Association of Crohn's disease with Foxp3 gene polymorphisms and its colonic expression in Chinese patients

Background Fork head/winged helix transcription factor (Foxp3) plays a pivotal role in regulatory T (Treg) cells. The present study aimed to assess the association of Crohn's disease (CD) with Foxp3 polymorphisms and its colonic expression in Chinese patients. Methods The Foxp3 polymorphisms, rs3761547, rs2232365, rs2294021, and rs3761548, were examined by SNaPshot in 268 CD patients and 490 controls. The colonic expression levels of Foxp3, IL‐2, and IL‐4 were detected in 31 CD patients and 31 controls using real‐time quantitative polymerase chain reaction, immunohistochemistry, and enzyme‐linked immunosorbent assay. Results Compared to male controls, the proportion of variant allele of rs3761547 was increased in male patients. The variant alleles of rs3761547, rs2232365, and rs2294021 were less in male patients with stricturing CD compared to those with non‐stricturing, non‐penetrating CD; however, these variants were frequently detected in male patients with colonic CD than in those with ileocolonic CD. The variant allele of rs3761548 was increased in male patients with penetrating CD compared to those with non‐stricturing, non‐penetrating CD. The colonic expression of Foxp3 was higher in CD patients than in controls (both males and females). Compared to male patients carrying wild‐type alleles, the colonic expression of Foxp3 was downregulated in male patients with variant alleles, rs3761547, rs2232365, rs2294021, and rs3761548, respectively. However, the Foxp3 polymorphisms were not significantly related with the colonic expression levels of IL‐2 and IL‐4 in CD patients (both males and females). Conclusion Foxp3 polymorphisms might increase the CD susceptibility by reducing the colonic expression of Foxp3 in male patients.


| INTRODUC TI ON
Crohn's disease (CD) is one of the leading clinical phenotypes of inflammatory bowel diseases (IBD). Although the exact etiology is not yet clearly understood, the genetic predisposition and immunological imbalance have been implicated as the cause of the disease. 1 Regulatory T (Treg) cells and Th17 cells have been described as two distinct subsets of Th1 and Th2 cells due to their opposite effects on autoimmunity. Chao et al 2 found that Th1 and Th17 subsets were significantly increased in blood circulation, while Treg cells were decreased in Chinese patients with CD as compared to healthy controls. Notably, recent studies have emphasized that the imbalance between Treg cells and Th17 cells is a key process in the intestinal inflammation and the onset of IBD. 3,4 Fork head/winged helix transcription factor 3 (Foxp3) is primarily expressed in Treg cells and has been demonstrated to program the differentiation and function of these cells. Previous studies have suggested that Foxp3 might switch on a complex transcriptional network responsible for the stabilization of Treg cells' phenotype. Some studies reported that Treg cells in peripheral blood in patients with active IBD were functionally normal but numerically deficient, and the transcriptional levels of Foxp3 increased in active IBD lesions as compared to those in non-inflammatory areas. 5 Moreover, several observations on murine colitis models deficit in Foxp3 indicated enhanced vulnerable to inflammation. Conversely, the induction of IBD in mouse models could be blocked by transduction with Foxp3 into CD4 + CD25 − Treg cells. 6 Furthermore, the suppressive function of Treg cells was found to be dependent on the high and stable expression of transcription factor Foxp3 along with the interaction of Foxp3 with other anti-inflammatory transcription factors, such as IL-2 and IL-4. Moreover, Foxp3 cooperates in a DNA-binding complex with the nuclear factor of activated T cells that modulate the T-cell activation and anergy to control the transcription of several key cytokine genes, including IL-2. 7 The human Foxp3 is mapped to chromosome Xp11.23, and it encodes the corresponding transcription factor possessing a fork head/winged helix domain, a C2H2 zinc finger domain, and a leucine zipper-like domain. Several single nucleotide polymorphisms (SNPs) in Foxp3 affect the expression level of the molecule, thereby resulting in a lack of functional CD4 + CD25 + Treg cells. This deficiency eventually contributes to an increased risk of autoimmune diseases, such as systemic lupus erythematosus, autoimmune thyroid diseases (AITD), and allergic rhinitis (AR). [8][9][10] Currently, the data from the human genome database indicated that the four SNPs, rs3761547, rs2232365, rs2294021, and rs3761548, are were reported to engender an increasing risk of vitiligo in a Chinese population. 12 Another study in Chinese population detected Foxp3 rs2232365, which represented a novel susceptibility locus for unexplained recurrent spontaneous abortion. 13 The present study aimed to ascertain whether the Foxp3 polymorphisms rs3761547, rs2232365, rs2294021, and rs3761548 were associated with the predisposition of CD in a Chinese population. In addition, the expression levels of Foxp3, IL-2, and IL-4 in colonic tissues were assessed to elucidate the function of Foxp3 polymorphisms in this cohort of patients with CD. (e) and IBD family history. The demographic data of CD patients and controls are presented in Table 1.

| Study subjects
A subset of participants (31 patients with CD and 31 controls) from each of the two groups (patients with CD and controls) was selected for detecting the expression levels of Foxp3, IL-2, and IL-4 in colonic tissue. However, before these measurements, the two subgroups were verified for any significant differences in terms of age or sex.
The specimens of inflamed and normal mucosa in the colon were uniformly collected from patients with CD and controls, respectively, during the colonoscopy examination.
The present study was approved by the Local Ethics Committee of Wenzhou Medical University, China, and informed consent was obtained from each patient.

| Genomic DNA extraction and genotyping
Genomic DNA was extracted from peripheral blood using the  The locations (B1, inflammatory-disease without evidence of stricture or penetrating disease; B2, stricturing-constant luminal narrowing with prestenotic dilation and/or obstructive symptoms but without penetrating disease; and B3, penetrating-bowel perforation, intra-abdominal fistula, inflammatory mass or abscess not related to a postoperative complication) and behaviors (L1, terminal ileum [TI] without colonic involvement; L2, colonic involvement without TI involvement; L3, ileocolonic disease involving both the TI and colon; and L4, disease proximal to the TI without TI or colonic involvement [coexisting with L1-L3 disease]) of CD were evaluated by the Montreal classification system. SD, standard deviation.

| Real-time quantitative PCR (qPCR) analysis for mRNA expression of FOXP3
TA B L E 1 Demographic characteristics of patients with Crohn's disease (CD) and the controls normalized to the expression of β-actin. Relative gene expression was calculated using the ΔCt method.

| Immunohistochemical analysis for the protein expression of FOXP3
Biopsy specimens were fixed in formalin and embedded in paraffin.
These embedded specimens were cut into 2-mm sections and boiled in Tris-EDTA buffer (pH 9.0) in a cooker-cooler for 25

| ELISA assay
To estimate the levels of IL-2 and IL-4 in the colonic tissue, the biopsy samples were homogenized mechanically in the buffer containing 1 mol/L Tris-HCl, 3 mol/L NaCl, and 10% Triton supplemented with protease inhibitor cocktail (Sigma-Aldrich). Then, the samples were then centrifuged at 18 300 g and 4°C for 30 minutes. The levels of IL-2 and IL-4 were examined by ELISA kits according to the manufacturer's instructions (Abcam).

| Statistical analysis
Continuous variables are presented as mean ± SD, and categorical vari-  (G) of rs3761547 was higher in male CD patients than in male controls (29.20% vs 19.82%, odds ratio [OR] = 1.688, 95% confidence interval [CI] = 1.017-2.735, P = 0.041). After adjusting for age, BMI, smoking, and treatment, the result described above reached a statistical significance (P = 0.013) ( Table 2). Nevertheless, no significant association of each Foxp3 polymorphisms with CD susceptibility was observed in females after the adjustment of these clinical characteristics in patients with CD (all P > 0.05; Table S2). Table 3, after Bonferroni correction (P < 0.05/4 = 0.0125), the variant alleles (G), (C), (G) of rs3761547, rs2232365, and rs2294021 were shown to be less frequent in male patients with B2-type CD than in those with B1-type CD (all P < 0.01).

As described in
Conversely, in male patients with B1-type CD, the variant allele (A) of rs3761548 was prevalent in male patients with B3-type CD (P = 0.001).
The variant alleles (G), (C), and (G) of rs3761547, rs2232365, and rs2294021 were more common in male patients with L2-type than in those with L3-type (all P < 0.001). However, no significant differences were observed in the comparisons of Foxp3 polymorphisms among the subgroups of female patients (all P > 0.0125; Table S3).
As illustrated in Figure 1, the male and female study subjects showed a strong LD between the SNPs rs3761547, rs2232365, rs2294021, and rs3761548 in Foxp3. Furthermore, we analyzed the associations between the haplotypes with CD susceptibility and the clinicopathological characteristics of CD patients. The frequencies of haplotypes due to the four SNPs are summarized in Table 4. Among the seven haplotypes, only (ATAC), (GCGC), and (ACGA) were evaluated, since their frequencies were more than 3% in both CD patients and the controls. When compared to the corresponding controls, the haplotype (GCGC) was more prevalent in male CD patients (P = 0.032), whereas the haplotype (ACGA) was less frequent in female CD patients (P = 0.046).
The proportion of haplotype (ATAC) was significantly higher in male patients with B2-type than in male patients with B1-type (P = 0.003). However, a converse conclusion was drawn for the haplotype (GCGC) when the above two subgroups were compared (P = 0.009). The haplotype (ACGA) was more prevalent in male patients with B3-type than in males with B1-type (P < 0.001).
In contrast to male patients with L3-type, the haplotype (ATAC) was remarkably reduced in the male L2-type subgroup (P < 0.001), while the haplotype (GCGC) was apparently increased in the male L2-type subgroup (P < 0.001).

| Correlation between Foxp3 polymorphisms and the colonic expression levels in patients with CD
The average expression of Foxp3 mRNA and protein was upregulated in patients with CD as compared to controls (in males or females; Figure 2). Moreover, the average expression of

| Association between Foxp3 polymorphisms and the expression levels of IL-2 and IL-4 in patients with CD
Previous studies reported that Foxp3 might be responsible for the modulation of several key cytokines in T-cell activation and anergy, such as IL-2 and IL-4. 7 Therefore, we further detected the colonic expression levels of the two cytokines by ELISA. The colonic expressions of IL-2 and IL-4 in patients with CD were lower than that in controls (both males and females). Nevertheless, no distinct correlation was established between the four SNPs and the expression levels of IL-2 and IL-4 in this cohort of patients with CD (in males and females; all P > 0.05; Figure 4).

| D ISCUSS I ON
Evidence from Foxp3-mutant scurfy mice and Foxp3-null mice has indicated that both were likely to suffer from autoimmune and inflammatory syndromes, mainly due to the deficiency of CD4 + CD25 + Treg cells. 16,17 Conversely, the retroviral gene transfer of Foxp3 into neonatal Foxp3-deficient mice successfully induced the differentiation of naive T cells into a Treg cell phenotype. [16][17][18] Moreover, the dysfunction of Foxp3 might not only give rise to the lack of Treg cells but also promote the activity of CD4 + CD25 − T cells, which expressed the increased levels of several inflammatory markers and cytokines, leading to autoimmune diseases. Several SNPs of Foxp3 that occur at high frequencies in the general population have been widely investigated in common multifactorial human diseases, such as AITD, immunodysregulation, polyendocrinopathy, enteropathy, X-linked (IPEX) syndrome, and type 1 diabetes. 19,20 The present study primarily suggested that the rs3761547 variation might confer an enhanced risk for CD in male individuals.
Moreover, the rs3761547, rs2232365, rs2294021, and rs3761548 variations, as well as the haplotypes GCGC, ACGA, and ATAC formed by the four SNPs, might exert a potential effect on the behaviors and/or locations of CD in male patients. These findings suggested that Foxp3 might be a candidate gene for CD in Chinese males.
Several genetic studies implicated the polymorphisms of Foxp3 in the risk of autoimmune and allergic diseases and demonstrated significant gender-based differences in the distribution of Foxp3 poly-

morphisms. For instance, a previous study in Hungarian population
showed that the variant homozygote (AA) of rs3761548 was a protective factor against AR in female patients. 9 However, the allele and genotype distributions of this SNP did not differ significantly between the male patients and controls. 9 Gao et al investigated a Subsequently, the present study analyzed the influence of  29 Furthermore, in a related biochemical assay, the minor allele of rs2294021 was reported to enhance the Foxp3 mRNA expression owing to the dramatically improved transcriptional activity of Foxp3. 29 Another previous study from Denmark reported that the rs3761548 variation might elevate the risk of CD due to skewed X chromosome inactivation (SXCI) in male individuals as opposed to females; the population differences were characterized by low expression levels of Foxp3 and the decreased numbers of Treg cells. 30 Moreover, the above studies explained the genetic variations of Foxp3 might contribute to the increased risk of CD in males.
Notably, in the current study, the females carrying haplotype (ACGA) were less susceptible to CD. However, we failed to draw similar conclusions when the four SNPs in the Foxp3 gene among female CD patients and the controls were investigated.
To date, none of the studies have associated the haplotypes of Foxp3 polymorphisms to CD susceptibility or its clinical outcome, thereby making the present study as the first to describe such a correlation.
In summary, the present study suggested that