Clinic Rev Allerg Immunol DOI 10.1007/s12016-015-8470-2

CD24: from a Hematopoietic Differentiation Antigen to a Genetic Risk Factor for Multiple Autoimmune Diseases Yixin Tan & Ming Zhao & Bo Xiang & Christopher Chang & Qianjin Lu

# Springer Science+Business Media New York 2015

Abstract The autoantibody is an essential characteristic of inflammatory disorders, including autoimmune diseases. Although the exact pathogenic mechanisms of these diseases remain elusive, accumulated evidence has implicated that genetic factors play important roles in autoimmune inflammation. Among these factors, CD24 was first identified as a heatstable antigen in 1978 and first successfully cloned in 1990. Thereafter, its functional roles have been intensively investigated in various human diseases, especially autoimmune diseases and cancers. It is currently known that CD24 serves as a costimulatory factor of T cells that regulate their homeostasis and proliferation, while in B cells, CD24 is functionally involved in cell activation and differentiation. CD24 can enhance autoimmune diseases in terms of its protective role in the clonal deletion of autoreactive thymocytes. Furthermore, CD24 deficiency has been linked to mouse experimental autoimmune encephalomyelitis. Finally, CD24 genetic variants, including single-nucleotide polymorphisms and deletions, are etiologically relevant to autoimmune diseases, such as multiple sclerosis and systemic lupus erythematosus. Therefore, CD24 is a promising biomarker and novel therapeutic target for autoimmune diseases. Y. Tan : M. Zhao : Q. Lu (*) Department of Dermatology, Second Xiangya Hospital, Hunan Key Laboratory of Medical Epigenetics, Central South University, #139 Renmin Middle Rd, Changsha, Hunan 410011, People’s Republic of China e-mail: [email protected] B. Xiang Cancer Research Institute, Xiangya School of Medicine, Central South University, #110 Xiangya Road, Changsha, Hunan 410078, People’s Republic of China C. Chang Division of Rheumatology, Allergy and Clinical Immunology, University of California at Davis, Suite 6510, 451 Health Sciences Drive, Davis, CA 95616, USA

Keywords CD24 . Hematopoietic differentiation antigen . Autoimmune diseases . Genetic susceptibility . Autoantibodies

Introduction When it was first discovered in 1978, CD24 was initially named heat-stable antigen (Ag), because it was discovered to be extremely stable upon heat inactivation [1]. The mouse Cd24 gene was then successfully cloned in 1990 [2] and mapped to chromosome 10 [3]. Later in the same year, the human CD24 gene at chromosome 6q21 was cloned [4]. As a small protein with 27 residues, mouse Cd24 has seven potential glycosylation sites and is localized to the plasma membrane through binding with a glycosyl-phosphatidylinositol (GPI) anchor [2]. In comparison, a mature human CD24 protein has 32 residues and is similarly linked to the cell membrane via a GPI anchor [4]. The molecular weight of both murine and human CD24 proteins ranges from 28 to 68 kDa due to the differential glycosylation in various tissues and cell types [5]. More than 20 organisms have been discovered to possess orthologs of the human CD24. However, CD24 is not evolutionally conserved among species mainly due to the fact that the protein core of CD24 is very short and comprises only approximately 30 amino acids. Autoimmune diseases are inflammatory disorders that are largely characterized by autoantibody production, although the target autoantigen has yet to be discovered in some of these diseases, such as psoriasis [6–12]. The precise mechanisms that underlie autoimmune diseases remain unclear, partly because of the diversity of autoimmune disease phenotypes, and treatment of autoimmune diseases has been an ongoing challenge [13–15]. Both T and B cells play separate and

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interacting roles in autoimmune diseases, and the interplay between various subsets of T cells, including CD4+ helper cells and their ever-increasing subtypes, CD8+ cytotoxic cells, and a myriad of T regulatory cells is becoming clear [16]. Accumulating evidence has found an association between CD24 and autoimmune responses [17]. Current knowledge of the biological functions of CD24 is very limited, but it is known that CD24 does possess diversified immunological functions, including critical regulation of lymphocyte maturation, neuronal cell differentiation, and physiological tissue renewal. These functions suggest that CD24 function is of a regulatory nature under normal conditions and appears to exert its effects on both T and B cell populations. Equally important is that CD24 has been proposed to be a genetic modifier associated with multiple autoimmune diseases.

The Distribution of CD24 Expression Normally, CD24 is expressed by diversified cell types, but mainly on immature cells. These include most hematopoietic cells, such as immature T cells [18–20] and B cells [4, 21], developing neuronal cells [22], and regenerating muscle cells [23]. CD 24 is also expressed on granulocytes [24], monocytes [25, 26], dendritic cells (DCs) [27], macrophages [28], keratinocytes [29], epidermal Langerhans cells [30], and epithelial cells in various tissues, including embryonic intestinal, nasal, salivary gland, and renal gland [31–33]. It can also be expressed on murine erythrocytes as well as on numerous cancer cell types [34] (Fig. 1). CD24 usually is not expressed on cells that are fully

Fig. 1 CD24 expression. CD24 is expressed by many cell types, including immature lymphocytes, developing neuronal cells, regenerating muscle cells, granulocytes, monocytes, dendritic cells, macrophages, keratinocytes, epidermal Langerhans cells, epithelial cells, and erythrocytes

differentiated. Therefore, during the maturation of T and B cells, the expression of CD24 is first strongly induced and then repressed as the cells mature [35]. The exceptions to this are erythrocytes, since these always express high levels of CD24.

Glycosylation Pattern, Ligands of CD24 CD24 is a mucin-type sialoglycoprotein with different glycosylation patterns. Edwin et al. analyzed oligosaccharides linked to human CD24 by the use of MALDI-TOF-MS. The data showed that both O- and N-glycosylation occurs. The major oligosaccharides linked to the CD24 protein core include the O-glycans Neu5Acα-2,3/6Galβ-1,3GalNAc and NeuAc2Gal β-1,3GalNAc1 and the N-glycans GalNAc2GlcNAc2Man3Fuc, Gal1GalNAc2GlcNAc2Man3Fuc1, and Gal2GalNAc2GlcNAc2Man3Fuc1. Among these oligosaccharides, Neu5Acα-2,3/6Galβ-1,3GalNAc (sialyl-tumor antigen, sT) was found to be the most abundant glycan associated with CD24 [36]. The function of CD24 is considered to be primarily mediated by the N- and/or O-glycans linked to the protein core. Two reports in 2009 identifying CD24 in mouse brain with structures containing α2,3-linked NeuAc, disialyl motifs, Lewisx sLex or HNK-1 epitopes in O-glycans [37], and α2, 3-linked N-acetylneuraminic acid (NeuAc), Lewisx H antigens, bisecting N-acetylglucosamine in N-glycans [38] (Table 1). Evidence shows that the glycosylation pattern seems to be variable in different tissues, which may account for the functional diversity of CD24 [39]. There are several ligands found to bind to CD24 protein, including P-selectin

Clinic Rev Allerg Immunol Table 1 Main categories of O-/ N-glycans of CD24 analyzed by MALDI-TOF-MS Human

Mice

N-glycans

O-glycans

Reference

GalNAc2GlcNAc2Man3Fuc Gal1GalNAc2GlcNAc2Man3Fuc1 Gal2GalNAc2GlcNAc2Man3Fuc1 α2,3-linked N-acetylneuraminic acid (NeuAc) Lewisx H antigens bisecting N-acetylglucosamine

Neu5Acα-2,3/6Galβ-1,3GalNAc NeuAc2Gal β-1,3GalNAc1

[36]

α2,3-linked NeuAc disialyl motifs Lewisx sLex HNK-1

[37, 38]

[40], sialic-acid-binding immunoglobulin-like lectin-G (Siglec-G, mouse) or Siglec 10 (human) [41], neural recognition molecule L1 [42], TAG-1, Contactin [43], and some damage-associated molecular patterns (DAMPs) such as HMGB1 [41]. Because the peptide core of CD24 is short, being only 27– 32 amino acids in length, it has been proposed that the ligand specificity of CD24 is mediated by its glycosylation pattern. For example, in mice brain, the binding of CD24 with the molecule L1 is mediated by α2,3-linked sialic acid on CD24 [37]. Besides α2,3-sialyl glycans, Lewisx glycans are also linked to the CD24 protein core. Unlike CD24 binding to L1 via its α2,3-sialyl glycans, the interactions of CD24 with TAG-1 and Contactin is mainly mediated by Lewisx glycans [43]. The function of CD24 is considered mainly if not exclusively mediated by its carbohydrates [43]. It has been proposed that CD24 induces neurite outgrowth by interaction of L1 with sialylated CD24 instead of GPI-linked CD24 [43]. Through the years, considerable research has been carried out by scientists from many regions to elucidate the correlations between glycosylation of CD24 and specific disease states. Some of these studies have confirmed that differences in glycosylation and specific carbohydrate structures are markers for diagnostic and prognostic evaluation in different cancers and autoimmune diseases. Increased sialylated antigenic determinants, such as sialyl-Lewisa (sLea NeuAcα2, 3Galβ3(Fucα4)GlcNAc) and sialyl-Lewisx (sLex NeuAcα2, 3-Galβ4(Fucα3)GlcNAc) are associated with tumor metastasis in breast and colon cancer, among others [44]. Other examples illustrate that sialyl-Tn (STn; NeuAcα2,6GalNAcSer/Thr), sialyl-3 T (S-3 T; NeuAcα2,3Galβ3GalNAc-Ser/ Thr), sialyl-6 T (S-6 T; Galβ3(NeuAcα2,6)GalNAc-Ser/ Thr), and disialyl-T (NeuAcα2,3-Galβ3(NeuAcα2, 6)GalNAc-Ser/Thr) are mucin-type O-glycans found in increasing prevalence in several other cancers [45, 46].

Signaling Pathways Mediated by CD24 Due to the absence of cytoplasmic domains, CD24 can only transduce intracellular signals through the interaction with its binding partners to activate bioprocesses, including protein

phosphorylation, intracellular calcium mobilization, and transcription factor activation [21]. Specifically, CD24-mediated signaling has been associated with glycolipid-enriched membrane (GEM) domains or lipid rafts, structures that have abundant glycosylsphingolipids (GSL), cholesterol, sphingomyelin, and GPI-anchored proteins. These components function together to form a micro-domain, which switches the cytoplasm redistribution of Src family protein tyrosine kinases (PTK); subsequently, the PTK auto-phosphorylation-induced activation of downstream signaling cascades can be initiated [21] (Fig. 2).

CD24 Regulates the Homeostatic Proliferation, Negative Selection, and Differentiation of T Cells CD24 is highly expressed on developing T cells, but its expression is reduced when T cells reach maturity [47]. CD24 can promote the differentiation of immature T cell and induce apoptosis of mature T cells, while serving as an adaptor in autoimmune diseases by setting thresholds for T cell responses. At a steady state, peripheral T cells have very low CD24 expression; however, after the formation of the TCRCD3 complex, expression of CD24 is greatly increased [18, 48]. While on antigen-presenting accessory cells, CD24 expression is an important costimulatory signal that is required for the T cell clonal expansion [18]. Without TCR–CD3 engagement, binding CD24 with mT-20, an anti-CD24 mAb, can mediate the cell death of both CD4−CD8− and CD4+CD8+ thymocytes as well as the thymic lymphoma Scid.adh cells [49]. CD24-induced mouse thymocyte apoptosis has been associated with reactive oxygen species (ROS) and mitochondrial regulation [49]. CD24 expression on activated B cells serves as a costimulatory factor as well, promoting the proliferation of CD4+ and CD8+ T cells [50]. However, CD24 mutations do not necessarily affect T cell priming in lymphoid organs [51, 52]. In addition, the proliferation rate of CD24-deficient T cells is significantly reduced upon adoptive transferred to syngeneic lymphopenic hosts [19]. In the same context, naïve T cells will undergo massive proliferation, which leads to a cytokine outburst and the quick death of recipient mice [53].

Clinic Rev Allerg Immunol Fig. 2 CD24 protein structure and CD24-mediated signaling transduction. CD24 is hooked onto the membrane by a GPI anchor. Both murine and human CD24 proteins are heavily and differentially glycosylated. CD24 serves as a signaling molecule and mediates signal transduction by recruiting Src family protein tyrosine kinases (PTKs). GPI glycosyl-phosphatidylinositol; Lyn, LYN proto-oncogene, Src family tyrosine kinase; Fgr FGR proto-oncogene, Src family tyrosine kinase; Lck LCK protooncogene, Src family tyrosine kinase

This may partially involve NFAT5, a transcription factor for CD24 that is required to sustain T cell expansion [54]. In addition, CD24 deficiency can synergize with CD28 deficiency to suppress the responses of CD4+ and CD8+ T cell [51]. The CD24- and CD28-related signaling can promote the development of memory T cells that are able to recall immune responses when secondarily exposed to an antigen [52]. Hence, CD24 expressed by non-T cells are responsible for negatively regulating T cell homeostasis. The clonal deletion of autoreactive T cells occurs in the thymus and is known as the process of negative selection. When T cells escape negative selection, autoimmune diseases may result. In this context, CD24 is required for the survival of autoreactive T cells in the thymus and promotes their escape from negative selection. By chimera experiments with bone marrow (BM), CD24 expression on radio-resistant stromal cells has been shown to amplify the resistance to the autoreactive T cell deletion [55]. Using 2D2-TCR-transgenic mice, a subsequent study confirmed that Cd24-deficient mice (2D2+Cd24/−) lack mature 2D2 T cells via a myelin oligodendrocyte glycoprotein (MOG) antigen-related mechanism. The CD24 expression on both medullary thymic epithelial cells (mTECs) and DCs, but not on thymocytes, is responsible for preventing the autoantigen-mediated clonal deletion of autoreactive thymocytes [56]. In addition, restoration of CD24 expression on DC, but not thymocytes, partly recovered the 2D2 + T cell generation [56]. A recent study showed that the formation of the Siglec-G (on host APCs)-CD24 (on donor T cells) axis can attenuate graft-versus-host disease (GVHD) [57].

CD24 and B Cell Development CD24 is a B cell differentiation antigen, and its abundance is correlated with B cell maturation [58]. High expression levels of CD24 can be observed starting from B cell progenitors to mature resting B cells. However, CD24 expression declines upon B cell activation and further maturation. Furthermore, it has been shown that during the transition from multipotent BM-derived Lin−CD43+B220low (B220low) progenitor cells to B cell progenitors, the loss of CD24 expression and silencing of c-kit occur simultaneously [59]. Indeed, CD24 expression in human B cells is an even earlier event than the CD19 expression [60]. In germinal center (GC) B cells, CD24 expression is prominently reduced [61]. In BM cultures, treatment with anti-CD24 mAb can mediate the apoptosis of precursor B cells [62]. Such a process does not affect the apoptosis of mature splenic B cells; however, anti-CD40+ IL-4-mediated stimulation of cell proliferation can be compromised, suggesting that CD24 is a negative regulator for B cell development [62]. The overexpression of Cd24 in the transgenic (Tg) mouse increases the apoptotic rate and population deletion of pre-B cells in mouse BM [63]. However, the apoptotic rate of pro-B cells and B cells were not considerably affected in the BM of Cd24-Tg mouse, which suggests that the apoptotic effect of Cd24 overexpression is highly selective for specific cell differentiation stages [63]. Surprisingly, Cd24-deficient mice have normal peripheral B cell frequencies and normal T and myeloid cell development and maintain normal immune functions in numerous immunization and infection models [64]. However, the immature B cell and late pre-B populations in the BM are decreased,

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suggesting that the Cd24 deficiency mediated block in B cell development is incomplete [64]. In particular, the Tg mouse with knockout of both CD24 alleles has normal peripheral B cell frequencies and normal IgM and IgG titers, suggesting that CD24 is not an absolute requirement for B cell maturation [65]. Suzuki et al. found that CD24 mediates intracellular signaling and induces apoptosis of Burkitt’s lymphoma cells through a GEM-dependent mechanism, which can be boosted by activating B cell receptor (BCR)-associated signaling [21]. Such a CD24-induced apoptosis can be inhibited by blocking BCR activation, as shown in pre-B and pro-B acute lymphoblastic leukemia (ALL) cell lines [66]. Mechanistically, the activation of p38 is largely responsible for the CD24-induced apoptosis of pre-B cells, whereas pre-BCR cross-linking mediates the activation of extracellular signal-regulated kinase 1 (ERK1) [66]. A recent work indicates that CD24 ligation induces human neutrophil apoptosis through mitochondrial inner membrane depolarization in a caspase-3- and caspase-9and ROS-dependent manner [67]. In addition, CD24 of pre-B cells also affects the function of the chemokine receptor CXCR4 by inhibiting the phosphorylation of ERK1, resulting in reduced SDF-1-mediated BM B cell migration and CXCR4 signaling [34].

CD24 Expression by APCs DCs are the most important APCs and include various subsets that develop from different cell lineages and have complex functions in adaptive and innate immunity [68, 69]. In mice, the spleen contains three main DC populations: plasmacytoid DCs, CD8+CD24+ DCs (CD8+ DCs), and CD8−CD24− DCs (CD8− DCs). The expression of CD24 is different in various DC subsets. CD24a is expressed by Langerhans cells (LCs) but not dermal dendritic cells (dDCs) that were primarily isolated from the skin. Moreover, increases in CD24a expression and major histocompatibility complex (MHC) class II level allow the differentiation of skin-resident LCs and dDCs from LCs and dDCs in skin-draining lymph nodes post antigen activation [70]. As compared with CD8+ DCs, CD4+ DCs are less efficient for both MHC-II-mediated antigen presentation and MHC-I-mediated cross presentation. CD24 may contribute to the superior antigen presentation function of CD8+ DCs. Evidence has shown that the CD24 overexpression on CD8+ DCs is a significant determinant of efficient antigen presentation [71]. Kim et al. reported that elevated CD24 expression on CD103+ respiratory DCs (RDCs) supports the differentiation of CD8+ T cells responsive to influenza virus by presenting high-mobility box 1 (HMGB1) on cell surface to modulate

CD8+ T cell activation [72]. It has been shown that CD24 expressed by DCs binds to Siglec-G/10 and HMGB1, an intracellular DNA-binding protein released by necrotic cells. This ternary complex has been shown to counteract TLR signaling induced by HMGB1. Chen et al. provided evidence that this inhibitory loop is important in sepsis, as mice deficient in either Cd24 or Siglec-G/10 exhibit significantly more mortality and inflammatory cytokine production [73]. Indeed, CD8+ DCs are the primary mediator of anti-viral cytotoxic T cell responses. However, by specifically deleting CD8+DCs, the only DC subset that can present viral Ag is the CD8− DC that expresses CD24. This population of cells can be significantly amplified in mice under treatment with DC growth factor FMS-like tyrosine kinase 3 ligand. The CD8−CD24+ DCs are a precursor of CD8+ DCs. Moreover, upon vaccination against HSV-1, CD8−CD24+ DCs induce stronger T cell responses and promote viral clearance as compared with CD8+ DCs. Thus, CD8−CD24+ DCs represent a promising population for DC-based immunotherapies [74].

CD24 and Autoimmune Diseases The apparent regulatory role of CD24 in the development of inflammation has led researchers to attribute a role for CD 24 in autoimmune diseases. Indeed, in vitro and animal studies have linked CD24 to the development and pathogenesis of numerous autoimmune diseases, such as systemic lupus erythematosus (SLE), multiple sclerosis (MS), experimental autoimmune encephalomyelitis (EAE), and rheumatoid arthritis (RA). Because CD24 has been consistently found to be expressed on immature cells, including both B and T cell progenitors, it has been proposed that CD24 is involved in the development of central and peripheral tolerance. But CD24 seems to have a BJekyll- and Hyde-^ type personality, in that while a functioning CD24 gene is necessary for the proliferation of T cells in a host who is lymphopenic, it has been found to be a negative regulator for T cell proliferation when expressed on dendritic cells. On the other hand, CD24 deficiency appears to improve efficiency of clonal deletion, and CD24 would thus be considered to inhibit the development of immune tolerance, leading to a higher incidence of autoimmunity. This is supported by the fact that CD24 and CD28 deficiency together leads to a suppression of CD4 and CD8 T cell responses. It is possible also to experimentally overexpress CD24, and when this is done in an EAE model, it was found that overexpression of CD24 was associated with a more progressive and severe disease presentation [75]. In both CD24 deficiency and wildtype mice, pathogenic T cells can be recruited to the central nervous system (CNS), but they proliferate and persist only in the wild-type CNS, suggesting that CD24

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deficiency has a protective effect against prolonged T cell-mediated inflammation [76]. In the inflammatory state triggered by a variety of DAMPs, CD24 seems to have a protective effect on the host response. This is based on its interaction with Siglec6 in the mouse or Siglec 10 in the human, as alluded to above. The CD24-Siglec pathway may play a role only in DAMP-triggered immune responses and not PAMP-triggered responses. Given what we now know about environmental triggers of autoimmunity and cancer, CD24 may play a significant role in the pathogenesis of both disease groups [77]. Polymorphisms in the CD24 gene have been associated with disease risk for developing autoimmune diseases, including SLE, RA, and MS. Interestingly, most CD24 polymorphisms have been associated with an increased incidence and/or severity of autoimmune diseases, suggesting that these genetic variations constitute gain of function mutations of CD24. The effects of CD24 genetic variants on multiple autoimmune diseases are summarized in Table 2.

Table 2

CD24 Polymorphisms Confer Susceptibility to SLE SLE is a multisystem autoimmune disease that primarily affects women [78]. The pathogenesis of SLE has been associated with multiple genetic and epigenetic factors [79–81]. SLE is characterized by the presence of autoantibodies, immune system malfunction, dysfunctional apoptosis, and inflammation in multiple organ systems, but the precise mechanism of disease is unknown [82, 83]. As indicated by recent genome-wide linkage studies, CD24 has been found to be relevant to SLE [84]. A non-conservative alanine to valine replacement at residue 57 relevant to the GPI anchor cleavage site results in a single-nucleotide polymorphism (SNP) in the human CD24 gene [85]. A study carried out in a Spanish cohort showed that more CD24V/V genotype subjects are found in the SLE patients as compared with controls [86]. Another study further confirmed that the CD24V/V and CD24A/V genotypes were associated with the increased risk for SLE in a Polish cohort. Interestingly, the CD24V allele significantly correlated with the presence of anti-Scl-70 and anti-snRNP autoantibodies in patients with SLE [87]. These

The effects of CD24 gene polymorphisms on the risk of developing autoimmune diseases

Disease

Polymorphism

Effect on CD24 expression in disease (patients vs controls)

Associations with the disease

Reference

SLE

SNP in the human CD24 gene at P226 (rs8734)

↑CD24V/V genotype and CD24V allele

[86, 87]

P1527del (rs3838646) SNP in the human CD24 gene at P226 (rs8734) P1527del (rs3838646) SNP in the human CD24 gene at P1626 (rs1058881)

↓CD24 mRNA stability ↑CD24V/V genotype and CD24V allele ↑efficiency of GPI- anchor cleavage. ↓CD24 mRNA stability ↑CD24A/A genotype

Haplotype of SNPs in the human CD24 gene P-534C, P-492G, and P-442C SNP in the human CD24 gene as rs8734 SNP in the human CD24 gene at P1626 (rs1058881) SNP in the human CD24 gene at P1056 (rs1058818) SNP in the human CD24 gene at P226 (rs8734) P1527del (rs3838646) SNP in the human CD24 gene at P226 (rs8734) rs8734/rs3838646

↑CD24 mRNA expression ↑CD24-SP1 interaction

↑risk for SLE ↑risk of production of anti-snRNP, anti-Scl70 and snRNP/Scl-70 autoantibodies ↓risk for SLE ↑risk for MS ↑rapid progression in MS ↓risk for MS ↑disease severity of MS ↑acceleration of the disease progression of MS ↑risk and progression of MS

MS

IBD

RA GCA

AITD

SNP in the human CD24 gene at P226 (rs8734)

↑risk for UC and CD ↑risk for UC

[88] [96, 97] [88] [98]

[99] [104]

↑risk for UC ↑CD24V/V genotype and CD24V allele

↑risk for RA

[115]

↓CD24 mRNA stability ↑CD24V/V genotype

↑risk for GCA ↑risk for GCA

[126]

↑C/del haplotype ↓C/TG haplotype

↑risk for GCA ↓risk for GCA

↑CD24V/V genotype and CD24V allele

↑risk for AITS

[134]

Abbreviations: (↓) decreased, (↑) increased, SLE systemic lupus erythematosus, MS multiple sclerosis, IBD inflammatory bowel disease, UC ulcerative colitis, CD Crohn’s disease, RA rheumatoid arthritis, GCA giant cell arteritis, AITD autoimmune thyroid diseases

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findings confirmed the relevance of CD24V/V and CD24A/V genotypes to the SLE pathogenesis. In contrast, reducing the stability of CD24 mRNA through a dinucleotide deletion at position 1527 can confer protection against both SLE and MS [88]. CD24 may also contribute to SLE by affecting the regulatory function of B cells. In a Chinese cohort with new-onset SLE, the number of primary circulating CD24hiCD27+CD19+ B cells was significantly and inversely correlated with the SLE disease activity index (SLEDAI) score [89]. Another study showed that CD40 stimulates the CD19+CD24hiCD38hi B cells to produce interleukin-10 (IL-10) and plays a suppressive role in T helper 1 cell differentiation. However, this regulatory pathway is disrupted in SLE patients. Evidence shows that the peripheral CD19+CD24hiCD38hi B cells in SLE patients could not be further stimulated by CD40 and produce less IL-10, which led to the loss of the suppressive capacity [90]. Thus, altered cellular functions within CD24-expressing B cells may be a critical factor in the aberrant immune responses of SLE and other autoimmune diseases.

CD24 as a Genetic Modifier for MS Risk MS is a severe and complex central nervous system (CNS) disease primarily characterized by demyelination [91]. Several genetic loci have been associated with MS or act as modifiers of disease progression, including HLA class II and the DR2 haplotype (HLADRB1*15, DQA1*01, DQB1*06) [92–94]. In addition, the IL-2 receptor and IL-7 receptor genes may also play roles in MS [95]. Recently, polymorphisms of the CD24 gene were found to be associated with the MS pathogenesis. The CD24V/V genotype is also associated with increased risk and more rapid progression of MS in a central Ohio cohort [96], and this observation was also supported by another study in a Spanish population [97]. Furthermore, González et al. detected a strong association between the CD24A/A genotype and disease severity and accelerated disease progression of MS [98]. A haplotype consist of the combination of three new SNPs (P-534C, P-492G, and P-442C) in the CD24 promoter constitute a haplotype that associates with increased risks and progression of MS [99]. Chromatin immunoprecipitation (ChIP) assays have shown that the transcription factor SP1 specifically binds to risk alleles; and in MS patients, the CD24 transcript abundance is positively correlated with the SP1-binding variants. Thus, this hypermorphic SP1binding CD24 variant may partially account for the risk and progression of MS [99] (Fig. 3).

Cd24 and Experimental Autoimmune Encephalomyelitis Experimental autoimmune encephalomyelitis (EAE) has been demonstrated to be an effective model to study MS. Etiologically, EAE develops when autoreactive T cells migrate into CNS where they become activated, followed by clonal expansion and persistence [100–102]. CD24 is abundantly expressed in the CNS, and Bai et al. have shown that an inactivating mutation of Cd24 decreases the odds of developing EAE in mice [100]. In comparison, in Cd24-deficient mice, adoptively transferred myelin oligodendrocyte glycoprotein (MOG)-reactive T cells can translocate into the CNS; however, these T cells are largely non-responsive [76]. Furthermore, Cd24 deficiency compromises the APC capacity of microglia and astrocytes in the CNS, which is associated with the down-regulation of their in vitro costimulatory activity [76]. In addition, overexpression of CD24 in mice can reconstitute the susceptibility to EAE [76]. Thus, CD24 is a key factor and/or checkpoint within the CNS for the development of EAE [76]. Recently, a study of Cd24-overexpression in mice revealed a potential pathogenic mechanism of CD24 in the development of EAE [75]. AstroCD24TG mice were generated using conventional microinjection technology, in which CD24 expression in the CNS was controlled by the glial fibrillary acidic protein (GFAP) promoter. Thereafter, the mice exhibited an abundant expression of CD24 in the CNS, as compared to the CNS of non-transgenic (NTG) mice. In addition, CD24 expression only co-localized with GFAP+ cells (astrocytes), and not with other cells in the CNS, for example, neurons (NeuN+), microglial cells (IB4+), and oligodendrocytes (CNPase+). After inducing EAE in these mice with MOG peptide and pertussis toxin, both AstroCD24TG mice and NTG mice developed the disease within 2 weeks, but after day 20, the paralysis of NTG mice gradually recovered to a lower level, whereas in AstroCD24TG mice, clinical signs continued at a high level throughout the entire experimental period. Large areas of inflammatory lesions and demyelination could be detected in the AstroCD24TG mice, and broad areas of axonal damage were demonstrated by axonal staining of the spinal cord sections, consistent with the hypothesis that overexpression of CD24 can enhance the progression of EAE. Further experiments demonstrating that these effects are correlated with higher expression of IL-17 and demyelination-associated marker P8 in AstroCD24TG mice compared with NTG mice. In addition, the CNS of AstroCD24TG mice had a higher percentage of CD4+ T cells and Th17 cells. A subsequent study revealed that the enhanced EAE may be related to astrocyte CD24 stimulation of pathogenic T cells such as MOG-specific, TCR-transgenic 2D2 T cells in the CNS. Since this process occurs during the effector phase of EAE, its inhibition may constitute a potential CD24-targeted therapy for EAE and MS (Fig. 4).

Clinic Rev Allerg Immunol Fig. 3 The association of CD24 haplotype and MS. The haplotype consists of the combination of three new SNPs (P-534C, P-492G, and P-442C) in the CD24 promoter region. The transcription factor SP1 binds to the risk allele and facilitates CD24 transcription. TSS transcription start site, MS multiple sclerosis

CD24 and Inflammatory Bowel Disease CD24 plays important roles in other autoimmune diseases as well, including inflammatory bowel diseases (IBD) such as Crohn disease (CD) and ulcerative colitis (UC) [103]. A case-controlled Israeli study of 117 IBD patients and 105 age- and gender-matched healthy controls showed that the CD24 rs8734 polymorphism is associated with the risk of UC and CD, while the A1626G (rs1058881) and A1056G (rs1058818) SNPs may only be associated with UC risk [104]. A case-controlled Spanish study of 369 CD patients, 323 UC patients, and 629 healthy matched controls showed the Bdel^ allele of the dinucleotide deletion in SNP rs3838646 was related to an increased risk of CD, but not with UC. Furthermore, this allele was found to be associated with the diagnosis of CD at ages between 17 and 40 years, as well as

the ileocolonic location, and the inflammatory behavior of CD. No difference was found for the A57V polymorphism (rs8734) in this study. In addition, SNPs of rs3838646 may be associated with a genetic predisposition to CD [105]. Together, these findings suggest that CD24 is of potential and novel value for IBD prognosis and therapy. Via its carbohydrate domains, CD24 plays a role in maintaining the interaction between myeloid cells and P-selectin on platelets and endothelial cells. Bretz’s study on CD24 knockout mice reported that these mice had fewer leukocytes in the colon than WT mice, and this was true across all subpopulations of leukocytes [106]. In colon and small intestine, CD24 can also be found expressed in EpCAM+ epithelial and CD31+ endothelial cells, and numbers of the latter are reduced in CD24−/− mice [106]. By leukocyte transfer experiments, they showed that the status of CD24 expression on non-immune cells (CD31+) is important for leukocyte recruitment to the colon [106]. Previous research has shown that CD24−/− cells have a reduction of α4β7-integrins and α4β1-integrinmediated leukocyte-endothelial interactions, therefore affecting the recruitment of lymphocytes into lymphoid organs and tissues [107–109]. It has also been suggested that CD24 expressed on colonic epithelial and endothelial cells is the key component of leukocyte retention and positioning, and since this affects integrin function, this may be one of the mechanisms by which CD24 plays a role in inducing IBD.

CD24 and Rheumatoid Arthritis

Fig. 4 Mechanisms of CD24 expression involved in the development of EAE. CD24 expression is increased in mice CNS after immunization with MOG peptide, which leads to enhanced cytokine IL-17 as well as higher numbers of Th17 and total CD4+ T cells. CD24 expression also enhances the costimulatory activity of astrocytes on encephalitogenic T cells

Rheumatoid arthritis (RA) is a chronic, systemic inflammatory disorder that primarily affects joints. It is associated with the induction of autoantibodies, such as rheumatoid factor (RF) or anti-citrullinated protein antibodies (ACPAs), and mediated by the action of proinflammatory mediators such as TNF-α [110, 111]. RA presents with painful and deformed joints leading to loss of joint function, but it may also have effects on other organs, including the skin, heart and blood vessels, lungs, etc. The pathogenesis of this disease is

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complex and is thought to involve genetic and environmental factors [112, 113]. Studies of gene polymorphisms have been carried out for years to investigate their role in defining susceptibility to RA. A database of rheumatoid arthritis-related polymorphisms has been developed by Zhang’s team, and it encompasses 3235 polymorphisms in RA within 636 genes and from 68 countries [114]. Among these, a non-synonymous variation (A57V), which has been confirmed to be associated with MS and SLE, has also been found to be related to RA. A study including 1015 patients with RA and 842 matched healthy controls showed that the CD24V/V genotype and CD24V allele were more common among patients with RA, but no significant differences were seen for the CD24 TG/del polymorphism between RA patients and controls [115]. Altered cellular functions of CD24-expressing regulatory B cells may also impact the maintenance of tolerance in patients with immune disorders, such as RA. Unlike those in healthy controls, CD19+CD24hiCD38hi B cells from RA patients lose the ability to inhibit the differentiation from naïve T cells into T helper 17 (Th17) cells and transform CD4+CD25− T cells into regulatory T (Treg) cells [116]. The Th17 and IL17 pathways have been implicated in a number of autoimmune diseases, including rheumatoid arthritis [117]. Moreover, progressive RA patients have less circulating CD19+CD24hiCD38hi B cells, when compared with less progressive RA subjects or healthy controls [116]. Although further investigations are warranted, all of these findings suggest that CD19+CD24hiCD38hi B cells in active RA patients have compromised regulatory function and are unable to contribute to suppressing autoreactive responses and aberrant inflammation [116].

CD24 and Giant Cell Arteritis Giant cell arteritis (GCA), appropriately named for its pathological features, has also been referred as temporal arteritis, cranial arteritis, or granuloma arteritis [118]. It is the most common systemic vasculitis in adults over 50 years of age, with the highest morbidity in Scandinavia and in Scandinavian immigrants in the USA [119, 120]. It usually involves large and medium blood vessels of the head, especially the extra-cranial branches of the carotid artery [120, 121]. Clinical features of this disease are the inflammation of the arterial internal elastic membrane, accompanied by endometrial hyperplasia and occlusion of the lumen [121]. The most feared complications of this vasculitis are severe ischemic complications, especially irreversible vision loss [122]. GCA is a polygenetic disease [119, 123]. It has been reported that HLA-DRB1*04, MICA A5, and HLA-B*15 [124] alleles are related to the disease, as are other polymorphisms of genes such as TNF- α, ICAM-1, IL-1 cluster, IL-6, and corticotropin-releasing hormone (CRH) [125]. A case-

controlled study of 120 GCA patients and 195 ethnically matched healthy controls showed that the CD24 rs3838646 TG/del polymorphisms are associated with an increased risk of GCA. The Bdel^ allele increases the risk of the disease whereas the TG allele has a protective effect against GCA. CD24 rs8734 Val/Val genotype frequency is also associated with a sevenfold increased risk of GCA, and this allele has also been found to correlate with susceptibility to SLE and MS. In addition, both rs8734 and rs3838646 polymorphisms of the C/del haplotype are found to be associated with an increased risk of GCA susceptibility, while the C/TG haplotype appears to convey a protective effect [126].

CD24 and Autoimmune Thyroiditis Autoimmune thyroiditis, also known as chronic lymphocytic thyroiditis or chronic autoimmune thyroiditis, is a disease in which the thyroid gland itself is being targeted, along with its exocrine products. In this way, T3, T4, and TSH are identified as threats or danger signals, leading to the production of specific autoantibodies that target and destroy thyroid tissue [127]. Autoimmune thyroiditis can be clinically divided into two categories: Hashimoto’s thyroiditis, which presents with goiters, and atrophic thyroiditis, in which the thyroid is atrophic [127]. The symptoms for these may be variable depending on whether there is hypothyroidism or hyperthyroidism. Unlike other autoimmune diseases, the effects of autoimmune thyroiditis are not lasting, and they may come and go depending on the type and success of treatment regimens. Rose and Witebsky [128] reproduced some of the disease’s features in 1956 by immunizing rabbits with thyroid extracts. This would be the first model of experimental autoimmune thyroiditis (EAT) [129, 130]. The role of CD24 in the EAT model has been studied by C.Y. Chen’s laboratory [131]. They extended the typical observation period of 35 days post immunization to 100 days and discovered that by day 100, follicles were restored and the thyroid gland had regained its normal morphology and function. They also found that CD24 is highly expressed on thyrocytes during this regeneration process. In Cd24deficient mice, they were able to induce a more aggressive EAT compared with wild-type controls during the initial phase, but in contrast, the regeneration process was even faster. This seemed to suggest that CD24 is important in maintaining a pathogenic infiltrate within the target organ, allowing the researchers to conclude that blocking CD24 may be potentially useful in treating autoimmune diseases. Autoimmune thyroiditis is found to be closely related to Grave disease (GD), and they are collectively known as autoimmune thyroid diseases (AITDs) [132]. Iwatani showed that the proportion of Th1 cells was higher in severe than in mild HD patients and that the reverse was true for the proportion of

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Th2 cells. It was concluded that the Th1/Th2 cell ratio is associated with the severity of the disease. They also noted that the numbers of Th17 cells were higher in AITD patients than in control. Moreover, Th17 cells and Th17-related cytokines are higher in intractable GD patients than in GD patients in remission, suggesting that Th17 cells may affect the intractability of GD [133]. Since CD24 may be associated with T cell homeostasis and pathogenesis in autoimmune disease, Iwatani’s later experiments focused on the T allele in the + 226C/T (Ala57Val) SNP of the CD24 gene (rs8734). They genotyped CD24 SNPs in a study of Japanese patients. There were 144 HD patients, 194 GD patients, and 103 healthy controls. There were a higher number of +226 T carriers (CT or TT genotype) in all AITDs patients than in controls, but the SNPs were not associated with a significant difference in severity and intractability of AITDs nor did they correlate with TRAb level, McAb titer, TgAb titer, or goiter size. It is still unclear whether or not functional SNPs of CD24+ 226C/T correlate with the development of AITDs [134].

CD24 and Other Autoimmune Diseases Furthermore, CD24hiCD27+ B cells might be a pathogenic factor in allergic asthma. T cells from allergic asthmatic patients have reduced IL-10 production post Der p1 treatment, which may be associated with compromised IL-10 induction by CD24hiCD27+ B cells, as discovered in a LPS treatment model [135]. Imbalance of regulatory B (Breg) cells may also be a key factor for the development of type 1 autoimmune pancreatitis (AIP). Sumimoto et al. [136] found that circulating CD19 + CD24 h i CD38 h i Breg cells increased, whereas CD19+CD24hiCD27+Breg cells decreased in AIP patients. These finding suggest that CD19+CD24hiCD38hiBreg cells have potentially suppressive functions, whereas the CD19+CD24hiCD27+Breg cells may have promoting roles in AIP.

CD24-Targeted Therapy of Autoimmune Diseases Overexpressed CD24 in mice can enhance the progression of experimental autoimmune disease such as EAE. Studies in human also reveal that SNPs of CD24, which are associated with increased risks and progression of autoimmune diseases, are mainly gain of function mutations and lead to increased production or activity of CD24. This suggests that blocking the expression and function of CD24 may have a therapeutic effect in autoimmune diseases. CD24-targeted treatment may be carried out by the following possible strategies: (1) cutting off CD24-mediated signal transduction by anti-CD24 antibodies or anti-CD24 receptor antibodies, (2) using small molecule

inhibitors to block the interactions between CD24 and its ligand, and (3) disruption of the CD24 glycosylation patterns by sialidases, which may be effective because of the likely dependence on high levels of glycosylation in the CD24ligand complex.

CD24 in Cancer Immune mediators and signaling pathways play a key role in immune surveillance and in the human body’s ability to ward off cancer cells. CD24 is expressed on many varieties of tumor cells. In general, CD24 is overexpressed in cancer [137, 138]. For example, CD24 expression is associated with various disease characteristics including lymph node metastases, survival, and tumor grade in esophageal squamous cell carcinoma [139]. These patterns have also been observed in other cancers as well, including ovarian cancer, prostate cancers, and cholangiocarcinomas [140–144]. Several experiments focused on the manipulation of CD24 expression and activity have been shown to inhibit factors that play a role in tumor growth and progression. For example, small-interfering RNA (siRNA) has been shown to be able to inhibit tumor cell proliferation and survival through silencing of CD24 expression on tumor cells [145]. Blocking of CD24 with antibody has been shown to inhibit growth of pancreatic cell lines [146]. And mutation of CD24 reduced the size of hepatitis B virus gene transfection-induced hepatocellular carcinomas [147].

Conclusions Since its discovery, CD24 has been intensively studied, with significant amounts of research targeting its roles in immune cells and autoimmune diseases. Existing evidence has demonstrated a strong genetic association between CD24 and the pathogenesis of autoimmune diseases. Current knowledge strongly suggests that CD24 plays a regulatory role in T and B cell homeostasis, but it is clear that future research to elucidate the pathogenic mechanisms of how the CD24 pathway impacts the regulatory axis of the immune system is necessary. This will improve our understanding of the pathogenesis of autoimmune diseases and spur development of the potential use of CD24 as a biomarker in terms of diagnosis, prognosis, and therapy as well as new CD24-related pharmacological agents to treat autoimmune and other diseases.

Conflicts of Interest Yixin Tan, Ming Zhao, Bo Xiang, Christopher Chang, and Qianjin Lu declare that they have no conflict of interest.

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