Macrophage Migration Inhibitory Factor (MIF) and D-Dopachrome Tautomerase (DDT): Pathways to Tumorigenesis and Therapeutic Opportunities.

Discovered as inflammatory cytokines, MIF and DDT exhibit widespread expression and have emerged as critical mediators in the response to infection, inflammation, and more recently, in cancer. In this comprehensive review, we provide details on their structures, binding partners, regulatory mechanisms, and roles in cancer. We also elaborate on their significant impact in driving tumorigenesis across various cancer types, supported by extensive in vitro, in vivo, bioinformatic, and clinical studies. To date, only a limited number of clinical trials have explored MIF as a therapeutic target in cancer patients, and DDT has not been evaluated. The ongoing pursuit of optimal strategies for targeting MIF and DDT highlights their potential as promising antitumor candidates. Dual inhibition of MIF and DDT may allow for the most effective suppression of canonical and non-canonical signaling pathways, warranting further investigations and clinical exploration.

Single-cell RNA sequencing reveals aberrant sphingolipid metabolism in non-small cell lung cancer impacts tumor-associated macrophages and stimulates angiogenesis via macrophage inhibitory factor signaling.

BACKGROUND: Sphingolipids not only serve as structural components for maintaining cell membrane fluidity but also function as bioactive molecules involved in cell signaling and the regulation of various biological processes. Their pivotal role in cancer cell development, encompassing cancer cell proliferation, migration, angiogenesis, and metastasis, has been a focal point for decades. However, the contribution of sphingolipids to the complexity of tumor microenvironment promoting cancer progression has been rarely investigated. METHODS: Through the integration of publicly available bulk RNA-seq and single-cell RNA-seq data, we conducted a comprehensive analysis to compare the transcriptomic features between tumors and adjacent normal tissues, thus elucidating the intricacies of the tumor microenvironment (TME). RESULTS: Disparities in sphingolipid metabolism (SLM)-associated genes were observed between normal and cancerous tissues, with the TME characterized by the enrichment of sphingolipid signaling in macrophages. Cellular interaction analysis revealed robust communication between macrophages and cancer cells exhibiting low SLM, identifying the crucial ligand-receptor pair, macrophage inhibitory factor (MIF)-CD74. Pseudo-time analysis unveiled the involvement of SLM in modulating macrophage polarization towards either M1 or M2 phenotypes. Categorizing macrophages into six subclusters based on gene expression patterns and function, the SPP1+ cluster, RGS1+ cluster, and CXCL10+ cluster were likely implicated in sphingolipid-induced M2 macrophage polarization. Additionally, the CXCL10+, AGER+, and FABP4+ clusters were likely to be involved in angiogenesis through their interaction with endothelial cells. CONCLUSION: Based on multiple scRNA-seq datasets, we propose that a MIF-targeted strategy could potentially impede the polarization from M1 to M2 and impair tumor angiogenesis in low-SLM non-small cell lung cancer (NSCLC), demonstrating its potent antitumor efficacy.

MIF-Modulated Spinal Proteins Associated with Persistent Bladder Pain: A Proteomics Study.

Bladder pain is a prominent symptom in Interstitial Cystitis/Bladder Pain Syndrome (IC/BPS). We studied spinal mechanisms of bladder pain in mice using a model where repeated activation of intravesical Protease Activated Receptor-4 (PAR4) results in persistent bladder hyperalgesia (BHA) with little or no bladder inflammation. Persistent BHA is mediated by spinal macrophage migration inhibitory factor (MIF), and is associated with changes in lumbosacral proteomics. We investigated the contribution of individual spinal MIF receptors to persistent bladder pain as well as the spinal proteomics changes associated with relief of persistent BHA by spinal MIF antagonism. Female mice with persistent BHA received either intrathecal (i.t.) MIF monoclonal antibodies (mAb) or mouse IgG1 (isotype control antibody). MIF antagonism temporarily reversed persistent BHA (peak effect: 2 h), while control IgG1 had no effect. Moreover, i.t. antagonism of the MIF receptors CD74 and C-X-C chemokine receptor type 4 (CXCR4) partially reversed persistent BHA. For proteomics experiments, four separate groups of mice received either repeated intravesical scrambled peptide and sham i.t. injection (control, no pain group) or repeated intravesical PAR4 and: sham i.t.; isotype IgG1 i.t. (15 µg); or MIF mAb (15 µg). L6-S1 spinal segments were excised 2 h post-injection and examined for proteomics changes using LC-MS/MS. Unbiased proteomics analysis identified and relatively quantified 6739 proteins. We selected proteins that showed significant changes compared to control (no pain group) after intravesical PAR4 (sham or IgG i.t. treatment) and showed no significant change after i.t. MIF antagonism. Six proteins decreased during persistent BHA (V-set transmembrane domain-containing protein 2-like confirmed by immunohistochemistry), while two proteins increased. Spinal MIF antagonism reversed protein changes. Therefore, spinal MIF and MIF receptors mediate persistent BHA and changes in specific spinal proteins. These novel MIF-modulated spinal proteins represent possible new targets to disrupt spinal mechanisms that mediate persistent bladder pain.

Blocking the MIF-CD74 axis augments radiotherapy efficacy for brain metastasis in NSCLC via synergistically promoting microglia M1 polarization.

BACKGROUND: Brain metastasis is one of the main causes of recurrence and death in non-small cell lung cancer (NSCLC). Although radiotherapy is the main local therapy for brain metastasis, it is inevitable that some cancer cells become resistant to radiation. Microglia, as macrophages colonized in the brain, play an important role in the tumor microenvironment. Radiotherapy could activate microglia to polarize into both the M1 and M2 phenotypes. Therefore, searching for crosstalk molecules within the microenvironment that can specifically regulate the polarization of microglia is a potential strategy for improving radiation resistance. METHODS: We used databases to detect the expression of MIF in NSCLC and its relationship with prognosis. We analyzed the effects of targeted blockade of the MIF/CD74 axis on the polarization and function of microglia during radiotherapy using flow cytometry. The mouse model of brain metastasis was used to assess the effect of targeted blockade of MIF/CD74 axis on the growth of brain metastasis. RESULT: Our findings reveals that the macrophage migration inhibitory factor (MIF) was highly expressed in NSCLC and is associated with the prognosis of NSCLC. Mechanistically, we demonstrated CD74 inhibition reversed radiation-induced AKT phosphorylation in microglia and promoted the M1 polarization in combination of radiation. Additionally, blocking the MIF-CD74 interaction between NSCLC and microglia promoted microglia M1 polarization. Furthermore, radiation improved tumor hypoxia to decrease HIF-1α dependent MIF secretion by NSCLC. MIF inhibition enhanced radiosensitivity for brain metastasis via synergistically promoting microglia M1 polarization in vivo. CONCLUSIONS: Our study revealed that targeting the MIF-CD74 axis promoted microglia M1 polarization and synergized with radiotherapy for brain metastasis in NSCLC.

Breast Cancer Stem Cells Secrete MIF to Mediate Tumor Metabolic Reprogramming That Drives Immune Evasion.

Reprogramming of energy metabolism exerts pivotal functions in cancer progression and immune surveillance. Identification of the mechanisms mediating metabolic changes in cancer may lead to improved strategies to suppress tumor growth and stimulate antitumor immunity. Here, it was observed that the secretomes of hypoxic breast cancer cells and breast cancer stem cells (BCSC) induced reprogramming of metabolic pathways, particularly glycolysis, in normoxic breast cancer cells. Screening of the BCSC secretome identified MIF as a pivotal factor potentiating glycolysis. Mechanistically, MIF increased c-MYC-mediated transcriptional upregulation of the glycolytic enzyme aldolase C by activating WNT/ß-catenin signaling. Targeting MIF attenuated glycolysis and impaired xenograft growth and metastasis. MIF depletion in breast cancer cells also augmented intratumoral cytolytic CD8+ T cells and proinflammatory macrophages while decreasing regulatory T cells and tumor-associated neutrophils in the tumor microenvironment. Consequently, targeting MIF improved the therapeutic efficacy of immune checkpoint blockade in triple-negative breast cancer. Collectively, this study proposes MIF as an attractive therapeutic target to circumvent metabolic reprogramming and immunosuppression in breast cancer. SIGNIFICANCE: MIF secreted by breast cancer stem cells induces metabolic reprogramming in bulk tumor cells and engenders an immunosuppressive microenvironment, identifying MIF targeting as a strategy to improve immunotherapy efficacy in breast cancer.

Single-cell RNA sequencing reveals the MIF-ACKR3 receptor-ligand interaction between iCAFs and tumor cells in esophageal squamous cell carcinoma.

BACKGROUND: Esophageal squamous cell carcinoma (ESCC) is a malignant tumor of the gastrointestinal tract with a high morbidity and mortality rate. The heterogeneity of ESCC poses challenges in treatment and contributes to the poor prognosis of patients. Therefore, it is crucial to gain a better understanding of the tumor microenvironment (TME) heterogeneity and identify novel therapeutic targets. METHODS: To solve this problem, we performed a single-cell RNA sequencing (scRNA-seq) analysis of ESCC samples obtained from the GEO database. RESULTS: A total of 31,283 single cells were categorized into nine cell types, which included four non-immune cells (epithelial cells, endothelial cells, fibroblasts, schwann cells) and five immune cells (T cells, macrophages, mast cells, neutrophils, B cells). Our study revealed the presence of immunosuppressive tumor microenvironments in ESCC. We have also identified not only inflammatory cancer-associated fibroblast (iCAFs) and myofibroblastic cancer-associated fibroblasts (myCAFs) but also a subset of antigen presenting cancer-associated fibroblasts (apCAFs) which express high levels of HLA class II molecules in ESCC. Furthermore, our analysis of cell communication showed up-regulation of MIF-ACKR3 interaction between iCAFs and tumor cells in tumors compared to normal tissues. Finally, it was demonstrated that macrophage migration inhibitory factor (MIF) facilitates tumor cell migration and invasion through interacting with ACKR3 in vitro. CONCLUSIONS: This study exposes the features of the tumor microenvironment of ESCC via scRNA-seq and examines the dynamics of various cellular subpopulations, thus facilitating the identification of future therapeutic targets for ESCC.

Sulforaphane effectively inhibits HBV by altering Treg/Th17 immune balance and the MIF-macrophages polarizing axis in vitro and in vivo.

BACKGROUND: Hepatitis B virus (HBV) infection is a major public health problem. After HBV infection, viral antigens shift the immune balance in favor of viral escape. Sulforaphane (SFN) is a traditional Chinese medicine.It regulates multi-biological activities, including anti-inflammation, anticancer, and antiviral. However, few studies reported that SFN can inhibit HBV infection before. METHODS: An immunocompetent HBV CBA/CaJ mouse model and a co-culture model were used to explore the effect of SFN on HBV and whether SFN altered the immune balance after HBV infection. RESULTS: We found that SFN was able to reduce HBV DNA, cccDNA, HBsAg, HBeAg, and HBcAg levels in serum and liver tissues of HBV-infected mice. In vitro and in vivo experiments showed that SFN could significantly increase the expression of Cd86 and iNOS and inhibit the expression of Arg1 on macrophages after HBV infection. After SFN administration, Th17 markers in liver tissue and serum were significantly increased. There was no significant changes in the proportion of Treg cells in peripheral blood, but a significant increase in the proportion of Th17 cells and decrease of the Treg/Th17 ratio. Using a network pharmacology approach, we predicted macrophage migration inhibitory factor (MIF) as a potential target of SFN and further validated that MIF expression was significantly increased after HBV infection and SFN significantly inhibited MIF expression both in vitro and in vivo. There was an upward trend in HBV markers (p>0.05) after MIF overexpression. Overexpression of MIF combined with the use of SFN resulted in a significant reversion in the expression of HBV markers and polarization of macrophages towards the M1 phenotype. CONCLUSION: Our results indicated that immunocompetent HBV CBA/CaJ mouse model is a good model to evaluate HBV infection. SFN could inhibit the expression of HBV markers, promote polarization of macrophages towards the M1 phenotype after HBV infection, change the proportion of Treg and Th17 cells. Our findings demonstrate that SFN inhibit HBV infection by inhibiting the expression of MIF and promoting the polarization of macrophages towards the M1 phenotype, which illustrates a promising therapeutic approach in HBV infection.

Murine double minute 2-mediated estrogen receptor 1 degradation activates macrophage migration inhibitory factor to promote vascular smooth muscle cell dedifferentiation and oxidative stress during thoracic aortic aneurysm progression.

Estrogen receptor 1 (ESR1) has been recently demonstrated as a potential diagnostic biomarker for thoracic aortic aneurysm (TAA). However, its precise role in the progression of TAA remains unclear. In this study, TAA models were established in ApoE-knockout mice and primary mouse vascular smooth muscle cells (VSMCs) through treatment with angiotensin (Ang) II. Our findings revealed a downregulation of ESR1 in Ang II-induced TAA mice and VSMCs. Upregulation of ESR1 mitigated expansion and cell apoptosis in the mouse aorta, reduced pathogenetic transformation of VSMCs, and reduced inflammatory infiltration and oxidative stress both in vitro and in vivo. Furthermore, we identified macrophage migration inhibitory factor (MIF) as a biological target of ESR1. ESR1 bound to the MIF promoter to suppress its transcription. Artificial MIF restoration negated the mitigating effects of ESR1 on TAA. Additionally, we discovered that murine double minute 2 (MDM2) was highly expressed in TAA models and mediated protein degradation of ESR1 through ubiquitination modification. Silencing of MDM2 reduced VSMC dedifferentiation and suppressed oxidative stress. However, these effects were reversed upon further silencing of ESR1. In conclusion, this study demonstrates that MDM2 activates MIF by mediating ESR1 degradation, thus promoting VSMC dedifferentiation and oxidative stress during TAA progression.

Periodontitis exacerbation during pregnancy in mice: Role of macrophage migration inhibitory factor as a key inductor.

OBJECTIVE: The present study was designed to investigate the role of macrophage migration inhibitory factor (MIF) in the exacerbation of pregestational periodontal disease (PGPD). BACKGROUND: Periodontitis (PT) is a severe stage of periodontal disease characterized by inflammation of the supporting tissues of the teeth, which usually worsens during pregnancy. MIF is a proinflammatory cytokine that is significantly elevated in periodontitis, both at the beginning and at the end of pregnancy. Although periodontitis usually presents with greater severity during pregnancy, the participation of MIF in the evolution of periodontitis has not been established. METHODS: To analyze the relevance of MIF in the exacerbation of PGPD, we employed a model of PGPD in WT and Mif-/- mice, both with a BALB/c genetic background. PT was induced with nylon suture ligatures placed supramarginally around the second upper right molar. For PGPD, PT was induced 2 weeks before mating. We evaluated histological changes and performed histometric analysis of the clinical attachment loss, relative expression of MMP-2 and MMP-13 by immunofluorescence, and relative expression of the cytokines mif, tnf-α, ifn-γ, and il-17 by quantitative real-time polymerase chain reaction (qRT-PCR). RESULTS: Our data revealed that periodontal tissue from PGPD WT mice produced a twofold increase in MIF compared with PT WT mice. Moreover, the evolution of periodontitis in Mif-/- mice was less severe than in PGDP WT mice. Periodontal tissue from Mif-/- mice with PGPD produced 80% less TNF-α and no IFN-γ, as well as 50% lower expression of matrix metalloproteinase (MMP)-2 and 25% less MMP-13 compared to WT PGDP mice. CONCLUSIONS: Our study suggests that MIF plays an important role in the exacerbation of periodontitis during pregnancy and that MIF is partially responsible for the inflammation associated with the severity of periodontitis during pregnancy.

The Tautomerase Activity of Tumor Exosomal MIF Promotes Pancreatic Cancer Progression by Modulating MDSC Differentiation.

Pancreatic cancer is a deadly disease that is largely resistant to immunotherapy, in part because of the accumulation of immunosuppressive cells in the tumor microenvironment (TME). Much evidence suggests that tumor-derived exosomes (TDE) contribute to the immunosuppressive activity mediated by myeloid-derived suppressor cells (MDSC) within the pancreatic cancer TME. However, the underlying mechanisms remain elusive. Herein, we report that macrophage migration inhibitory factor (MIF) in TDEs has a key role in inducing MDSC formation in pancreatic cancer. We identified MIF in both human and murine pancreatic cancer-derived exosomes. Upon specific shRNA-mediated knockdown of MIF, the ability of pancreatic cancer-derived exosomes to promote MDSC differentiation was abrogated. This phenotype was rescued by reexpression of the wild-type form of MIF rather than a tautomerase-null mutant or a thiol-protein oxidoreductase-null mutant, indicating that both MIF enzyme activity sites play a role in exosome-induced MDSC formation in pancreatic cancer. RNA sequencing data indicated that MIF tautomerase regulated the expression of genes required for MDSC differentiation, recruitment, and activation. We therefore developed a MIF tautomerase inhibitor, IPG1576. The inhibitor effectively inhibited exosome-induced MDSC differentiation in vitro and reduced tumor growth in an orthotopic pancreatic cancer model, which was associated with decreased numbers of MDSCs and increased infiltration of CD8+ T cells in the TME. Collectively, our findings highlight a pivotal role for MIF in exosome-induced MDSC differentiation in pancreatic cancer and underscore the potential of MIF tautomerase inhibitors to reverse the immunosuppressive pancreatic cancer microenvironment, thereby augmenting anticancer immune responses.

Single-cell RNA-seq reveals MIF-(CD74 + CXCR4) dependent inhibition of macrophages in metastatic papillary thyroid carcinoma.

BACKGROUND: The mechanism promoting papillary thyroid carcinoma (PTC) metastasis remains unclear. We aimed to investigate the potential metastatic mechanisms at a single-cell resolution. METHODS: We performed single-cell RNA-seq (scRNA-seq) profiling of thyroid tumour (TT), adjacent normal thyroid (NT) and lymph node metastasized tumour (LN) from a young female with PTC. Validation of our results was conducted in 31 tumours with metastasis and 30 without metastasis. RESULTS: ScRNA-seq analysis generated data on 38,215 genes and 0.14 billion transcripts from 28,839 cells, classified into 18 clusters, each annotated to represent 10 cell types. PTC cells were found to originate from epithelial cells. Epithelial cells and macrophages emerged as the strongest signal emitters and receivers, respectively. After reclustering epithelial cells and macrophages, our analysis, incorporating gene set variation analysis (GSVA), SCENIC analysis, and pseudotime trajectory analysis, indicated that subcluster 0 of epithelial cells (EP_0) showed a more malignant phenotype, and subclusters 3 and 4 of macrophages (M_3 and M_4) demonstrated heightened activity. Further analysis suggested that EP_0 may suppress the activity of M_3 and M_4 via MIF - (CD74 + CXCR4) in the MIF pathway. After analysing the expression of the 4 genes in the MIF pathway in both the TCGA cohort and our cohort (n = 61), CD74 was identified as significantly overexpressed in PTC tumours particularly those with lymph node metastasis. CONCLUSION: Our study revealed that PTC may facilitate lymph node metastasis by inhibiting macrophages via MIF signalling. It is suggested that malignant PTC cells may suppress the immune activity of macrophages by consistently releasing signals to them via MIF-(CD74 + CXCR4).

Phosphorylation of MIF by PIP4K2a is necessary for cilia biogenesis.

Primary cilia are microtubule-based organelles that play important roles in development and tissue homeostasis. Macrophage migration inhibitory factor (MIF) has long been recognized as a secreted cytokine in the pathogenesis of various human diseases, including cancer and autosomal dominant polycystic kidney disease (ADPKD). Unlike other cytokines, unique functional characteristics of intracellular MIF have emerged. In this study, we show that MIF is localized and formed a ring like structure at the proximal end of centrioles, where it regulates cilia biogenesis through affecting 1) the recruitment of TTBK2 to basal body and the removal of CP110 from mother centriole, 2) the accumulation of CEP290 at centriolar satellites, and 3) the trafficking of intraflagellar transport (IFT) related proteins. We also show that MIF functions as a novel transcriptional factor to regulate the expression of genes related to ciliogenesis via binding on the promotors of those genes. MIF also binds chromatin and regulates transcription of genes involved in diverse homeostatic signaling pathways. We identify phosphatidylinositol-5-phosphate 4-kinase type 2 alpha (PIP4K2a) as an upstream regulator of MIF, which interacts with and phosphorylates MIF at S91 to increase its interaction with 14-3-3ζ, resulting in its nuclear translocation and transcription regulation. This study suggests that MIF is a key player in cilia biogenesis and a novel transcriptional regulator in homeostasis, which forward our understanding of how MIF is able to carry out several nonoverlapping functions.

Macrophage migration inhibitory factor (MIF) promotes intervertebral disc degeneration through the NF-κB pathway, and the MIF inhibitor CPSI-1306 alleviates intervertebral disc degeneration in a mouse model.

Lumbar intervertebral disc degeneration(IDD) is a prevalent inflammatory disease caused by many proinflammatory factors, such as TNF and IL-1ß. Migration inhibitory factor (MIF) is an upstream inflammatory factor widely expressed in vivo that is associated with a variety of inflammatory diseases or malignant tumors and has potential therapeutic value in many diseases. We explored the role of MIF in intervertebral disc degeneration by regulating the content of exogenous MIF or the expression of MIF in cells. Upon inducing degeneration of nucleus pulposus (NP) cells with IL-1ß, we found that the increase in intracellular and exogenous MIF promoted the catabolism induced by proinflammatory factors in NP cells, while silencing of the MIF gene alleviated the degeneration to some extent. In a mouse model, the intervertebral disc degeneration of MIF-KO mice was significantly less than that of wild-type mice. To explore the treatment of intervertebral disc degeneration, we selected the small-molecular MIF inhibitor CPSI-1306. CPSI-1306 had a therapeutic effect on intervertebral disc degeneration in the mouse model. In summary, we believe that MIF plays an important role in intervertebral disc degeneration and is a potential therapeutic target for the treatment of intervertebral disc degeneration.

Macrophage migration inhibitory factor (MIF) and its homolog D-dopachrome tautomerase (D-DT) are significant promotors of UVB- but not chemically induced non-melanoma skin cancer.

Non-melanoma skin cancer (NMSC) is the most common cancer in Caucasians worldwide. We investigated the pathophysiological role of MIF and its homolog D-DT in UVB- and chemically induced NMSC using Mif-/-, D-dt-/- and Mif-/-/D-dt-/- mice on a hairless SKH1 background. Knockout of both cytokines showed similar attenuating effects on inflammation after acute UVB irradiation and tumor formation during chronic UVB irradiation, without additive protective effects noted in double knockout mice, indicating that both cytokines activate a similar signaling threshold. In contrast, genetic deletion of Mif and D-dt had no major effects on chemically induced skin tumors. To get insight into the contributing mechanisms, we used an in vitro 3D skin model with incorporated macrophages. Application of recombinant MIF and D-DT led to an accumulation of macrophages within the epidermal part that could be reversed by selective inhibitors of MIF and D-DT pathways. In summary, our data indicate that MIF and D-DT contribute to the development and progression of UVB- but not chemically induced NMSC, a role at least partially accounted by effects of both cytokines on epidermal macrophage accumulation. These data highlight that MIF and D-DT are both potential therapeutic targets for the prevention of photocarcinogenesis but not chemical carcinogenesis.

15-deoxy-Δ12,14-prostaglandin J2 relieved acute liver injury by inhibiting macrophage migration inhibitory factor expression via PPARγ in hepatocyte.

15-deoxy-Δ12,14-prostaglandin J2 (15d-PGJ2) exhibited potential to alleviate liver inflammation in chronic injury but was less studied in acute injury. Acute liver injury was associated with elevated macrophage migration inhibitory factor (MIF) levels in damaged hepatocytes. This study aimed to investigate the regulatory mechanism of hepatocyte-derived MIF by 15d-PGJ2 and its subsequent impact on acute liver injury. In vivo, mouse models were established by carbon tetrachloride (CCl4) intraperitoneal injection, with or without 15d-PGJ2 administration. 15d-PGJ2 treatment reduced the necrotic areas induced by CCl4. In the same mouse model constructed using enhanced green fluorescent protein (EGFP)-labeled bone marrow (BM) chimeric mice, 15d-PGJ2 reduced CCl4 induced BM-derived macrophage (BMM, EGFP+F4/80+) infiltration and inflammatory cytokine expression. Additionally, 15d-PGJ2 down-regulated liver and serum MIF levels; liver MIF expression was positively correlated with BMM percentage and inflammatory cytokine expression. In vitro, 15d-PGJ2 inhibited Mif expression in hepatocytes. In primary hepatocytes, reactive oxygen species inhibitor (NAC) showed no effect on MIF inhibition by 15d-PGJ2; PPARγ inhibitor (GW9662) abolished 15d-PGJ2 suppressed MIF expression and antagonists (troglitazone, ciglitazone) mimicked its function. In Pparg silenced AML12 cells, the suppression of MIF by 15d-PGJ2 was weakened; 15d-PGJ2 promoted PPARγ activation in AML 12 cells and primary hepatocytes. Furthermore, the conditioned medium of recombinant MIF- and lipopolysaccharide-treated AML12 respectively promoted BMM migration and inflammatory cytokine expression. Conditioned medium of 15d-PGJ2- or siMif-treated injured AML12 suppressed these effects. Collectively, 15d-PGJ2 activated PPARγ to suppress MIF expression in injured hepatocytes, reducing BMM infiltration and pro-inflammatory activation, ultimately alleviating acute liver injury.

MIF Increases sFLT1 Expression in Early Uncomplicated Pregnancy and Preeclampsia.

Insufficient immune tolerance during pregnancy is associated with pathological conditions such as preeclampsia (PE). Soluble fms-like tyrosine kinase-1 (sFLT1), which exerts a role in the late stage of PE, has shown its beneficial anti-inflammatory effects in inflammation-associated diseases. Macrophage migration inhibitory factor (MIF) was reported to upregulate sFLT1 production in experimental congenital diaphragmatic hernia. However, the placental sFLT1 expression in early uncomplicated pregnancy and whether MIF can regulate sFLT1 expression in uncomplicated and preeclamptic pregnancy are unclear. We collected first-trimester placentas and term placentas from uncomplicated and preeclamptic pregnancies to investigate sFLT1 and MIF expression in vivo. Primary cytotrophoblasts (CTBs) and a human trophoblast cell line (Bewo) were used to study the regulation of MIF on sFLT1 expression in vitro. In placentas from first-trimester pregnancy, we observed a high expression of sFLT1, specifically in extravillous trophoblasts (EVTs) and syncytiotrophoblast (STB) cells. MIF mRNA levels strongly correlated with sFLT1 expression in term placentas from preeclamptic pregnancies. In in vitro experiments, sFLT1 and MIF levels increased significantly in CTBs during their differentiation to EVTs and STBs, and MIF inhibitor (ISO-1) significantly reduced sFLT1 expression in a dose-dependent manner during this process. sFLT1 showed significant upregulation with increasing doses of MIF in Bewo cells. Our results show that sFLT1 is highly expressed at the maternal-fetal interface during early pregnancy and that MIF can increase sFLT1 expression in early uncomplicated pregnancy and PE, which suggests that sFLT1 plays an essential role in the modulation of inflammation in pregnancy.

Protocol for purification and enzymatic characterization of members of the human macrophage migration inhibitory factor superfamily.

Macrophage migration inhibitory factor (MIF) and D-dopachrome tautomerase (D-DT or MIF-2) are two proteins serving a key role in the pathogenesis of multiple disorders, including cancer.1 Here, we present a protocol for the purification and enzymatic characterization of MIF and D-DT using keto-enol tautomerase activity. This approach measures enzymatic activity through the formation of an enol-borate complex. We describe steps for expressing and purifying proteins, preparing the 96-well microplate, and assay implementation including monitoring of keto-enol tautomerase activity. For complete details on the use and execution of this protocol, please refer to Parkins et al.2,3.

Allosteric Inhibitors of Macrophage Migration Inhibitory Factor (MIF) Interfere with Apoptosis-Inducing Factor (AIF) Co-Localization to Prevent Parthanatos.

Macrophage migration inhibitory factor (MIF) is a multifunctional cytokine and essential signaling protein associated with inflammation and cancers. One of the newly described roles of MIF is binding to apoptosis-inducing factor (AIF) that "brings" cells to death in pathological conditions. The interaction between MIF and AIF and their nuclear translocation stands as a central event in parthanatos. However, classical competitive MIF tautomerase inhibitors do not interfere with MIF functions in parthanatos. In this study, we employed a pharmacophore-switch to provide allosteric MIF tautomerase inhibitors that interfere with the MIF/AIF co-localization. Synthesis and screening of a focused compound collection around the 1,2,3-triazole core enabled identification of the allosteric tautomerase MIF inhibitor 6y with low micromolar potency (IC50 = 1.7 ± 0.1 µM). This inhibitor prevented MIF/AIF nuclear translocation and protects cells from parthanatos. These findings indicate that alternative modes to target MIF hold promise to investigate MIF function in parthanatos-mediated diseases.

Microphysiological head and neck cancer model identifies novel role of lymphatically secreted monocyte migration inhibitory factor in cancer cell migration and metabolism.

Regional metastasis of head and neck cancer (HNC) is prevalent (approximately 50% of patients at diagnosis), yet the underlying drivers and mechanisms of lymphatic spread remain unclear. The complex tumor microenvironment (TME) of HNC plays a crucial role in disease maintenance and progression; however, the contribution of the lymphatics remains underexplored. We created a primary patient cell derived microphysiological system that incorporates cancer-associated-fibroblasts from patients with HNC alongside a HNC tumor spheroid and a lymphatic microvessel to create an in vitro TME platform to investigate metastasis. Screening of soluble factor signaling identified novel secretion of macrophage migration inhibitory factor (MIF) by lymphatic endothelial cells conditioned in the TME. Importantly, we also observed patient-to-patient heterogeneity in cancer cell migration similar to the heterogeneity observed in clinical disease. Optical metabolic imaging at the single cell level identified a distinct metabolic profile of migratory versus non-migratory HNC cells in a microenvironment dependent manner. Additionally, we report a unique role of MIF in increasing HNC reliance on glycolysis over oxidative phosphorylation. This multicellular, microfluidic platform expands the tools available to explore HNC biology in vitro through multiple orthogonal outputs and establishes a system with enough resolution to visualize and quantify patient-to-patient heterogeneity.

Tumor Necrosis Factor α-Dependent Lung Inflammation Promotes the Progression of Lung Adenocarcinoma Originating From Alveolar Type II Cells by Upregulating MIF-CD74.

Lung adenocarcinoma is the most common type of lung cancer. We recently reported that inflammation-driven lung adenocarcinoma (IDLA) originates from alveolar type (AT)-II cells, which depend on major histocompatibility complex (MHC) class II to promote the expansion of regulatory T cells. The MHC class II-associated invariant chain (CD74) binds to the macrophage migration inhibitory factor (MIF), which is associated with promoting tumor growth and invasion. However, the role of MIF-CD74 in the progression of lung adenocarcinoma and the underlying mechanisms remain unclear. We aimed to explore the role of MIF-CD74 in the progression of lung adenocarcinoma and elucidate the mechanisms by which tumor necrosis (TNF)-α-mediated inflammation regulates CD74 and MIF expression in IDLA. In human lung adenocarcinoma, CD74 was upregulated on the surface of tumor cells originating from AT-II cells, which correlated positively with lymph node metastasis, tumor origin/nodal involvement/metastasis stage, and TNF-α expression. MIF interaction with CD74 promoted the proliferation and migration of A549 and H1299 cells in vitro. Using a urethane-induced IDLA mouse model, we observed that CD74 was upregulated in tumor cells and macrophages. MIF expression was upregulated in macrophages in IDLA. Blocking TNF-α-dependent inflammation downregulated CD74 expression in tumor cells and CD74 and MIF expression in macrophages in IDLA. Conditioned medium from A549 cells or activated mouse AT-II cells upregulated MIF in macrophages by secreting TNF-α. TNF-α-dependent lung inflammation contributes to the progression of lung adenocarcinoma by upregulating CD74 and MIF expression, and AT-II cells upregulate MIF expression in macrophages by secreting TNF-α. This study provides novel insights into the function of CD74 in the progression of IDLA.