S3I-201, a selective Stat3 inhibitor, restores neuroimmune function through upregulation of Treg signaling in autistic BTBR T+ Itpr3tf/J mice
a,⁎
Sheikh F. Ahmad , Mushtaq A. Ansaria, Ahmed Nadeema, Saleh A. Bakheeta,
Musaad A. Alshammaria, Mohammad R. Khana, Abdulaziz M.S. Alsaada, Sabry M. Attiaa,b
aDepartment of Pharmacology and Toxicology, College of Pharmacy, King Saud University, Riyadh, Saudi Arabia
bDepartment of Pharmacology and Toxicology, College of Pharmacy, Al-Azhar University, Cairo, Egypt

A R T I C L E I N F O

Keywords:
Autism spectrum disorder Stat3 inhibitor
Th1/Th17 cells T regulator cells
BTBR and B6 mice
A B S T R A C T

Autism spectrum disorder (ASD) is a neurodevelopmental disorder whose symptoms include communication deficits, a lack of social skills, and stereotyped repetitive behaviors. We used BTBR T+ Itpr3tf/J (BTBR) mice, a model that demonstrates most of the core behavioral features of ASD, such as decreased sociability and high levels of repetitive behaviors. Currently, there is no treatment available that is able to improve most of the ASD disorder symptoms; thus, fi nding novel therapies is immediately required. Stat3 inhibitors are potential targets in the treatment of several immune disorders. The aim of the present study was to investigate the effects of S3I- 201, a selective Stat3 inhibitor, to determine its potential mechanism in BTBR mice. In this study, we fi rst examined the effects of S3I-201 on repetitive behavior and marble burying. We also examined the treatment of S3I-201 on Th1 (IFN-γ and T-bet), Th17 (IL-17A, RORγt, Stat3, IL-21, and IL-22), and T regulatory (Treg, Foxp3 and Helios) production in spleen CD4+ T cells. We further assessed Th1, Th17, and Treg mRNA and protein expression levels in brain tissues. S3I-201 treatment in BTBR mice significantly prevents marble burying and repetitive behavior. Furthermore, S3I-201 administration causes 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 CD4+ T cells in BTBR mice. Additionally, S3I-201 treatment also significantly decreases Th1 and Th17 levels, and increases Treg mRNA and protein expression levels. Therefore, these results suggest that S3I-201 could be considered as a therapeutic option for ASD.

1.Introduction

Autism spectrum disorder (ASD) is a devastating neurodevelop- mental disability, described by stereotypical patterns of behavior, in- terests, or activities, and defi cits in social communication [1]. The pa- thophysiology and etiology of ASD is still largely unclear, and, even with major eff orts, there are no known eff ective treatments for the core symptoms of ASD [2]. It has been recognized that children with ASD show dysregulated immune profiles that are associated with behavioral deficits [3]. Several studies have provided evidence that children with ASD suff er from ongoing neuroinfl ammatory processes in diff erent re- gions of the brain [4,5]. Indications of neuroinflammation, which in- clude activated astrocytes and microglia, are evident in the proin- fl ammatory cytokine profiles of children with ASD [5–7]. Recently, we have shown that there are alterations to cytokine and transcription factor signaling in BTBR mice and children with ASD [8,9]. There is also evidence that increased chemokine receptor expression is

associated in ASD [10]. However, the exact mechanism requires further investigation.
Changes in cytokine levels have provided evidence of immune dysfunction associated with the impaired behavioral outcomes in in- dividuals with ASD [11]. An upregulation of IFN-γ levels is associated with several neurological disorders [12]. Mothers of children with autism have a confirmed increased IFN-γ level, which may suggest an atypical immune state during gestation [13]. Children with autism have significantly increased IFN-γ levels [14]. T box expressed in T cells (T- bet) is a determinant of Th1 [15], and it has been detected in the brain and spinal cord [16]. Previously, we showed that T-bet expression is significantly higher in children with autism and BTBR mice [8,9].
Previously, it was confi rmed that levels of the cytokine IL-17A were increased in individuals with ASD; high IL-17A levels are associated with severe behavioral symptoms [17,18]. Several studies have also shown increased IL-17A levels in children with autism [17,18]. In ad- dition, recent studies have shown that IL-17A expression has an

⁎ Corresponding author.
E-mail address: [email protected] (S.F. Ahmad). https://doi.org/10.1016/j.cellsig.2018.09.006
Received 20 June 2018; Received in revised form 2 September 2018; Accepted 10 September 2018

0898-6568/ ©2018 Elsevier Inc. All rights reserved.

important role in the development of autistic disorders [19,20]. RORγt is a key transcriptional factor of Th17 cells and is involved in neuro- degeneration [21].
Stat3 is involved in the differentiation of Th17 cells [22]. Previously, it has been shown that neuroinflammation precedes the activation of the Stat3 pathway [23]. Increased IL-21 cytokine expression has been ob- served in lymphocytes and neurons [24]. Recently, we showed that IL-21 expression is significantly increased in autistic children [25]. It has been reported that the IL-21 level is highly upregulated in the mouse brain [26], whereas IL-22 promotes leukocyte infiltration into the brain [27]. Our study on IL-22 indicated that its level was considerably increased in children with autism [25]. Taken together, these results highlight the potential of Th17 biology in neuroimmune dysfunction. On the other hand, the protective effects of Foxp3 have been shown to be mediated through a reduction in glial cell activation and proinflammatory cytokine production and elevated neuronal survival [28]. Helios binds to the FoxP3 promoter and triggers FoxP3 synthesis [29]. Therefore, the reg- ulation of Th17/Treg differentiation could be a key event in the mani- festation of immune dysregulation in autism.
S3I-201, a selective Stat3 inhibitor, specifi cally inhibits Stat3 DNA- binding activity and reduces Stat3 phosphorylation [30,31]. Previously, it has been demonstrated that administration of S3I-201 inhibits spinal neuroinflammation and induces SOCS3 expression [32]. Furthermore, the treatment of S3I-201 signifi cantly attenuates stress-induced reduc- tion of splenocytes [33]. Since activation of Stat3 is involved in Th17 diff erentiation and subsequent release of IL-17A/IL-22, and suppression of Treg/Foxp3 [34], it is likely that inhibition of Stat3 would tilt the balance in favor of Treg development through suppression of Th17 development. Therefore, this present study explored the effects of S3I- 201 on pathways connected with Th17 and Treg development in BTBR T+ Itpr3tf/J (BTBR) and C57BL/6 (B6) mice.

2.Materials and methods

2.1.Reagents and antibodies

Stat3 inhibitor VI, S3I-201 (#sc-204,304), and antibodies used in this study β-actin (#sc-12,742), IL-17A (#sc-374,218), IFN-γ (#sc- 59,992), Stat3 (#sc-8019), IL-22 (#sc-14,436), RORγt (#sc-28,559), and Foxp3 (#sc-130,666) were purchased from Santa Cruz Biotech (Dallas, USA). Phorbol 12-myristate13-acetate (PMA) (#P8139-1MG) and ionomycin # (M7145-100ML) were obtained from Sigma-Aldrich (St. Louis, USA). Mouse anti-CD4-APC (#100412), mouse anti-CD4- FITC (#100406), mouse anti-CD4-PE (#100408), mouse anti-CD25- FITC (#101908), mouse anti-IFN-γ-FITC (#505806), mouse anti-IL- 17A-APC (#506916), mouse anti-IL-21-APC (#131910), mouse anti-IL- 22-PE (#516404), mouse anti-Stat3-PE (#678008), mouse anti-RORγT- PE (#562607), mouse T-bet-APC (#644814), and mouse anti-Foxp3-PE (#126404) were purchased from BioLegend (San Diego, USA). RBC lysis buffer (#420301), fixation (#420801) and permeabilization (#421002) buff ers, and GolgiStop (#554724) were purchased from BioLegend and BD Biosciences (San Diego, USA. RPMI 1640 medium (#R8758) was purchased from Sigma-Aldrich (St. Louis, USA). Primers were synthesized from GenScript (Piscataway, USA). Nitrocellulose membranes were obtained from Bio-Rad Laboratories (Hercules, USA). To detect western blots, a chemiluminescence kit was obtained from Millipore (Billerica, USA). TRIzol reagent (#15596018) was obtained from Life Technologies (Paisley, UK). SYBR Green Master Mix (#4364346) and High-Capacity cDNA Reverse Transcription (#4368814) kit were purchased from Applied Biosystems (Foster City, USA).

2.2.Animals and treatment

Male C57BL/6 (B6) and BTBR T+ Itpr3tf/J (BTBR) mice aged 5–7 weeks old and weighing 20–25 g were purchased from Jackson

Laboratory (Bar Harbor, ME USA). All mice were maintained in the College of Pharmacy, Department of Pharmacology and Toxicology, King Saud University (KSU). The KSU Committee on Animal Care ap- proved all animal procedures. B6 and BTBR mice were treated with Stat3 inhibitor VI, S3I-201 (Stat3 inhibitor VI, cat# sc-204,304, Santa Cruz Biotech, USA) was initially dissolved in dimethyl sulfoxide (DMSO; 0.05%) and diluted in saline just prior to once daily in- traperitoneal (i.p) injection for 7 days at a dose of 10 mg/kg body weight. The vehicle received equal volume of 0.05% DMSO. The dose of S3I-201 was selected based on results of a previous study [33]. All mice were sacrifi ced on day 8; the spleen and brain were isolated for fl ow cytometric, RT-PCR, and western blotting analyses.

2.3.Self-grooming

Mice were scored for spontaneous self-grooming behavior, as de- scribed previously [35]. Each mouse was placed separately into a 50 × 50 cm square arena made from non-glare Perspex, illuminated at 40 lx. After a 10-min habituation period, each mouse was scored using the parameter of cumulative time spent grooming during a 20-min session.

2.4.Marble burying test

Repetitive marble burying was measured, as described previously [36]. The apparatus comprised non-glare Perspex (20 × 40 cm). Twenty green glass marbles (15 mm in diameter) were arranged in a 4 × 5 grid that covered 2/3 of the apparatus on top of 5 cm clean bedding. Each mouse was placed in the corner that did not contain the marbles and was given a 30-min exploration period, after which the number of marbles that were buried was counted. “Buried” was defi ned as 2/3 covered by bedding. Testing was performed under dim light.

2.5.Flow cytometric analysis

Flow cytometric analysis was performed to assess the IFN-γ, T-bet, IL-17A, RORγt, Stat3, IL-21, IL-22, Foxp3, and Helios production in CD4+ and CD25+ T cells. Briefly, splenocytes were incubated with PMA/ionomycin (Sigma-Aldrich) and GolgiStop (BD Biosciences) were added before staining [37]. Cells were washed and surface staining of CD4 and CD25 surface receptors (BioLegend) was then performed. After fi xation and permeabilization (BioLegend), the cells were stained with intracellular cytokines (anti-IL-17A, anti-21, anti-IL-22, and anti-IFN-γ BioLegend) and transcription factors (anti-Stat3, anti-RORγt, anti-T-bet, anti-Helios, and anti-Foxp3 BioLegend). Ten thousand cell events were acquired on flow cytometric analysis (Beckman Coulter, USA) and analyzed using CXP software (Beckman Coulter, USA).

2.6.Western blot analysis

Western blot analysis was conducted, as previously described [38]. Briefly, protein was extracted from the mouse brain and quantifi cation was carried out using the Direct Detect Spectrometer (Millipore, Bill- erica, USA) [39]. Primary mouse monoclonal antibodies (IFN-γ, IL-17A, RORγt, IL-22, Stat3, and Foxp3), followed by incubation for 2 h with peroxidase-conjugated secondary antibodies (Santa Cruz, USA), at room temperature. The IFN-γ, IL-17A, RORγt, IL-22, Stat3, and Foxp3 bands were visualized using western blotting band intensity was detected using a Chemiluminescence kit (Millipore, USA), and presented relative to β-actin [40].

2.7.RT-PCR analysis

RNA was extracted with TRIzol reagent as previously described [41,42] and reversed to cDNA and subjected to quantitative PCR, which was performed with the Applied Biosystems 7500 Real-Time PCR

system (Applied Biosystems, USA) using SYBR® Green PCR master mix. The primer sequences used in this study were: β-actin, 5′- AGGGAAA TCGTGCGTGACAT-3′ (forward) and 5′-GGAAAAGAGCCTCAGGG CAT-3′ (reverse). IFN-γ, 5′-AGGAAGCGGAAAAGGAGTCG-3′ (forward) and 5′-GGGTCACTGCAGCTCTGAAT-3′ (reverse); T-bet, 5′-AGTGTAAT
GTGGCCTACTCCT-3′ (forward) and 5′-GCTGCTGTTGCAGTTGTT TCT-3′ (reverse); IL-17A, 5′-GGACTCTCCACCGCAATGAA-3′ (forward) and 5′- GGGTTTCTTAGGGGTCAGCC-3′ (reverse); RORγt, 5′-AGTGTA ATGTGGCCTACTCCT-3′ (forward) and 5′-GCTGCTGTTGCAGTTGTT TCT-3′ (reverse); IL-22, 5′-GGGGAGAAACTGTTCCGAGG-3′ (forward) and 5′-GGCAGGAAGGAGCAGTTCTT-3′ (reverse); Stat3, 5′-CCCCAGG
AATAGGGAGGACA-3′ (forward) and 5′-TGGTATTGCTGCAGGTC GTT-3′ (reverse); Foxp3, 5′-GGTATATGCTCCCGGCAACT-3′ (forward) and 5′-CACTGCCCTGAGTACTGGTG-3′ (reverse); Helios, 5′-ATGCCTT CCCTATCCCCACT-3′ (forward) and 5′-GCCTGCAGGTGTTCACAAAG-3′ (reverse). The data were presented as the fold change in mRNA ex- pression levels normalized to an endogenous reference gene, β-actin.

2.8.Statistical analysis

All results are reported as mean ± SEM of six animals. Data were analyzed with two-way ANOV, strain (B6 versus BTBR) x treatment (control versus S3I-201 treatment). If there were significant diff erences, the post-hoc Bonferroni’s test was used. The results were considered significant at p < .05. All the analyses were performed using GraphPad Prism 5.

3.Results

3.1.S3I-201 treatment attenuates marble burying and repetitive behaviors

The effects of S3I-201 treatment on marble burying behavior in B6 and BTBR mice are shown in Fig. 1A. S3I-201 treatment reduced marble burying in BTBR mice in comparison to that in saline-treated (control) mice (Fig. 1A), which suggests a decrease in repetitive behaviors. Fig. 1B illustrates the fi ndings for spontaneous self-grooming in B6 and BTBR mice. S3I-201 treatment reduced self-grooming behavior in BTBR mice, as compared to that in control mice (Fig. 1B). Therefore, these results showed that S3I-201 treatment reduced marble burying and self- grooming behavior in BTBR mice.

3.2.S3I-201 treatment deceases Th1 response

Flow cytometric studies were conducted to examine IFN-γ and T-bet production in CD4+ T cells in the spleen. We found that the percentage
of IFN-γ-producing CD4+ T cells was lower in S3I-201-treated BTBR mice than in control mice (Fig. 2A). The S3I-201-treated BTBR mice also showed markedly diminished T-bet producing-CD4+ T cells, as compared to control mice (Fig. 2B). Real-time PCR analysis showed that the mRNA expression of IFN-γ was significantly decreased in S3I-201- treated BTBR mice as compared to that in BTBR control mice (Fig. 2C). Furthermore, the S3I-201-treated mice showed signifi cantly decreased T-bet mRNA expression in brain tissue, when compared with the control mice (Fig. 2D). To confi rm these results, we further examined IFN-γ protein expression in the brain tissue. Our results showed that there was a signifi cant diff erence in IFN-γ protein expression between the BTBR and B6 mice (Fig. 2E). S3I-201-treated BTBR mice showed a significant decrease in IFN-γ protein expression, as compared to BTBR control mice (Fig. 2E). These results indicate that S3I-201, a Stat3 inhibitor, at- tenuates neuroinflammation.

3.3.S3I-201 treatment inhibits Th17 response

We observed the eff ects of S3I-201 on IL-17A, RORγt, Stat3, IL-21, and IL-22 production in CD4+ T cells in the spleen. S3I-201-treated BTBR mice showed decreased IL-17A-producing CD4+ T cells, com- pared to the spleen cells of BTBR control mice (Fig. 3A). To further elucidate the role of Stat3 inhibitors in RORγt signaling, S3I-201 strongly suppressed RORγt-producing CD4+ T cells, when compared to control mice (Fig. 3B). To further explain the mechanism of S3I-201 treatment in BTBR mice, we used RT-PCR to define the changes in gene expression of IL-17A and RORγt in brain tissue. In this assessment, S3I- 201-treated BTBR mice showed a decrease in IL-17A gene expression, as compared to BTBR control mice (Fig. 3C). Additionally, we also as- sessed the effects of S3I-201 on RORγt mRNA expression. RORγt mRNA expression was diminished in S3I-201-treated BTBR mice (Fig. 3D). Western blotting results showed that the IL-17A protein expression was significantly decreased in S3I-201-treated BTBR mice, as compared to that in BTBR control mice (Fig. 3E). RORγt protein expression was also suppressed in S3I-201-treated BTBR mice in the brain tissue, when compared to that in BTBR control mice (Fig. 3F). These results showed that administration of S3I-201 could control the mechanism that pro- tects against ASD.
The S3I-201-treated BTBR mice showed a lower number of Stat3- producing CD4+ T cells than the BTBR control mice (Fig. 4A). We further measured intracellular IL-21 cytokine production by CD4+ T cells. The number of IL-21-producing CD4+ T cells was lower in the spleen cells of S3I-201-treated BTBR mice, and higher in BTBR control mice (Fig. 4B). Moreover, treating BTBR mice with S3I-201 signifi cantly decreased the number of IL-22+CD4+ T cells in the spleen, compared
Fig. 1. Eff ects of S3I-201 on (A) marble burying and (B) re- petitive behavior in B6 and BTBR mice. Control B6 and BTBR mice were given dimethyl sulfoxide (DMSO) 0.05% in saline by intraperitoneal (i.p) injection. The treated B6 and BTBR mice were administered S3I-201 at a dose of 10 mg/kg by i.p injection dissolved in DMSO (0.05%) daily for 7 days. All data are shown as mean ± SEM (n = 6). ⁎P < .05 compared to the B6 saline-treated control mice; aP < .05 compared to BTBR saline-treated control mice. Signifi cant eff ect of S3I-201 treatment; +P < .05 and ++P < .01 (two-way ANOVA with the Bonferroni's test for multiple comparisons).

Fig. 2. A and B Effects of S3I-201 on intracellular IFN-γ and T-bet production in CD4+ T cells were analyzed by fl ow cytometry in the spleen cells. C and D IFN-γ and T-bet mRNA expression were measured by RT-PCR in the brain tissue. E IFN-γ protein expression level was measured by western blotting in the brain tissue. F Representative dot plots of one mouse from each group. Control B6 and BTBR mice were given dimethyl sulfoxide (DMSO) 0.05% in saline by intraperitoneal (i.p) injection. The treated B6 and BTBR mice were ad- ministered S3I-201 at a dose of 10 mg/
kg by i.p injection dissolved in DMSO (0.05%) daily for 7 days. All data are shown as mean ± SEM (n = 6). ⁎P < .05 compared to the B6 saline- treated control mice; aP < .05 com- pared to BTBR saline-treated control mice. Signifi cant effect of S3I-201 treatment; +P < .05 and ++P < .01 (two-way ANOVA with the Bonferroni's test for multiple comparisons).

Fig. 3. A and B Effects of S3I-201 on in- tracellular IL-17A and RORγt production in CD4+ T cells were analyzed by flow cyto- metry in the spleen cells. C and D IL-17A and RORγt mRNA expression levels were measured by RT-PCR in the brain tissue. E and F IL-17A and RORγt protein expression levels were measured by western blotting in the brain tissue. G Representative dot plots of one mouse from each group. Control B6 and BTBR mice were given di- methyl sulfoxide (DMSO) 0.05% in saline by intraperitoneal (i.p) injection. The treated B6 and BTBR mice were adminis- tered S3I-201 at a dose of 10 mg/kg by i.p injection dissolved in DMSO (0.05%) daily for 7 days. All data are shown as mean ± SEM (n = 6). ⁎P < .05 compared to the B6 saline-treated control mice; aP < .05 compared to BTBR saline-treated control mice. Significant effect of S3I-201 treatment; +P < .05 and ++P < .01 (two-way ANOVA with the Bonferroni's test for multiple comparisons).

Fig. 4. A, B, and C Effects of S3I-201 on intracellular Stat3, IL-21, and IL-22 production in CD4+ T cells were analyzed by fl ow cy- tometry in the spleen cells. D and E Stat3 and IL-21 mRNA ex- pression were measured by RT-PCR in the brain tissue. F and G Stat3 and IL-22 protein expression levels were measured by wes- tern blotting in the brain tissue. H Representative dot plots of one mouse from each group. Control B6 and BTBR mice were given dimethyl sulfoxide (DMSO) 0.05% in saline by intraperitoneal (i.p) injection. The treated B6 and BTBR mice were administered S3I-201 at a dose of 10 mg/kg by i.p injection dissolved in DMSO (0.05%) daily for 7 days. All data are shown as mean ± SEM (n = 6). ⁎P < .05 compared to the B6 saline-treated control mice; aP < .05 compared to BTBR saline-treated control mice. Significant effect of S3I-201 treatment; +P < .05 and ++P < .01 (two-way ANOVA with the Bonferroni's test for multiple com- parisons).

Fig. 5. A, B, and C Effects of S3I-201 on intracellular CD4+Foxp3+, CD25+Foxp3+, and CD4+Helios+ production were ana- lyzed by flow cytometry in spleen cells. D and E Foxp3 and Helios mRNA expression levels were measured by RT-PCR in the brain tissue. F Foxp3 protein expression level was measured by western blotting in the brain tissue. G Representative dot plots of one mouse from each group. Control B6 and BTBR mice were given dimethyl sulf- oxide (DMSO) 0.05% in saline by in- traperitoneal (i.p) injection. The treated B6 and BTBR mice were administered S3I-201 at a dose of 10 mg/kg by i.p injection dis- solved in DMSO (0.05%) daily for 7 days. All data are shown as mean ± SEM (n = 6). ⁎P < .05 compared to the B6 saline-treated control mice; aP < 0.05 compared to BTBR saline-treated control mice. Significant effect of S3I-201 treat- ment; +P < 0.05 and ++P < .01 (two- way ANOVA with the Bonferroni's test for multiple comparisons).

with that in control mice (Fig. 4C). RT-PCR analysis of the brain tissue on BTBR treated mice with S3I-201 showed significantly reduced Stat3 mRNA induction, compared to that in controls (Fig. 4D). IL-21 mRNA level was lower in the brain tissue of S3I-201-treated BTBR mice than in control mice (Fig. 4E). Protein levels of Stat3 and IL-22 were measured in brain tissue from both BTBR and B6 mice, with or without S3I-201 treatment. BTBR control mice presented increased Stat3 and IL-22 protein expression, as compared with B6 control mice (Fig. 4F and G). In the BTBR mice administered with S3I-201, a significant inhibition of stat3 and IL-22 protein levels were revealed in brain tissue, when compared with BTBR control mice (Fig. 4F and G). These data suggest that S3I-201 attenuates Th17 related signaling in BTBR mice.

3.4. S3I-201 upregulates Treg signaling

To elucidate the role of Stat3 inhibitors in Foxp3 and Helios sig- naling, we assessed the effects of S3I-201 on BTBR mice. We demon- strated that the production of CD4+Foxp3+ cells strongly increased in the spleen of BTBR S3I-201-treated mice, as compared with that in control mice (Fig. 5A). To confi rm further the role of Stat3 inhibitors in the Treg pathway, the S3I-201-treated BTBR mice showed a significant increase in Foxp3+CD25+ T cell production in the spleen compared to that in controls (Fig. 5B). To further understand the molecular me- chanism of S3I-201 by which Foxp3 expression is regulated, we char- acterized the role of S3I-201 in the Helios signaling pathway. BTBR mice treated with S3I-201 showed enhanced levels of Helios-producing CD4+ T cells, as compared with controls (Fig. 5C). RT-PCR analysis was used to confi rm induction of the Foxp3 gene expression in brain tissue. The administration of S3I-201 to BTBR mice increased the mRNA level of Foxp3 in brain tissue (Fig. 6D). Furthermore, BTBR S3I-201-treated mice showed a signifi cant increase in Helios mRNA expression (Fig. 6E). Measuring Foxp3 protein levels in the brain tissue further corroborated these fi ndings. Administration of S3I-201 to BTBR mice caused a significant increase in Foxp3 protein level, as compared to BTBR saline-treated mice (Fig. 6F). In summary, our results demon- strate that S3I-201 enhances Treg signaling while suppressing Th17 diff erentiation which suggests that Stat3 inhibition could be a potential therapeutic target for the treatment of ASD.

4.Discussion

Autism spectrum disorders (ASD) are described by impairments in social interaction, presence of stereotyped behaviors, and restricted interests [43]. Immune abnormalities in ASD include evidence of neu- roinfl ammation, increased immune cell activation, elevated proin- fl ammatory cytokines, and decreased anti-inflammatory cytokines [3,5,44]. Persistent immune dysfunctions are assumed to contribute to behavioral development associated with many neurodevelopmental and psychiatric disorders [45]. Immune activation is involved in the regulation of the development of homeostatic mechanisms in social interaction [46]. Stat-3 inhibitors regulate Th17 and Treg cells levels and are also known to prevent autoimmune infl ammation [47]. An evaluation of S3I-201 in a cell-based study showed that it brought about potent antitumor activities via mechanisms involved in the inhibition of aberrant Stat3 activity [30]. It has been observed that treatment with S3I-201 inhibits the infi ltration of monocytes, neutrophils, as well as suppressing the expression levels of IL-1β, TNF-α, and ICAM-1 [31]. In addition, S3I-201 inhibits Stat3 and decreases cell proliferation and migration [48]. A previous study revealed that Stat3 inhibitors were considered a possible therapeutic treatment option for immune-medi- ated diseases, where they have been shown to block Stat3 signaling [49]. In this study, we revealed the role of Stat3 inhibitor on neu- roimmune function and autism-like behavior in BTBR mice.
BTBR mice showed abnormal behavior that resembles the symptoms of autism [50]. The elevated levels of immune markers in BTBR mice

are associated with the severity of social impairments and repetitive behaviors [51]. In our recent studies, we showed that BTBR mice had distinct immune profiles and increased cytokine and chemokine levels [9,19]. Previous results also indicated that the altered immune profi le of BTBR mice was caused by proinfl ammatory cytokines, which are associated with social interaction dysfunction [51]. In our present study, we confirmed that S3I-201 eff ectively reduced the number of marbles buried, as well as repetitive behavior in the BTBR mice. The eff ectiveness of S3I-201 treatment in reducing repetitive behavior and number of marbles buried is a novel finding that could help to elucidate potential therapeutic indications for the treatment of ASD.
Upregulation of proinflammatory cytokines predominantly showed a severe disease form in children with autism [3]. IFN-γ levels are in- creased in the cerebrospinal fluid and plasma of psychotic patients [52], and it has been shown that social interaction deficiency occurs as a result of increased IFN-γ levels in early childhood [53]. Furthermore, it has been demonstrated that IFN-γ is increased in the brains of autistic chil- dren [14] and in pregnant women who gave birth to children subse- quently diagnosed with autism [54]. T-bet has been shown to be critical for several neurological disorders [55,56]. The transcription factor, T-bet, is also a potential therapeutic target in inflammatory and neurological disease development [57]. In this study, we investigated the therapeutic efficiency of S3I-201 in BTBR animals through downregulation of IFN-γ and T-bet production in CD4+ cells. Our results indicated that treatment of BTBR mice with S3I-201 reduces IFN-γ and T-bet expression levels in the brain tissue. Our results suggest that IFN-γ and T-bet could have an important impact on the development of neuroimmune dysfunction.
Blocking of Stat3 signaling has been shown to lead to reduction of MIA-induced neuropathological abnormalities and abnormal behavior [58]. Previously, our results have shown increased Stat3 signaling in children with autism and BTBR mice [8,9]. Since STAT3 is required for Th17 diff erentiation, its inhibition may be beneficial in the reduction of autism-like symptoms. This is based on previous observations which have shown that increased IL-17 levels are associated with behavioral impairments in both mice and humans [17,18,59]. Moreover, selective elimination of Th17 cells in the pregnant mice during immune activa- tion has been shown to restrain the development of autism-like beha- viors in off spring [59]. Mediators related to Th17 related signaling such as IL-21 and IL-22 are also involved in neuroinflammation [21,24,26]. Our results demonstrated downregulation of markers related to Th17 biology such as RORγt, IL-17A, IL-21, and IL-22 in BTBR mice. This indicates that S3I-201 could be responsible for attenuation of neu- roinflammation and concurrent improvement in autism-like behavior in BTBR mice through inhibition of Th17 suppression.
Alterations in Treg cells also play an important role in autoimmune neuroinfl ammatory diseases [64]. Previously, it had been proposed that modulation of Treg cell number was as a potential therapeutic approach for neuroinflammation-mediated disorders [32]. Furthermore, Treg cells are shown to be defi cient in children with autism [60]. The defi - ciency of Treg cells could contribute to immune dysregulation in ASD. A previous study has indicated a role of Helios in Treg differentiation [61,62]. In the present study, we found that S3I-201 treatment caused upregulation of Foxp3 and Helios in BTBR mice This could be due to inhibition of Th17 related signaling by S3I-201 as shown in the current study. It is reported earlier that suppression of Th17 differentiation usually favors the formation of Treg cells [34,63].

5.Conclusion

S3I-201 treatment led to suppression of Th17 related signaling while enhancing Treg related signaling with concomitant improvement in autism-like behavior in BTBR mice. This suggests that STAT3 in- hibition could be developed into a potential therapeutic approach for the treatment of autism.

Acknowledgments

The authors extend their appreciation to the Deanship of Scientific Research at King Saud University for funding this work through re- search group project No. RGP-120.

Conflict of interest

The authors declare no confl ict of interest. References
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