Th1-mediated Autoimmune Diseases Research Articles

Epigenetic activation during T helper 17 cell differentiation is mediated by Tripartite motif containing 28

Epigenetic regulation is important for T-cell fate decision. Although STAT3 is known to initiate Th17 differentiation program, the downstream mechanism is unclear. Here we show that Tripartite motif containing 28 (Trim28) expression in Th17 cells is required for Th17-mediated cytokine production and experimental autoimmune diseases. Genome-wide occupancy analysis reveals that TRIM28-bound regions overlap with almost all Th17-specific super-enhancers (SE), and that those SEs are impaired by the deficiency of STAT3 or TRIM28, but not of RORγt. Importantly, IL-6-STAT3 signaling facilitates TRIM28 binding to the Il17-Il17f locus, and this process is required for epigenetic activation and high-order chromosomal interaction. TRIM28 also forms a complex with STAT3 and RORγt, and promotes the recruitment of RORγt to its target cytokine genes. Our study thus suggests TRIM28 to be important for the epigenetic activation during Th17 cell differentiation, and prompts the potential use of epigenetic interventions for Th17-related autoimmune diseases.

Intranuclear delivery of the transcription modulation domain of Tbet-improved lupus nephritis in (NZB/NZW) F1 lupus-prone mice

Excessive expression of Tbet and IFNγ is evidence of systemic lupus erythematosus (SLE) in lupus patients. In this study, the nucleus-transducible form of Transcription Modulation Domain (TMD) of Tbet (ntTbet-TMD), which is a fusion protein between Protein Transduction Domain Hph-1 (Hph-1-PTD) and the TMD of Tbet comprising DNA binding domain and isotype-specific domain, was generated to inhibit Tbet-mediated transcription in the interactomic manner. ntTbet-TMD was effectively delivered into the nucleus of the cells and specifically inhibited Tbet-mediated transcription without influencing the differentiation of other T cell subsets and signaling events for T cell activation. The severity of nephritis was significantly reduced by ntTbet-TMD as effectively as methylprednisolone in lupus-prone mice. The number of Th1, Th2 or Th17 cells and the secretion of their cytokines substantially decreased in the spleen and kidney of lupus-prone mice by ntTbet-TMD treatment. In contrast to methylprednisolone, the marked increase of Treg cells and the secretion of their immunosuppressive cytokine were detected in the spleen of (NZB/NZW) F1 mice treated withntTbet-TMD. Thus, ntTbet-TMD can improve nephritisin lupus-prone mice by modulating the overall proinflammatory microenvironment and rebalancing Tcell subsets, leading to new immune therapeutics for Th1-mediated autoimmune diseases.

Vitamin D deficiency in patients with alopecia areata: A systematic review and meta-analysis

Vitamin D deficiency in patients with alopecia areata: A systematic review and meta-analysis

PO150 Memory b cells are key for immunotherapy in multiple sclerosis

Although multiple sclerosis (MS) is considered to be a CD4+,Th17-mediated autoimmune disease, supportive evidence is largely circumstantial and often based on animal studies. Whilst alemtuzumab inhibits relapsing MS and depletes CD4+ T cells, it also selectively depletes CD19+,CD27+ memory B cells. Although CD4-depletion is considered to have failed in MS, we found following extensive review of the relevant literature and data mining that current treatments including: beta-interferons, glatiramer acetate, cytostatic agents, dimethyl fumarate, fingolimod, cladribine, daclizumab, rituximab/ocrelizumab physically, or in the case of natalizumab functionally, depleted CD19+CD27+ memory B cells. This depletion was substantial and long-term following CD20-depletion, and like with alemtuzumab, also led to long-term inhibition of MS with few treatment cycles, indicating induction-therapy activity. Importantly, memory B cells were augmented by B cell activating factor (atacicept) and tumour necrosis factor (infliximab) blockade that are known to worsen MS. This creates a unifying concept centred on memory B cells that is consistent with therapeutic, histopathological and aetiological aspects of MS.

PTEN drives Th17 cell differentiation by preventing IL-2 production.

T helper 17 (Th17) cells are a CD4+ T cell subset that produces IL-17A to mediate inflammation and autoimmunity. IL-2 inhibits Th17 cell differentiation. However, the mechanism by which IL-2 is suppressed during Th17 cell differentiation remains unclear. Here, we show that phosphatase and tensin homologue (PTEN) is a key factor that regulates Th17 cell differentiation by suppressing IL-2 production. Th17-specific Pten deletion (Ptenfl/flIl17acre ) impairs Th17 cell differentiation in vitro and ameliorated symptoms of experimental autoimmune encephalomyelitis (EAE), a model of Th17-mediated autoimmune disease. Mechanistically, Pten deficiency up-regulates IL-2 and phosphorylation of STAT5, but reduces STAT3 phosphorylation, thereby inhibiting Th17 cell differentiation. PTEN inhibitors block Th17 cell differentiation in vitro and in the EAE model. Thus, PTEN plays a key role in Th17 cell differentiation by blocking IL-2 expression.

Metabolic control of TH17 and induced Treg cell balance by an epigenetic mechanism.

Metabolism has been shown to integrate with epigenetics and transcription to modulate cell fate and function. Beyond meeting the bioenergetic and biosynthetic demands of T-cell differentiation, whether metabolism might control T-cell fate by an epigenetic mechanism is unclear. Here, through the discovery and mechanistic characterization of a small molecule, (aminooxy)acetic acid, that reprograms the differentiation of T helper 17 (TH17) cells towards induced regulatory T (iTreg) cells, we show that increased transamination, mainly catalysed by GOT1, leads to increased levels of 2-hydroxyglutarate in differentiating TH17 cells. The accumulation of 2-hydroxyglutarate resulted in hypermethylation of the Foxp3 gene locus and inhibited Foxp3 transcription, which is essential for fate determination towards TH17 cells. Inhibition of the conversion of glutamate to α-ketoglutaric acid prevented the production of 2-hydroxyglutarate, reduced methylation of the Foxp3 gene locus, and increased Foxp3 expression. This consequently blocked the differentiation of TH17 cells by antagonizing the function of transcription factor RORγt and promoted polarization into iTreg cells. Selective inhibition of GOT1 with (aminooxy)acetic acid ameliorated experimental autoimmune encephalomyelitis in a therapeutic mouse model by regulating the balance between TH17 and iTreg cells. Targeting a glutamate-dependent metabolic pathway thus represents a new strategy for developing therapeutic agents against TH17-mediated autoimmune diseases.

RORγt and RORα signature genes in human Th17 cells.

RORγt and RORα are transcription factors of the RAR-related orphan nuclear receptor (ROR) family. They are expressed in Th17 cells and have been suggested to play a role in Th17 differentiation. Although RORγt signature genes have been characterized in mouse Th17 cells, detailed information on its transcriptional control in human Th17 cells is limited and even less is known about RORα signature genes which have not been reported in either human or mouse T cells. In this study, global gene expression of human CD4 T cells activated under Th17 skewing conditions was profiled by RNA sequencing. RORγt and RORα signature genes were identified in these Th17 cells treated with specific siRNAs to knock down RORγt or RORα expression. We have generated selective small molecule RORγt modulators and they were also utilized as pharmacological tools in RORγt signature gene identification. Our results showed that RORγt controlled the expression of a very selective number of genes in Th17 cells and most of them were regulated by RORα as well albeit a weaker influence. Key Th17 genes including IL-17A, IL-17F, IL-23R, CCL20 and CCR6 were shown to be regulated by both RORγt and RORα. Our results demonstrated an overlapping role of RORγt and RORα in human Th17 cell differentiation through regulation of a defined common set of Th17 genes. RORγt as a drug target for treatment of Th17 mediated autoimmune diseases such as psoriasis has been demonstrated recently in clinical trials. Our results suggest that RORα could be involved in same disease mechanisms and gene signatures identified in this report could be valuable biomarkers for tracking the pharmacodynamic effects of compounds that modulate RORγt or RORα activities in patients.

RGC-32 Promotes Th17 Cell Differentiation and Enhances Experimental Autoimmune Encephalomyelitis.

Th17 cells play a critical role in autoimmune diseases, including multiple sclerosis and its animal model, experimental autoimmune encephalomyelitis. Response gene to complement (RGC)-32 is a cell cycle regulator and a downstream target of TGF-β that mediates its profibrotic activity. In this study, we report that RGC-32 is preferentially upregulated during Th17 cell differentiation. RGC-32-/- mice have normal Th1, Th2, and regulatory T cell differentiation but show defective Th17 differentiation in vitro. The impaired Th17 differentiation is associated with defects in IFN regulatory factor 4, B cell-activating transcription factor, retinoic acid-related orphan receptor γt, and SMAD2 activation. In vivo, RGC-32-/- mice display an attenuated experimental autoimmune encephalomyelitis phenotype accompanied by decreased CNS inflammation and reduced frequency of IL-17- and GM-CSF-producing CD4+ T cells. Collectively, our results identify RGC-32 as a novel regulator of Th17 cell differentiation in vitro and in vivo and suggest that RGC-32 is a potential therapeutic target in multiple sclerosis and other Th17-mediated autoimmune diseases.

The nuclear receptor REV-ERBα modulates Th17 cell-mediated autoimmune disease

Abstract T helper 17 cells (Th17) play an important role in controlling extracellular bacteria and yeast infections; however, dysregulated Th17 response can lead to detrimental autoimmune diseases such as multiple sclerosis. Th17 cell differentiation relies on IL6/TGF-β-induced expression of retinoic acid receptor-related orphan nuclear receptor RORγt, which is able to induce Th17 effector cytokines such as IL17A. Therefore, antagonizing RORγt activity may prove to be one of the therapeutic strategies for ameliorating Th17-mediated inflammation. Here we identify REV-ERBα (encoded by Nr1d1), a member of the nuclear hormone receptor family, as a transcriptional repressor that antagonizes RORγt-induced Il17a expression. Both RORγt and REV-ERBα bind to the same ROR response element (RORE) in Il17a promoter region. Elevated expression of REV-ERBα prevents RORγt binding to RORE and reduces Il17a expression. Furthermore, treatment with a synthetic REV-ERBα agonist, SR9009, significantly delays the onset and impedes the progression of experimental autoimmune encephalomyelitis (EAE), a Th17-mediated autoimmune disease. These results suggest that modulating REV-ERBα activity holds therapeutic potential for treatment of Th17 cell-mediated autoimmune diseases.

Negative regulation of Th17 cell development and function by the REV-ERBs

Abstract A significant amount of work has identified key factors that drive Th17 cell development, including the nuclear receptors (NRs) RORα and RORγ. However, cell-intrinsic mechanisms that negatively regulate Th17 cell development and inflammation have received less attention. Two other members of the NR superfamily, the REV-ERBs (REV-ERBα and REV-ERBβ), are often co-expressed in the same tissues as the RORs, bind the same DNA response elements, and co-regulate their shared target genes. The REV-ERBs have well described roles in the regulation of the circadian rhythm and metabolism, but their roles in immune cell function are poorly understood. Therefore, we hypothesize that the REV-ERBs are key negative regulators of Th17 cell development and function. We we aim to understand the role of the REV-ERBs in Th17 cell development as well as determine whether targeting these NRs is a viable therapeutic option for the treatment of Th17-mediated autoimmune diseases. Our results indicate that the REV-ERBs are differentially expressed during Th17 cell development. Overexpression and pharmacological modulation of REV-ERB activity suppresses Th17 cell development whereas genetic deletion of the REV-ERBs perturbs Th17 cell development in vitro and induces autoimmunity in vivo. Using REV-ERB-specific small molecules that we have developed, we have found that pharmacological modulation of REV-ERB activity suppresses Th17 cell development in vitro and development of autoimmunity in vivo. Collectively, our data suggest that the REV-ERBs are key negative regulators of Th17 cell development and function and represent a novel therapeutic target for the treatment of Th17-mediated autoimmune diseases. Funding support – NIH (R01AI116885-01A1)

Th17 cells modulate the LN microenvironment through IL-17 dependent regulation of fibroblastic reticular cells

Abstract Integrins play a critical role in the function and migration of Th17 cells in CNS, and blockade of inflammatory T cell migration is used therapeutically in multiple sclerosis. Our previously published data demonstrated a critical role for αvβ3 in driving encephalitogenic Th17 cell migration in experimental autoimmune encephalomyelitis (EAE), in an IL-23R-dependent manner. Herein, we show that fibronectin, a ligand for integrin αvβ3, is significantly upregulated in the dLNs of EAE mice. Strikingly, the expression of fibronectin in dLN was significantly reduced in IL23R−/− mice following immunization. Fibronectin expression increased in dLNs during the progression of EAE with similar kinetics but delayed peak compared to IL-17. Since IL23R regulates IL-17 production, we analyzed LN from IL-17−/− and IL17Ra−/− mice immunized for EAE, and observed a similar defect in fibronectin expression in the absence of IL-17 signaling. CD45−gp38+CD31− fibroblastic reticular cells (FRCs) in the T cell zone are the major producers of fibronectin in the dLNs, and these stromal cells expand during EAE. Although IL-17RA−/− LN appeared similarly enlarged following immunization, indicating an inflammatory response, the number of FRCs was decreased in immunized IL17Ra−/− LN, corresponding to decreased fibronectin. Taken together, our data suggests that during priming of the immune response, and before migrating to their peripheral target tissue, Th17 cells modulate the local LN environment in which they are activated through production of IL-17 to promote FRCs expansion and induction of ECM. This could have long-term consequences in the host upon future infectious challenge, as well as influencing chronicity of Th17-mediated autoimmune disease.

Inhibition of Th17 by targeted degradation of HIF1-α

Abstract Death-associated protein kinase (DAPK) is a tumor suppressor, while hypoxia-inducible factor 1α (HIF-1α) promotes tumorigenesis in various type of cancers. Here we report a targeted degradation of HIF-1α mediated by DAPK in T helper 17 (Th17) cells. HIF-1α is a transcription factor critical for Th17 development. We found that DAPK inhibits Th17 differentiation and prevents Th17-mediated autoimmune diseases. DAPK suppresses the expression of RORγt and IL-17, which could be attributed to a specific suppression of HIF-1α. In contrast, Th1 cell differentiation and T-bet expression is not affected by DAPK. We demonstrate that DAPK specifically downregulates HIF-1α in a unique process. While HIF-1α is predominant nuclear localized in many cancer cells, HIF-1α is located in both cytosol and nucleus in T cells. This leads to an interaction between HIF-1α and DAPK in T cell cytoplasm. The simultaneous binding of DAPK to prolyl hydroxylase domain protein 2 (PHD2) increases the association between HIF-1α and PHD2. Consequently, DAPK enhances the proline hydroxylation and proteasome degradation of HIF-1α in Th17 cells. DAPK-deficiency thus results in excess HIF-1α accumulation, enhanced Th17 generation, and exacerbated experimental autoimmune encephalomyelitis (EAE). Transgenic DAPK expression, in contrast, suppresses Th17 differentiation and prevents EAE generation. The overproduction of Th17 cells and exacerbated EAE development in Dapk−/− mice is fully reversed by knockout of HIF-1α. Our results demonstrate a new process involving DAPK-mediated degradation of cytoplasmic HIF-1α, and suggest that a potential therapy for Th17-associated inflammatory diseases by targeted elevation of DAPK.

Expansion of IL-6+ Th17-like cells expressing TLRs correlates with microbial translocation and neurological disabilities in NMOSD patients

Different microbial antigens, by signaling through toll-like receptors (TLR), may contribute to Th17-mediated autoimmune diseases, such as neuromyelitis optica spectrum disorder (NMOSD). The objective of this study was to determine the proportion of different Th17-like cell subsets that express TLR in NMOSD patients. For this study, the frequency of different Th17 cell subsets expressing TLR subsets in healthy individuals (n=20) and NMOSD patients (n=20) was evaluated by cytometry. The peripheral levels of soluble CD14 (sCD14) and cytokines were determined by ELISA. Our results demonstrated that the proportion of peripheral CD4+ T cells expressing TLR2, 4 and 9 was significantly higher in NMOSD samples than in healthy subjects. In NMOSD, these cells are CD28+PD-1−CD57− and produce elevated levels of IL-17. Among different TLRs+ Th17-like subsets, the proportion of those that co-express IL-17 and IL-6 was significantly higher in NMOSD patients, which was positively correlated with sCD14 levels and EDSS score. By contrast, the percentage of TLRs+Treg17 cells (IL-10+IL-17+) was negatively related to sCD14 and the severity of NMOSD. In conclusion, the expansion of peripheral IL-6-producing TLR+ Th17-like cells in NMOSD patients was associated with both bacterial translocation and disease severity.

Nutritional control of IL-23/Th17-mediated autoimmune disease through HO-1/STAT3 activation

The nutritional curcumin (CUR) is beneficial in cell-mediated autoimmune diseases. The molecular mechanisms underlying this food-mediated silencing of inflammatory immune responses are poorly understood. By investigating antigen-specific immune responses we found that dietary CUR impairs the differentiation of Th1/Th17 cells in vivo during encephalomyelitis and instead promoted Th2 cells. In contrast, feeding CUR had no inhibitory effect on ovalbumin-induced airway inflammation. Mechanistically, we found that CUR induces an anti-inflammatory phenotype in dendritic cells (DC) with enhanced STAT3 phosphorylation and suppressed expression of Il12b and Il23a. On the molecular level CUR readily induced NRF2-sensitive heme oxygenase 1 (HO-1) mRNA and protein in LPS-activated DC. HO-1 enhanced STAT3 phosphorylation, which enriched to Il12b and Il23a loci and negatively regulated their transcription. These findings demonstrate the underlying mechanism through which a nutritional can interfere with the immune response. CUR silences IL-23/Th17-mediated pathology by enhancing HO-1/STAT3 interaction in DC.

New roles for CD14 and IL-β linking inflammatory dendritic cells to IL-17 production in memory CD4+ T cells.

Interleukin (IL)-1β has proven to be crucial in the differentiation of human and mouse Th17 cells. Although it has become evident that IL-1β has potent IL-17-inducing effects on CD4+ T cells directly, it has not yet been explored whether IL-1β can also prime dendritic cells (DCs) for a Th17 instruction program. Here, we show that human immature DCs exposed to IL-1β promote IL-17 production in human memory CD4+ T cells. IL-1β-primed DCs express high levels of CD14 that mediate IL-17 production through direct interaction with T cells. Moreover, culturing human CD4+CD45RO+ memory T cells with soluble CD14 is sufficient for the upregulation of retinoic acid-related orphan receptor-γ thymus and IL-17 production. In addition, in a human in situ model using tissue-resident skin DCs, upregulation of CD14 expression induced by IL-1β on skin residents DCs promotes IL-17 production in memory T cells; strongly suggesting the in vivo relevance of this mechanism. Our findings uncover new roles for IL-1β and CD14, and may therefore have important consequences for the development of new therapies for Th17-mediated autoimmune diseases and bacterial and fungal pathogenic infections.

GM-CSF as a therapeutic target in autoimmune diseases.

Granulocyte-macrophage colony-stimulating factor (GM-CSF) has been known as a hematopoietic growth factor and immune modulator. Recent studies revealed that GM-CSF also had pro-inflammatory functions and contributed to the pathogenicity of Th17 cells in the development of Th17-mediated autoimmune diseases. GM-CSF inhibition in some animal models of autoimmune diseases showed significant beneficial effects. Therefore, several agents targeting GM-CSF are being developed and are expected to be a useful strategy for the treatment of autoimmune diseases. Particularly, in clinical trials for rheumatoid arthritis (RA) patients, GM-CSF inhibition showed rapid and significant efficacy with no serious side effects. This article summarizes recent findings of GM-CSF and information of clinical trials targeting GM-CSF in autoimmune diseases.

Discovery and structural optimization of 4-(4-(benzyloxy)phenyl)-3,4-dihydropyrimidin-2(1H)-ones as RORc inverse agonists.

Retinoic acid receptor-related orphan nuclear receptors (RORs) are orphan nuclear receptors that show constitutive activity in the absence of ligands. Among 3 subtypes of RORs, RORc is a promising therapeutic target for the treatment of Th17-mediated autoimmune diseases. Here, we report novel RORc inverse agonists discovered through structure-based drug design. Based on the structure of compound 8, a previously described agonist of RORa, a series of 4-(4-(benzyloxy)phenyl)-3,4-dihydropyrimidin-2(1H)-one derivatives were designed and synthesized. The interaction between the compounds and RORc was detected at molecular level using AlphaScreen assay. The compounds were further examined in 293T cells transfected with RORc and luciferase reporter gene. Thermal stability shift assay was used to evaluate the effects of the compounds on protein stability. A total of 27 derivatives were designed and synthesized. Among them, the compound 22b was identified as the most potent RORc inverse agonist. Its IC50 values were 2.39 μmol/L in AlphaScreen assay, and 0.82 μmol/L in inhibition of the cell-based luciferase reporter activity. Furthermore, the compound 22b displayed a 120-fold selectivity for RORc over other nuclear receptors. Moreover, a molecular docking study showed that the structure-activity relationship was consistent with the binding mode of compound 22b in RORc. 4-(4-(Benzyloxy)phenyl)-3,4-dihydropyrimidin-2(1H)-one derivatives are promising candidates for the treatment of Th17-mediated autoimmune diseases, such as rheumatoid arthritis, psoriasis, and multiple sclerosis.

T-bet promotes the accumulation of encephalitogenic Th17 cells in the CNS

T-bet enhances the encephalitogenicity of myelin-reactive CD4+ T cells, however its mechanism of action is unknown. In this study we show that T-bet confers a competitive advantage for the accumulation of IL-23 conditioned Th17 effector cells in the central nervous system (CNS). Impaired migration of T-bet deficient Th17 cells to the CNS is associated with altered expression of adhesion molecules and chemokine receptors on their cell surface. Our data suggest that therapeutic targeting of T-bet in individuals with Th17-mediated autoimmune demyelinating disease may inhibit inflammatory infiltration of the CNS and, hence, clinical exacerbations.

Anti-inflammatory effects of interleukin-23 receptor cytokine-binding homology region rebalance T cell distribution in rodent collagen-induced arthritis

IL-23 is an important cytokine to regulate Th17 cell differentiation and promote the proliferation of inflammatory cells in Th17-mediated autoimmune diseases. The collagen-induced arthritis (CIA) in rat is a model of rheumatoid arthritis characterized by pronounced inflammatory auto-responses from B and T cells, especially Th17 cells in lesions. In the present study, we used rhIL23R-CHR to block the IL-23 signaling pathway to probe the importance of IL-23 in misbalancing the ratio of Th17/Th9/Treg cells in CIA rats. After treatments with rhIL23R-CHR, the CIA rats showed a significant decrease of secretions of IL-17 and IL-9, whereas FoxP3 was activated in the process, indicating that IL-23 can manipulate the balance of Th17/Th9/Treg cells. Similar to the animal model, IL-23 also possessed remarkable proinflammatory effects on human fibroblast-like synoviocyte cells (HFLS), showing synergetic outcomes with TNF-α. Together, IL-23 could act as a modulator to imbalance the ratio of Th17/Th9/Treg cells, and rhIL23R-CHR could serve as a potential therapeutic agent for RA patients.

Carbon nanospheres mediated delivery of nuclear matrix protein SMAR1 to direct experimental autoimmune encephalomyelitis in mice.

Owing to the suppression of immune responses and associated side effects, steroid based treatments for inflammatory encephalitis disease can be detrimental. Here, we demonstrate a novel carbon nanosphere (CNP) based treatment regime for encephalomyelitis in mice by exploiting the functional property of the nuclear matrix binding protein SMAR1. A truncated part of SMAR1 ie, the DNA binding domain was conjugated with hydrothermally synthesized CNPs. When administered intravenously, the conjugate suppressed experimental animal encephalomyelitis in T cell specific conditional SMAR1 knockout mice (SMAR−/−). Further, CNP-SMAR1 conjugate delayed the onset of the disease and reduced the demyelination significantly. There was a significant decrease in the production of IL-17 after re-stimulation with MOG. Altogether, our findings suggest a potential carbon nanomaterial based therapeutic intervention to combat Th17 mediated autoimmune diseases including experimental autoimmune encephalomyelitis.