Retinoic Acid Receptor-related Orphan Research Articles

Therapeutic effects and mechanism of action of lavender essential oil on atopic dermatitis by modulating the STAT3/RORγt pathway

ObjectivesAtopic dermatitis (AD) is a common skin disorder characterized by skin inflammation caused by an imbalance in the immune response. The efficacy of lavender essential oil in treating atopic dermatitis has been demonstrated; however, its specific mechanism of action and active components remain unknown. Therefore, this study investigated the therapeutic effects and possible mechanism of action of LEO on AD in a 2,4-dinitrochlorobenzene (DNCB)-induced dermatitis mice model. MethodsTo determine the efficacy of LEO, serum levels of the cytokines IL-6 and TNF-α were tested in Kunming mice, and network pharmacology was used to predict the targets and mechanisms of LEO in AD treatment, after which network pharmacology was combined with metabolomics to construct complex response–enzyme–gene networks and investigate their potential associations. Based on the predicted mechanisms, skin tissues were further examined by immunohistochemistry and immunoblotting analysis, and the skin epidermis was stained with hematoxylin-eosin (HE) and toluidine blue (TB). ResultsLEO significantly suppressed the basal levels of IL-6 and TNF-α in the DNCB-induced mice model and predicted that Th17 cell differentiation is a critical pathway for LEO-based network pharmacology in AD treatment. As for the cytokines associated with the Th17 cell differentiation pathway, further experiments verified that LEO significantly reduced the protein expression of interleukin-17A (IL-17A)、Phosphorylated JAK2 (p-JAK)、Phosphorylated STAT3 (p-STAT3), and Retinoic acid-related orphan receptor γt (RORγt) but increased the expression of Foxp3. Additionally, the results of combined network pharmacology and metabolomics analysis showed that LEO could improve two metabolic pathways, namely, linoleic acid metabolism and arachidonic acid metabolism, by regulating the Th17 cell differentiation pathway; identify two key metabolites (linoleic acid, arachidonic acid); and regulate two differential genes (PTGS1, HPGD). ConclusionLEO may alleviate DNCB-induced skin inflammation by inhibiting the STAT3/RORγt pathway in Th17 cell differentiation, and reducing the expression of associated inflammatory cytokines and chemokines and improving the metabolism of linoleic and arachidonic acid in vivo.

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.

Increased gene expression of CCR6 and RORγt in peripheral blood cells of rheumatoid arthritis patients and their correlation with anti-cyclic citrullinated peptide and disease activity.

The significance of T helper 17 (Th17) cells in the pathogenesis of rheumatoid arthritis (RA) has recently been demonstrated in many studies. Retinoic acid receptor-related orphan receptor γt (RORγt) is a transcription factor that is specifically involved in the generation of Th17 cells. Besides, the chemokine receptor CCR6, the receptor for CCL20, is characteristically expressed by these cells. Considering the pivotal roles of Th17 cells in RA pathogenesis, in this study, we assessed the gene expression of CCR6 and RORγt in the peripheral blood leukocytes of new case RA patients. Also, we evaluated their association with anticyclic citrullinated peptide (anti-CCP) antibodies and disease activity. Forty-five new case RA patients and 45 healthy persons have been recruited in this investigation. The gene expression of CCR6 and RORγt was evaluated by quantitative real-time PCR (qRT-PCR), and anti-CCP antibodies plasma levels were measured using the enzyme-linked immunosorbent assay (ELISA) technique. Disease activity was measured according to the disease activity score-28 (DAS-28) formula. The gene expression of CCR6 and RORγt increased remarkably in new case RA patients compared to healthy controls (p < .05 and p < .01, respectively). Moreover, there was a positive correlation between RORγt gene expression and parameters, including gene expression of CCR6 (p = .001, r = .461), plasma levels of CCL20 (p = .0009, r = .477), ESR (p = .004, r = .419), DAS-28 (p = .006, r = .402), anti-CCP (p = .019, r = .346), and RF (p = .001, r = .451). Also, CCR6 gene expression was positively associated with the DAS-28 (p = .037, r = .310), plasma levels of anti-CCP (p = .037, r = .312), and ESR (p = .029, r = .327). Increased gene expression of CCR6 and RORγt in peripheral blood leukocytes of new case RA patients may contribute to the exacerbation and pathogenesis of RA.

Discovery of Biaryl Amide Derivatives as Potent, Selective, and Orally Bioavailable RORγt Agonists for Cancer Immunotherapy.

The master transcription factor receptor retinoic acid receptor-related orphan receptor γt (RORγt) regulates the differentiation of T-helper 17 (Th17) cells and the production of interleukin-17 (IL-17). Activation of RORγt+ T cells in the tumor microenvironment promotes immune infiltration to more effectively inhibit tumor growth. Therefore, RORγt agonists provide a reachable approach to cancer immunotherapy. Herein, a series of biaryl amide derivatives as novel RORγt agonists were designed, synthesized, and evaluated. Starting from the reported RORγt inverse agonist GSK805 (1), "functionality switching" and structure-based drug optimization led to the discovery of a promising RORγt agonist lead compound 14, which displayed potent and selective RORγt agonist activity and significantly improved metabolic stability. With excellent in vivo pharmacokinetic profiles, compound 14 demonstrated robust efficacy in preclinical tumor models of mouse B16F10 melanoma and LLC lung adenocarcinoma. Taken together, current studies indicate that 14 deserves further investigation as a potential lead RORγt agonist for cancer immunotherapy.

(±)-Salvicatone A: A Pair of C27-Meroterpenoid Enantiomers with Skeletons from the Roots and Rhizomes of Salvia castanea Diels f. tomentosa Stib.

(±)-Salvicatone A (1), a C27-meroterpenoid featuring a unique 6/6/6/6/6-pentacyclic carbon skeleton with a 7,8,8a,9,10,10a-hexahydropyren-1 (6H)-one motif, was isolated from the roots and rhizomes of Salvia castanea Diels f. tomentosa Stib. Its structure was characterized by comprehensive spectroscopic analyses along with computer-assisted structure elucidation, including ACD/structure elucidator and quantum chemical calculations with 1H/13C NMR and electronic circular dichroism. Biogenetically, compound 1 was constructed from decarboxylation following [4 + 2] Diels-Alder cycloaddition reaction between caffeic acid and miltirone analogue. Bioassays showed that (-)-1 and (+)-1 inhibited nitric oxide production in lipopolysaccharide-induced RAW264.7 macrophage cells with an IC50 value of 6.48 ± 1.25 and 15.76 ± 5.55 μM, respectively. The structure-based virtual screening based on the pharmacophores in ePharmaLib, as well as the molecular docking and molecular dynamics simulations study, implied that (-)-1 and (+)-1 may potentially bind to retinoic acid receptor-related orphan receptor C to exert anti-inflammatory activities.

The Effect of BI 730357 (Retinoic Acid-Related Orphan Receptor Gamma t Antagonist, Bevurogant) on the Pharmacokinetics of a Transporter Probe Cocktail, Including Digoxin, Furosemide, Metformin, and Rosuvastatin: An Open-Label, Non-randomized, 2-Period Fixed-Sequence Trial in Healthy Subjects.

Evaluating Drug-Drug Interactions (DDIs) for new investigational compounds requires several trials evaluating different drugs with different transporter specificities. By using a cocktail of drugs with different transporter specificities, a single trial could evaluate the pharmacokinetics (PKs) of each cocktail drug simultaneously, reducing the number of clinical DDI trials required for clinical development. We aimed to investigate the effect of steady-state Boehringer Ingelheim (BI) 730357 (bevurogant) on the PKs of a validated and optimized 4-component transporter cocktail. This open-label, non-randomized, 2-period fixed-sequence phase I trial compared transporter cocktail (0.25mg digoxin/1mg furosemide/10mg metformin hydrochloride/10mg rosuvastatin) with and without BI 730357 in healthy subjects aged 18-55 years with body mass index 18.5-29.9kg/m2 . During reference treatment/period 1, transporter cocktail was administered 90 minutes after breakfast. After a washout period, during test treatment/period 2, BI 730357 was dosed twice daily for 13 days, with transporter cocktail administered on day 1. The primary endpoints were the area under the concentration-time curve of the analyte in plasma over the time interval from 0 extrapolated to infinity (AUC0-∞ ) and the maximum measured concentration of the analyte in plasma (Cmax ), and the secondary endpoint was the area under the concentration-time curve of the analyte in plasma over the time interval from 0 to the last quantifiable data point (AUC0-tz ). Steady-state BI 730357 increased digoxin (+48% to +94%), minimally affected metformin (-2% to -9%), furosemide (+12% to +18%), and rosuvastatin (+19% to +39%) exposure. Therefore, no clinically relevant inhibition of transporters OCT2/MATE-1/MATE-2K, OAT1/OAT3, OATP1B1/OATP1B3 was observed. Potential inhibition of breast cancer resistance protein noted as PK parameters of coproporphyrin I/III (OATP1B1/OATP1B3 biomarkers) remained within bioequivalence boundaries while rosuvastatin PK parameters (AUC0-∞ /Cmax /AUC0-tz ) exceeded the bioequivalence boundary. BI 730357 was safe and well tolerated. This trial confirms the usefulness and tolerability of the transporter cocktail consisting of digoxin, furosemide, metformin, and rosuvastatin in assessing drug-transporter interactions in vivo.

The role of retinoic acid receptor-related orphan receptors in skeletal diseases.

Bone homeostasis, depending on the balance between bone formation and bone resorption, is responsible for maintaining the proper structure and function of the skeletal system. As an important group of transcription factors, retinoic acid receptor-related orphan receptors (RORs) have been reported to play important roles in bone homeostasis by regulating the transcription of target genes in skeletal cells. On the other hand, the dysregulation of RORs often leads to various skeletal diseases such as osteoporosis, rheumatoid arthritis (RA), and osteoarthritis (OA). Herein, we summarized the roles and mechanisms of RORs in skeletal diseases, aiming to provide evidence for potential therapeutic strategies.

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.

Hydrogen sulfide promoted retinoic acid-related orphan receptor α transcription to alleviate diabetic cardiomyopathy

Diabetic cardiomyopathy (DCM) is one serious and common complication in diabetes without effective treatments. Hydrogen sulfide (H2S) fights against a variety of cardiovascular diseases including DCM. Retinoic acid-related orphan receptor α (RORα) has protective effects on cardiovascular system. However, whether RORα mediates the protective effect of H2S against DCM remains unknown. The present research was to explore the roles and mechanisms of RORα in H2S against DCM. The study demonstrated that H2S donor sodium hydrosulfide (NaHS) alleviated cell injury but enhanced RORα expression in high glucose (HG)-stimulated cardiomyocytes. However, NaHS no longer had the protective effect on attenuating cell damage and oxidative stress, improving mitochondrial membrane potential, inhibiting necroptosis and enhanced signal transducer and activator of transcription 3 (STAT3) Ser727 phosphorylation in HG-stimulated cardiomyocytes after RORα siRNA transfection. Moreover, NaHS improved cardiac function, attenuated myocardial hypertrophy and fibrosis, alleviated oxidative stress, inhibited necroptosis, but increased STAT3 phosphorylation in wild type (WT) mice but not in RORα knockout mice (a spontaneous staggerer mice, sg/sg mice) with diabetes. Additionally, NaHS increased RORα promoter activity in cardiomyocytes with HG stimulation, which was related to the binding sites of E2F transcription factor 1 (E2F1) in the upstream region of RORα promoter. NaHS enhanced E2F1 expression and increased the binding of E2F1 to RORα promoter in cardiomyocytes with HG stimulation. In sum, H2S promoted RORα transcription via E2F1 to alleviate necroptosis and protect against DCM. It is helpful to propose a novel therapeutic implication for DCM.

Aryl hydrocarbon receptors improve migraine-like pain behaviors in rats through the regulation of regulatory T cell/T-helper 17 cell-related homeostasis.

To investigate the effect of the aryl hydrocarbon receptor (AHR)/regulatory T cell (Treg)/T-helper 17 (Th17) cell pathway on the pathogenesis of migraine. Migraine is a disabling neurovascular disease that imposes an enormous burden on both individuals and society. The pathophysiological mechanisms of migraine remain controversial. Recent studies have suggested that immune dysfunction may be involved in the pathogenesis of migraine. The AHR, a receptor expressed on most immune cells, has been implicated in the occurrence of many autoimmune diseases; however, whether it is involved in the pathogenesis of migraine is unclear. A chronic migraine rat model was established through repeated intraperitoneal injection of nitroglycerin (NTG). The mechanical and thermal pain thresholds were assessed using von Frey filaments and radiant heat. Next, the protein expression levels of AHR in the trigeminal nucleus caudalis (TNC) region of chronic migraine (CM)-like rats were quantified and the changes in Treg/Th17-related transcription factors and inflammatory factors in the TNC were explored. To determine the role of AHR in CM, we examined the effects of the AHR agonist 2-(1'-indole-3'-carbonyl)-thiazole-4-carboxylic acid methyl ester (ITE), and AHR antagonist CH-223191 on pain behavior, c-Fos, calcitonin gene-related peptide (CGRP), AHR, and Treg/Th17-related factor expression in CM-like rats. Repeated administration of NTG significantly enhanced nociceptive hypersensitivity and increased expression of c-Fos and CGRP in rats, while AHR was significantly decreased in the TNC. In addition, the expression of the transcription factor forkhead box protein P3 and the signal transducer and activator of transcription 5 decreased significantly. In contrast, the expression of the transcription factor retinoic acid receptor-related orphan receptor γ t and signal transducer and activator of transcription 3 were significantly increased. Moreover, the mRNA level of transforming growth factor beta-1 was decreased, while that of interleukin (IL)-10 and IL-22 was increased in the TNC. The AHR agonist ITE alleviated migraine-like pain behaviors in rats, activated the AHR signaling pathway, and improved the imbalance of Treg/Th17-related transcription factors and inflammatory factors. Conversely, the AHR antagonist CH-223191 did not alleviate migraine-like pain behaviors in rats; and even exacerbated them. The AHR participates in the development of CM by regulating Treg/Th17-related homeostasis. Therefore, treatments targeting the AHR/Treg/Th17 signaling pathway could be new effective interventions for CM treatment.

Neuroprotective role for RORA in Parkinson’s disease revealed by analysis of post-mortem brain and a dopaminergic cell line

Parkinson’s disease (PD) is almost twice as prevalent in men, which has largely been attributed to neuroprotective effect of oestradiol in women. RORA (retinoic acid receptor-related orphan receptor alpha) regulates the transcription of central aromatase, the enzyme responsible for local oestradiol synthesis, simultaneously, RORA expression is regulated by sex hormones. Moreover, RORA protects neurones against oxidative stress, a key mechanism contributing to the loss of dopaminergic neurones in PD. Therefore, we hypothesized that there would be sex differences in RORA expression in the substantia nigra pars compacta (SNpc), which could contribute to sex differences observed in PD prevalence and pathogenesis. In a case control study, qPCR and western blot analyses were used to quantify gene and protein expression in the SNpc of post-mortem brains (n = 14 late-stage PD and 11 age and sex matched controls). The neuroprotective properties of a RORA agonist were then investigated directly using a cell culture toxin-based model of PD coupled with measures of viability, mitochondrial function and apoptosis. RORA was expressed at significantly higher levels in the SNpc from control females’ brains compared to males. In PD, we found a significant increase in SNpc RORA expression in male PD compared to female PD. Treatment with a RORA agonist showed a significant neuroprotection in our cell culture model of PD and revealed significant effects on intracellular factors involved in neuronal survival and demise. This study is the first to demonstrate a sex specific pattern of RORA protein and gene expression in the SNpc of controls post-mortem human brains, and to show that this is differentially altered in male and female PD subjects, thus supporting a role for RORA in sex-specific aspects of PD. Furthermore, our in vitro PD model indicates mechanisms whereby a RORA agonist exerts its neuroprotective effect, thereby highlighting the translational potential for RORA ligands in PD.

The emerging family of RORγt+ antigen-presenting cells.

Antigen-presenting cells (APCs) are master regulators of the immune response by directly interacting with T cells to orchestrate distinct functional outcomes. Several types of professional APC exist, including conventional dendritic cells, B cells and macrophages, and numerous other cell types have non-classical roles in antigen presentation, such as thymic epithelial cells, endothelial cells and granulocytes. Accumulating evidence indicates the presence of a new family of APCs marked by the lineage-specifying transcription factor retinoic acid receptor-related orphan receptor-γt (RORγt) and demonstrates that these APCs have key roles in shaping immunity, inflammation and tolerance, particularly in the context of host-microorganism interactions. These RORγt+ APCs include subsets of group 3 innate lymphoid cells, extrathymic autoimmune regulator-expressing cells and, potentially, other emerging populations. Here, we summarize the major findings that led to the discovery of these RORγt+ APCs and their associated functions. We discuss discordance in recent reports and identify gaps in our knowledge in this burgeoning field, which has tremendous potential to advance our understanding of fundamental immune concepts.

Yin Yang 1 impacts upon preeclampsia by regulating Treg/TH17 cells and PI3K/AKT pathway

Preeclampsia (PE) is a common obstetric syndrome with an unclear etiology and pathogenesis. The study here aimed to investigate the role of Yin Yang 1 (YY1) in PE, and to reveal any YY1-regulated mechanisms in PE. Peripheral blood, placenta, and endometrial tissues of PE patients, healthy volunteers, and patients who had undergone an elective Cesarean section and had a scarred uterus (control group) were collected for analyses. Rat PE models were established by lipopolysaccharide induction. Subsets of these rats were then made to over-express YY1. At 18 d after the PE was established, urine, blood, and placental tissues from all rats were collected. Levels of regulatory-T (Treg) and helper T-type 17 (TH17) cells in both human and rat blood were measured by flow cytometry. ELISA kits were used to evaluate blood levels of inflammatory factors (i.e. IL-6, IL-10, and IL-17) as well. RT-qPCR and Western blot assays were performed to quantify levels of forkhead box P3 (Foxp3), retinoic acid-related orphan receptor C (RORc), and YY1 in the human and rat placenta and endometrial tissues. Expressions of PI3K/AKT pathway-related proteins were also evaluated by Western blots. The results indicated that the PE patients, relative to levels in control group and the healthy control subjects, had decreased circulating levels of Treg cells/increased TH17 cells; tissues from these patients also had relatively-decreased FoxP3 mRNA and protein expressions and elevated RORc mRNA and protein expressions. YY1 was expressed only at low levels in the PE patient placenta and endometrial tissues. In rats, PE rats treated with over-expressed YY1 had (relative to in PE rats without over-induced YY1) increased circulating levels of Treg cells/decreased TH17 cells; tissues from these rats had elevated FoxP3 mRNA and protein expressions and reduced mRNA and protein RORc expressions, as well as indications of alleviated inflammation. In the rat placenta samples, YY1 was also determined to activate the PI3K/AKT pathway. In summary, YY1 regulates the balance among Treg/TH17 cells and so affect the PE process in part through activation of the PI3K/AKT pathway.

Inhibition of Nuclear Receptor Related Orphan Receptor γ Ameliorates Mechanical Hypersensitivity Through the Suppression of Spinal Microglial Activation

Microglia are crucial in induction of central sensitization under a chronic pain state. Therefore, control of microglial activity is important to ameliorate nociceptive hypersensitivity. The nuclear receptor retinoic acid related orphan receptor γ (RORγ) contributes to the regulation of inflammation-related gene transcription in some immune cells, including T cells and macrophages. Their role and function in regulation of microglial activity and nociceptive transduction have yet to be elaborated. Treatment of cultured microglia with specific RORγ inverse agonists, SR2211 or GSK2981278, significantly suppressed lipopolysaccharide (LPS)-induced mRNA expression of pronociceptive molecules interleukin-1β (IL-1β), interleukin-6 (IL-6), and tumor necrosis factor (TNF). Intrathecal treatment of naïve male mice with LPS markedly induced mechanical hypersensitivity and upregulation of ionized calcium-biding adaptor molecule (Iba1) in the spinal dorsal horn, indicating microglial activation. In addition, intrathecal treatment with LPS significantly induced mRNA upregulation of IL-1β and IL-6 in the spinal dorsal horn. These responses were prevented by intrathecal pretreatment with SR2211. In addition, intrathecal administration of SR2211 significantly ameliorated established mechanical hypersensitivity and upregulation of Iba1 immunoreactivity in the spinal dorsal horn of male mice following peripheral sciatic nerve injury. The current findings demonstrate that blockade of RORγ in spinal microglia exerts anti-inflammatory effects, and that RORγ may be an appropriate target for the treatment of chronic pain.

Retinoic Acid-Related Orphan Receptor α Is Required for Generation of Th2 Cells in Type 2 Pulmonary Inflammation.

The transcription factor retinoic acid-related orphan receptor α (RORα) is important in regulating several physiological functions, such as cellular development, circadian rhythm, metabolism, and immunity. In two invivo animal models of type 2 lung inflammation, Nippostrongylus brasiliensis infection and house dust mite (HDM) sensitization, we show a role for Rora in Th2 cellular development during pulmonary inflammation. N. brasiliensis infection and HDM challenge induced an increase in frequency of Rora-expressing GATA3+CD4 T cells in the lung. Using staggerer mice, which have a ubiquitous deletion of functional RORα, we generated bone marrow chimera mice, and we observed a delayed worm expulsion and reduced frequency in the expansion of Th2 cells and innate lymphoid type 2 cells (ILC2s) in the lungs after N. brasiliensis infection. ILC2-deficient mouse (Rorafl/flIl7raCre) also had delayed worm expulsion with associated reduced frequency of Th2 cells and ILC2s in the lungs after N. brasiliensis infection. To further define the role for Rora-expressing Th2 cells, we used a CD4-specific Rora-deficient mouse (Rorafl/flCD4Cre), with significantly reduced frequency of lung Th2 cells, but not ILC2, after N. brasiliensis infection and HDM challenge. Interestingly, despite the reduction in pulmonary Th2 cells in Rorafl/flCD4Cre mice, this did not impact the expulsion of N. brasiliensis after primary and secondary infection, or the generation of lung inflammation after HDM challenge. This study demonstrates a role for RORα in Th2 cellular development during pulmonary inflammation that could be relevant to the range of inflammatory diseases in which RORα is implicated.

Astragalus regulates the intestinal immune response during sepsis by mediating ILC3 proliferation through RORγt

BackgroundSepsis is a common complication of many diseases and is associated with high morbidity and mortality rates. Astragalus can improve humoral and innate immunity, inhibit inflammatory responses, and protect immune cells and organs from damage. However, to the best of our knowledge there are no reports on whether astragalus can regulate intestinal innate immune function during sepsis. MethodsIn this study, a rat cecal ligation and puncture model of sepsis was used to investigate the effects of astragalus treatment, following which the apoptosis rate of lymphocytes from Peyer’s patches (PP) was determined. Type 3 innate lymphoid cells (ILC3) were cultured in vitro to further evaluate the effects and mechanisms of astragalus. ResultsThe apoptosis level of lymphocytes from PP in rats with sepsis was significantly increased, and the number of ILC3 was significantly reduced, compared with the sham operation group, which aggravated intestinal injury and ultimately led to the death of rats. Astragalus treatment significantly inhibited the apoptosis of lymphocytes from PP, increased the number of ILC3, and improved the intestinal inflammatory environment compared to the sepsis group. RT-PCR revealed that astragalus and the retinoic acid-related orphan receptor γt (RORγt) agonist LYC-55716 both promote the expression of interleukin (IL)-17A, IL-17F, IL-22, interferon-γ, and granulocyte-macrophage colony-stimulating factor mRNA. Mechanistically, astragalus promotes the proliferation of ILC3 through RORγt, thereby reducing intestinal inflammatory damage. ConclusionAstragalus, via RORγt, promotes the generation of ILC3, improves the inflammatory environment in rats with sepsis.

Cholesterol sulfate inhibits osteoclast differentiation and survival by regulating the AMPK-Sirt1-NF-κB pathway.

Cholesterol sulfate (CS) is an activator of retinoic acid-related orphan receptor α (RORα). CS treatment or RORα overexpression attenuates osteoclastogenesis in a collagen-induced arthritis mouse model. However, the mechanism by which CS and RORα regulate osteoclast differentiation remains largely unknown. Thus, we aimed to investigate the role of CS and RORα in osteoclastogenesis and their underlying mechanism. CS inhibited osteoclast differentiation, but RORα deficiency did not affect osteoclast differentiation and CS-mediated inhibition of osteoclastogenesis. CS enhanced adenosine monophosphate-activated protein kinase (AMPK) phosphorylation and sirtuin1 (Sirt1) activity, leading to nuclear factor-κB (NF-κB) inhibition by decreasing acetylation at Lys310 of p65. The NF-κB inhibition was restored by AMPK inhibitor, but the effects of CS on AMPK and NF-κB were not altered by RORα deficiency. CS also induced osteoclast apoptosis, which may be due to sustained AMPK activation and consequent NF-κB inhibition, and the effects of CS were significantly reversed by interleukin-1β treatment. Collectively, these results indicate that CS inhibits osteoclast differentiation and survival by suppressing NF-κB via the AMPK-Sirt1 axis in a RORα-independent manner. Furthermore, CS protects against bone destruction in lipopolysaccharide- and ovariectomy-mediated bone loss mouse models, suggesting that CS is a useful therapeutic candidate for treating inflammation-induced bone diseases and postmenopausal osteoporosis.

Liraglutide ameliorates hepatic steatosis via retinoic acid receptor-related orphan receptor α-mediated autophagy pathway.

Liraglutide, an analog of human glucagon-like peptide-1 (GLP-1), has been found to improve hepatic steatosis in clinical practice. However, the underlying mechanism remains to be fully defined. Increasing evidence suggests that retinoic acid receptor-related orphan receptor α (RORα) is involved in hepatic lipid accumulation. In the current study, we investigated whether the ameliorating impact of liraglutide on lipid-induced hepatic steatosis is dependent on RORα activity and examined the underlying mechanisms. Cre-loxP-mediated, liver-specific Rorα knockout (Rora LKO) mice, and littermate controls with a Roraloxp/loxp genotype were established. The effects of liraglutide on lipid accumulation were evaluated in mice challenged with a high-fat diet (HFD) for 12 weeks. Moreover, mouse AML12 hepatocytes expressing small interfering RNA (siRNA) of Rora were exposed to palmitic acid to explore the pharmacological mechanism of liraglutide. The results showed that liraglutide treatment significantly alleviated HFD-induced liver steatosis, marked by reduced liver weight and triglyceride accumulation, improved glucose tolerance and serum levels of lipid profiles and aminotransferase. Consistently, liraglutide also ameliorated lipid deposits in a steatotic hepatocyte model in vitro. In addition, liraglutide treatment reversed the HFD-induced downregulation of Rora expression and autophagic activity in mouse liver tissues. However, the beneficial effect of liraglutide on hepatic steatosis was not observed in Rora LKO mice. Mechanistically, the ablation of Rorα in hepatocytes diminished liraglutide-induced autophagosome formation and the fusion of autophagosomes and lysosomes, resulting in weakened autophagic flux activation. Thus, our findings suggest that RORα is essential for the beneficial impact of liraglutide on lipid deposition in hepatocytes and regulates autophagic activity in the underlying mechanism.

Comprehensive analysis of the prognosis and biological significance of RORβ in head and neck squamous cell carcinoma.

Recent studies have shown that abnormal expression of the core circadian clock gene, retinoic acid-related orphan receptor β (RORβ ), is closely associated with the occurrence and progression of various malignant tumors. However, the expression and function of RORβ in head and neck squamous cell carcinoma (HNSCC) remains unclear. Here, we comprehensively investigated the altered expression, clinical significance, prognostic value, and biological functions of RORβ in HNSC, as well as its correlation with changes in the tumor immune microenvironment. We found that RORβ expression was decreased in HNSC and 19 other cancers. Low RORβ expression was significantly associated with tumor size, clinical stage, and survival time in HNSC patients, indicating that it may have diagnostic and prognostic value in HNSCC. Epigenetic analysis showed that the promoter methylation level of RORβ was significantly higher in HNSCC compared to adjacent noncancerous tissues. Furthermore, RORβ hypermethylation was significantly associated with low expression levels of RORβ and poor prognosis in HNSCC patients (p < 0.05). Enrichment analysis found that RORβ was involved in immune system regulation and T-cell activation, as well as the PI3K/AKT and ECM receptors interaction pathways. In vitro assays revealed that RORβ regulated the proliferation, migration and invasion ability of HNSCC cells. Additionally, we found that RORβ expression was significantly correlated with changes in the tumor immune microenvironment, suggesting it may affect prognosis by regulating immune infiltration in HNSC patients. Therefore, RORβ may serve as a potential prognostic biomarker and therapeutic target for HNSCC patients.

Escherichia coli enhances Th17/Treg imbalance via TLR4/NF-κB signaling pathway in oral lichen planus

Oral lichen planus (OLP) is a T-cell-mediated immunoinflammatory disease. Several studies have proposed that Escherichia coli (E. coli) may participate in the progress of OLP. In this study, we examined the functional role of E. coli and its supernatant via toll-like receptor 4 (TLR4)/nuclear factor-kappab (NF-κB) signaling pathway in regulating T helper (Th) 17/ regulatory T (Treg) balance and related cytokines and chemokines profile in OLP immune microenvironment. We discovered that E. coli and supernatant could activate the TLR4/NF-κB signaling pathway in human oral keratinocytes (HOKs) and OLP-derived T cells and increase the expression of interleukin (IL)-6, IL-17, C-C motif chemokine ligand (CCL) 17 and CCL20, thereby increasing the expression of retinoic acid-related orphan receptor (RoRγt) and the proportion of Th17 cells. Furthermore, the co-culture experiment revealed that HOKs treated with E. coli and supernatant increased T cell proliferation and migration, which promoted HOKs apoptosis. TLR4 inhibitor (TAK-242) successfully reversed the effect of E. coli and its supernatant. Consequently, E. coli and supernatant activated the TLR4/NF-κB signaling pathway in HOKs and OLP-derived T cells, leading to increased cytokines and chemokines expression and Th17/Treg imbalance in OLP.