Retinoid-related Orphan Receptor Γt Research Articles

Abstract MP02: Inhibition of RORγt Ameliorates Angiotensin II-Induced Blood-Brain Barrier Disruption and Microglia Activation by Reducing Interleukin-17A Production in Rats

Inflammation-induced blood-brain barrier (BBB) disruption and microglial activation in the autonomic and cardiovascular brain centers contribute largely to sympathetic excitation and hypertension. We recently reported that interleukin (IL)-17A, a major mediator of tissue inflammation, promotes BBB disruption, neuroinflammation, and sympathetic activation in angiotensin (ANG) II-induced hypertension and myocardial infarction-induced heart failure. The nuclear receptor retinoid-related orphan receptor γt (RORγt) has been identified as a master transcription factor regulating the differentiation of T helper 17 cells, the primary producer of IL-17A. The present study sought to determine whether inhibition of RORγt to suppress IL-17A production can ameliorate BBB disruption and attenuate microglia activation in ANG II-induced hypertension model. SD rats were treated with a 2-week subcutaneous (SC) infusion of ANG II (150 ng/kg/min) via mini-pumps, combined with daily SC injections of RORγt inhibitor digoxin or vehicle (VEH). Compared to the VEH control, IL-17A levels in plasma and cerebrospinal fluid were significantly (*P<0.05) elevated in ANG II+VEH rats and were reduced in ANG II + digoxin rats. In ANG II+VEH rats, BBB permeability, measured by fluorescein isothiocyanate-dextran (FITC) 10 kDa, was elevated (6.82±0.61%* vs. 2.36±0.28% FITC10 extravasation), along with increased mRNA levels of caveolin-1 (a transcytosis marker, 2.26±0.39* vs. 1.07±0.17) and decreased mRNA levels of mfsd2a (a BBB-specific lipid transporter and inhibitor of transcytosis, 0.55±0.04* vs. 1.06±0.09) in the hypothalamic paraventricular nucleus (PVN). However, BBB permeability and mRNA levels of caveolin-1 and mfsd2a in the PVN were reversed in ANG II + digoxin rats. Immunostaining revealed that both caveolin-1 and mfsd2a were primarily located in the endothelial BBB within the PVN. Microglia activation, assessed by the number of branches (26±2* vs. 37±4) and total branch length (184±15* vs. 250±23), as well as the mRNA levels of tumor necrosis factor-α (2.78±0.45* vs. 1.04±0.12) and IL-1β (2.57±0.53* vs. 1.03±0.14) in the PVN were augmented in ANG II+VEH rats but were attenuated (40-58%*) in ANG II + digoxin rats. Additionally, ANG II-induced hypertension and sympathetic tone were attenuated by digoxin. These data suggest that inhibiting RORγt diminishes BBB disruption, microglial activation and sympathetic excitation induced by ANG II, likely via reducing IL-17A production.

Electroacupuncture of scalp acupoint alleviates cerebral ischemic inflammatory injury by down-regulating RORγt and promoting balance of IL-17A+Th17/FOXP3+Treg in MCAO rats.

To observe the effect of electroacupuncture (EA) of scalp acupoint (Dingnieqian-xiexian, MS6) on expression of retinoid-related orphan receptor γT (ROR γ t), interleukin (IL)-17A, IL-10, transfor-ming growth factor-β1 (TGF-β1), IL-6, IL-21, and IL-17A+ Thelper cells(Th) 17 and forkhead transcription factor P3 (FOXP3)+ regulatory T cells (Treg) differentiation of ischemic cortex in ischemic stroke rats, so as to explore its molecular mechanisms underlying relief of inflammatory injury of ischemic stroke. A total of 120 male SD rats were randomly assigned to sham operation, model, EA, inhibitor, agonist and EA+agonist groups, with 15 rats in each group. The ischemic stroke model was established by occlusion of the left middle cerebral artery according to Longa's methods. For rats of the EA group and EA+agonist group, EA (2 Hz/100 Hz, 1 mA) was applied to bilateral MS6 for 30 min, once daily for 7 days. Rats of the inhibitor group received intraperitoneal injection of solution of SR1001 (RORγt inhibitor) (2.5 mg/mL, 10 mg/kg), once daily for 7 days. Rats of the agonist and EA+agonist groups received intraperitoneal injection of solution of SR1078 (RORγt agonist) (5 mg/mL, 5 mg/kg) before EA, once daily for 7 days. Rats of the sham operation and model groups were grabbed and fixed in the same way with the other groups. The Zea-longa's score, modified neurological severity score (mNSS) and the neurobehavioral score were assessed before and after the intervention. At the end of experiments, the ischemic cortex tissue was collected. The 2, 3, 5-Triphenyltetrazolium chloride (TTC) staining was used to detect the volume of cerebral infarction. The expression of RORγt mRNA was detected by real-time quantitative PCR；the protein expression levels of RORγt, IL-17A, IL-10 and TGF-β1 were detected by Western blot；the immunoactivity of IL-6 and IL-21 were detected by immunohistochemistry；the fluorescence areas of IL-17A+Th17 and FOXP3+Treg cells were measured by immunofluorescence and their ratio was calculated in the tissue of ischemic cortex. Relevant to the sham operation group, the model group had a significant increase in the Zea-Longa's score, mNSS score, neurobehavioral score, cerebral infarct volume, expression levels of RORγt mRNA and protein, IL-17A protein, IL-6 and IL-21 immunoactivity, IL-17A+Th17 immunofluorescence intensity, and the ratio of IL-17A+Th17/FOXP3+Treg (P<0.01), and an obvious decrease in the expression levels of TGF-β1 and IL-10 proteins and FOXP3+Treg immunofluorescence intensity (P<0.01). In contrast to the model group, both EA and inhibitor groups had a significant decrease in the Zea-Longa's score, mNSS score, neurobehavioral score, cerebral infarct volume, expression levels of RORγt mRNA and protein, IL-17A protein, IL-6 and IL-21 immunoactivity, IL-17A+Th17 immunofluorescence intensity, and the ratio of IL-17A+Th17/FOXP3+Treg (P<0.01, P<0.05), and a marked increase in the expression levels of TGF-β1 and IL-10 proteins and FOXP3+Treg immunofluorescence intensity (P<0.05, P<0.01), while the above indicators of the agonist group were all reversed (P<0.01, P<0.05). Comparison between the agonist and EA+agonist groups showed that the Zea-Longa's score, mNSS score, neurobehavioral score, cerebral infarct volume, expression levels of RORγt mRNA and protein, IL-17A protein, IL-6 and IL-21 immunoactivity, IL-17A+Th17 immunofluorescence intensity, and the ratio of IL-17A+Th17/FOXP3+Treg were significantly lower (P<0.01, P<0.05), and the expression of TGF-β1 and IL-10 proteins and FOXP3+Treg immunofluorescence intensity were obviously higher (P<0.01, P<0.05) in the EA+agonist group than in the agonist group, suggesting that EA intervention can effectively weaken the effects of RORγt agonist. EA of scalp acupoint MS6 can effectively improve the neurological function, behavior reaction and reduce cerebral infarct volume in ischemic stroke rats, which may be associated with its functions in down-regulating the expression of RORγt and promoting the balance of IL-17A+Th17/FOXP3+Treg to alleviate inflammatory injury after ischemic stroke.

HMGB1 regulates Th17 cell differentiation and function in patients with psoriasis.

Psoriasis is an immune-mediated chronic inflammatory skin disease, in which T helper 17 (Th17) cells and its effective cytokine interleukin (IL)-17A play a pivotal pathogenic role. High mobility group box 1 (HMGB1) is an important proinflammatory cytokine, which has been confirmed to be highly expressed in the peripheral circulation and epidermis tissues of psoriasis patients. The regulatory effect of HMGB1 on IL-17A expression and function has been reported in some inflammatory and autoimmune diseases by theHMGB1-Toll-like receptor 4 (TLR4)-interleukin (IL)-23-IL-17A pathway. While, in the pathological environment of psoriasis, whether HMGB1 can exert the regulatory effect on IL-17A is not clear. We aimed to evaluate the role of HMGB1-TLR4-IL-23-IL-17A pathway in the pathogenesis of psoriasis and explore the possible regulatory mechanism of HMGB1 on Th17 cell differentiation. Serum levels of HMGB1, TLR4, IL-23, and IL-17A were quantified in 50 patients with moderate-to-severe plaque psoriasis and 30 healthy controls. Peripheral blood mononuclear cells were acquired from 10 severe psoriasis patients and administrated by different concentrations of recombinant-HMGB1 (rHMGB1) to detect the Th17 cell percentage, mRNA and protein levels of TLR4, IL-23, IL-17A and retinoid-related orphan receptor γt (RORγt). The serum levels of HMGB1, TLR4, IL-23, and IL-17A in psoriasis patients were significantly higher than healthy controls, especially in severe patients, and positively correlated with the severity index. There were also positive correlations between every two detected indicators of HMGB1, TLR4, IL-23, and IL-17A. In vitro study, rHMGB1 can promote the elevated expression of Th17 cell percentage as well as TLR4, IL-23, IL-17A, and RORγt in a dose-dependent manner. HMGB1 can contribute to the pathogenesis of psoriasis by regulating Th17 cell differentiation through HMGB1-TLR4-IL-23-RORγt pathway, then promotesIL-17A production and aggravates inflammation process. Targeting HMGB1 may be a possible potential candidate for the immunotherapy of psoriasis.

Prostaglandin E2 may clinically alleviate dry eye disease by inducing Th17 cell differentiation.

Dry eye (DE) is a multifactorial ocular surface disease characterised by an imbalance in tear homeostasis. The pathogenesis of DE is complex and related to environmental, immunological (e.g., T helper 17 cells) and other factors. However, the DE disease pathogenesis remains unclear, thereby affecting its clinical treatment. This study aimed to explore the mechanism through which prostaglandin E2 (PGE2) affects DE inflammation by regulating Th17. The DE mouse model was established through subcutaneous injection of scopolamine hydrobromide. The tear secretion test and break-up time (BUT) method were used to detect tear secretion and tear film BUT, respectively. Enzyme-linked immunosorbent assay (ELISA) was used to detect the concentrations of PGE2, interleukin (IL)-17, IL-6 and tumour necrosis factor (TNF-α) in tear fluid and those of PGE2 and IL-17 in the serum. RT-qPCR and western blotting were used to test the mRNA and protein expression levels of IL-17 and retinoid-related orphan receptor-γt (RORγt). PGE2 was highly expressed in the DE mouse model. The mRNA and protein levels of IL-17 and the key Th17 transcription factor RORγt were increased in tissues of the DE mice. Moreover, PGE2 promoted tear secretion, reduced the BUT, increased the IL-17 concentration in tears and increased the Th17 cell proportion in DE, whereas the PGE2 receptor inhibitor AH6809 reversed the effects of PGE2 on tear secretion, BUT, and the Th17 cell proportion in draining lymph node (DLN) cells. Taken together, the study findings indicate that PGE2 could induce DE-related symptoms by promoting Th17 differentiation.

Regulating Th17/Treg Balance Contributes to the Therapeutic Effect of Ziyuglycoside I on Collagen-Induced Arthritis

To investigate the therapeutic effect and primary pharmacological mechanism of Ziyuglycoside I (Ziyu I) on collagen-induced arthritis (CIA) mice. CIA mice were treated with 5, 10, or 20 mg/kg of Ziyu I or 2 mg/kg of methotrexate (MTX), and clinical manifestations, as well as pathological changes, were observed. T cell viability and subset type were determined, and serum levels of transforming growth factor-beta (TGF-β) and interleukin-17 (IL-17) were detected. The mRNA expression of retinoid-related orphan receptor-γt (RORγt) and transcription factor forkhead box protein 3 (Foxp3) in mouse spleen lymphocytes was ascertained by the real-time reverse transcriptase-polymerase chain reaction (RT-qPCR). Molecular docking was used to detect whether there was a molecular interaction between Ziyu I and protein kinase B (Akt). The activation of mechanistic target of rapamycin (mTOR) in T cells was verified by Western blotting or immunofluorescence. Ziyu I treatment effectively alleviated arthritis symptoms of CIA mice, including body weight, global score, arthritis index, and a number of swollen joints. Similarly, pathological changes of joints and spleens in arthritic mice were improved. The thymic index, T cell activity, and RORγt production of Ziyu I-treated mice were significantly reduced. Notably, through molecular docking, western blotting, and immunofluorescence data analysis, it was found that Ziyu I could interact directly with Akt to reduce downstream mTOR activation and inhibit helper T cell 17 (Th17) differentiation, thereby regulating Th17/regulatory T cell (Treg) balance and improving arthritis symptoms. Ziyu I effectively improves arthritic symptoms in CIA mice by inhibiting mTOR activation, thereby affecting Th17 differentiation and regulating Th17/Treg balance.

Effect of baotaiyin on IL-23 /Th17 immune inflammatory axis in mouse model of spontaneous abortion.

The mechanism for traditional Chinese medicine in treating of recurrent spontaneous abortion is not clear. This study aims to explore the mechanism of baotaiyin in the treatment of recurrent abortion by regulating the immune inflammatory axis of interleukin (IL)-23/helper T cell (Th)17. Spontaneous abortion model mice were randomly divided into a model group, 3 dose (low, medium, and high) groups of baotaiyin, with 10 mice in each group. After 14 days of medication, the levels of IL-17, IL-23, IL-10, and TGF-β in serum were detected with enzyme-linked immunosorbent assay. The proportion of Th17 and regulatory T cells (Treg) cells in spleen lymphocytes was tested with flow cytometry. The expressions of (retinoid-related orphan receptor γt, ROR-γt) and forkhead box P3 (FOXP3) mRNA in decidua tissues was detected with RT-PCR. Embryo absorption rate was counted. Compared with the model group, the absorption rate of embryo and Th17/Treg cell ratio in baotaiyin medium- and high-dose groups were decreased significantly (all P<0.05); the levels of IL-17 and IL-23 in serum were decreased (both P<0.05), while the levels of TGF-β and IL-10 in baotaiyin medium- and high-dose groups were increased (P<0.05, P<0.01, respectively); the expression of ROR-γt mRNA was decreased and the expression of FOXP3 mRNA was increased (all P<0.01) in decidua tissues of baotaiyin medium- and high-dose groups. Baotaiyin inhibits the positive feedback cycle of IL-23/Th17 immune inflammatory axis, which regulates Th17/Treg cell balance, mediates the maternal and fetal immune tolerance, and prevents the recurrent abortion.

Natural Compound 2,2′,4′-Trihydroxychalcone Suppresses T Helper 17 Cell Differentiation and Disease Progression by Inhibiting Retinoid-Related Orphan Receptor Gamma T

Retinoid-related orphan receptor γt (RORγt), a vital transcription factor for the differentiation of the pro-inflammatory Th17 cells, is essential to the inflammatory response and pathological process mediated by Th17 cells. Pharmacological inhibition of the nuclear receptor RORγt provides novel immunomodulators for treating Th17-driven autoimmune diseases and organ transplant rejection. Here, we identified 2,2',4'-trihydroxychalcone (TDC), a natural chalcone derivant, binds directly to the ligand binding domain (LBD) of RORγt and inhibited its transcriptional activation activity. Using three mice models of Th17-related diseases, it was found that the administration of TDC effectively alleviated the disease development of experimental autoimmune encephalomyelitis (EAE), experimental colitis, and skin allograft rejection. Collectively, these results demonstrated TDC targeting RORγt to suppress Th17 cell polarization, as well as its activity, thus, indicating the potential of this compound in treating of Th17-related autoimmune disorders and organ transplant rejection disorders.

Abstract P345: Inhibition Of Nuclear Receptor Retinoid-related Orphan Receptor γt Attenuates Interleukin-17A Production And Ameliorates Angiotensin II-induced Neuroinflammation And Hypertension In Rats

Interleukin (IL)-17A, a key inflammatory mediator produced primarily by T helper (Th) 17 cells, has been implicated in the pathogenesis of cardiovascular diseases including hypertension and heart failure. IL-17A can access the brain by disrupting blood-brain barrier (BBB) integrity to induce neuroinflammation. We previously reported that IL-17A contributes to angiotensin (ANG) II-induced hypertension via promoting neuroinflammation and sympathetic excitation. The nuclear receptor retinoid-related orphan receptor γt (RORγt) is a master transcription factor regulating Th17 cell differentiation. Here, we sought to determine whether systemic inhibition of RORγt attenuates IL-17A production and diminishes ANG II-induced neuroinflammation and hypertension. Sprague-Dawley rats received a 2-week subcutaneous (sc) infusion of ANG II (150 ng/kg/min) combined with daily sc injection of a RORγt inhibitor digoxin or vehicle (VEH). Compared with the control animals, blood pressure (162±5* vs 118±3 mmHg, *P&lt;0.05) and sympathetic tone as indicated by blood pressure change in response to ganglionic blockade (55±5* vs 23±3 mmHg) were increased in ANG II+VEH rats, which were reduced (38-41%*) in ANG II+digoxin rats. ANG II+VEH rats also had elevated RORγt mRNA in peripheral blood mononuclear cells (PBMCs: 3.38±0.70* vs 1.12±0.22) and higher IL-17A levels in plasma (23.22±5.85 * vs 4.30±0.66 pg/mL), along with increased IL-17A levels in cerebrospinal fluid (CSF: 11.89±1.91* vs 2.01±0.55 pg/mL), mRNA of IL-17A (3.02±0.48* vs 1.07±0.17), cytokines tumor necrosis factor (TNF)-α (2.78±0.45* vs 1.04±0.12) and IL-1β (2.57±0.53* vs 1.03±0.14) in the hypothalamic paraventricular nucleus (PVN), a key cardiovascular-related center in the brain. Although RORγt mRNA was unchanged in PBMCs, levels of IL-17A in plasma and CSF, mRNA of IL-17A, TNF-α and IL-1β in the PVN was reduced (41-62 %*) in ANG II+digoxin rats. Additionally, mRNA of caveolin-1 (2.26±0.39* vs 1.06±0.17), a marker of the BBB permeability, was increased in the PVN in ANG II+VEH rats and decreased (43%*) in ANG II+digoxin rats. These data suggest that RORγt inhibition improves ANG II-induced hypertension and sympathetic activation probably by reducing IL-17A production and neuroinflammation.

Blockade of NLRP3/Caspase-1/IL-1β Regulated Th17/Treg Immune Imbalance and Attenuated the Neutrophilic Airway Inflammation in an Ovalbumin-Induced Murine Model of Asthma

Asthma is a heterogeneous inflammatory disorder of the airways, and multiple studies have addressed the vital role of the nucleotide-binding oligomerization domain-like receptor family pyrin domain containing 3 (NLRP3)/caspase-1/interleukin-1β (IL-1β) pathway in asthma, but its impact on ovalbumin- (OVA-) induced neutrophilic asthma remains unclear. Here, we explored this pathway's effect on airway inflammation in neutrophilic asthma to clarify whether blocking this signaling could alleviate asthmatic airway inflammation. Using an established OVA-induced neutrophilic asthma mouse model, we provided asthmatic mice with a highly selective NLRP3 inhibitor, MCC950, and a specific caspase-1 inhibitor, Ac-YVAD-cmk. Our results indicated that asthmatic mice exhibited increased airway hyperresponsiveness, neutrophil infiltration, and airway mucus hypersecretion, upregulated retinoid-related orphan receptor-γt (RORγt) mRNA expression, and downregulated fork head box p3 (Foxp3) mRNA expression, which was concurrent with NLRP3 inflammasome activation and upregulation of caspase-1, IL-1β, and IL-18 expression in lung. Treatment of NLRP3 inflammasome inhibitors significantly attenuated airway hyperresponsiveness, airway inflammation, and reversed T helper 17 (Th17)/regulatory T (Treg) cell imbalance in asthmatic mice. We propose that the NLRP3/caspase-1/IL-1β pathway plays an important role in the pathological process of neutrophilic asthma and provides evidence that blocking this pathway could potentially be a treatment strategy to ameliorate airway inflammation in asthma after validation with future experimental and clinical studies.

LncRNA STAT4-AS1 Inhibited TH17 Cell Differentiation by Targeting RORγt Protein.

Objective From our previous study, we obtained long noncoding RNA (lncRNA) STAT4-AS1, which is related to asthma through high-throughput screening. However, we could not determine the specific mechanism involved and in response to this. We further designed this study. Results First, we found that lncRNA STAT4-AS1 was downregulated in T cells from patients with asthma when compared to healthy controls. Next, we confirmed that lncRNA STAT4-AS1 was significantly negatively correlated with T helper 17 (TH17) differentiation in vitro experiments. The decreases of STAT4-AS1 promoted TH17 differentiation, while the increases of STAT4-AS1 inhibited TH17 differentiation. Subsequently, through RNA pull-down, RNA-binding protein immunoprecipitation (RIP), and dual luciferase reporter assay, we found that STAT4-AS1 could inhibit the binding of retinoid-related orphan receptor-γt (RORγt) protein with an IL-17A promoter after binding with RORγt protein. Fluorescence in situ hybridization (FISH) and nuclear-cytoplasmic separation assay showed that STAT4-AS1 is bonded to RORγt in the cytoplasm, preventing RORγt from entering the nucleus. Conclusion Overall, STAT4-AS1 directly targets RORγt protein, inhibits the mutual binding of RORγt and IL-17 gene promoter, and eventually inhibits TH17 differentiation. To this end, STAT4-AS1 as a potential target may confer applications in the clinical treatment and diagnosis of TH17-related diseases.

Parthenolide Suppresses T Helper 17 and Alleviates Experimental Autoimmune Encephalomyelitis.

T helper (Th) cells play crucial roles in inflammation and adaptive immune system. Importantly, Th17 cells, a major pathogenic Th cell subset, are involved in the pathogenesis of multiple sclerosis (MS) and its classical animal modal experimental autoimmune encephalomyelitis (EAE). Previous studies have shown that parthenolide (PTL), a sesquiterpene lactone, possesses potent anti-cancer and anti-inflammatory activities. However, the immunosuppressive effect of PTL on the pathogenic Th17 cell and MS is unclear. In this study, we showed that PTL treatment could alleviate clinical symptoms by inhibiting inflammatory cell infiltration, reducing inflammation and demyelination of CNS. In addition, the mRNA expression of cytokines and inflammatory factors in CD4+ T cells, especially Th1 and Th17 cells, reduced in both CNS and peripheral immune tissue of EAE mice. Furthermore, PTL could inhibit the reactivation of MOG-specific T cells and the differentiation of naïve CD4+ T cells into Th17 cells in vitro. We also found that PTL inhibited nuclear factor kappa B (NF-κB) signaling and retinoid-related orphan receptor-γt (RORγt) in mouse Th17 cell and human Jurkat cell line. Taken together, our data demonstrated a critical immune-suppressive effect of PTL on autoimmune inflammation through regulating Th17 cells and the NF-κB/RORγt pathway.

Taxifolin ameliorates sepsis-induced lung capillary leak through inhibiting the JAK/STAT3 pathway

As a systemic inflammatory reaction, sepsis is associated with various organ dysfunctions. The capillary leakage and the imbalance between T helper 17 and regulatory T (Th17/Treg) cells are associated with sepsis-induced lung injury. Taxifolin (TXL) has been found to play a vital role in regulating this diverse disease. However, the detailed functioning and mechanism of TXL in regulating sepsis-induced lung capillary leak remain elusive. Balb/c mice were used to establish sepsis-induced lung injury model through administration of lipopolysaccharide (LPS). The structure of lung tissues was observed by using hematoxylin & eosin staining. Protein level and total cells in bronchoalveolar lavage fluid (BALF) were measured by bicinchoninic acid (BCA) protein assay kit and hematimetry assay, respectively. Quantitative real-time reverse transcription polymerase chain reaction and enzyme-linked immunosorbent assay were employed to detect the level of inflammatory cytokines. The content of Th17 and Treg cells were measured by flow cytometry analysis. Western blot assay was used to determine the protein level of retinoid-related orphan receptor-γt (RORγt), Forkhead box P3 (Foxp3), Janus kinase 2 (JAK2), phospho(p)-JAK2, signal transducer and activator of transcription 3 (STAT3), and phospho(p)-STAT3. Taxifolin effectively prolonged the survival period of sepsis mice and alleviated LPS-induced lung injury in a dose-dependent manner. Moreover, TXL reduced LPS-induced increase in protein levels and T cell content in BALF, and effectively restored the wet:dry ratio of lung tissue and tissue permeability. Treating with TXL notably inhibited the production of pro-inflammatory cytokines induced by sepsis and influenced the balance between Th17 and Treg cells. Furthermore, TXL treatment suppressed the activation of JAK/STAT3 signaling pathway in a dose-dependent manner. Our findings revealed that TXL alleviated sepsis-induced capillary leak in the lungs of mice by regulating JAK/STAT3 signaling pathway.

MAF bZIP Transcription Factor B (MAFB) Protected Against Ovalbumin-Induced Allergic Rhinitis via the Alleviation of Inflammation by Restoring the T Helper (Th) 1/Th2/Th17 Imbalance and Epithelial Barrier Dysfunction.

PurposeThis work aimed to investigate the effects of MAF bZIP transcription factor B (MAFB) on the progression of allergic rhinitis (AR).Patients and MethodsNasal mucosa was isolated from AR patients and healthy individuals from Shengjing Hospital of China Medical University. The experimental procedures were approved by the Medical Ethics Committee of Shengjing Hospital of China Medical University (2019PS341K) in accordance with the Declaration of Helsinki. Informed consents were signed by participants or a parent/legal guardian of the participants under 18 years old of age. Then, an AR mouse model with MAFB overexpression was established with 25 μg ovalbumin (OVA) sensitization on day 0, 7, 14, followed by an injection with 1×107 TU/mL lentivirus MAFB on day 19 and a nasal challenge with 500 μg OVA from day 21 to 27.ResultsThe results revealed that MAFB was down-regulated in the nasal mucosa of AR patients. The up-regulation of MAFB protected the AR mice against the OVA-induced allergic symptoms (sneezing and nasal rubbing) by alleviating the OVA-induced epithelial thicknesses, goblet cell hyperplasia, and inflammation including the eosinophil and mast cell infiltration. Moreover, MAFB facilitated the T helper (Th) 1 response and inhibited the Th2 and Th17 responses by the down-regulation of T-box transcription factor 21 and the up-regulation of GATA binding protein-3 as well as retinoid-related orphan receptor-γt in the splenocytes of AR mice. MAFB was found to repress the differentiation of naive CD4+ T cells into Th2 cells. Subsequently, MAFB overexpression reversed the OVA-induced enhancement of epithelial permeability, downregulation of tight junctions, and upregulation of cadherin-26, indicating the protective role of MAFB on epithelial barrier integrity.ConclusionMAFB protected against OVA-induced AR via the alleviation of inflammation by restoring the Th1/Th2/Th17 imbalance and epithelial barrier dysfunction.

WSDTNBI: a novel network-based inference method for virtual screening.

In recent years, the rapid development of network-based methods for the prediction of drug–target interactions (DTIs) provides an opportunity for the emergence of a new type of virtual screening (VS), namely, network-based VS. Herein, we reported a novel network-based inference method named wSDTNBI. Compared with previous network-based methods that use unweighted DTI networks, wSDTNBI uses weighted DTI networks whose edge weights are correlated with binding affinities. A two-pronged approach based on weighted DTI and drug–substructure association networks was employed to calculate prediction scores. To show the practical value of wSDTNBI, we performed network-based VS on retinoid-related orphan receptor γt (RORγt), and purchased 72 compounds for experimental validation. Seven of the purchased compounds were confirmed to be novel RORγt inverse agonists by in vitro experiments, including ursonic acid and oleanonic acid with IC50 values of 10 nM and 0.28 μM, respectively. Moreover, the direct contact between ursonic acid and RORγt was confirmed using the X-ray crystal structure, and in vivo experiments demonstrated that ursonic acid and oleanonic acid have therapeutic effects on multiple sclerosis. These results indicate that wSDTNBI might be a powerful tool for network-based VS in drug discovery.

Effect of different orthodontic forces on the expression of T helper cell 17 cell-related cytokines in the pressure side of periodontal tissue in rats

This study aimed to explore the changes in the expression of the characteristic transcription factor retinoid related orphan receptor γt (RORγt) and the cytokine interleukin-17 (IL-17) of T helper cell 17 (Th17) in the pressure side of the periodontal tissue of rats under different orthodontic forces. Their effects on the expression of osteoprotegerin (OPG) and the quantity of osteoclast (OC) were also explored. The role of Th17 cell in alveolar bone remodeling under different forces was preliminarily investigated. A total of 108 rats were chosen and randomly divided into three groups. Mesial forces of 0, 50, and 100 g were loaded on the maxillary first molar in the three groups. The rats were executed at 0, 1, 3, 5, 7, and 14 days. The expression of RORγt mRNA was quantified by real-time quantitative polymerase chain reaction. The expression of IL-17 protein was quantified by enzyme linked immunosorbent assay. The expression levels of RORγt and OPG proteins were quantified, and the quantity of OC was counted via immunohistochemistry. The expression levels of RORγt and IL-17 and the quantity of OC increased first and then decreased in the 50 and 100 g groups, and the peak values of the two groups were on days 5 and 7, respectively. The expression levels in the 50 g group basically recovered to normal level on day 14, while that in the 100 g group remained at a high level. The expression levels in the 50 g group were higher than those in the 0 g group and lower than those in the 100 g group. The expression of OPG in the 50 g group decreased first, then increased, and finally decreased. It basically recovered to normal level on day 14. The expression of OPG in the 100 g group decreased first and then increased. It remained at a high level on day 14. The expression in the 50 g group was significantly higher than that in the 0 g group on day 7, while the expression in the 100 g group was significantly higher than that in the 0 g group on day 14. RORγt, IL-17, and OPG were expressed regularly over time under different orthodontic forces, indicating that Th17 participated in the process of bone resorption on the pressure side of periodontal tissue by secreting IL-17.

ILC3-derived acetylcholine promotes protease-driven allergic lung pathology

ILC3-derived acetylcholine promotes protease-driven allergic lung pathology

Correlation of Vitamin D3 with the Expression of RORγt and Foxp3 mRNAs in the Peripheral Blood of Myasthenia Gravis Patients

Objectives: to investigate the expression levels of 1,25(OH)₂D₃ in the peripheral blood from patients with myasthenia gravis (MG) and to correlate levels with retinoid-related orphan receptor γt (RORγt) and forkhead or winged-helix transcription factor 3 (Foxp3) mRNA expression. Methods: Sixty-seven patients with MG were enrolled in the experimental group, and 50 normal subjects were selected as the control group. The expression levels of 1,25(OH)₂D₃ and RORγt and Foxp3 mRNAs were measured in the serum of the 2 patient groups and the relationship between factors were correlated with the severity score of MG. The relationship between the levels of 1,25(OH)₂D₃ and the relative expressions of RORγt and Foxp3 mRNAs was determined. Results: There were no differences between groups regarding patient’s baseline data. 1,25(OH)₂D₃ and RORγt and Foxp3 mRNAs are differentially expressed in the MG group and the control group (p < 0.05). QMG score is negatively correlated with the expression level of peripheral blood 1,25(OH)₂D₃ and Foxp3 mRNA (r = −0.797, −0.543; p < 0.01) and positively correlated with the relative expression level of RORγt mRNA (r = 0.539; p < 0.01). 1,25(OH)₂D₃ expression level was negatively correlated with the relative expression of RORγt mRNA (r = −0.559; p < 0.01) and positively correlated with the relative expression of Foxp3 mRNA (r = 0.390; p < 0.01). Conclusions: The levels of 1,25(OH)₂D₃ were shown to be lower in patients with MG compared to normal controls. The observed low levels of 1,25(OH)₂D₃ may lead to changes in the expression of RORγt and Foxp3 mRNAs involved in MG.

Exosomes derived from thymic stromal lymphopoietin-treated dendritic cells regulate T helper 17/regulatory T cell differentiation via miR-21/Smad7 axis

Thymic stromal lymphopoietin (TSLP) is associated with fungal keratitis. This work aims to investigate whether TSLP can regulate T helper (Th) 17 and regulatory T cell (Treg) differentiation. We separated dendritic cells (DCs) from peripheral blood of healthy volunteers. DCs were treated with TSLP to activate DCs, and exosomes were obtained. CD+ T cells were incubated with exosomes from TSLP-treated DCs. We found that exosomes from TSLP-treated DCs notably promoted the proportions of Th17 cells and inhibited the proportions of Tregs in the CD4+ T cells. Moreover, exosomes from TSLP-treated DCs enhanced the expression of retinoid-related orphan receptor γt (RORγt) and interleukin 17 (IL-17), and repressed the expression of forkhead box protein P3 (Foxp3) and interleukin 10 (IL-10) in the CD4+ T cells. Furthermore, miR-21 was highly expressed in exosomes from TSLP-treated DCs. Exosomes from TSLP-treated miR-21-silenced DCs promoted Treg differentiation and suppressed Th17 differentiation. Smad7 up-regulation repressed Th17 differentiation and enhanced Treg differentiation, which was abolished by miR-21 overexpression. Smad7 overexpression rescued the effect of exosomes from TSLP-treated DCs on Th17/Treg differentiation. In conclusion, our article confirms that TSLP induces DCs to deliver miR-21 by secreting exosomes, and thus miR-21 regulates Th17/Treg differentiation by inhibiting Smad7. Thus, this work further reveals the biological role of miR-21 in fungal keratitis.

Asymmetric Synthesis of a 5,6,7,8-Tetrahydro-1,6-naphthyridine Scaffold Leading to Potent Retinoid-Related Orphan Receptor γt Inverse Agonist TAK-828F.

An asymmetric synthesis of the tetrahydronaphthyridine scaffold of TAK-828F as a RORγt inverse agonist has been developed. The synthesis features a newly discovered atom-economical protocol for Heck-type vinylation of chloropyridine using ethylene gas, an unprecedented formation of dihydronaphthyridine directly from 2-vinyl-3-acylpyridine mediated by ammonia, and a ruthenium-catalyzed enantioselective transfer hydrogenation as key steps. This represents the first example of the enantioselective synthesis of a 5,6,7,8-tetrahydro-1,6-naphthyridine compound. The new synthesis is also free of chromatography or distillation purification processes and therefore qualifies for extension to large-scale manufacture.

Lactobacillus rhamnosus 2016SWU.05.0601 regulates immune balance in ovalbumin-sensitized mice by modulating expression of the immune-related transcription factors and gut microbiota.

Probiotics regulate host immune balance, which may reduce immune-related diseases. The effects and mechanisms of Lactobacillus rhamnosus 2016SWU.05.0601 (Lr-0601) on the immune response in ovalbumin (OVA)-sensitized mice were explored. Lr-0601 reduced serum immunoglobulin (Ig)E and OVA-IgE and attenuated the alteration in lung pathology in OVA-sensitized mice. Lr-0601 blocked OVA-induced up-regulation in serum T helper (Th) 2 and Th17 cytokines but increased the serum levels of Th1 and regulatory T (Treg) cytokines in OVA-sensitized mice. OVA also markedly reduced the protein levels of spleen T-box transcription factor and forkhead/winged helix transcription factor p3, leading to the reduced mRNA expression of interferon-γ and interleukin (IL)-10. By contrast, OVA markedly increased the protein expression of spleen GATA-binding protein 3 and retinoid-related orphan receptor γt, as well as the mRNA expression of spleen IL-4 and IL-17. These changes induced by OVA were reversed by Lr-0601. Moreover, Lr-0601 helped alleviate OVA-induced intestinal microbiota dysbiosis. A correlation was found between specific genera and immune-associated cytokines. The combined results indicate that Lr-0601 modulated the balance of Th1/Th2 and Treg/Th17 in OVA-sensitized mice, which was associated with the regulation of immune-related transcription factors and gut microbiota. Lr-0601 can potentially be used as a probiotic for preventing immune-related diseases. © 2020 Society of Chemical Industry.