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1.
Cell Mol Life Sci ; 81(1): 296, 2024 Jul 11.
Artigo em Inglês | MEDLINE | ID: mdl-38992165

RESUMO

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.


Assuntos
Antígenos de Diferenciação de Linfócitos B , Linfócitos T CD4-Positivos , COVID-19 , Antígenos de Histocompatibilidade Classe II , Oxirredutases Intramoleculares , Ativação Linfocitária , Fatores Inibidores da Migração de Macrófagos , SARS-CoV-2 , Humanos , Antígenos de Diferenciação de Linfócitos B/metabolismo , Linfócitos T CD4-Positivos/metabolismo , Linfócitos T CD4-Positivos/imunologia , Antígenos de Histocompatibilidade Classe II/metabolismo , Antígenos de Histocompatibilidade Classe II/imunologia , Fatores Inibidores da Migração de Macrófagos/metabolismo , Fatores Inibidores da Migração de Macrófagos/genética , Ativação Linfocitária/imunologia , SARS-CoV-2/metabolismo , SARS-CoV-2/imunologia , COVID-19/imunologia , COVID-19/metabolismo , COVID-19/patologia , Oxirredutases Intramoleculares/metabolismo , Oxirredutases Intramoleculares/genética , Receptores CXCR4/metabolismo , Receptores CXCR4/genética , Movimento Celular , Masculino , Feminino , Pessoa de Meia-Idade , Receptores Imunológicos
2.
Sci Signal ; 16(812): eadg2621, 2023 11 21.
Artigo em Inglês | MEDLINE | ID: mdl-37988455

RESUMO

Mammalian macrophage migration inhibitory factor (MIF) and its paralog, D-dopachrome tautomerase, are multifunctional inflammatory cytokines. Plants have orthologous MIF and D-dopachrome tautomerase-like (MDL) proteins that mimic some of the effects of MIF on immune cells in vitro. We explored the structural and functional similarities between the three Arabidopsis thaliana MDLs and MIF. X-ray crystallography of the MDLs revealed high structural similarity between MDL and MIF homotrimers and suggested a potential explanation for the lack of tautomerase activity in the MDLs. MDL1 and MDL2 interacted with each other and with MIF in vitro, in yeast, and in plant leaves and formed hetero-oligomeric complexes with MIF in vitro. The MDLs stimulated signaling through the MIF receptors CXCR2 or CXCR4 and enhanced the responses to MIF in a yeast reporter system, in human neutrophils, and in human lung epithelial cells. Pharmacological inhibitors that disrupted MIF activity or prevented the formation of MIF-MDL hetero-oligomers blocked the observed synergism. These findings demonstrate that MDLs can enhance cellular responses to MIF, which may have functional implications in tissues exposed to MDLs from the diet or environment.


Assuntos
Fatores Inibidores da Migração de Macrófagos , Animais , Humanos , Fatores Inibidores da Migração de Macrófagos/genética , Fatores Inibidores da Migração de Macrófagos/química , Proteínas de Plantas , Receptores CXCR4/genética , Receptores CXCR4/metabolismo , Saccharomyces cerevisiae/metabolismo , Neutrófilos/metabolismo , Mamíferos/metabolismo , Oxirredutases Intramoleculares/genética
3.
FASEB J ; 36(11): e22601, 2022 11.
Artigo em Inglês | MEDLINE | ID: mdl-36269019

RESUMO

Macrophage migration inhibitory factor (MIF) as well as its more recently described structural homolog D-dopachrome tautomerase (D-DT), now also termed MIF-2, are atypical cytokines and chemokines with key roles in host immunity. They also have an important pathogenic role in acute and chronic inflammatory conditions, cardiovascular diseases, lung diseases, adipose tissue inflammation, and cancer. Although our mechanistic understanding of MIF-2 is relatively limited compared to the extensive body of evidence available for MIF, emerging data suggests that MIF-2 is not only a functional phenocopy of MIF, but may have differential or even oppositional activities, depending on the disease and context. In this review, we summarize and discuss the similarities and differences between MIF and MIF-2, with a focus on their structures, receptors, signaling pathways, and their roles in diseases. While mainly covering the roles of the MIF homologs in cardiovascular, inflammatory, autoimmune, and metabolic diseases, we also discuss their involvement in cancer, sepsis, and chronic obstructive lung disease (COPD). A particular emphasis is laid upon potential mechanistic explanations for synergistic or cooperative activities of the MIF homologs in cancer, myocardial diseases, and COPD as opposed to emerging disparate or antagonistic activities in adipose tissue inflammation, metabolic diseases, and atherosclerosis. Lastly, we discuss potential future opportunities of jointly targeting MIF and MIF-2 in certain diseases, whereas precision targeting of only one homolog might be preferable in other conditions. Together, this article provides an update of the mechanisms and future therapeutic avenues of human MIF proteins with a focus on their emerging, surprisingly disparate activities, suggesting that MIF-2 displays a variety of activities that are distinct from those of MIF.


Assuntos
Doenças Cardiovasculares , Inflamação , Oxirredutases Intramoleculares , Humanos , Quimiocinas/metabolismo , Inflamação/metabolismo , Oxirredutases Intramoleculares/metabolismo , Fatores Inibidores da Migração de Macrófagos/metabolismo
5.
Cell Mol Life Sci ; 79(10): 512, 2022 Sep 12.
Artigo em Inglês | MEDLINE | ID: mdl-36094626

RESUMO

To fulfil its orchestration of immune cell trafficking, a network of chemokines and receptors developed that capitalizes on specificity, redundancy, and functional selectivity. The discovery of heteromeric interactions in the chemokine interactome has expanded the complexity within this network. Moreover, some inflammatory mediators, not structurally linked to classical chemokines, bind to chemokine receptors and behave as atypical chemokines (ACKs). We identified macrophage migration inhibitory factor (MIF) as an ACK that binds to chemokine receptors CXCR2 and CXCR4 to promote atherogenic leukocyte recruitment. Here, we hypothesized that chemokine-chemokine interactions extend to ACKs and that MIF forms heterocomplexes with classical chemokines. We tested this hypothesis by using an unbiased chemokine protein array. Platelet chemokine CXCL4L1 (but not its variant CXCL4 or the CXCR2/CXCR4 ligands CXCL8 or CXCL12) was identified as a candidate interactor. MIF/CXCL4L1 complexation was verified by co-immunoprecipitation, surface plasmon-resonance analysis, and microscale thermophoresis, also establishing high-affinity binding. We next determined whether heterocomplex formation modulates inflammatory/atherogenic activities of MIF. Complex formation was observed to inhibit MIF-elicited T-cell chemotaxis as assessed by transwell migration assay and in a 3D-matrix-based live cell-imaging set-up. Heterocomplexation also blocked MIF-triggered migration of microglia in cortical cultures in situ, as well as MIF-mediated monocyte adhesion on aortic endothelial cell monolayers under flow stress conditions. Of note, CXCL4L1 blocked binding of Alexa-MIF to a soluble surrogate of CXCR4 and co-incubation with CXCL4L1 attenuated MIF responses in HEK293-CXCR4 transfectants, indicating that complex formation interferes with MIF/CXCR4 pathways. Because MIF and CXCL4L1 are platelet-derived products, we finally tested their role in platelet activation. Multi-photon microscopy, FLIM-FRET, and proximity-ligation assay visualized heterocomplexes in platelet aggregates and in clinical human thrombus sections obtained from peripheral artery disease (PAD) in patients undergoing thrombectomy. Moreover, heterocomplexes inhibited MIF-stimulated thrombus formation under flow and skewed the lamellipodia phenotype of adhering platelets. Our study establishes a novel molecular interaction that adds to the complexity of the chemokine interactome and chemokine/receptor-network. MIF/CXCL4L1, or more generally, ACK/CXC-motif chemokine heterocomplexes may be target structures that can be exploited to modulate inflammation and thrombosis.


Assuntos
Aterosclerose , Fatores Inibidores da Migração de Macrófagos , Trombose , Aterosclerose/metabolismo , Células HEK293 , Humanos , Inflamação/metabolismo , Oxirredutases Intramoleculares , Fatores Inibidores da Migração de Macrófagos/metabolismo , Fator Plaquetário 4 , Receptores de Interleucina-8B/química , Receptores de Interleucina-8B/genética , Receptores de Interleucina-8B/metabolismo
6.
Structure ; 30(6): 787-790, 2022 06 02.
Artigo em Inglês | MEDLINE | ID: mdl-35660241

RESUMO

In this issue of Structure, Skeens et al. provide insights into the structure and dynamics of an oxidized form of the atypical cytokine macrophage migration-inhibitory factor (MIF). The study unveils a surprising conformational susceptibility of MIF to ambient redox alterations and identifies redox-sensitive residues and latent allostery sites with functional relevance.


Assuntos
Fatores Inibidores da Migração de Macrófagos , Sítio Alostérico , Fatores Inibidores da Migração de Macrófagos/química , Fatores Inibidores da Migração de Macrófagos/metabolismo , Oxirredução
7.
Front Physiol ; 12: 638448, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34366876

RESUMO

Background: Adipose-derived stem cells (ASCs) are multipotent mesenchymal stem cells characterized by their strong regenerative potential and low oxygen consumption. Macrophage migration inhibitory factor (MIF) is a multifunctional chemokine-like cytokine that is involved in tissue hypoxia. MIF is not only a major immunomodulator but also is highly expressed in adipose tissue such as subcutaneous adipose tissue of chronic non-healing wounds. In the present study, we investigated the effect of hypoxia on MIF in ASCs isolated from healthy versus inflamed adipose tissue. Methods: Human ASCs were harvested from 17 patients (11 healthy adipose tissue samples, six specimens from chronic non-healing wounds). ASCs were treated in a hypoxia chamber at <1% oxygen. ASC viability, MIF secretion as well as expression levels of MIF, its receptor CD74, hypoxia-inducible transcription factor-1α (HIF-1α) and activation of the AKT and ERK signaling pathways were analyzed. The effect of recombinant MIF on the viability of ASCs was determined. Finally, the effect of MIF on the viability and production capacity of ASCs to produce the inflammatory cytokines tumor necrosis factor (TNF), interleukin (IL)-6, and IL-1ß was determined upon treatment with recombinant MIF and/or a blocking MIF antibody. Results: Hypoxic treatment inhibited proliferation of ASCs derived from healthy or chronic non-healing wounds. ASCs from healthy adipose tissue samples were characterized by a low degree of MIF secretion during hypoxic challenge. In contrast, in ASCs from adipose tissue samples of chronic non-healing wounds, secretion and expression of MIF and CD74 expression were significantly elevated under hypoxia. This was accompanied by enhanced ERK signaling, while AKT signaling was not altered. Recombinant MIF did stimulate HIF-1α expression under hypoxia as well as AKT and ERK phosphorylation, while no effect on ASC viability was observed. Recombinant MIF significantly reduced the secretion of IL-1ß under hypoxia and normoxia, and neutralizing MIF-antibodies diminished TNF-α and IL-1ß release in hypoxic ASCs. Conclusions: Collectively, MIF did not affect the viability of ASCs from neither healthy donor site nor chronic wounds. Our results, however, suggest that MIF has an impact on the wound environment by modulating inflammatory factors such as IL-1ß.

8.
Chembiochem ; 22(6): 1012-1019, 2021 03 16.
Artigo em Inglês | MEDLINE | ID: mdl-33125165

RESUMO

Macrophage migration inhibitory factor (MIF) is an inflammatory cytokine and atypical chemokine with a key role in inflammatory diseases including atherosclerosis. Key atherogenic functions of MIF are mediated by noncognate interaction with the chemokine receptor CXCR2. The MIF N-like loop comprising the sequence 47-56 is an important structural determinant of the MIF/CXCR2 interface and MIF(47-56) blocks atherogenic MIF activities. However, the mechanism and critical structure-activity information within this sequence have remained elusive. Here, we show that MIF(47-56) directly binds to CXCR2 to compete with MIF receptor activation. By using alanine scanning, essential and dispensable residues were identified. Moreover, MIF(cyclo10), a designed cyclized variant of MIF(47-56), inhibited key inflammatory and atherogenic MIF activities in vitro and in vivo/ex vivo, and exhibited strongly improved resistance to proteolytic degradation in human plasma in vitro, thus suggesting that it could serve as a promising basis for MIF-derived anti-atherosclerotic peptides.


Assuntos
Fatores Inibidores da Migração de Macrófagos/química , Peptídeos Cíclicos/metabolismo , Receptores de Interleucina-8B/metabolismo , Sequência de Aminoácidos , Animais , Adesão Celular , Fluoresceínas/química , Células HEK293 , Humanos , Leucócitos/química , Leucócitos/citologia , Leucócitos/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Peptídeos Cíclicos/sangue , Peptídeos Cíclicos/química , Ligação Proteica , Estabilidade Proteica , Receptores de Interleucina-8B/antagonistas & inibidores , Espectrometria de Fluorescência , Ácidos Sulfônicos/química
9.
Nat Commun ; 11(1): 5981, 2020 11 25.
Artigo em Inglês | MEDLINE | ID: mdl-33239628

RESUMO

Targeting a specific chemokine/receptor axis in atherosclerosis remains challenging. Soluble receptor-based strategies are not established for chemokine receptors due to their discontinuous architecture. Macrophage migration-inhibitory factor (MIF) is an atypical chemokine that promotes atherosclerosis through CXC-motif chemokine receptor-4 (CXCR4). However, CXCR4/CXCL12 interactions also mediate atheroprotection. Here, we show that constrained 31-residue-peptides ('msR4Ms') designed to mimic the CXCR4-binding site to MIF, selectively bind MIF with nanomolar affinity and block MIF/CXCR4 without affecting CXCL12/CXCR4. We identify msR4M-L1, which blocks MIF- but not CXCL12-elicited CXCR4 vascular cell activities. Its potency compares well with established MIF inhibitors, whereas msR4M-L1 does not interfere with cardioprotective MIF/CD74 signaling. In vivo-administered msR4M-L1 enriches in atherosclerotic plaques, blocks arterial leukocyte adhesion, and inhibits atherosclerosis and inflammation in hyperlipidemic Apoe-/- mice in vivo. Finally, msR4M-L1 binds to MIF in plaques from human carotid-endarterectomy specimens. Together, we establish an engineered GPCR-ectodomain-based mimicry principle that differentiates between disease-exacerbating and -protective pathways and chemokine-selectively interferes with atherosclerosis.


Assuntos
Aterosclerose/tratamento farmacológico , Oxirredutases Intramoleculares/antagonistas & inibidores , Fatores Inibidores da Migração de Macrófagos/antagonistas & inibidores , Fragmentos de Peptídeos/farmacologia , Receptores CXCR4/metabolismo , Idoso , Animais , Antígenos CD/metabolismo , Aterosclerose/genética , Aterosclerose/patologia , Aterosclerose/cirurgia , Sítios de Ligação , Artéria Carótida Primitiva/patologia , Artéria Carótida Primitiva/cirurgia , Quimiocina CXCL12/metabolismo , Cristalografia por Raios X , Modelos Animais de Doenças , Desenho de Fármacos , Avaliação Pré-Clínica de Medicamentos , Endarterectomia das Carótidas , Feminino , Humanos , Oxirredutases Intramoleculares/metabolismo , Fatores Inibidores da Migração de Macrófagos/metabolismo , Masculino , Camundongos , Camundongos Knockout para ApoE , Pessoa de Meia-Idade , Fragmentos de Peptídeos/uso terapêutico , Receptores CXCR4/química , Receptores CXCR4/ultraestrutura , Sialiltransferases/metabolismo , Transdução de Sinais/efeitos dos fármacos
10.
J Biol Chem ; 295(3): 850-867, 2020 01 17.
Artigo em Inglês | MEDLINE | ID: mdl-31811089

RESUMO

Human macrophage migration-inhibitory factor (MIF) is an evolutionarily-conserved protein that has both extracellular immune-modulating and intracellular cell-regulatory functions. MIF plays a role in various diseases, including inflammatory diseases, atherosclerosis, autoimmunity, and cancer. It serves as an inflammatory cytokine and chemokine, but also exhibits enzymatic activity. Secreted MIF binds to cell-surface immune receptors such as CD74 and CXCR4. Plants possess MIF orthologs but lack the associated receptors, suggesting functional diversification across kingdoms. Here, we characterized three MIF orthologs (termed MIF/d-dopachrome tautomerase-like proteins or MDLs) of the model plant Arabidopsis thaliana Recombinant Arabidopsis MDLs (AtMDLs) share similar secondary structure characteristics with human MIF, yet only have minimal residual tautomerase activity using either p-hydroxyphenylpyruvate or dopachrome methyl ester as substrate. Site-specific mutagenesis suggests that this is due to a distinct amino acid difference at the catalytic cavity-defining residue Asn-98. Surprisingly, AtMDLs bind to the human MIF receptors CD74 and CXCR4. Moreover, they activate CXCR4-dependent signaling in a receptor-specific yeast reporter system and in CXCR4-expressing human HEK293 transfectants. Notably, plant MDLs exert dose-dependent chemotactic activity toward human monocytes and T cells. A small molecule MIF inhibitor and an allosteric CXCR4 inhibitor counteract this function, revealing its specificity. Our results indicate cross-kingdom conservation of the receptor signaling and leukocyte recruitment capacities of human MIF by its plant orthologs. This may point toward a previously unrecognized interplay between plant proteins and the human innate immune system.


Assuntos
Antígenos de Diferenciação de Linfócitos B/genética , Antígenos de Histocompatibilidade Classe II/genética , Imunidade Inata/genética , Oxirredutases Intramoleculares/genética , Fatores Inibidores da Migração de Macrófagos/genética , Receptores CXCR4/genética , Antígenos de Diferenciação de Linfócitos B/química , Arabidopsis/genética , Arabidopsis/imunologia , Quimiotaxia/genética , Quimiotaxia/imunologia , Sequência Conservada/genética , Sequência Conservada/imunologia , Citocinas/genética , Citocinas/imunologia , Células HEK293 , Antígenos de Histocompatibilidade Classe II/química , Humanos , Oxirredutases Intramoleculares/química , Oxirredutases Intramoleculares/imunologia , Fatores Inibidores da Migração de Macrófagos/química , Fatores Inibidores da Migração de Macrófagos/imunologia , Monócitos/química , Monócitos/metabolismo , Ligação Proteica/genética , Receptores CXCR4/química , Homologia de Sequência , Linfócitos T/química , Linfócitos T/metabolismo
11.
Sci Rep ; 8(1): 5171, 2018 03 26.
Artigo em Inglês | MEDLINE | ID: mdl-29581527

RESUMO

MIF is a chemokine-like cytokine that plays a role in the pathogenesis of inflammatory and cardiovascular disorders. It binds to the chemokine-receptors CXCR2/CXCR4 to trigger atherogenic leukocyte migration albeit lacking canonical chemokine structures. We recently characterized an N-like-loop and the Pro-2-residue of MIF as critical molecular determinants of the CXCR4/MIF binding-site and identified allosteric agonism as a mechanism that distinguishes CXCR4-binding to MIF from that to the cognate ligand CXCL12. By using peptide spot-array technology, site-directed mutagenesis, structure-activity-relationships, and molecular docking, we identified the Arg-Leu-Arg (RLR) sequence-region 87-89 that - in three-dimensional space - 'extends' the N-like-loop to control site-1-binding to CXCR4. Contrary to wildtype MIF, mutant R87A-L88A-R89A-MIF fails to bind to the N-terminal of CXCR4 and the contribution of RLR to the MIF/CXCR4-interaction is underpinned by an ablation of MIF/CXCR4-specific signaling and reduction in CXCR4-dependent chemotactic leukocyte migration of the RLR-mutant of MIF. Alanine-scanning, functional competition by RLR-containing peptides, and molecular docking indicate that the RLR residues directly participate in contacts between MIF and CXCR4 and highlight the importance of charge-interactions at this interface. Identification of the RLR region adds important structural information to the MIF/CXCR4 binding-site that distinguishes this interface from CXCR4/CXCL12 and will help to design MIF-specific drug-targeting approaches.


Assuntos
Oxirredutases Intramoleculares/genética , Fatores Inibidores da Migração de Macrófagos/genética , Ligação Proteica/genética , Receptores CXCR4/genética , Relação Estrutura-Atividade , Sítios de Ligação , Doenças Cardiovasculares/genética , Doenças Cardiovasculares/patologia , Quimiotaxia/genética , Humanos , Inflamação/genética , Inflamação/patologia , Oxirredutases Intramoleculares/química , Ligantes , Fatores Inibidores da Migração de Macrófagos/química , Simulação de Acoplamento Molecular , Peptídeos/química , Peptídeos/genética , Receptores CXCR4/química , Receptores de Interleucina-8B/química , Receptores de Interleucina-8B/genética
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