Retinoic Acid-related Orphan Nuclear Receptor Research Articles

Thiostrepton alleviates experimental colitis by promoting RORγt ubiquitination and modulating dysbiosis.

Thiostrepton (TST) is a natural antibiotic with pleiotropic properties. This study aimed to elucidate the therapeutic effect of TST on experimental colitis and identify its targets. The effect of TST on colon inflammation was evaluated in a dextran sulfate sodium (DSS)-induced colitis model and a T-cell transfer colitis model. The therapeutic targets of TST were investigated by cytokine profiling, immunophenotyping and biochemical approaches. The effect of TST on the gut microbiota and its contribution to colitis were evaluated in mice with DSS-induced colitis that were subjected to gut microbiota depletion and fecal microbiota transplantation (FMT). Alterations in the gut microbiota caused by TST were determined by 16S rDNA and metagenomic sequencing. Here, we showed that TST treatment significantly ameliorated colitis in the DSS-induced and T-cell transfer models. Specifically, TST targeted the retinoic acid-related orphan nuclear receptor RORγt to reduce the production of IL-17A by γδ T cells, type 3 innate lymphoid cells (ILC3s) and Th17 cells in mice with DSS-induced colitis. Similarly, TST selectively prevented the development of Th17 cells in the T-cell transfer colitis model and the differentiation of naïve CD4+ T cells into Th17 cells in vitro. Mechanistically, TST induced the ubiquitination and degradation of RORγt by promoting the binding of Itch to RORγt. Moreover, TST also reversed dysbiosis to control colonic inflammation. Taken together, these results from our study describe the previously unexplored role of TST in alleviating colonic inflammation by reducing IL-17A production and modulating dysbiosis, suggesting that TST is a promising candidate drug for the treatment of IBD.

Saikosaponin D inhibits nasal inflammation by regulating the transcription factors T-box protein expressed in T cells/GATA-3 and retinoic acid-related orphan nuclear receptor γt in a murine model of allergic rhinitis

ContextSaikosaponin D (SSD) is a commonly prescribed agent against inflammatory diseases in Asian countries. However, the anti-allergic inflammatory effect of SSD in allergic rhinitis (AR) model is not well known. ObjectiveWe investigated the anti-allergic and anti-inflammatory effects of SSD on the ovalbumin (OVA)-induced AR model. Materials and methodBALB/c mice were divided into the control, OVA, OVA + SSD, and OVA + dexamethasone (Dex) groups. AR was established by intraperitoneal injection with OVA adsorbed to aluminum hydroxide, and intranasal challenge with OVA. Thereafter, the mice were treated with 10 mg/kg BW (Body weight) of OVA + SSD and 2.5 mg/kg BW of Dex orally for 11 days before being challenged. Subsequently, the mice were challenged with OVA 1 h after SSD or Dex treatment. The Control group was treated with saline only. ResultsThe addition of 10 mg/kg BW of OVA + SSD significantly ameliorated the nasal symptoms including sneezing and rubbing from 30 ± 5.2 times in OVA group to 20 ± 5.8 times. Moreover, OVA + SSD group decreased the production of TNF-α, IL-4, IL-5, IL-17, GATA-3 and RORγ about 1.2–1.4-fold compared to the OVA-induced AR mice near to 2.5 mg/kg BW of Dex levels. Meanwhile OVA + SSD group slightly increased the levels of INF-γ, IL-12 and T-bet about 1.8–2.0-fold compared to the OVA group near to control group. Notably, OVA + SSD group also reduced the levels of OVA-specific IgE and IgG1 about 0.5–2.5-fold compared OVA group but increased the levels of IgG2a in serum. The results were analyzed using Graph Pad Prism software (v5.0, La Jolla, CA, USA). ConclusionSSD may represent an alternative therapeutic approach for the treatment of patients with AR through the regulation of transcription factors T-bet, GATA-3, and RORγ in inflammatory cells.

Negative Correlation between Serum NLRP3 and the Ratio of Treg/Th17 in Patients with Obstructive Coronary Artery Disease

Background: Regulatory T (Treg) cells are a class of anti-inflammatory lymphocyte subpopulations with a potential protective effect against atherosclerosis, whereas T helper 17 (Th17) cells have been reported to possess proatherogenic activity. It was believed that disturbed circulating Treg/Th17 balance was associated with the onset and progression of atherosclerosis. This study is designed to probe the regulative action of serum Nod-like receptor protein 3 (NLRP3) on the Treg/Th17 balance in patients with atherosclerosis. Methods: Fifty-two patients with coronary atherosclerosis and stenosis degrees of more than 50% were assigned to the coronary artery disease (CAD) group, and an equal number of people without coronary atherosclerosis were assigned to the control group (assessed by coronary angiography). Peripheral blood mononuclear cells (PBMCs) from two group patients were extracted and cultivated. The calculation of the Treg/Th17 ratio and quantitative analysis of the Treg and Th17 cell frequencies were performed through flow cytometry. Real-time fluorescence quantitative polymerase chain reaction (RT-PCR) was executed for the quantitative mRNA detection of the fork head-winged helix transcription factor (Foxp3) and the retinoic acid-related orphan nuclear receptor C (RORC) in PBMCs. Enzyme-linked immunosorbent assays were applied to measure the serum level of NLRP3, interleukin (IL)-10, IL-1β, IL-17A, IL-23, and transforming growth factor (TGF)-β1. Additionally, the connection between serum Treg/Th17 ratio and NLRP3 levels was analyzed using the Pearson correlation coefficient. Results: The baseline parameters, including sex, age, or blood biochemical indices had no difference in both groups (p > 0.05). The CAD group showed higher Th17 cell frequency, lower Treg cell frequency, and a lower Treg/Th17 ratio when compared to the control (p < 0.05). Consistent with the variation in the T-cell subset ratio, in patients with atherosclerosis, the Th17-cell-related transcription factor RORC showed a markedly higher mRNA level (p < 0.05), conversely, the mRNA expression of the Treg cell-related transcription factor Foxp3 was notably reduced (p < 0.05). Similarly, the serum levels of NLRP3, IL-17A, IL-1, and IL-23 were significantly enhanced in CAD group but IL-10 and TGF-β1 were reduced (p < 0.05). Additionally, a negative correlation was found between NLRP3 and the Treg/Th17 ratio (r = –0.69, p < 0.001). Conclusions: Due to the potential impact on the serum Treg/Th17 ratio, NLRP3 may act as an aggravator in the onset and progression of atherosclerotic disease.

Effects of astragaloside Ⅳ on inflammatory response and percentage of peripheral blood Th17 cells in mice with ulcerative colitis

This study aimed to investigate the anti-inflammatory effect of astragaloside Ⅳ in mice with ulcerative colitis(UC) and its effect on the percentage of peripheral blood T helper(Th17) cells. Following the establishment of UC mouse model with 2% sodium dextran sulfate(DSS), mice in the positive control group and low-and high-dose astragaloside Ⅳ groups were treated with corresponding drugs by gavage. Disease activity index(DAI) was calculated, and serum interleukin-17(IL-17), tumor necrosis factor-α(TNF-α), and transforming growth factor-β(TGF-β) levels were assayed by ELISA. The pathological changes in colon tissue were observed by HE staining, and Th17/regulatory T cells(Treg) ratio in the peripheral blood was determined by flow cytometry. Western blot was conducted for detecting the relative protein expression levels of forkhead box protein P3(Foxp3) and retinoic acid-related orphan nuclear receptor γT(ROR-γt). The findings demonstrated that in normal mice, the colonic structure was intact. The goblet cells were not reduced and the glands were neatly arranged, with no mucosal erosion, bleeding, or positive cell infiltration. In the model group, the colonic mucosal structure was seriously damaged, manifested as disordered arrangement or missing of glands, vascular dilatation, congestion, and massive inflammatory cell infiltration. The pathological injury of colon tissue was alleviated to varying degrees in drug treatment groups. Compared with the normal group, the model group exhibited elevated percentage of Th17 cells, increased IL-17 and TNF-α content, up-regulated relative ROR-γt protein expression, lowered TGF-β, reduced percentage of Treg cells, and down-regulated relative Foxp3 protein expression. The comparison with the model group showed that DAI score, pathological score, percentage of Th17 cells, IL-17 and TNF-α content, and relative ROR-γt protein expression in the positive control group, low-dose astragaloside Ⅳ group, and high-dose astragaloside Ⅳ group were decreased, while TGF-β content, percentage of Treg cells, and relative Foxp3 protein expression were increased. The DAI score, pathological score, percentage of Th17 cells, IL-17 and TNF-α content, and relative ROR-γt protein expression in the low-dose astragaloside Ⅳ group were higher than those in the positive control group, whereas the content of TGF-β, percentage of Treg cells, and relative Foxp3 protein expression were lower. DAI score, pathological score, percentage of Th17 cells, IL-17 and TNF-α content, relative ROR-γt protein expression in the high-dose astragaloside Ⅳ group declined in contrast to those in the low-dose astragaloside Ⅳ group, while the TGF-β content, percentage of Treg cells, and relative Foxp3 protein expression rose. There was no significant difference between the positive control group and the high-dose astragaloside Ⅳ group. Astragaloside Ⅳ is able to inhibit inflammatory response and diminish the percentage of Th17 cells in mice with UC.

Morphological and neurochemical characterization of glycinergic neurons in laminae I-IV of the mouse spinal dorsal horn.

A growing body of experimental evidence shows that glycinergic inhibition plays vital roles in spinal pain processing. In spite of this, however, our knowledge about the morphology, neurochemical characteristics, and synaptic relations of glycinergic neurons in the spinal dorsal horn is very limited. The lack of this knowledge makes our understanding about the specific contribution of glycinergic neurons to spinal pain processing quite vague. Here we investigated the morphology and neurochemical characteristics of glycinergic neurons in laminae I-IV of the spinal dorsal horn using a GlyT2::CreERT2-tdTomato transgenic mouse line. Confirming previous reports, we show that glycinergic neurons are sparsely distributed in laminae I-II, but their densities are much higher in lamina III and especially in lamina IV. First in the literature, we provide experimental evidence indicating that in addition to neurons in which glycine colocalizes with GABA, there are glycinergic neurons in laminae I-II that do not express GABA and can thus be referred to as glycine-only neurons. According to the shape and size of cell bodies and dendritic morphology, we divided the tdTomato-labeled glycinergic neurons into three and six morphological groups in laminae I-II and laminae III-IV, respectively. We also demonstrate that most of the glycinergic neurons co-express neuronal nitric oxide synthase, parvalbumin, the receptor tyrosine kinase RET, and the retinoic acid-related orphan nuclear receptor β (RORβ), but there might be others that need further neurochemical characterization. The present findings may foster our understanding about the contribution of glycinergic inhibition to spinal pain processing.

Orphan nuclear receptor RORγ confers doxorubicin resistance in prostate cancer.

Prostate cancer (PCa) is a malignant tumor with an extremely high prevalence. Doxorubicin is the first-line clinical treatment for castration-resistant PCa. Clinically, relapse is almost inevitable due to the cancer cells' increasing resistance to doxorubicin. Our previous studies have revealed that retinoic acid-related orphan nuclear receptor γ (RORγ) is a key protein for cancer progression and a promising target for PCa therapy. Though, RORγ's role and mechanism in doxorubicin-resistant PCa remain unclear. To study the mechanism of doxorubicin resistance, we generated a doxorubicin-resistant PCa cell line C4-2B (C4-2B DoxR) in this study, by culturing cells in an increasing doxorubicin concentration. Here, we show that RORγ expression was upregulated in C4-2B DoxR cells compared with that in normal C4-2B cells. The RORγ-stably-overexpressing PCa cell line constructed by lentiviral transfection showed an obvious improvement in doxorubicin resistance and a trend toward castration resistance. Furthermore, RORγ-specific small molecule inhibitors XY018, GSK805, and SR2211 can significantly inhibit the proliferation of C4-2B DoxR cells and promote their apoptosis. Collectively, these results have demonstrated the correlation between the upregulation of RORγ and the development of PCa's doxorubicin resistance, thus providing new ideas for solving the problem of chemotherapy drug resistance in PCa.

Melatonin inhibits apoptosis in mouse Leydig cells via the retinoic acid-related orphan nuclear receptor α/p53 pathway

Melatonin is an endogenous indoleamine hormone involved in various physiological processes. However, the mechanism of melatonin in mediating Leydig cells function has not been fully explained. In this study, we investigated the mechanism through which melatonin inhibits apoptosis in mouse Leydig cells by activating the retinoic acid-related orphan nuclear receptor (ROR) α/p53 signaling pathway. We confirmed the expression and localization of RORα in mouse Leydig cells using immunofluorescence. After treatment with 10 ng/mL melatonin for 36 h, RORα mRNA and protein levels were significantly increased (P < 0.01). TUNEL and flow cytometry showed that melatonin significantly decreased the TUNEL-positive cell ratio and apoptosis rate (P < 0.05). Moreover, melatonin decreased BAX expression and increased BCL-2 expression (P < 0.05). However, the RORα inhibitor SR1001 reversed the inhibitory effects of melatonin on apoptosis (P < 0.05). Additionally, analysis of p53 expression showed that melatonin inhibited p53 mRNA and protein expression (P < 0.05), whereas SR1001 reversed these effects. p53 reversed the anti-apoptotic process involving RORα-mediated melatonin in mouse Leydig cells. Collectively, our findings suggested that melatonin inhibited apoptosis via the RORα/p53 pathway.

Saikosaponin A ameliorates nasal inflammation by suppressing IL-6/ROR-γt/STAT3/IL-17/NF-κB pathway in OVA-induced allergic rhinitis

Allergic rhinitis (AR) is a type I hypersensitivity immune response and is a common chronic allergic respiratory disorder characterized by one or more nasal symptoms. Despite many studies on AR therapy, the drugs of treatment for AR remain limited in effect. In the present study, we aimed to elucidate the effects of saikosaponin A (SSA) on nasal inflammation, T helper (Th)2 and Th17 cytokines, retinoic acid-related orphan nuclear receptor (ROR)-γt, signal transducer and activator of transcription (STAT)3, and nuclear factor (NF)-κB signalings in ovalbumin (OVA)-induced AR mice model. OVA-induced AR mice exhibited increase in nasal symptoms, histological alteration, OVA-specific immunoglobulin (Ig)E/IgG1, ROR-γt, STAT3 and NF-κB signalings. However, the administration of SSA significantly decreased allergic symptoms including nasal rubbing and sneezing. Additionally, histological alterations such as mucosa layer thickness, goblet cell hyperplasia, eosinophils and mast cell infiltration in nasal tissues dramatically improved by treatment with SSA. Also, SSA treatment decreased the levels of OVA-specific IgE/IgG1 in serum and the levels of Th2 and Th17 cytokines such as interleukin (IL)-6 and IL-17 in nasal lavage fluid (NALF). Moreover, SSA inhibited the activation of transcription factor ROR-γt, STAT3 and phosphorylated STAT3 in both NALF and lung. Futher, SSA could also significantly inhibit the expressions of NF-κB p65 and phosphorylated NF-κB p65 in NALF and lung. These present results suggested that SSA may attenuate OVA-induced allergic rhinitis through regulating the expression of IL-6/ROR-γt/STAT3/IL-17/NF-κB signaling. The results indicate that SSA may be used as a therapeutic candidate for allergic rhinitis disease.

Research progress of peripheral blood regulatory T cell/helper T cell 17 imbalance and relevant gene methylation in immune thrombocytopenia

Immune thrombocytopenia (ITP) is an autoimmune hemorrhagic disease. The classic pathogenesis of ITP is that the production of autoantibodies against platelet membrane glycoproteins, which mediates platelet destruction. In recent years, it has been found that there are still many other mechanisms in ITP inducing thrombocytopenia and platelet damage, among which abnormal cellular immunity plays an important role. In cellular immunity, CD4+ CD25high CD127- regulatory T cells (Treg) are a group of T cell subsets that exist in human peripheral blood and spleen and can inhibit auto-reactive T cell responses; T helper cells (Th)17 is a newly discovered T cell subset that secretes interleukin (IL)-17, involved in mediating inflammatory response, which is of great significance in autoimmune diseases. Several studies showed that Treg/Th17 imbalance and abnormal expression of specific transcription factors folkhead box protein (Foxp)3 and retinoic acid-related orphan nuclear receptor (ROR)-γt were observed in peripheral blood of ITP patients. DNA methylation induces Foxp3 gene silencing and abnormal activation of IL-6/STAT3 signaling pathway, which may be the mechanism leading to Treg/Th17 imbalance and causes of ITP. The proposed demethylation treatment may provide new ideas for the molecular targeted treatment of ITP, but its scientificity, effectiveness and safety still need to be confirmed by further studies. The author reviews literature on the role of Treg/Th17 imbalance and relevant gene methylation mechanism in the occurrence, development and treatment of ITP. Key words: T-lymphocytes, regulatory; T-lymphocyte subsets; DNA methylation; Thrombocytopenia; Helper T lymphocyte 17; Decitabine

The changes of expressive levels of IL-17A, STAT3, and RORγt in different invasive pulmonary aspergillosis mice.

BackgroundT helper 17 (Th17) lymphocytes play an important role in Aspergillus adaptive immune response against Aspergillus fumigatus, but there is little attention focused on the different types of immunosuppressive models in which invasive pulmonary aspergillosis (IPA) develops. In addition, the expression levels of signal transducer and activator of transcription 3 (STAT3)/retinoic acid-related orphan nuclear receptor gamma (RORγt)/interleukin (IL)-17A signaling pathway, which is involved in the regulation of Th17 cells, as well as whether there are differences between two types of IPA mice models, remain unknown.Materials and methodsSix to eight weeks old female BALB/c mice were treated with cortisone acetate or cyclophosphamide to establish the immunosuppressive mice models, and then, A. fumigatus inoculum was injected to form the IPA groups and sterile saline was injected to form the control groups. Flow cytometry was performed to measure the proportion of Th17 cells in CD4+ T cells in the peripheral blood, spleen, and lung of the mice. The expression of IL-17A, RORγt, and STAT3 mRNA was detected by real-time polymerase chain reaction. Concentrations of IL-6 in the plasma and bronchoalveolar lavage fluid were measured by enzyme-linked immunosorbent assay.ResultsThe proportion of Th17 in the peripheral blood and lung tissue in neutropenic IPA mice showed a more significant increase than in non-neutropenic IPA mice (P<0.01). The IL-6 protein also showed the same trend in plasma and bronchoalveolar lavage fluid (P<0.01). Compared with the control groups, the expression of IL-17A at mRNA level in the lung was significantly increased, while RORγt/STAT3 mRNA was significantly decreased in the IPA groups (P<0.01).ConclusionThe expression of RORγt and STAT3 mRNA in the lung tissue in both groups was significantly decreased. IL-17 may play a negative role in the defense against Aspergillus through uprating IL-6.

Inhibition of the Nuclear Receptor RORγ and Interleukin-17A Suppresses Neovascular Retinopathy: Involvement of Immunocompetent Microglia.

Although inhibitors of vascular endothelial growth factor (VEGF) provide benefit for the management of neovascular retinopathies, their use is limited to end-stage disease and some eyes are resistant. We hypothesized that retinoic acid-related orphan nuclear receptor γ (RORγ) and its downstream effector, interleukin (IL)-17A, upregulate VEGF and hence are important treatment targets for neovascular retinopathies. Utilizing a model of oxygen-induced retinopathy, confocal microscopy and flow cytometry, we identified that retinal immunocompetent cells, microglia, express IL-17A. This was confirmed in primary cultures of rat retinal microglia, where hypoxia increased IL-17A protein as well as IL-17A, RORγ, and tumor necrosis factor-α mRNA, which were reduced by the RORγ inhibitor, digoxin, and the RORα/RORγ inverse agonist, SR1001. By contrast, retinal macroglial Müller cells and ganglion cells, key sources of VEGF in oxygen-induced retinopathy, did not produce IL-17A when exposed to hypoxia and IL-1β. However, they expressed IL-17 receptors, and in response to IL-17A, secreted VEGF. This suggested that RORγ and IL-17A inhibition might attenuate neovascular retinopathy. Indeed, digoxin and SR1001 reduced retinal vaso-obliteration, neovascularization, and vascular leakage as well as VEGF and VEGF-related placental growth factor. Digoxin and SR1001 reduced microglial-derived IL-17A and Müller cell and ganglion cell damage. The importance of IL-17A in oxygen-induced retinopathy was confirmed by IL-17A neutralization reducing vasculopathy, VEGF, placental growth factor, tumor necrosis factor-α, microglial density and Müller cell, and ganglion cell injury. Our findings indicate that an RORγ/IL-17A axis influences VEGF production and neovascular retinopathy by mechanisms involving neuroglia. Inhibition of RORγ and IL-17A may have potential for the improved treatment of neovascular retinopathies.

HMGB1Modulates the Treg/Th17 Ratio in Atherosclerotic Patients.

Atherosclerosis (AS) characterized as a chronic inflammatory disease. Multiple immune cells and inflammatory cytokines, such as high mobility group protein (HMGB1), regulatory T (Treg) cells, T helper (Th17) cells, and inflammation-related cytokines, play a key role in its pathophysiology. A large number of studies report that HMGB1 and Th17 cells may promote atherosclerosis progression, whereas Treg cells may play a protective role in atherosclerosis; thus, alterations in the Treg/Th17 ratio may exist in atherosclerosis diseases. Up till now, the relationships between HMGB1 levels and the Treg/Th17 ratio remain incompletely understood. The major purpose of this study was to investigate the relationship between HMGB1 levels and the Treg/Th17 ratio in patients with coronary artery atherosclerotic plaques. We enrolled patients with coronary atherosclerosis and normal coronary artery as the research subjects. Flow cytometry was used to analyze the Treg cells, the Th17 cells frequency, and the Treg/Th17 ratio. Otherwise, real-time polymerase chain reaction was used for assays the mRNA expressions of HMGB1, retinoic acid-related orphan nuclear receptor C (RORC), and forkhead-winged helix transcription factor (Foxp3). Moreover, enzyme-linked immunosorbent assays were used to detect the level of protein and cytokines, such as HMGB1, IL-10, TGF-β1, IL-17A, and IL-23. Using flow cytometry, we observed a significantly increased of Th17 cell frequency, whereas Treg cell frequency significantly decreased in atherosclerotic patients. Consistently, the levels of RORC mRNA were significantly increased in coronary atherosclerosis (AS) group compared to normal coronary artery (NCA) group (P＜0.01). In contrast, the expression of Foxp3 mRNA was markedly lower in the AS group than in the NCA group (P＜0.01). Furthermore, we observed the serum concentrations of HMGB1, IL-17A, and IL-23 were significantly higher in the AS group than in the NCA group (P＜0.01, respectively), whereas the concentrations of serum IL-10 and TGF-β1 were significantly lower in the AS group than in the NCA group (P＜0.01, respectively). In addition, we also found that HMGB1 levels showed negative correlation with the Treg/Th17 ratio in the two groups (r=－0.6984, P＜0.01). The data in our study indicated that HMGB1 may promote atherosclerosis progression via modulating the imbalance in the Treg/Th17 ratio.

How Can We Manipulate the IL-23/IL-17 Axis?

There is compelling evidence that interleukin (IL)-23/IL-17 axis plays a critical role in the pathogenesis of inflammatory spondyloarthritides including ankylosing spondylitis, reactive arthritis, psoriatic arthritis, and inflammatory bowel disease-associated arthritis. IL-17 is a family of cytokines. Of the six members of IL-17, IL-17A and IL-17F are the most closely related proinflammatory cytokines and effector molecules. A distinct subset of CD4+ T helper (Th) cells producing IL-17A and IL-17F is designated Th17 cells. Many cytokines are involved in Th17 cell differentiation and therefore are therapeutic targets. IL-6 in combination with transforming growth factor (TGF)-β mediates initiation of Th17 cell differentiation. IL-23 is not required for the initial differentiation of Th17 cells, but is indispensable for stabilizing differentiated Th17 clones in vivo. Clinical trials are undergoing to assess therapeutic efficacy of several monoclonal antibodies against these cytokines to suppress Th17 cells. Ustekinumab, a monoclonal antibody against p40 subunit which is shared by IL-12 and IL-23, has been approved to treat psoriasis and psoriatic arthritis. The therapeutic effect of ustekinumab is thought to be via inhibition of Th17 cells. Monoclonal antibodies to IL-17A and IL-17F and their receptors are also being evaluated in clinical trials. Retinoic acid-related orphan nuclear receptor (ROR)-γt is the master transcription factor for Th17 cells and hence a target. Recently, small molecules blocking ROR-γt activity have been identified and are potentially to be developed as drugs to inhibit Th17 cells. Small interfering RNA (siRNA) and small hairpin RNA (shRNA) targeting Rorc gene can be specifically delivered into CD4+ T cells to suppress IL-17 production for even more precise inhibition of Th17 cells. Technology advancement shows promise for these nucleotide-based therapeutics to be developed for clinical use.

The E3 Deubiquitinase USP17 Is a Positive Regulator of Retinoic Acid-related Orphan Nuclear Receptor γt (RORγt) in Th17 Cells

Stable retinoic acid-related orphan nuclear receptor γt (RORγt) expression is pivotal for the development and function of Th17 cells. Here we demonstrate that expression of the transcription factor RORγt can be regulated through deubiquitination, which prevents proteasome-mediated degradation. We establish that USP17 stabilizes RORγt protein expression by reducing RORγt polyubiquitination at its Lys-360 residue. In contrast, knockdown of endogenous USP17 in Th17 cells resulted in decreased RORγt protein levels and down-regulation of Th17-related genes. Furthermore, USP17 expression was up-regulated in CD4(+) T cells from systemic lupus erythematosus patients. Our data reveal a molecular mechanism in which RORγt expression in Th17 cells can be positively regulated by USP17, thereby modulating Th17 cell functions.

Effect of hydrodynamics-based delivery of IL-18BP fusion gene on rat experimental autoimmune myocarditis

Interleukin-18 (IL-18) is a powerful and important cytokine in myocarditis. IL-18-binding protein (IL-18BP), a naturally occurring antagonist of IL-18, is presumed to play a vital regulatory function in IL-18-mediated immune responses. The purpose of this study was to evaluate the alterations of IL-18 and its related protein expressions and the effect of hydrodynamics-based delivery of the IL-18BP gene for treatment of rat experimental autoimmune myocarditis (EAM).Rats were immunized on Day 0 and killed on 2, 3 and 4 weeks to determine IL-18 and its related protein expression and target cells in EAM hearts. On Day 6, rats were injected with a recombinant plasmid encoding IL-18BP-Ig or SP-Ig. On Day 17, rats were detected with echocardiography and then be killed. IL-18BP gene therapy was effective in controlling EAM, as monitored by a decreased ratio of heart weight to body weight, reduced myocarditis areas, reduced expression of atrial natriuretic peptide, brain natriuretic peptide, IL-17, IFN-γ, IL-6 and IL-10. Furthermore, the effect of serum containing IL-18BP on the expression of immune-relevant genes in IL-1α-stimulated NC cells and splenocytes cultured from EAM rats was examined. The results showed that IL-18BP significantly suppressed the expression of IL-17 as well as other proinflammatory genes such as transforming growth factor-β, prostaglandin E2 synthase, cyclooxygenase-2 in IL-1α-stimulated NC cells, and IL-18BP also significantly suppressed the expression of IL-17, IL-17R, IL-21 and IL-17-related transcriptional factor retinoic acid-related orphan nuclear receptor, signal transducer and activator of transcription-3 and Foxp3 in IL-1α-stimulated splenocytes cultured from EAM rats. IL-18 and its related protein played an important role on the development of EAM. IL-18BP effectively prevented progression of EAM by blocking IL-17 and related inflammatory genes expression. This might be a possible mechanism of the amelioration of EAM by IL-18BP treatment.

Hydrodynamics-based delivery of an interleukin-1 receptor II fusion gene ameliorates rat autoimmune myocarditis by inhibiting IL-1 and Th17 cell polarization

Type II interleukin-1 receptor (IL-1RII) is a non-signaling decoy receptor that blocks the activity of interleukin-1 (IL-1), a pro-inflammatory cytokine involved in experimental autoimmune myocarditis (EAM). The aim of this study was to examine the effects of hydrodynamics-based delivery of a recombinant plasmid encoding IL-1RII-Ig and to elucidate the role of IL-1RII in EAM rats. Rats were immunized on day 0 and injected with a recombinant plasmid encoding IL-1RII-Ig or pCAGGS-SP-Ig (control plasmid) on day 6. IL-1RII-Ig gene therapy effectively controlled EAM as indicated by a decreased heart weight-to-body weight ratio, reduced areas of myocarditis, reduced expression of genes encoding atrial natriuretic peptide and brain natriuretic peptide in the heart, and improved cardiac function. IL-1RII-Ig significantly inhibited the expression of IL-1-related cytokines such as IL-1β, prostaglandin E2 synthase, cyclooxygenase, and monocyte chemotactic protein-1 in EAM hearts. Furthermore, the effect of serum containing IL-1RII-Ig on the expression of immune-related genes in IL-1-stimulated splenocytes cultured from EAM rats was examined. The results showed that the expression of IL-6, transforming growth factor-β, retinoic acid-related orphan nuclear receptor (RORγt) and IL-17, was significantly decreased upon exposure to serum containing IL-1RII-Ig. In conclusion, hydrodynamics-based delivery of a recombinant plasmid encoding IL-1RII-Ig effectively prevented progression of left ventricular remodeling and myocardial damage in EAM rats. Moreover, IL-1RII may ameliorate experimental autoimmune myocarditis by blocking IL-1 and inhibiting production of the cytokines important for the polarization of T cells toward a Th17 phenotype.

Leukocyte Function-associated Antigen-1/Intercellular Adhesion Molecule-1 Interaction Induces a Novel Genetic Signature Resulting in T-cells Refractory to Transforming Growth Factor-β Signaling

The immunesuppressive cytokine TGF-β plays crucial regulatory roles in the induction and maintenance of immunologic tolerance and prevention of immunopathologies. However, it remains unclear how circulating T-cells can escape from the quiescent state maintained by TGF-β. Here, we report that the T-cell integrin leukocyte function-associated antigen-1 (LFA-1) interaction with its ligand intercellular adhesion molecule-1 (ICAM-1) induces a genetic signature associated with reduced TGF-β responsiveness via up-regulation of SKI, E3 ubiquitin-protein ligase SMURF2, and SMAD7 (mothers against decapentaplegic homolog 7) genes and proteins. We confirmed that the expression of these TGF-β inhibitory molecules was dependent on STAT3 and/or JNK activation. Increased expression of SMAD7 and SMURF2 in LFA-1/ICAM-1 cross-linked T-cells resulted in impaired TGF-β-mediated phosphorylation of SMAD2 and suppression of IL-2 secretion. Expression of SKI caused resistance to TGF-β-mediated suppression of IL-2, but SMAD2 phosphorylation was unaffected. Blocking LFA-1 by neutralizing antibody or specific knockdown of TGF-β inhibitory molecules by siRNA substantially restored LFA-1/ICAM-1-mediated alteration in TGF-β signaling. LFA-1/ICAM-1-stimulated human and mouse T-cells were refractory to TGF-β-mediated induction of FOXP3(+) (forkhead box P3) and RORγt(+) (retinoic acid-related orphan nuclear receptor γt) Th17 differentiation. These mechanistic data suggest an important role for LFA-1/ICAM-1 interactions in immunoregulation concurrent with lymphocyte migration that may have implications at the level of local inflammatory response and for anti-LFA-1-based therapies.

Structural Basis of Digoxin That Antagonizes RORγt Receptor Activity and Suppresses Th17 Cell Differentiation and Interleukin (IL)-17 Production

The retinoic acid-related orphan nuclear receptor γt (RORγt)/RORγ2 is well known as a master regulator of interleukin 17 (IL-17)-producing helper T (Th17) cell development. To develop a therapeutic agent against Th17-mediated autoimmune diseases, we screened chemical compounds and successfully found that digoxin inhibited IL-17 production. Further studies revealed that digoxin bound to the ligand binding domain of RORγt and suppressed Th17 differentiation without affecting Th1 differentiation. To better understand the structural basis for the inhibitory activity of digoxin, we determined the crystal structure of the RORγt ligand-binding domain in complex with digoxin at 2.2 Å resolution. The structure reveals that digoxin binds to the ligand-binding pocket protruding between helices H3 and H11 from the pocket. In addition, digoxin disrupts the key interaction important for the agonistic activity, resulting in preventing the positioning of helix H12 in the active conformation, thus antagonizing coactivator interaction. Functional studies demonstrated that digoxin inhibited RORγt activity and decreased IL-17 production but not RORα activity. Digoxin inhibited IL-17 production in CD4(+) T cells from experimental autoimmune encephalomyelitis mice. Our data indicates that RORγt is a promising therapeutic target for Th17-derived autoimmune diseases and our structural data will help to design novel RORγt antagonists.

Differentiation Imbalance of Th1/ Th17 in Peripheral Blood Mononuclear Cells Might Contribute to Pathogenesis of Hashimoto’s Thyroiditis

T helper 17(Th17) cell is a new subset of CD4(+) T cells that produce a proinflammatory cytokine interleukin-17 (IL-17). Th17 cells have recently been shown to play a critical role in many autoimmune diseases that had previously been thought to be Th1 dominant. Although Hashimoto's thyroiditis (HT) was thought to be a Th1-type disease, the contributions of Th17 cells to the pathogenesis remain unclear. In this study, we investigated the expression levels of Th1/Th17 cell-associated factors in peripheral blood mononuclear cells (PBMC) and plasma from patients with HT by quantitative real-time polymerase chain reaction (RT-qPCR) and enzyme-linked immunosorbent assay (ELISA). Our results showed that the expression levels of Th1 cells-related T-bet and interferon-gamma (IFN-gamma) mRNA in PBMC from HT significantly decreased. However, the mRNA of Th17 coherent retinoic acid-related orphan nuclear receptor gamma t (RORgammat) and IL-17 in patients with HT increased. In addition, a negative correlation between T-bet and RORgammat mRNA expression was found in patients with HT, and the similar phenomena also appeared on the levels of mRNA and plasma concentration between IFN-gamma and IL-17. It suggested that Th17 cells rather than Th1 cells predominated among patients suffering from HT, and Th17 cells might be involved in the pathogenesis of HT.