0.0001 (****). All calculations were performed using the GraphPad Prism software (GraphPad Software Inc., San Diego, CA, USA).

3.1. Temporal Profiles of IDO Induction and 5-HT Concentration Alteration in Balb/c Mice Infected with T. gondii

We assessed the time course of changes in the serum levels of serotonin and IDO1 mRNA expression in the brains of Balb/c mice infected with T. gondii. It is observed that the serotonin serum concentration in the infected Balb/c mice was substantially higher than the non-infected groups on the day 10th, 20th, 30th and 40th post-infection, while the serotonin serum was dramatically diminished in the infected groups rather than non-infected mice on day 60th. Serotonin serum levels in the infected and non-Infected mice at different time points are depicted in Fig. 2.

The mRNA levels of the two IDO isoforms (IDO1 and IDO2) were assessed in these mice. Notably, the expression of the IDO1 gene in brain cells increased by 5.65-fold on day 10 post-infection, followed by a slight decrease until day 30th. On day 40th, a drastic reduction of 1.91-fold was observed, but an increase of 4.8-fold taken place on day 50th. However, by day 60th, a considerable attenuation of 1.29-fold occurred (Fig. 3). Notably, no mRNA expression of IDO2 was detected.

3.2. Pathological Results

Twenty-day post-infection, tissue cysts were detected in the brains of Balb/c mice. By day 40th, some mice exhibited ruptured tissue cysts (Fig. 4). In contrast, no cysts were observed in any mice from the control group.

The findings of this study revealed that not only there is a significant increase in the serotonin serum concentration in the T.gondii infected mice group by the 40th day post-infection compared to the control group but also the IDO1 mRNA expression folding change was noticeable in the brain of Balb/c infected mice pertinent to the control group. The surge in serotonin may be linked to the activation of platelets, known to store serotonin and release it upon activation. The initial boost in serotonin levels suggests that the immune response to tachyzoites in the bloodstream may be intense during this stage, as platelets play an essential role in immune defense and inflammation. On the 10th day after infection, tachyzoites increased the concentration of serum serotonin by entering the blood and probably by the induction of platelets, which are the storage source of serotonin, and during that period of time, a part of tachyzoites probably went to the brain and interacted with cells. Cerebral induction caused the expression of IDO1. The increase in the expression of this gene was accompanied by a decrease in serum serotonin levels on the 20th and 30th days. On the 20th and 30th days, the expression of IDO 1 decreased, and on the 40th day, we observed the amplification in the concentration of serum serotonin levels, which was associated with a more decrease in IDO1 gene in this period. Continuing to measure serum serotonin and IDO1 gene expression in the brain showed that on the 50th day, the level of serum serotonin decreased and the expression of the IDO1 gene increased. By examining the pathology of the brain tissue, we observed that tissue cysts were ruptured on the 40th day, and this is probably the cause and this was probably the reason for the IDO1 gene expression to be reactivated and show a significant increase after 10 days of measurement, on the 50th day. After that and on the 60th day, the measurements of the two mentioned parameters showed that they both decreased on the 60th day. This finding aligns with numerous studies investigating the effects of T.gondii on blood serotonin levels and related gene expression. For instance, research by Castello et al. demonstrated that serotonin levels in the blood of 17 sheep with anti-Toxoplasma antibodies were significantly higher than in the control group, with infected sheep exhibiting greater platelet and monocyte counts [22]. Similarly, Pastre et al. reported elevated expression of the 5-HT, ICAM1, Type II collagen, and total mast cell genes in rats infected with sporulated oocysts of T. gondii [23]. This study somehow highlighted a strong correlation between mast cell migration to the infection site and increased expression of ICAM1 and 5-HT during peak infection.

In another study, Pastre et al. found that acute infection with T. gondii oocysts in mice preferentially activates non-neuronal cells to secrete serotonin [24]. Their findings suggested that T. gondii manipulates the jejunal epithelium to activate the immune system, including mast cells. An increase in enterochromaffin cells and mast cells expressing serotonin was also noted in the jejunum wall. According to Casagrande et al., serotonin expression levels are associated with moderate intestinal immunomodulation following acute infection with T. gondii oocysts in C57BL/6 mice [25]. Mucus from infected mice showed a marked increase in serotonin-immunoreactive cells.

Strikingly, T. gondii multiplies as a tachyzoite within nucleated cells after entering the host. Once the immune response is overcome, it transforms into an inactive bradyzoite, forming tissue cysts in various organs, primarily the brain, where they can remain dormant [26]. In response to infection, macrophages produce the cytokine IL-12, which prompts natural killer cells to produce interferon gamma (IFN-γ) [27]. IFN-γ inhibits the intracellular proliferation of the parasite in human and mouse cells, with mice lacking IFN-γ being unable to control T. gondii growth. Various studies have demonstrated that IFN-γ exerts anti-T. gondii effects in human cells, such as macrophages, fibroblasts, and microglial cells, by inducing indoleamine 2,3-dioxygenase (IDO). IDO is expressed in microglial cells in the brain and is responsible for tryptophan degradation, serving as a defense mechanism against T. gondii [28].

In current study throughout the 60-day period, complex interactions between the parasite and host responses were observed. In the acute phase of toxoplasmosis, IDO1 expression appears to be stimulated, while its levels decline when T. gondii enters the chronic phase. Ufermann et al. reported a definite induction of IDO1 during acute murine toxoplasmosis in their experimental model [29]. Other studies have also demonstrated IDO1 expression in mice infected with T. gondii, with IDO activation leading to increased and earlier mortality in Huntington disease mice [30]. Fujigak et al. found significantly higher levels of IDO enzyme activity and IDO mRNA in the brains and lungs of C57BL/6 mice compared to those lacking IFN-γ [28]. Their findings suggest that inducible nitric oxide synthase (iNOS) and IDO are cross-regulated in an anti-T. gondii manner.

In this study, we observed a significant increase in IDO1 gene expression shortly after infection, followed by fluctuations over the subsequent 10-day intervals. This pattern suggests that the initial entry of T. gondii tachyzoites into the brain triggers an immune response, activating the kynurenine pathway. The upregulation of the IDO1 enzyme appears to be a strategy to deprive the parasite of tryptophan, an amino acid essential for parasite growth [31]. IDO1 has been shown to play a significant role in immune regulation during infection, as its upregulation can lead to tryptophan catabolism, thus limiting the parasite's availability of this critical resource. The decline in gene expression on days 20th, 30th, and 40th likely corresponds to the parasite transitioning into its chronic and inactive phase. The increase in expression on day 50th suggests reactivation of the parasite, overcoming the host's immune defenses, which can often occur after a period of dormancy or immune evasion [32] .

Examination of the brains of infected mice revealed ruptured cysts on day 40th, indicating parasite reactivation, which was associated with increased IDO1 expression that subsequently decreased again after 10 days [33]. This temporal pattern of IDO1 regulation has been linked to the host's immune response and parasite reactivation dynamics [34].

This timeline demonstrates how Toxoplasma gondii infection influences host biochemistry, particularly serotonin levels, through an immune-mediated mechanism involving IDO1. These fluctuations in serotonin, driven by immune responses and parasite behavior, could have significant physiological and neurological consequences. For instance, as serotonin is a key neurotransmitter involved in mood regulation, its depletion during infection phases might explain behavioral changes in infected hosts. Additionally, the study provides profound insights into how the immune response adapts from an acute phase, characterized by high serotonin turnover and IDO1 activation, to a chronic phase where the immune response stabilizes as tissue cysts form.

Similar to other studies, this study includes some strength and limitation points. The current study is the first one, within which we checked the serotonin concentration and relative expression of IDO1 in T.gondii infected Balb/c mice by day 60 post-infection, which can take into account as a novelty of this investigation. However, some limitations and suggestions are observed as follows: 1) the measurement of inflammatory cytokines, particularly IFN-γ in the blood and brain, could not be conducted; 2) Regarding the IDO can impact on anti-inflammatory cytokines secretion such as tumor growth factor beta and interleukin 10, the assessment of these cytokine fluctuations in the blood and brain of the infected mice during the post-infection period may have a beneficiary for evaluation and modulation of inflammatory response. 3) Given that regulatory T cells occupy a prominent role in IDO secretion, the evaluation of this immune cell subset frequency can be fruitful in this context.

Overall, this infection timeline reflects the complex interactions between a pathogen and host defenses, affecting both the brain and systemic biochemistry. It is revealed that the serum level of serotonin and IDO1 mRNA expression were significantly higher in T.gondii infected Balb/c mice than normal control group. However, future investigations could explore therapeutic interventions targeting IDO1 or serotonin pathways to mitigate neurological impacts in chronic T.gondii infections.

Ethics statement

The Animal Care and Use Committee of Ahvaz Jundishapur University of Medical Sciences (IR.AJUMS.ABHC.REC.1401.021) approved this study and all procedures used for animal experiments.

Consent to participate

All authors participated to the research described in this study.

Consent for publication

All authors gave their consent for the research described in this study to be published.

CRediT authorship contribution statement

Elham Kordserkeche: Conducting experiments, writing – review & editing, writing original draft; Jasem Saki: Conceptualization; Reza Arjmand: Data curation; Mohammad Amin Behmanesh: Methodology; Saeedeh Shojaee: Methodology.

Declaration of competing interest

None.

Funding

This research was supported by Ahvaz Jundishapur University of Medical Sciences, Ahvaz, Iran (grant number: CMRC-0106).

Data availability

The datasets used and/or analyzed during the current study are available from the corresponding author on reasonable request.

Acknowledgments

This study originates from the thesis of a Ph.D. student, Elham Kordserkeche, granted by Ahvaz Jundishapur University of Medical Sciences (CMRC-0106). We feel it necessary to express our sincere gratitude to Dr. Tabandeh from Shahid Chamran Ahvaz University for his valuable assistance.

Transparency Statement

The corresponding author, Dr. Jasem Saki approve that this manuscript is an honest, accurate, and transparent account of the study being reported; that no important aspects of the study have been discarded; and that any discrepancies from the study as planned (and, if relevant, registered) have been explained.

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No competing interests reported.

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Toxoplasma gondii: Effects on Serum Serotonin Concentration and Indoleamine 2, 3-Dioxygenase Expression

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Version 1

Abstract

Background

Methods

Results

Conclusion

Figures

1. Introduction

2. Methods

2.1. Animals

2.2. Inoculation of Cyst-free T. gondii Parasites

2.3. Sample Collection

2.4. Determination of Anti-T. gondii Antibodies

2.5. Histopathological Studies

2.6. Analysis of Serum 5-HT Concentration

2.7. Gene expression analysis

2.8. Statistical Analysis

3. Results

3.2. Pathological Results

4. Discussion

5. Conclusion

Declarations

References

Additional Declarations

Supplementary Files

Status:

Version 1