ABSTRACT
Cholestatic liver diseases, such as primary biliary cholangitis (PBC) and primary sclerosing cholangitis (PSC), lead to inflammation and severe hepatic damage with limited therapeutic options. This study assessed the efficacy of the inverse RORγt agonist, GSK805, both in vitro using the hepatic stellate cell-line LX-2 and in vivo using male bile duct-ligated BALB/c mice. In vitro, 0.3 µM GSK805 reduced alpha-smooth muscle actin expression in LX-2 cells. In vivo, GSK805 significantly decreased IL-23R, TNF-α, and IFN-γ expression in cholestatic liver. Despite high concentrations of GSK805 in the liver, no significant reduction in fibrosis was noticed. GSK805 significantly increased aspartate aminotransferase and alanine aminotransferase activity in the blood, while levels of glutamate dehydrogenase, alkaline phosphatase, and bilirubin were not substantially increased. Importantly, GSK805 did neither increase an animal distress score nor substantially reduce body weight, burrowing activity, or nesting behavior. These results suggest that a high liver concentration of GSK805 is achieved by daily oral administration and that this drug modulates inflammation in cholestatic mice without impairing animal well-being.
Subject(s)
Mice, Inbred BALB C , Animals , Mice , Male , Humans , Actins/metabolism , Liver/metabolism , Liver/drug effects , Hepatic Stellate Cells/metabolism , Hepatic Stellate Cells/drug effects , Nuclear Receptor Subfamily 1, Group F, Member 3/metabolism , Cell Line , Inflammation/metabolism , Inflammation/drug therapy , Cholestasis/metabolism , Cholestasis/drug therapyABSTRACT
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 αß 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 in vitro. 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.
Subject(s)
Enhancer Elements, Genetic , Introns , Nuclear Receptor Subfamily 1, Group F, Member 3 , Receptors, Antigen, T-Cell, gamma-delta , Animals , Mice , Introns/genetics , Receptors, Antigen, T-Cell, gamma-delta/genetics , Receptors, Antigen, T-Cell, gamma-delta/metabolism , Enhancer Elements, Genetic/genetics , Nuclear Receptor Subfamily 1, Group F, Member 3/genetics , Nuclear Receptor Subfamily 1, Group F, Member 3/metabolism , Glucose/metabolism , Receptors, Interleukin-7/genetics , Receptors, Interleukin-7/metabolism , Mice, Inbred C57BL , Th17 Cells/immunology , Interleukin-17/metabolism , Interleukin-17/genetics , Humans , Adipose Tissue/metabolism , Adipose Tissue/immunologyABSTRACT
Neuroimmune cross-talk participates in intestinal tissue homeostasis and host defense. However, the matrix of interactions between arrays of molecularly defined neuron subsets and of immunocyte lineages remains unclear. We used a chemogenetic approach to activate eight distinct neuronal subsets, assessing effects by deep immunophenotyping, microbiome profiling, and immunocyte transcriptomics in intestinal organs. Distinct immune perturbations followed neuronal activation: Nitrergic neurons regulated T helper 17 (TH17)-like cells, and cholinergic neurons regulated neutrophils. Nociceptor neurons, expressing Trpv1, elicited the broadest immunomodulation, inducing changes in innate lymphocytes, macrophages, and RORγ+ regulatory T (Treg) cells. Neuroanatomical, genetic, and pharmacological follow-up showed that Trpv1+ neurons in dorsal root ganglia decreased Treg cell numbers via the neuropeptide calcitonin gene-related peptide (CGRP). Given the role of these neurons in nociception, these data potentially link pain signaling with gut Treg cell function.
Subject(s)
Calcitonin Gene-Related Peptide , Ganglia, Spinal , Neuroimmunomodulation , Nociceptors , T-Lymphocytes, Regulatory , TRPV Cation Channels , Th17 Cells , Animals , Mice , Calcitonin Gene-Related Peptide/metabolism , Calcitonin Gene-Related Peptide/genetics , Cholinergic Neurons/metabolism , Ganglia, Spinal/metabolism , Ganglia, Spinal/cytology , Gastrointestinal Microbiome , Intestines/immunology , Intestines/cytology , Macrophages/immunology , Macrophages/metabolism , Mice, Inbred C57BL , Nociception , Nociceptors/metabolism , Nuclear Receptor Subfamily 1, Group F, Member 3/metabolism , T-Lymphocytes, Regulatory/immunology , T-Lymphocytes, Regulatory/metabolism , Th17 Cells/immunology , TRPV Cation Channels/metabolism , TRPV Cation Channels/geneticsABSTRACT
Objective: To investigate the roles of histone H3K27me3 methylation and its regulatory enzymes JMJD3 and EZH2 in the differentiation of Th17 cells in ankylosing spondylitis (AS), to unveil their potential involvement in the pathogenesis of AS, and to provide new strategies and targets for the clinical treatment of AS by analyzing the methylation state of H3K27me3 and its interactions with Th17-related factors. Methods: A total of 84 AS patients (42 active AS patiens and 42 patients in the stable phase of AS) were enrolled for the study, while 84 healthy volunteers were enrolled as the controls. Blood samples were collected. Peripheral blood mononuclear cells were isolated. ELISA assay was performed to examine Th17 cells and the relevant cytokines IL-21, IL-22, and IL-17. The mRNA expressions of RORc, JAK2, and STAT3 were analyzed by RT-PCR, the protein expressions of RORc, JAK2/STAT3 pathway protein, H3K27me3 and the relevant protease (EZH2 and JMJD3) were determined by Western blot. Correlation between H3K27me3, EZH2 and JMJD3 and the key signaling pathway molecules of Th cell differentiation was analyzed by Pearson correlation analysis. Results: The mRNA expressions of RORc, JAK2, and STAT3 were significantly higher in the active phase group than those in the stable phase group ( P<0.05). The relative grayscale values of H3K27me3 and EZH2 in the active phase group were lower than those of the stable phase group, which were lower than those of the control group, with the differences being statistically significant ( P<0.05). The relative grayscale values of JMJD3, RORc, JAK2, pJAK2, STAT3, and pSTAT3 proteins were significantly higher in the active phase group than those in the stable phase group, which were higher than those in the control group (all P<0.05). The proportion of Th17 and the expression level of inflammatory factors in the active period group were higher than those in the other two groups (P<0.05). H3K27me3 was negatively correlated with RORc, JAK2, STAT3, and IL-17, JMJD3 was positvely correlated with JAK2, STAT3, and IL-17, and EZH2 was negatively correlated with JAK2, STAT3, and IL-17 (all P<0.05). Conclusion: The low expression of H3K27me3 in AS is influenced by the gene loci JMJD3 and EZH2, which can regulate the differentiation of Th17 cells and thus play a role in the pathogenesis and progression of AS.
Subject(s)
Cell Differentiation , Enhancer of Zeste Homolog 2 Protein , Epigenesis, Genetic , Histones , Interleukin-17 , Jumonji Domain-Containing Histone Demethylases , Nuclear Receptor Subfamily 1, Group F, Member 3 , STAT3 Transcription Factor , Spondylitis, Ankylosing , Th17 Cells , Humans , Spondylitis, Ankylosing/genetics , Spondylitis, Ankylosing/metabolism , Th17 Cells/metabolism , Th17 Cells/cytology , Th17 Cells/immunology , Jumonji Domain-Containing Histone Demethylases/metabolism , Jumonji Domain-Containing Histone Demethylases/genetics , Histones/metabolism , STAT3 Transcription Factor/metabolism , STAT3 Transcription Factor/genetics , Enhancer of Zeste Homolog 2 Protein/metabolism , Enhancer of Zeste Homolog 2 Protein/genetics , Interleukin-17/metabolism , Interleukin-17/genetics , Nuclear Receptor Subfamily 1, Group F, Member 3/metabolism , Nuclear Receptor Subfamily 1, Group F, Member 3/genetics , Janus Kinase 2/metabolism , Janus Kinase 2/genetics , Methylation , Interleukins/metabolism , Interleukins/genetics , Interleukin-22 , Male , Female , AdultABSTRACT
Objective To investigate the effect of Terminalia chebula water extract (TCWE) on the cellular immunity and PD-1/PD-L1 pathway in rats with collagen-induced arthritis (CIA). Methods SD rats were randomly divided into four groups: a control group, a CIA group, a TCWE group and a methotrexate (MTX) group, with 15 rats in each group. Except for the control group, SD rats in other groups were subcutaneously injected with type II collagen to establish the model of collagen-induced arthritis (CIA). The rats in the TCWE group were treated with 20 mg/(kg.d) TCWE and the rats in the MTX group were treated with 1.67 mg/(kg.d) MTX. After 14 days of treatment, the cartilage morphology was examined using hematoxylin-eosin (HE) staining, and splenic T lymphocyte apoptosis and Treg/Th17 cell ratio were detected by flow cytometry. The mRNA expressions of retinoid-related orphan nuclear receptor γt (RORγt), forkhead box P3 (FOXP3), PD-1 and PD-L1 in spleen were detected by reverse transcription PCR. The expression and localization of RORγt and FOXP3 were detected by immunohistochemical staining. The protein expressions of PD-1 and PD-L1 in splenic lymphocytes were detected by Western blot, and the levels of serum interleukin 17 (IL-17) and transforming growth factor ß (TGF-ß) in rats were detected by ELISA. Results Compared with CIA group, the pathological changes of cartilage and synovium were significantly alleviated in the TCWE group and the MTX group. Both the apoptosis rate of T lymphocytes in spleen and the ratio of Treg/Th17 cells increased. The expression of RORγt decreased, while the expressions of FOXP3, PD-1 and PD-L1 increased in spleen lymphocytes. The level of serum IL-17 decreased, while the level of serum TGF-ß increased. Conclusion TCWE treatment may activate PD-1/PD-L1 pathway in spleen cells to regulate cellular immunity, thus reducing cartilage injury in CIA rats.
Subject(s)
Arthritis, Experimental , B7-H1 Antigen , Programmed Cell Death 1 Receptor , Rats, Sprague-Dawley , Spleen , Terminalia , Animals , Arthritis, Experimental/immunology , Arthritis, Experimental/drug therapy , Arthritis, Experimental/metabolism , Spleen/drug effects , Spleen/immunology , Spleen/metabolism , B7-H1 Antigen/genetics , B7-H1 Antigen/metabolism , Programmed Cell Death 1 Receptor/genetics , Programmed Cell Death 1 Receptor/metabolism , Rats , Terminalia/chemistry , Male , Immunity, Cellular/drug effects , Up-Regulation/drug effects , Plant Extracts/pharmacology , Nuclear Receptor Subfamily 1, Group F, Member 3/genetics , Nuclear Receptor Subfamily 1, Group F, Member 3/metabolism , Inflammation/drug therapy , Inflammation/immunology , Inflammation/metabolism , T-Lymphocytes, Regulatory/immunology , T-Lymphocytes, Regulatory/drug effects , T-Lymphocytes, Regulatory/metabolism , Th17 Cells/immunology , Th17 Cells/drug effects , Th17 Cells/metabolismABSTRACT
Cardiac glycosides, derived from plants and animals, have been recognized since ancient times. These substances hinder the function of the sodium-potassium pump within eukaryotic cells. Many reports have shown that these compounds influence the activity of nuclear receptors. Thus, we assessed the effects of various cardiac glycosides at nontoxic concentrations on RORγ and RORγT. RORγT is a crucial protein involved in the differentiation of Th17 lymphocytes. Sixteen analyzed cardiac glycosides exhibited varying toxicities in HepG2 cells, all of which demonstrated agonistic effects on RORγ, as confirmed in the RORγ-HepG2 reporter cell line. The overexpression of both the RORγ and RORγT isoforms intensified the effects of these compounds. Additionally, these glycosides induced the expression of G6PC, a gene regulated by RORγ, in HepG2 cells. Subsequently, the effects of two endogenous cardiac glycosides (marinobufagenin and ouabain) and the three most potent glycosides (bufalin, oleandrin, and telecinobufagenin) were evaluated in Th17 primary lymphocytes. All of these compounds increased the expression of the IL17A, IL17F, IFNG, and CXCL10 genes, but they exhibited varying effects on GZMB and CCL20 expression. Molecular docking analysis revealed the robust binding affinity of cardiac glycosides for the ligand binding domain of the RORγ/RORγT receptors. Thus, we demonstrated that at nontoxic concentrations, cardiac glycosides have agonistic effects on RORγ/RORγT nuclear receptors, augmenting their activity. This potential can be harnessed to modulate the phenotype of IL17-expressing cells (e.g., Th17 or Tc17 lymphocytes) in adoptive therapy for combating various types of cancer.
Subject(s)
Cardiac Glycosides , Molecular Docking Simulation , Nuclear Receptor Subfamily 1, Group F, Member 3 , Th17 Cells , Humans , Nuclear Receptor Subfamily 1, Group F, Member 3/metabolism , Nuclear Receptor Subfamily 1, Group F, Member 3/agonists , Nuclear Receptor Subfamily 1, Group F, Member 3/genetics , Hep G2 Cells , Cardiac Glycosides/pharmacology , Cardiac Glycosides/chemistry , Th17 Cells/metabolism , Th17 Cells/drug effects , Th17 Cells/immunologyABSTRACT
OBJECTIVE: To investigate the changes of Notch signaling molecules and Th22 cells in adult patients with infectious mononucleosis (IM), and assess the regulatory function of Notch signaling inhibition to Th22 cells. METHODS: Forty-two IM patients and twenty-one healthy controls were enrolled in this study. Their peripheral blood was collected, from which plasma and peripheral blood mononuclear cells (PBMCs) were isolated. Plasma interleukin (IL)-17 and IL-22 were measured by enzyme-linked immunosorbent assay. The percentages of CD3+ CD4+ IL-17+ Th17 cells and CD3+ CD4+ IL-22+ Th22 cells were investigated by flow cytometry. The mRNA relative levels corresponding to Th17 transcription factor retinoic acid related orphan receptor γt (RORγt), Th22 transcription factor aryl hydrocarbon receptor (AhR), and Notch signaling pathway molecules (including Notch receptors, Notch ligands, Notch downstream molecules) were semi-quantified by real-time PCR. CD4+ T cells were purified and stimulated with γ-secretase inhibitor (GSI). Cellular proliferation, Th17 and Th22 percentage, IL-17 and IL-22 secretion, transcription factor mRNA were measured in response to GSI stimulation. RESULTS: The relative expression levels of Notch1 and Notch2 mRNA in PBMCs of IM group were 13.58±3.18 and 4.73±1.16, respectively, which were significantly higher than 1.09±0.12 and 1.07±0.15 in PBMCs of control group (both P < 0.001). However, there were no significant differences in relative expression levels of Notch3 and Notch4 mRNA between IM group and control group (P >0.05). The relative expression levels of Notch ligands (including DLL1 and Jagged1 ) mRNA and Notch downstream molecules (including Hes1, Hes5, and Hey1 ) were increased in IM group compared with control group (all P < 0.001). In IM group, the Th17 and Th22 percentage were 5.03%±1.15% and 4.48%±1.29%, respectively, which were both higher than 4.36%±0.82% and 3.83%±0.55% in control group (both P < 0.05). In IM group, the IL-17 and IL-22 level were (301.1±53.82) and (101.2±16.45) pg/ml, respectively, which were both higher than (237.2±72.18) and (84.75±11.83) pg/ml in control group (both P < 0.001). In IM group, the relative expression levels of RORγt and AhR mRNA were 1.25±0.22 and 1.21±0.12, respectively, which were both higher than 0.99±0.15 and 1.04±0.11 in control group (both P < 0.001). There were no remarkable differences in CD4+ T cell proliferation, Th17 percentage, IL-17 secretion, and relative expression level of RORγt mRNA between cells with GSI stimulation and without GSI stimulation (P >0.05). GSI stimulation reduced Th22 percentage, IL-22 secretion, and relative expression level of AhR mRNA compared with non-stimulation (all P < 0.05). CONCLUSION: Notch signaling pathway regulates IL-22 secretion by CD4+ T cells via AhR in IM patients. Notch-AhR-Th22 pathway may take part in the pathogenesis of IM.
Subject(s)
Infectious Mononucleosis , Interleukin-17 , Interleukin-22 , Interleukins , Nuclear Receptor Subfamily 1, Group F, Member 3 , Receptors, Notch , Signal Transduction , Th17 Cells , Humans , Adult , Th17 Cells/metabolism , Receptors, Notch/metabolism , Interleukin-17/metabolism , Nuclear Receptor Subfamily 1, Group F, Member 3/metabolism , Infectious Mononucleosis/metabolism , Interleukins/metabolism , Herpesvirus 4, Human , Leukocytes, Mononuclear/metabolism , Receptor, Notch1/metabolism , Receptors, Aryl Hydrocarbon/metabolism , CD4-Positive T-Lymphocytes/metabolismABSTRACT
Diet composition impacts metabolic health and is now recognized to shape the immune system, especially in the intestinal tract. Nutritional imbalance and increased caloric intake are induced by high-fat diet (HFD) in which lipids are enriched at the expense of dietary fibers. Such nutritional challenge alters glucose homeostasis as well as intestinal immunity. Here, we observed that short-term HFD induced dysbiosis, glucose intolerance and decreased intestinal RORγt+ CD4 T cells, including peripherally-induced Tregs and IL17-producing (Th17) T cells. However, supplementation of HFD-fed male mice with the fermentable dietary fiber fructooligosaccharides (FOS) was sufficient to maintain RORγt+ CD4 T cell subsets and microbial species known to induce them, alongside having a beneficial impact on glucose tolerance. FOS-mediated normalization of Th17 cells and amelioration of glucose handling required the cDC2 dendritic cell subset in HFD-fed animals, while IL-17 neutralization limited FOS impact on glucose tolerance. Overall, we uncover a pivotal role of cDC2 in the control of the immune and metabolic effects of FOS in the context of HFD feeding.
Subject(s)
Dendritic Cells , Diet, High-Fat , Homeostasis , Mice, Inbred C57BL , Oligosaccharides , Animals , Oligosaccharides/pharmacology , Diet, High-Fat/adverse effects , Dendritic Cells/immunology , Dendritic Cells/drug effects , Dendritic Cells/metabolism , Male , Mice , Th17 Cells/immunology , Th17 Cells/metabolism , Th17 Cells/drug effects , Glucose/metabolism , Interleukin-17/metabolism , Dietary Fiber/pharmacology , Glucose Intolerance/immunology , Glucose Intolerance/metabolism , Nuclear Receptor Subfamily 1, Group F, Member 3/metabolism , Dysbiosis/immunology , Gastrointestinal Microbiome/drug effectsABSTRACT
Th17 cells mediated immune response is the basis of a variety of autoimmune diseases, including multiple sclerosis and its mouse model of immune aspects, experimental autoimmune encephalomyelitis (EAE). The gene network that drives both the development of Th17 and the expression of its effector program is dependent on the transcription factor RORγt. In this report, we showed that Peptidylprolyl Cis/Trans Isomerase, NIMA-Interacting 1 (Pin1) formed a complex with RORγt, and enhanced its transactivation activity, thus sustained the expression of the effector genes as well as RORγt in the EAE-pathogenic Th17 cells. We first found out that PIN1 was highly expressed in the samples from patients of multiple sclerosis, and the expression of Pin1 by the infiltrating lymphocytes in the central nerve system of EAE mice was elevated as well. An array of experiments with transgenic mouse models, cellular and molecular assays was included in the study to elucidate the role of Pin1 in the pathology of EAE. It turned out that Pin1 promoted the activation and maintained the effector program of EAE-pathogenic Th17 cells in the inflammation foci, but had little effect on the priming of Th17 cells in the draining lymph nodes. Mechanistically, Pin1 stabilized the phosphorylation of STAT3 induced by proinflammatory stimuli, and interacted with STAT3 in the nucleus of Th17 cells, which resulted in the increased expression of Rorc. Moreover, Pin1 formed a complex with RORγt, and enhanced the transactivation of RORγt to the +11 kb enhancer of Rorc, which enforced and maintained the expression of both Rorc and the effector program of pathogenic Th17 cells in EAE. Finally, the inhibition of Pin1, by genetic knockdown or by small molecule inhibitor, deceased the population of Th17 cells and the neuroinflammation, and alleviated the symptoms of EAE. These findings suggest that Pin1 is a potential therapeutic target for MS and other autoimmune inflammatory diseases.
Subject(s)
Encephalomyelitis, Autoimmune, Experimental , Multiple Sclerosis , NIMA-Interacting Peptidylprolyl Isomerase , Nuclear Receptor Subfamily 1, Group F, Member 3 , Th17 Cells , Th17 Cells/immunology , Th17 Cells/metabolism , Animals , NIMA-Interacting Peptidylprolyl Isomerase/metabolism , NIMA-Interacting Peptidylprolyl Isomerase/genetics , Encephalomyelitis, Autoimmune, Experimental/immunology , Encephalomyelitis, Autoimmune, Experimental/metabolism , Mice , Nuclear Receptor Subfamily 1, Group F, Member 3/metabolism , Nuclear Receptor Subfamily 1, Group F, Member 3/genetics , Humans , Multiple Sclerosis/immunology , STAT3 Transcription Factor/metabolism , Disease Models, Animal , Mice, Transgenic , Mice, Inbred C57BL , FemaleABSTRACT
The retinoic acid receptor-related orphan receptor γ (RORγ) is regarded as an attractive therapeutic target for the treatment of prostate cancer. Herein, we report the identification, optimization, and evaluation of 1,2,3,4-tetrahydroquinoline derivatives as novel RORγ inverse agonists, starting from high throughput screening using a thermal stability shift assay (TSA). The representative compounds 13e (designated as XY039) and 14a (designated as XY077) effectively inhibited the RORγ transcriptional activity and exhibited excellent selectivity against other nuclear receptor subtypes. The structural basis for their inhibitory potency was elucidated through the crystallographic study of RORγ LBD complex with 13e. Both 13e and 14a demonstrated reasonable antiproliferative activity, potently inhibited colony formation and the expression of AR, AR regulated genes, and other oncogene in AR positive prostate cancer cell lines. Moreover, 13e and 14a effectively suppressed tumor growth in a 22Rv1 xenograft tumor model in mice. This work provides new and valuable lead compounds for further development of drugs against prostate cancer.
Subject(s)
Antineoplastic Agents , Cell Proliferation , Nuclear Receptor Subfamily 1, Group F, Member 3 , Prostatic Neoplasms , Quinolines , Male , Animals , Humans , Prostatic Neoplasms/drug therapy , Prostatic Neoplasms/pathology , Prostatic Neoplasms/metabolism , Quinolines/pharmacology , Quinolines/chemistry , Quinolines/therapeutic use , Nuclear Receptor Subfamily 1, Group F, Member 3/metabolism , Nuclear Receptor Subfamily 1, Group F, Member 3/agonists , Nuclear Receptor Subfamily 1, Group F, Member 3/antagonists & inhibitors , Antineoplastic Agents/pharmacology , Antineoplastic Agents/chemistry , Antineoplastic Agents/therapeutic use , Cell Line, Tumor , Cell Proliferation/drug effects , Structure-Activity Relationship , Drug Inverse Agonism , Mice , Mice, Nude , Drug Discovery , Xenograft Model Antitumor Assays , Mice, Inbred BALB CABSTRACT
In mice, skin-resident type 2 innate lymphoid cells (ILC2s) exhibit some ILC3-like characteristics. However, the underlying mechanism remains elusive. Here, we observed lower expression of the ILC2 master regulator GATA3 specifically in cutaneous ILC2s (cILC2s) compared with canonical ILC2s, in line with its functionally divergent role in transcriptional control in cILC2s. Decreased levels of GATA3 enabled the expansion of RORγt fate-mapped (RORγtfm+) cILC2s after postnatal days, displaying certain similarities to ILC3s. Single-cell trajectory analysis showed a sequential promotion of the RORγtfm+ cILC2 divergency by RORγt and GATA3. Notably, during hair follicle recycling, these RORγtfm+ cILC2s accumulated around the hair follicle dermal papilla (DP) region to facilitate the process. Mechanistically, we found that GATA3-mediated integrin α3ß1 upregulation on RORγtfm+ cILC2s was required for their positioning around the DP. Overall, our study demonstrates a distinct regulatory role of GATA3 in cILC2s, particularly in promoting the divergence of RORγtfm+ cILC2s to facilitate hair follicle recycling.
Subject(s)
GATA3 Transcription Factor , Hair Follicle , Immunity, Innate , Lymphocytes , Skin , Animals , GATA3 Transcription Factor/metabolism , GATA3 Transcription Factor/genetics , Hair Follicle/metabolism , Mice , Lymphocytes/metabolism , Lymphocytes/immunology , Skin/metabolism , Mice, Inbred C57BL , Nuclear Receptor Subfamily 1, Group F, Member 3/metabolism , Nuclear Receptor Subfamily 1, Group F, Member 3/genetics , Cell DifferentiationABSTRACT
Inflammatory epithelial diseases are spurred by the concomitant dysregulation of immune and epithelial cells. How these two dysregulated cellular compartments simultaneously sustain their heightened metabolic demands is unclear. Single-cell and spatial transcriptomics (ST), along with immunofluorescence, revealed that hypoxia-inducible factor 1α (HIF1α), downstream of IL-17 signaling, drove psoriatic epithelial remodeling. Blocking HIF1α in human psoriatic lesions ex vivo impaired glycolysis and phenocopied anti-IL-17 therapy. In a murine model of skin inflammation, epidermal-specific loss of HIF1α or its target gene, glucose transporter 1, ameliorated epidermal, immune, vascular, and neuronal pathology. Mechanistically, glycolysis autonomously fueled epithelial pathology and enhanced lactate production, which augmented the γδ T17 cell response. RORγt-driven genetic deletion or pharmacological inhibition of either lactate-producing enzymes or lactate transporters attenuated epithelial pathology and IL-17A expression in vivo. Our findings identify a metabolic hierarchy between epithelial and immune compartments and the consequent coordination of metabolic processes that sustain inflammatory disease.
Subject(s)
Glycolysis , Hypoxia-Inducible Factor 1, alpha Subunit , Interleukin-17 , Animals , Humans , Interleukin-17/metabolism , Interleukin-17/immunology , Mice , Hypoxia-Inducible Factor 1, alpha Subunit/metabolism , Skin/immunology , Skin/pathology , Skin/metabolism , Th17 Cells/immunology , Th17 Cells/metabolism , Glucose Transporter Type 1/metabolism , Glucose Transporter Type 1/genetics , Psoriasis/immunology , Psoriasis/metabolism , Epithelium/immunology , Epithelium/metabolism , Mice, Knockout , Signal Transduction/immunology , Nuclear Receptor Subfamily 1, Group F, Member 3/metabolism , Nuclear Receptor Subfamily 1, Group F, Member 3/genetics , Disease Models, Animal , Lactic Acid/metabolism , Chronic Disease , Inflammation/immunology , Mice, Inbred C57BLABSTRACT
Environmental air irritants including nanosized carbon black (nCB) can drive systemic inflammation, promoting chronic obstructive pulmonary disease (COPD) and emphysema development. The let-7 microRNA (Mirlet7 miRNA) family is associated with IL-17-driven T cell inflammation, a canonical signature of lung inflammation. Recent evidence suggests the Mirlet7 family is downregulated in patients with COPD, however, whether this repression conveys a functional consequence on emphysema pathology has not been elucidated. Here, we show that overall expression of the Mirlet7 clusters, Mirlet7b/Mirlet7c2 and Mirlet7a1/Mirlet7f1/Mirlet7d, are reduced in the lungs and T cells of smokers with emphysema as well as in mice with cigarette smoke (CS)- or nCB-elicited emphysema. We demonstrate that loss of the Mirlet7b/Mirlet7c2 cluster in T cells predisposed mice to exaggerated CS- or nCB-elicited emphysema. Furthermore, ablation of the Mirlet7b/Mirlet7c2 cluster enhanced CD8+IL17a+ T cells (Tc17) formation in emphysema development in mice. Additionally, transgenic mice overexpressing Mirlet7g in T cells are resistant to Tc17 and CD4+IL17a+ T cells (Th17) development when exposed to nCB. Mechanistically, our findings reveal the master regulator of Tc17/Th17 differentiation, RAR-related orphan receptor gamma t (RORγt), as a direct target of Mirlet7 in T cells. Overall, our findings shed light on the Mirlet7/RORγt axis with Mirlet7 acting as a molecular brake in the generation of Tc17 cells and suggest a novel therapeutic approach for tempering the augmented IL-17-mediated response in emphysema.
Subject(s)
Cell Differentiation , Down-Regulation , MicroRNAs , Nuclear Receptor Subfamily 1, Group F, Member 3 , Pulmonary Emphysema , Th17 Cells , Animals , Female , Humans , Male , Mice , Interleukin-17/metabolism , Interleukin-17/genetics , Lung/pathology , Lung/metabolism , Mice, Inbred C57BL , MicroRNAs/genetics , MicroRNAs/metabolism , Nuclear Receptor Subfamily 1, Group F, Member 3/metabolism , Nuclear Receptor Subfamily 1, Group F, Member 3/genetics , Pulmonary Emphysema/genetics , Pulmonary Emphysema/metabolism , Pulmonary Emphysema/pathology , Th17 Cells/immunology , Th17 Cells/metabolismABSTRACT
Osteosarcoma (OS) is the most common malignant bone tumor with a poor prognosis. Here, we show that the nuclear receptor RORγ may serve as a potential therapeutic target in OS. OS exhibits a hyperactivated oxidative phosphorylation (OXPHOS) program, which fuels the carbon source to promote tumor progression. We found that RORγ is overexpressed in OS tumors and is linked to hyperactivated OXPHOS. RORγ induces the expression of PGC-1ß and physically interacts with it to activate the OXPHOS program by upregulating the expression of respiratory chain component genes. Inhibition of RORγ strongly inhibits OXPHOS activation, downregulates mitochondrial functions, and increases ROS production, which results in OS cell apoptosis and ferroptosis. RORγ inverse agonists strongly suppressed OS tumor growth and progression and sensitized OS tumors to chemotherapy. Taken together, our results indicate that RORγ is a critical regulator of the OXPHOS program in OS and provides an effective therapeutic strategy for this deadly disease.
Subject(s)
Bone Neoplasms , Mitochondria , Nuclear Receptor Subfamily 1, Group F, Member 3 , Osteosarcoma , Oxidative Phosphorylation , Osteosarcoma/metabolism , Osteosarcoma/pathology , Osteosarcoma/genetics , Humans , Oxidative Phosphorylation/drug effects , Mitochondria/metabolism , Mitochondria/pathology , Nuclear Receptor Subfamily 1, Group F, Member 3/metabolism , Nuclear Receptor Subfamily 1, Group F, Member 3/genetics , Cell Line, Tumor , Animals , Bone Neoplasms/metabolism , Bone Neoplasms/pathology , Bone Neoplasms/genetics , Bone Neoplasms/drug therapy , Mice , Reactive Oxygen Species/metabolism , Apoptosis/drug effects , Gene Expression Regulation, Neoplastic , Ferroptosis/genetics , Ferroptosis/drug effects , Mice, Nude , Male , Cell Proliferation , RNA-Binding ProteinsABSTRACT
The immune response induced by respiratory syncytial virus (RSV) infection is closely related to changes in the composition and function of gastrointestinal microorganisms. However, the specific mechanism remains unknown and the pulmonary-intestinal axis deserves further study. In this study, the mRNA levels of ROR-γt and Foxp3 in the lung and intestine increased first and then decreased. IL-17 and IL-22 reached the maximum on the third day after infection in the lung, and on the second day after infection in the small intestine and colon, respectively. Reg⠢γ in intestinal tissue reached the maximum on the third day after RSV infection. Moreover, the genus enriched in the RSV group was Aggregatibacter, and Proteus was reduced. RSV infection not only causes Th17/Treg cell imbalance in the lungs of mice but also leads to the release of excessive IL-22 from the lungs through blood circulation which binds to IL-22 receptors on the intestinal surface, inducing Reg⠢γ overexpression, impaired intestinal Th17/Treg development, and altered gut microbiota composition. Our research reveals a significant link between the pulmonary and intestinal axis after RSV infection. IMPORTANCE: RSV is the most common pathogen causing acute lower respiratory tract infections in infants and young children, but the complex interactions between the immune system and gut microbiota induced by RSV infection still requires further research. In this study, it was suggested that RSV infection in 7-day-old BALB/c suckling mice caused lung inflammation and disruption of Th17/Treg cells development, and altered the composition of gut microbiota through IL-22 induced overexpression of Reg⠢γ, leading to intestinal immune injury and disruption of gut microbiota. This research reveals that IL-22 may be the link between the lung and gut. This study may provide a new insight into the intestinal symptoms caused by RSV and other respiratory viruses and the connection between the lung and gut axis, as well as new therapeutic ideas for the treatment of RSV-infected children.
Subject(s)
Gastrointestinal Microbiome , Interleukin-22 , Interleukins , Lung , Mice, Inbred BALB C , Respiratory Syncytial Virus Infections , Respiratory Syncytial Viruses , T-Lymphocytes, Regulatory , Th17 Cells , Animals , Respiratory Syncytial Virus Infections/immunology , Respiratory Syncytial Virus Infections/microbiology , Gastrointestinal Microbiome/immunology , T-Lymphocytes, Regulatory/immunology , Mice , Th17 Cells/immunology , Lung/immunology , Lung/microbiology , Lung/virology , Lung/pathology , Interleukins/metabolism , Interleukins/genetics , Interleukins/immunology , Respiratory Syncytial Viruses/immunology , Nuclear Receptor Subfamily 1, Group F, Member 3/metabolism , Nuclear Receptor Subfamily 1, Group F, Member 3/genetics , Interleukin-17/metabolism , Interleukin-17/genetics , Interleukin-17/immunology , Female , Pancreatitis-Associated Proteins/genetics , Pancreatitis-Associated Proteins/immunology , Pancreatitis-Associated Proteins/metabolism , Intestines/immunology , Intestines/microbiology , Forkhead Transcription Factors/metabolism , Forkhead Transcription Factors/geneticsABSTRACT
RORγt+ group 3 innate lymphoid cells (ILC3s) are essential for intestinal homeostasis. Dysregulation of ILC3s has been found in the gut of patients with inflammatory bowel disease and colorectal cancer, yet the specific mechanisms still require more investigation. Here we observe increased ß-catenin in intestinal ILC3s from inflammatory bowel disease and colon cancer patients compared with healthy donors. In contrast to promoting RORγt expression in T cells, activation of Wnt/ß-catenin signaling in ILC3s suppresses RORγt expression, inhibits its proliferation and function, and leads to a deficiency of ILC3s and subsequent intestinal inflammation in mice. Activated ß-catenin and its interacting transcription factor, TCF-1, cannot directly suppress RORγt expression, but rather alters global chromatin accessibility and inhibits JunB expression, which is essential for RORγt expression in ILC3s. Together, our findings suggest that dysregulated Wnt/ß-catenin signaling impairs intestinal ILC3s through TCF-1/JunB/RORγt regulation, further disrupting intestinal homeostasis, and promoting inflammation and cancer.
Subject(s)
Inflammatory Bowel Diseases , beta Catenin , Humans , Mice , Animals , beta Catenin/metabolism , Nuclear Receptor Subfamily 1, Group F, Member 3/genetics , Nuclear Receptor Subfamily 1, Group F, Member 3/metabolism , Immunity, Innate , Lymphocytes/metabolism , Wnt Signaling Pathway , Inflammatory Bowel Diseases/genetics , InflammationABSTRACT
This study aims to investigate the effect of Xixin Decoction on the T helper 17 cell(Th17)/regulatory T cell(Treg) ba-lance of intestinal mucosa and the expression of related transcription factors in the senescence-accelerated mouse-prone 8(SAMP8) model. Fifty 14-week male mice of SAMP8 were randomized by the random number table method into model group, probiotics group, and high-, medium-, and low-dose Xixin Decoction groups, with 10 mice in each group. Ten 14-week male mice of senescence-acce-lerated mouse-resistant 1(SAMR1) served as control group. After 10 weeks of feeding, the mice were administrated with correspon-ding drugs for 10 weeks. Morris water maze test was carried out to examine the learning and memory abilities of mice. Enzyme-linked immunosorbent assay(ELISA) was employed to determine the content of secretory immunoglobulin A(SIgA) in the intestinal mucosa, and flow cytometry to detect the percentage content of Th17 and Treg in the intestinal mucosa. Western blot was performed to determine the protein levels of retinoid-related orphan receptor gamma t(RORγt) and forkhead box p3(Foxp3) in the mouse colon tissue. Compared with control group, the escape latency of mice in model group was significantly prolonged(P<0.01), and the number of times of crossing the platform and the residence time in the target quadrant were significantly reduced within 60 s(P<0.01), intestinal mucosal SIgA content was significantly decreased(P<0.01), Th17 content was increased(P<0.05), Treg content was decreased(P<0.01), the expression of RORγt protein was increased and Foxp3 protein was decreased in colon(P<0.01). Compared with the model group, high-dose Xixin Decoction group improved the learning and memory ability(P<0.05 or P<0.01). Probiotics group and high-and medium-dose Xixin Decoction group increased the content of SIgA in intestinal mucosa(P<0.05 or P<0.01), decreased percentage content of Th17 and increased the percentage content of Treg in intestinal mucosa(P<0.05 or P<0.01). Furthermore, they down-regulated the protein level of RORγt and up-regulated the protein level of Foxp3 in the intestinal mucosa(P<0.01). In conclusion, Xixin Decoction may act on intestinal mucosal immune barrier, affect gut-brain information exchange, and improve the learning and memory ability of SAMP8 by promoting SIgA secretion and regulating the Th17/Treg balance and the expression of RORγt and Foxp3.
Subject(s)
T-Lymphocytes, Regulatory , Th17 Cells , Mice , Male , Animals , Nuclear Receptor Subfamily 1, Group F, Member 3/genetics , Nuclear Receptor Subfamily 1, Group F, Member 3/metabolism , Forkhead Transcription Factors/genetics , Forkhead Transcription Factors/metabolism , Immunoglobulin A, Secretory/pharmacologyABSTRACT
Multiple sclerosis (MS) is a condition where the central nervous system loses its myelin coating due to autoimmune inflammation. The experimental autoimmune encephalomyelitis (EAE) simulates some aspects of human MS. Boswellic acids are natural compounds derived from frankincense extract, known for their anti-inflammatory properties. The purpose of this research was to investigate therapeutic potential of boswellic acids. Mice were divided into three groups: low-dose (LD), high-dose (HD), and control groups (CTRL). Following EAE induction, the mice received daily doses of boswellic acid for 25 days. Brain tissue damage, clinical symptoms, and levels of TGF-ß, IFN-γ, and IL-17 cytokines in cell cultured supernatant of lymphocytes were assessed. Gene expression of transcription factors in brain was measured using real-time PCR. The levels of brain demyelination were significantly lower in the treatment groups compared to the CTRL group. Boswellic acid reduced the severity and duration of EAE symptoms. Furthermore, boswellic acid decreased the amounts of IFN-γ and IL-17, also the expression of T-bet and ROR-γt in brain. On the contrary, it increased the levels of TGF-ß and the expression FoxP3 and GATA3. Our findings suggest that boswellic acids possess therapeutic potential for EAE by modulating the immune response and reducing inflammation.
Subject(s)
Encephalomyelitis, Autoimmune, Experimental , Triterpenes , Animals , Encephalomyelitis, Autoimmune, Experimental/drug therapy , Encephalomyelitis, Autoimmune, Experimental/immunology , Encephalomyelitis, Autoimmune, Experimental/pathology , Triterpenes/pharmacology , Triterpenes/therapeutic use , Mice , Female , Mice, Inbred C57BL , Brain/drug effects , Brain/pathology , Brain/metabolism , Brain/immunology , Cytokines/metabolism , Transforming Growth Factor beta/metabolism , Transforming Growth Factor beta/genetics , Immunomodulating Agents/pharmacology , Immunomodulating Agents/therapeutic use , Interleukin-17/metabolism , Nuclear Receptor Subfamily 1, Group F, Member 3/metabolism , Nuclear Receptor Subfamily 1, Group F, Member 3/genetics , Anti-Inflammatory Agents/pharmacology , Anti-Inflammatory Agents/therapeutic useABSTRACT
Peripherally-induced regulatory T cells (pTregs) expressing the retinoic acid receptor-related orphan-receptor gamma t (RORγt) are indispensable for intestinal immune homeostasis. Nuclear factor kappa family members regulate the differentiation of thymic Tregs and promote their survival in the periphery. However, the Treg intrinsic molecular mechanisms controlling the size of the pTregs in the intestine and associated lymphoid organs remain unclear. Here, we provide direct evidence that B-cell lymphoma 3 (Bcl3) limits the development of pTregs in a T cell-intrinsic manner. Moreover, the absence of Bcl3 allowed for the formation of an unusual intestinal Treg population co-expressing the transcription factors Helios and RORγt. The expanded RORγt+ Treg populations in the absence of Bcl3 displayed an activated phenotype and secreted high levels of the anti-inflammatory cytokines interleukin (IL)-10 and transforming growth factor beta. They were fully capable of suppressing effector T cells in a transfer colitis model despite an intrinsic bias to trans-differentiate toward T helper 17-like cells. Finally, we provide a Bcl3-dependent gene signature in pTregs including altered responsiveness to the cytokines IL-2, IL-6, and tumor necrosis factor alpha. Our results demonstrate that Bcl3 acts as a molecular switch to limit the expansion of different intestinal Treg subsets and may thus serve as a novel therapeutic target for inflammatory bowel disease by restoring intestinal immune tolerance.
Subject(s)
B-Cell Lymphoma 3 Protein , Cell Differentiation , Mice, Knockout , Nuclear Receptor Subfamily 1, Group F, Member 3 , T-Lymphocytes, Regulatory , Animals , B-Cell Lymphoma 3 Protein/metabolism , B-Cell Lymphoma 3 Protein/genetics , T-Lymphocytes, Regulatory/immunology , Mice , Nuclear Receptor Subfamily 1, Group F, Member 3/metabolism , Nuclear Receptor Subfamily 1, Group F, Member 3/genetics , Transcription Factors/metabolism , Transcription Factors/genetics , Colitis/immunology , Colitis/metabolism , T-Lymphocyte Subsets/immunology , T-Lymphocyte Subsets/metabolism , Mice, Inbred C57BL , Intestinal Mucosa/immunology , Intestinal Mucosa/metabolism , Intestines/immunology , DNA-Binding Proteins/metabolism , DNA-Binding Proteins/genetics , Cells, Cultured , Th17 Cells/immunologyABSTRACT
Bronchopulmonary dysplasia (BPD) is characterized by alveolar dysplasia, and evidence indicates that interferon regulatory factor 4 (IRF4) is involved in the pathogenesis of various inflammatory lung diseases. Nonetheless, the significance and mechanism of IRF4 in BPD remain unelucidated. Consequently, we established a mouse model of BPD through hyperoxia exposure, and ELISA was employed to measure interleukin-17 A (IL-17 A) and interleukin-6 (IL-6) expression levels in lung tissues. Western blotting was adopted to determine the expression of IRF4, surfactant protein C (SP-C), and podoplanin (T1α) in lung tissues. Flow cytometry was utilized for analyzing the percentages of FOXP3+ regulatory T cells (Tregs) and FOXP3+RORγt+ Tregs in CD4+ T cells in lung tissues to clarify the underlying mechanism. Our findings revealed that BPD mice exhibited disordered lung tissue structure, elevated IRF4 expression, decreased SP-C and T1α expression, increased IL-17 A and IL-6 levels, reduced proportion of FOXP3+ Tregs, and increased proportion of FOXP3+RORγt+ Tregs. For the purpose of further elucidating the effect of IRF4 on Treg phenotype switching induced by hyperoxia in lung tissues, we exposed neonatal mice with IRF4 knockout to hyperoxia. These mice exhibited regular lung tissue structure, increased proportion of FOXP3+ Tregs, reduced proportion of FOXP3+RORγt+ Tregs, elevated SP-C and T1α expression, and decreased IL-17 A and IL-6 levels. In conclusion, our findings demonstrate that IRF4-mediated Treg phenotype switching in lung tissues exacerbates alveolar epithelial cell injury under hyperoxia exposure.