RORγt Inhibitors 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.

Digoxin attenuates bisphosphonate related osteonecrosis of the jaws by RORγt-dependent Th17 response in male rats

The study aimed to evaluate digoxin, an RORγt inhibitor, in Medication-Related Osteonecrosis of the Jaws (MRONJ) in male rats treated with zoledronic acid (ZA). Forty male Wistar rats were divided into a negative control group (0.1 mL/kg saline), a positive control group (ZA, 0.20 mg/kg), and three test groups treated with ZA and digoxin at 1 (DG1), 2 (DG2), or 4 (DG4) mg/kg. These groups received treatment three times weekly. ZA was administered intravenously on days 0, 7, and 14, followed by extraction of the left lower first molar on day 42, a final ZA dose on day 49, and euthanasia on day 70. Analyses included radiographic, histomorphometric, and immunohistochemical evaluation of the mandibles, western blotting of gingiva, and mechanical tests on femurs. Statistical analysis was performed using ANOVA/Bonferroni tests (P < .05). Digoxin reduced radiolucency of MRONJ (P < .001), inflammatory cells, empty osteocyte lacunae (P < .001), apoptotic osteoclasts (P < .001), and Caspase-3-positive osteocytes (P = .021). ZA increased immunoreactivity for most markers except c-Fos, while digoxin reduced interleukin 17, TNF-α, IL-6, IL-2, FOXP3, c-Jun, NFκB (P < .001), TGF-β (P = .009), RANKL (P = .035), and OPG (P = .034). Digoxin also reversed RORγt expression (P < .001), increased diarrhea scores (P = .028), renal and cardiac indexes (P < .001), and enhanced femur mechanical properties (P < .013). Digoxin attenuated MRONJ by inhibiting RORγt and reducing the Th17 response.

Lipid metabolism: a central modulator of RORγt-mediated Th17 cell differentiation.

Among the T helper cell subsets, Th17 cells contribute to the development of various inflammatory and autoimmune diseases, including psoriasis, rheumatoid arthritis, inflammatory bowel disease, steroid-resistant asthma, and multiple sclerosis. Retinoid-related orphan receptor gamma t (RORγt), a nuclear hormone receptor, serves as a master transcription factor for Th17 cell differentiation. Recent findings have shown that modulating the metabolic pathway is critical for Th17 cell differentiation, particularly through the engagement of de novo lipid biosynthesis. Suppression of lipid biosynthesis, either through the pharmacological inhibition or gene deletion of related enzymes in CD4+ T cells, results in significant impairment of Th17 cell differentiation. Mechanistic studies indicate that metabolic fluxes through both the fatty acid and cholesterol biosynthetic pathways have a pivotal role in the regulation of RORγt activity through the generation of endogenous RORγt lipid ligands. This review discusses recent discoveries highlighting the importance of lipid metabolism in Th17 cell differentiation and function, as well as exploring specific molecular pathways involved in RORγt activation through cellular lipid metabolism. We further elaborate on a pioneering therapeutic approach to improve inflammatory and autoimmune disorders via the inhibition of RORγt.

Carbon-14 Labeling Synthesis of RORγt Inhibitor JNJ-61803534.

Carbon-14 labeling synthesis of RORγt inhibitor JNJ-61803534 (1) was accomplished in four steps with the C14 label located at the thiazole-2-carboxamide carbon. The synthesis featured a highly efficient conversion of nitrile [14C]-12 to ester [14C]-17 under mild conditions via an imidate intermediate, overcoming the unsuccessful direct hydrolysis of nitrile 12 under either acidic or basic conditions. Since carbon-14 labeling via [14C]-nitrile installation and subsequent conversion to [14C]-carboxylic acid derivatives is a common labeling strategy, an efficient conversion of a nitrile to an ester under mild conditions could be of use for the future C14 labeling syntheses.

Thiazole Derivatives as RORγt Inhibitors: Synthesis, Biological Evaluation, and Docking Analysis

Background: Cancer is a serious concern in developing countries. Due to various worldwide impacts, it has surpassed coronary disorders becoming the leading cause of mortality. In cancer progression, the involvement of RORs (retinoic acid receptor-related orphan receptors) has gained attention in the search for versatile agents. Thiazole has the potential to be used as a scaffold in the development of anticancer drugs. Objective: This work’s major goal was to design and synthesize promising anticancer candidates. Methods: Docking investigations concerning the RORγt receptor were utilized in the design of the compounds. A series of novel thiazole derivatives was synthesized employing Hantsch synthesis. Melting points, TLC development, and spectral analyses were considered for the synthesized compounds' characterization. Corresponding spectra were analyzed and determined to be consistent with the assigned structure. On 60 human cancer cell lines, selected compounds were investigated in vitro by NCI (National Cancer Institute), USA, for anticancer action. Results: Varying ratios of anticancer potential were presented by the compounds in in vitro studies. Compound 3c was determined to be the most active compound in the series, with a -15.03 mean growth percent. In silico study revealed that the synthesized compounds could have anticancer possibilities. In the targeted pdb id (6q7a) binding site, compound 3c demonstrated excellent docking scores in molecular docking analyses, supporting its ability to be utilized as a lead compound in rational drug design. Conclusion: Compounds with satisfactory docking scores within the binding pocket of chosen pdb id (6q7a) validated their suitability for usage as lead compounds in rational drug design. Outcomes of in silico investigations were in agreement with the findings of the cytotoxicity experiments. Leukemia, renal, breast, and lung cancer cell lines were most sensitive to the substance-treated tumor cells. Electronwithdrawing groups were essential for antiproliferative action, according to the findings.

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.

Challenges and Future Trends in the Treatment of Psoriasis.

Psoriasis is a chronic inflammatory skin disorder, and current treatments include topical therapies, phototherapy, systemic immune modulators, and biologics, aiming to alleviate symptoms and improve quality of life. However, challenges persist, such as adverse effects, treatment resistance, high costs, and variability in response among individuals. The future of psoriasis treatment shows promising emerging trends. New biologic agents targeting novel pathways, such as interleukin 23 inhibitors like mirikizumab, offer enhanced efficacy. Small molecule inhibitors like RORγt inhibitors and ROCK2 inhibitors provide additional treatment options. Combination therapies, including biologics with methotrexate, may improve treatment response. Advancements in topical treatments utilizing microneedles and nanoparticle-based carriers can enhance drug delivery and improve therapeutic outcomes. Biomarkers and multi-omics technologies hold potential for personalized treatment approaches, thus aiding in diagnosis, predicting treatment response, and guiding therapeutic decisions. Collaboration among researchers, clinicians, and industry stakeholders is crucial to translating these scientific breakthroughs into clinical practice. By addressing current challenges and exploring these promising trends, we can optimize psoriasis management and improve the lives of those affected by this chronic condition.

Topotecan alleviates acetic acid-induced ulcerative colitis in rats via attenuation of the RORγT transcription factor

AimsUlcerative colitis is characterized as a chronic immune-mediated inflammatory condition, affecting the intestinal gastroenteric tissue. Previous studies revealed that Th-17 cells are key players in the pathogenesis of ulcerative colitis. RORγT (Retinoic-acid-receptor-related orphan receptor-gamma T) is a lineage-specific transcription factor of Th-17 cells and thus has a role in their differentiation. Transient inhibition of RORγT has been reported to attenuate the differentiation of Th-17 cells and secretion of interleukin-17 (IL-17). Here, we investigated the efficacy of topotecan in ameliorating ulcerative colitis in rodents, via inhibition of the RORγT transcription factor. Main methods and key findingsExperimental ulcerative colitis was induced in rats by intrarectal acetic acid administration. Topotecan attenuated the severity of ulcerative colitis in rats by revoking neutrophils and macrophage infiltration to the colon. It also alleviated diarrhea and rectal bleeding and improved body weight. Further, attenuation of RORγT and IL-17 expression was observed in topotecan treated animals. Levels of pro-inflammatory cytokines TNF-α, IL-6, and IL-1β in the colon tissue were reduced by topotecan treatment. Significant reduction in malondialdehyde level, elevation of superoxide dismutase (SOD) and catalase activity was observed in the colon tissue of rats treated with topotecan compared to the diseased group. SignificanceThis study shows the therapeutic potential of topotecan in attenuating ulcerative colitis in rats probably via inhibition of the RORγT transcription factor and downstream mediators of Th-17 cells.

Virtual Screening Strategy to Identify Retinoic Acid-Related Orphan Receptor γt Modulators.

Molecular docking is a key method used in virtual screening (VS) campaigns to identify small-molecule ligands for drug discovery targets. While docking provides a tangible way to understand and predict the protein-ligand complex formation, the docking algorithms are often unable to separate active ligands from inactive molecules in practical VS usage. Here, a novel docking and shape-focused pharmacophore VS protocol is demonstrated for facilitating effective hit discovery using retinoic acid receptor-related orphan receptor gamma t (RORγt) as a case study. RORγt is a prospective target for treating inflammatory diseases such as psoriasis and multiple sclerosis. First, a commercial molecular database was flexibly docked. Second, the alternative docking poses were rescored against the shape/electrostatic potential of negative image-based (NIB) models that mirror the target's binding cavity. The compositions of the NIB models were optimized via iterative trimming and benchmarking using a greedy search-driven algorithm or brute force NIB optimization. Third, a pharmacophore point-based filtering was performed to focus the hit identification on the known RORγt activity hotspots. Fourth, free energy binding affinity evaluation was performed on the remaining molecules. Finally, twenty-eight compounds were selected for in vitro testing and eight compounds were determined to be low μM range RORγt inhibitors, thereby showing that the introduced VS protocol generated an effective hit rate of ~29%.

RORγt inhibition ameliorates IL-23 driven experimental psoriatic arthritis by predominantly modulating γδ-T cells.

Divergent therapeutic outcomes on different disease domains have been noted with IL-23 and IL-17A-blockade in PsA. Therefore, elucidating the role of RORγt, the master regulator of type 17 immune responses, is of potential therapeutic interest. To this end, RORγt inhibition was assessed in combined skin, joint and gut inflammation in vivo, using a PsA model. We tested the efficacy of a RORγt antagonist in B10.RIII mice challenged with systemic overexpression of IL-23 by hydrodynamic injection of IL-23 enhanced episomal vector (IL-23 EEV). Clinical outcomes were evaluated by histopathology. Bone density and surface erosions were examined using micro-computed tomography. Cytokine production was measured in serum and by intracellular flow cytometry. Gene expression in PsA-related tissues was analysed by qPCR. RORγt-blockade significantly ameliorated psoriasis, peripheral arthritis and colitis development in IL-23 EEV mice (improvement of clinical scores and weight loss respectively by 91.8%, 58.2% and 7.0%, P < 0.001), in line with profound suppression of an enhanced type IL-17 immune signature in PsA-affected tissues. Moreover, inflammation-induced bone loss and bone erosions were reduced (P < 0.05 in calcaneus, P < 0.01 in tibia). Sustained IL-23 overexpression resulted in only mild signs of sacroiliitis. Gamma-delta (γδ)-T cells, the dominant source of T cell-derived IL-17A and IL-22, were expanded during IL-23 overexpression, and together with Th17 cells, clearly countered by RORγt inhibition (P < 0.001). RORγt-blockade shows therapeutic efficacy in a preclinical PsA model with protection towards extra-musculoskeletal manifestations, reflected by a clear attenuation of type 17 cytokine responses by γδ-T cells and Th17 cells.

Interleukin-17 plays a role in dental pulp inflammation mediated by zoledronic acid: a mechanism unrelated to the Th17 immune response?

To evaluate the influence of RORγT inhibition by digoxin on inflammatory changes related to interleukin-17 (IL-17) in the pulp of rats treated with zoledronate (ZOL). Forty male Wistar rats were divided into a negative control group (NCG) treated with saline solution, a positive control group (PCG) treated with ZOL (0.20 mg/kg), and three groups treated with ZOL and co-treated with digoxin 1, 2, or 4 mg/kg (DG1, 2, and 4). After four intravenous administrations of ZOL or saline solution in a 70-day protocol, the right molars were evaluated by histomorphometry (number of blood vessels, blood vessels/µm2, cells/µm2, total blood vessel area, and average blood vessel area) and immunohistochemistry (IL-17, TNF-α, IL-6, and TGF-β). The Kruskal-Wallis/Dunn test was used for statistical analysis. PCG showed an increase in total blood vessel area (p=0.008) and average blood vessel area (p=0.014), and digoxin treatment reversed these changes. DG4 showed a reduction in blood vessels/µm2 (p<0.001). In PCG odontoblasts, there was an increase in IL-17 (p=0.002) and TNF-α (p=0.002) immunostaining, and in DG4, these changes were reversed. Odontoblasts in the digoxin-treated groups also showed an increase in IL-6 immunostaining (p<0.001) and a reduction in TGF-β immunostaining (p=0.002), and all ZOL-treated groups showed an increase in IL-17 (p=0.011) and TNF-α (p=0.017) in non-odontoblasts cells. ZOL induces TNF-α- and IL-17-dependent vasodilation and ectasia, and the classical Th17 response activation pathway does not seem to participate in this process.

α-Synuclein induces Th17 differentiation and impairs the function and stability of Tregs by promoting RORC transcription in Parkinson's disease

BackgroundParkinson’s disease (PD) is characterized by the loss of dopaminergic neurons (DA) and the accumulation of Lewy body deposits composed of alpha-Synuclein (α-Syn), which act as antigenic epitopes to drive cytotoxic T-cell responses in PD. Increased T helper 17 (Th17) cells and dysfunctional regulatory T cells (Tregs) have been reported to be associated with the loss of DA in PD. However, the mechanism underlying the Th17/Treg imbalance remains unknown. MethodsHere, we examined the percentage of Th17 cells, the percentage of Tregs and the α-Syn level and analysed their correlations in the peripheral blood of PD patients and in the substantia nigra pars compacta (SNpc) and spleen of MPTP-treated mice and A53 transgenic mice. We assessed the effect of α-Syn on the stability and function of Tregs and the differentiation of Th17 cells and evaluated the role of retinoid-related orphan nuclear receptor (RORγt) upregulation in α-Syn stimulation in vivo and in vitro. ResultsWe found that the α-Syn level and severity of motor symptoms were positively correlated with the increase in Th17 cells and decrease in Tregs in PD patients. Moreover, α-Syn stimulation led to the loss of Forkhead box protein P3 (FOXP3) expression in Tregs, accompanied by the acquisition of IL-17A expression. Increased Th17 differentiation was detected upon α-Syn stimulation when naïve CD4+ T cells were cultured under Th17-polarizing conditions. Mechanistically, α-Syn promotes the transcription of RORC, encoding RORγt, in Tregs and Th17 cells, leading to increased Th17 differentiation and loss of Treg function. Intriguingly, the increase in Th17 cells, decrease in Tregs and apoptosis of DA were suppressed by a RORγt inhibitor (GSK805) in MPTP-treated mice. ConclusionTogether, our data suggest that α-Syn promotes the transcription of RORC in circulating CD4+ T cells, including Tregs and Th17 cells, to impair the stability of Tregs and promote the differentiation of Th17 cells in PD. Inhibition of RORγt attenuated the apoptosis of DA and alleviated the increase in Th17 cells and decrease in Tregs in PD.

INKT17 cells play a pathogenic role in ethinylestradiol-induced cholestatic hepatotoxicity.

IL-17 is closely associated with inflammation in intrahepatic cholestasis (IHC). Targeting IL-17 ameliorates IHC in mice. Invariant natural killer T (iNKT) cells are predominantly enriched in the liver and they mediate drug-induced liver injury through their secreted cytokines. However, whether iNKT17 cells are involved in ethinylestradiol (EE)-induced IHC remains unclear. In the present study, the administration of EE (10mg/kg in vivo and 6.25μM in vitro) promoted the activation and expansion of iNKT17 cells, which contributed to a novel hepatic iNKT17/Treg imbalance. iNKT cell-deficient Jα18-/- mice and the RORγt inhibitor digoxin (20μg) alleviated EE-induced cholestatic hepatotoxicity and downregulated the IL-17 signalling pathway. In contrast, the co-administration of EE with recombinant IL-17 (1μg) to Jα18-/- mice induced cholestatic hepatotoxicity and increased the infiltration of hepatic neutrophils and monocytes. Importantly, the administration of IL-17-/- iNKT cells (3.5 × 105) to Jα18-/- mice resulted in the attenuation of hepatotoxicity and the recruitment of fewer hepatic neutrophils and monocytes than the adoptive transfer of wild-type iNKT cells. These results indicated that iNKT17 cells couldexert pathogenic effects. The recruitment and activation of iNKT17 cells could be attributed to the high level of CXCR3 expression on their surface. CXCL10 deficiency ameliorated EE-induced cholestatic liver damage, reduced hepatic CXCR3+ iNKT cells and inhibited RORγt expression. These findings suggest that iNKT17 cells play a key role in EE-induced cholestatic liver injury via CXCR3-mediated recruitment and activation. Our study provides new insights and therapeutic targets for cholestatic diseases.

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.

Alcohol-driven metabolic reprogramming promotes development of RORγt-deficient thymic lymphoma.

RORγt is a master regulator of Th17 cells. Despite evidence linking RORγt deficiency/inhibition with metastatic thymic T cell lymphomas, the role of RORγt in lymphoma metabolism is unknown. Chronic alcohol consumption plays a causal role in many human cancers. The risk of T cell lymphoma remains unclear in humans with alcohol use disorders (AUD) after chronic RORγt inhibition. Here we demonstrated that alcohol consumption accelerates RORγt deficiency-induced lymphomagenesis. Loss of RORγt signaling in the thymus promotes aerobic glycolysis and glutaminolysis and increases allocation of glutamine carbon into lipids. Importantly, alcohol consumption results in a shift from aerobic glycolysis to glutaminolysis. Both RORγt deficiency- and alcohol-induced metabolic alterations are mediated by c-Myc, as silencing of c-Myc decreases the effects of alcohol consumption and RORγt deficiency on glutaminolysis, biosynthesis, and tumor growth in vivo. The ethanol-mediated c-Myc activation coupled with increased glutaminolysis underscore the critical role of RORγt-Myc signaling and translation in lymphoma.

Novel Therapeutic Target(s) for Psoriatic Disease.

Psoriasis and psoriatic arthritis, together known as psoriatic disease, is highly prevalent chronic relapsing inflammatory disease affecting skin, joints or both and is associated with several comorbidities such as cardiovascular, metabolic, psychiatric, renal disease etc. The etiopathogenesis of psoriasis is complex and mainly driven by aberrant immune response owing to the genetic susceptibility and various environmental factors such as trauma, infections and drugs. Recent advances in understanding molecular and cellular pathways have identified tumor necrosis factor-α (TNF-α), interleukin-17 (IL-17), IL-23, IL-22 as major contributors in psoriasis pathogenesis. Advances in the knowledge of pathophysiology, the interaction of autoinflammation and clinical phenotypes have led to the development of highly effective targeted therapeutic agents which include TNF-α, IL-17, IL-23, IL-1 α/β or IL-36 inhibitors or receptor blockers, small molecule drugs like phosphodiesterase-4 inhibitors (apremilast), Janus kinase (JAK) inhibitors, retinoic acid receptor-related orphan receptor γt (RORγt) inhibitors. These novel drugs have promised the potential of improved disease control. In recent years, the transition from biologics to biosimilars especially with TNF-α inhibitors had significant impact on decreasing health care cost and increasing therapeutic options to the patients. However, selection of right treatment for an individual patient still remains challenging. Moreover, interplay between different epigenetic mechanisms such as the DNA methylation, chromatin modifications and noncoding RNA regulation has recently been started to be deciphered. Enzymes inhibitors involved in epigenetic pathways such as DNA methyltransferases and histone deacetylases demonstrated to restore normal epigenetic patterns in clinical settings and have provided the potential as novel therapeutic targets for psoriasis. In this review, we will discuss novel biologic agents and newer therapeutic approaches in treatment of psoriatic disease.

Tuberous Sclerosis Complex 1 Deficiency in Macrophages Promotes Unclassical Inflammatory Response to Lipopolysaccharide In Vitro and Dextran Sodium Sulfate-Induced Colitis in Mice.

Human tuberous sclerosis (TSC) is mainly caused by genetic mutations of tuberous TSC1or TSC2. Recent studies found that TSC1 deficiency promoted classical M1 macrophage polarization. However, whether TSC1 regulates other inflammatory cytokine expression in lipopolysaccharidem (LPS)-stimulated macrophages is unknown. Herein, we studied the cytokine expression profile of wild-type (WT) and TSC1-deleted macrophages after LPS stimulation in vitro and the pathogenesis of dextran sodium sulfate (DSS)-induced colitis in mice with myeloid-specific TSC1 deletion (TSC1cKO mice). We found that TSC1-deficient macrophages exhibited the enhanced secretion of interleukin-17A (IL-17A), IL-17F, and interferon-gamma (IFN-γ) in response to LPS stimulation in vitro. This is in contrast to LPS-stimulated WT macrophages, which usually do not. Importantly, TSC1cKO mice exhibited exacerbated DSS-induced acute colitis with severer symptoms. MTOR deletion or rapamycin treatment significantly reversed the enhanced expressions of IL-17A, IL-17F, and IFN-γ in LPS-stimulated TSC1-deficient macrophages in vitro and rescued the enhanced DSS-induced colitis in TSC1cKO mice, indicating that TSC1 deficiency increased these cytokine productions in an mTOR-dependent manner. RNA-sequencing and molecular studies indicated that TSC1 deficiency enhanced the aerobic glycolysis process and the activities of mTOR-STAT3-RORγT pathway in LPS-stimulated macrophages. Inhibition of aerobic glycolysis, STAT3, or RORγT reversed IL-17 and IFN-γ expression in LPS-treated TSC1-deficient macrophages. Thus, TSC1 is essential for macrophages to shut down IL-17A, IL-17F, and IFN-γ expression during LPS stimulation by suppressing the aerobic glycolysis process and mTOR-STAT3, RORγT, and T-bet pathways. The present study uncovered the key role of TSC1 in shutting down IL-17A, IL-17F, and IFN-γ expressions in LPS-treated macrophages.

Microbial Cgr2 will let your Th17 cells ROR(γT)

In this issue of Cell Host & Microbe, Alexander etal. show that the enzyme cardiac glycoside reductase 2 (cgr2), which is produced by Eggerthella lenta, metabolizes RORγT inhibitors, resulting in an increased Th17 response and more severe inflammation in colitis models. The effect of cgr2 can be neutralized by a diet rich in arginine.

Asymmetric Synthesis of the Cyclohexyl Fragment in RORγt Inhibitor (BMS-986251) Enabled by a Dynamic Kinetic Resolution of Hageman’s Ester

The cyclohexyl fragment 1 in BMS-986251 was synthesized starting from Hagemann’s ester 2 in 7 steps and 5 isolations. The route is highlighted by a dynamic kinetic resolution (DKR), a telescoped en...

Small molecule allosteric inhibitors of RORγt block Th17-dependent inflammation and associated gene expression in vivo.

Retinoic acid receptor-related orphan nuclear receptor (ROR) γt is a member of the RORC nuclear hormone receptor family of transcription factors. RORγt functions as a critical regulator of thymopoiesis and immune responses. RORγt is expressed in multiple immune cell populations including Th17 cells, where its primary function is regulation of immune responses to bacteria and fungi through IL-17A production. However, excessive IL-17A production has been linked to numerous autoimmune diseases. Moreover, Th17 cells have been shown to elicit both pro- and anti-tumor effects. Thus, modulation of the RORγt/IL-17A axis may represent an attractive therapeutic target for the treatment of autoimmune disorders and some cancers. Herein we report the design, synthesis and characterization of three selective allosteric RORγt inhibitors in preclinical models of inflammation and tumor growth. We demonstrate that these compounds can inhibit Th17 differentiation and maintenance in vitro and Th17-dependent inflammation and associated gene expression in vivo, in a dose-dependent manner. Finally, RORγt inhibitors were assessed for efficacy against tumor formation. While, RORγt inhibitors were shown to inhibit tumor formation in pancreatic ductal adenocarcinoma (PDAC) organoids in vitro and modulate RORγt target genes in vivo, this activity was not sufficient to delay tumor volume in a KP/C human tumor mouse model of pancreatic cancer.