ABSTRACT
Head and neck squamous cell carcinoma (HNSCC) is a significant public health concern worldwide. Immunomodulatory targets in the HNSCC tumor microenvironment are crucial to enhance the efficacy of HNSCC immunotherapy. Macrophage migration inhibitory factor (MIF) is a pro-inflammatory cytokine that has been linked to poor prognosis in many cancers, but the mechanistic role of MIF in HNSCC remains unclear. Using a murine orthotopic oral cancer model in Mif+/+ or Mif-/- mice, we determined the function of host derived MIF in HNSCC tumor development, metastasis as well as localized and systemic tumor immune responses. We observed that Mif-/- mice have decreased tumor growth and tumor burden compared to their wild-type counterparts. Flow cytometric analysis of immune populations within the primary tumor site revealed increased Th1 and cytotoxic T cell recruitment to the HNSCC tumor microenvironment. Within the tumors of Mif-/- mice, MIF deletion also enhanced the effector function of anti-tumoral effector CD8+ T cells as well as Th1 cells and decreased the accumulation of granulocytic myeloid derived suppressor cells (g-MDSCs) in the tumor microenvironment. Furthermore, MDSCs isolated from tumor bearing mice chemotactically respond to MIF in a dose dependent manner. Taken together, our results demonstrate a chemotactic and immunomodulatory role for host derived MIF in promoting HNSCC and suggest that MIF targeted immunomodulation is a promising approach for HNSCC treatment.
Subject(s)
Head and Neck Neoplasms , Intramolecular Oxidoreductases , Macrophage Migration-Inhibitory Factors , Squamous Cell Carcinoma of Head and Neck , Tumor Microenvironment , Macrophage Migration-Inhibitory Factors/genetics , Macrophage Migration-Inhibitory Factors/metabolism , Animals , Tumor Microenvironment/immunology , Mice , Squamous Cell Carcinoma of Head and Neck/immunology , Squamous Cell Carcinoma of Head and Neck/pathology , Squamous Cell Carcinoma of Head and Neck/metabolism , Squamous Cell Carcinoma of Head and Neck/genetics , Head and Neck Neoplasms/immunology , Head and Neck Neoplasms/pathology , Head and Neck Neoplasms/metabolism , Head and Neck Neoplasms/genetics , Intramolecular Oxidoreductases/genetics , Intramolecular Oxidoreductases/metabolism , Mice, Knockout , Myeloid-Derived Suppressor Cells/metabolism , Myeloid-Derived Suppressor Cells/immunology , Myeloid-Derived Suppressor Cells/pathology , Humans , Cell Line, Tumor , Mice, Inbred C57BL , Immune Tolerance , Th1 Cells/immunology , Th1 Cells/metabolism , CD8-Positive T-Lymphocytes/immunology , CD8-Positive T-Lymphocytes/metabolismABSTRACT
Next to its classical role in MHC II-mediated antigen presentation, CD74 was identified as a high-affinity receptor for macrophage migration inhibitory factor (MIF), a pleiotropic cytokine and major determinant of various acute and chronic inflammatory conditions, cardiovascular diseases and cancer. Recent evidence suggests that CD74 is expressed in T cells, but the functional relevance of this observation is poorly understood. Here, we characterized the regulation of CD74 expression and that of the MIF chemokine receptors during activation of human CD4+ T cells and studied links to MIF-induced T-cell migration, function, and COVID-19 disease stage. MIF receptor profiling of resting primary human CD4+ T cells via flow cytometry revealed high surface expression of CXCR4, while CD74, CXCR2 and ACKR3/CXCR7 were not measurably expressed. However, CD4+ T cells constitutively expressed CD74 intracellularly, which upon T-cell activation was significantly upregulated, post-translationally modified by chondroitin sulfate and could be detected on the cell surface, as determined by flow cytometry, Western blot, immunohistochemistry, and re-analysis of available RNA-sequencing and proteomic data sets. Applying 3D-matrix-based live cell-imaging and receptor pathway-specific inhibitors, we determined a causal involvement of CD74 and CXCR4 in MIF-induced CD4+ T-cell migration. Mechanistically, proximity ligation assay visualized CD74/CXCR4 heterocomplexes on activated CD4+ T cells, which were significantly diminished after MIF treatment, pointing towards a MIF-mediated internalization process. Lastly, in a cohort of 30 COVID-19 patients, CD74 surface expression was found to be significantly upregulated on CD4+ and CD8+ T cells in patients with severe compared to patients with only mild disease course. Together, our study characterizes the MIF receptor network in the course of T-cell activation and reveals CD74 as a novel functional MIF receptor and MHC II-independent activation marker of primary human CD4+ T cells.
Subject(s)
Antigens, Differentiation, B-Lymphocyte , CD4-Positive T-Lymphocytes , COVID-19 , Histocompatibility Antigens Class II , Intramolecular Oxidoreductases , Lymphocyte Activation , Macrophage Migration-Inhibitory Factors , SARS-CoV-2 , Humans , Antigens, Differentiation, B-Lymphocyte/metabolism , CD4-Positive T-Lymphocytes/metabolism , CD4-Positive T-Lymphocytes/immunology , Histocompatibility Antigens Class II/metabolism , Histocompatibility Antigens Class II/immunology , Macrophage Migration-Inhibitory Factors/metabolism , Macrophage Migration-Inhibitory Factors/genetics , Lymphocyte Activation/immunology , SARS-CoV-2/metabolism , SARS-CoV-2/immunology , COVID-19/immunology , COVID-19/metabolism , COVID-19/pathology , Intramolecular Oxidoreductases/metabolism , Intramolecular Oxidoreductases/genetics , Receptors, CXCR4/metabolism , Receptors, CXCR4/genetics , Cell Movement , Male , Female , Middle Aged , Receptors, ImmunologicABSTRACT
Despite recent advances in the treatment of cancer, the issue of therapy resistance remains one of the most significant challenges in the field. In this context, signaling molecules, such as cytokines have emerged as promising targets for drug discovery. Examples of cytokines include macrophage migration inhibitory factor (MIF) and its closely related analogue D-dopachrome tautomerase (D-DT). In this study we aim to develop a new chemical class of D-DT binders and subsequently create a dual-targeted inhibitor that can potentially trigger D-DT degradation via the Proteolysis Targeting Chimera (PROTAC) technology. Here we describe the synthesis of a novel library of 1,2,3-triazoles targeting D-DT. The most potent derivative 19c (IC50 of 0.5 ± 0.04 µM with high selectivity toward D-DT) was attached to a cereblon (CRBN) ligand through aliphatic amides, which were synthesized by a remarkably convenient and effective solvent-free reaction. Enzyme inhibition experiments led to the discovery of the compound 10d, which exhibited moderate inhibitory potency (IC50 of 5.9 ± 0.7 µM), but unfortunately demonstrated no activity in D-DT degradation experiments. In conclusion, this study offers valuable insight into the SAR of D-DT inhibition, paving the way for the development of novel molecules as tools to study D-DT functions in tumor proliferation and, ultimately, new therapeutics for cancer treatment.
Subject(s)
Enzyme Inhibitors , Intramolecular Oxidoreductases , Triazoles , Triazoles/pharmacology , Triazoles/chemistry , Triazoles/chemical synthesis , Intramolecular Oxidoreductases/antagonists & inhibitors , Intramolecular Oxidoreductases/metabolism , Humans , Structure-Activity Relationship , Enzyme Inhibitors/pharmacology , Enzyme Inhibitors/chemical synthesis , Enzyme Inhibitors/chemistry , Molecular Structure , Dose-Response Relationship, Drug , Antineoplastic Agents/pharmacology , Antineoplastic Agents/chemical synthesis , Antineoplastic Agents/chemistryABSTRACT
Interaction of mast cells (MCs) with fibroblasts is essential for MC maturation within tissue microenvironments, although the underlying mechanism is incompletely understood. Through a phenotypic screening of >30 mouse lines deficient in lipid-related genes, we found that deletion of the lysophosphatidic acid (LPA) receptor LPA1, like that of the phospholipase PLA2G3, the prostaglandin D2 (PGD2) synthase L-PGDS, or the PGD2 receptor DP1, impairs MC maturation and thereby anaphylaxis. Mechanistically, MC-secreted PLA2G3 acts on extracellular vesicles (EVs) to supply lysophospholipids, which are converted by fibroblast-derived autotaxin (ATX) to LPA. Fibroblast LPA1 then integrates multiple pathways required for MC maturation by facilitating integrin-mediated MC-fibroblast adhesion, IL-33-ST2 signaling, L-PGDS-driven PGD2 generation, and feedforward ATX-LPA1 amplification. Defective MC maturation resulting from PLA2G3 deficiency is restored by supplementation with LPA1 agonists or PLA2G3-modified EVs. Thus, the lipid-orchestrated paracrine circuit involving PLA2G3-driven lysophospholipid, eicosanoid, integrin, and cytokine signaling fine-tunes MC-fibroblast communication, ensuring MC maturation.
Subject(s)
Anaphylaxis , Fibroblasts , Lysophospholipids , Mast Cells , Mice, Knockout , Paracrine Communication , Phosphoric Diester Hydrolases , Receptors, Lysophosphatidic Acid , Signal Transduction , Animals , Mast Cells/immunology , Mast Cells/metabolism , Anaphylaxis/immunology , Anaphylaxis/metabolism , Mice , Fibroblasts/metabolism , Lysophospholipids/metabolism , Receptors, Lysophosphatidic Acid/metabolism , Receptors, Lysophosphatidic Acid/genetics , Phosphoric Diester Hydrolases/metabolism , Phosphoric Diester Hydrolases/genetics , Prostaglandin D2/metabolism , Extracellular Vesicles/metabolism , Interleukin-33/metabolism , Intramolecular Oxidoreductases/metabolism , Intramolecular Oxidoreductases/genetics , Receptors, Prostaglandin/metabolism , Receptors, Prostaglandin/genetics , Cell Differentiation , Mice, Inbred C57BL , Interleukin-1 Receptor-Like 1 Protein , LipocalinsABSTRACT
Chemical protein knockdown technology using proteolysis-targeting chimeras (PROTACs) to hijack the endogenous ubiquitin-proteasome system is a powerful strategy to degrade disease-related proteins. This chapter describes in silico design of a hematopoietic prostaglandin D synthase (H-PGDS) degrader, PROTAC(H-PGDS), using a docking simulation of the ternary complex of H-PGDS/PROTAC/E3 ligase as well as the synthesis of the designed PROTAC(H-PGDS)s and evaluation of their H-PGDS degradation activity.
Subject(s)
Intramolecular Oxidoreductases , Lipocalins , Molecular Docking Simulation , Proteolysis , Intramolecular Oxidoreductases/metabolism , Intramolecular Oxidoreductases/chemistry , Intramolecular Oxidoreductases/antagonists & inhibitors , Humans , Lipocalins/metabolism , Lipocalins/chemistry , Computer Simulation , Drug Design , Ubiquitin-Protein Ligases/metabolism , Proteasome Endopeptidase Complex/metabolism , Proteasome Endopeptidase Complex/chemistryABSTRACT
Damage to renal tubular epithelial cells (RTECs) signaled the onset and progression of sepsis-associated acute kidney injury (SA-AKI). Recent research on mitochondria has revealed that mitophagy plays a crucial physiological role in alleviating injury to RTECs and it is suppressed progressively by the inflammation response in SA-AKI. However, the mechanism by which inflammation influences mitophagy remains poorly understood. We examined how macrophage migration inhibitory factor (MIF), a pro-inflammatory protein, influences the PINK1-Parkin pathway of mitophagy by studying protein-protein interactions when MIF was inhibited or overexpressed. Surprisingly, elevated levels of MIF were found to directly bind to PINK1, disrupting its interaction with Parkin. This interference hindered the recruitment of Parkin to mitochondria and impeded the initiation of mitophagy. Furthermore, this outcome led to significant apoptosis of RTECs, which could, however, be reversed by an MIF inhibitor ISO-1 and/or a new mitophagy activator T0467. These findings highlight the detrimental impact of MIF on renal damage through its disruption of the interaction between PINK1 and Parkin, and the therapeutic potential of ISO-1 and T0467 in mitigating SA-AKI. This study offers a fresh perspective on treating SA-AKI by targeting MIF and mitophagy.
Subject(s)
Acute Kidney Injury , Macrophage Migration-Inhibitory Factors , Mitophagy , Protein Kinases , Sepsis , Ubiquitin-Protein Ligases , Macrophage Migration-Inhibitory Factors/metabolism , Macrophage Migration-Inhibitory Factors/genetics , Acute Kidney Injury/metabolism , Acute Kidney Injury/pathology , Ubiquitin-Protein Ligases/metabolism , Ubiquitin-Protein Ligases/genetics , Protein Kinases/metabolism , Sepsis/complications , Sepsis/metabolism , Animals , Humans , Mitochondria/metabolism , Kidney Tubules/metabolism , Kidney Tubules/pathology , Epithelial Cells/metabolism , Epithelial Cells/pathology , Apoptosis , Protein Binding , Male , Intramolecular Oxidoreductases/metabolismABSTRACT
Macrophage Migration Inhibitory Factor (MIF) and its homolog D-dopachrome Tautomerase (DDT) have been implicated as drivers of tumor progression across a variety of cancers. Recent evidence suggests MIF as a therapeutic target in immune checkpoint inhibition (ICI) resistant melanomas, however clinical evidence of MIF and particularly of DDT remain limited. This retrospective study analyzed 97 patients treated at Yale for melanoma between 2002-2020. Bulk-RNA sequencing of patient tumor samples from the Skin Cancer SPORE Biorepository was used to evaluate for differential gene expression of MIF, DDT, CD74, and selected inflammatory markers, and gene expression was correlated with patient survival outcomes. Our findings revealed a strong correlation between MIF and DDT levels, with no statistically significant difference across common melanoma mutations and subtypes. Improved survival was associated with lower MIF and DDT levels and higher CD74:MIF and CD74:DDT levels. High CD74:DDT and CD74:MIF levels were also associated with enrichment of infiltrating inflammatory cell markers. These data suggest DDT as a novel target in immune therapy. Dual MIF and DDT blockade may provide synergistic responses in patients with melanoma, irrespective of common mutations, and may overcome ICI resistance. These markers may also provide prognostic value for further biomarker development.
Subject(s)
Antigens, Differentiation, B-Lymphocyte , Biomarkers, Tumor , Histocompatibility Antigens Class II , Intramolecular Oxidoreductases , Macrophage Migration-Inhibitory Factors , Melanoma , Humans , Macrophage Migration-Inhibitory Factors/metabolism , Macrophage Migration-Inhibitory Factors/genetics , Melanoma/drug therapy , Melanoma/genetics , Melanoma/metabolism , Melanoma/pathology , Melanoma/mortality , Intramolecular Oxidoreductases/genetics , Intramolecular Oxidoreductases/metabolism , Antigens, Differentiation, B-Lymphocyte/metabolism , Antigens, Differentiation, B-Lymphocyte/genetics , Histocompatibility Antigens Class II/genetics , Histocompatibility Antigens Class II/metabolism , Prognosis , Male , Female , Biomarkers, Tumor/metabolism , Biomarkers, Tumor/genetics , Middle Aged , Retrospective Studies , Aged , Adult , Skin Neoplasms/drug therapy , Skin Neoplasms/genetics , Skin Neoplasms/pathology , Skin Neoplasms/metabolism , Skin Neoplasms/mortality , Mutation , Immune Checkpoint Inhibitors/therapeutic use , Immune Checkpoint Inhibitors/pharmacology , Aged, 80 and overABSTRACT
Macrophages are a rich source of macrophage migration inhibitory factor (MIF). It is well established that macrophages and MIF play a pathogenic role in anti-glomerular basement membrane crescentic glomerulonephritis (anti-GBM CGN). However, whether macrophages mediate anti-GBM CGN via MIF-dependent mechanism remains unexplored, which was investigated in this study by specifically deleting MIF from macrophages in MIFf/f-lysM-cre mice. We found that compared to anti-GBM CGN induced in MIFf/f control mice, conditional ablation of MIF in macrophages significantly suppressed anti-GBM CGN by inhibiting glomerular crescent formation and reducing serum creatinine and proteinuria while improving creatine clearance. Mechanistically, selective MIF depletion in macrophages largely inhibited renal macrophage and T cell recruitment, promoted the polarization of macrophage from M1 towards M2 via the CD74/NF-κB/p38MAPK-dependent mechanism. Unexpectedly, selective depletion of macrophage MIF also significantly promoted Treg while inhibiting Th1 and Th17 immune responses. In summary, MIF produced by macrophages plays a pathogenic role in anti-GBM CGN. Targeting macrophage-derived MIF may represent a novel and promising therapeutic approach for the treatment of immune-mediated kidney diseases.
Subject(s)
Anti-Glomerular Basement Membrane Disease , Antigens, Differentiation, B-Lymphocyte , Histocompatibility Antigens Class II , Intramolecular Oxidoreductases , Macrophage Migration-Inhibitory Factors , Macrophages , Macrophage Migration-Inhibitory Factors/metabolism , Animals , Macrophages/immunology , Macrophages/metabolism , Mice , Anti-Glomerular Basement Membrane Disease/immunology , Anti-Glomerular Basement Membrane Disease/metabolism , Intramolecular Oxidoreductases/metabolism , Intramolecular Oxidoreductases/genetics , Histocompatibility Antigens Class II/metabolism , Histocompatibility Antigens Class II/immunology , Antigens, Differentiation, B-Lymphocyte/metabolism , Disease Models, Animal , NF-kappa B/metabolism , Mice, Knockout , p38 Mitogen-Activated Protein Kinases/metabolism , T-Lymphocytes, Regulatory/immunology , T-Lymphocytes, Regulatory/metabolism , Mice, Inbred C57BL , Th17 Cells/immunology , Th17 Cells/metabolism , Proteinuria/immunology , Signal TransductionABSTRACT
Melanoma is an aggressive tumour with poor prognosis that arises from the malignant transformation of melanocytes. Over the past few decades, intense research into the pathogenesis of melanoma has led to the development of BRAF and immune checkpoint inhibitors, including antibodies against programmed cell death protein 1 (PD-1) and cytotoxic T lymphocyte-associated protein 4 (CTLA-4), which have shown clinically significant efficacy. However, some tumours do not respond to these therapies initially or become treatment resistant. Most melanoma tissues appear to possess biological characteristics that allow them to evade these treatments, and identifying these characteristics is one of the major challenges facing cancer researchers. One such characteristic that has recently gained attention is the role of macrophage migration inhibitory factor (MIF) and its receptor CD74. This review outlines the cellular and molecular functions of CD74, MIF and their family of proteins. We then review their roles in tumours based on previous reports, highlight their pathological significance in melanoma and discuss their potential as therapeutic targets.
Subject(s)
Antigens, Differentiation, B-Lymphocyte , Histocompatibility Antigens Class II , Intramolecular Oxidoreductases , Macrophage Migration-Inhibitory Factors , Melanoma , Humans , Melanoma/metabolism , Melanoma/pathology , Melanoma/etiology , Macrophage Migration-Inhibitory Factors/metabolism , Antigens, Differentiation, B-Lymphocyte/metabolism , Histocompatibility Antigens Class II/metabolism , Intramolecular Oxidoreductases/metabolism , Skin Neoplasms/metabolism , Skin Neoplasms/pathology , Skin Neoplasms/etiology , AnimalsABSTRACT
Sigma-class glutathione transferase (GST) proteins with dual GST and prostaglandin synthase (PGS) activities play a crucial role in the establishment of Clonorchis sinensis infection. Herein, we analyzed the structural and enzymatic properties of sigma-class GST (CsGST-σ) proteins to obtain insight into their antioxidant and immunomodulatory functions in comparison with mu-class GST (CsGST-µ) proteins. CsGST-σ proteins conserved characteristic structures, which had been described in mammalian hematopoietic prostaglandin D2 synthases. Recombinant forms of these CsGST-σ and CsGST-µ proteins expressed in Escherichia coli exhibited considerable degrees of GST and PGS activities with substantially different specific activities. All recombinant proteins displayed higher affinities toward prostaglandin H2 (PGS substrate; average Km of 30.7 and 3.0 µm for prostaglandin D2 [PGDS] and E2 synthase [PGES], respectively) than those toward CDNB (GST substrate; average Km of 1,205.1 µm). Furthermore, the catalytic efficiency (Kcat/Km) of the PGDS/PGES activity was higher than that of GST activity (average Kcat/Km of 3.1, 0.7, and 7.0×10-3 s-1µm-1 for PGDS, PGES, and GST, respectively). Our data strongly suggest that the C. sinensis sigma- and mu-class GST proteins are deeply involved in regulating host immune responses by generating PGD2 and PGE2 in addition to their roles in general detoxification.
Subject(s)
Clonorchis sinensis , Glutathione Transferase , Intramolecular Oxidoreductases , Glutathione Transferase/metabolism , Glutathione Transferase/chemistry , Glutathione Transferase/genetics , Clonorchis sinensis/enzymology , Clonorchis sinensis/genetics , Animals , Intramolecular Oxidoreductases/metabolism , Intramolecular Oxidoreductases/chemistry , Intramolecular Oxidoreductases/genetics , Recombinant Proteins/metabolism , Recombinant Proteins/genetics , Recombinant Proteins/chemistry , Lipocalins/metabolism , Lipocalins/genetics , Lipocalins/chemistry , Lipocalins/immunology , Escherichia coli/genetics , Prostaglandin H2/metabolism , Prostaglandin H2/chemistry , KineticsABSTRACT
Endometritis is a common disease in animals, leading to disruption of reproductive processes and economic losses. Noradrenergic control of prostaglandin (PG)I2 formation by inflamed endometrium is unknown. We determined the involvement of α1-, α2- and ß-adrenoreceptors (ARs) in noradrenaline-influenced PGI synthase (PGIS) protein abundance and PGI2 release from porcine (1) endometrial explants with Escherichia coli (E. coli)-induced inflammation in vivo, and (2) E. coli lipopolysaccharide (LPS)-treated endometrial epithelial cells. Experiment 1. E. coli suspension (E. coli group) or saline (CON group) was injected into the uterine horns. In both groups, noradrenaline increased endometrial PGIS abundance and PGI2 release versus the control values, and it was higher in the E. coli group than in the CON group. In the CON group, a noradrenaline stimulating effect on both parameters takes place through α1D-, α2C- and ß2-ARs. In the E. coli group, noradrenaline increased PGIS abundance and PGI2 release via α1A-, α2(B,C)- and ß(1,2)-ARs, and PGI2 release also by α2A-ARs. Experiment 2. LPS and noradrenaline augmented the examined parameters in endometrial epithelial cells versus the control value. In LPS-treated cells, ß(1,2)-ARs mediate in noradrenaline excitatory action on PGIS protein abundance and PGI2 release. ß3-ARs also contribute to PGI2 release. Under inflammatory conditions, noradrenaline via ARs increases PGI2 synthesis and release from the porcine endometrium, including epithelial cells. Our findings suggest that noradrenaline may indirectly affect processes regulated by PGI2 in the inflamed uterus.
Subject(s)
Endometrium , Epoprostenol , Norepinephrine , Animals , Female , Norepinephrine/metabolism , Endometrium/metabolism , Endometrium/pathology , Swine , Epoprostenol/metabolism , Receptors, Adrenergic/metabolism , Lipopolysaccharides , Inflammation/metabolism , Inflammation/pathology , Escherichia coli , Endometritis/metabolism , Endometritis/pathology , Epithelial Cells/metabolism , Epithelial Cells/drug effects , Intramolecular Oxidoreductases/metabolism , Cytochrome P-450 Enzyme SystemABSTRACT
The hallmarks of spondyloarthritis (SpA) are type 3 immunity-driven inflammation and new bone formation (NBF). Macrophage migration inhibitory factor (MIF) was found to be a key driver of the pathogenesis of SpA by amplifying type 3 immunity, yet MIF-interacting molecules and networks remain elusive. Herein, we identified hypoxia-inducible factor-1 alpha (HIF1A) as an interacting partner molecule of MIF that drives SpA pathologies, including inflammation and NBF. HIF1A expression was increased in the joint tissues and synovial fluid of SpA patients and curdlan-injected SKG (curdlan-SKG) mice compared to the respective controls. Under hypoxic conditions in which HIF1A was stabilized, human and mouse neutrophils exhibited substantially increased expression of MIF and IL-23, an upstream type 3 immunity-related cytokine. Similar to MIF, systemic overexpression of IL-23 induced SpA pathology in SKG mice, while the injection of a HIF1A-selective inhibitor (PX-478) into curdlan-SKG mice prevented or attenuated SpA pathology, as indicated by a marked reduction in the expression of MIF and IL-23. Furthermore, genetic deletion of MIF or HIF1A inhibition with PX-478 in IL-23-overexpressing SKG mice did not induce evident arthritis or NBF, despite the presence of psoriasis-like dermatitis and blepharitis. We also found that MIF- and IL-23-expressing neutrophils infiltrated areas of the NBF in curdlan-SKG mice. These neutrophils potentially increased chondrogenesis and cell proliferation via the upregulation of STAT3 in periosteal cells and ligamental cells during endochondral ossification. Together, these results provide supporting evidence for an MIF/HIF1A regulatory network, and inhibition of HIF1A may be a novel therapeutic approach for SpA by suppressing type 3 immunity-mediated inflammation and NBF.
Subject(s)
Chondrogenesis , Disease Models, Animal , Hypoxia-Inducible Factor 1, alpha Subunit , Macrophage Migration-Inhibitory Factors , Neutrophils , Animals , Macrophage Migration-Inhibitory Factors/metabolism , Macrophage Migration-Inhibitory Factors/genetics , Hypoxia-Inducible Factor 1, alpha Subunit/metabolism , Neutrophils/immunology , Neutrophils/metabolism , Humans , Mice , Spondylarthritis/immunology , Spondylarthritis/pathology , Intramolecular Oxidoreductases/metabolism , Intramolecular Oxidoreductases/genetics , Interleukin-23/metabolism , beta-Glucans/pharmacology , Mice, Inbred C57BL , Male , Female , ImmunityABSTRACT
Functional variants of the gene for the cytokine macrophage migration inhibitory factor (MIF) are defined by a 4-nucleotide promoter microsatellite (-794 CATT5-8, rs5844572) and confer risk for autoimmune, infectious, and oncologic diseases. We describe herein the discovery of a prototypic, small molecule inhibitor of MIF transcription with selectivity for high microsatellite repeat number and correspondingly high gene expression. Utilizing a high-throughput luminescent proximity screen, we identify 1-carbomethoxy-5-formyl-4,6,8-trihydroxyphenazine (CMFT) to inhibit the functional interaction between the transcription factor ICBP90 (namely, UHRF1) and the MIF -794 CATT5-8 promoter microsatellite. CMFT inhibits MIF mRNA expression in a -794 CATT5-8 length-dependent manner with an IC50 of 470 nM, and preferentially reduces ICBP90-dependent MIF mRNA and protein expression in high-genotypic versus low-genotypic MIF-expressing macrophages. RNA expression analysis also showed CMFT to downregulate MIF-dependent, inflammatory gene expression with little evidence of off-target metabolic toxicity. These findings provide proof-of-concept for advancing the pharmacogenomic development of precision-based MIF inhibitors for diverse autoimmune and inflammatory conditions.
Subject(s)
Intramolecular Oxidoreductases , Macrophage Migration-Inhibitory Factors , Macrophage Migration-Inhibitory Factors/genetics , Macrophage Migration-Inhibitory Factors/antagonists & inhibitors , Macrophage Migration-Inhibitory Factors/metabolism , Macrophage Migration-Inhibitory Factors/immunology , Humans , Intramolecular Oxidoreductases/genetics , Intramolecular Oxidoreductases/antagonists & inhibitors , Intramolecular Oxidoreductases/metabolism , Alleles , Microsatellite Repeats , Promoter Regions, Genetic , Animals , Macrophages/metabolism , Macrophages/immunology , Macrophages/drug effects , Transcription, Genetic/drug effects , Mice , CCAAT-Enhancer-Binding Proteins/genetics , CCAAT-Enhancer-Binding Proteins/metabolismABSTRACT
BACKGROUND: Inflammatory macrophage infiltration plays a critical role in acute kidney disease induced by ischemia-reperfusion (IRI-AKI). Calycosin is a natural flavone with multiple bioactivities. This study aimed to investigate the therapeutic role of calycosin in IRI-AKI and its underlying mechanism. METHODS: The renoprotective and anti-inflammatory effects of calycosin were analyzed in C57BL/6 mice with IRI-AKI and lipopolysaccharide (LPS)-stimulated RAW 264.7 cells. RNA-seq was used for mechanism investigation. The molecular target of calycosin was screened by in silico methods and validated by surface plasmon resonance (SPR). Macrophage chemotaxis was analyzed using Transwell and agarose gel spot assays. RESULTS: Calycosin treatment significantly reduced serum creatinine and urea nitrogen and attenuated tubular destruction in IRI-AKI mice. Additionally, calycosin markedly suppressed NF-κB signaling activation and the expression of inflammatory mediators IL-1ß and TNF-α in IRI-AKI kidneys and LPS-stimulated RAW 264.7 cells. Interestingly, RNA-seq revealed calycosin remarkably downregulated chemotaxis-related pathways in RAW 264.7 cells. Among the differentially expressed genes, Ccl2/MCP-1, a critical chemokine mediating macrophage inflammatory chemotaxis, was downregulated in both LPS-stimulated RAW 264.7 cells and IRI-AKI kidneys. Consistently, calycosin treatment attenuated macrophage infiltration in the IRI-AKI kidneys. Importantly, in silico target prediction, molecular docking, and SPR assay demonstrated that calycosin directly binds to macrophage migration inhibitory factor (MIF). Functionally, calycosin abrogated MIF-stimulated NF-κB signaling activation and Ccl2 expression and MIF-mediated chemotaxis in RAW 264.7 cells. CONCLUSIONS: In summary, calycosin attenuates IRI-AKI by inhibiting MIF-mediated macrophage inflammatory chemotaxis, suggesting it could be a promising therapeutic agent for the treatment of IRI-AKI.
Subject(s)
Acute Kidney Injury , Chemotaxis , Isoflavones , Macrophage Migration-Inhibitory Factors , Macrophages , Reperfusion Injury , Animals , Male , Mice , Acute Kidney Injury/drug therapy , Acute Kidney Injury/metabolism , Anti-Inflammatory Agents/pharmacology , Anti-Inflammatory Agents/therapeutic use , Chemotaxis/drug effects , Intramolecular Oxidoreductases/metabolism , Intramolecular Oxidoreductases/genetics , Isoflavones/pharmacology , Isoflavones/therapeutic use , Kidney/drug effects , Kidney/pathology , Lipopolysaccharides , Macrophages/drug effects , Mice, Inbred C57BL , NF-kappa B/metabolism , RAW 264.7 Cells , Reperfusion Injury/drug therapyABSTRACT
BACKGROUNDS: Melanogenesis, regulated by genetic, hormonal, and environmental factors, occurs in melanocytes in the basal layer of the epidermis. Dysregulation of this process can lead to various skin disorders, such as hyperpigmentation and hypopigmentation. Therefore, the present study investigated the effect of ultrasonic-assisted ethanol extract (SHUE) from Sargassum horneri (S. horneri), brown seaweed against melanogenesis in α-melanocyte-stimulating hormone (MSH)-stimulated B16F10 murine melanocytes. METHODS: Firstly, yield and proximate compositional analysis of the samples were conducted. The effect of SHUE on cell viability has been evaluated by using 3-(4,5-Dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide (MTT) assay. After that, the melanin content and cellular tyrosinase activity in α-MSH-stimulated B16F10 murine melanocytes were examined. Western blot analysis was carried out to investigate the protein expression levels of microphthalmia-associated transcription factor (MITF), tyrosinase, tyrosinase-related protein-1 (TRP1), and tyrosinase-related protein-2 (TRP2). In addition, the effect of extracellular signal-regulated kinase (ERK) on the melanogenesis process was assessed via Western blotting. RESULTS: As per the analysis, SHUE contained the highest average yield on a dry basis at 28.70 ± 3.21%. The findings showed that SHUE reduced the melanin content and cellular tyrosinase activity in α-MSH-stimulated B16F10 murine melanocytes. Additionally, the expression levels of MITF, TRP1, and TRP2 protein were significantly downregulated by SHUE treatment in α-MSH-stimulated B16F10 murine melanocytes. Moreover, SHUE upregulated the phosphorylation of ERK and AKT in α-MSH-stimulated B16F10 murine melanocytes. In addition, experiments conducted using the ERK inhibitor (PD98059) revealed that the activity of SHUE depends on the ERK signaling cascade. CONCLUSION: These results suggest that SHUE has an anti-melanogenic effect and can be used as a material in the formulation of cosmetics related to whitening and lightening.
Subject(s)
Ethanol , Melanins , Melanocytes , Monophenol Monooxygenase , Sargassum , Animals , Sargassum/chemistry , Melanins/biosynthesis , Melanins/metabolism , Monophenol Monooxygenase/metabolism , Monophenol Monooxygenase/antagonists & inhibitors , Melanocytes/drug effects , Melanocytes/metabolism , Mice , Ethanol/chemistry , Microphthalmia-Associated Transcription Factor/metabolism , alpha-MSH/metabolism , Plant Extracts/pharmacology , Plant Extracts/chemistry , Cell Survival/drug effects , Melanoma, Experimental/metabolism , Cell Line, Tumor , Intramolecular Oxidoreductases/metabolismABSTRACT
Abnormal melanogenesis can lead to hyperpigmentation. Tyrosinase (TYR), a key rate-limiting enzyme in melanin production, is an important therapeutic target for these disorders. We investigated the TYR inhibitory activity of hydrolysates extracted from the muscle tissue of Takifugu flavidus (TFMH). We used computer-aided virtual screening to identify a novel peptide that potently inhibited melanin synthesis, simulated its binding mode to TYR, and evaluated functional efficacy in vitro and in vivo. TFMH inhibited the diphenolase activities of mTYR, reducing TYR substrate binding activity and effectively inhibiting melanin synthesis. TFMH indirectly reduced cAMP response element-binding protein phosphorylation in vitro by downregulating melanocortin 1 receptor expression, thereby inhibiting expression of the microphthalmia-associated transcription factor, further decreasing TYR, tyrosinase related protein 1, and dopachrome tautomerase expression and ultimately impeding melanin synthesis. In zebrafish, TFMH significantly reduced black spot formation. TFMH (200 µg/mL) decreased zebrafish TYR activity by 43% and melanin content by 52%. Molecular dynamics simulations over 100 ns revealed that the FGFRSP (T-6) peptide stably binds mushroom TYR via hydrogen bonds and ionic interactions. T-6 (400 µmol/L) reduced melanin content in B16F10 melanoma cells by 71% and TYR activity by 79%. In zebrafish, T-6 (200 µmol/L) inhibited melanin production by 64%. TFMH and T-6 exhibit good potential for the development of natural skin-whitening cosmetic products.
Subject(s)
Melanins , Melanoma, Experimental , Monophenol Monooxygenase , Takifugu , Zebrafish , Animals , Melanins/biosynthesis , Takifugu/metabolism , Monophenol Monooxygenase/antagonists & inhibitors , Monophenol Monooxygenase/metabolism , Mice , Melanoma, Experimental/drug therapy , Melanoma, Experimental/metabolism , Cell Line, Tumor , Microphthalmia-Associated Transcription Factor/metabolism , Muscles/drug effects , Muscles/metabolism , Intramolecular Oxidoreductases/metabolism , Receptor, Melanocortin, Type 1/metabolism , Molecular Dynamics Simulation , Cyclic AMP Response Element-Binding Protein/metabolismABSTRACT
BACKGROUND: Postpartum depression (PPD) is a serious psychiatric disorder that has significantly adverse impacts on maternal health. Metabolic abnormalities in the brain are associated with numerous neurological disorders, yet the specific metabolic signaling pathways and brain regions involved in PPD remain unelucidated. METHODS: We performed behavioral test in the virgin and postpartum mice. We used mass spectrometry imaging (MSI) and targeted metabolomics analyses to investigate the metabolic alternation in the brain of GABAAR Delta-subunit-deficient (Gabrd-/-) postpartum mice, a specific preclinical animal model of PPD. Next, we performed mechanism studies including qPCR, Western blot, immunofluorescence staining, electron microscopy and primary astrocyte culture. In the specific knockdown and rescue experiments, we injected the adeno-associated virus into the central amygdala (CeA) of female mice. RESULTS: We identified that prostaglandin D2 (PGD2) downregulation in the CeA was the most outstanding alternation in PPD, and then validated that lipocalin-type prostaglandin D synthase (L-PGDS)/PGD2 downregulation plays a causal role in depressive behaviors derived from PPD in both wild-type and Gabrd-/- mice. Furthermore, we verified that L-PGDS/PGD2 signaling dysfunction-induced astrocytes atrophy is mediated by Src phosphorylation both in vitro and in vivo. LIMITATIONS: L-PGDS/PGD2 signaling dysfunction may be only responsible for the depressive behavior rather than maternal behaviors in the PPD, and it remains to be seen whether this mechanism is applicable to all depression types. CONCLUSION: Our study identified abnormalities in the L-PGDS/PGD2 signaling in the CeA, which inhibited Src phosphorylation and induced astrocyte atrophy, ultimately resulting in the development of PPD in mice.
Subject(s)
Astrocytes , Atrophy , Depression, Postpartum , Disease Models, Animal , Prostaglandin D2 , Signal Transduction , Animals , Astrocytes/pathology , Astrocytes/metabolism , Female , Depression, Postpartum/pathology , Depression, Postpartum/metabolism , Mice , Signal Transduction/physiology , Prostaglandin D2/metabolism , Central Amygdaloid Nucleus/metabolism , Intramolecular Oxidoreductases/genetics , Intramolecular Oxidoreductases/metabolism , Lipocalins/genetics , Lipocalins/metabolism , src-Family Kinases/metabolism , Mice, KnockoutABSTRACT
ETHNOPHARMACOLOGICAL RELEVANCE: Tokishakuyakusan (TSS), a traditional Kampo medicine, can effectively alleviate symptoms unique to women, such as menstrual pain and menopausal symptoms, and this effect is believed to be related to its ability to increase the secretion of female hormones. TSS is also believed to be effective against skin pigmentation. However, no studies have examined the effect of TSS on pigmentation. AIM OF THE STUDY: In this study, we conducted basic research to determine the effects of TSS on pigmentation. MATERIALS AND METHODS: Female HRM-2 mice were given free access to a normal diet or a TSS-containing diet for 7 weeks. For 3 weeks starting from the 4th week of treatment, the back of the skin was irradiated with ultraviolet (UV) light, and the melanin level was measured. The expression levels of melanogenesis-related genes and inflammatory markers in the skin were analyzed. RESULTS: The melanin level in the skin of the mice exposed to UV radiation was approximately three times greater than that in the skin of the mice in the non-UV-irradiated group, confirming pigmentation due to UV irradiation. The protein expression levels of tyrosinase (Tyr), tyrosinase-related protein-1 (Tyrp1), and dopachrome tautomerase (Dct), which are important for melanin production, were significantly greater in the UV irradiation group than in the non-UV irradiation group. In contrast, the amount of skin melanin in the mice treated with TSS was significantly lower than that in the UV-irradiated group, and the expression levels of melanogenesis-related enzymes were also lower. Furthermore, TSS significantly decreased the expression of microphthalmia transcription factor (Mitf), a transcription factor for melanogenesis-related enzymes, and the inflammatory cytokines interleukin-1ß and interleukin-6. CONCLUSIONS: TSS inhibits melanin production in melanocytes by suppressing the increase in the expression of melanogenesis-related enzymes caused by UV irradiation. These findings suggested that this effect of TSS is exerted through the combined regulation of MITF expression and anti-inflammatory responses.
Subject(s)
Drugs, Chinese Herbal , Melanins , Monophenol Monooxygenase , Skin Pigmentation , Ultraviolet Rays , Animals , Ultraviolet Rays/adverse effects , Melanins/biosynthesis , Melanins/metabolism , Skin Pigmentation/drug effects , Skin Pigmentation/radiation effects , Female , Mice , Monophenol Monooxygenase/metabolism , Drugs, Chinese Herbal/pharmacology , Skin/drug effects , Skin/radiation effects , Skin/metabolism , Microphthalmia-Associated Transcription Factor/metabolism , Microphthalmia-Associated Transcription Factor/genetics , Medicine, Kampo , Intramolecular Oxidoreductases/metabolism , Intramolecular Oxidoreductases/genetics , Mice, Hairless , Melanogenesis , Membrane Glycoproteins , OxidoreductasesABSTRACT
BACKGROUND: The role of the macrophage migration inhibitory factor (MIF) has recently attracted considerable attention in cancer research; nonetheless, the insights provided by current investigations remain constrained. Our main objective was to investigate its role and the latent mechanisms within the pan-cancer realm. METHODS: We used comprehensive pan-cancer bulk sequencing data and online network tools to investigate the association between MIF expression and patient prognosis, genomic instability, cancer cell stemness, DNA damage repair, and immune infiltration. Furthermore, we validated the relationship between MIF expression and M0 macrophages using single-cell datasets, the SpatialDB database, and fluorescence staining. Additionally, we assessed the therapeutic response using the ROC plotter tool. RESULTS: We observed the upregulation of MIF expression across numerous cancer types. Notably, elevated MIF levels were associated with a decline in genomic stability. We found a significant correlation between increased MIF expression and increased expression of mismatch repair genes, stemness features, and homologous recombination genes across diverse malignancies. Subsequently, through an analysis using ESTIMATE and cytokine results, we revealed the involvement of MIF in immune suppression. Then, we validated MIF as a hallmark of the M0 macrophages involved in tumor immunity. Our study suggests an association with other immune-inhibitory cellular populations and restraint of CD8 + T cells. In addition, we conducted a comparative analysis of MIF expression before and after treatment in three distinct sets of therapy responders and non-responders. Intriguingly, we identified notable disparities in MIF expression patterns in bladder urothelial carcinoma and ovarian cancer following particular therapeutic interventions. CONCLUSION: Comprehensive pan-cancer analysis revealed notable enrichment of MIF within M0 macrophages, exerting a profound influence on tumor-associated immunosuppression and the intricate machinery of DNA repair.
Subject(s)
Biomarkers, Tumor , Intramolecular Oxidoreductases , Macrophage Migration-Inhibitory Factors , Macrophages , Neoplasms , Humans , Macrophage Migration-Inhibitory Factors/genetics , Macrophage Migration-Inhibitory Factors/metabolism , Intramolecular Oxidoreductases/genetics , Intramolecular Oxidoreductases/metabolism , Neoplasms/immunology , Neoplasms/genetics , Neoplasms/metabolism , Biomarkers, Tumor/genetics , Biomarkers, Tumor/metabolism , Macrophages/immunology , Macrophages/metabolism , Gene Expression Regulation, Neoplastic , Prognosis , Genomic Instability , Tumor Microenvironment/immunologyABSTRACT
Nucleus pulposus cells (NPCs) apoptosis and inflammation are the extremely critical factors of intervertebral disc degeneration (IVDD). Nevertheless, the underlying procedure remains mysterious. Macrophage migration inhibitory factor (MIF) is a cytokine that promotes inflammation and has been demonstrated to have a significant impact on apoptosis and inflammation. For this research, we employed a model of NPCs degeneration stimulated by lipopolysaccharides (LPS) and a rat acupuncture IVDD model to examine the role of MIF in vitro and in vivo, respectively. Initially, we verified that there was a significant rise of MIF expression in the NP tissues of individuals with IVDD, as well as in rat models of IVDD. Furthermore, this augmented expression of MIF was similarly evident in degenerated NPCs. Afterwards, it was discovered that ISO-1, a MIF inhibitor, effectively decreased the quantity of cells undergoing apoptosis and inhibited the release of inflammatory molecules (TNF-α, IL-1ß, IL-6). Furthermore, it has been shown that the PI3K/Akt pathway plays a vital part in the regulation of NPCs degeneration by MIF. Ultimately, we showcased that the IVDD process was impacted by the MIF inhibitor in the rat model. In summary, our experimental results substantiate the significant involvement of MIF in the degeneration of NPCs, and inhibiting MIF activity can effectively mitigate IVDD.