S3I-201, a Selective Stat3 Inhibitor, Restores Neuroimmune Function Through Upregulation of Treg Signaling in Autistic BTBR T+ Itpr3tf/J Mice

Abstract
Autism spectrum disorder (ASD) is a neurodevelopmental disorder characterized by communication deficits, lack of social skills, and stereotyped repetitive behaviors. BTBR T+ Itpr3tf/J (BTBR) mice serve as a model for ASD, displaying decreased sociability and high levels of repetitive behaviors. Currently, there is no treatment available that improves most ASD symptoms; thus, novel therapies are urgently needed. Stat3 inhibitors are potential targets for several immune disorders. This study investigated the effects of S3I-201, a selective Stat3 inhibitor, and its potential mechanism in BTBR mice. We examined the effects of S3I-201 on repetitive behavior and marble burying, as well as its impact on Th1 (IFN-γ and T-bet), Th17 (IL-17A, RORγt, Stat3, IL-21, and IL-22), and T regulatory (Treg, Foxp3 and Helios) cell production in spleen CD4+ T cells. Th1, Th17, and Treg mRNA and protein expression levels were also assessed in brain tissues. S3I-201 treatment in BTBR mice significantly prevented marble burying and repetitive behavior. Furthermore, S3I-201 administration caused a considerable decrease in IFN-γ, T-bet, IL-17A, RORγt, Stat3, IL-21, and IL-22 levels, and increases in Foxp3 and Helios production in CD4+ T cells. Additionally, S3I-201 significantly decreased Th1 and Th17 levels, and increased Treg mRNA and protein expression levels. These results suggest that S3I-201 could be considered as a therapeutic option for ASD.

Keywords: Autism spectrum disorder, Stat3 inhibitor, Th1/Th17 cells, T regulator cells, BTBR and B6 mice

Introduction
Autism spectrum disorder (ASD) is a devastating neurodevelopmental disability, described by stereotypical patterns of behavior, interests, or activities, and deficits in social communication. The pathophysiology and etiology of ASD remain largely unclear, and no effective treatments for the core symptoms have been identified. Children with ASD often show dysregulated immune profiles associated with behavioral deficits. Evidence suggests ongoing neuroinflammatory processes in different brain regions of children with ASD, including activated astrocytes and microglia, and proinflammatory cytokine profiles. Increased chemokine receptor expression and alterations in cytokine and transcription factor signaling have also been observed in ASD.

Upregulation of IFN-γ is associated with several neurological disorders, including ASD, and increased maternal IFN-γ levels may indicate an atypical immune state during gestation. Children with autism have significantly increased IFN-γ levels, and T-bet, a determinant of Th1 cells, is also elevated in both children with autism and BTBR mice. IL-17A levels are increased in individuals with ASD and are associated with severe behavioral symptoms. RORγt, a key transcriptional factor of Th17 cells, is involved in neurodegeneration. Stat3 is crucial for Th17 cell differentiation, and neuroinflammation often precedes Stat3 pathway activation. Increased IL-21 and IL-22 cytokine expression has been observed in ASD, further implicating Th17 biology in neuroimmune dysfunction. Conversely, Foxp3 and Helios are markers of Treg cells, which have protective effects via reduction of glial activation and proinflammatory cytokine production.

S3I-201 is a selective Stat3 inhibitor that specifically inhibits Stat3 DNA-binding activity and reduces Stat3 phosphorylation. Previous studies have shown that S3I-201 administration inhibits neuroinflammation and induces SOCS3 expression. As Stat3 activation is involved in Th17 differentiation and suppression of Treg/Foxp3, inhibiting Stat3 may favor Treg development and suppress Th17 cells. This study explored the effects of S3I-201 on Th17 and Treg pathways in BTBR and C57BL/6 (B6) mice.

Materials and Methods
Reagents and Antibodies:
S3I-201 (Stat3 inhibitor VI) and all antibodies were purchased from Santa Cruz Biotechnology and BioLegend. Other reagents included PMA, ionomycin, flow cytometry buffers, RPMI 1640 medium, PCR primers, nitrocellulose membranes, chemiluminescence kits, TRIzol reagent, SYBR Green Master Mix, and cDNA synthesis kits.

Animals and Treatment:
Male C57BL/6 (B6) and BTBR T+ Itpr3tf/J (BTBR) mice (5–7 weeks old, 20–25 g) were used. Mice were treated with S3I-201 (10 mg/kg, i.p.) daily for 7 days. Controls received equal volumes of DMSO (0.05%) in saline. All procedures were approved by the King Saud University Committee on Animal Care.

Behavioral Tests:

Self-grooming: Mice were placed in a 50 x 50 cm arena, and cumulative grooming time was recorded during a 20-min session after a 10-min habituation.

Marble Burying: Mice were placed in an arena with 20 marbles arranged on bedding. After 30 min, the number of marbles buried (≥2/3 covered) was counted.

Flow Cytometry:
Splenocytes were stimulated with PMA/ionomycin and GolgiStop, stained for surface CD4 and CD25, then fixed, permeabilized, and stained for intracellular cytokines and transcription factors (IFN-γ, IL-17A, IL-21, IL-22, Stat3, RORγt, T-bet, Helios, Foxp3). Ten thousand events were acquired and analyzed.

Western Blot Analysis:
Protein was extracted from brain tissue, quantified, and analyzed by SDS-PAGE and immunoblotting for IFN-γ, IL-17A, RORγt, IL-22, Stat3, and Foxp3, with β-actin as a loading control.

RT-PCR Analysis:
Total RNA was extracted from brain tissue, converted to cDNA, and subjected to quantitative PCR for IFN-γ, T-bet, IL-17A, RORγt, IL-22, Stat3, Foxp3, Helios, and β-actin.

Statistical Analysis:
Results are reported as mean ± SEM (n=6). Data were analyzed with two-way ANOVA (strain x treatment), followed by Bonferroni’s post-hoc test. Significance was set at p < .05.

Results
S3I-201 Reduces Repetitive Behaviors in BTBR Mice:
S3I-201 treatment significantly reduced marble burying and self-grooming behaviors in BTBR mice compared to controls, indicating decreased repetitive behaviors.

S3I-201 Decreases Th1 Response:
Flow cytometry revealed that S3I-201 reduced the percentage of IFN-γ- and T-bet-producing CD4+ T cells in the spleen. RT-PCR showed decreased IFN-γ and T-bet mRNA expression in brain tissue. Western blot confirmed reduced IFN-γ protein in S3I-201-treated BTBR mice, indicating attenuation of neuroinflammation.

S3I-201 Inhibits Th17 Response:
S3I-201-treated BTBR mice had fewer IL-17A- and RORγt-producing CD4+ T cells in the spleen. IL-17A and RORγt mRNA and protein expression in brain tissue were also significantly decreased. Stat3, IL-21, and IL-22 production by CD4+ T cells, as well as their mRNA and protein levels in the brain, were reduced following S3I-201 treatment. These results indicate that S3I-201 suppresses Th17 cell differentiation and associated cytokine production.

S3I-201 Enhances Treg Cell Markers:
S3I-201 treatment increased Foxp3 and Helios expression in CD4+ T cells from the spleen, as determined by flow cytometry. RT-PCR and western blot analyses showed elevated Foxp3 and Helios mRNA and protein expression in brain tissue, indicating an upregulation of regulatory T cell signaling.

Discussion
This study demonstrates that S3I-201, a selective Stat3 inhibitor, significantly ameliorates core behavioral deficits in the BTBR mouse model of ASD, including repetitive behaviors. S3I-201 exerts its effects by suppressing Th1 and Th17 cell responses and their associated proinflammatory cytokines and transcription factors (IFN-γ, T-bet, IL-17A, RORγt, Stat3, IL-21, IL-22). Meanwhile, S3I-201 enhances Treg cell markers (Foxp3, Helios), shifting the immune balance toward regulatory, anti-inflammatory pathways. Given the role of immune dysfunction and neuroinflammation in ASD, these findings suggest that Stat3 inhibition may represent a promising therapeutic approach for ASD.

Conclusion
S3I-201 restores neuroimmune function in BTBR mice by downregulating Th1/Th17 responses and upregulating Treg signaling, resulting in improved behavioral outcomes.NSC 74859 These results support further investigation of Stat3 inhibitors as potential treatments for ASD.