MicroRNAs targeting TGF-ß signaling exacerbate central nervous system autoimmunity by disrupting regulatory T cell development and function.

Transforming growth factor beta (TGF-ß) signaling is essential for a balanced immune response by mediating the development and function of regulatory T cells (Tregs) and suppressing autoreactive T cells. Disruption of this balance can result in autoimmune diseases, including multiple sclerosis (MS). MicroRNAs (miRNAs) targeting TGF-ß signaling have been shown to be upregulated in naïve CD4 T cells in MS patients, resulting in a limited in vitro generation of human Tregs. Utilizing the murine model experimental autoimmune encephalomyelitis, we show that perinatal administration of miRNAs, which target the TGF-ß signaling pathway, enhanced susceptibility to central nervous system (CNS) autoimmunity. Neonatal mice administered with these miRNAs further exhibited reduced Treg frequencies with a loss in T cell receptor repertoire diversity following the induction of experimental autoimmune encephalomyelitis in adulthood. Exacerbated CNS autoimmunity as a result of miRNA overexpression in CD4 T cells was accompanied by enhanced Th1 and Th17 cell frequencies. These findings demonstrate that increased levels of TGF-ß-associated miRNAs impede the development of a diverse Treg population, leading to enhanced effector cell activity, and contributing to an increased susceptibility to CNS autoimmunity. Thus, TGF-ß-targeting miRNAs could be a risk factor for MS, and recovering optimal TGF-ß signaling may restore immune homeostasis in MS patients.

TGF-ß regulation of encephalitogenic and regulatory T cells in multiple sclerosis.

Transforming growth factor beta (TGF-ß) is a pleiotropic cytokine that has been shown to influence the differentiation and function of T cells. The role that TGF-ß plays in immune-mediated disease, such as multiple sclerosis (MS), has become a major area of investigation since CD4+ T cells appear to be a major mediator of autoimmunity. This review provides an analysis of the literature on the role that TGF-ß plays in the generation and regulation of encephalitogenic and regulatory T cells (Treg) in experimental autoimmune encephalomyelitis (EAE), an animal model of MS, as well as in T cells of MS patients. Since TGF-ß plays a major role in the development and function of both CD4+ effector and Treg, which are defective in MS patients, recent studies have found potential mechanisms to explain the basis for these T-cell defects to establish a foundation for potentially modulating TGF-ß signaling to restore normal T-cell function in MS patients.

MicroRNAs targeting TGFß signalling underlie the regulatory T cell defect in multiple sclerosis.

Transforming growth factor beta (TGFß) signalling is critical for regulatory T cell development and function, and regulatory T cell dysregulation is a common observation in autoimmune diseases, including multiple sclerosis. In a comprehensive miRNA profiling study of patients with multiple sclerosis naïve CD4 T cells, 19 differentially expressed miRNAs predicted to target the TGFß signalling pathway were identified, leading to the hypothesis that miRNAs may be responsible for the regulatory T cell defect observed in patients with multiple sclerosis. Patients with multiple sclerosis had reduced levels of TGFß signalling components in their naïve CD4 T cells. The differentially expressed miRNAs negatively regulated the TGFß pathway, resulting in a reduced capacity of naïve CD4 T cells to differentiate into regulatory T cells. Interestingly, the limited number of regulatory T cells, that did develop when these TGFß-targeting miRNAs were overexpressed, were capable of suppressing effector T cells. As it has previously been demonstrated that compromising TGFß signalling results in a reduced regulatory T cell repertoire insufficient to control autoimmunity, and patients with multiple sclerosis have a reduced regulatory T cell repertoire, these data indicate that the elevated expression of multiple TGFß-targeting miRNAs in naïve CD4 T cells of patients with multiple sclerosis impairs TGFß signalling, and dampens regulatory T cell development, thereby enhancing susceptibility to developing multiple sclerosis.

Regulation of effector function of CNS autoreactive CD4 T cells through inhibitory receptors and IL-7Rα.

BACKGROUND: Multiple sclerosis (MS) is a chronic CNS autoimmune disease characterized by inflammation, demyelination, and neuronal degeneration, where myelin-specific CD4 T cells play critical roles in the formation of acute MS lesions and disease progression. The suppression of IL-7Rα expression and the upregulation of inhibitory receptors (PD-1, etc.) are essential parts of the cell-intrinsic immunosuppressive program regulating T effector functions to prevent autoimmunity. However, little is known on the factors regulating IL-7Rα/PD-1 balance in myelin-specific CD4 T effector/memory cells during the development of CNS autoimmunity. METHODS: We analyzed the roles of the transcription factor T-bet in regulating the expression of IL-7Rα and inhibitory receptors in myelin-specific CD4 T cells. Furthermore, we compared the effects of different inflammatory cytokines that are crucial for Th1 and Th17 development in regulating the IL-7Rα/PD-1 balance. RESULTS: We discovered that T-bet suppresses the expression of inhibitory receptors (PD-1 and LAG-3) and promotes IL-7Rα expression in myelin-specific CD4 T cells in vitro and in vivo. As a result, T-bet skews IL-7Rα/PD-1 balance towards IL-7Rα and promotes enhanced effector function. Furthermore, IL-12 enhances IL-7Rα expression in a T-bet independent manner in myelin-specific Th1 cells. Meanwhile, IL-6, the cytokine inducing highly encephalitogenic Th17 differentiation, suppresses PD-1 while upregulating IL-7Rα, skewing IL-7Rα/PD-1 balance towards IL-7Rα, and promoting enhanced effector function. Moreover, blocking IL-7 signaling in myelin-specific CD4 T cells by αIL-7Rα significantly delays experimental autoimmune encephalomyelitis (EAE) onset and reduces disease severity. CONCLUSIONS: T-bet is a major transcription factor regulating IL-7Rα/PD-1 balance in myelin-specific CD4 T cells during EAE development, and there is a positive correlation between several major determinants promoting T cell encephalitogenicity (T-bet, IL-6, IL-12) and an IL-7Rα/PD-1 balance skewed towards IL-7Rα. Furthermore, IL-7 signaling inhibits PD-1 expression in myelin-specific CD4 T cells and blocking IL-7 signaling suppresses T cell encephalitogenicity. Therefore, interference with inhibitory pathways and IL-7Rα expression may suppress the encephalitogenic potential of myelin-specific CD4 T cells and have therapeutic benefits for MS patients.

Analysis of TGF-ß1 and TGF-ß3 as regulators of encephalitogenic Th17 cells: Implications for multiple sclerosis.

The phenotype of the CD4(+) T cells that mediate the CNS pathology in multiple sclerosis is still unclear, and yet a vital question for developing therapies. One of the conundrums is the role of TGF-ß in the development of encephalitogenic Th17 cells. In the present study, TGF-ß1 and TGF-ß3 were directly compared in their capacity to promote the differentiation of myelin-specific Th17 cells that could induce experimental autoimmune encephalomyelitis (EAE). Myelin-specific CD4(+) T cell receptor transgenic cells differentiated with antigen in the presence of IL-6+TGF-ß1 or IL-6+TGF-ß3 generated T cells that produced robust amounts of IL-17, but were incapable of inducing EAE when transferred into mice. Further analysis of these non-encephalitogenic Th17 cells found that they expressed lower amounts of GM-CSF or IL-23R, both molecules necessary for encephalitogenicity. Thus, TGF-ß, irrespective of isoform, negatively regulates the differentiation of encephalitogenic Th17 cells.

Vitamin D as a Risk Factor for Multiple Sclerosis: Immunoregulatory or Neuroprotective?

Vitamin D insufficiency during childhood has been linked to the development of multiple sclerosis (MS), typically an adult-onset inflammatory demyelinating disease of the central nervous system (CNS). Since vitamin D was known to have immunoregulatory properties on both innate and adaptive immunity, it was hypothesized that low vitamin D resulted in aberrant immune responses and the development of MS. However, vitamin D receptors are present on many cell types, including neurons, oligodendrocytes, astrocytes and microglia, and vitamin D has profound effects on development and function of the CNS. This leads to the possibility that low vitamin D may alter the CNS in a manner that makes it vulnerable to inflammation and the development of MS. This review analysis the role of vitamin D in the immune and nervous system, and how vitamin D insufficiency in children may contribute to the development of MS.

Neuron-Specific Vitamin D Signaling Attenuates Microglia Activation and CNS Autoimmunity.

Low vitamin D during childhood is associated with an increased risk of developing multiple sclerosis (MS) as an adult. Given that vitamin D has anti-inflammatory properties, it has been postulated that the relationship between MS and low vitamin D is due to immune dysregulation. Since the vitamin D receptor (VDR) is expressed in many cell types, this study investigated an alternative hypothesis-neuron-specific VDR signaling induces anti-inflammatory molecules that protect the central nervous system from autoimmunity. Using media from neurons treated with calcitriol, the active form of vitamin D3, LPS-activated microglia had a reduction in pro-inflammatory molecules, and a reciprocal induction of anti-inflammatory molecules. Since IL-34 is critical to the homeostasis of microglia, and was previously shown to be induced in endothelial cells by vitamin D, we investigated IL-34 as the potential anti-inflammatory molecule induced in neurons by vitamin D. Treatment of LPS-activated microglia with IL-34 reduced pro-inflammatory cytokine production and enhanced the expression of anti-inflammatory transcripts. However, neutralizing IL-34 in vitamin D neuronal conditioned media only impacted IL-6 and not the broader anti-inflammatory phenotype of microglia. To mimic low vitamin D in children, we used a neuron-specific inducible mouse model in which VDR was partially deleted in juvenile mice. Partial deletion of VDR in neurons during early life resulted in exacerbated CNS autoimmunity in adult mice. Overall, the study illustrated that vitamin D signaling in neurons promotes an anti-inflammatory state in microglia, and low vitamin D in early life may enhance CNS autoimmunity.

IL-3 Is a Marker of Encephalitogenic T Cells, but Not Essential for CNS Autoimmunity.

Identifying molecules that are differentially expressed in encephalitogenic T cells is critical to the development of novel and specific therapies for multiple sclerosis (MS). In this study, IL-3 was identified as a molecule highly expressed in encephalitogenic Th1 and Th17 cells, but not in myelin-specific non-encephalitogenic Th1 and Th17 cells. However, B10.PL IL-3-deficient mice remained susceptible to experimental autoimmune encephalomyelitis (EAE), a mouse model of MS. Furthermore, B10.PL myelin-specific T cell receptor transgenic IL-3-/- Th1 and Th17 cells were capable of transferring EAE to wild-type mice. Antibody neutralization of IL-3 produced by encephalitogenic Th1 and Th17 cells failed to alter their ability to transfer EAE. Thus, IL-3 is highly expressed in myelin-specific T cells capable of inducing EAE compared to activated, non-encephalitogenic myelin-specific T cells. However, loss of IL-3 in encephalitogenic T cells does not reduce their pathogenicity, indicating that IL-3 is a marker of encephalitogenic T cells, but not a critical element in their pathogenic capacity.

IL-23R-activated STAT3/STAT4 is essential for Th1/Th17-mediated CNS autoimmunity.

The factors that promote the differentiation of pathogenic T cells in autoimmune diseases are poorly defined. Use of genetically modified mice has provided insight into molecules necessary for the development of autoimmunity, but the sum of the data has led to contradictory observations based on what is currently known about specific molecules in specific signaling pathways. To define the minimum signals required for development of encephalitogenic T cells that cause CNS autoimmunity, myelin-specific T cells were differentiated with various cytokine cocktails, and pathogenicity was determined by transfer into mice. IL-6+IL-23 or IL-12+IL-23 generated encephalitogenic T cells and recapitulated the essential cytokine signals provided by antigen-presenting cells, and both IL-6 and IL-12 induced IL-23 receptor expression on both mouse and human naive T cells. IL-23 signaled through both STAT3 and STAT4, and disruption in STAT4 signaling impaired CNS autoimmunity independent of IL-12. These data explain why IL-12-deficient mice develop CNS autoimmunity, while STAT4-deficient mice are resistant. CD4+ memory T cells from multiple sclerosis patients had significantly higher levels of p-STAT3/p-STAT4, and p-STAT3/p-STAT4 heterodimers were observed upon IL-23 signaling, suggesting that p-STAT3/p-STAT4 induced by IL-23 signaling orchestrate the generation of pathogenic T cells in CNS autoimmunity, regardless of Th1 or Th17 phenotype.