Retinoic Acid-related Orphan Receptor Γt Research Articles

Dihydroberberine alleviates Th17/Treg imbalance in premature ovarian insufficiency mice via inhibiting Rheb/mTOR signaling

BackgroundPremature ovarian insufficiency (POI) is an immune-related condition. Dihydroberberine (dhBBR) plays a regulatory role in maintaining the T-helper 17 (Th17)/regulatory T (Treg) cell balance. This study aimed to explore the action mechanisms of dhBBR on POI.MethodsIn vivo, female BALB/c mice were used as POI models, treated with dhBBR, or injected with recombinant interleukin (rIL)-17 and anti-CD25 monoclonal antibody. Hematoxylin and eosin staining was used to validate the model and assess the therapeutic effects of dhBBR. mRNA expression levels of cytochrome P450 (Cyp)-17a1, Cyp19a1, Cyp11a1, steroidogenic acute regulatory protein, and luteinizing hormone receptor in mouse ovaries were quantified via quantitative polymerase chain reaction (qPCR). Enzyme-linked immunosorbent assay was used to determine the cytokine and sex hormone levels. Immunohistochemical staining for cleaved-caspase 3 and Ki-67 were performed to assess ovarian cell apoptosis and proliferation. Flow cytometry was used to analyze the Th17/Treg cell balance in the ovary and spleen. In vitro cytotoxicity of dhBBR was measured using the cell counting kit-8 assay. GTP-Ras homolog enriched in brain (Rheb) activity was determined via immunofluorescence assay. Co-immunoprecipitation was performed to assess Rheb activity, Th17 or Treg induction, and binding between Rheb and mammalian target of rapamycin (mTOR) after dhBBR treatment. Flow cytometry and qPCR assays were used to verify the effect of dhBBR on CD4 + cell differentiation. Finally, Rheb/mTOR pathway activation was confirmed via western blotting of proteins, including mTOR, p-mTOR, p70S6K, p-p70S6K, 4E-BP1, and p-4E-BP1.ResultsdhBBR improved the ovarian function in a dose-dependent manner. It also decreased ovarian cell apoptosis and increased cell proliferation. It decreased Th1 and Th17 cell proportions but increased Treg cell proportions in the ovaries and spleens of POI model mice. Cell experiments revealed that dhBBR promoted CD4 + cell differentiation into Treg cells. Co-immunoprecipitation revealed Rheb as the dhBBR target that bound to mTOR. However, MHY1485 restored dhBBR-induced changes in forkhead box P3, IL-10, transforming growth factor-β1, IL-17, IL-22, retinoic acid-related orphan receptor-γt and p-mTOR levels in Th17- and Treg-induced CD4 + cells.ConclusionOverall, dhBBR targeted the Rheb/mTOR pathway to promote CD4 + cell differentiation into Treg cells and alleviate POI.

A SAR Study on a Class of 6-(Trifluoromethyl)-pyridine Derivatives as RORγt Inverse Agonists

Background: The nuclear retinoic acid-related orphan receptor γt (RORγt) is an important transcription factor in immune cells. Functionally, RORγt plays an important role in promoting the differentiation of T helper 17 cells and regulating the expression of proinflammatory factors, such as interleukin 17. Therefore, RORγt is considered a promising target for the treatment of the autoimmune disorder. Currently, 21 RORγt inverse agonists with various scaffolds have entered clinical trials. Objective: To discover novel and potent RORγt inverse agonists, a series of novel 6-(trifluoromethyl) pyridine derivatives were designed and synthesized. Methods: We designed and synthesized a series of potent RORγt inverse agonists W1~W16 based on VTP-43742. Molecular docking, molecular dynamics (MD) simulation, and MM/GBSA were used to study the structure-activity relationship (SAR) of the derivatives. Results: The biological activity evaluation indicated that the target compounds showed potent RORγt inhibitory activity. The most active compound, W14, exhibited low nanomolar inhibitory activity (IC50 = 7.5 nM) in the luciferase reporter assay, which was superior to the clinical compound VTP-43742. Analysis of the binding mode of W14 demonstrated that the interaction of -CF3 with Leu324, Leu396, and His479 has an important contribution to the binding. Furthermore, W14 broke the H-bond formed by His479 and Tyr502 via a “push-pull” mechanism. Conclusion: Compound W14 could be used as a potential RORγt inverse agonist for further modification.

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

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

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

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

E. coli LPS/TLR4/NF-κB Signaling Pathway Regulates Th17/Treg Balance Mediating Inflammatory Responses in Oral Lichen Planus.

Oral lichen planus (OLP) is a chronic inflammatory autoimmune disease mediated by T cells. The imbalance of microflora has potential impacts on the onset and development of OLP, but the mechanism is still unclear. Here, we investigated the effects of Escherichia coli (E. coli) lipopolysaccharide (LPS) simulating the microbial enrichment state of OLP on T cell immune functions in vitro. Effect of E. coli LPS on the viability of T cell using CCK8 assay. After E. coli LPS pretreatment, the expression of the toll-like receptor 4 (TLR4), nuclear factor-kappa B p65 (NF-κB p65), cytokines, retinoic acid-related orphan receptor γt (RORγt), and forkhead box p3 (Foxp3) in the peripheral blood of OLP patients and normal controls (NC) were assessed using quantitative RT-PCR (qRT-PCR), western blot, and enzyme-linked immunosorbent assay (ELISA). Finally, Th17 and Treg cells were detected by flow cytometry. We found that the TLR4/NF-κB pathway was activated and the expression of interleukin (IL)-6 and IL-17 was increased in both groups after E. coli LPS stimulation. CC chemokine ligand (CCL)20 and CC chemokine receptor (CCR)4 expression was increased in OLP after E. coli LPS treatment, while no difference was found in CCR6 and CCL17 expression of both groups. Moreover, E. coli LPS treatment enhanced the proportion of Th17 cells, Th17/Treg ratio, and RORγt/Foxp3 ratio in OLP. In conclusion, E. coli LPS regulated Th17/Treg balance to mediate the inflammatory responses of OLP through the TLR4/NF-κB pathway in vitro, indicating that oral microbiota dysbiosis affected the chronic inflammatory state of OLP.

Synthesis of A-9758, an Inverse Agonist of Retinoic Acid-Related Orphan Receptor γt.

A-9758 is an inverse agonist of retinoic acid-related orphan receptor γt with well-characterized in vitro and in vivo anti-inflammatory activity. A chromatography-free decagram-scale synthesis of this compound was developed to support pre-clinical research activities. This route was designed to enable late-stage structure-activity relationship studies of the amide moiety and convergently uses a reductive alkylation sequence between indole and benzaldehyde intermediates. A key advantage of this strategy is the fact that the indole precursor can be alkylated at C2, as required for A-9758, or at C3 to provide access to an isomeric chemical series. Access to the critical indole fragment was expedited via an underutilized SnAr/reductive cyclization cascade sequence, and the benzaldehyde fragment was prepared in two steps from inexpensive 2,4-dichlorobenzoic acid.

Gancao Fuzi decoction regulates the Th17/Treg cell imbalance in rheumatoid arthritis by targeting Foxp3 via miR-34a

Ethnopharmacological relevanceDuring the Eastern Han Dynasty, Zhang Zhongjing first recorded the Gancao Fuzi decoction (GCFZD) formula in the “Synopsis of the Golden Chamber”, which is reportedly an effective and safe treatment for rheumatoid arthritis (RA). However, the mechanism underlying the observed improvement in the T helper 17 (Th17)/regulatory T (Treg) cell imbalance in RA obtained with GCFZD has not been reported. Aim of the studyThis study aimed to demonstrate whether GCFZD ameliorated RA by modulating the Th17/Treg imbalance in RA mice. Materials and methodsCollagen was used to induce a model of collagen-induced arthritis (CIA) in mice. GCFZD was administered by gavage, and the arthritis index score, imaging and histopathological changes of the ankle joints, and the levels of the immunoglobulin G (IgG) class antibodies and proinflammatory factors in serum were determined. In addition, the frequencies of Th17 and Treg cells, the levels of relevant transcription factors and functional factors and the miR-34a gene in the spleen and the levels of interleukin-17A (IL-17A) and IL-10 in serum were determined. ResultsGCFZD significantly reduced the arthritis score, improved joint swelling and bone damage, reduced the pathological score, and decreased the serum levels of IgG class antibody (IgG and IgG2a) and proinflammatory factor [tumour necrosis factor-alpha (TNF-α), IL-1β and IL-6]. Moreover, the Th17-cell proportion, the expression level of the Th17-specific transcription factor retinoic acid-related orphan receptor γt (RORγt) and functional factor IL-17A in the spleen, and the serum IL-17A level were decreased, whereas the Treg cell proportion, expression levels of the Treg-specific transcription factor forkhead box P3 (Foxp3) and functional factor IL-10 in the spleen, and the serum IL-10 level were increased. Furthermore, GCFZD inhibited miR-34a gene expression while promoting Foxp3 protein expression. ConclusionsThe findings of this study demonstrate the therapeutic effect of GCFZD on mice with CIA, and the mechanism is related to an improvement in the Th17/Treg cell imbalance by targeting Foxp3 via miR-34a.

Impact of MyD88, Microbiota, and Location on Type 1 and Type 3 Innate Lymphoid Cells during Toxoplasma gondii Infection.

Toxoplasma gondii induces strong IFN-γ-based immunity. Innate lymphoid cells (ILC), in particular ILC1, are an important innate source of this protective cytokine during infection. Our objective was to determine how MyD88-dependent signaling influences ILC function during peroral compared with i.p. infection with T. gondii. MyD88 +/+ and MyD88 -/- mice were orally inoculated with ME49 cysts, and small intestinal lamina propria ILC were assessed using flow cytometry. We observed T-bet+ ILC1, retinoic acid-related orphan receptor γt+ ILC3, and a population of T-bet+retinoic acid-related orphan receptor γt+ double-positive ILC. In MyD88 -/- mice, IFN-γ-producing T-bet+ ILC1 frequencies were reduced compared with wild-type. Treatment of MyD88 -/- mice with an antibiotic mixture to deplete microflora reduced IFN-γ+ ILC1 frequencies. To examine ILC responses outside of the mucosal immune system, peritoneal exudate cells were collected from wild-type and knockout mice after i.p. inoculation with ME49 cysts. In this compartment, ILC were highly polarized to the ILC1 subset that increased significantly and became highly positive for IFN-γ over the course of infection. Increased ILC1 was associated with expression of the Ki67 cell proliferation marker, and the response was driven by IL-12p40. In the absence of MyD88, IFN-γ expression by ILC1 was not maintained, but proliferation remained normal. Collectively, these data reveal new aspects of ILC function that are influenced by location of infection and shaped further by MyD88-dependent signaling.

Interleukin-17 governs hypoxic adaptation of injured epithelium.

Mammalian cells autonomously activate hypoxia-inducible transcription factors (HIFs) to ensure survival in low-oxygen environments. We report here that injury-induced hypoxia is insufficient to trigger HIF1α in damaged epithelium. Instead, multimodal single-cell and spatial transcriptomics analyses and functional studies reveal that retinoic acid-related orphan receptor γt+ (RORγt+) γδ T cell-derived interleukin-17A (IL-17A) is necessary and sufficient to activate HIF1α. Protein kinase B (AKT) and extracellular signal-regulated kinase 1/2 (ERK1/2) signaling proximal of IL-17 receptor C (IL-17RC) activates mammalian target of rapamycin (mTOR) and consequently HIF1α. The IL-17A-HIF1α axis drives glycolysis in wound front epithelia. Epithelial-specific loss of IL-17RC, HIF1α, or blockade of glycolysis derails repair. Our findings underscore the coupling of inflammatory, metabolic, and migratory programs to expedite epithelial healing and illuminate the immune cell-derived inputs in cellular adaptation to hypoxic stress during repair.

Elevated Th17 cell proportion, related cytokines and mRNA expression level in patients with hypertension-mediated organ damage: a case control study.

BackgroundImmune abnormalities and inflammatory responses play critical roles in progression of hypertension. Basic studies have confirmed that Th17 cell and related cytokines are important in promoting hypertension-mediated organ damage, but few clinical evidences have been published. Therefore, our study aimed to investigate the relationship between Th17 cell and its related cytokines and hypertension-mediated organ damage in human.MethodsThis study enrolled 179 patients with hypertension (including 92 with hypertension-mediated organ damage and 87 without hypertension-mediated organ damage) and 63 healthy participants. The proportion of Th17 cells in peripheral blood mononuclear cells was measured by flow cytometry. The concentrations of interleukin-17 and interleukin-23 were detected by enzyme-linked immunosorbent assay. Real time-polymerase chain reaction was used to detect the mRNA expression levels of interleukin-17, retinoic acid-related orphan receptor (ROR) γt and signal transducer and activator of transcription-3 (STAT-3).ResultsThe proportion of Th17 cells, the concentration of interleukin-17 and interleukin-23 and the mRNA expression levels of interleukin-17, retinoic acid-related orphan receptor γt and signal transducer and activator of transcription-3 were significantly increased in hypertension-mediated organ damage group compared with those in non-hypertension-mediated organ damage group and control group (P < 0.005).ConclusionTh17 cells and their associated cytokines may be involved in hypertension-mediated organ damage formation and may be able to serve as new biomarkers of hypertension-mediated organ damage and potential therapeutic targets.

RORγt-Expressing Pathogenic CD4+ T Cells Cause Brain Inflammation during Chronic Colitis.

Neurobehavioral disorders and brain abnormalities have been extensively reported in both Crohn's disease and ulcerative colitis patients. However, the mechanism causing neuropathological disorders in inflammatory bowel disease patients remains unknown. Studies have linked the Th17 subset of CD4+ T cells to brain diseases associated with neuroinflammation and cognitive impairment, including multiple sclerosis, ischemic brain injury, and Alzheimer's disease. To better understand how CD4+ T lymphocytes contribute to brain pathology in chronic intestinal inflammation, we investigated the development of brain inflammation in the T cell transfer model of chronic colitis. Our findings demonstrate that CD4+ T cells infiltrate the brain of colitic Rag1 -/- mice in proportional levels to colitis severity. Colitic mice developed hypothalamic astrogliosis that correlated with neurobehavioral disorders. Moreover, the brain-infiltrating CD4+ T cells expressed Th17 cell transcription factor retinoic acid-related orphan receptor γt (RORγt) and displayed a pathogenic Th17 cellular phenotype similar to colonic Th17 cells. Adoptive transfer of RORγt-deficient naive CD4+ T cells failed to cause brain inflammation and neurobehavioral disorders in Rag1 -/- recipients, with significantly less brain infiltration of CD4+ T cells. The finding is mirrored in chronic dextran sulfate sodium-induced colitis in Rorcfl/fl Cd4-Cre mice that showed lower frequency of brain-infiltrating CD4+ T cells and astrogliosis despite onset of significantly more severe colitis compared with wild-type mice. These findings suggest that pathogenic RORγt+CD4+ T cells that aggravate colitis migrate preferentially into the brain, contributing to brain inflammation and neurobehavioral disorders, thereby linking colitis severity to neuroinflammation.

PRMT5 in T Cells Drives Th17 Responses, Mixed Granulocytic Inflammation, and Severe Allergic Airway Inflammation.

Severe asthma is characterized by steroid insensitivity and poor symptom control and is responsible for most asthma-related hospital costs. Therapeutic options remain limited, in part due to limited understanding of mechanisms driving severe asthma. Increased arginine methylation, catalyzed by protein arginine methyltransferases (PRMTs), is increased in human asthmatic lungs. In this study, we show that PRMT5 drives allergic airway inflammation in a mouse model reproducing multiple aspects of human severe asthma. We find that PRMT5 is required in CD4+ T cells for chronic steroid-insensitive severe lung inflammation, with selective T cell deletion of PRMT5 robustly suppressing eosinophilic and neutrophilic lung inflammation, pathology, airway remodeling, and hyperresponsiveness. Mechanistically, we observed high pulmonary sterol metabolic activity, retinoic acid-related orphan receptor γt (RORγt), and Th17 responses, with PRMT5-dependent increases in RORγt's agonist desmosterol. Our work demonstrates that T cell PRMT5 drives severe allergic lung inflammation and has potential implications for the pathogenesis and therapeutic targeting of severe asthma.

IL-22 Plays a Dual Role in the Amniotic Cavity: Tissue Injury and Host Defense against Microbes in Preterm Labor.

IL-22 is a multifaceted cytokine with both pro- and anti-inflammatory functions that is implicated in multiple pathologies. However, the role of IL-22 in maternal-fetal immunity in late gestation is poorly understood. In this study, we first showed that IL-22+ T cells coexpressing retinoic acid-related orphan receptor γt (ROR-γt) are enriched at the human maternal-fetal interface of women with preterm labor and birth, which was confirmed by in silico analysis of single-cell RNA sequencing data. T cell activation leading to preterm birth in mice was preceded by a surge in IL-22 in the maternal circulation and amniotic cavity; however, systemic administration of IL-22 in mice did not induce adverse perinatal outcomes. Next, using an ex vivo human system, we showed that IL-22 can cross from the choriodecidua to the intra-amniotic space, where its receptors (Il22ra1, Il10rb, and Il22ra2) are highly expressed by murine gestational and fetal tissues in late pregnancy. Importantly, amniotic fluid concentrations of IL-22 were elevated in women with sterile or microbial intra-amniotic inflammation, suggesting a dual role for this cytokine. The intra-amniotic administration of IL-22 alone shortened gestation and caused neonatal death in mice, with the latter outcome involving lung maturation and inflammation. IL-22 plays a role in host response by participating in the intra-amniotic inflammatory milieu preceding Ureaplasma parvum-induced preterm birth in mice, which was rescued by the deficiency of IL-22. Collectively, these data show that IL-22 alone is capable of causing fetal injury leading to neonatal death and can participate in host defense against microbial invasion of the amniotic cavity leading to preterm labor and birth.

Activation of retinoic acid-related orphan receptor γ(t) by parabens and benzophenone UV-filters

Retinoic acid-related orphan receptor γt (RORγt) regulates immune responses and its impaired function contributes to inflammatory and autoimmune diseases and may promote skin cancer. Synthetic inverse RORγt agonists block the production of Th17-associated cytokines including interleukin (IL)-17A and IL-22 and are under investigation for treatment of such pathologies. Unintentional RORγt activation in skin, following exposure to environmental chemicals, may promote inflammatory skin disease. Parabens and UV-filters, frequently used as additives in cosmetics and body care products, are intensively inspected for endocrine disrupting properties. This study assessed whether such compounds can interfere with RORγ activity using a previously established tetracycline-inducible reporter gene assay in CHO cells. These transactivation experiments revealed hexylparaben, benzylparaben and benzophenone-10 as RORγ agonists (EC50 values: 144 ± 97 nM, 3.39 ± 1.74 µM and 1.67 ± 1.04 µM, respectively), and they could restore RORγ activity after suppression by an inverse agonist. Furthermore, they enhanced RORγt-dependent transcription of the pro-inflammatory IL-17A and/or IL-22 genes in the murine T-cell model EL4. Virtual screening of a cosmetics database for structurally similar chemicals and in vitro testing of the most promising hits revealed benzylbenzoate, benzylsalicylate and 4-methylphenylbenzoate as RORγ agonists (low micromolar EC50 values). Moreover, an analysis of mixtures of the newly identified RORγ agonists suggested additive effects. This study presents novel RORγ(t) agonistic structural scaffolds. By activating RORγ(t) the identified parabens and UV-filters may potentially aggravate pathophysiological conditions, especially skin diseases where highest exposure of such chemicals can be expected. Follow-up studies should assess whether such compounds, either alone or as mixtures, can reach relevant concentrations in tissues and target cells to activate RORγ(t) in vivo.

Crosstalk between bile acid-activated receptors and microbiome in entero-hepatic inflammation.

Bile acids are potent signaling molecules exerting diverse actions through bile acid-activated receptors. Among them, the Farnesoid X receptor (FXR) and the Takeda G protein-coupled receptor 5 (TGR5; GPBAR1), modulate the inflammation occurring in chronic/acute hepatitis, cholestasis, and inflammatory bowel disease. A role for other bile acid-responsive receptors in this context is emerging. This review aims to summarize recent advances on the immune-modulatory actions of the bile acid-responsive receptors Shingosine-1-phosphate receptor 2 (S1PR2), pregnane X receptor (PXR), constitutive androstane receptor (CAR), vitamin D receptor (VDR), and retinoic acid-related orphan receptor γt (RORγt). How microbiota-derived bile acids contribute to intestinal and hepatic inflammation, potentially through these receptors, is also discussed. These concepts pave the way to novel and innovative strategies aiming at modulating the gut microbiota to tackle inflammatory syndromes.

IL-23 Promotes Neutrophil Extracellular Trap Formation and Bacterial Clearance in a Mouse Model of Alcohol and Burn Injury.

Our previous studies have shown that ethanol intoxication combined with burn injury increases intestinal bacterial growth, disrupts the intestinal barrier, and enhances bacterial translocation. Additionally, studies show that Th17 effector cytokines IL-17 and IL-22, which are dependent on IL-23, play important roles in maintaining intestine mucosal barrier integrity. Recent findings suggest neutrophils are a significant source of IL-17 and IL-22. We determined the effect of ethanol and burn injury on neutrophil IL-17 and IL-22 production, as well as their ability to phagocytose and in bacterial clearance, and whether these effects are modulated by IL-23. Mice were given ethanol 4 h prior to receiving ∼12.5% total body surface area burn and were euthanized day 1 after injury. We observed that intoxication combined with burn injury significantly decreases blood neutrophil phagocytosis and bacteria killing, as well as their ability to produce IL-17 and IL-22, compared with sham vehicle mice. The treatment of neutrophils with rIL-23 significantly increases IL-22 and IL-17 release and promotes expression of IL-23R, retinoic acid-related orphan receptor γt, Lipocalin2, and Nod-like receptor 2 following ethanol and burn injury. Furthermore, IL-22- and IL-17-producing neutrophils have enhanced neutrophil extracellular trap formation and bacterial killing ability, which are dependent on IL-23. Finally, although we observed that peritoneal neutrophils harvested after casein treatment are functionally different from blood neutrophils, both blood and peritoneal neutrophils exhibited the same response to rIL-23 treatment. Together these findings suggest that IL-23 promotes neutrophil IL-22 and IL-17 production and their ability to kill bacteria following ethanol and burn injury.

RORγt agonist synergizes with CTLA-4 antibody to inhibit tumor growth through inhibition of Treg cells via TGF-β signaling in cancer

To date, the overall response rate to checkpoint blockade remains unsatisfactory, partially due to the limited understanding of the tumor immune microenvironment. The retinoic acid-related orphan receptor γt (RORγt) is the key transcription factor of T helper cell 17 (Th17) cells and plays an essential role in tumor immunity. In this study, we used JG-1, a potent and selective small-molecule RORγt agonist to evaluate the therapeutic potential and mechanism of action of targeting RORγt in tumor immunity. JG-1 promotes Th17 cells differentiation and inhibition of regulatory T (Treg) cells differentiation. JG-1 demonstrates robust tumor growth inhibition in multiple syngeneic models and shows a synergic effect with the Cytotoxic T-Lymphocyte Antigen 4 (CTLA-4) antibody. In tumors, JG-1 not only promotes Th17 cells differentiation and increases C-C Motif Chemokine Receptor 6 (CCR6)- Chemokine (C-C motif) ligand 20 (CCL20) expression, but also inhibits both the expression of transforming growth factor-β1 (TGF-β1) and the differentiation and infiltration of Treg cells. In summary, JG-1 is a lead compound showing a potent activity in vitro and robust tumor growth inhibition in vivo with synergetic effects with anti-CTLA-4.

Overexpression of Hepatocyte Growth Factor in Dental Pulp Stem Cells Ameliorates the Severity of Psoriasis by Reducing Inflammatory Responses.

Psoriasis is an autoimmune disease still lacking standard treatment, and it has been demonstrated that mesenchymal stem cells (MSCs) are capable of immunoregulation. The underlying mechanism might involve the secretion of soluble cytokines, such as hepatocyte growth factor (HGF). This study aims to investigate the therapeutic effect of HGF-overexpressed dental pulp stem cells (DPSCs) [DPSCs; HGF overexpressed DPSCs (HGF-DPSCs)] on imiquimod-induced psoriasis. DPSCs were isolated and transfected by adenovirus vector carrying HGF gene (Ad-HGF). The immunoregulatry abilities of DPSCs and HGF-DPSCs were investigated by coculture of the MSCs with peripheral blood mononuclear cells (PBMCs) under appropriated stimulation. The psoriatic mice were treated with saline control, DPSCs, or HGF-DPSCs. Then the mice spleens were collected and weighted. The psoriatic skin lesions were analyzed by Hematoxylin/Eosin and immunohistochemical staining for histopathological changes, and quantitative real-time polymerase chain reaction to detect the expression levels of CD4+ T cell-related transcription factors and cytokines. The mice blood serum was measured by MILLIPLEX analysis and enzyme-linked immunosorbent assay to evaluate the expression levels of inflammation cytokines. The coculture experiments showed HGF overexpression enhanced the immunoregulation abilities of DPSCs not by suppressing PBMCs' proliferation, but by downregulating T helper 1 (Th1), Th17 cells, and upregulating regulatory T (Treg) cells. In psoriatic skin lesions, the psoriasis-like erythema, scaling, and thickening were ameliorated; and the expression of cytokeratin 6 (CK6), and cytokeratin 17 (CK17) were downregulated by DPSCs and HGF-DPSCs treatment. HGF overexpression enhanced the decrease of spleen masses; enhanced the downregulation of the expression levels of interferon-gamma (IFN-γ), tumor necrosis factor-α, and interleukin (IL)-17A in the blood serums; enhanced the downregulation of T-box transcription factor 21 (T-bet), IFN-γ, retinoic acid-related orphan receptor-γt (RORγt), IL-17A, IL-17F, IL-23, and upregulation of Foxp3 and IL-10 in the psoriatic skin lesions. Therefore, HGF overexpression enhanced DPSCs' treatment effect on psoriasis mainly by reducing inflammatory responses. These findings might provide new immunoregulation strategies for psoriasis treatment.

Dendritic Cells Require TMEM176A/B Ion Channels for Optimal MHC Class II Antigen Presentation to Naive CD4+ T Cells.

Intracellular ion fluxes emerge as critical actors of immunoregulation but still remain poorly explored. In this study, we investigated the role of the redundant cation channels TMEM176A and TMEM176B (TMEM176A/B) in retinoic acid-related orphan receptor γt+ cells and conventional dendritic cells (DCs) using germline and conditional double knockout mice. Although Tmem176a/b appeared surprisingly dispensable for the protective function of Th17 and group 3 innate lymphoid cells in the intestinal mucosa, we found that they were required in conventional DCs for optimal Ag processing and presentation to CD4+ T cells. Using a real-time imaging method, we show that TMEM176A/B accumulate in dynamic post-Golgi vesicles preferentially linked to the late endolysosomal system and strongly colocalize with HLA-DM. Taken together, our results suggest that TMEM176A/B ion channels play a direct role in the MHC class II compartment of DCs for the fine regulation of Ag presentation and naive CD4+ T cell priming.

Substituted diaryl ether compounds as retinoic acid-related orphan Receptor-γt (RORγt) agonists

The discovery of a series of substituted diarylether compounds as retinoic acid related orphan receptor γt (RORγt) agonists is described. Compound 1 was identified from deck mining as a RORγt agonist. Hit-to-lead optimization led to the identification of lead compound 5, which possesses improved potency (10x). Extensive SAR exploration led to the identification of a potent and selective compound 22, that demonstrated an improved pharmacokinetic profile and a dose-dependent pharmacodynamic response. However, when dosed in a MC38 syngeneic tumor model, no evidence of efficacy was observed. ©2020 Elsevier Science Ltd. All rights reserved.