Molecular feature and therapeutic perspectives of immune dysregulation, polyendocrinopathy, enteropathy, X-linked syndrome

Journal of Genetics and Genomics xxx (xxxx) xxx

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Review

Molecular feature and therapeutic perspectives of immune dysregulation, polyendocrinopathy, enteropathy, X-linked syndrome Qianru Huang, Xu Liu, Yujia Zhang, Jingyao Huang, Dan Li*, Bin Li* Shanghai Institute of Immunology, Department of Immunology and Microbiology, Shanghai Jiao Tong University School of Medicine, Shanghai Jiao Tong University, Shanghai, 200025, China

a r t i c l e i n f o

a b s t r a c t

Article history: Received 19 July 2019 Received in revised form 2 November 2019 Accepted 10 November 2019 Available online xxx

Regulatory T (Treg) cells, a subtype of immunosuppressive CD4þ T cells, are vital for maintaining immune homeostasis in healthy people. Forkhead box protein P3 (FOXP3), a member of the forkheadewingedhelix family, is the pivotal transcriptional factor of Treg cells. The expression, post-translational modifications, and protein complex of FOXP3 present a great impact on the functional stability and immune plasticity of Treg cells in vivo. In particular, the mutation of FOXP3 can result in immune dysregulation, polyendocrinopathy, enteropathy, X-linked (IPEX) syndrome, which is a rare genetic disease mostly diagnosed in early childhood and can soon be fatal. IPEX syndrome is related to several manifestations, including dermatitis, enteropathy, type 1 diabetes, thyroiditis, and so on. Here, we summarize some recent findings on FOXP3 regulation and Treg cell function. We also review the current knowledge about the underlying mechanism of FOXP3 mutanteinduced IPEX syndrome and some latest clinical prospects. At last, this review offers a novel insight into the role played by the FOXP3 complex in potential therapeutic applications in IPEX syndrome. Copyright © 2020, The Authors. Institute of Genetics and Developmental Biology, Chinese Academy of Sciences, and Genetics Society of China. Published by Elsevier Limited and Science Press. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).

Keywords: IPEX syndrome Regulatory T cell Immune cell therapy FOXP3 Post-translational modification Transcriptional complex ensemble

1. Introduction Regulatory T (Treg) cells are a class of immunosuppressive T lymphocytes that express the key transcription factor, forkhead box P3 (FOXP3), as well as cell surface markers including CD4 and CD25 (Sakaguchi et al., 2008; Josefowicz et al., 2012). In healthy individuals, Treg cells are of special importance in maintaining immune tolerance and homeostasis. Moreover, FOXP3 can greatly influence the immunosuppressive phenotype of Treg cells (AartsRiemens et al., 2008). Disruption of FOXP3 expression results in inflammation, autoimmunity, and metabolic dysregulation owing to the development of unstable Treg cells and acquisition of effector T (Teff) cellelike function (Zhou et al., 2009; Beres et al., 2011; Bailey-Bucktrout et al., 2013; Chen et al., 2013; Li et al., 2016). Foxp3 gene mutations in mice lead to severe systemic autoimmune inflammatory diseases (scurfy phenotype) (Godfrey et al., 1991; Khattri et al., 2003). Scurfy is a spontaneous, sex-linked, recessive

* Corresponding authors. E-mail addresses: [email protected] (D. Li), [email protected], [email protected]. cn (B. Li).

mutation resulting in lethality in hemizygous males approximately 3 weeks after birth. Immunological studies showed lymphocytes and myeloid cell infiltration in multiple organs and the hyperproliferation of CD4þ CD8-T lymphocytes with elevated cytokine levels (Blair et al., 1994; Kanangat et al., 1996). Brunkow et al. (2001) used large-scale sequence analysis in sf mice to show that a 2-bp insertion can lead to the deletion of a forkhead (FKH) domain in Foxp3. They also verified the incomplete Foxp3 as the main genetic cause of the autoimmune phenotype through genetic complementation (Brunkow et al., 2001). Meantime, mutations in counterpart human FOXP3 are associated with immune dysregulation, polyendocrinopathy, enteropathy, X-linked (IPEX) syndrome (Wildin and Freitas, 2005). IPEX syndrome is one of the autoimmune polyendocrine syndromes, which happens owing to the destruction of immune tolerance and can lead to multiple endocrine gland dysfunction. The pathological changes in affected male infants often appear as lymphocyte infiltration and multiorgan inflammation. Powell et al. (1982) discovered a family in which 17 male infants died of refractory diarrhea, hemolytic anemia, diabetes mellitus, eczema, or thyroid autoimmunity in their first year of life. Data showed that for half of them, T-cell numbers, B-lymphocyte function, polymorphonuclear

https://doi.org/10.1016/j.jgg.2019.11.011 1673-8527/Copyright © 2020, The Authors. Institute of Genetics and Developmental Biology, Chinese Academy of Sciences, and Genetics Society of China. Published by Elsevier Limited and Science Press. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).

Please cite this article as: Huang, Q et al., Molecular feature and therapeutic perspectives of immune dysregulation, polyendocrinopathy, enteropathy, X-linked syndrome, Journal of Genetics and Genomics, https://doi.org/10.1016/j.jgg.2019.11.011

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leukocyte chemotaxis, and complement concentrations were normal. Hence, they speculated that the disorder was caused by the overactivity of the autoimmune system, which was X-linked recessive (Powell et al., 1982). Subsequently, similar cases were gradually noticed. These cases emphasized the diversity in the clinical manifestations of untreated immune disorders, multiple endocrine diseases, enteropathy, and IPEX syndrome. However, the underlying pathogenetic mechanism is still unclear. In short, IPEX syndrome is thought to be generated by hemizygous mutations of the FOXP3 gene, associated with severe organspecific autoimmunity destruction due to the absence or dysfunction of Treg cells. Here, we provide an overview of the current knowledge toward FOXP3 in immune dysregulation, mainly focusing on the results of FOXP3 mutations and potential treatments related to IPEX syndrome.

NFAT:FOXP3 complex functions as a repressor of IL-2 and an activator of CTLA-4 (Wu et al., 2006). A FOXP3:TIP60:HDAC7 complex is necessary for IL-2 production in Treg cells (Li et al., 2007a). Besides, FOXP1 assists FOXP3 binding to DNA through heterodimerization, and the deletion of FOXP1 can impair IL-2 signaling (Konopacki et al., 2019). In response to transforming growth factor b (TGF-b), the interaction of FOXP3 and RORgt can repress RORgt-induced transcription, resulting in limited TH17 cell differentiation (Zhou et al., 2008a). Previous studies identified AML1 (acute myeloid leukemia 1), an activator of IL-2 and IFN-g in conventional T (Tconv) cells, could interact with FOXP3 to suppress IL-2 and IFN-g and upregulate Treg-associated genes (Ono et al., 2007). These complexes shown previously may be the potential therapeutic targets useful for controlling immune responses regulated by Treg cells. 2.2. Epigenetic modifications of FOXP3

2. The regulation of FOXP3 Immunosuppressive Treg cells are distinguished by the existence of the interleukin (IL) 2 receptor a-chain (CD25), glucocorticoid-induced tumor necrosis factor (TNF) receptor (GITR), and cytotoxic T-lymphocyteeassociated antigen 4 (CTLA-4). But above all, FOXP3 plays a master role in regulating the differentiation and function of Treg cells, although not essential for thymic development (Gavin et al., 2007; Sakaguchi et al., 2008; Josefowicz et al., 2012). Furthermore, the study revealed that naive T cells could be converted to Treg cells by transferring the retroviral gene of Foxp3 with immunosuppressive phenotypes (Hori et al., 2003). Furthermore, a series of assays used gene-edited cells or mice to elucidate the dominant character of FOXP3 in immune regulation (Kim and Rudensky, 2006; Gavin et al., 2007; Lin et al., 2007; Zhou et al., 2008b). 2.1. Gene composition and the protein structure of FOXP3 FOXP3 is situated in the X chromosome short arm zone (Xp11.23) (Bennett et al., 2000). The immensely conserved FOXP3 comprises 12 exons in humans, which transcribes in a centromeric to telomeric orientation. The first exon, 50 part of exon 2, and 30 part of exon 12 belong to noncoding sequences. In humans, the fulllength open reading frame of FOXP3 encodes a 431eamino acid (aa) protein with a molecular weight of 47.25 kDa. FOXP3 protein contains four distinct domains, including N-terminal proline-rich region (PRR), central zinc finger (ZF; aa 199d222), leucine zipper (LZ; aa 239d260), and FKH domain (aa 335d418) (Mailer et al., 2009). The first three domains are important for protein-protein interactions and the last domain is obligatory for nuclear localization and DNA-binding activity (Ziegler, 2006). The N-terminal region belongs to the repressor domain (aa 67d132), which is required to repress nuclear factor of activated Tcells (NFAT)-mediated transcriptional activity (Lopes et al., 2006). To date, vital functional mutations are not found in the ZF domain. It demonstrated the deletion of glutamic acid (DeltaE250) in the LZ domain limited the ability to suppress transcription and impaired the homodimerization of Foxp3 (Chae et al., 2006). Noticeably, the FKH domain with DNA-binding activity is the most common target in patients with IPEX syndrome (Gambineri et al., 2018) (Fig. 1). When FOXP3 binds its cognate sequence GTAAACA motifs via the FKH domain, it governs the transcription of target genes, including genes that are promoted (CD25, CTLA-4, GITR, CD103, and TNFRSF18) and genes that are suppressed (IL-2, Interferon-g, IL-4, and PTPN22), leading to immunosuppression (IS). FOXP3 interacts with additional transcription factors to regulate the expression of target genes. For example, FOXP3 can target NFAT:AP-1 complexes to repress transcription of the latter target genes. Besides, the

The character of Treg cells is greatly determined by the stability of its transcriptional factor FOXP3, which can be regulated by epigenetic modifications (Lal and Bromberg, 2009). A few epigenetic targets have been reported at the FOXP3 locus, including DNA methylation and histone modifications (Floess et al., 2007; Kitagawa et al., 2015). The pattern of DNA methylation of FOXP3 usually occurs within the FOXP3 promoter. There are several conserved noncoding sequences (CNSs) that are located in the FOXP3 promoter region, including CNS1, CNS2, and CNS3. It identified CNS2, also named as Treg-specific demethylated region (TSDR), was indispensable for FOXP3 expression in the offspring of Treg cells (Zheng et al., 2010). The structure of CNS2 includes several CpG motifs that are demethylated within the 5-terminal region of FOXP3 in Treg cells (Baron et al., 2007). Studies have shown that CpG motif demethylation in CNS2 is of great importance in FOXP3 stabilization in Treg cells, while the methylation of CpG leads to transitory expression of FOXP3, which only happens in Teff cells (Huehn et al., 2009). The DNA methyltransferase (DNMT) family is largely found in the TSDR of Treg cells (Shevach and Thornton, 2014), while inducing the demethylation of CpG motifs by the DNMT inhibitor Aza brings about the induction of stable expression of FOXP3 (Lal et al., 2009). Therefore, it is feasible to generate functional FOXP3þ Treg cells under the control of DNMT inhibitors. In addition, the recent study found that Satb1 can bind to a super-enhancer located approximately 8-kb upstream of the region of the transcriptional start site. Satbl acts as a pioneer factor in Treg cells to alter chromatin accessibility for histone modifications (Kitagawa et al., 2017). The study conducted by Nagata et al. (2015) determines the role of an H3K4 histone methyltransferase, SMYD3, without which can lead to the decrease of H3K4me3 in CNS1 of the Foxp3 locus. Meanwhile, some trans-acting factors also play essential roles for FOXP3 induction, including binding of Smad3 and NFAT to CNS1 and binding of CREB and STAT5 to CNS2 (Kim and Leonard, 2007; Tone et al., 2008). Furthermore, a study conducted by Zheng et al. (2010) shows that CNS3 binds c-Rel to facilitate opening of the FOXP3 locus after T-cell receptor (TCR) and CD28 stimulating, which is able to enhance the frequency of both thymus Treg (tTreg) and peripheral Treg (pTreg) cells. On the contrary, CNS1, which is responsive to TGF-b-NFAT, can only induce the expression of pTreg instead of tTreg in lymphoid tissues (Tone et al., 2008). The aforementioned findings raise our awareness of the importance of epigenetic modifications in the regulation of FOXP3 expression. 2.3. Noncoding RNA regulation of FOXP3 Noncoding RNAs (ncRNAs) have been discovered as a novel research hotspot involved in the regulation of FOXP3. MicroRNAs

Please cite this article as: Huang, Q et al., Molecular feature and therapeutic perspectives of immune dysregulation, polyendocrinopathy, enteropathy, X-linked syndrome, Journal of Genetics and Genomics, https://doi.org/10.1016/j.jgg.2019.11.011

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(miRNAs) are small RNAs of about 22 nucleotides and are responsible for RNA silencing and post-transcriptional regulation through binding to 30 -UTR of the target mRNA or limiting the translational expression of target mRNA. At first, Dicer and Drosha, two proteins associated with the processing of miRNAs, were conditionally knocked out in Treg cell lineages, resulting in downregulated Foxp3 and abolished suppressor capacity. The results from mouse models implicated that miRNAs played a central role in maintaining the function of Treg cells (Chong et al., 2008; Liston et al., 2008; Zhou et al., 2008b). Furthermore, specific miRNAs and their contribution to disturb Treg homeostasis are illuminated. It reports that silencing of miR-126 by antisense oligonucleotides can upregulate the PI3K/Akt pathway, leading to reduced expression of Foxp3 and loss of suppressive function of Treg cells (Qin et al., 2013). MiR-34a can downregulate FOXP3 expression, which provides a way to attenuate allergic asthma by adding resveratrol to control miR-34a (Alharris et al., 2018). MiR-15a/16 as an alternation switch may affect Treg/Tconv cell plasticity. Overexpression of mi-15a/16 can decrease Foxp3 expression in umbilical cord blood (CB)ederived Treg cells, and knockdown of miR-15a/16 can confer Foxp3 expression in CB Tconv cells in the Graft vs Host Disease (GVHD) mouse model (Liu et al., 2014). Overexpression of miR-138 can inhibit the expression of FOXP3, CTLA-4, and programmed cell death protein 1 (PD-1) in human CD4þ T cells (Wei et al., 2016). There are other miRNA-mediating Treg cell functions without directly targeting FOXP3, such as miR-17 and miR-99a (Warth et al., 2015; Yang et al., 2016). Meanwhile, FOXP3 can control the expression of some miRNAs to maintain function of Treg cells. During development and proliferation of Treg cells, elevated miR155 expression controlled by FOXP3 was important to target SOCS1 (Lu et al., 2009). Long ncRNAs (lncRNAs), as the largest group of ncRNAs, have been implicated to participate in regulation of FOXP3. In Jurkat cells, lncRNA DQ786243 related to Crohn's disease can upregulate CREB and FOXP3 expression (Qiao et al., 2013). It was found that the lncRNA Flicr (Foxp3 long intergenic ncRNA), encoded within the FOXP3 promoter region, moderately decreases FOXP3 expression by modifying chromatin accessibility in CNS3/accessible region 5 (AR5) of FOXP3. The process is regulated by IL-2, resulting in impaired suppressive function of Treg cells (Zemmour et al., 2017). In hepatocellular carcinoma, lnc-epidermal growth factor receptor correlates positively with expression of FOXP3, and it could promote tumor growth by stimulating Treg cell differentiation (Jiang et al., 2017). It was identified that a lncRNA Flatr (Foxp3-specific lncRNA anticipatory of Treg cells) could indicate Foxp3 expression during Treg cell differentiation and subtly regulate the peripheral conversion process (Brajic et al., 2018). 2.4. Post-translational modifications of FOXP3 Besides the epigenetic modification of the FOXP3 locus, the expression level of FOXP3 can be affected by post-translational modifications. Acetylation of FOXP3, reciprocally managed by the histone acetyltransferase p300 and the histone deacetylase SIRT1, has been shown to stabilize the protein level and enhance the DNA-binding ability (van Loosdregt et al., 2010). Another study shows that the lack of histone deacetylase 11 (HDAC11) in FOXP3þ Treg cells results in the enhancement of Treg cells' suppressive function and increased expression of FOXP3, which is a potential drug target for autoimmune diseases. Likewise, the data show that using the HDAC11 inhibitor to target HDAC11 can promote longterm survival in fully MHC-disparate cardiac allografts (Huang et al., 2017). Phosphorylation of FOXP3 has different consequences for Treg cell function, which depends on the modification of distinct residues and induction signals (Chunder et al., 2012; Nie

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et al., 2013). Ubiquitination may be one of the most intensely studied regulatory modifications. It is considered that FOXP3 suffered from K48-type polyubiquitination for precipitating the degradation via proteasome. However, Ni et al. (2019) showed the E3 ligase TRAF6 as a crucial Treg cell regulator can stabilize its function by regulating K63-linked ubiquitination at lysine 262 loci on FOXP3. We have also identified a few key post-translational regulation of FOXP3 involving phosphorylation, ubiquitination, acetylation, methylation, and poly(ADP-ribosyl)ation. It was found that the residue S422 of human FOXP3 can be phosphorylated by PIM1, which leads to decrease in its DNA-binding activity (Li et al., 2014). The E3 ubiquitin ligase ring finger protein 31 can stabilize FOXP3 by conjugating atypical ubiquitin chains (Zhu et al., 2018). Using point mutations determined the relationship between arginine methyltransferase PRMT5 and FOXP3, suggesting the potential of PRMT5 in cancer therapy (Nagai et al., 2019). Luo et al. (2015) determined the role of poly(ADP-ribose) polymerase 1 in downregulating the suppressive function of Treg cells through Foxp3 poly(ADP-ribosyl) action. The function of E3 ubiquitin ligase STUB1 and E3 deubiquitinase USP21 in regulating the stability of FOXP3 was determined (Chen et al., 2013; Li et al., 2016). The different enzymes could, therefore, provide new therapeutic targets to control inappropriate immune responses. 3. The function of Treg cells 3.1. Classic immunosuppressive function Treg cells as a natural brake protect the host from autoimmune reactions by suppressing overstimulated immune responses. The mechanisms about how Treg cellssuppress immune responses have been explored in an ample number ofstudies. (1) The Treg cell inhibits Teff cells and natural killer (NK) cells by the secretion of immunosuppressive cytokines, including IL-10, IL-35, TGF-b, and so on (Collison et al., 2007; Li et al., 2007c; Rubtsov et al., 2008; Frimpong-Boateng et al., 2010). TGF-b inhibits the secretion of IL-2, thereby inhibiting T-cell proliferation (Das and Levine, 2008). Ectopic expression of IL-35 via retroviral transduction confers naive T cells with regulatory activity, whereas recombinant IL-35 can inhibit T-cell proliferation (Collison et al., 2007). (2) The cytotoxic effects of the Treg cell are elicited by releasing perforin and granzyme B to kill B cells, NK cells, and cytotoxic lymphocytes (Cao et al., 2007). (3) Treg cells can upregulate the expression of IL-2 receptor, competitively binding IL-2 leading to IL-2 depletion in the microenvironment. The process can prevent the binding of other T cells to IL-2 and affect T-cell activation and proliferation (Yu et al., 2009). (4) The cosuppressor molecule CTLA-4 expressed on Treg cells can bind with the high affinity to surface molecules CD80 and CD86 of antigen-presenting cells (APCs) to initiate inhibition signals (Wing et al., 2008). Meantime, the process also induces dendritic cells to produce indoleamine 2,3-dioxygenase, and it catalyzes the breakdown of tryptophan into canine urea, leading to nearby cell death (Puccetti and Grohmann, 2007). (5) The Treg cell modulates the activity of APCs and Teff cells through inhibitory receptors, involving GITR, CTLA-4, LAG-3, and PD-1 (Huang et al., 2004; Walker, 2013; Asano et al., 2017). In addition, some research studies have reported that Treg cells promoted T-cell apoptosis by tumor necrosis factorerelated apoptosiseinducing ligand/death receptor 5 pathway (Ren et al., 2007). (6) Molecules such as surface proteins, CD39 and CD73, reduce extracellular ATP concentration by hydrolyzing extracellular ATP and ADP, thereby reducing ATP-regulated proinflammation and development of dendritic cells (Deaglio et al., 2007). (7) Treg cells promote the second messenger, cAMP, transferring to Teff cells via gap junction

Please cite this article as: Huang, Q et al., Molecular feature and therapeutic perspectives of immune dysregulation, polyendocrinopathy, enteropathy, X-linked syndrome, Journal of Genetics and Genomics, https://doi.org/10.1016/j.jgg.2019.11.011

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and directly inhibit the Teff cell's immune function (Vang et al., 2013). (8) It has been demonstrated that Treg cellederived exosomes containing miRNA could suppress IFN-g production and the proliferation of Th1 effector cells (Okoye et al., 2014). Besides, Treg cells actively release exosomes to promote the transformation of other T cells to Treg cell phenotype (Aiello et al., 2017). In brief, various strategies are used by Treg cells to maintain immune homeostasis and tolerance (Fig. 2). 3.2. Nonclassical tissue-specific function Notably, researchers discovered that Treg cells in peripheral tissues played a different role in regulating tissue metabolism, stem cell maintenance, and wound healing by nonclassical phenotype. In visceral adipose tissue, resident Treg cells expressing PPAPg were found to be implicated in control of tissue metabolism and insulin resistance (Cipolletta et al., 2012). In the acutely injured skeletal muscle of mice, a phenotypically and functionally special population of Treg cells can accumulate and express the growth factor Amphiregulin. The protein can act straightaway on muscle satellite cells to potentiate muscle recovering and wound healing (Burzyn et al., 2013). Besides, the researchers demonstrated that the skinresident Treg cells tended to express a mass of the Notch ligand family member, Jagged 1, which can augment proliferation, and differentiation of hair follicles, a subset of skin stem cells (Ali et al., 2017). Similarly, Treg cells take part in protecting intestinal epithelial cells and bone marrow stem cells (Agudo et al., 2018). Finally, in the brain, Treg cells can inhibit astrocyte proliferation and promote neurological recovery after ischemic stroke (Ito et al., 2019). In summary, the understanding of the biological functions of Treg cells is rapidly evolving. Treg cells are indispensable to maintain immune homeostasis and tolerance; thereby, the dysfunctional Treg cell could have fatal effects. 4. Manifestations and pathological mechanisms of IPEX syndrome

desquamation over the limbs (Halabi-Tawil et al., 2009), whereas other skin disorders include bullae, urticaria, alopecia universalis, trachyonychia, and so on (Nieves et al., 2004). Most affected patients can also develop other types of autoimmune phenomena including cytopenia, autoimmune hepatitis, and nephropathy (Lopez et al., 2011). What is more, severe invasive infections, which may be the result of immunosuppressive therapy or the disease itself, including sepsis, meningitis, pneumonia, and osteomyelitis, can play a big role (Barzaghi et al., 2012). The outcomes of these patients are usually poor, resulting in metabolic derangements, severe malabsorption, or sepsis and even leading to death in no more than 2 years. IPEX syndrome mostly affects males as a result of the X-linked recessive disorder of FOXP3. The diagnosis of IPEX syndrome mainly depends on several typical clinical symptoms and the identification of a hemizygous pathogenic variant in FOXP3. Owing to X-chromosome inactivation (XCI), researchers analyzed the distribution of wild-type and mutated FOXP3 in normal female with heterozygous FOXP3 mutations. FOXP3 allele profiles a random choice of XCI in peripheral blood mononuclear cell (PBMC) including naive and Tconv cells; however, natural Treg solely expresses active WTFOXP3 (Di Nunzio et al., 2009). In females, the mutation must occur in both copies of the gene to cause immune disorder. However, some exceptions have been noted. During the process of pregnancy, fetal tissue needs rapid development of fetomaternal tolerance, which is associated with recurrent male miscarriage with fetal akinesia (Rae et al., 2015). Besides, in some cases, heterozygous females showed a lower expression level of FOXP3 mRNA than normal groups or the monogenic diabetes mellitus group (Bennett et al., 2001; Alkorta-Aranburu et al., 2014). The immune system of patients with IPEX syndrome with FOXP3 mutation may go wrong and even attack their tissues and organs. Up to now, around two hundred patients have been reported in the literature, and half of them were diagnosed in recent years. This suggests that a deeper knowledge of FOXP3 and Treg cell function in the maintenance of immune tolerance helps a better understanding of IPEX syndrome.

4.1. Clinical features in IPEX syndrome 4.2. Genetic changes of FOXP3 in IPEX syndrome IPEX syndrome is distinguished by the existence of multiple autoimmune diseases in males, which usually begins to occur in the first year of life. However, there are also some patients with lateonset IPEX syndrome, which appears to be a milder disease phenotype (Ge et al., 2017). There are three typical clinical manifestations in IPEX syndrome: enteropathy, endocrinopathy, and dermatitis. Usually, the first symptom in IPEX syndrome is intractable diarrhea, which belongs to autoimmune enteropathy. Villous atrophy infiltrated with activated T cells in the lamina propria was discovered. On the other hand, B cells can elevate the secretion of IgA and IgE as well as produce specific antibodies in IPEX syndrome, such as antiharmonin autoantibodies (HAAs) and antivillin autoantibodies (VAAs) (Lampasona et al., 2013). Theses antigens are distributed in the small intestine and proximal renal tubules, consisting of high prevalence in enteropathy. The disorders in intestines can lead to the symptom of malabsorption, maldevelopment, weight loss, and so on owing to the lack of total parenteral nutrition (Tan et al., 1993). Toward endocrinopathy, type 1 diabetes mellitus can occur around enteritis in most cases, and autoimmune thyroid disease may also happen (Wildin et al., 2002). The imaging studies which showed immune-mediated damage to the pancreas may explain why sugar content could not be controlled in the body of patients with IPEX syndrome (Rubio-Cabezas et al., 2009). The majority of the dermatitis cases are eczematous, with abnormal red patches and irritated skin. The elevated levels of IgE may result in skin

The occurrence of symptoms of IPEX syndrome is closely related to the genetic changes in FOXP3. The pathogenic variants in FOXP3 include nonsense variants, missense variants, small in-frame amino acid deletions/insertions, and splice site variants. Surprisingly, similar genotypes can result in dramatically different phenotypes with regard to disease severity, even from the same family. Functional loss variants (frameshifts), which predict missing FKH domains, are indicated in both fetal and nonviable cases. They can also be described in individuals who survive to adolescence (Kobayashi et al., 2001; Louie et al., 2017). Meanwhile, compared with the expression level of FOXP3, the activity of expressed FOXP3 is more important for disease severity. 4.2.1. Mutation of FOXP3 Although the incidence of the IPEX mutant is quite rare, so far, a large number of FOXP3 mutations associated with this disease have been reported. The c.1150G>A mutation, which has been predicted to cause the missing of the FKH domain, is observed to be the most common mutation in several studies. It has been reported in fetalonset and nonviable cases, as well as in individuals who might survive to adolescence (Barzaghi et al., 2012). Another highfrequency mutation is c.1189C>T, which has been reported in four neonatal or fetal deaths, also is located in the FKH domain (Nieves et al., 2004; Gambineri et al., 2018). These two FKH domainerelated mutations are of great importance to DNA binding and are both

Please cite this article as: Huang, Q et al., Molecular feature and therapeutic perspectives of immune dysregulation, polyendocrinopathy, enteropathy, X-linked syndrome, Journal of Genetics and Genomics, https://doi.org/10.1016/j.jgg.2019.11.011

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highly lethal, generally. What is more, the single IPEX mutation A384T lying within a helix H3 can influence DNA contact (Wu et al., 2006), while the mutation in other location is sometimes highly risky. C319-320del stands for deleting two-base pair in the N-terminal domain, resulting in four neonatal or fetal deaths (Bacchetta et al., 2018). Two mutations located in exon 3 are thought to result in frameshifts and premature stop codons (227DT and 303_304DTT) (Kobayashi et al., 2001). Among these identified mutations, nearly half are located in the sequence encoding the Cterminal FKH DNA-binding domain, followed by the PRR domain, LZ-FKH loop, and even some in the noncoding region. The LZ region is vital for FOXP3 to form dimers. An IPEX deletion mutation (DE251) in this region can neither homo-oligomerize with itself nor hetero-associate with FOXP1, which impairs its capability to inhibit IL-2 transcription (Li et al., 2007b). The mutations DE251 and DK250 may directly influence the intersubunit salt bridge, which leads to a disruption of FOXP3 homodimerization and an impaired inhibiting ability (Song et al., 2012). The FKH domain of FOXP3, besides, can shape a domain-swapped dimer, while three IPEX mutations are found within or near this region, including F371C, F373A, and R347H. As a result, the ability of FOXP3 to inhibit transcription can be impaired by these domain-swap mutations (Bandukwala et al., 2011). When mutations occur in the promoter and 50 untranslated region of FOXP3, they will lead to a milder clinical phenotype. For instance, a 1388-bp deletion (g.del-62474859) encompassing the upstream noncoding exon and the adjacent intron of FOXP3 leads to the accumulation of unsliced premRNA and alternatively spliced mRNA. The large deletion can reduce the amount of Treg cells, resulting in enteropathy, impressive allergic phenotype, and elevated IgE levels while without endocrinopathy (Torgerson et al., 2007). Another group of researchers has found that some FOXP3 variants may only result in insulin-dependent diabetes without other characters of IPEX syndrome (Hwang et al., 2018). Specific mutations can also cause unique clinical phenotypes. For instance, a patient with G>A (1150G>a) at the DNA-binding site can suffer from psoriasiform dermatitis and alopecia universalis (Nieves et al., 2004). Regarding the most common mutation in IPEX syndrome, p.A384T, Bin Dhuban et al. (2017) have found that this mutation on the Treg cell can disintegrate the linkage between FOXP3 and histone acetyltransferase TIP60. This disruption results in the selective impairment of Treg cell suppressive function and can be revised by certain allosteric modifiers, which provides a potential target for Treg cell treatment of IPEX syndrome. The development of modern sequencing technology can help identify more mutation sites related to IPEX syndrome. However, the exact relationship between the genotype and phenotype of IPEX syndrome still has not been fully understood. 4.2.2. Alternative splicing of FOXP3 There is evidence indicating the conservative occurrence of FOXP3 regulation, interaction, and function within several species, but alternative splicing of FOXP3 is only discovered in humans, which may play a key role in IPEX syndrome. Notably, the structure of the FOXP3 gene controls the number of potential exon deletions. Alternative splicing is a conservative process relying on particular exons with ribonucleoproteins-identified sequence. As blunt 50 and 30 -tails only in exon 3 and exon 8, it is currently known that alternative splicing sequences can express human FOXP3 splice isoforms in CD4þ T cells: full-length FOXP3 (FOXP3fl) and exon 3 (FOXP3D3; D shows the deleted exon in the protein), exon 8 (FOXP3D8), and exon 3 and exon 8 (FOXP3D3D8) deleting isoforms (Allan et al., 2005; Mailer et al., 2009). Several cases involve exon 3 as exon 2 and exon 8 as exon 7 because the noncoding exon E1 is neglected. The function of exon 3 (aa 72d106) is shown to be

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related to blocking retinoic aciderelated orphan receptors a and gt (Ichiyama et al., 2008). Exon 8 encoding a LZ motif is considered to be essentially necessary and sufficient to FOXP3 function. Generally, FOXP3D3 is coexpressed with FOXP3fl in comparable amounts, while the activation of Treg cells can promote the excision of exon 3 of FOXP3 mRNA, and FOXP3D8 expression is at a low level. Some results also suggest the association between FOXP3fl/FOXP3D3 and autoimmune as well as inflammatory diseases (Ziegler, 2006; Joly et al., 2018). It has been reported that human FOXP3D3D8 cannot confer suppressive ability to Treg cells and does not affect proteinprotein interaction experimented by generating mice exclusively expressing FOXP3D3D8 (Joly et al., 2015). Besides, the increasing expression of FOXP3D3D8 may promote Th17 cell differentiation (Mailer et al., 2015). However, the lack of suitable animal models may hinder the study of the function of different FOXP3 isoforms in human beings. The mutations from patients with IPEX syndrome that affect alternative splicing could help to understand the function of FOXP3 exons. A de novo substitution (c.210þ1G>T) is identified as a medical replacement, leading to abnormal splicing of FOXP3 (Otsubo et al., 2011). Especially, Treg cells in patients with IPEX syndrome have not been found to express FOXP3D3 alone, detected by flow cytometry (Fuchizawa et al., 2007). A report about the molecular phenotype of a family with two patients and one carrier shows the opposite result. One patient with a milder IPEX phenotype is a result of selective deletion in FOXP3 exon 3 expression. It speculates that the retained expression of FOXP3D3 can support Treg cell growth and partly protect against IPEX syndrome despite lacking FOXP3fl (Frith et al., 2019). The result suggests a new option for IPEX syndrome treatment and provides potent patient-derived evidence for FOXP3D3 functional studies. In addition, changes in the expression of FOXP3 isoforms have also been studied in a variety of diseases, including Crohn's disease, carcinoma, and so on. 5. Clinical diagnosis and treatment of IPEX 5.1. Diagnosis of IPEX syndrome IPEX syndrome is classified by the International Union of Immunological Societies as a disease of immune dysregulation (Bousfiha et al., 2018). First, the diagnosis of IPEX syndrome is based on the typical clinical findings in male patients. Second, it is important to identify a heterozygous pathogenic variant in FOXP3 by molecular genetic testing, such as single-gene testing, exome sequencing, and genome sequencing. When the symptoms of IPEX syndrome are typical, sequence analysis of FOXP3 can help detect small pathogenic variant and intragenic deletions or duplications. However, if it is difficult to distinguish between symptoms of IPEX syndrome and many other inherited disorders, comprehensive genomic testing (exome sequencing and genome sequencing) is recommended to confirm disease types. Because some evidence show that some FOXP3 variants only result in insulindependent diabetes without other clinical characteristics of IPEX syndrome, such as p.Lys393Met and c.1044_5G>A (Hwang et al., 2018). The scope of application of genetic testing needs to be carefully considered. A recent study reported prenatal ultrasound was associated with diagnose of IPEX syndrome. It broadens the phenotypic spectrum of IPEX syndrome and proves the feasibility of prenatal ultrasound diagnosis (Louie et al., 2017). Especially, it has demonstrated the feasibility of screening and clinical monitoring of patients with IPEX syndrome by detecting HAAs and VAAs. A fanciful Luminescent-Immuno-Precipitation-System quantitative test was used to detect HAAs and VAAs to help diagnose IPEX syndrome (Lampasona et al., 2013).

Please cite this article as: Huang, Q et al., Molecular feature and therapeutic perspectives of immune dysregulation, polyendocrinopathy, enteropathy, X-linked syndrome, Journal of Genetics and Genomics, https://doi.org/10.1016/j.jgg.2019.11.011

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5.2. Therapy for IPEX syndrome There are three major barriers, including autoimmunity, inflammation due to autoimmune damage, and the seriousness of the overview, in the treatment of patients with IPEX syndrome (Barzaghi et al., 2012). Currently, IS drugs and bone hematopoietic stem cell transplantation (HSCT) are main treatments of IPEX syndrome. Allogeneic HSCT is the primary therapeutic approach for IPEX syndrome. For those patients without HSCT, IS therapy and long-term supportive care are considered. Because large damage can be caused by the autoimmune disorder, supportive care in the hospital is essential for patients. Nutritional supports, including total parenteral nutrition or elemental or low-carbohydrate-containing formula and fluid, are necessary for patients with severe enteropathy. Other replacement therapies for some complications such as autoimmune cytopenia and endocrinopathy are also needed. HSCs have the competence to self-renew and differentiate into various kinds of mature blood cells, including Treg cells. Allogeneic HSCT has been widely applied to treat diseases related to blood cells, using cells from either human leukocyte antigen (HLA)identical siblings or other HLA-matched donors (Hatzimichael and Tuthill, 2010). Baud et al. (2001) carried out an allogeneic bone marrow transplantation from an HLA-identical sister to the patients with IPEX syndrome. The genotype of 95% of white cells is recovered successfully after the transplantation. Although the patient died of hemophagocytic syndrome, at last, main IPEX syndromes have been overcome (Baud et al., 2001). After that, more patients received HSCT from either CB, peripheral blood, or the bone marrow (Lucas et al., 2007; Zhan et al., 2008). According to a recent long-term flow-up multicenter retrospective study, which evaluated 58 patients who received HSCT, the survival of patients after HSCT is strongly affected by patients' manifestation before the transplantation (severity of patients' condition may contribute to a high death rate in early years after transplantation) but has a poor correlation with the type of donors, stem cell source, and chimerism. It suggests prompt diagnosis and transplantation and the optimization of pre-transplant condition might be crucial (Barzaghi et al., 2018). IS therapies are used to control the acute phase of autoimmune in IPEX syndrome, usually with combinations of several IS agents. Most patients who received transplantation also received IS therapy. Most frequently used IS drugs are calcineurin inhibitors,

cyclosporine A and tacrolimus, providing direct suppression to T cells, while some also received concomitant steroids. Rapamycin, noncalcineurin inhibitor, is now considered to be the primary choice of IS drugs either used alone or in combination (Barzaghi et al., 2018). Most IS therapies are beneficial to patients' manifestation (one-third of the patients totally overcome autoimmunity); however, IS therapies without transplantation do not have changed disease progression, and manifestation (organ involvement score) is decreasing in long-term flow-up, which have a negative impact on survival. Although HSCT might be the only curative treatment for IPEX syndrome, with the development of genetically reprogrammed techniques, we might engineer Treg-like cells from patients' autologous cells through replacing or repairing the FOXP3 mutants with wild-type ones. There have been some meaningful attempts of gene therapy based on either Treg cells or stem cells (Passerini et al., 2014). Potent and stable human Treg cells can be produced from both naive and memory CD4þ T cells by lentivirus FOXP3 transfer (Aarts-Riemens et al., 2008; Allan et al., 2008). It is also possible to develop gene therapies for IPEX syndrome by generating CD4þ T cells expressing wild-type FOXP3. Researchers have successfully converted CD4þ T cells from patients with IPEX syndrome into Treg-like CD4FOXP3 cells by transferring the wild-type FOXP3 gene with the lentiviral vector and established potent suppressor function both in vitro and in the humanized murine model (Passerini et al., 2013). Whether these generated Treg-like cells can establish sufficient suppression function in patients with severe autoimmune needs evaluation and further study (Dawson et al., 2017). Because roles of FOXP3 are not fully understood, it might be insufficient to generate only Treg cells expressing wild-type FOXP3. Gene therapies based on engineered HSCs have been applied to some diseases such as Wiskott-Aldrich syndrome (Aiuti et al., 2013) and severe combined immunodeficiency (SCID) due to adenosine deaminase deficiency (Aiuti et al., 2009). With the inspiration of successes in HSCT treatment, generating wild-type FOXP3eexpressing HSCs could be considered but is also more challenged because overexpression of FOXP3 could be detrimental in hematopoietic precursors (Passerini et al., 2014). Apart from the lentivirus gene transfer approaches, the genome editing approach is also promising and might have a better long-term therapeutic effect than gene transfer. Genome editing uses a site-specific endonuclease to produce a DNA double-strand break (DSB), and sequence in the DSB can be repaired by a donor sequence or, if no

Fig. 1. Schematic diagram of the human FOXP3 gene structure, mRNA structure, and functional features. The human FOXP3 gene comprises 12 exons in humans, in which the first exon, 50 part of exon 2, and 30 part of exon 12 belong to noncoding sequences. FOXP3 protein contains four distinct domains, including the N-terminal proline-rich region, central zinc finger, leucine zipper, and forkhead (FKH) domain. These domains can interact with different proteins to regulate transcriptional activity and affect Treg cell function.

Please cite this article as: Huang, Q et al., Molecular feature and therapeutic perspectives of immune dysregulation, polyendocrinopathy, enteropathy, X-linked syndrome, Journal of Genetics and Genomics, https://doi.org/10.1016/j.jgg.2019.11.011

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conducted by Barzaghi et al. (2018). This study including 96 patients (34 patients treated with IS therapy, 58 patients treated with HSCT and 4 patients improved spontaneously) reveals similar overall survival but diverse disease-free survival for patients receiving IS or HSCT. Although most patients' clinical manifestation improved after IS therapy and rapamycin is proven to be the most effective inhibitors, IS therapy shows a limitation in disease progression control (showing low and decreasing disease-free survival). Patients who received HSCT showed slightly lower overall survival but much higher disease-free survival than patients who received only IS therapy, which drops fast with disease progression. In conclusion, the only and primary choice for curing IPEX syndrome was HSCT. Because it might be not easy to receive prompt transplantation from HLA-matched donors, gene therapy is promising. And efforts to solve this FOXP3 monogenetic disorder can also provide experience and inspire cell therapy based on Treg cells (Fig. 3).

6. Perspectives

Fig. 2. Molecular mechanisms of the immunosuppressive function of Treg cells. Treg cells use diverse strategies to exert their immunosuppressive influence: (1) secreting exosomes with immunosuppressive miRNAs; (2) secreting immunosuppressive cytokines, including TGF-b, IL-10, and IL-35; (3) adenosine generated by CD39 and CD73 to inhibit DC cells; (4) cosuppressor molecule CTLA-4 expressing on Tregs can bind with high affinity to surface molecules CD80 and CD86 of antigen-presenting cells (APCs) to initiate inhibition signals. The process also induces dendritic cells to produce IDO, which catalyzes the breakdown of tryptophan into canine urea, leading to nearby cell death; (5) upregulating IL-2 receptor expression and competitively binding IL-2, leading to IL-2 depletion in the microenvironment; and (6) cytotoxic effects elicited by perforin and granzyme. CTL, cytotoxic T-lymphocyte; Teff, effector T; Treg, regulatory T; IL, interluekin; CTLA-4, cytotoxic T-lymphocyteeassociated antigen 4; miRNA, microRNA; TGF, transforming growth factor; TCR, T cell receptor; DC, dendritic cells; IDO, indoleamine 2,3-dioxygenase.

donor sequence, nonhomologous end joining (Kohn, 2018). Gene editing on HSCs from patients with SCID-XI has been applied and successfully corrected the IL2RG gene mutation (Genovese et al., 2014). For mutations of FOXP3 in IPEX syndrome scattered throughout the whole gene, functional correction approaches are considered more suitable (Passerini et al., 2014). Because the IPEX syndrome cases reported are really rare, the two main therapeutic approaches were not compared and evaluated until a long-term flow-up multicenter retrospective study was

IPEX syndrome is a rare disease, with an extremely high mortality rate if untreated. The incidence of disease may be underestimated because of previous immature sequencing technology. The diagnosis of the disease depends on the detection of genetic mutations. Now, widespread genetic tests and large cohort studies provide new ideas for disease occurrence and treatment. But the research of pathogenesis stays on finding the class of abnormal FOXP3 proteins, while the disorder mechanism evoked by different FOXP3 proteins is unclear. The association of FOXP3 with IPEX syndrome highlights the significance of FOXP3 in maintaining normal immune homeostasis. Different FOXP3 mutations greatly affect the severity of IPEX syndrome, which provides an effective way to study the importance of the different residues in FOXP3 protein and their various roles in interactions. Meantime, an in-depth understanding of the molecular mechanisms underlying the function of FOXP3 in Treg cells can help discover new therapies for autoimmune diseases and cancer. Bin Dhuban et al. (2017) uncovered that a small-molecule modifier of TIP60 rescued the dysfunction of Treg cells carrying the A384T mutation by enhancing FOXP3-TIP60 interaction. It provides potential avenues not only for IPEX syndrome but also for achieved disease control in mouse models of collagen-induced arthritis and colitis. Charbonnier et al. (2019) demonstrated that the limited set of pathways, including mTORC2 and glycolysis, are main regulators during the skewing of Treg cells toward a Teff-like phenotype. And

Fig. 3. The diagnosis of and therapy for IPEX syndrome. First, the diagnosis of IPEX syndrome is based on the typical clinical findings in male patients. Second, it is important to identity a heterozygous pathogenic variant in FOXP3 by molecular genetic testing. Currently, IPEX syndrome is treated with either hematopoetic stem cell transplantation or immunosuppression (IS) therapy with cyclosporin A or FK506. For those patients without HSCT, IS therapy and long-term supportive care are considered. IPEX, immune dysregulation polyendocrinopathy enteropathy X-linked; Teff, effector T; Treg, regulatory T; FOXP3, forkhead box P3.

Please cite this article as: Huang, Q et al., Molecular feature and therapeutic perspectives of immune dysregulation, polyendocrinopathy, enteropathy, X-linked syndrome, Journal of Genetics and Genomics, https://doi.org/10.1016/j.jgg.2019.11.011

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using mTOR inhibitors can restore Treg cell function in IPEX syndrome, with decreased cytokine expression and elevated suppression ability (Charbonnier et al., 2019). It provides a new opportunity to reinstate immune tolerance. On the other hand, IPEX syndrome, as a monogenic autoimmune disease, is a suitable model for evaluating the new immunomodulatory drug efficacy and the safety of gene therapy. At present, the most successful treatment for IPEX syndrome might be HSCT (Barzaghi et al., 2018). Nevertheless, the treatment for patients with limited donor availability or a mild disease phenotype needs to be further explored. In the future, the researchers should raise concerns on different immunosuppressive treatments targeting the regulation of gene expression and protein stability of FOXP3. 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Please cite this article as: Huang, Q et al., Molecular feature and therapeutic perspectives of immune dysregulation, polyendocrinopathy, enteropathy, X-linked syndrome, Journal of Genetics and Genomics, https://doi.org/10.1016/j.jgg.2019.11.011