Toxoplasma gondii excreted‐secreted antigens suppress Foxp3 promoter activity via a SP1‐dependent mechanism

Abstract Toxoplasma gondii excreted‐secreted antigens (ESA) could result in adverse outcomes of pregnancy including abortion, stillbirth, foetal infection or teratogenesis in mice during early stage of pregnancy. Defective generation or function of regulatory T cells (Tregs) may account for those adverse pregnancy outcomes. Forkhead box p3 (Foxp3), which is the key transcriptional factor of Tregs, modulates its development and maintains inhibitory function. We previously demonstrated that ESA inhibited Foxp3 expression by attenuating transforming growth factor β RII/Smad2/Smad3/Smad4 pathway. In this study, we propose to study the role of ESA on the activity of Foxp3 promoter and explore potential mechanisms. We demonstrated that ESA suppressed Foxp3 promoter activity using dual‐luciferase reporter assay. ESA functioned at −443/−96 region of Foxp3 promoter to suppress its activity using truncated fragments of Foxp3 promoter. Further analysis revealed that suppressive role of ESA on Foxp3 promoter activity is related to specificity protein 1 (SP1). Transfection of expression plasmid of pcDNA3.1‐SP1 could restore the down‐regulation of Foxp3 induced by ESA. In conclusion, this study provides a new mechanism by which ESA could inhibit the Foxp3 promoter activity via SP1.


| INTRODUC TI ON
Toxoplasmosis, which is caused by Toxoplasma gondii (T. gondii), is regarded as the common zoonotic disease with a worldwide prevalence. 1 T. gondii mainly infects humans through the ingestion of contaminated water or food with oocysts. Sometimes, it can also be transmitted to humans vertically during pregnancy. T. gondii infection in healthy individuals is regularly asymptomatic and self-limited, while infection in pregnant individual may develop pathological conditions including abortion or congenital infection. 2 Additionally, the severity of congenital toxoplasmosis is closely linked to infection timing during pregnancy. 3 Maternal infection with T. gondii during early gestation could cause severe congenital toxoplasmosis; however, the infection during the late gestation invariably generates low infective rate of newborns. 4,5 Previous reports have demonstrated that immunopathological effects caused by T. gondii antigens are mainly responsible for the congenital toxoplasmosis. 6 Consistent with those studies, our previous study suggested that ESA accounts for foetal absorption and teratogenesis during the early pregnancy stage in mice. 7 A successful pregnancy relies on the maternal immune system that provides tolerance towards the semi-allogeneic foetus without eliciting an immunopathological reaction. 8 Regulatory T cells (Tregs) are an essential regulator on maintaining immune tolerance status of pregnancy. 9 Our previous study indicated that Tregs number and function were down-regulated in pregnant mice after ESA treatment. Foxp3, which belongs to the forkhead family and is a transcription factor encoded by X chromosome, can activate or repress its target genes to cooperatively regulate the development, function and homeostasis of Tregs. 10,11 In abortion-prone mice, the decreased Foxp3 expression is associated with reduction of Tregs (CD4 + CD25 + ) and may account for immune barrier disruption at foetal-maternal interface. 12 The key to initiate and regulate Foxp3 transcription is the conserved promoter sequence of Foxp3, which is located upstream of the transcriptional starting site (TSS). 13 Our previous work demonstrated that ESA could inhibit Foxp3 expression via IL-2Rγ/JAK3/Stats pathway 14 and PI3K/AKT/mTOR signalling pathway. 15 We propose to further study the effect of ESA on the activity of Foxp3 promoter and explore its potential mechanisms.

| Preparation of excreted-secreted antigens
1 × 10 5 tachyzoites of T. gondii (Chinese I strain) infected female C57BL/6 mice every 3 days. ESA preparation was conducted according to our previous study. 6 Briefly, tachyzoite was cultured for 3 hours in medium 1640 without 10% foetal bovine serum (FBS; Thermo Fisher Scientific). Collected cell supernatants were further concentrated with Amicon Ultra-15 centrifugal filter devices by EMD Millipore. For the removal of endotoxin from ESA, Detoxi-Gel Affinity Pak prepacked columns (Thermo Fisher Scientific) were utilized according to the manufacture's instruction.

| Bioinformatics analysis of Foxp3 promoter and plasmids construction
A major mRNA start site was mapped and defined as position +1.
The Foxp3 promoter sequence (−1711 bp to +179 bp) got from the National Center for Biotechnology Information (NCBI). Both PROMO network platform and JASPAR network tool software were used to analyse transcription factor binding sites. 16

| Chromatin immunoprecipitation
Simple ChIP enzymatic chromatin immunoprecipitation kit (CST, Cell Signaling Technology) was utilized to perform chromatin immunoprecipitation (ChIP) following the manufacturer's instructions.
In brief, for one chromatin preparation, 4 × 10 6 cells were treated with 1% methanol-free formaldehyde for 10 minutes. To block the reaction, glycine was added. The chromatin was harvested and fragmented using enzymatic digestion and sonication. Chromatin immunoprecipitation was performed with anti-histone H3 (CST), anti-SP1 (Santa Cruz Biotechnology) or anti-IgG (CST). Anti-histone H3 antibody and mouse IgG were utilized as positive and negative control, respectively. The immunoprecipitated chromatins were eluted with ChIP elution buffer and then treated with ribonuclease A and proteinase K. The DNA was amplified by two pairs of site-specific primers (Table 1) by PCR.

| Western blot
Proteins were extracted from cells treated with lysis buffer con- peroxidase-conjugated goat anti-mouse IgG was secondary antibody, and then, immunoreactive proteins were revealed with enhanced chemiluminescence (Merck).

| Statistical analysis
Statistical analyses were performed with Prism7 (Graphpad). For comparisons between only two groups, an unpaired two tailed t test was used to assess statistical significance. Statistical analyses for experiments with more than two groups were conducted with a oneway ANOVA. P < .05 indicated statistical significance.

| ESA suppressed the activity of Foxp3 promoter in EL4 cells
Foxp3, an essential molecular marker of Tregs, is a critical regulator in development, differentiation and maintenance of Tregs. [17][18][19] To  Figure 1A). In our previous study, ESA exhibited an inhibitory effect on the mRNA level of Foxp3. 7 Foxp3 promoter, located in the upstream of transcription start site, is a conserved sequence in the Foxp3 gene and is involved in initiating and regulating Foxp3 transcription. 13 To further study the effect of ESA on Foxp3 at the gene level, EL4 cells were transfected with Foxp3 promoter luciferase reporter vectors by electroporation, and then treated with ESA for 24 hours. Unexpectedly, no activity of Foxp3 promoter was detected in Foxp3 + EL4 cells transfected with PB-Foxp3 vector ( Figure 1B).
However, PE-Foxp3 vector did enhance luciferase activity of Foxp3 promoter. It suggested that Foxp3 gene expression could be regulated through PE-Foxp3 vector ( Figure 1B). Therefore, we chose PE-

Foxp3 vector for subsequent experiments. A decrease in luciferase
activity was observed when Foxp3 promoter was stimulated with ESA ( Figure 1B). These results showed that ESA suppressed the activity of Foxp3 promoter in EL4 cells.

| ESA functioned at −443/−96 region of Foxp3 promoter to inhibit its activity
Aiming to narrow down the activity region, PE-Foxp3 A and PE- analysis was conducted to analyse SP1 and P65/NF-κB expression after ESA treatment. We observed that ESA failed to down-regulate P65/NF-κB expression, but did diminish SP1 expression ( Figure 3A). promoter at the −146/−155 activity region ( Figure 3B). These data suggested that the inhibitory effect of ESA on Foxp3 promoter activity may be correlated to SP1 in EL4 cells.

| ESA suppressed Foxp3 promoter activity via inhibiting SP1 expression
It has been demonstrated that SP1 is a critical regulator in Foxp3 gene transactivation through recruitment on Foxp3 promoter. 20 We

| D ISCUSS I ON
Toxoplasma gondii infection during pregnancy frequently causes abnormal pregnancy outcomes including spontaneous abortion, stillbirth, macro or microcephalus, hydrocephalus, and retinochoroiditis, though it is often self-limited and asymptomatic in the mother. 22 Maternal immune system providing tolerance towards the semi-allogeneic foetus plays a vital role in a successful pregnancy. 8 Tregs is key regulator in the development of immune-tolerant environment. 9 Studies have shown that the percentage and absolute number of circulating maternal Tregs enhance progressively during human pregnancy initiating from the first trimester. 23 And then, levels reduce in the post-natal period, though they are still higher than in pregnant control group. And it has proved that the increased levels of Tregs are linked with normal pregnancy, 23,24 whereas a reduced number of circulating Tregs is responsible for the immunological rejection of the foetus. 25 Indeed, adoptive transfer with expanded Tregs isolated from pregnant mice could reduce abortion rate in abortion-prone mice. 26 Our previous studies have noted that ESA treatment in pregnant mice during the early pregnant stage resulted in spontaneous abortion, accompanied by decreased number and its function of Tregs. 7 Accordingly, it seems that Tregs is critical to maintain maternal immune tolerance during pregnancy.
Foxp3, a lineage specification factor for Tregs plays an indispensable role on generating and maintaining regulatory T-cell phenotypes. 27 Foxp3 is expressed in Tregs precursors from thymus or signals. It has proved that ectopic Foxp3 expression in T cells could improve autoimmune symptoms in CD25 + T cell-depleted mice. 18,19 Mutation of Foxp3 in mice leads to severe autoimmunity disease and multi-organ infiltration owing to Tregs deficiency. 17,18 Foxp3 mutation in humans results in a similar autoimmune syndrome termed IPEX (immunodysregulation, polyendocrinopathy, enteropathy and X-linked syndrome) with symptoms of insulin-dependent diabetes, thyroiditis, enteropathy, infections, endocrinopathy and eczema. 28,29 In addition, Foxp3 ablation or attenuation in matured Tregs dysregulates Foxp3 target genes and compromised Tregs inhibitory function, 30 39,40 Foxp3 promoter has identified a GC-rich region via sequence scanning, and the region is bound by SP1. 20 Additionally, the positive effect of SP1 on Foxp3 was observed, as the deficiency of SP1 blocked Foxp3 expression in CD4 + T cells. 41 In conclusion, our current study reveals that ESA inhibits SP1 expression, thereby reducing its binding to Foxp3 promoter, ultimately resulting in Foxp3 loss. Our past and current studies provided potential mechanisms by which ESA regulated Foxp3 expression and therefore revealed an important pathway for targeted therapy of adverse pregnancy outcomes.

ACK N OWLED G EM ENTS
This study was supported by National Natural Science Foundation of China (No. 81401683) and Priority Academic Program Development of Jiangsu Higher Education Institutions (PAPD).

CO N FLI C T O F I NTE R E S T
All authors state that they have no conflicts of interest.

DATA AVA I L A B I L I T Y S TAT E M E N T
The data that support the findings of this study are available from the corresponding author upon reasonable request.