Foxp3 Mutant Mice Research Articles

Splicing factor SRSF1 controls autoimmune-related molecular pathways in regulatory T cells distinct from FoxP3

Regulatory T cells (Tregs) are vital for maintaining immune self-tolerance, and their impaired function leads to autoimmune disease. Mutations in FoxP3, the master transcriptional regulator of Tregs, leads to immune dysregulation, polyendocrinopathy, enteropathy, X-linked (IPEX) syndrome in humans and the early lethal “scurfy” phenotype with multi-organ autoimmune disease in mice. We recently identified serine/arginine-rich splicing factor 1 (SRSF1) as an indispensable regulator of Treg homeostasis and function. Intriguingly, Treg-conditional SRSF1-deficient mice exhibit early lethal systemic autoimmunity with multi-organ inflammation reminiscent of the scurfy mice. Importantly, SRSF1 is decreased in T cells from patients with the autoimmune disease systemic lupus erythematosus (SLE), and low SRSF1 levels inversely correlate with disease severity. Given that the Treg-specific deficiency of SRSF1 causes similarly profound autoimmune disease outcomes in mice as the deficiency/mutation in FoxP3, we aimed to evaluate the genes and molecular pathways controlled by these two indispensable regulatory proteins. We performed comparative bioinformatic analyses of transcriptomic profiles of Tregs from Srsf1-knockout mice and two Foxp3 mutant mice––the FoxP3-deficient ΔFoxp3 and the Foxp3 M370I mutant mice. We identified 132 differentially expressed genes (DEGs) unique to Srsf1-ko Tregs, 503 DEGs unique to Foxp3 M370I Tregs, and 1367 DEGs unique to ΔFoxp3 Tregs. Gene set enrichment and pathway analysis of DEGs unique to Srsf1-ko Tregs indicate that SRSF1 controls cytokine and immune response pathways. Conversely, FoxP3 controls pathways involved in DNA replication and cell cycle. Besides the distinct gene signatures, we identified only 30 shared genes between all three Treg mutants, mostly contributing to cytokine and immune defense pathways. Prominent genes included the chemokines CXCR6 and CCL1 and the checkpoint inhibitors FASLG and PDCD1. Thus, we demonstrate that SRSF1 and FoxP3 control common and distinct molecular pathways implicated in autoimmunity. Our analyses suggest that SRSF1 controls crucial immune functions in Tregs contributing to immune tolerance, and perturbations in its levels lead to systemic autoimmunity via mechanisms that are largely distinct from FoxP3.

FOXP3 and Tip60 Structural Interactions Relevant to IPEX Development Lead to Potential Therapeutics to Increase FOXP3 Dependent Suppressor T Cell Functions.

Regulatory T (Treg) cells play a role in the maintenance of immune homeostasis and are critical mediators of immune tolerance. The Forkhead box P3 (FOXP3) protein acts as a regulator for Treg development and function. Mutations in the FOXP3 gene can lead to autoimmune diseases such as Immunodysregulation, polyendocrinopathy, enteropathy, and X-linked (IPEX) syndrome in humans, often resulting in death within the first 2 years of life and a scurfy like phenotype in Foxp3 mutant mice. We discuss biochemical features of the FOXP3 ensemble including its regulation at various levels (epigenetic, transcriptional, and post-translational modifications) and molecular functions. The studies also highlight the interactions of FOXP3 and Tat-interacting protein 60 (Tip60), a principal histone acetylase enzyme that acetylates FOXP3 and functions as an essential subunit of the FOXP3 repression ensemble complex. Lastly, we have emphasized the role of allosteric modifiers that help stabilize FOXP3:Tip60 interactions and discuss targeting this interaction for the therapeutic manipulation of Treg activity.

Treg-specific deletion of NKAP results in severe, systemic autoimmunity due to peripheral loss of Tregs

Regulatory T cells are critical for the generation and maintenance of peripheral tolerance. Conditional deletion of the transcriptional repressor NKAP in Tregs using Foxp3-YFP-cre NKAP conditional knockout mice causes aggressive autoimmunity characterized by thymic atrophy, lymphadenopathy, peripheral T cell activation, generation of autoantibodies, immune infiltration into several organs, and crusty skin at 3 weeks of age, similar to that of “scurfy” Foxp3-mutant mice. While Treg development in the thymus proceeds normally in the absence of NKAP, there is a severe loss of thymically-derived Tregs in the periphery. NKAP-deficient Tregs have a recent thymic emigrant phenotype, and are attacked by complement in a cell-intrinsic manner in the periphery. Previously, we demonstrated that NKAP is required for conventional T cell maturation as it prevents complement-mediated attack in the periphery. We now show that Tregs undergo a similar maturation process as conventional T cells, requiring NKAP to acquire complement resistance after thymic egress.

Treg mediated immune system homeostasis requires NKAP association with HDAC3

Abstract NKAP is a transcriptional regulator essential for conventional T cell maturation. It does not have any previously defined DNA binding domains and is thought to interact with HDAC3 to exert its functions. The deletion of NKAP at the double positive stage (DP) in T cell development using the CD4-cre NKAP conditional knockout (cKO) mouse model renders thymocytes unable to incorporate α2,8-linked sialic acids and upregulate the complement inhibitory protein CD55, leading to rapid elimination of RTEs by complement. Here, we interrogate the importance of NKAP in Tregs. To address this, we created the Foxp3-YFP-cre NKAP cKO mouse model to delete NKAP only in the Treg lineage. Loss of NKAP in Tregs results in their disappearance, culminating in systemic autoimmunity, similar to the “scurfy” phenotype of Foxp3 mutant mice. Mice with Tregs lacking NKAP have increased conventional T cell proliferation, lymphocytic infiltration into various organs, and crusty skin on tail and ears, which necessitate euthanasia by three weeks. Preliminary results indicate that the disappearance of Tregs is a result of increased complement deposition. Transgenic expression of wildtype NKAP in a cre-inducible manner in cKO mice ameliorates the lymphoproliferative disease by restoring normal Treg numbers, and the mice are healthy. Interestingly, transgenic expression of a mutant version of NKAP that has lost its ability to bind HDAC3 is unable to prevent autoimmunity. Previous studies have demonstrated that HDAC3 is important for the generation and function of Tregs. Our results indicate that NKAP’s binding with HDAC3 is important for its role in Treg mediated immune system homeostasis.

Protein SUMOylation Is Required for Regulatory T Cell Expansion and Function

Foxp3-expressing regulatory T (Treg) cells are essential for immune tolerance; however, the molecular mechanisms underlying Treg cell expansion and function are still not well understood. SUMOylation is a protein post-translational modification characterized by covalent attachment of SUMO moieties to lysines. UBC9 is the only E2 conjugating enzyme involved in this process, and loss of UBC9 completely abolishes the SUMOylation pathway. Here, we report that selective deletion of Ubc9 within the Treg lineageresults in fatal early-onset autoimmunity similar to Foxp3 mutant mice. Ubc9-deficient Treg cells exhibit severe defects in TCR-driven homeostatic proliferation, accompanied by impaired activation and compromised suppressor function. Importantly, TCR ligation enhanced SUMOylation of IRF4, a critical regulator of Treg cell function downstream of TCR signals, which regulates its stability in Treg cells. Our data thus have demonstrated an essential role of SUMOylation in the expansion and function of Treg cells.

FOXP3-miR-146-NF-κB Axis and Therapy for Precancerous Lesions in Prostate.

The tumor-suppressive activity of FOXP3 has been observed in tumor initiation, but the underlying mechanism still remains largely unknown. Here, we identified a FOXP3-microRNA-146 (miR-146)-NF-κB axis in vitro and in vivo in prostate cancer cells. We observed that FOXP3 dramatically induced the expression of miR-146a/b, which contributed to transcriptional inhibition of IRAK1 and TRAF6, in prostate cancer cell lines. Tissue-specific deletion of Foxp3 in mouse prostate caused a significant reduction of miR-146a and upregulation of NF-κB activation. In addition, prostatic intraepithelial neoplasia lesions were observed in miR-146a-mutant mice as well as in Foxp3-mutant mice. Notably, the NF-κB inhibitor bortezomib inhibited cell proliferation and induced apoptosis in prostate epithelial cells, attenuating prostatic intraepithelial neoplasia formation in Foxp3-mutant mice. Our data suggest that the FOXP3-miR-146-NF-κB axis has a functional role during tumor initiation in prostate cancer. Targeting the miR-146-NF-κB axis may provide a new therapeutic approach for prostate cancers with FOXP3 defects.

The Forkhead Transcription Factor, FOXP3: A Critical Role in Male Fertility in Mice1

Fertility is dependent on the hypothalamic-pituitary-gonadal axis. Each component of this axis is essential for normal reproductive function. Mice with a mutation in the forkhead transcription factor gene, Foxp3, exhibit autoimmunity and infertility. We have previously shown that Foxp3 mutant mice have significantly reduced expression of pituitary gonadotropins. To address the role of Foxp3 in gonadal function, we examined the gonadal phenotype of these mice. Foxp3 mutant mice have significantly reduced seminal vesicle and testis weights compared with Foxp3(+/Y) littermates. Spermatogenesis in Foxp3 mutant males is arrested prior to spermatid elongation. Activation of luteinizing hormone signaling in Foxp3 mutant mice by treatment with human chorionic gonadotropin significantly increases seminal vesicle and testis weights as well as testicular testosterone content and seminiferous tubule diameter. Interestingly, human chorionic gonadotropin treatments rescue spermatogenesis in Foxp3 mutant males, suggesting that their gonadal phenotype is due primarily to a loss of pituitary gonadotropin stimulation rather than an intrinsic gonadal defect.

Aberrant Muscle Antigen Exposure in Mice Is Sufficient to Cause Myositis in a Treg Cell–Deficient Milieu

ObjectiveMyositis is associated with muscle-targeted inflammation and is observed in some Treg cell–deficient mouse models. Because an autoimmune pathogenesis has been strongly implicated, the aim of this study was to investigate the hypothesis that abnormal exposure to muscle antigens, as observed in muscle injury, can induce autoimmune-mediated myositis in susceptible hosts.MethodsFoxP3 mutant (scurfy) mice were mated to synaptotagmin VII (Syt VII) mutant mice, which resulted in a new mouse strain that combines impaired membrane resealing with Treg cell deficiency. Lymphocyte preparations from double-mutant mice were adoptively transferred intraperitoneally, with or without purified Treg cells, into recombination-activating gene 1 (RAG-1)–null recipients. Lymph node cells from mice with the FoxP3 mutation were transferred into RAG-1–null mice either 1) intraperitoneally in conjunction with muscle homogenate or purified myosin protein or 2) intramuscularly with or without cotransfer of purified Treg cells.ResultsFoxP3-deficient mouse lymph node cells transferred in conjunction with myosin protein or muscle homogenate induced robust skeletal muscle inflammation. The infiltrates consisted predominantly of CD4+ and CD8+ T cells, a limited number of macrophages, and no B cells. Significant inflammation was also seen in similar experiments using lymph node cells from FoxP3/Syt VII double-mutant mice but was absent in experiments using adoptive transfer of FoxP3 mutant mouse cells alone. The cotransfer of Treg cells completely suppressed myositis.ConclusionThese data, derived from a new, reproducible model, demonstrate the critical roles of Treg cell deficiency and aberrant muscle antigen exposure in the priming of autoreactive cells to induce myositis. This mouse system has multifaceted potential for examining the interplay in vivo between tissue injury and autoimmunity.

The forkhead transcription factor, FOXP3, is required for normal pituitary gonadotropin expression in mice.

The hypothalamic-pituitary-gonadal axis is central to normal reproductive function. This pathway begins with the release of gonadotropin-releasing hormone in systematic pulses by the hypothalamus. Gonadotropin-releasing hormone is bound by receptors on gonadotroph cells in the anterior pituitary gland and stimulates the synthesis and secretion of luteinizing hormone and, to some extent, follicle-stimulating hormone. Once stimulated by these glycoprotein hormones, the gonads begin gametogenesis and the synthesis of sex hormones. In humans, mutations of the forkhead transcription factor, FOXP3, lead to an autoimmune disorder known as immunodysregulation, polyendocrinopathy, and enteropathy, X-linked syndrome. Mice with a mutation in the Foxp3 gene have a similar autoimmune syndrome and are infertile. To understand why FOXP3 is required for reproductive function, we are investigating the reproductive phenotype of Foxp3 mutant mice (Foxp3(sf/Y)). Although the gonadotroph cells appear to be intact in Foxp3(sf/Y) mice, luteinizing hormone beta (Lhb) and follicle-stimulating hormone beta (Fshb) expression are significantly decreased, demonstrating that these mice exhibit a hypogonadotropic hypogonadism. Hypothalamic expression of gonadotropin-releasing hormone is not significantly decreased in Foxp3(sf/Y) males. Treatment of Foxp3(sf/Y) males with a gonadotropin-releasing hormone receptor agonist does not rescue expression of Lhb or Fshb. Interestingly, we do not detect Foxp3 expression in the pituitary or hypothalamus, suggesting that the infertility seen in Foxp3(sf/Y) males is a secondary effect, possibly due to loss of FOXP3 in immune cells. Pituitary expression of glycoprotein hormone alpha (Cga) and prolactin (Prl) are significantly reduced in Foxp3(sf/Y) males, whereas the precursor for adrenocorticotropic hormone, pro-opiomelanocortin (Pomc), is increased. Human patients diagnosed with IPEX often exhibit thyroiditis due to destruction of the thyroid gland by autoimmune cells. We find that Foxp3(sf/Y) mice have elevated expression of thyroid-stimulating hormone beta (Tshb), suggesting that they may suffer from thyroiditis as well. Expression of the pituitary transcription factors, Pitx1, Pitx2, Lhx3, and Egr1, is normal; however, expression of Foxl2 and Gata2 is elevated. These data are the first to demonstrate a defect at the pituitary level in the absence of FOXP3, which contributes to the infertility observed in mice with Foxp3 loss of function mutations.

MyD88 is critically involved in immune tolerance breakdown at environmental interfaces of Foxp3-deficient mice

Tregs expressing the transcription factor Foxp3 suppress self-reactive T cells, prevent autoimmunity, and help contain immune responses to foreign antigens, thereby limiting the potential for inadvertent tissue damage. Mutations in the FOXP3 gene result in Treg deficiency in mice and humans, which leads to the development of a multisystem autoimmune inflammatory disease. The contribution of dysregulated innate immune responses to the pathogenesis of Foxp3 deficiency disease is unknown. In this study, we examined the role of microbial signals in the pathogenesis of Foxp3 deficiency disease by studying Foxp3 mutant mice that had concurrent deficiencies in TLR signaling pathways. Global deficiency of the common TLR adaptor MyD88 offered partial protection from Foxp3 deficiency disease. Specifically, it protected from disease at the environmental interfaces of the skin, lungs, and gut. In contrast, systemic disease, in the form of unrestrained lymphoproliferation, continued unabated. The effect of MyD88 deficiency at environmental interfaces involved the disruption of chemokine gradients that recruit effector T cells and DCs, resulting in their entrapment in secondary lymphoid tissues. These results suggests that Tregs have a key role in maintaining tolerance at host-microbial interfaces by restraining tonic MyD88-dependent proinflammatory signals. Moreover, microbial factors may play a substantial role in the pathogenesis of human autoimmune disease resulting from Treg deficiency.

Foxp3 is a novel repressor of microglia activation

Forkhead transcription factor 3 (Foxp3) is critical for generating CD4(+)CD25(+) regulatory T cells. However, its role in microglia has not been identified. Here, we show that Foxp3 is expressed in microglia and is upregulated upon activation. In Foxp3 mutant mice (Foxp3(sf)), microglia release higher levels of inflammatory cytokines and mediators such as NO, MCP-1, CXCL10, and ROS upon liposaccharide treatment than the wild type, while TNF-alpha and IL-1 beta were not significantly different between wild and mutant microglial cells. In addition, Foxp3 silencing enhances inflammatory responses, suggesting that the major role of Foxp3 in microglia is that of a repressor of activation. Similarly, Foxp3 overexpression reduces inflammatory responses in microglia. We also demonstrate that Foxp3 interacts directly with NF-kappaB and modulates its transcriptional activities. These findings point to the importance of Foxp3 in NF-kappaB mediated inflammatory responses in microglia.

Homeostatic Proliferation in the Mice with Germline FoxP3 Mutation and its Contribution to Fatal Autoimmunity

FoxP3 has emerged as a critical regulator for the development and function of regulatory T cells. Recent studies by several groups have demonstrated that FoxP3 is expressed outside T cell lineages. In this context, we have reported that germline mutation of FoxP3 caused defective thymopoiesis, although its potential contribution to autoimmune diseases has not been analyzed. In this study, we report that, during perinatal period, germline mutation of FoxP3 in scurfy mice caused lymphopenia in the spleen and massive homeostatic proliferation, characterized by the independence from cognate Ags and expression of bona fide markers for homeostatic proliferation. The homeostatic proliferation is suppressed by increases in T cell numbers but not by adoptive transfer of regulatory T cells (Treg). Adoptive transfer of Treg-containing bulk T cells was dramatically more effective than transfer of either Treg alone or Treg-depleted CD4 T cells in curing the scurfy mice. Our data demonstrated that FoxP3 mutation not only ablates Treg, but also dramatically increased homeostatic proliferation during the perinatal period. Homeostatic proliferation acts in concert with Treg defects in causing acute and fatal autoimmune diseases in the FoxP3 mutant mice. These results demonstrated that germline mutation of FoxP3 caused two defects that work in concert to cause lethal autoimmunity.

Where FoxP3‐dependent regulatory T cells impinge on the development of inflammatory arthritis

Regulatory T cells play a suppressive role in many autoimmune diseases and can potentially affect various steps in the progression of disease. The purpose of this study was to analyze the role of Treg cells in the control of arthritis development. Using crosses and cell transfers, we tested the effect of the scurfy loss-of-function mutation of the Foxp3 gene in the K/BxN mouse model. In this model, arthritis develops as the result of the production of high levels of pathogenic autoantibodies. The absence of Treg cells in K/BxN mice led to faster and more aggressive arthritis. Strikingly, disease also spread to joints not normally affected in this model. The absence of Treg cells resulted in an acceleration of the immunologic phase of disease, with significantly earlier autoantibody production. However, the broadened spectrum of affected joints in Foxp3-mutant mice was not due to the earlier appearance of autoantibodies and could not be reproduced by increasing the anti-glucose-6-phosphate isomerase antibody load, which demonstrates an impact of Treg cells on effector phase manifestations. In addition, FoxP3+,CD25+ Treg cells accumulated in inflamed joints, even in nontransgenic animals. This preferential localization mimics that in human arthritides and indicates a preferential homing/retention of Treg cells to sites of inflammation. These results indicate that Treg cells play a role in antibody-mediated arthritis at several levels. Treg cells are involved in constraining the immune phase of disease, as well as limiting the articular damage provoked by the pathogenic autoantibodies in terms of severity and of the range of affected joints, which may contribute to the limited distal predominance of many arthritides.

Foxp3 controls autoreactive T cell activation through transcriptional regulation of early growth response genes and E3 ubiquitin ligase genes, independently of thymic selection

To elucidate the mechanisms of autoreactive T cell activation and expansion, we used endogenous viral superantigens (VSAg)-reactive T cells as a model of self-antigens in two strains of Foxp3-mutant mice. These two strains, together with wild-type mice, provided us with an advantage to simultaneously study the positively and negatively selected as well as rescued autoreactive T cells. We show here that while both VSAg-reactive and non-VSAg-reactive T cells are equally activated in Foxp3-mutant mice, only the VSAg-reactive T cells are preferentially expanded independently of their selected states in the thymus. The T cell activation appears to be controlled by Foxp3 through transcriptional regulation of early growth response (Egr) genes Egr-2 and Egr-3, and E3 ubiquitin (Ub) ligase genes Cblb, Itch and GRAIL, subsequently affecting degradation of two key signaling proteins, PLCγ1 and PKC-θ. Physiologically, the positively, but not negatively selected VSAg-reactive T cells are spontaneously activated without significant expansion. The results suggest that autoreactive T cell activation is controlled by Foxp3 through transcriptional regulation of early growth response genes and E3 ubiquitin ligase genes, independently of thymic selection.