Receptor-related Orphan Receptor Research Articles

Nobiletin Stimulates Adrenal Hormones and Modulates the Circadian Clock in Mice.

Polymethoxyflavonoids, such as nobiletin (abundant in Citrus depressa), have been reported to have antioxidant, anti-inflammatory, anticancer, and anti-dementia effects, and are also a circadian clock modulator through retinoic acid receptor-related orphan receptor (ROR) α/γ. However, the optimal timing of nobiletin intake has not yet been determined. Here, we explored the time-dependent treatment effects of nobiletin and a possible novel mechanistic idea for nobiletin-induced circadian clock regulation in mice. In vivo imaging showed that the PER2::LUC rhythm in the peripheral organs was altered in accordance with the timing of nobiletin administration (100 mg/kg). Administration at ZT4 (middle of the light period) caused an advance in the peripheral clock, whereas administration at ZT16 (middle of the dark period) caused an increase in amplitude. In addition, the intraperitoneal injection of nobiletin significantly and potently stimulated corticosterone and adrenaline secretion and caused an increase in Per1 expression in the peripheral tissues. Nobiletin inhibited phosphodiesterase (PDE) 4A1A, 4B1, and 10A2. Nobiletin or rolipram (PDE4 inhibitor) injection, but not SR1078 (RORα/γ agonist), caused acute Per1 expression in the peripheral tissues. Thus, the present study demonstrated a novel function of nobiletin and the regulation of the peripheral circadian clock.

Validation of nuclear receptor RORγ isoform 1 as a novel host-directed antiviral target based on the modulation of cholesterol levels

Currently, the clinically approved repertoire of antiviral drugs predominantly comprises direct-acting antivirals (DAAs). However, the use of DAAs is frequently limited by adverse effects, restriction to individual virus species, or the induction of viral drug resistance. These issues will likely be resolved by the introduction of host-directed antivirals (HDAs) targeting cellular proteins crucial for viral replication. However, experiences with the development of antiviral HDAs and clinical applications are still in their infancy. With the present study, we explored the human nuclear receptor and transcription factor RORγ isoform 1 (RORγ1), a member of the retinoic acid receptor-related orphan receptor (ROR) family, as a putative target of antiviral HDAs. To this end, cell culture models were used to investigate major viral human pathogens, i.e. the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), human cytomegalovirus (HCMV), varicella zoster virus (VZV) and human immunodeficiency virus 1 (HIV-1). Our results demonstrated (i) an antiviral activity of the clinically relevant RORγ modulators cedirogant and others, (ii) that isoform RORγ1 acts as the responsible determinant and drug target in the analyzed cell culture-based models, (iii) a selectivity of the antiviral effect for RORγ1 over related receptors RORα and RORβ, (iv) a late-phase inhibition exerted by cedirogant in HCMV replication and (v) a mechanistic link to the cellular cholesterol biosynthesis. Combined, the data highlight this novel RORγ-specific antiviral targeting concept and the developmental potential of RORγ-directed small molecules.

Tfh and Tfr Cells in Autoimmune Diseases

An immunological condition known as autoimmunity causes the excessive generation of autoantibodies against self-antigen and is characterized by enhanced T-cell activation and extra-stimulated B-cells. The development of lymphatic follicle germinal centers (GCs), the maturation of B cells, and differentiation into plasma cells are all significantly aided by follicular helper T cells (Tfh). Tfh cells express the transcriptional regulator B cell lymphoma 6 (BCL-6), C-X-C chemokine receptor 5 (CXCR5), inducible T cell co-stimulator (ICOS), and programmed cell death protein 1 (PD-1). The production of interleukin (IL)-21 and low expression of the chemokine (C-C motif) receptor 7 (CCR7) define Tfh cells. Additionally, Tfh cells are a diverse population of cells with the potential to co-express minute quantities of transcription factors, such as T-box expressed in T cells (T-bet), GATA-binding protein 3 (GATA-3), and retinoic acid receptor-related orphan receptor (ROR-t). Tfh cells that also produce IL-21, IL-4, IL-17, and IFN-γ are referred to as Tfh1, Tfh2, and Tfh17 cells, respectively. The control of humoral immunity is carried out by follicular regulatory (Tfr) cells that express Forkhead box protein 3 (Foxp3). Tfr cells can, however, decrease T-B cell interactions through the cytotoxic T lymphocyte-associated protein 4 (CTLA-4) while promoting B cell maturation through IL-10. In the context of autoimmunity, the role of Tfh and Tfr cells is still not fully understood. We intend to present the most recent data on the characteristics and function of Tfh and Tfr cells under conditions of autoimmunity in this review.

Abstract P3084: T Helper Cell Polarity Determines Salt-sensitivity And Development Of Hypertension

Background: Sodium chloride drives the stable induction of pathogenic T helper (Th) 17 cells and development of hypertension. However, high-salt intake induces hypertension in salt-sensitive persons but not in salt-resistant persons. Pathogenic Th17 cell plays an important role in the development of hypertension whereas suppressive Treg cell plays an important role in the prevention of hypertension. Objective: We tested the hypothesis that . Methods and Results: Eight-week-old male SS and SR rats were fed chow (NS, 0.4% NaCl) or high-salt (HS, 4% NaCl) diet for 4 weeks. Systolic blood pressure was measured by the tail-cuff method. Cultured splenic T cells from Dahl salt-sensitive (SS) and salt-resistant (SR) rats were exposed to hypertonic salt solution with or without IL-6 or TGFβ. T helper cell (Th17 and Treg) polarity was determined via flow cytometry. The expression of Th17-related genes such as interleukin (IL)-17A, IL-23R and retinoic acid receptor-related orphan receptor (ROR) γt) as well as Treg-related genes such as IL-10, CD25 and forkhead box (Fox) P3 were measured by ELISA or qRT-PCR. Adoptive transfer of Th17 cells isolated from SS or SR donors fed HS were performed intraperitoneally into SS and SR recipients. Upon chow diet, SR has higher Treg cells than SS does. HS intake induced hypertension and increased proinflammatory Th17 cells in SS but not in SR. Hypertonic salt solution induced differentiation of cultured splenic T cells preferentially into proinflammatory Th17 cells and anti-inflammatory Treg cells in SS and SR, respectively. Polarizing cytokine IL-6 also induced differentiation of cultured splenic T cells preferentially into proinflammatory Th17 cells and anti-inflammatory Treg cells in SS and SR, respectively, which were further potentiated by hypertonic salt solution. Adoptive transfer of activated Th17 cells isolated from hypertensive SS or normotensive SR, both of which had been fed HS diet, induced hypertension in SS recipients, but not SR recipients. Conclusions: These results indicate that T helper cell polarity after HS intake determines salt-sensitivity and development of hypertension in SS and SR rats. Th17 cell polarity predicts salt-sensitivity and development of hypertension, whereas Treg cell polarity predicts salt-resistance and maintenance of normotension. SR rats protect against hypertension after HS intake through anti-inflammatory Treg cell polarity.

596 Retinoic acid receptor-related orphan receptor (ROR) agonists impact cellular responses to UV radiation in human keratinocytes

Retinoic acid receptor-related orphan receptor (ROR) agonists have been shown to regulate circadian rhythms in cultured cells and to improve various disease outcomes in animal models. Because expression of the core nucleotide excision repair protein XPA is governed by the circadian clock, we examined whether a natural and synthetic ROR agonist (nobiletin and SR1078, respectively) impact XPA expression and cellular responses to UV radiation in human keratinocytes. Interestingly, though these compounds have little effect on total XPA expression, they both reduce the expression of the endopeptidase cathepsin L (CTSL), which is known to act on several nuclear proteins, including at the C-terminus of XPA under defined cell lysis conditions.

Orphan receptors in prostate cancer.

The identification of new cellular receptors has been increasing rapidly. A receptor is called "orphan" if an endogenous ligand has not been identified yet. Here we review receptors that contribute to prostate cancer and are considered orphan or partially orphan. This means that the full spectrum of their endogenous ligands remains unknown. The orphan receptors are divided into two major families. The first group includes G protein-coupled receptors. Most are orphan olfactory receptors. OR51E1 inhibits cell proliferation and induces senescence in prostate cancer. OR51E2 inhibits prostate cancer growth, but promotes invasiveness and metastasis. GPR158, GPR110, and GPCR-X play significant roles in prostate cancer development and progression. However, GPR160 induces cell cycle arrest and apoptosis. The other major subset of orphan receptors are nuclear receptors. Receptor-related orphan receptor α (RORα) inhibits tumor growth, but RORγ stimulates androgen receptor signaling. PXR contributes to metabolic deactivation of androgens and inhibits cell proliferation. TLX has protumorigenic effects in prostate cancer, while its knockdown triggers cellular senescence and growth arrest. Estrogen-related receptor ERRγ can inhibit tumor growth but ERRα is protumorigenic. Dax1 and short heterodimeric partner are also inhibitory in prostate cancer. There is a "zoo" of relatively underappreciated orphan receptors that play key roles in prostate cancer.

Discovery and Characterization of Benzimidazole Derivative XY123 as a Potent, Selective, and Orally Available RORγ Inverse Agonist.

Receptor-related orphan receptor γ (RORγ) has emerged as an attractive therapeutic target for the treatment of cancer and inflammatory diseases. Herein, we report our effort on the discovery, optimization, and evaluation of benzothiazole and benzimidazole derivatives as novel inverse agonists of RORγ. The representative compound 27h (designated as XY123) potently inhibited the RORγ transcription activity with a half-maximal inhibitory concentration (IC50) value of 64 nM and showed excellent selectivity against other nuclear receptors. 27h also potently suppressed cell proliferation, colony formation, and the expression of androgen receptor (AR)-regulated genes in AR-positive prostate cancer cell lines. In addition, 27h demonstrated good metabolic stability and a pharmacokinetic property with reasonable oral bioavailability (32.41%) and moderate half-life (t1/2 = 4.98 h). Significantly, oral administration of compound 27h achieved complete and long-lasting tumor regression in the 22Rv1 xenograft tumor model in mice. Compound 27h may serve as a new valuable lead compound for further development of drugs for the treatment of prostate cancer.

Thiomyristoyl ameliorates colitis by blocking the differentiation of Th17 cells and inhibiting SIRT2-induced metabolic reprogramming

BackgroundThe pathogenesis of inflammatory bowel diseases (IBD), including Crohn’s disease (CD) and ulcerative colitis (UC) has not been fully elucidated. However, a strong correlation between IBD and high T helper 17 (Th17) levels has been found. Sirtuin 2 (SIRT2) has recently been found to play an important role in metabolic reprogramming, but its potential anti-inflammatory properties remain unclear. MethodsThe expression levels of SIRT2 and glucose metabolism-related proteins in peripheral blood mononuclear cells (PBMCs) of IBD patients and healthy volunteers were detected. Human PBMCs were differentiated into Th17 cells in vitro and were treated with TM simultaneously. The ratio of Th17 cells and apoptotic cells and the production of Interleukin (IL)-17A and the expression levels of transcription factors of classical signaling pathway related to Th17 differentiation were determined. The acetylation of LDHA and glucose metabolism was assessed. Subsequently, C57BL/6J colitis mice induced by 2.5% dextran sulfatesodiumsalt (DSS) were treated with or without TM, Disease activity index, T cell subsets in the mice spleen, relevant inflammatory cytokines in serum, specific mRNA, and proteins in mice colon were evaluated respectively. ResultsSIRT2 and glucose metabolism-related proteins in PBMCs of patients were overexpressed. Compared with the positive control group, human PBMCs treated with TM had lower levels of IL-17A, percentage of Th17 cells, levels of phospho-signal transducer and activator of transcription (p-STAT) 3 and phospho-nuclear transcription factor-κB (p-NF-κB), but higher levels of acetylated LDHA. Compared with colitis mice, TM-treated colitis mice had longer colons, reduced weight-losses, and lower disease activity index and histopathologic scores. Interestingly, although the expression levels of interferon (IFN)-γ, IL-17A, and retinoic acid receptor-related orphan receptor (ROR)-γt were inhibited in the colons of TM-treated colitis mice, the expression of forkhead box protein P3 (FOXP3) didn’t change. Consistently, relative to the high percentage of splenic Th17 cells in colitis mice, the percentage of splenic Th17 cells in TM-treated colitis mice was as normal as PBS-treated mice, while the percentage of Treg cells was not affected. Additionally, the TM group had reduced levels of IL-23 and hypoxiainduciblefactor-1α (HIF-1α), and an increased level of IL-10 in the colon, compared with the colitis group. ConclusionOur results indicate that TM reduces UC progression by reducing metabolic reprogramming and T cell differentiation. Specifically, TM prevented Th17 differentiation by reducing the expression of related transcription factors and promoting acetylation of LDHA (weakening glycolysis). SIRT2 may be a potential target for IBD treatment.

ROR-α-1 inhibits the proliferation, invasion, and migration of hepatocellular carcinoma MHCC97H via downregulation of chemokine CXCL5

Hepatocarcinogenesis is a complicated process that is affected by a variety of microenvironmental factors, such as secretory chemokines and cell-extracellular matrix (ECM). Retinoic acid receptor-related orphan receptor (ROR)-α has been shown to attenuate tumor invasiveness by inducing suppressive cell microenvironment, and its low expression was associated with a worse prognosis in HCC patients. In the present study, we attempted to investigate the role and mechanism of the dominant transcript of ROR-α, ROR-α-1, in HCC development and progression. Among the four transcripts (ROR-α-1/-2/-3/-4), overexpression of ROR-α-1 dramatically suppressed the capacity of MHCC97H cells to proliferate, migrate and invade. We analyzed the differentially expressed genes in ROR-α-1-overexpressed and non-overexpressed MHCC97H cells, performed Gene Ontology (GO) enrichment analysis on these differentially-expressed genes, and found out that factors involved in the tumor microenvironment and ECM are related to the anti-tumor effects of ROR-α-1. Among these factors, chemokine CXCL5 was significantly downregulated by ROR-α-1 overexpression. Overexpression of ROR-α-1 remarkably inhibited the capacity of HCC cells to proliferate, migrate, invade, and downregulated the protein levels of β-catenin, c-Myc, Cyclin D1, and N-cadherin, suggesting the tumor-suppressive role of ROR-α-1 in MHCC97H cells. Moreover, overexpression of CXCL5 dramatically attenuated the suppressive effects of cell proliferation, migration and invasion induced by ROR-α-1 overexpression in MHCC97H, suggesting that ROR-α-1 exerts its anti-tumor effects via downregulating CXCL5. In conclusion, we demonstrate the tumor-suppressive role of ROR-α-1 in MHCC97H cells and that ROR-α-1 might play a tumor-suppressive role via regulation of chemokine CXCL5.

Molecular dynamics simulations on RORγt: insights into its functional agonism and inverse agonism.

The retinoic acid receptor-related orphan receptor (ROR) γt receptor is a member of nuclear receptors, which is indispensable for the expression of pro-inflammatory cytokine IL-17. RORγt has been established as a drug target to design and discover novel treatments for multiple inflammatory and immunological diseases. It is important to elucidate the molecular mechanisms of how RORγt is activated by an agonist, and how the transcription function of RORγt is interrupted by an inverse agonist. In this study we performed molecular dynamics simulations on four different RORγt systems, i.e., the apo protein, protein bound with agonist, protein bound with inverse agonist in the orthosteric-binding pocket, and protein bound with inverse agonist in the allosteric-binding pocket. We found that the orthosteric-binding pocket in the apo-form RORγt was mostly open, confirming that apo-form RORγt was constitutively active and could be readily activated (ca. tens of nanoseconds scale). The tracked data from MD simulations supported that RORγt could be activated by an agonist binding at the orthosteric-binding pocket, because the bound agonist helped to enhance the triplet His479-Tyr502-Phe506 interactions and stabilized H12 structure. The stabilized H12 helped RORγt to form the protein-binding site, and therefore made the receptor ready to recruit a coactivator molecule. We also showed that transcription function of RORγt could be interrupted by the binding of inverse agonist at the orthosteric-binding pocket or at the allosteric-binding site. After the inverse agonist was bound, H12 either structurally collapsed, or reorientated to a different position, at which the presumed protein-binding site was not able to be formed.

Bob1 enhances RORγt-mediated IL-17A expression in Th17 cells through interaction with RORγt

POU domain class 2-associating factor 1 (also called Bob1), which is mainly expressed in B cells, regulates B cell homeostasis and controls humoral immune responses. Although Bob1 is known to function reliably in T cell subsets including follicular helper T cells, Th1 cells and Th2 cells, it is unknown whether Bob1 functions in other T cell subsets. In this study, we found that Bob1 knock out (KO) mice are resistant to experimental autoimmune encephalomyelitis (EAE) induced by MOG35-55 peptide and that Bob1 KO T cells are defective in Th17 differentiation. Importantly, Bob1 interacts with retinoid acid receptor-related orphan receptor (ROR) gamma t (RORγt), a signature transcription factor for Th17 cells, through the ligand-binding domain of RORγt, thereby enhancing IL-17A transcription activity. IL-17A induction by Bob1 requires the ability for its formation of a DNA-Oct1-Bobl ternary complex. Thus, our findings demonstrate that Bob1 enhances IL-17A expression in vivo and in vitro by interacting with RORγt in Th17 cells, suggesting that Bob1 plays a pivotal role in Th17-mediated autoimmune disease.

Type 3 innate lymphoid cells are altered in colons of C57BL/6 mice with dioxin exposure

Type 3 innate lymphoid cells (ILC3s) are distributed in the gut and regulate inflammation by secreting cytokines, including interferon (IFN)-γ and interleukin (IL)-17. The maintenance and function of ILC3s involve the activity of aryl hydrocarbon receptor (AhR), a potent ligand of which is 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), one of the most toxic dioxin congeners. Thus, TCDD exposure might affect ILC3s. To obtain in vivo evidence supporting this notion, we exposed female C57BL/6 mice orally to TCDD (low/high doses: 0.1/10 μg/kg body weight) during pregnancy and lactation periods, and after the exposure, evaluated the mothers and offspring for alterations in ILC3 differentiation and function in the colon. ILC3 frequency among colonic lamina propria lymphocytes was preferentially diminished in the offspring, and, in parallel, the median fluorescence intensity (MFI) of retinoic acid receptor-related orphan receptor (ROR)γt, which is associated with ILC3 differentiation, was also decreased in ILC3s. Conversely, the percentages of two subsets of the cells, one positive for natural cytotoxicity receptor NKp46 and the other for IL-17a, were increased in TCDD-exposed mothers and offspring. Moreover, the percentage of IFN-γ+ ILC3s was increased specifically in the mothers, but this was in conjunction with a significant decrease in the MFI of IFN-γ, which suggests that the IFN-γ+ ILC3 subset was functionally altered. In conclusion, maternal exposure to TCDD suppresses ILC3 differentiation in the offspring and influences ILC3 function in distinct manners in the mother and offspring. Our study provides new insights into the intergenerational interference of dioxins in colonic ILC3s.

Baicalein stimulates fibroblast growth factor 21 expression by up-regulating retinoic acid receptor-related orphan receptor α in C2C12 myotubes

Retinoic acid receptor-related orphan receptor (ROR) α has been implicated in various physiological functions, including the immune system, inflammation, and circadian rhythms. In the present study, the synthetic RORα/γ agonist SR1078 stimulated the production and gene expression of fibroblast growth factor 21 (FGF21) in C2C12 myotubes. FGF21, a member of the FGF family, plays important roles in the regulation of peripheral glucose tolerance and lipid metabolism and improves metabolic health. The mRNA expression and secretion of FGF21 was significantly weaker in Rora-silenced cells than in cells transfected with non-targeting control siRNA. SR1078 significantly up-regulated C/EBP homologous protein (CHOP), an established marker of ER stress, in a dose-dependent manner in C2C12 myotubes, while CHOP expression was decreased in Rora-silenced C2C12 cells, suggesting that RORα is involved in the regulation of FGF21 expression and stimulates ER stress in C2C12 myotubes. The naturally occurring compound baicalein up-regulated FGF21 expression and secretion in C2C12 myotubes. Additionally, the up-regulation of CHOP mRNA and protein expression was observed in C2C12 myotubes after the baicalein treatment. Furthermore, the knockdown of RORα prevented the augmentation of FGF21 and up-regulation of CHOP in response to baicalein in C2C12 cells. Collectively, these results suggest that baicalein stimulates the ER stress response and FGF21 expression through an RORα-dependent mechanism in C2C12 myotubes, and indicate the potential of baicalein as an effective anti-obesity therapy via its ability to enhance FGF21 production.

A fourth subtype of retinoic acid receptor-related orphan receptors is activated by oxidized all-trans retinoic acid in medaka (Oryzias latipes)

BackgroundThe three known subtypes of the retinoic acid receptor-related orphan receptor (ROR) have been implicated in the control of immunity, brain function, and circadian rhythm in mammals. Here, we demonstrate by phylogenetic analysis that there were originally four subtypes of RORs in vertebrates. One of the novel ror paralogs, rord1 (rorca in the Ensembl database), is conserved among teleosts, but absent in mammals. Using medaka (Oryzias latipes) as a model teleost, we evaluated the expression pattern of this gene, its transactivational properties for endogenic chemicals, and its ability to activate the promoters of putative target genes.ResultsIn eyes, the transcript of rord1 was expressed at higher levels during the day than at night. Interestingly, cholesterol derivatives, which are well-known ligands for mammalian RORs, did not efficiently promote transcriptional activity via RORd1. Thus we sought to identify the ligands that regulate the transcriptional activity of RORd1 using a luciferase reporter cell-based screening system. Using this system, we identified two metabolites of all-trans retinoic acid (ATRA), 4OH-ATRA and 4-keto ATRA, as potential ligands of RORd1. Moreover, RORd1 activated the promoter of cyp26a1 in a 4OH-ATRA -dependent manner.ConclusionsA novel ror subtype, rord has two paralogs, rord1 and rord2, in teleost. Rord1 mRNA is highly abundant in the eyes of medaka during light periods, suggesting that rord1 expression is involved in the regulation of circadian rhythm. We identified two ATRA metabolites, 4OH-ATRA and 4 K–ATRA, as endogenous candidate ligands of RORd1. We also show that 4-oxygenated ATRA metabolites have the potential to activate cyp26a1, the metabolic enzyme of ATRA. Our results support the notion that RORd1 is involved in the metabolism of ATRA in medaka.

SiRNA‑mediated knockdown of T‑bet and RORγt contributes to decreased inflammation in pre‑eclampsia.

Abnormal immune response resulting from disordered T helper (Th)1/Th2 and Th17/regulatory T cells (Treg) cytokine expression has been demonstrated to serve an important role in the pathogenesis of preeclampsia (PE). However, the role of transcription factors regulating Th cell differentiation contributing to PE remain unclear. To determine whether a decrease in the expression of the T cell lineage transcription factor T‑bet can restore immune balance and alleviate the systemic inflammatory response present in PE, 30patients diagnosed with PE were assessed and compared with healthy pregnant controls. The expression of the transcription factors T‑bet and retinoic acid receptor‑related orphan receptor (ROR)γt were increased in the peripheral blood mononuclear cells of PE patients compared with controls, consistent with the presence of abnormally high T‑bet:GATA3 and RORγt:forkhead box (FOX) P3 ratios. The present study additionally identified a high‑efficiency, specific small interfering (si)RNA that can downregulate RORγt and T‑bet mRNA levels and inhibit protein expression. This effective siRNA was transfected into activated CD4+ Tcells derived from patients with PE to observe the changes to transcription factor expression and attempt to elucidate the regulatory mechanism of T cell subsets. It was identified that knockdown of RORγt induced increased expression of FOXP3 and that the ratios of RORγt:FOXP3 and interleukin (IL)‑17A:IL‑10 were subsequently decreased. The results suggested that siRNA‑mediated knockdown of T‑bet regulated the immune balance of Th17/Tregs via changes to RORγt and FOXP3. When siRNA against RORγt and T‑bet were used in combination, a stronger ability to regulate immune balance was observed. These results imply that Th1‑ and Th17‑type immunity is dominant in PE and that the siRNA‑mediated knockdown of certain Th1 and Th17 cell transcription factors may be an effective therapeutic target for promoting immune balance in CD4+ T cell subgroups and ameliorating local and generalized inflammation in PE.

Effect of interleukin (IL)-35 on IL-17 expression and production by human CD4+T cells.

BackgroundInterleukin (IL)-17 produced by mainly T helper 17 (Th17) cells may play an important destructive role in chronic periodontitis (CP). Thus, anti-inflammatory cytokines, such as IL-35, might have a beneficial effect in periodontitis by inhibiting differentiation of Th17 cells. Th17 differentiation is regulated by the retinoic acid receptor-related orphan receptor (ROR) α (encoded by RORA) and RORγt (encoded by RORC). However, the role of IL-35 in periodontitis is not clear and the effect of IL-35 on the function of Th17 cells is still incompletely understood. Therefore, we investigated the effects of IL-35 on Th17 cells.MethodsPeripheral blood mononuclear cells (PBMCs) were sampled from three healthy volunteers and three CP patients and were analyzed by flow cytometry for T cell population. Th17 cells differentiated by a cytokine cocktail (recombinant transforming growth factor-β, rIL-6, rIL-1β, anti-interferon (IFN)-γ, anti-IL-2 and anti-IL-4) from PBMCs were cultured with or without rIL-35. IL17A (which usually refers to IL-17), RORA and RORCmRNA expression was analyzed by quantitative polymerase chain reaction, and IL-17A production was determined by enzyme-linked immunosorbent assay.ResultsThe proportion of IL-17A+CD4+ slightly increased in CP patients compared with healthy controls, however, there were no significant differences in the percentage of IL-17A+CD4+ as well as IFN-γ+CD4+ and Foxp3+CD4+ T cells between healthy controls and CP patients. IL17A, RORA and RORC mRNA expression was significantly increased in Th17 cells induced by the cytokine cocktail, and the induction was significantly inhibited by addition of rIL-35 (1 ng/mL). IL-17A production in Th17 cells was significantly inhibited by rIL-35 addition (1 ng/mL).DiscussionThe present study suggests that IL-35 could directly suppress IL-17 expression via RORα and RORγt inhibition and might play an important role in inflammatory diseases such as periodontitis.

Effect of Curcumin on Systemic T Helper 17 Cell Response; Gingival Expressions of Interleukin-17 and Retinoic Acid Receptor-Related Orphan Receptor γt; and Alveolar Bone Loss in Experimental Periodontitis.

Curcumin has anti-inflammatory and antioxidant effects and is reported to have many biologic activities. The current study examines effect of curcumin on: 1) systemic T helper 17 (Th17) cell response; 2) gingival expressions of interleukin (IL)-17 and retinoic acid receptor-related orphan receptor (ROR) γt; and 3) alveolar bone loss (ABL) in experimental periodontitis. Thirty-eight male albino Wistar rats were divided into four groups: 1) group 1 = periodontitis; 2) group 2 = periodontitis with curcumin treatment; 3) group 3 = periodontally healthy with curcumin treatment; and 4) group 4 = periodontally healthy. Curcumin was administered via oral gavage (30 mg/kg/d) for 15 days. After sacrifice via exsanguination, the following serum levels were determined using enzyme-linked immunosorbent assay: 1) IL-1β; 2) IL-6; 3) IL-17A; 4) IL-23; and 5) transforming growth factor- β. Morphometric evaluation of ABL was conducted and expression levels of IL-17 and RORγt in gingival tissues were evaluated immunohistochemically. Group 2 had significantly lower ABL than group 1 (P <0.0125). Highest expression levels of IL-17 and RORγt were observed in group 1 and were significantly higher than those in all other groups (P <0.0125). The only serum biochemical parameter significantly different among groups was level of IL-23 (P <0.05). Serum IL-23 levels were higher in groups 1 and 2 than groups 3 and 4 (P <0.0125); however, they were not significantly different for groups 1 and 2 (P >0.0125). Curcumin seems to be a promising host modulatory agent in periodontal disease pathogenesis regarding IL-17/IL-23 axis, with a decreasing effect on ABL and gingival expressions of IL-17 and RORγt.

Altered activity profile of a tertiary silanol analog of multi-targeting nuclear receptor modulator T0901317

We report the design, synthesis, and physicochemical/biological evaluation of novel silanol derivative 6 (sila-T) as a silanol analog of multi-target nuclear receptor modulator T0901317 (5). Compound 6 showed intermediate hydrophobicity between the corresponding alcohol 13 and perfluoroalcohol 5. While 5 exhibited potent activities toward liver X receptor α and β, farnesoid X receptor, pregnane X receptor (PXR) and retinoic acid receptor-related orphan receptor (ROR)γ, silanol 6 exhibited activity only toward PXR and RORs. Incorporation of silanol instead of perfluoroalcohol is a promising option for developing novel target-selective, biologically active compounds.

ROR nuclear receptors: structures, related diseases, and drug discovery.

Nuclear receptors (NRs) are ligand-regulated transcription factors that regulate metabolism, development and immunity. The NR superfamily is one of the major classes of drug targets for human diseases. Retinoic acid receptor-related orphan receptor (ROR) α, β and γ belong to the NR superfamily, and these receptors are still considered as 'orphan' receptors because the identification of their endogenous ligands has been controversial. Recent studies have demonstrated that these receptors are regulated by synthetic ligands, thus emerge as important drug targets for the treatment of multiple sclerosis, rheumatoid arthritis, psoriasis, etc. Studying the structural basis and ligand development of RORs will pave the way for a better understanding of the roles of these receptors in human diseases. Here, we review the structural basis, disease relevance, strategies for ligand identification, and current status of development of therapeutic ligands for RORs.

Melatonin in sperm biology: breaking paradigms.

Melatonin is a ubiquitous molecule, present in a wide range of organisms, and involved in multiple functions. Melatonin relays the information about the photoperiod to the tissues that express melatonin-binding sites in both central and peripheral nervous systems. This hormone has a complex mechanism of action. It can cross the cell plasma membrane and exert its actions in all cells of the body. Certain melatonin actions are mediated by receptors that belong to the superfamily of G-protein-coupled receptors (GPCRs), the MT1 and MT2 membrane. Melatonin can also bind to calmodulin as well as to nuclear receptors of the retinoic acid receptor family, RORα1, RORα2 and RZRβ. The purpose of this review is to report on recent developments in the physiological role of melatonin and its receptors. Specific issues concerning the biological function of melatonin in mammalian seasonal reproduction and spermatozoa are considered. The significance of the continuous presence of melatonin in seminal plasma with a fairly constant concentration is also discussed.