Macrophage Migration Inhibitory Factor Signaling Research Articles

Exosomes originating from neural stem cells undergoing necroptosis participate in cellular communication by inducing TSC2 upregulation of recipient cells following spinal cord injury.

We previously demonstrated that inhibiting neural stem cells necroptosis enhances functional recovery after spinal cord injury. While exosomes are recognized as playing a pivotal role in neural stem cells exocrine function, their precise function in spinal cord injury remains unclear. To investigate the role of exosomes generated following neural stem cells necroptosis after spinal cord injury, we conducted single-cell RNA sequencing and validated that neural stem cells originate from ependymal cells and undergo necroptosis in response to spinal cord injury. Subsequently, we established an in vitro necroptosis model using neural stem cells isolated from embryonic mice aged 16-17 days and extracted exosomes. The results showed that necroptosis did not significantly impact the fundamental characteristics or number of exosomes. Transcriptome sequencing of exosomes in necroptosis group identified 108 differentially expressed messenger RNAs, 104 long non-coding RNAs, 720 circular RNAs, and 14 microRNAs compared with the control group. Construction of a competing endogenous RNA network identified the following hub genes: tuberous sclerosis 2 (Tsc2), solute carrier family 16 member 3 (Slc16a3), and forkhead box protein P1 (Foxpl). Notably, a significant elevation in TSC2 expression was observed in spinal cord tissues following spinal cord injury. TSC2-positive cells were localized around SRY-box transcription factor 2-positive cells within the injury zone. Furthermore, in vitro analysis revealed increased TSC2 expression in exosomal receptor cells compared with other cells. Further assessment of cellular communication following spinal cord injury showed that Tsc2 was involved in ependymal cellular communication at 1 and 3 days post-injury through the epidermal growth factor and midkine signaling pathways. In addition, Slc16a3 participated in cellular communication in ependymal cells at 7 days post-injury via the vascular endothelial growth factor and macrophage migration inhibitory factor signaling pathways. Collectively, these findings confirm that exosomes derived from neural stem cells undergoing necroptosis play an important role in cellular communication after spinal cord injury and induce TSC2 upregulation in recipient cells.

Single-cell transcriptomics unveiled that early life BDE-99 exposure reprogrammed the gut-liver axis to promote a proinflammatory metabolic signature in male mice at late adulthood.

Polybrominated diphenyl ethers (PBDEs) are legacy flame retardants that bioaccumulate in the environment. The gut microbiome is an important regulator of liver functions including xenobiotic biotransformation and immune regulation. We recently showed that neonatal exposure to polybrominated diphenyl ether-99 (BDE-99), a human breast milk-enriched PBDE congener, up-regulated proinflammation-related and down-regulated drug metabolism-related genes predominantly in males in young adulthood. However, the persistence of this dysregulation into late adulthood, differential impact among hepatic cell types, and the involvement of the gut microbiome from neonatal BDE-99 exposure remain unknown. To address these knowledge gaps, male C57BL/6 mouse pups were orally exposed to corn oil (10 ml/kg) or BDE-99 (57 mg/kg) once daily from postnatal days 2-4. At 15 months of age, neonatal BDE-99 exposure down-regulated xenobiotic and lipid-metabolizing enzymes and up-regulated genes involved in microbial influx in hepatocytes. Neonatal BDE-99 exposure also increased the hepatic proportion of neutrophils and led to a predicted increase of macrophage migration inhibitory factor signaling. This was associated with decreased intestinal tight junction protein (Tjp) transcripts, altered gut environment, and dysregulation of inflammation-related metabolites. ScRNA-seq using germ-free (GF) mice demonstrated the necessity of a normal gut microbiome in maintaining hepatic immune tolerance. Microbiota transplant to GF mice using large intestinal microbiome from adults neonatally exposed to BDE-99 down-regulated Tjp transcripts and up-regulated several cytokines in large intestine. In conclusion, neonatal BDE-99 exposure reprogrammed cell type-specific gene expression and cell-cell communication in liver towards proinflammation, and this may be partly due to the dysregulated gut environment.

A small molecule macrophage migration inhibitory factor agonist ameliorates age-related myocardial intolerance to ischemia-reperfusion insults via metabolic regulation

Macrophage migration inhibitory factor (MIF) is an innate cytokine that regulates both inflammatory and homeostatic responses. MIF is expressed by cardiomyocytes, where it exerts a protective action against ischemia-reperfusion (I/R) injury by activating AMP-activated protein kinase (AMPK). This effect is attenuated in the senescent heart due to an intrinsic, age-related reduction in MIF expression. We hypothesized that treating the aged heart with the small molecule MIF agonist (MIF20) can reinforce protective MIF signaling in cardiomyocytes, leading to a beneficial effect against I/R stress. The administration of MIF20 at the onset of reperfusion was found to not only decrease myocardial infarct size but also preserves systolic function in the aged heart. Protection from I/R injury was reduced in mice with cardiomyocyte-specific Mif deletion, consistent with the mechanism of action of MIF20 to allosterically increase MIF affinity for its cognate receptor CD74. We further found MIF20 to contribute to the maintenance of mitochondrial fitness and to preserve the contractile properties of aged cardiomyocytes under hypoxia/reoxygenation. MIF20 augments protective metabolic responses by reducing the NADH/NAD ratio, leading to a decrease in the accumulation of reactive oxygen species (ROS) in the aged myocardium under I/R stress. We also identify alterations in the expression levels of the downstream effectors PDK4 and LCAD, which participate in the remodeling of the cardiac metabolic profile. Data from this study demonstrates that pharmacologic augmentation of MIF signaling provides beneficial homeostatic actions on senescent myocardium under I/R stress.

Single-cell transcriptomics reveals ferrimagnetic vortex iron oxide nanoring-mediated mild magnetic hyperthermia exerts antitumor effects by alleviating macrophage suppression in breast cancer

The potential of Ferrimagnetic vortex iron oxide nanoring-mediated mild magnetic hyperthermia (FVIO-MHT) in solid tumor therapy has been demonstrated. However, the impact of FVIO-MHT on the tumor microenvironment (TME) remains unclear. This study utilized single-cell transcriptome sequencing to examine the alterations in the TME in response to FVIO-MHT in breast cancer. The results revealed the cellular composition within the tumor microenvironment (TME) was primarily modified due to a decrease in tumor cells and an increased infiltration of myeloid cells. Subsequently, an enhancement in active oxygen (ROS) metabolism was observed, indicating oxidative damage to tumor cells. Interestingly, FVIO-MHT reprogrammed the macrophages’ phenotypes, as evidenced by alterations in the transcriptome characteristics associated with both classic and alternative activated phenotypes. And an elevated level of ROS generation and oxidative phosphorylation suggested that activated phagocytosis and inflammation occurred in macrophages. Additionally, cell–cell communication analysis revealed that FVIO-MHT attenuated the suppression between tumor cells and macrophages by inhibiting phagocytic checkpoint and macrophage migration inhibitory factor signaling pathways. Inhibition of B2m, an anti-phagocytosis checkpoint, could promote macrophage-mediated phagocytosis and significantly inhibit tumor growth. These data emphasize FVIO-MHT may promote the antitumor capabilities of macrophages by alleviating the suppression between tumor cells and macrophages.

Abstract 13208: A Small Molecule Macrophage Migration Inhibitory Factor Agonist Ameliorates Age-Related Myocardial Intolerance to Ischemia-Reperfusion Insults via Metabolic Regulation

Introduction: The macrophage migration inhibitory factor (MIF) is emerged as a critical mediator during myocardia ischemia and reperfusion (I/R). An impaired MIF signaling is occurred in aging. MIF agonist, MIF20, can argument cardiac MIF signaling through increasing the binding affinity between MIF and its receptor, CD74/CD44. Hypothesis: Administration of MIF20 can increase the resistance of aged myocardium to ischemic insults caused by I/R via modulating the MIF signaling regulated energy metabolism. Methods: Young (3-5 months) and aged (24-26 months) wild-type C57BL/6J mice, cardiomyocyte specific MIF knock out C57BL/6J mice (cMIF-/-) and wild type littermates (MIF flox/flox ) were injected with MIF20 (0.15 μg/kg, I.V.) 5 minutes before reperfusion after 45 minutes of ischemia. The left ventricular myocardium was for immunoblotting, NAD/NADH assay, and metabolomics analysis. The working heart perfusion and the SeaHorse system were for metabolic measurements. Results: The small molecule MIF agonist, MIF20, which binds to MIF to increase signaling through its cognate receptor CD74/CD44 not only decreases infarct size but also preserves cardiac systolic function in the aged versus young hearts. MIF20 contributes to the maintenance of mitochondrial fitness and preserves the contractility of cardiomyocytes under hypoxia and reoxygenation stress. These beneficial effects of MIF20 are reduced in cardiomyocyte-specific MIF knock-out (cMIF -/- ) versus littermates (MIF flox/flox ) mice, supporting the autocrine role of MIF in mediating ischemic cardiac protection. The age-related disruption of metabolic homeostasis in aged versus young hearts was modulated by MIF20’s ability to upregulate glucose oxidation and downregulate the oxidation of fatty acid. These actions were associated with an upregulation of pyruvate dehydrogenase kinase 4 (PDK4) and downregulation of Long-chain acyl-CoA dehydrogenase (LCAD). MIF20 as MIF signaling agonism modulation of metabolic adaptation in aged hearts further led to reduced generation of reactive oxygen species (ROS) in the myocardium. Conclusions: MIF agonists such as MIF20 may have a therapeutic role by limiting cardiac damage and rescuing cardiac dysfunctions of senescent hearts to I/R stress.

Single-cell RNA sequencing of intestinal crypts reveals vital events in damage repair and the double-edged sword effect of the Wnt3/β-catenin pathway in irradiated mice

In this study, we executed single-cell RNA sequencing of intestinal crypts. We analyzed the differentially expressed genes (DEGs) at different time points (the first, third, and fifth days) after 13 Gy and 15 Gy abdominal body radiation (ABR) exposure and then executed gene ontology (GO) enrichment analysis, RNA velocity analysis, cell communication analysis, and ligand‒receptor interaction analysis to explore the vital events in damage repair and the multiple effects of the Wnt3/β-catenin pathway on irradiated mice. Results from bioinformatics analysis were confirmed by a series of biological experiments. Results showed that the antibacterial response is a vital event during the damage response process after 13 Gy ABR exposure; ionizing radiation (IR) induced high heterogeneity in the transient amplification (TA) cluster, which may differentiate into mature cells and stem cells in irradiated small intestine (SI) crypts. Conducting an enrichment analysis of the DEGs between mice exposed to 13 Gy and 15 Gy ABR, we concluded that the Wnt3/β-catenin and MIF-CD74/CD44 signaling pathways may contribute to 15 Gy ABR-induced mouse death. Wnt3/β-catenin promotes the recovery of irradiated SI stem/progenitor cells, which may trigger macrophage migration inhibitory factor (MIF) release to further repair IR-induced SI injury; however, with the increase in radiation dose, activation of CD44 on macrophages provides the receptor for MIF signal transduction, initiating the inflammatory cascade response and ultimately causing a cytokine release syndrome. In contrast to previous research, we confirmed that inhibition of the Wnt3/β-catenin pathway or blockade of CD44 on the second day after 15 Gy ABR may significantly protect against ABR-induced death. This study indicates that the Wnt3/β-catenin pathway plays multiple roles in damage repair after IR exposure; we also propose a novel point that the interaction between intestinal crypt stem cells (ISCs) and macrophages through the MIF-CD74/CD44 axis may exacerbate SI damage in irradiated mice.

The early life immune dynamics and cellular drivers at single-cell resolution in lamb forestomachs and abomasum

BackgroundFour-chambered stomach including the forestomachs (rumen, reticulum, and omasum) and abomasum allows ruminants convert plant fiber into high-quality animal products. The early development of this four-chambered stomach is crucial for the health and well-being of young ruminants, especially the immune development. However, the dynamics of immune development are poorly understood.ResultsWe investigated the early gene expression patterns across the four-chambered stomach in Hu sheep, at 5, 10, 15, and 25 days of age. We found that forestomachs share similar gene expression patterns, all four stomachs underwent widespread activation of both innate and adaptive immune responses from d 5 to 25, whereas the metabolic function were significantly downregulated with age. We constructed a cell landscape of the four-chambered stomach using single-cell sequencing. Integrating transcriptomic and single-cell transcriptomic analyses revealed that the immune-associated module hub genes were highly expressed in T cells, monocytes and macrophages, as well as the defense-associated module hub genes were highly expressed in endothelial cells in the four-stomach tissues. Moreover, the non-immune cells such as epithelial cells play key roles in immune maturation. Cell communication analysis predicted that in addition to immune cells, non-immune cells recruit immune cells through macrophage migration inhibitory factor signaling in the forestomachs.ConclusionsOur results demonstrate that the immune and defense responses of four stomachs are quickly developing with age in lamb's early life. We also identified the gene expression patterns and functional cells associated with immune development. Additionally, we identified some key receptors and signaling involved in immune regulation. These results help to understand the early life immune development at single-cell resolution, which has implications to develop nutritional manipulation and health management strategies based on specific targets including key receptors and signaling pathways.

Intermittent fasting activates macrophage migration inhibitory factor and alleviates high-fat diet-induced nonalcoholic fatty liver disease

Switching to normal diet (ND) is the regular therapy for high-fat diet (HFD)-induced nonalcoholic fatty liver disease (NAFLD). Intermittent fasting (IF) is a unique treatment which may exhibits better therapeutic efficacy. Thus, we aim to investigate the therapeutic effects of these treatments and exploring the mechanisms. In the present study, NAFLD mouse model was induced by a 10-week HFD. Thereafter, mice adopted continued HFD, ND, or IF for the next 12 weeks. Finally, the liver was then harvested to assess lipid deposition, lipid metabolism, apoptosis, and autophagy, while blood was collected to determine blood glucose and insulin. The results showed that IF and ND treatment improved lipid deposition and metabolic disorder of NAFLD mice; the increasing body weight, liver weight, and HOMA-IR index of HFD mice were also alleviated by IF and ND. Furthermore, IF and ND treatment activated the macrophage migration inhibitory factor (MIF)/AMPK pathway and regulated its downstream autophagy and apoptosis. However, the efficacy of IF was better than ND. Both IF and ND activates MIF signaling and alleviate the lipotoxicity of NAFLD while IF therapy is more effective than ND. The different MIF up-regulation might be the underlying mechanism of why IF benefits more than ND.

Abstract 25: MIF and NR3C2 interactively regulate glucose metabolism in pancreatic cancer

Abstract Inflammation and altered cellular metabolism are described as hallmarks of cancer. Although inflammatory signaling and metabolic reprogramming are highly integrated and play critical role in the development and progression of pancreatic ductal adenocarcinoma (PDAC). The role of inflammatory mediators in the regulation of metabolic reprogramming is not adequately studied in pancreatic cancer and remains to be explored. Here we show that macrophage migration inhibitory factor (MIF) collaborates with its downstream transcriptional factor nuclear-receptor-subfamily-3, group-C, member-2 (NR3C2) regulate metabolic reprogramming to support cancer growth and progression in PDAC. MIF enhances glucose uptake and lactate efflux through enhancing HK1 (hexokinase 1), HK2 (hexokinase 2) and LDHA (lactate dehydrogenase) expression in pancreatic cancer cell in vitro and in an orthotopic mouse model. Furthermore, a decreased HK1, HK2, LDHA expression, pyruvate and lactate production are found in patient tumors with lower MIF expression and MIF-deficiency tumors from genetically engineered KPC mouse model. Conversely, NR3C2 suppresses HK1, HK2, and LDHA expression and decreases glucose uptake and lactate efflux in pancreatic cancer. Mechanistically, MIF-mediated regulation of glycolytic metabolism involves MAPK-ERK Kinase pathway while NR3C2 interacts with AP-1 transcriptional factor in regulation of glycolysis metabolism. Taken together, our result demonstrates an interactive role of MIF and NR3C2 signaling in regulation cancer metabolism and therapeutic approaches target this axis may improve disease outcome in patients with PDAC. Citation Format: Shouhui Yang, Wei Tang, Azadeh Azizian, Philipp Ströbel, Yuuki Ohara, Helen Cawley, Nader Hanna, B. Michael Ghadimi, Nagireddy Putluri, S. Perwez Hussain. MIF and NR3C2 interactively regulate glucose metabolism in pancreatic cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 25.

MP60-01 MACROPHAGE MIGRATION INHIBITORY FACTOR AND TLR4 SIGNALING ARE INVOLVED IN PAR4-INDUCED PERSISTENT BLADDER PAIN

You have accessJournal of UrologyCME1 Apr 2023MP60-01 MACROPHAGE MIGRATION INHIBITORY FACTOR AND TLR4 SIGNALING ARE INVOLVED IN PAR4-INDUCED PERSISTENT BLADDER PAIN Shaojing Ye, Fei Ma, Dlovan F. D. Mahmood, and Pedro Vera Shaojing YeShaojing Ye More articles by this author , Fei MaFei Ma More articles by this author , Dlovan F. D. MahmoodDlovan F. D. Mahmood More articles by this author , and Pedro VeraPedro Vera More articles by this author View All Author Informationhttps://doi.org/10.1097/JU.0000000000003318.01AboutPDF ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareFacebookLinked InTwitterEmail Abstract INTRODUCTION AND OBJECTIVE: Interstitial cystitis/Bladder pain syndrome is a painful condition with unknown etiology. Repeated intravesical PAR4 induced persistent bladder hyperalgesia (BHA) in mice that lasts several days after the last stimulus with little or no evidence of bladder inflammation. Our studies showed that spinal macrophage migration inhibitory factor (MIF) and High Mobility Group Box 1 (HMGB1) are critical causative factors. We aimed to dissect this persistent pain pathway and explore effective targets for treating persistent bladder pain. METHODS: Persistent BHA was induced in female mice (WT, TLR4 null, Trif null) by three intravesical instillations (days 0, 2 and 4) with PAR4 agonist (100 uM; 1hr, scrambled peptide as control) under anesthesia. Lower abdominal hypersensitivity was measured by von Frey filaments on days 0-4 and 7-9. Intraperitoneal treatment was performed on days 2-8 and included: ISO-1 (MIF antagonist); 10% DMSO; ethyl pyruvate (EP; inhibits HMGB1 release); and PBS. Awake micturition parameters (voided volume; frequency) were assessed on day 9. Bladder histology assessed inflammation/edema. R was used for all analyses. RESULTS: Inhibition of MIF activity with ISO-1 robustly reversed the PAR4-induced persistent BHA in WT mice (Figure 1A) while treatment with EP partially reduced BHA (Figure 1B). TLR4 deficiency substantially damped persistent pain (Figure 2A) while lack of Trif did not affect PAR4-induced persistent BHA (Figure 2B). No significant effects were noted on micturition volume, frequency, inflammation, or edema. CONCLUSIONS: MIF, HMGB1, and TLR4 signaling activation are required for PAR4-induced persistent BHA. However, Trif may not be critical for persistent bladder pain. Further dissecting TLR4 downstream signaling pathway is necessary to identify important mediators and whether they act centrally or peripherally. This may lead to the discovery of novel potential therapeutic targets to treat persistent bladder pain. Source of Funding: DK121695 © 2023 by American Urological Association Education and Research, Inc.FiguresReferencesRelatedDetails Volume 209Issue Supplement 4April 2023Page: e842 Advertisement Copyright & Permissions© 2023 by American Urological Association Education and Research, Inc.MetricsAuthor Information Shaojing Ye More articles by this author Fei Ma More articles by this author Dlovan F. D. Mahmood More articles by this author Pedro Vera More articles by this author Expand All Advertisement PDF downloadLoading ...

Thrombin acts as inducer of proinflammatory macrophage migration inhibitory factor in astrocytes following rat spinal cord injury

BackgroundThe danger-associated molecular patterns (DAMPs) are critical contributors to the progressive neuropathology and thereafter affect the functional outcomes following spinal cord injury (SCI). Up to now, the regulatory mechanisms on their inducible production from the living cells remain elusive, aside from their passive release from the necrotic cells. Thrombin is immediately activated by the damaged or stressed central nervous system (CNS), which potently mediates inflammatory astrocytic responses through proteolytic cleavage of protease-activated receptors (PARs). Therefore, SCI-activated thrombin is conceived to induce the production of DAMPs from astrocytes at lesion site.MethodsRat SCI model was established by the cord contusion at T8–T10. The expression of thrombin and macrophage migration inhibitory factor (MIF) was determined by ELISA and Western blot. The PAR1, PAR3, and PAR4 receptors of thrombin were examined by PCR and immunohistochemistry. Primary astrocytes were isolated and purified from the spinal cord, followed by stimulation with different concentrations of thrombin either for transcriptome sequencing or for analysis of thrombin-mediated expression of MIF and related signal pathways in the presence or absence of various inhibitors. The post-injury locomotor functions were assessed using the Basso, Beattie, and Bresnahan (BBB) locomotor scale.ResultsMIF protein levels were significantly elevated in parallel with those of thrombin induced by SCI. Immunostaining demonstrated that PAR1 receptor, together with MIF, was abundantly expressed in astrocytes. By transcriptome sequencing and bioinformatical analysis of thrombin-stimulated primary astrocytes, MIF was identified to be dynamically regulated by the serine protease. Investigation of the underlying mechanism using various inhibitors revealed that thrombin-activated PAR1 was responsible for the MIF production of astrocytes through modulation of JNK/NFκB pathway. Administration of PAR1 inhibitor at lesion sites following SCI significantly reduced the protein levels of MIF and ameliorated functional deficits of rat locomotion.ConclusionSCI-activated thrombin is a robust inducer of MIF production from astrocytes. Exploring the roles of thrombin in promoting the production of DAMPs from astrocytes at lesion site will provide an alternative strategy for the clinical therapy of CNS inflammation.

TPT1 Supports Proliferation of Neural Stem/Progenitor Cells and Brain Tumor Initiating Cells Regulated by Macrophage Migration Inhibitory Factor (MIF).

One of the key areas in stem cell research is the identification of factors capable of promoting the expansion of Neural Stem Cell/Progenitor Cells (NSPCs) and understanding their molecular mechanisms for future use in clinical settings. We previously identified Macrophage Migration Inhibitory Factor (MIF) as a novel factor that can support the proliferation and/or survival of NSPCs based on in vitro functional cloning strategy and revealed that MIF can support the proliferation of human brain tumor-initiating cells (BTICs). However, the detailed downstream signaling for the functions has largely remained unknown. Thus, in the present study, we newly identified translationally-controlled tumor protein-1 (TPT1), which is expressed in the ventricular zone of mouse embryonic brain, as a downstream target of MIF signaling in mouse and human NSPCs and human BTICs. Using gene manipulation (over or downregulation of TPT1) techniques including CRISPR/Cas9-mediated heterozygous gene disruption showed that TPT1 contributed to the regulation of cell proliferation/survival in mouse NSPCs, human embryonic stem cell (hESC) derived-NSPCs, human-induced pluripotent stem cells (hiPSCs) derived-NSPCs and BTICs. Furthermore, gene silencing of TPT1 caused defects in neuronal differentiation in the NSPCs in vitro. We also identified the MIF-CHD7-TPT1-SMO signaling axis in regulating hESC-NSPCs and BTICs proliferation. Intriguingly, TPT1suppressed the miR-338 gene, which targets SMO in hESC-NSPCs and BTICs. Finally, mice with implanted BTICs infected with lentivirus-TPT1 shRNA showed a longer overall survival than control. These results also open up new avenues for the development of glioma therapies based on the TPT1 signaling pathway.

Bladder Oxidative Stress and HMGB1 Release Contribute to PAR4-Mediated Bladder Pain in Mice.

Activation of intravesical PAR4 receptors leads to bladder hyperalgesia (BHA) through release of urothelial macrophage migration inhibitory factor (MIF) and urothelial high mobility group box-1 (HMGB1). MIF deficiency and/or MIF antagonism at the bladder block BHA in mice yet the mechanisms are not clear. Since oxidative stress and ERK phosphorylation are involved in MIF signaling we hypothesized that oxidative stress and/or ERK signaling, activated by MIF release, promote intravesical HMGB1 release to induce BHA. We induced BHA by intravesical PAR4 infusion in female C57BL/6 mice. Mechanical sensitivity was evaluated by measuring abdominal von Frey (VF) 50% thresholds before (baseline) and 24 h post-infusion. Intravesical pre-treatment (10 min infusion prior to PAR4) with N-acetylcysteine amide (NACA; reactive-oxygen species scavenger; 3 mg in 50 μl), FR180204 (selective ERK1/2 inhibitor; 200 μg in 50 μl), ethyl pyruvate (EP; HMGB1 release inhibitor; 600 μg in 50 μl), or diluent controls (50 μl) tested the effects of pre-treatment on PAR4-induced BHA. Intravesical fluid was collected after each treatment and HMGB1 concentration was measured using ELISA. Awake micturition parameters (volume and frequency) were assessed at the end of the experiments. Bladders were collected and examined for histological signs of edema and inflammation. Pre-treatment with PBS followed by PAR4 induced BHA in mice but PBS followed by scrambled peptide did not. Pre-treatment with NACA or EP partially blocked PAR4-induced BHA while FR180204 had no effect. A significant correlation between intravesical HMGB1 levels and 50% VF thresholds was observed. All PAR4 treated groups had increased levels of HMGB1 in the intravesical fluid compared to PBS-Scrambled group although not statistically significant. No significant effects were noted on awake micturition volume, micturition frequency or histological evidence of bladder edema or inflammation. Our results show that intravesical antagonism of bladder reactive-oxygen species accumulation was effective in reducing PAR4-induced bladder pain. The correlation between intravesical levels of HMGB1 and bladder pain indicates that released HMGB1 is pivotal to bladder pain. Thus, modulating events in the MIF signaling cascade triggered by PAR4 activation (including bladder oxidative stress and HMGB1 release) warrant further investigation as possible therapeutic strategies.

Macrophage Migration Inhibitory Factor (MIF) as a Stress Molecule in Renal Inflammation.

Renal inflammation is an initial pathological process during progressive renal injury regardless of the initial cause. Macrophage migration inhibitory factor (MIF) is a truly proinflammatory stress mediator that is highly expressed in a variety of both inflammatory cells and intrinsic kidney cells. MIF is released from the diseased kidney immediately upon stimulation to trigger renal inflammation by activating macrophages and T cells, and promoting the production of proinflammatory cytokines, chemokines, and stress molecules via signaling pathways involving the CD74/CD44 and chemokine receptors CXCR2, CXCR4, and CXCR7 signaling. In addition, MIF can function as a stress molecule to counter-regulate the immunosuppressive effect of glucocorticoid in renal inflammation. Given the critical position of MIF in the upstream inflammatory cascade, this review focuses on the regulatory role and molecular mechanisms of MIF in kidney diseases. The therapeutic potential of targeting MIF signaling to treat kidney diseases is also discussed.

Single-Cell RNA-Seq Analysis Reveals Macrophage Involved in the Progression of Human Intervertebral Disc Degeneration

Intervertebral disc degeneration (IDD) has been considered as the primary pathological mechanism that underlies low back pain. Understanding the molecular mechanisms underlying human IDD is imperative for making strategies to treat IDD-related diseases. Herein, we report the molecular programs, lineage progression patterns, and paths of cellular communications during the progression of IDD using single-cell RNA sequencing (scRNA-seq) on nucleus pulposus (NP) cells from patients with different grades of IDD undergoing discectomy. New subtypes of cells and cell-type-specific gene signatures of the metabolic homeostatic NP cells (Met NPC), adhesive NP cells (Adh NPC), inflammatory response NP cells (IR NPC), endoplasmic reticulum stress NP cells (ERS NPC), fibrocartilaginous NP cells (Fc NPC), and CD70 and CD82+ progenitor NP cells (Pro NPC) were identified. In the late stage of IDD, the IR NPC and Fc NPC account for a large proportion of NPC. Importantly, immune cells including macrophages, T cells, myeloid progenitors, and neutrophils were also identified, and further analysis showed that significant intercellular interaction between macrophages and Pro NPC occurred via MIF (macrophage migration inhibitory factor) and NF-kB signaling pathways during the progression of IDD. In addition, dynamic polarization of macrophage M1 and M2 cell subtypes was found in the progression of IDD, and gene set functional enrichment analysis suggested a significant role of the macrophage polarization in regulating cell metabolism, especially the Pro NPC. Finally, we found that the NP cells in the late degenerative stage were mainly composed of the cell types related to inflammatory and endoplasmic reticulum (ER) response, and fibrocartilaginous activity. Our results provided new insights into the identification of NP cell populations at single-cell resolution and at the relatively whole-transcriptome scale, accompanied by cellular communications between immune cells and NP cells, and discriminative markers in relation to specific cell subsets. These new findings present clues for effective and functional manipulation of human IDD-related bioremediation and healthcare.

Canonical (CD74/CD44) and Non-Canonical (CXCR2, 4 and 7) MIF Receptors Are Differentially Expressed in Rheumatoid Arthritis Patients Evaluated by DAS28-ESR.

Macrophage migration inhibitory factor (MIF) significantly contributes to rheumatoid arthritis (RA) pathogenesis. We aimed to evaluate the canonical (CD74/CD44) and non-canonical MIF receptors (CXCR2,4 and 7) expression and sCD74 to establish their association with RA clinical activity according to DAS28-ESR. Methodology: 101 RA patients with different clinical activities (remission (n = 27), low (n = 16), moderate (n = 35) and high (n = 23)) and 9 control subjects (CS) were included. Expression was evaluated by flow cytometry and levels of soluble CD74 (sCD74) by ELISA. Data analysis was performed with FlowJov10.0, STATAv12.0, and GraphPad Prism v7.0. Results: According to disease activity, CXCR7 expression (percentage of expression and mean fluorescence intensity (MFI)) was higher in granulocytes from patients in remission, while the expression of CXCR4 was higher in patients with high disease activity (p < 0.05). The expression of CD74 was higher in B cells (p < 0.05) and monocytes (p < 0.01) from patients in remission. Regarding sCD74 levels these were higher in patients with high disease activity when compared to those in remission (p <0.05). Conclusions: The results support the need for further study of the role of sCD74 as a soluble MIF decoy receptor, sequestering it to negatively regulate MIF signaling though its membrane receptors. The expression patterns of CXCR4 and CXCR7 show that the latter is a scavenger-type receptor that prevents endocytosis and even degradation of CXCR4 under inflammatory conditions.

Recombinant Mycobacterium smegmatis delivering a fusion protein of human macrophage migration inhibitory factor (MIF) and IL-7 exerts an anticancer effect by inducing an immune response against MIF in a tumor-bearing mouse model

BackgroundMacrophage migration inhibitory factor (MIF) is a pleotropic inflammatory cytokine that is overexpressed in a number of cancer types including most types of human cancer. Inhibition of MIF signaling can...

Targeting Macrophage Migration Inhibitory Factor in Acute Pancreatitis and Pancreatic Cancer.

Macrophage migration inhibitory factor (MIF) is a pleiotropic cytokine implicated in the pathogenesis of inflammation and cancer. It is produced by various cells and circulating MIF has been identified as a biomarker for a range of diseases. Extracellular MIF mainly binds to the cluster of differentiation 74 (CD74)/CD44 to activate downstream signaling pathways. These in turn activate immune responses, enhance inflammation and can promote cancer cell proliferation and invasion. Extracellular MIF also binds to the C-X-C chemokine receptors cooperating with or without CD74 to activate chemokine response. Intracellular MIF is involved in Toll-like receptor and inflammasome-mediated inflammatory response. Pharmacological inhibition of MIF has been shown to hold great promise in treating inflammatory diseases and cancer, including small molecule MIF inhibitors targeting the tautomerase active site of MIF and antibodies that neutralize MIF. In the current review, we discuss the role of MIF signaling pathways in inflammation and cancer and summarize the recent advances of the role of MIF in experimental and clinical exocrine pancreatic diseases. We expect to provide insights into clinical translation of MIF antagonism as a strategy for treating acute pancreatitis and pancreatic cancer.

Revisiting the secretion mechanism(s) of macrophage migration inhibitory factor-welcome to the "UPS club".

Revisiting the secretion mechanism(s) of macrophage migration inhibitory factor-welcome to the "UPS club".

Glioblastoma Myeloid-Derived Suppressor Cell Subsets Express Differential Macrophage Migration Inhibitory Factor Receptor Profiles That Can Be Targeted to Reduce Immune Suppression.

The application of tumor immunotherapy to glioblastoma (GBM) is limited by an unprecedented degree of immune suppression due to factors that include high numbers of immune suppressive myeloid cells, the blood brain barrier, and T cell sequestration to the bone marrow. We previously identified an increase in immune suppressive myeloid-derived suppressor cells (MDSCs) in GBM patients, which correlated with poor prognosis and was dependent on macrophage migration inhibitory factor (MIF). Here we examine the MIF signaling axis in detail in murine MDSC models, GBM-educated MDSCs and human GBM. We found that the monocytic subset of MDSCs (M-MDSCs) expressed high levels of the MIF cognate receptor CD74 and was localized in the tumor microenvironment. In contrast, granulocytic MDSCs (G-MDSCs) expressed high levels of the MIF non-cognate receptor CXCR2 and showed minimal accumulation in the tumor microenvironment. Furthermore, targeting M-MDSCs with Ibudilast, a brain penetrant MIF-CD74 interaction inhibitor, reduced MDSC function and enhanced CD8 T cell activity in the tumor microenvironment. These findings demonstrate the MDSC subsets differentially express MIF receptors and may be leveraged for specific MDSC targeting.