Retinoic Acid Receptor-related Orphan Receptor Research Articles

Discovery of 1-(Phenylsulfonyl)-1,2,3,4-tetrahydroquinoline Derivative as Orally Bioavailable and Safe RORγt Inverse Agonists for Potential Treatment of Rheumatoid Arthritis.

The retinoic acid receptor-related orphan receptor γt (RORγt) is a key regulator of Th17 cells, associated with autoimmune diseases, making it a significant drug target. Herein, we designed and synthesized 1-(phenylsulfonyl)-1,2,3,4-tetrahydroquinoline derivatives, improving upon GSK2981278 to enhance bioavailability. Of which, D4 exhibited superior bioavailability (F = 48.1 and 32.9% in mice and rats, respectively) compared to GSK2981278 (F = 6.2 and 4.1%, respectively), and effectively treated psoriasis in mice at lower doses. Moreover, for the first time, we report the efficacies of a RORγt inverse agonist in mouse models of rheumatoid arthritis. (R)-D4, the eutomer of D4, matched or exceeded GSK2981278's therapeutic effects at lower doses, with no adverse effects observed after 2 weeks of administration. These results position (R)-D4 as a promising and orally bioavailable candidate for Th17-mediated autoimmune disease treatment.

MNAM enhances Blautia abundance and modulates Th17/Treg balance to alleviate diabetes in T2DM mice

This study investigated the therapeutic effects of N1-Methylnicotinamide (MNAM), a metabolic derivative, on T2DM mice induced by a high-fat diet and streptozotocin (STZ), focusing on its impact on the gut microbiome and immune modulation. MNAM significantly reduced hyperglycemia and enhanced insulin secretion, effects that were dependent on the presence of gut microbiota. It also mitigated STZ-induced weight loss and improved islet cell morphology, reducing islet cell mortality and increasing insulin (INS) levels. Flow cytometry analysis showed a decrease in T helper 17 cells (Th17) and an increase in Treg cells after MNAM treatment, corresponding to the upregulation of Treg markers [interleukin (IL)-10, forkhead box P3 (FOXP3)] and downregulation of Th17 markers [IL17A, RAR-related orphan receptor gamma (RORγt)]. Additionally, MNAM raised anti-inflammatory IL-10 levels while reducing pro-inflammatory cytokines [IL-17α, tumor necrosis factor (TNF-α), IL-6]. Microbiome analysis revealed decreased diversity and increased Blautia abundance post-MNAM administration. Treatment with Blautia not only reversed diabetes indicators but also modulated the Th17/Treg balance and reduced inflammation, with its metabolite sodium acetate mimicking these effects through the G protein-coupled receptor 43 (GPR43) pathway. These findings suggest that MNAM’s mitigation of diabetes operates through modulation of the gut microbiota and immune regulation, highlighting Blautia and its metabolite as potential therapeutic agents and providing a theoretical foundation for novel treatment strategies in T2DM.

Orally bioavailable RORγ/DHODH dual host-targeting small molecules with broad-spectrum antiviral activity

Host-directed antivirals (HDAs) represent an attractive treatment option and a strategy for pandemic preparedness, especially due to their potential broad-spectrum antiviral activity and high barrier to resistance development. Particularly, dual-targeting HDAs offer a promising approach for antiviral therapy by simultaneously disrupting multiple pathways essential for viral replication.Izumerogant (IMU-935) targets two host proteins, (i) the retinoic acid receptor-related orphan receptor γ isoform 1 (RORγ1), which modulates cellular cholesterol metabolism, and (ii) the enzyme dihydroorotate dehydrogenase (DHODH), which is involved in de novo pyrimidine synthesis. Here, we synthesized optimized derivatives of izumerogant and characterized their antiviral activity in comparison to a recently described structurally distinct RORγ/DHODH dual inhibitor. Cell culture-based infection models for enveloped and non-enveloped DNA and RNA viruses, as well as a retrovirus, demonstrated high potency and broad-spectrum activity against human viral pathogens for RORγ/DHODH dual inhibitors at nanomolar concentrations. Comparative analyses with equipotent single-target inhibitors in metabolite supplementation approaches revealed that the dual-targeting mode represents the mechanistic basis for the potent antiviral activity. For SARS-CoV-2, an optimized dual inhibitor completely blocked viral replication in human airway epithelial cells at 5 nM and displayed a synergistic drug interaction with the nucleoside analog molnupiravir. In a SARS-CoV-2 mouse model, treatment with a dual inhibitor alone, or in combination with molnupiravir, reduced the viral load by 7- and 58-fold, respectively.Considering the clinical safety, oral bioavailability, and tolerability of izumerogant in a recent Phase I study, izumerogant-like drugs represent potent dual-targeting antiviral HDAs with pronounced broad-spectrum activity for further clinical development.

UBE2T promotes stage I lung adenocarcinoma progression through PBX1 ubiquitination and PBX1/RORA regulation

BackgroundPost-translational modification pathway of protein ubiquitination is intricately associated with tumorigenesis. We previously reported elevated ubiquitin-conjugating enzyme 2T (UBE2T) as an independent risk factor in stage I lung adenocarcinoma and promoting cellular proliferation. However, its underlying mechanisms needed further investigation.MethodsImmunohistochemistry was used to assess the expression of UBE2T and retinoic acid receptor-related orphan receptor α (RORA) in stage I LUAD. Cell proliferation, migration, and invasion of LUAD cell lines were measured by Cell Counting Kit-8 assay (CCK-8), Colony-forming assay and Transwell assay, respectively. Western blot analysis was performed to determine the expression of epithelial-mesenchymal transition (EMT) markers. A xenograft model was established to evaluate the proliferative capacity of UBE2T and its interaction with RORA in promoting LUAD. Mechanistic insights into the promotion of early-stage LUAD by UBE2T were obtained through luciferase reporter assay, chromatin immunoprecipitation and co-immunoprecipitation.ResultsUBE2T and RORA expression was significantly up- and down-regulated in early-stage LUAD patients which’s proved to be associated with unfavorable outcomes, strengthened cell proliferation, migration, EMT and invasion through its interaction with RORA both in vivo and in vitro. The growth NSCLC xenografts was reduced by down-expression of UBE2T but was suppressed by RORA knockout. Mechanistically, UBE2T mediated the ubiquitination of the intermediate transcription factor PBX1, which played a transcriptional role in downstream regulation of RORA.ConclusionThe oncogenic role of UBE2T and the UBE2T-PBX1-RORA axis in driving malignant progression in Stage I LUAD had been established. UBE2T might be a novel and promising therapeutic target for LUAD treatment.

Exosomal miR-205–5p contributes to the immune liver injury induced by trichloroethylene: Pivotal role of RORα mediating M1 Kupffer cell polarization

Trichloroethylene (TCE) is a common environmental contaminant that can induce occupational dermatitis medicamentosa-like TCE (ODMLT), where the liver damage is the most common complication. The study aims to uncover the underlying mechanism of TCE-sensitization-induced liver damage by targeting specific exosomal microRNAs (miRNAs). Among the enriched serum exosomal miRNAs of ODMLT patients, miR-205–5p had a significant correlation coefficient with the liver function damage indicators. Moreover, retinoic acid receptor-related orphan receptor α (RORα) was identified as a direct target of miR-205–5p via specific binding. Further experiments showed that kupffer cells (KCs) underwent M1 phenotypic and functional changes in liver injury induced by TCE which were alleviated by reducing the expression of miR-205–5p. However, this alleviation was reversed by the RORα antagonist SR1001. In vitro experiments showed that miR-205–5p promoted M1 polarization of macrophages and enhanced the secretion of inflammatory factors by regulating RORα. An increase in RORα reversed the polarization direction of M1-type macrophages and reduced the secretion of proinflammatory factors. In addition, pretreatment of mice with SR1078, a specific RORα agonist, effectively blocked M1 polarization of KCs and reduced the severity of TCE-induced liver injury. Our study uncovers that miR-205–5p regulates KC M1 polarization by targeting RORα in immune liver injury induced by TCE sensitization, providing new insight into the molecular mechanisms and new therapeutic targets for ODMLT.

A High-Quality Photoswitchable Probe that Selectively and Potently Regulates the Transcription Factor RORγ.

Retinoic acid receptor-related orphan receptor γ (RORγ) is a nuclear hormone receptor with multiple biological functions. As an experimental therapeutic target in inflammation and immunity, there is great interest in spatially-localised RORγ inhibition; and its cyclic temporal role in circadian rhythms also makes it an intriguing target for time-resolved pharmacology. To create tools that can study RORγ biology with appropriate spatial and temporal resolution, we designed light-dependent inverse RORγ agonists by building azobenzene photoswitches into ligand consensus structures. Optimizations gave photoswitchable RORγ inhibitors with a large degree of potency photocontrol, plus remarkable on-target potency, plus excellent selectivity over related off-target receptors. This still-rare, but urgently-needed combination of performance features, distinguishes them as high quality photopharmaceutical probes; and they can now serve as high precision tools to study the spatial and dynamic intricacies of RORγ action in signaling and in inflammatory disorders.

Regulation of Bacteroides acidifaciens by the aryl hydrocarbon receptor in IL-22-producing immune cells has sex-dependent consequential impact on colitis.

Colitis is an inflammatory bowel disease (IBD) characterized by immune cell dysregulation and alterations in the gut microbiome. In our previous report, we showed a natural product in cruciferous vegetables and ligand of the aryl hydrocarbon receptor (AhR), indole-3-carbinol (I3C), was able to reduce colitis-induced disease severity and microbial dysbiosis in an interleukin-22 (IL-22) dependent manner. In the current study, we performed single-cell RNA sequencing (scRNAseq) from colonocytes during colitis induction and supplementation with I3C and show how this treatment alters expression of genes involved in IL-22 signaling. To further define the role of IL-22 signaling in I3C-mediated protection during colitis and disease-associated microbial dysbiosis, we generated mice with AhR deficiency in RAR-related orphan receptor c (Rorc)-expressing cells (AhR ΔRorc ) which depletes this receptor in immune cells involved in production of IL-22. Colitis was induced in wildtype (WT), AhR ΔRorc , and littermate (LM) mice with or without I3C treatment. Results showed AhR ΔRorc mice lost the efficacy effects of I3C treatment which correlated with a loss of ability to increase IL-22 by innate lymphoid type 3 (ILC3s), not T helper 22 (Th22) cells. 16S rRNA microbiome profiling studies showed AhR ΔRorc mice were unable to regulate disease-associated increases in Bacteroides, which differed between males and females. Lastly, inoculation with a specific disease-associated Bacteroides species, Bacteroides acidifaciens (B. acidifaciens), was shown to exacerbate colitis in females, but not males. Collectively, this report highlights the cell and sex-specific role of AhR in regulating microbes that can impact colitis disease.

Single-nucleus multiome analysis of human cerebellum in Alzheimer's disease-related dementia.

Although human cerebellum is known to be neuropathologically impaired in Alzheimer's disease (AD) and AD-related dementias (ADRD), the cell type-specific transcriptional and epigenomic changes that contribute to this pathology are not well understood. Here, we report single-nucleus multiome (snRNA-seq and snATAC-seq) analysis of 103,861 nuclei isolated from cerebellum from 9 human cases of AD/ADRD and 8 controls, and with frontal cortex of 6 AD donors for additional comparison. Using peak-to-gene linkage analysis, we identified 431,834 significant linkages between gene expression and cell subtype-specific chromatin accessibility regions enriched for candidate cis-regulatory elements (cCREs). These cCREs were associated with AD/ADRD-specific transcriptomic changes and disease-related gene regulatory networks, especially for RAR Related Orphan Receptor A (RORA) and E74 Like ETS Transcription Factor 1 (ELF1) in cerebellar Purkinje cells and granule cells, respectively. Trajectory analysis of granule cell populations further identified disease-relevant transcription factors, such as RORA, and their regulatory targets. Finally, we prioritized two likely causal genes, including Seizure Related 6 Homolog Like 2 (SEZ6L2) in Purkinje cells and KAT8 Regulatory NSL Complex Subunit 1 (KANSL1) in granule cells, through integrative analysis of cCREs derived from snATAC-seq, genome-wide AD/ADRD loci, and Hi-C looping data. This first cell subtype-specific regulatory landscape in the human cerebellum identified here offer novel genomic and epigenomic insights into the neuropathology and pathobiology of AD/ADRD and other neurological disorders if broadly applied.

MiR-143-5p regulates the proangiogenic potential of human dental pulp stem cells by targeting HIF-1α/RORA under hypoxia: A laboratory investigation in pulp regeneration.

Angiogenesis is a key event in the successful healing of pulp injuries, and hypoxia is the main stimulator of pulpal angiogenesis. In this study, we investigated the effect of hypoxia on the proangiogenic potential of human dental pulp stem cells (hDPSCs) and the role of miR-143-5p in the process. Human dental pulp stem cells were isolated, cultured and characterized invitro. Cobalt chloride (CoCl2) was used to induce hypoxia in hDPSCs. CCK-8 and Transwell assays were used to determine the effect of hypoxia on hDPSCs proliferation and migration. Quantitative real-time polymerase chain reaction (qRT-PCR), Western blotting (WB) and ELISA were performed to assess the mRNA and protein levels of HIF-1α and angiogenic cytokines in hDPSCs. The effect of hypoxia on hDPSCs proangiogenic potential was measured invitro using Matrigel tube formation and chick chorioallantoic membrane (CAM) assays. Recombinant lentiviral vectors were constructed to stably overexpress or inhibit miR-143-5p in hDPSCs, and the proangiogenic effects were assessed using qRT-PCR, WB, and tube formation assays. miR-143-5p target genes were identified and verified using bioinformatics prediction tools, dual-luciferase reporter assays and RNA pull-down experiments. Finally, a subcutaneous transplantation model in nude mice was used to determine the effects of hypoxia treatment and miR-143-5p overexpression/inhibition in hDPSCs in dental pulp regeneration. Hypoxia promotes hDPSCs proliferation, migration and proangiogenic potential. The invivo experiments showed that hypoxia treatment (50 and 100 μM CoCl2) promoted pulp angiogenesis and dentine formation. In contrast to the levels of proangiogenic factors, miR-143-5p levels decreased with increasing CoCl2 concentration. miR-143-5p inhibition significantly promoted proangiogenic potential of hDPSCs, whereas miR-143-5p overexpression inhibited angiogenesis invitro. Dual-luciferase reporter assay identified retinoic acid receptor-related orphan receptor alpha (RORA) as an miR-143-5p target gene in hDPSCs. RNA pull-down experiments demonstrated that HIF-1α and RORA were pulled down by biotin-labelled miR-143-5p, and the levels of HIF-1α and RORA bound to miR-143-5p in the hypoxia group were lower than those in the normoxia group. Inhibition of miR-143-5p expression in hDPSCs promoted ectopic dental pulp tissue regeneration. CoCl2-induced hypoxia promotes hDPSCs-driven paracrine angiogenesis and pulp regeneration. The inhibition of miR-143-5p upregulates the proangiogenic potential of hDPSCs under hypoxic conditions by directly targeting HIF-1α and RORA.

Anti-Inflammatory and Immunoregulatory Effects of Xiaoqinglong Decoction on a Murine Model of Combined Allergic Rhinitis and Asthma Syndrome

Background Xiaoqinglong Decoction (XQLD) is a traditional Chinese medicine formula used for the treatment of allergic rhinitis and asthma, including combined allergic rhinitis and asthma syndrome (CARAS), due to its anti-inflammatory and anti-seizure properties. However, the pharmacological activities and the underlying molecular mechanisms of XQLD remain to be elucidated . Hence, we investigated the effects of XQLD against inflammation in an ovalbumin (OVA)-induced CARAS model in BALB/c mice. Methods BALB/c mice were sensitized by intraperitoneal injection and aerosol inhalation of OVA. XQLD or dexamethasone was administered by oral gavage prior to OVA challenge for 7 consecutive days. Specific airway resistance (sRAW) was evaluated 24 h after the final challenge with OVA. Enzyme-linked immunosorbent assay was used to measure the levels of serum inflammatory factors. The nasal mucosa and lung tissue were examined for general morphology and goblet hyperplasia using hematoxylin and eosin or periodic acid Schiff staining. Flow cytometry was employed to analyze the frequencies of helper T lymphocytes in peripheral blood. Immunohistochemistry was performed to determine the expression of transcription factors in helper T lymphocytes and γδ TCR. Results XQLD significantly ameliorated the symptoms of CARAS model mice, suppressed serum levels of interferon-γ, interleukin (IL)-2, IL-4, IL-5, IL-17, while increasing levels of IL-10, transforming growth factor-β. Epithelium impairment, cilia loss, eosinophil infiltration, and goblet cell metaplasia were significantly reduced in the nasal mucosa and lung tissue sections. XQLD restored the balance of Th1/Th2 and Th17/Treg cell frequency in peripheral blood. Furthermore, XQLD down-modulated the expression of GATA-binding protein 3 (GATA3), retinoic acid receptor-related orphan receptor γt, and γδ T cell receptor (TCR) in the nasal mucosa, along with GATA3 and γδ TCR in the lung tissue. Conclusions XQLD alleviated OVA-induced CARAS in BALB/c mice through its anti-allergic effects and modulation of the immune system.

Renal IL-23-Dependent Type 3 Innate Lymphoid Cells Link Crystal-induced Intrarenal Inflammasome Activation with Kidney Fibrosis.

Chronic inflammasome activation in mononuclear phagocytes (MNPs) promotes fibrosis in various tissues, including the kidney. The cellular and molecular links between the inflammasome and fibrosis are unclear. To address this question, we fed mice lacking various immunological mediators an adenine-enriched diet, which causes crystal precipitation in renal tubules, crystal-induced inflammasome activation, and renal fibrosis. We found that kidney fibrosis depended on an intrarenal inflammasome-dependent type 3 immune response driven by its signature transcription factor Rorc (retinoic acid receptor-related orphan receptor C gene), which was partially carried out by type 3 innate lymphoid cells (ILC3s). The role of ILCs in the kidney is less well known than in other organs, especially that of ILC3. In this article, we describe that depletion of ILCs or genetic deficiency for Rorc attenuated kidney inflammation and fibrosis. Among the inflammasome-derived cytokines, only IL-1β expanded ILC3 and promoted fibrosis, whereas IL-18 caused differentiation of NKp46+ ILC3. Deficiency of the type 3 maintenance cytokine, IL-23, was more protective than IL-1β inhibition, which may be explained by the downregulation of the IL-1R, but not of the IL-23R, by ILC3 early in the disease, allowing persistent sensing of IL-23. Mechanistically, ILC3s colocalized with renal MNPs invivo as shown by multiepitope-ligand cartography. Cell culture experiments indicated that renal ILC3s caused renal MNPs to increase TGF-β production that stimulated fibroblasts to produce collagen. We conclude that ILC3s link inflammasome activation with kidney inflammation and fibrosis and are regulated by IL-1β and IL-23.

OLFM4 modulates intestinal inflammation by promoting IL-22+ILC3 in the gut

Group 3 innate lymphoid cells (ILC3s) play key roles in intestinal inflammation. Olfactomedin 4 (OLFM4) is highly expressed in the colon and has a potential role in dextran sodium sulfate-induced colitis. However, the detailed mechanisms underlying the effects of OLFM4 on ILC3-mediated colitis remain unclear. In this study, we identify OLFM4 as a positive regulator of IL-22+ILC3. OLFM4 expression in colonic ILC3s increases substantially during intestinal inflammation in humans and mice. Compared to littermate controls, OLFM4-deficient (OLFM4−/−) mice are more susceptible to bacterial infection and display greater resistance to anti-CD40 induced innate colitis, together with impaired IL-22 production by ILC3, and ILC3s from OLFM4−/−mice are defective in pathogen resistance. Besides, mice with OLFM4 deficiency in the RORγt compartment exhibit the same trend as in OLFM4−/−mice, including colonic inflammation and IL-22 production. Mechanistically, the decrease in IL-22+ILC3 caused by OLFM4 deficiency involves the apoptosis signal-regulating kinase 1 (ASK1)- p38 MAPK signaling-dependent downregulation of RAR-related orphan receptor gamma (RORγt) protein. The OLFM4-metadherin (MTDH) complex upregulates p38/RORγt signaling, which is necessary for IL-22+ILC3 activation. The findings indicate that OLFM4 is a novel regulator of IL-22+ILC3 and essential for modulating intestinal inflammation and tissue homeostasis.

Retinoic acid receptor-related orphan receptor alpha and synthetic RORα agonist against invasion and metastasis in tongue squamous cell carcinoma

Retinoic acid receptor-related orphan receptor alpha (RORα), an essential tumor suppressor in a range of human malignancies, is classified as a member of the orphan nuclear receptor family. The most prevalent form of oral cancer, tongue squamous cell carcinoma (TSCC) is characterized by its severe malignancy and unfavorable prognosis. However, the extent to which its tumorigenesis mechanisms are associated with RORα expression levels is still not fully understood. The objective of this study was to examine the molecular mechanisms by which RORα is involved in TSCC. Through the use of immunohistochemistry (IHC), it was discovered that the expression level of RORα was significantly downregulated in TSCC tissues when compared to adjacent normal tissues in this study. To further investigate the role of RORα in TSCC, we activated the expression of RORα in human TSCC cell line (SCC9 cells) by transfecting RORα cDNA and using the selective RORα agonist SR1078. The results show that RORα can significantly inhibit the invasion, migration, proliferation, and adhesion of TSCC cells and induce cell apoptosis. In addition, xenograft models confirmed the conclusion that stable activation or treatment with SR1078 to increase RORα content significantly inhibited tumor growth and development. Taken together, this study provides solid evidence for the inhibitory role of RORα in the progression of TSCC. In addition, the preliminary application results of SR1078 in TSCC show that SR1078 is expected to be a potential therapeutic medication for TSCC. These findings provide innovative perspectives on the development of potential biomarkers and agents for TSCC therapy. The objective is to introduce novel strategy and alternatives for the prevention and treatment of TSCC.

A systematic search for RNA structural switches across the human transcriptome

RNA structural switches are key regulators of gene expression in bacteria, but their characterization in Metazoa remains limited. Here, we present SwitchSeeker, a comprehensive computational and experimental approach for systematic identification of functional RNA structural switches. We applied SwitchSeeker to the human transcriptome and identified 245 putative RNA switches. To validate our approach, we characterized a previously unknown RNA switch in the 3ʹ untranslated region of the RORC (RAR-related orphan receptor C) transcript. In vivo dimethyl sulfate (DMS) mutational profiling with sequencing (DMS-MaPseq), coupled with cryogenic electron microscopy, confirmed its existence as two alternative structural conformations. Furthermore, we used genome-scale CRISPR screens to identify trans factors that regulate gene expression through this RNA structural switch. We found that nonsense-mediated messenger RNA decay acts on this element in a conformation-specific manner. SwitchSeeker provides an unbiased, experimentally driven method for discovering RNA structural switches that shape the eukaryotic gene expression landscape.

Tumoral periprostatic adipose tissue exovesicles-derived miR-20a-5p regulates prostate cancer cell proliferation and inflammation through the RORA gene

BackgroundFrom the first steps of prostate cancer (PCa) initiation, tumours are in contact with the most-proximal adipose tissue called periprostatic adipose tissue (PPAT). Extracellular vesicles are important carriers of non-coding RNA such as miRNAs that are crucial for cellular communication. The secretion of extracellular vesicles by PPAT may play a key role in the interactions between adipocytes and tumour. Analysing the PPAT exovesicles (EVs) derived-miRNA content can be of great relevance for understanding tumour progression and aggressiveness.MethodsA total of 24 samples of human PPAT and 17 samples of perivesical adipose tissue (PVAT) were used. EVs were characterized by western blot and transmission electron microscopy (TEM), and uptake by PCa cells was verified by confocal microscopy. PPAT and PVAT explants were cultured overnight, EVs were isolated, and miRNA content expression profile was analysed. Pathway and functional enrichment analyses were performed seeking potential miRNA targets. In vitro functional studies were evaluated using PCa cells lines, miRNA inhibitors and target gene silencers.ResultsWestern blot and TEM revealed the characteristics of EVs derived from PPAT (PPAT-EVs) samples. The EVs were up taken and found in the cytoplasm of PCa cells. Nine miRNAs were differentially expressed between PPAT and PVAT samples. The RORA gene (RAR Related Orphan Receptor A) was identified as a common target of 9 miRNA-regulated pathways. In vitro functional analysis revealed that the RORA gene was regulated by PPAT-EVs-derived miRNAs and was found to be implicated in cell proliferation and inflammation.ConclusionTumour periprostatic adipose tissue is linked to PCa tumour aggressiveness and could be envisaged for new therapeutic strategies.

Effects of dietary Chitosan oligosaccharides supplementation on Th17/Treg balance and gut microbiota of early weaned pigeon squabs

Our previous study found that early weaning is associated with decreased growth performance, intestinal barrier impairment, and an imbalance in Th17/Treg in pigeon squabs. Chitosan oligosaccharides (COS) has been substantiated to regulate gut microbiota and restore Th17/Treg equilibrium in mammals, thereby ameliorating growth performance. However, the potential effects of COS in altricial birds remain unclear. Three hundred healthy 7-day-old American king pigeon squabs were selected with similar body weights and randomly divided into 5 groups. The 5 treatment groups were as follows: the control group (CON), fed with artificial pigeon milk; 4 supplementation groups, fed with artificial pigeon milk +100 (COS1), 150 (COS2), 200 (COS3), and 250 (COS4) mg/kg COS, respectively. Results showed that dietary supplementation of COS significantly enhanced the growth performance of weaned squabs. Compared to the CON group, the COS groups exhibited increased villus length and villus area in the jejunum and ileum, accompanied by improvements in morphological structure and mucosal permeability. COS was found to reduce the levels of Th17-associated cytokines and increase the levels of Treg-associated cytokines. COS downregulated the expression of retinoic acid receptor-related orphan receptor C (RORC), a key transcription factor of Th17 cells, while upregulated the expression of Forkhead box protein P3 (FOXP3), a key transcription factor of Treg cells. Dietary COS supplementation increased gut bacterial diversity, altered the relative abundance of several bacteria taxa and enhanced the concentration of short-chain fatty acids (SCFA). Correlation analysis demonstrated a close association between gut microbiota, SCFAs, and indicators related to the Th17/Treg balance. Moreover, we found that SCFAs correlated more strongly with Th17/Treg-related indexes than gut microbiota. These results demonstrated that COS could relieve early weaning stress in pigeon squabs and the optimal dosage of dietary COS supplementation was suggested to be 200 mg/kg. In addition, COS had a protective effect on maintaining intestinal immune balance by modulating microbiota and Th17/Treg related signaling pathways, in which SCFAs might play a crucial role as messengers.

GATA2‑miR‑374a axis promotes vascular smooth muscle cells proliferation, migration via targeting circTADA2A/RORA axis.

Evidence has shown that microRNAs (miRNAs/miRs) play key roles in biological functions of vascular smooth muscle cells (VSMCs). However, the role of miR-374a in VSMCs remains to be elucidated. The present study aimed to explore the influence of miR-374a on VSMCs and its molecular mechanism. The expression level of miR-374a was measured by reverse transcription-quantitative (RT-q) PCR. MTT and Transwell assay were employed to assess the role of miR-374a in proliferation and migration of VSMCs. To order to determine miR-374a targets, a dual-luciferase reporter assay was conducted, which was further verified by rescue experiments. Chromatin Immunoprecipitation Assay and JASPAR databases were applied to explore the regulatory association between GATA binding protein 2 (GATA2) and miR-374a. Western blotting or RT-qPCR were employed to detect the protein expression levels of GATA2 or RAR-related orphan receptor A (RORA). The present study found that miR-374a was elevated in VSMCs following treatment with platelet-derived growth factor-BB (PDGF-BB) compared with that in control group. In addition, the results demonstrated that a higher expression of a miR-374a could promote proliferation and migration of VSMCs while miR-374a inhibitor suppressed the PDGF-BB-induced proliferation and migration of VSMCs in vitro. Furthermore, circTADA2A bound to miR-374a and then upregulated RORA expression, which resulted in inhibition in VSMCs proliferation and migration. On the other hand, the result indicated that GATA2 overexpression could augment the proliferation, migration of PDGF-bb-induced VSMCs, which could be rescued by miR-374a inhibitor. The findings suggested that the GATA2/circTADA2A-miR-374a axis promoted the proliferation and migration of VSMCs by targeting RORA, which were closely related to atherosclerosis (AS). Thus the results might offer a new therapeutic target for AS.

ILC2s induce Treg but not Th2-type immunity through IL-33/ST2 pathway in pulmonary tuberculosis.

We investigated the function of type 2 innate lymphoid cells (ILC2s) and IL-33 in pulmonary tuberculosis (PTB). Peripheral blood samples were collected from PTB patients and healthy controls. The cytometric bead array was used to detect plasma IL-33, TGF-β, IL-4, IL-5, IL-6, IL-10, IL-13, and soluble ST2 (sST2). ILC2s, Th2, and Treg cells were detected with flow cytometry. Quantitative real-time PCR was used to measure mRNA levels. ILC2s were co-cultured with peripheral blood mononuclear cells and then intervened with IL-33 or anti-ST2 antibody + IL-33 in vitro. IL-4, IL-6, IL-5, IL-10, IL-13, and TGF-β levels were measured by enzyme-linked immunosorbent assay. Compared with healthy controls, the levels of IL-33, sST2, TGF-β, IL-10, and IL-6 in the plasma of PTB patients were significantly higher. No significant difference was found in the plasma IL-4, IL-5, and IL-13 levels. Patients with PTB had significantly increased ILC2s proportion and mRNA levels of RAR-related orphan receptor α and GATA binding protein 3. After 48 h of IL-33 stimulation in vitro, Treg cell proportion significantly increased and the IL-10 level was significantly elevated. Treatment with anti-ST2 abolished these effects. No significant difference was found in cytokines of IL-4, IL-6, IL-5, IL-13, and TGF-β, or Th2 cells before and after IL-33 treatment. ILC2s proportion in peripheral blood was increased and plasma IL-33 was upregulated in PTB patients. IL-33 may promote the growth of ILC2s and the production of Treg-related cell cytokines, but not Th2-related cell cytokines, to participate in immune response to PTB.

A RORE-dependent Intronic Enhancer in the IL-7 Receptor-α Locus Controls Glucose Metabolism via Vγ4+ γδT17 Cells.

The IL-7R regulates the homeostasis, activation, and distribution of T cells in peripheral tissues. Although several transcriptional enhancers that regulate IL-7Rα expression in αβ T cells have been identified, enhancers active in γδ T cells remain unknown. In this article, we discovered an evolutionarily conserved noncoding sequence (CNS) in intron 2 of the IL-7Rα-chain (IL-7Rα) locus and named this region CNS9. CNS9 contained a conserved retinoic acid receptor-related orphan receptor (ROR)-responsive element (RORE) and exerted RORγt-dependent enhancer activity invitro. Mice harboring point mutations in the RORE in CNS9 (CNS9-RORmut) showed reduced IL-7Rα expression in IL-17-producing Vγ4+ γδ T cells. In addition, the cell number and IL-17A production of Vγ4+ γδ T cells were reduced in the adipose tissue of CNS9-RORmut mice. Consistent with the reduction in IL-17A, CNS9-RORmut mice exhibited decreased IL-33 expression in the adipose tissue, resulting in fewer regulatory T cells and glucose intolerance. The CNS9-ROR motif was partially responsible for IL-7Rα expression in RORγt+ regulatory T cells, whereas IL-7Rα expression was unaffected in RORγt-expressing Vγ2+ γδ T cells, Th17 cells, type 3 innate lymphoid cells, and invariant NKT cells. Our results indicate that CNS9 is a RORΕ-dependent, Vγ4+ γδ T cell-specific IL-7Rα enhancer that plays a critical role in adipose tissue homeostasis via regulatory T cells, suggesting that the evolutionarily conserved RORΕ in IL-7Rα intron 2 may influence the incidence of type 2 diabetes.

Impairment of Gal-9 and Tim-3 crosstalk between Tregs and Th17 cells drives tobacco smoke-induced airway inflammation.

Overexpression of T-cell immunoglobulin and mucin-domain containing-3 (TIM-3) on T cells has been observed in smokers. However, whether and how galectin-9 (Gal-9)/TIM-3 signal between T-regulatory cells (Tregs) and type 17 helper (Th17) cells contributes to tobacco smoke-induced airway inflammation remains unclear. Here, we aimed to explore the role of the Gal-9/TIM-3 signal between Tregs and Th17 cells during chronic tobacco smoke exposure. Tregs phenotype and the expression of TIM-3 on CD4+ T cells were detected in a mouse model of experimental emphysema. The role of TIM-3 in CD4+ T cells was explored in a HAVCR2-/- mouse model and in mice that received recombinant anti-TIM3. The crosstalk between Gal-9 and Tim-3 was evaluated by coculture Tregs with effector CD4+ T cells. We also invested the expression of Gal-9 in Tregs in patients with COPD. Our study revealed that chronic tobacco smoke exposure significantly reduces the frequency of Tregs in the lungs of mice and remarkably shapes the heterogeneity of Tregs by downregulating the expression of Gal-9. We observed a pro-inflammatory but restrained phenotypic transition of CD4+ T cells after tobacco smoke exposure, which was maintained by TIM-3. The restrained phenotype of CD4+ T cells was perturbed when TIM-3 was deleted or neutralised. Tregs from the lungs of mice with emphysema displayed a blunt ability to inhibit the differentiation and proliferation of Th17 cells. The inhibitory function of Tregs was partially restored by using recombinant Gal-9. The interaction between Gal-9 and TIM-3 inhibits the differentiation of Th17 cells and promotes apoptosis of CD4+ T cells, possibly by interfering with the expression of retinoic acid receptor-related orphan receptor gamma t. The expression of Gal-9 in Tregs was reduced in patients with COPD, which was associated with Th17 response and lung function. These findings present a new paradigm that impairment of Gal-9/Tim-3 crosstalk between Tregs and Th17 cells during chronic tobacco smoke exposure promotes tobacco smoke-induced airway/lung inflammation.