RORγt Protein Research Articles

Protective Effect of Thalidomide on 2,4,6-Trinitrobenzenesulfonic Acid-Induced Experimental Colitis in Rats via the Inhibition of T Helper 17 Cells

Objective To observe the effects of thalidomide on 2,4,6-trinitrobenzenesulfonic acid- (TNBS-) induced experimental colitis in rats and to explore the possible mechanism of thalidomide in the treatment of CD. Methods Forty SD rats were randomly assigned into a healthy control group and TNBS-induced colitis groups, including an untreated TNBS-induced colitis group, a low-dose thalidomide group, and a high-dose thalidomide group, with 10 rats in each. After 7 days, the disease activity index (DAI), colon macroscopic damage index (CMDI), and tissue damage index (TDI) were evaluated. The colonic protein and mRNA expression levels of interleukin-6 (IL-6), IL-17, IL-23, and retinoic acid receptor-related orphan nuclear receptor gamma t (RORγt) were determined using immunohistochemistry, western blot, and qRT-PCR. Results Relative to the untreated TNBS-induced colitis group, the DAI, CMDI, and TDI were all reduced following the administration of thalidomide. Analytical testing (immunohistochemistry, western blot, and qRT-PCR) shows that IL-6, IL-17, IL-23, and RORγt protein and mRNA expression levels were significantly reduced by thalidomide (p < 0.05 for all) and that these levels were significantly lower in the high-dose thalidomide group than in the low-dose thalidomide group (p < 0.05 for all). Conclusions Thalidomide effectively alleviated the symptoms and intestinal inflammatory injury induced by TNBS in rats, the effect of which was dose-dependent. The underlying mechanism may be a reduction in the expression levels of IL-6, IL-17, IL-23, and RORγt in colonic tissue and then subsequent inhibition of the differentiation and function of Th17 cells, thus further alleviating the intestinal inflammatory response.

The effect of 6-gingerol on inflammatory response and Th17/Treg balance in DSS-induced ulcerative colitis mice.

BackgroundUlcerative colitis (UC) is a non-specific chronic intestinal inflammatory disease with unclear etiology. Previous studies have suggested that the imbalance of Treg/Thl7 cells may be involved in the development of UC. It was found that 6-gingerol can alleviate the intestinal inflammatory damage and improve the weight loss of colitis mice. However, whether 6-gingerol can regulate the balance of Th17/Treg cells and inhibit the intestinal inflammatory response remains to be clarified.MethodsIn this study, a dextran sulfate sodium (DSS)-induced colitis mouse model was established, and the effects of 6-gingerol on cytokines and the balance of Th17/Treg cells were observed usingserial assays, including enzyme-linked immunosorbent assay (ELISA), quantitative real time-polymerase chain reaction (qPCR), and Western blotting.ResultsDSS caused the damage of bowel tissue and a 100% weight loss rate in colitis mice. The treatment of 6-gingerol can significantly relieve bowel damage and reduce incidence of weight loss to 16.7% at a low or high dose (P<0.05), which was similar to the therapeutic effect of mesalazine. It was found that DSS can up-regulate the mRNA levels of IL-6 and IL-17 in serum (by qPCR), and the serum and bowel levels of IL-6 and IL-17 (by ELISA); these levels were significantly different from those of the blank group (P<0.05). Furthermore, 6-gingerol was found to inhibit the increase of mRNA levels and serum and bowel levels of IL-6 and IL-17 induced by DSS, which is similar with mesalazine. It was also found that DSS can down-regulate the mRNA level of IL-10 in serum, along with the serum and bowel level of IL-10, with this being significantly different from the levels of the blank group (P<0.05). 6-gingerol could also inhibit the decrease of mRNA levels and serum and bowel levels of IL-10 induced by DSS, which is also similar to mesalazine. In addition, DSS could increase Th17 cell count and decrease Treg cell count in blood, with significant difference from that of the blank group (P<0.05). 6-gingerol could significantly (P<0.05) inhibit the increase of Th17 cells and the decrease of Treg cells induced by DSS, which is similar to the effect of mesalazine. The detection of expression levels of transcription factors RORγT for Th17 and FOXP3 for Treg at both mRNA and protein levels showed that DSS can up-regulate the mRNA and protein levels of RORγT, and down-regulate the mRNA and protein levels of FOXP3. Furthermore, 6-gingerol could significantly (P<0.05) inhibit the up-regulation of RORγT mRNA and protein, and the down-regulation of FOXP3 mRNA and protein induced by DSS, which is similar to the effect of mesalazine.Conclusions6-gingerol showed efficacy in the treatment of DSS-induced UC in mice, by regulating the cell balance of Th17/Treg, and by relieving inflammatory responses both systematically and locally.

Artesunate attenuates 2, 4-dinitrochlorobenzene-induced atopic dermatitis by down-regulating Th17 cell responses in BALB/c mice

Steroidal agent is a standard clinical treatment of atopic dermatitis; however, have serious side effects. Artesunate is reported to exhibit anti-inflammatory properties although its effect on atopic eczema remains unknown. We investigated the therapeutic effects and possible mechanism of systemic artesunate on DNCB-induced atopic dermatitis in a BALB/c mouse model. To ascertain artesunate (5 and 10 mg/kg) efficacy, skin dermatitis severity and ear, spleen, and lymph node weight were evaluated. Skin tissue mRNA and protein expression and serum cytokine levels were examined. Artesunate significantly improved atopic dermatitis symptoms, decreasing the dermatitis score, ear weight difference, spleen weight, and lymph node weight compared with those following DNCB treatment. Artesunate reduced ear and skin epidermal thickness and mast cell infiltration, as determined using hematoxylin-eosin and toluidine blue staining, respectively. The basal level of IgE (287.67 ± 70.41 ng/ml) and TNF-α (19.94 ± 3.98 pg/ml) were Significantly elevated by DNCB (IgE: 1273.23 ± 176.53 ng/ml; TNF-α: 57.53 ± 3.87 pg/ml), while markedly been suppressed in the treatment group (AS-L: IgE: 1100.25 ± 135.32 ng/ml; TNF-α: 38.47 ± 3.26 pg/ml; AS-H: IgE: 459.46 ± 74.75 ng/ml; TNF-α: 24.38 ± 3.85 pg/ml). Among Th17 cell-related factors, DNCB treatment increased mRNA expression of IL-6, IL-17, IL-23, STAT3, and ROR-γt, but reduced TGF-β and SOCS 3; While artesunate reverse these changes. Compared with the model group, artesunate promoted SOCS3 protein and significantly inhibited ROR-γt protein and STAT3 phosphorylation. Thus, artesunate attenuates DNCB-induced atopic dermatitis by inhibiting the release of inflammatory cytokines and downregulating Th17 cell responses in atopic dermatitis mice.

The imbalance of Treg/Th17 cells induced by perinatal bisphenol A exposure is associated with activation of the PI3K/Akt/mTOR signaling pathway in male offspring mice

Bisphenol A (BPA) can inhibit the differentiation and function of regulatory T cells (Treg), and affect the balance of helper T cell (Th) 1/Th2, therefore, the immunotoxicity of BPA has attracted widespread attention in recent years, but its mechanism is not clear. The main aim of this study was to explore the regulatory mechanism of the PI3K/Akt/mTOR signaling pathway in the context of perinatal exposure to BPA-induced Treg/Th17 imbalance in male offspring mice through a combination of in vivo and in vitro methods. Our results showed that perinatal exposure to BPA could increase the number of Th17 cells while decreasing Treg cell numbers, which was consistent with the expression levels of up-regulation of RORγt protein and a down-regulation FOXP3 protein in the splenocytes of the male offspring mice. BPA could activate the PI3K/Akt/mTOR signaling pathway and increase the inflammatory response, as evidenced by higher serum IL-17 and TNF-α levels by inducing the activation of the AhR and TLR4/NF-κB signaling pathways. Moreover, our results also supported the hypothesis whereby the Treg/Th17 imbalance, induced by perinatal exposure to BPA, was associated with the activation of PI3K/Akt/mTOR signaling in vitro-cultured peripheral blood mononuclear cells by using rapamycin as an inhibitor of mTOR.

Th17 Cell Subset levels in Peripheral Blood of Patients with Aplastic Anemia and Their Clinical Significance

To study the level and clinical value of Th17 cell subset in the peripheral blood of the patients with aplastic anemia (AA). Eighty-five patients with aplastic anemia in our hospital from May 2017 to February 2018 were selected. Among them, 51 patients with poor curative effect were enrolled in group A. and the 34 patients with good curative effect were enrolled in group B, the 35 healthy persons were selected as controls. The levels of serum IL-17, IL-6, IL-4 and IFN-β were analyzed by ELISA; the levels of peripheral blood Th17 cell subset were analyzed by flow cytometry; the expression levels of RORγt and STAT3 mRNA in lymphocytes were analyzed by RT-PCR; the expression levels of RORγt and STAT3 protein in lymphocytes were analyzed by Western blot. There was no significant difference in sex, age, WBC count and complications between group A and group B (P＞0.05). Non-severe aplastic anemia (NSAA) in group B accounted for 23 cases, and the NSAA ratio (23/34) was significantly higher than that in group A (20/51) (P＜0.05). The Hb level (86.25±7.9 g/L) and Plt count (54.7 ± 6.3×109/L) in group B were significantly higher than those in group A (P＜0.05). ELISA showed that the levels of IL-17 and IL-6 in group A were significantly higher than those in control group (P＜0.05); the levels of IL-17 and IL-6 in group B were significantly lower than those in group A (P＜0.05); the levels of IL-4 and IFN-β in group A were significantly lower than those in control group (P＜0.05); the levels of IL-4 and IFN-βin group B were significantly higher than those in group A (P＜0.05). Flow cytometry showed that the proportion of Th17 cells in peripheral blood of group A was higher than that of group B (P＜0.05). RT-PCR showed that the levels of RORγt and STAT3 mRNA in group A were significantly higher than those in control group (P＜0.05), and the mRNA levels of RORγt and STAT3 in group B were significantly lower than those in group A (P＜0.05). Western blot showed that the protein levels of RORγt and STAT3 in group A were significantly higher than those in control group (P＜0.05), and the protein levels of RORγt and STAT3 in group B were significantly lower than those in group A (P＜0.05). The Th17 cells in peripheral blood of AA patients increase, and the inflammatory reaction in the patients are enhanced. It may be related with the rise of RORγt and STAT3 gene expression, which provides a research direction for clinical treatment of AA.

Th17 Cell Subset levels in Peripheral Blood of Patients with Aplastic Anemia and Their Clinical Significance

To study the level and clinical value of Th17 cell subset in the peripheral blood of the patients with aplastic anemia (AA). Eighty-five patients with aplastic anemia in our hospital from May 2017 to February 2018 were selected. Among them, 51 patients with poor curative effect were enrolled in group A. and the 34 patients with good curative effect were enrolled in group B, the 35 healthy persons were selected as controls. The levels of serum IL-17, IL-6, IL-4 and IFN-β were analyzed by ELISA; the levels of peripheral blood Th17 cell subset were analyzed by flow cytometry; the expression levels of RORγt and STAT3 mRNA in lymphocytes were analyzed by RT-PCR; the expression levels of RORγt and STAT3 protein in lymphocytes were analyzed by Western blot. There was no significant difference in sex, age, WBC count and complications between group A and group B (P＞0.05). Non-severe aplastic anemia (NSAA) in group B accounted for 23 cases, and the NSAA ratio (23/34) was significantly higher than that in group A (20/51) (P＜0.05). The Hb level (86.25±7.9 g/L) and Plt count (54.7 ± 6.3×109/L) in group B were significantly higher than those in group A (P＜0.05). ELISA showed that the levels of IL-17 and IL-6 in group A were significantly higher than those in control group (P＜0.05); the levels of IL-17 and IL-6 in group B were significantly lower than those in group A (P＜0.05); the levels of IL-4 and IFN-β in group A were significantly lower than those in control group (P＜0.05); the levels of IL-4 and IFN-βin group B were significantly higher than those in group A (P＜0.05). Flow cytometry showed that the proportion of Th17 cells in peripheral blood of group A was higher than that of group B (P＜0.05). RT-PCR showed that the levels of RORγt and STAT3 mRNA in group A were significantly higher than those in control group (P＜0.05), and the mRNA levels of RORγt and STAT3 in group B were significantly lower than those in group A (P＜0.05). Western blot showed that the protein levels of RORγt and STAT3 in group A were significantly higher than those in control group (P＜0.05), and the protein levels of RORγt and STAT3 in group B were significantly lower than those in group A (P＜0.05). The Th17 cells in peripheral blood of AA patients increase, and the inflammatory reaction in the patients are enhanced. It may be related with the rise of RORγt and STAT3 gene expression, which provides a research direction for clinical treatment of AA.

Abnormal differentiation of regulatory T cells and Th17 cells induced by perinatal bisphenol A exposure in female offspring mice

Bisphenol A (BPA) is an environmental estrogen widely exposed to human beings, and there are more studies on its reproductive toxicity, endocrine disruption and neurobehavioral disorders. Recent few studies have found that BPA has immunotoxicity, and its mechanism is not clear. Therefore, the effects of BPA on immune system have attracted extensive attention. The aim of this study was to investigate the effect and mechanism of perinatal exposure to BPA on regulatory T cells (Treg) and Th17 cells in female offspring mice. Twenty-one pregnant C57BL/6 mice were randomly divided into three groups: a control group, low-dose BPA (0.2 µg/mL) and high-dose BPA (2.0 µg/mL) exposure group. All received BPA exposure via drinking water from gestational day 6 to the end of lactation. Female offspring were fed a normal diet and drinking water for 1 month. The percentages of Treg and Th17 cells, the levels of Foxp3 and RORγt protein and IL-17 and TGF-β from spleen tissue or blood were measured in female offspring. The percentage of Treg cells and levels of Foxp3 protein decreased, while the percentage of Th17 cells and levels of RORγt protein increased, which showed a dose–effect relationship. The levels of serum TGF-β were significantly lower and the levels of serum IL-17 were statistically higher in BPA-exposed female offspring compared with controls (P 0.05). BPA exposure during pregnancy and lactation could cause abnormal differentiation and function of Treg and Th17 cells in female offspring mice, which was associated with down-regulated Foxp3 and up-regulated RORγt protein, respectively. Our findings indicated that BPA exposure during early development may play an important role in the development of autoimmune diseases later.

Design and Synthesis of Conformationally Constrained RORγt Inverse Agonists.

Retinoic-acid-related orphan receptor γt (RORγt) inverse agonists could be used for the treatment of autoimmune diseases. Previously, we reported a novel quinazolinedione 1 a with a flexible linear linker as a novel RORγt inverse agonist. A U-shaped conformation in the complex structure of 1 a with RORγt protein was confirmed. Further improvement of the pharmacokinetic (PK) profiles was required because of the low drug exposure in mice upon oral administration (mouse AUC of 1 a: 27 ng ⋅ h ⋅ mL-1 at 1 mg ⋅ kg-1 , p.o.). To improve the PK profiles, conformationally constrained U-shaped scaffolds were investigated. As a result, morpholine analogues with improved PK profiles and high potency were successfully identified. The substituent at the N1 position of the quinazoline moiety was also modified, leading to an enhancement of reporter activity. Consequently, compound 43 (N2 -(3-chloro-4-cyanophenyl)-N4 -(3-(cyclopropylmethyl)-1-isopropyl-2,4-dioxo-1,2,3,4-tetrahydroquinazolin-6-yl)morpholine-2,4-dicarboxamide) exhibited improved drug exposure (mouse AUC: 1289 ng ⋅ h ⋅ mL-1 at 1 mg ⋅ kg-1 , p.o.). In addition, suppression of IL-17A gene expression by IL-23 stimulation in a mouse pharmacodynamics model was observed for 43. The conformation of 43 with RORγt protein was also confirmed as U-shape by X-ray co-crystal structure analysis. The key interaction that boosts potency is also discussed.

Immunomodulatory effect of human umbilical cord mesenchymal stem cells on T lymphocytes in rheumatoid arthritis

Rheumatoid arthritis (RA) is an autoimmune disease which is lack of effective therapies. Abnormal activation, proliferation, and differentiation of T lymphocytes are closely related to RA. Mesenchymal stem cells (MSCs) can be used for RA treatment due to their immunoregulatory effects. However, the specific molecular mechanisms have not been fully elucidated and the therapeutic effect has been inconsistent. This study investigated the immunomodulatory effect of human umbilical cord MSCs (hUCMSCs) on T lymphocytes in collagen-induced arthritis (CIA) rats and RA patients to clarify the possible mechanism of hUCMSCs in RA treatment. The effects of hUCMSCs on arthritis index, radiological and synovial pathological changes, T lymphocyte proliferation and apoptosis, RORγt and Foxp3 expression, Th17 and Treg cell ratios, and IL-17 and TGF-β levels were assessed in CIA rats. Further, we verified the effect of hUCMSCs in RA patients, and compared the effect of hUCMSCs with that of hUCMSC derived extracellular vesicles (EVs). The results showed that hUCMSCs inhibited the proliferation and promoted apoptosis in T lymphocytes, downregulated RORγt mRNA and protein expression, decreased Th17 cell ratio, upregulated Foxp3 mRNA and protein expression, and increased Treg cell ratio in the spleen. Furthermore, they downregulated RORγt and Foxp3 expression in the joints, and inhibited IL-17 and promoted TGF-β expression in the serum, thereby improving arthritis, delaying radiological progression, and inhibiting synovial hyperplasia in CIA rats. In vitro the effects of hUCMSCs and EVs were consistent with those in vivo. Therefore, hUCMSCs may be expected to serve as a new therapy for RA.

Deregulation of Long Intergenic Non-coding RNAs in CD4+ T Cells of Lamina Propria in Crohn's Disease Through Transcriptome Profiling.

The aetiology of Crohn's disease [CD] involves immune dysregulation in a genetically susceptible individual. Genome-wide association studies [GWAS] have identified 200 loci associated with CD, ulcerative colitis, or both, most of which fall within non-coding DNA regions. Long non-coding RNAs [lncRNAs] regulate gene expression by diverse mechanisms and have been associated with disease activity in inflammatory bowel disease. However, disease-associated lncRNAs have not been characterised in pathogenic immune cell populations. Terminal ileal samples were obtained from 22 CD patients and 13 controls. RNA from lamina propria CD4+ T cells was sequenced and long intergenic non-coding RNAs [lincRNAs] were detected. Overall expression patterns, differential expression [DE], and pathway and gene enrichment analyses were performed. Knockdown of novel lincRNAs XLOC_000261 and XLOC_000014 was performed. Expression of Th1 or Th17-associated transcription factors, T-bet and RORγt, respectively, was assessed by flow cytometry. A total of 6402 lincRNAs were expressed, 960 of which were novel. Unsupervised clustering and principal component analysis showed that the lincRNA expression discriminated patients from controls. A total of 1792 lincRNAs were DE, and 295 [79 novel; 216 known] mapped to 267 of 5727 DE protein-coding genes. The novel lincRNAs were enriched in inflammatory and Notch signalling pathways [p <0.05]. Furthermore, DE lincRNAs in CD patients were more frequently found in DNA regions with known inflammatory bowel disease [IBD]-associated loci. The novel lincRNA XLOC_000261 negatively regulated RORγt expression in Th17 cells. We describe a novel set of DE lincRNAs in CD-associated CD4+ cells and demonstrate that novel lincRNA XLOC_000261 appears to negatively regulate RORγt protein expression in Th17 cells.

Acupoint injection improves allergic rhinitis by balancing Th17/Treg in allergic rhinitis rats

To observe the effect of acupoint injection on expression of fork head/winged helix protein 3 (Foxp3), retinoic acid-related orphan receptor γt (RORγt) in nasal mucosa and serum interleukin-17 (IL-17) level in allergic rhinitis (AR) rats, so as to explore its mechanism underlying improvement of AR in terms of balancing Th17/Treg. Thirty-two SD rats (half male and half female) were randomized into normal control, AR model, acupoint injection and non-acupoint injection groups (n＝8 in each group). The AR model was established by ovalbumin sensitization. In the acupoint injection group, "Yintang" (EX-HN3) and bilateral "Yingxiang"(LI20) were selected for injection of mixture solution of dexamethasone (DEX) and transfer factor and lidocaine (0.1 mL/acupoint), once every 3 days for a total of 4 times. The non-acupoints, located at the mid-point between the "Houhai" (GV1) and "Huantiao"(GB30) on the bilateral hips and the sites 5 cm inferior to the axillary were injected with the same dose of mixture solution as that in the acupoint injection. The AR severity was assessed by cumulative quantification scoring methods (including the numbers of nose-catching and sneezes, and the amount of nasal secretions in 30 min). The expressions of Foxp3 and RORγt in the nasal mucosa were detected by immunohistochemistry. The serum IL-17 content was detected by enzyme linked immunosorbent assay (ELISA)．. The AR symptom score and serum IL-17 content were significantly higher in the AR model group than those in the normal control group (P<0.05), and significantly down-regulated in the acupoint injection group (not in the non-acupoint group) relevant to the AR model group (P<0.05). Following modeling, the expression levels of nasal Foxp3 protein was significantly down-regulated while that of RORγt protein markedly up-regulated in the AR model group relevant to the normal control group (P<0.05), indicating an imbalance between Foxp3 and RORγt activity(P<0.05). After EA intervention, the increased expression of Foxp3 and the down-regulated expression of RORγt were revised in the acupoint injection group (P<0.05) but not in the non-acupoint group (P>0.05). The percentage of the Foxp3 positive cells and the ratio of Foxp3/RORγt were negatively correlated with the AR symptom score(P<0.05), the expression of RORγt and the content of IL-17 were positively correlated with the symptom score (P<0.05)．. Acupoint injection is able to improve symptoms of RA rats, which may be related with its function in up-regulating the level of nasal mucosal Foxp3 and suppressing the levels of nasal RORγt and serum IL-17 to correct the imbalance of Th17/Treg.

DAPTA, a C-C chemokine receptor 5 (CCR5) antagonist attenuates immune aberrations by downregulating Th9/Th17 immune responses in BTBR T+ Itpr3tf/J mice

Autism spectrum disorder (ASD) is a prevalent neurodevelopmental disorder characterized by deficits in social interaction, communication, and repetitive behaviors. BTBR T+ Itpr3tf/J (BTBR) mice, a preclinical autistic model featuring ASD symptoms as defined by social relations, was used in this study. We evaluated the potentially protective effect of D-Ala-peptide T-amide (DAPTA), a selective C-C chemokine receptor 5 (CCR5) antagonist, in BTBR mice. CCR5 is considered a potential therapeutic target in different neurodegenerative disorders. BTBR and C57 mice were intraperitoneally (i.p) treated with the DAPTA (0.01 mg/kg, i.p, once daily) for 7 days. We examined the effect of DAPTA by evaluating marble burying and administering repetitive behavior tests. We employed flow cytometry to assess the effect of DAPTA on CCR5+, CD4+CCR5+, CCR5+IL-6+, CCR5+IL-9+, CCR5+IL-17A+, CCR5+RORγT+, CCR5+IL-10+, and CCR5+Foxp3+ in spleen cells. We further explored the effects of DAPTA on IL-6, IL-9, IL-17A, RORγT, IL-10, and Foxp3 protein and mRNA expression levels in the brain tissues. DAPTA administration significantly decreased marble burying and repetitive behavior in BTBR mice. Additionally, DAPTA treatment inhibited CCR5+, CD4+CCR5+, CCR5+IL-6+, CCR5+IL-9+, CCR5+IL-17A+, CCR5+RORγT+, and upregulated CCR5+IL-10+, and CCR5+Foxp3+ production. We further observed that DAPTA downregulated IL-6, IL-9, IL-17A, and RORγT, and increased IL-10 and Foxp3 protein and mRNA expression. Therefore, our results suggest that DAPTA administration represents a potential treatment strategy for patients with ASD.

LncRNA-MEG3 functions as a competing endogenous RNA to regulate Treg/Th17 balance in patients with asthma by targeting microRNA-17/ RORγt

BackgroundTreg/Th17 imbalance plays an essential role in the pathogenesis of asthma. Disordered LncRNAs were observed in asthma, however, whether LncRNAs can regulate the Treg/Th17 balance and its mechanism still needs to be investigated. MethodsMicroarrays were performed to identify the differentially expressed lncRNAs and microRNAs in peripheral blood CD4 + T cells from patients with asthma and healthy controls. Bioinformatical evidence was used to select candidate lncRNAs and microRNAs which may involve in regulation of Treg/Th17 balance. The function of LncRNA-MEG3 and microRNA-17 on the alteration of the CD4 + T cell population were determined in vitro experiments. Meanwhile, the regulatory effect of LncRNA-MEG3 and microRNA-17 on RORγt or Foxp3 was estimated. The interaction of LncRNA-MEG3 with microRNA-17 was confirmed by dual luciferase reporter assay and RNA pull-down. Results25 lncRNAs and 19 microRNAs were selected as candidate genes which differentially expressed in CD4 + T cells from patients with asthma compared with healthy controls and had potential to control Treg/Th17 balance by regulating RORγt or Foxp3. Alternation of LncRNA-MEG3 changed the function and increased the percentage of Th17. LncRNA-MEG3 could regulate the RORγt mRNA and protein level. LncRNA-MEG3 could inhibit the level of microRNA-17 as a competing endogenous RNA (ceRNA). microRNA-17 suppressed Th17 though targeting RORγt directly. ConclusionLncRNA-MEG3 can sponge microRNA-17 as a ceRNA, thereby regulating RORγt and ultimately affecting Treg/Th17 balance in asthma. The lncRNA/microRNA axis may have potential application in clinical treatment and diagnosis of the disease.

Cholesterol Unbound RORγt Protein Enables a Sensitive Inverse Agonist Screening.

The retinoic acid-related orphan receptor gamma T (RORγt) plays an important role in Th17 cell proliferation and functionality. Thus, RORγt inverse agonists are thought to be potent therapeutic agents for Th17-mediated autoimmune diseases, such as rheumatoid arthritis, asthma, inflammatory bowel disease, and psoriasis. Although RORγt has constitutive activity, it is recognized that the receptor is physiologically regulated by various cholesterol derivatives. In this study, we sought to identify RORγt inverse agonists through a high-throughput screening campaign. To this end, we compared an apo-RORγt protein from Escherichia coli and a cholesterol-bound RORγt protein from insect cells. The IC50 of the known RORγt inverse agonist TO901317 was significantly lower for the apoprotein than for the cholesterol-bound RORγt. Through high-throughput screening using a fluorescence-based cholesterol binding assay with the apoprotein, we identified compound 1 as a novel cholesterol-competitive RORγt inverse agonist. Compound 1 inhibited the RORγt-TopFluor cholesterol interaction, coactivator recruitment, and transcriptional activity of RORγt. Cell-based reporter gene assay demonstrated that compound 1 showed higher potency by lipid depletion treatment. Collectively, our findings indicate that eliminating cholesterol from the RORγt protein is suitable for sensitive high-throughput screening to identify RORγt inverse agonists.

Pim-1 inhibitor attenuates trinitrobenzene sulphonic acid induced colitis in the mice

Pim-1 kinase has been implicated in inflammatory bowel disease (IBD). This study aimed to evaluate the application of Pim-1 inhibitor (PIM-Inh) for the treatment of IBD. Mouse model of IBD was established by the treatment with trinitrobenzene sulphonic acid (TNBS). The results showed that disease activity index score was significantly decreased, colon length was significantly increased while Wallace score and pathological score were significantly decreased after PIM-Inh treatment compared to TNBS model group. In addition, GATA3 and ROR-γt mRNA and protein levels significantly increased but Foxp3 mRNA and protein levels significantly decreased in mice with TNBS treatment compared to mice without TNBS treatment. Administration of PIM-Inh caused significant decreases in GATA3, T-bet and ROR-γt mRNA and protein levels as well as significant increases in FOXP3 mRNA and protein levels. In conclusion, our data suggest that Pim-1 kinase inhibitor could attenuate IBD by promoting T-cell differentiation into Foxp3+ regulatory T-cells and is a promising agent for IBD therapy.

FOXP3, RORγt and IL-10 cytokine profile in chronic heart failure.

Distinct subsets of T cells play crucial regulatory roles in inflammatory processes of chronic heart failure (CHF). Retinoic acid receptor-related orphan receptor-γt (Ror-γt) and Forkhead box P3 (Foxp3) have been defined as the "master regulators" of Th17 cells and Treg cells, respectively. At the same time, anti-inflammatory cytokines such as IL-10 may neutralize inflammation in CHF. The current study was designed to compare FOXP3, RORγt and IL-10 protein expression in the blood and IL-10 in supernatant PBMCs in CHF patients versus normal subjects. Our study population consisted of 42 patients with CHF in four different function classes and 42 healthy subjects who served as controls. RNA extraction and cDNA synthesis was performed and mRNA expression for genes FOXP3, RORγt, IL-10 was determined by RT-PCR. The amount of IL-10 protein in supernatant of PBMCs was measured by ELISA technique. There was no significant difference in FOXP3, RORγt, IL-10 protein expression and supernatant PBMCs IL-10 in CHF patients as compared to control. The level of Foxp3 was significantly lower in CHF patients with ischemic vs non-ischemic cause (p = 0.04). Although inflammation plays a central role in the pathophysiology of CHF, the roles of FOXp3, RORγt, and IL-10 remain to be determined (Tab. 3, Ref. 33).

Identification of novel quinazolinedione derivatives as RORγt inverse agonist

Novel small molecules were synthesized and evaluated as retinoic acid receptor-related orphan receptor-gamma t (RORγt) inverse agonists for the treatment of inflammatory and autoimmune diseases. A hit compound, 1, was discovered by high-throughput screening of our compound library. The structure–activity relationship (SAR) study of compound 1 showed that the introduction of a chlorine group at the 3-position of 4-cyanophenyl moiety increased the potency and a 3-methylpentane-1,5-diamide linker is favorable for the activity. The carbazole moiety of 1 was also optimized; a quinazolinedione derivative 18i suppressed the increase of IL-17A mRNA level in the lymph node of a rat model of experimental autoimmune encephalomyelitis (EAE) upon oral administration. These results indicate that the novel quinazolinedione derivatives have great potential as orally available small-molecule RORγt inverse agonists for the treatment of Th17-driven autoimmune diseases. A U-shaped bioactive conformation of this chemotype with RORγt protein was also observed.

Fibrinogen-like protein-2 causes deterioration in cardiac function in experimental autoimmune myocarditis rats through regulation of programmed death-1 and inflammatory cytokines.

Programmed death-1 (PD-1) plays an important role in protecting against inflammation and myocyte damage in T-cell-mediated myocarditis. To understand whether fibrinogen-like protein-2 (FGL2) can affect the role of the PD-1/PD-L1 pathway in experimental autoimmune myocarditis (EAM), we investigated cardiac function in EAM rats over-expressing FGL2. Over-expression of FGL2 significantly decreased PD-1 and deteriorated cardiac function in rats with autoimmune myocarditis. Histopathology revealed increased inflammatory cell infiltrate in EAM-FGL2 rats compared with the control groups (EAM, EAM-GFP and NC). Notably, transcription factor forkhead box P3 (Foxp3) and retinoic acid-related orphan receptor γt (RORγt) protein and mRNA levels were statistically (P<0·05) increased in EAM rats. We also found that interferon-γ, interleukin-6, interleukin-17 and brain natriuretic peptide levels were profoundly increased in serum of FGL2 over-expressing EAM rats. Hence, FGL2 plays an important role in the pathogenesis of autoimmune myocarditis that also involves the PD-1/PD-L1 pathway. Our findings may provide novel therapeutic targets for the treatment of immune-induced heart injury.

RORγt-expressing cells attenuate cardiac remodeling after myocardial infarction.

AimsRetinoic acid receptor-related orphan nuclear receptor γt (RORγt) is a transcriptional factor responsible for IL-17-producing T-cell differentiation. Although it was demonstrated that RORγt plays essential roles in the onset of autoimmune myocarditis, pathophysiological significance of RORγt in cardiac remodeling after myocardial infarction (MI) remains to be fully elucidated.Methods and resultsMI was generated by ligating coronary artery. The expression of RORγt and IL-17A transcripts increased in murine hearts after MI. Additionally, immunohistochemical staining revealed that RORγt-expressing cells infiltrated in the border zone after MI. Flow cytometric analysis showed that RORγt-expressing cells were released from the spleen at day 1 after MI. Though RORγt-expressing cells in spleen expressed γδTCR or CD4, γδTCR+ cells were major population of RORγt-expressing cells that infiltrated into post-infarct myocardium. To address the biological functions of RORγt-expressing cells in infarcted hearts, we used mice with enhanced GFP gene heterozygously knocked-in at RORγt locus (RORγt+/- mice), which physiologically showed reduced expression of RORγt mRNA in thymus. Kaplan-Meier analysis showed that MI-induced mortality was higher in RORγt+/- mice than wild-type (WT) mice. Masson’s trichrome staining demonstrated that cardiac injury was exacerbated in RORγt+/- mice 7 days after MI (Injured area: RORγt+/-; 42.1±6.5%, WT; 34.0±3.7%, circumference of injured myocardium: RORγt+/-; 61.8±4.8%, WT; 49.6±5.1%), accompanied by exacerbation of cardiac function (fractional shortening: RORγt+/-; 32.9±2.9%, WT; 38.3±3.6%). Moreover, immunohistochemical analyses revealed that capillary density in border zone was significantly reduced in RORγt+/- mice after MI, compared with WT mice, associated with the reduced expression of angiopoietin 2. Finally, the mRNA expression of RORγt, IL-17A, IL-17F and IL-23 receptor (IL-23R) mRNA and protein expression of IL-10 were decreased in RORγt+/- hearts.ConclusionsHeterozygous deletion of RORγt gene resulted in aggravated cardiac remodeling, accompanied by reduced capillary density, after MI, suggesting that RORγt-expressing cells contribute to tissue repair in infarcted myocardium.

Adenosine A2A receptor modulates neuroimmune function through Th17/retinoid-related orphan receptor gamma t (RORγt) signaling in a BTBR T+ Itpr3tf/J mouse model of autism

Autism spectrum disorder (ASD) is a complex neurodevelopmental disorder characterized by abnormal social interactions, repetitive behaviors that impair social communication, and circumscribed interests. BTBR T+tf/J (BTBR) inbred mice are generally used as a model for ASD, as they show repetitive behaviors and social deficits that resemble signs of ADS in humans. Adenosine A2A receptors (A2ARs) are considered as potential targets in the treatment of immune, inflammatory, and neurodegenerative diseases. In this study, we investigated the potential effects of the A2A adenosine receptor (A2AR) antagonist SCH 5826 (SCH) and agonist CGS 21680 (CGS) on behavior (self-grooming), hot plate test results, and expression levels of IL-17A+, RORγt+, Foxp3+, and IL-10+ in CD4+ T spleen cells in BTBR and C57BL/6 (B6) mice. We also assessed IL-17A, RORγt, Stat3, pStat3, Foxp3, and IL-10 mRNA and protein expression levels in the brain tissue. The CGS-treated mice showed a significantly altered self-grooming score and a reduced response to the hot plate test. The results further revealed that the SCH efficiently increased the IL-17A+ and RORγt+ expression levels and decreased the Foxp3+ and IL-10+ expression levels in CD4+ cells. However, the treatment with CGS significantly reversed these effects. In addition, CGS significantly decreased the IL-17A, RORγt, Stat3, and pStat3 levels and increased the Foxp3 and IL-10 mRNA and protein expression levels as compared with the BTBR control and SCH treatments. Our results clearly indicate that the CGS A2AR agonist may represent a unique target for future therapeutic strategies for neuroimmune dysfunction.