CD4⁺ T cell differentiation is a tightly controlled process requiring cytokine signaling pathways, which activates distinct transcription factors. During the course of this differentiation, several coordinated changes happen at the chromatin level leading to differential expression of genes specific to the functional aspects of the effector cells (39). Lineage-specific transcriptional factors and other chromatin proximal proteins interplay and mediate the activation of cytokine subsets marking a particular lineage commitment while repressing others (1, 40). Our lab provided the evidence that the expression of Th1-specific lineage commitment transcriptional factor T-bet could be regulated by SMAR1 and enhanced expression of SMAR1 caused defective Th1 response with a reciprocal increase in Th2 cell commitment (41). This inverse correlation of Th1/Th2 axis has been substantiated by many previous reports describing the differential function of proteins involved in the lineage specifications of T cell development (42, 43).

A large group of evidence has provided a clear insight into the involvement of chromatin changes associated with the naïve T cell differentiation into effector cells (44). IFN-γ and Th2 cytokine locus (IL-4, IL-5, and IL-13) undergo substantial changes in the chromatin conformation during Th1 and Th2 differentiation, respectively, orchestrated by interchromosomal and intrachromosomal interactions (45–47). These long range interactions and chromatin loop formations are consequence of temporal binding between the cis elements and many associated nuclear proteins (48–50). Many MAR-binding proteins are well characterized and described including CDP/Cux, SATB1, PARP, SAFs, and ARBP (30). Recently, a thymus-enriched MARBP, SATB1, has been shown to play a crucial role in the lineage determination and maintenance of Th2 (51, 52) and T_reg cells (53), respectively.

High throughput technologies including full genomic microarray has assisted the investigation and identification of many novel factors that are crucial for the differentiation of T cells (54, 55). Lineage-specific transcriptional factor T-bet induces the expression of IFN-γ through the chromatin remodeling of its gene along with CTCF and establishes a Th1 phenotype (56). Similarly, GATA3 induces chromatin changes at the Th2 locus and repressive changes at the IFN-γ locus (57). Thus, the function of lineage-specific factors and master regulators is to establish a particular lineage by inducing specific genes and at the same time repressing others (44). Many nuclear proteins such as IRF4 (58, 59), Gfi-1 (60, 61), Ikaros (62), and Dec 2 (9) have been documented to be selectively expressed in Th2 differentiated cells, and these proteins function either by upregulating the genes involved in the Th2 lineage commitment or by repressing the genes involved in the establishment of other cell lineages. We observed the role of SMAR1 particularly in the Th2 cells when its expression is selectively induced. In this condition, the expression of GATA3 is induced that results in activation of Th2 cytokine genes along with suppression of gene subsets that are committed to other lineages (63). Previous reports also suggested a reciprocal regulation of genes involved in the effector T cells differentiation (40), and we observed T-bet as a target of SMAR1 in Th2 differentiated cells. Our lab demonstrated an inverse correlation of T-bet expression in T cells from SMAR1 transgenic and SMAR1^−/− mice, showing the regulation of SMAR1 at the T-bet axis (41).

T-bet is important for the differentiation of Th1 cells (64). Therefore, regulation of T-bet gene expression is important to establish Th1 and maintain Th1/Th2 axis as evidenced by the abnormal disease conditions correlated with the deregulation of T-bet (65). Previous studies on the regulation of T-bet promoter revealed an indispensable function of Notch in the transactivation of T-bet (66). Many putative cleaved activated Notch (CSL)-binding sites were characterized on the T-bet promoter crucial for the activation of T-bet in a Th1 specific condition. These binding sites function not only as enhancer elements but also as a regulatory region by an interplay of differential protein binding (67, 68). Notch1 activation is required for both Th1 (66) and Th2 cell lineage differentiation (68, 69), but SMAR1 is induced in Th2 differentiated cells. We noticed that GATA box-binding elements on SMAR1 promoter bind GATA3 and positively activate SMAR1 in Th2 differentiated condition. Furthermore, SMRT/HDAC complex has been demonstrated to mediate effective regulation at Notch target sites by functioning as corepressor (70). In agreement with these reports, we observed SMAR1-mediated downregulation of T-bet expression by directly interacting to the distal CSL-binding site on the T-bet promoter (41). In addition, the binding of SMAR1 to this region recruits the SMRT/HDAC corepressor complex on its promoter, and this corepressor complex competes with the Notch-mediated transactivation of T-bet in Th2 cells even at induced levels of Notch signaling (Figure 2). Moreover, the recruitment of corepressor complex modifies the chromatin at this region into a silencer mark by reduced histone acetylation and increased methylation. Thus, SMAR1 functions as an “adaptor molecule” crucial for the regulation of T-bet in Th2 cells at the chromatin level through differential binding to MAR sequences that mediates chromatin looping associated with necessary repressive modifications (41) (Figure 2).

T-bet expression drives aggressive and inflammatory processes by regulating such responses, which are essential for the prevention of organ specific autoimmunity (65). T-bet deficiency is correlated with increased hypersensitivity to allergen in airway (71). Tbx21^−/− CD4⁺ T cells showed Th2 biasness with signature hyper-acetylation of IL-4 promoter reflecting the suppressive effect of T-bet on the IL-4 locus (57). Moreover, T-bet over expression attenuates airway hypersensitivity by shifting the cytokine balance to Th1 response (65, 72). SMAR1^−/− mice showed a significantly reduced hypersensitivity response with lower frequency of IL-4-producing Th2 cells and eosinophilia in the BAL fluid. SMAR1^−/− mice were resistant to ovalbumin induced allergic airway inflammation. Since naïve CD4⁺ T cells from SMAR1^−/− mice have impaired Th2 differentiation in the lung, allergic inflammation leads to aberrant expression of genes that are responsible for Th1 and Th17 commitment, which in turn suppresses Th2 response in vivo. This observation is in line with the previous reports suggesting the elevated T-bet expression in SMAR1^−/− mice after chronic allergic antigen exposure (41, 71, 73). It shows SMAR1 is a novel and essential factor for the establishment of Th2 cells by functioning as a Th1-specific transcriptional gene repressor.

Regulatory T cells are central to controlling immune tolerance and maintaining immune homeostasis. Foxp3 is recognized as a single gene determinant essential for T_reg cell function (74). Alteration in Foxp3 expression, even the slightest, often leads to impaired T_reg cell function and is associated with various autoimmune and inflammatory disorders (75, 76). Many factors including Runx–CBFβ complexes, NF-κB, FOXO1, and FOXO3 are known to be important for Foxp3 expression (77). Deletion of either of these genes causes abrogation of Foxp3 expression. Other transcription factors have recently been shown to regulate Foxp3 expression including Bcl11b and TCF3 that bind to the Foxp3 promoter and induce its expression in response to TGF-β. TCF3 requires ID3 for GATA3-mediated repressive activity upon Foxp3 expression (78). Recent studies have revealed involvement of mTOR signaling pathways in the process of T cell fate determination, including the differentiation of naïve T cells into effector or T_reg cells. When activated, an mTOR-deficient T cell becomes Foxp3⁺ T_reg cells (79, 80). The main role of mTOR in regulating Foxp3⁺ T_reg cell responsiveness or stability has been implicated by studies using mTOR inhibitor, rapamycin (79). More recent studies of Rheb- or Rictor-deficient mice suggest that distinct mTORC1 or mTORC2 activities selectively regulate each subset of effector T cells, but inhibition of both is required for the spontaneous generation on Foxp3⁺ T_reg cells (81). Regarding the mechanism underlying the repression of Foxp3 in developing T_reg cells, IL-6- or IL-23-mediated activation of STAT3 has been shown to play a central role. STAT3 binds to CNS2 region of Foxp3 promoter and represses its expression (82). With regards to downstream pathways of STAT3, several genes including Rora, Rorc, Batf, Irf4, and HIF-1α have been demonstrated to be activated by STAT3 and are implicated in the Th17 cells differentiation (83, 84). Recent reports show a degree of plasticity through the acquisition of specific transcription factors in T_reg cell, which is required for controlling a defined polarized condition. In extreme inflammatory condition or in defined compartments, T_reg cells can also express effector cytokines (85, 86). For instance addition of RORγt in T_reg cell can produce IL-17A (85–87), indicating sustained expression of Foxp3 in T_reg cell is essential for maintaining its regulatory function.

Effector CD4⁺ T cells are responsible for the production of the pro-inflammatory cytokines that cause tissue damage. Conversely, T_reg cells are responsible for maintaining peripheral tolerance of effector T cells and keeping these cells in check (88). Our lab for the first has shown that the role of a MAR-binding protein, SMAR1 in maintaining the balance between Th17 and T_reg cells and its role in inflammatory diseases. In absence of SMAR1, T_reg cells lose their suppressive activity that leads to increased production of pro-inflammatory cytokine through T cells in the colon. These T cells showed upregulation of gut homing markers, integrin α4β7, and CCR9 that help them to accumulate in the gut during colonic inflammation. This observation revealed an indispensable role of SMAR1 in regulating T_reg cell function and immune tolerance that maintain the balance between T_reg and Th17 cells. Deletion of SMAR1 in T cells enhances Th17 cells activity in experimental colitis, and the increased number of Th17 cells is thought to be the reason for the progression of the disease (89, 90). SMAR1-deficient T_reg cells are not able to prevent IBD in Rag^−/− mice, indicating that the suppressive function of T_reg cell is severely compromised. However, SMAR1-deficient T_reg cells showed reduced levels of IL-10 and upregulation of pro-inflammatory cytokines, including TNF-α, IL-17, and IFN-γ (89). Studies have shown that IL-10-deficient mice lack T_reg cells and are not capable of controlling inflammatory responses in the intestine (91, 92). In the absence of SMAR1, T_reg cells fail to suppress the reactive CD4⁺ T cells, as a result the whole balance among CD4⁺ T cells is severely damaged leading to the progression of the disease (89, 90, 93).

We found constitutive expression of SMAR1 in natural T_reg cells as well as induced T_reg (iT_reg) cells. Data from our lab support the idea that IL-2 contributes to the expression of SMAR1 through T_reg. Recent reports suggest that the T_reg cell require acquisition of specific transcription factors to exhibit control in defined polarized situation (94, 95). Previous reports demonstrated that increased expression of RORγt in T_reg can produce IL-17A (85) that leads to compromised T_reg functions. We have shown that in the colon, expression of RORγt was influenced by SMAR1 in T_reg and increased expression of IL-17A compared to WT (89, 93). The expression of Foxp3 is reduced by genetic alteration causing upregulation of RORγt, followed by increased levels of IL-17A production and generation of effector Th17 cells (86, 87, 96). Therefore, the loss of SMAR1 has severe loss on Foxp3 expression, leading to the induction of IL-17 conferring a T_reg phenotype to Th17 phenotype (Figure 3).

Understanding of how SMAR1 regulates immune function is still unclear. Indeed, our work opens a new role of SMAR1 in controlling T_reg cell function. The predominant cell type that expresses Foxp3 is CD4⁺CD25⁺ T cell; the same population that has been reported to suppress proliferation and cytokine production in conventional CD4⁺ T cells (97). Foxp3 appears to function through the transcriptional repression of many genes including the effector cytokines (98, 99). The factors that mediates the trans-activation or trans-repression are critical to delineate the molecular mechanisms involved in controlling regulation of Foxp3. Previous reports suggest that TGF-β mediates enrichment of SMAD2/3 at the Foxp3 promoter and the activation of Foxp3 transcription (100, 101). On the other hand, STAT3 is reported to bind to silencer regions of Foxp3 promoter (100, 102) and suppresses its expression. Deficiency of SMAR1 in T_reg cells leads to uncontrolled STAT3 production and results in the production of IL-17. Additionally, IL-6-mediated suppression of SMAR1 has a direct effect on the enrichment of STAT3 at Foxp3 promoter. Inhibition of SMAR1 restores STAT3 enrichment in Foxp3 promoter in response to TGF-β1 in SMAR1^−/− CD4⁺ T cells (89). Finally, IL-6 influences Foxp3 epigenetically by loosening the chromatin allowing the access of STAT3 to the Foxp3 promoter. These observations support the idea that over expression of STAT3 is a key factor in defective Foxp3 induction in SMAR1^−/− CD4⁺ T cells. It is known from earlier studies that SMAR1 affect transcriptional activity mainly through DNA binding (34, 35). Presence of several MAR-binding regions at the promoter of STAT3 suggests that SMAR1 can potentially bind to these sites and influence the STAT3 expression. SMAR1 bound to regulatory regions of STAT3 locus could inhibit the activity of STAT3, a negative regulator for Foxp3 (103, 104) (Figure 3). In support of this idea, we observed lower expression of SMAR1 in Th17 cells (41) and was unable to bind to STAT3 locus. However, in T_reg cells, SMAR1 binds at a position -660 to -840 associated with strong MAR and -229 to -478 associated with IL-6 response elements from the transcription start site of STAT3 locus (89). Our lab showed that in the WT cells treated with TGF-β1, SMAD2/3 bind to the Foxp3 promoter. At the same time, SMAR1 was found to bind STAT3 promoter suggesting a positive role for SMAR1 in the transcriptional regulation of STAT3. This activity was regulated through TGF-β signaling. This observation also suggests a role of SMAR1 in regulating Foxp3 expression in TGF-β1 iT_reg cells and thus SMAR1 ultimately decides the plasticity of T_reg cells (Figure 3).

Reports on plasticity of T_reg cells in inflammatory response showed the control of T_reg cells by specific transcription factors in polarized condition, and loss of this polarity leads to expression of effector cytokines (42, 87). We found that expression of SMAR1 in T_reg cells is downregulated during colonic inflammation and SMAR1-deficient T_reg cells produced large amount of pro-inflammatory cytokine IL-17 compared to WT. This clearly shows effector cytokine production by T_reg cells occurred in a condition in which SMAR1 was either reduced or absent (93).

We also assessed the relative contribution of IL-10 in mediating T_reg cell immunosuppressive function. Neutralization of IL-10 in SMAR1^−/− mice led to an interesting finding that compensation for T_reg cell defects also depend on IL-10 signaling. Foxp3 amount in SMAR1^−/− mice is greatly diminished upon anti-IL-10 treatment following DSS administration; we demonstrated that T_reg cells are a major source of IL-10 in SMAR1^−/− mice. It is proposed that IL-10-secreting T_reg cells are a critical component of immune-mediated protection during increased intestinal inflammation in SMAR1^−/− mice (93). In the context of T_reg cell biology, the current study reveals a novel role of SMAR1 in controlling T_reg physiology during inflammation. Therefore, to study the factors that are modulating the regulation and function of T_reg is an interesting target for immunotherapy in inflammatory disorders.

Scaffold matrix attachment region-binding protein 1 has emerged as important factor for gene expression by regulating epigenetic modifications. SMAR1 seems to coordinate cytokine-dependent gene expression in CD4⁺ T cells. We have shown SMAR1 is downregulated under Th1 and Th17 differentiation, and we have observed IL-6, a major cytokine involved in generation of Th17 cells, downregulates SMAR1 expression (89). Our lab has also reported that SMAR1 regulates STAT3 gene expression by directly binding to STAT3 promoter and recruiting the repressor complex (89). In agreement with this, a deficiency of SMAR1 in T cells renders the mice susceptible to myelin oligodendrocyte glycoprotein peptide-driven EAE disease with higher pro-inflammatory IL-17-producing T cells (105). EAE is an animal model of human CNS demyelinating disease, including multiple sclerosis (MS). Researches have shown that IL-17-secreting Th17 cells are the causative mediator of the disease. Thus, EAE is considered to be Th17-driven auto-inflammatory disease (106). EAE disease progression can be controlled by signaling molecules or transcription factor that prevents Th17 generation (106, 107). We have shown using a nanoparticle-mediated delivery that SMAR1 controls the Th17 generation and EAE disease progression (105). The small size and high surface volumes of nanoparticles makes them a convenient route of drugs/proteins delivery inside the cell (108). Currently, diverse types of nanoparticle including carbon-based nanoparticles, metal-based nanoparticles, and dendrimers are used for protein delivery (109, 110). We used carbon nanospheres (CNPs) as a carrier for delivery of SMAR1 at the site of inflammation. We observed CNP-mediated delivery of SMAR1 represses the EAE disease progression by inhibiting the IL-17 expression from T cells (Figure 4). Recent study from our lab has shown that nanoparticle-mediated SMAR1 delivery could potentially be used to suppress auto-inflammatory diseases (105). As a treatment for MS, CNP-SMAR1 has three therapeutic values (i) opposition of Th17 differentiation, (ii) increment in the anti-inflammatory IL-10 production by favoring T_reg differentiation, and (iii) promotion of the self-tolerance to myelin. Controlling inflammation by treating inflammatory Th17 cells during EAE by CNP-SMAR1 provides a virus and drug free option to current strategies of MS treatments (Figure 4).

Abnormal inflammatory responses cause many adverse effects to the body as observed in many disease conditions. Pro-inflammatory Th1 and Th17 cells are attributed to many of these disease conditions and targeting the pro-inflammatory cells is now assumed to be a pivotal option. Most therapies in autoimmune and inflammatory disorders are aimed at general supersession of the inflammatory responses (111, 112). Since autoimmune and inflammatory disorders are the result of an imbalance in immune regulation, a different approach that modulates T_reg population could potentially be a target (113, 114). The main strategy in treating IBD is to halt the ongoing inflammation and prevent permanent tissue damage. T_reg cells play a key role in regulation of IBD, and recently, the number of studies has described the presence and function of T_reg cells in patients with IBD (115, 116). In the colon, a target organ of IBD, T_reg cells, was shown to be decreased by 40–50% compared to peripheral blood from IBD patients (117, 118). Therefore, regulating the functions of T_reg is an interesting target for immunotherapy in IBD. We elaborate the role of MARs and SMAR1 in CD4⁺ T cell gene regulation by altering the local chromatin structure that governs the Foxp3⁺ T_reg-mediated immune response. Therefore, not only T cell-modulating cytokine but also MARs and MAR-binding proteins such as SMAR1 could be an interesting target to reduce pro-inflammatory IFN-γ- and IL-17-producing Th1 and Th17 cells. We have shown that SMAR1 regulates some essential genes that dictate the CD4⁺ T cell phenotype and that the SMAR1 aberrant expression leads to dysregulated T cell polarization. SMAR1 level gradually decreases during the development of auto-inflammatory disorders (90, 93), it can be therefore used as a marker for diagnosis of T cell-mediated auto-inflammatory disorders. Identifying the epigenetic modifications of SMAR1-targeting pro-inflammatory cytokine genes in T_reg cells leads to its role as a potential candidate for the use as anti-inflammatory drugs. Though the studies so far have elucidated the role of SMAR1 with respect to tumor suppressor, our recent studies initiated to establish the anti-inflammatory function of SMAR1 in autoimmune disorders like EAE and IBD.