Macrophage Migration Inhibitory Factor Secretion Research Articles

The role of PAR2 in regulating MIF release in house dust mite-induced atopic dermatitis.

Atopic dermatitis (AD) is a chronic disease characterized by relapsed eczema and intractable itch, and is often triggered by house dust mites (HDM). PAR2 is a G-protein coupled receptor on keratinocytes and may be activated by HDM to affect AD processes. We first established a HDM-derived AD mouse model in wild-type (WT) and Par2-/- mice. Single cell RNA sequencing of the diseased skins found a stronger cellular communication between the ligand macrophage migration inhibitory factor (MIF) from keratinocytes and its receptors on antigen-presenting cells, suggesting the critical role of MIF in AD. HDM-WT mice showed severer skin lesions and pathological changes with stronger immunofluorescence MIF signals in skin sections than HDM-Par2-/- mice. Primary keratinocytes from WT mice stimulated with HDM or SLIGRL (PAR2 agonist) secreted more MIF in cultured medium and induced stronger immunofluorescence MIF signals than those from Par2-/- mice. The skin section of HDM-WT mice showed higher immunofluorescence signals of P115 (relating to MIF secretion) and KIF13B (possibly relating to intracellular trafficking of MIF) than that of HDM-Par2-/- mice. Acetylation of α-tubulin increased after stimulation by SLIGRL in WT keratinocytes but not in Par2-/- keratinocytes. HDM-WT mice treated with the MIF antagonist ISO-1 displayed improvement of AD-like presentations and lower expressions of IL-4, IL-13, TSLP and Arg1 (a biomarker of M2 macrophage) mRNAs. We conclude that MIF is an important cytokine and is significantly increased in the AD model. PAR2 affects AD changes by regulating the expression, intracellular trafficking, and secretion of MIF in epidermis.

Depression exacerbates myocardial ischemia-reperfusion injury in mice via CNR2 gene and MIF-AMPK signaling pathway

BackgroundMyocardial ischemia-reperfusion(I/R)injury constitute the fundamental pathophysiology of acute myocardial infarction (AMI). Ischemic heart releases macrophage migration inhibitory factor (MIF), which activates MIF- AMPK signaling pathway. Depression is a significant risk factor for AMI. In a state of depression, peripheral expression of cannabinoid receptor 2 (CNR2) genes was downregulated. AimsWe investigated the mechanism by which depression exacerbates myocardial I/R injury through the CNR2 and MIF-AMPK signaling pathways. MethodsWe established mouse models of depression and myocardial I/R. Left ventricular function was assessed using cardiac ultrasound and TTC staining. The protein levels of myocardial CNR2, MIF, AMPK, and ACC were determined by Western blot, while the expression level of CNR2 was measured using RT-qPCR. Additionally, MIF content in peripheral blood was quantified using ELISA. ResultsAfter I/R, the expression level of CNR2 was found to be lower in the depression group, leading to a deterioration in left heart function. Depressed mice exhibited lower secretion of MIF, accompanied by a decrease in the activation of the MIF-AMPK signaling pathway. However, injection of CNR2 agonist JWH133 prior to ischemia increased the activation of the MIF-AMPK signaling pathway, while CNR2 inhibitor AM630 decreased the activation. LimitationsFurther research is needed to investigate the specific neuroendocrine mechanism affecting myocardial CNR2 expression in depression. And these experimental conclusions require further verification at the cellular level. ConclusionsThe activation of CNR2 in myocardium following I/R is impeded by depression, thereby exacerbating myocardial I/R injury through attenuation of the MIF-AMPK signaling pathway activation.

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

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. 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. 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. Our study revealed that targeting the MIF-CD74 axis promoted microglia M1 polarization and synergized with radiotherapy for brain metastasis in NSCLC.

Elevated MIF identified by multiple cytokine analyses facilitates macrophage M2 polarization contributing to postoperative recurrence in chronic rhinosinusitis with nasal polyps.

Chronic rhinosinusitis with nasal polyps (CRSwNP) is characterized by tissue heterogeneity and high postoperative recurrence risk. This study aims to employ cytokine analyses to identify serum biomarkers associated with postoperative CRSwNP recurrence and elucidate underlying recurrent mechanisms. A prospective cohort study was conducted on CRSwNP patients undergoing functional endoscopic sinus surgery. Serum and tissue samples were collected and analyzed for multiple cytokines. Participants were followed for 3 years and categorized into recurrent and non-recurrent groups. Cytokine profiles were compared, and potential markers for recurrence were further assessed. Macrophage migration inhibitory factor (MIF) expression in macrophages was modulated, and their polarization and cytokine secretion were assessed. In the discovery cohort (21 recurrent and 40 non-recurrent patients), circulating cytokine profiles differed significantly, with 8 cytokines showing differential expression between the two groups. Among them, serum eotaxin, MIF, RANTES, and TRAIL exhibited promise in predicting recurrence. In the validation cohort (24 recurrent and 44 non-recurrent patients), serum eotaxin, MIF, and TRAIL levels were higher in recurrent cases. Tissue MIF was elevated in recurrent cases and had a strong predictive value for recurrence. Moreover, tissue MIF was co-expressed with CD206 in recurrent cases. Mechanistically, MIF overexpression promoted macrophage M2 polarization and TGF-β, CCL-24, and MIF secretion, and MIF recombinant protein facilitated M2 polarization, and TGF-β1 and CCL-24 production, contributing to CRSwNP recurrence. Serum-specific cytokine signatures were associated with postoperative recurrence risk in CRSwNP. Elevated MIF enhanced macrophage M2 polarization and cytokine secretion, contributing to the recurrent mechanisms of CRSwNP.

Host cell-type and pathogen-specific immunomodulatory functions of macrophage migration inhibitory factor (MIF) in infectious keratitis

Therapeutic management of inflammation in infectious keratitis (IK) requires new strategy and targets for selective immunomodulation. Targeting host cell-type specific inflammatory responses might be a viable strategy to curtail unnecessary inflammation and reduce tissue damage without affecting pathogen clearance. This study explores the possibility of pathogen and host cell-type dependent differences in the inflammatory pathways relevant in the pathogenesis of IK. Human corneal epithelial cell line (HCEC) and phorbol 12-myristate-13 acetate (PMA) differentiated THP-1 macrophage line were infected with either Aspergillus flavus conidia or Acanthamoeba castellanii trophozoites and the elicited inflammatory responses were studied in terms of gene expression and secretion of proinflammatory factors interleukin-8 (IL-8) and tumor necrosis factor-alpha (TNF-α) and an upstream inflammatory regulator and mediator protein-the Macrophage Migration Inhibitory Factor (MIF). Given the pleotropic mode of MIF function in diverse cell types relevant in many human diseases, we tested if MIF driven responses to infection is different in HCECs and THP-1 macrophages by studying its expression, secretion and involvement in inflammation by siRNA mediated knockdown. We also examined IK patient tear samples for MIF levels. Infection with A. flavus or A. castellanii induced IL-8 and TNF-α responses in HCECs and THP-1 macrophages but to different levels. Our preliminary human data showed that the level of secreted MIF protein was elevated in IK patient tear, however, MIF secretion by the two cell types were strikingly different in-vitro, under both normal and infected conditions. We found that HCECs released MIF constitutively, which was significantly inhibited with infection, whereas THP-1 macrophages were stimulated to release MIF during infection. MIF gene expression remained largely unaffected by infection in both the cell lines. Although MIF in HCECs appeared to be intracellularly captured during infection, MIF knockdown in HCECs associated with a partial reduction of the IL-8 and TNF-α expression produced by either of the pathogens, suggesting a pro-inflammatory role for MIF in HCECs, independent of its canonical cytokine like function. In contrast, MIF knockdown in THP-1 macrophages accompanied a dramatic increase in IL-8 and TNF-α expression during A. castellanii infection, while the responses to A. flavus infection remained unchanged. These data imply a host cell-type and pathogen specific distinction in the MIF- related inflammatory signaling and MIF as a potential selective immunomodulatory target in infectious keratitis.

MAPK4 silencing in gastric cancer drives liver metastasis by positive feedback between cancer cells and macrophages

Liver metastasis is a major cause of death in gastric cancer patients, but the underlying mechanisms are poorly understood. Through a combination of in vivo screening and transcriptome profiling followed by quantitative RT-PCR and tissue array analyses, we found that mitogen-activated protein kinase 4 (MAPK4) downregulation in gastric cancer tissues from patients is significantly associated with liver metastasis and poor prognosis. The knockdown of MAPK4 in gastric cancer cells promotes liver metastasis in orthotopic mouse models. MAPK4 depletion in gastric cancer cells induces the secretion of macrophage migration inhibitory factor (MIF) to polarize tumor-associated macrophages (TAMs) in orthotopic xenograft tumors. Moreover, TAMs activate epithelial–mesenchymal transition of gastric cancer cells to suppress MAPK4 expression, which further increases MIF secretion to polarize TAMs. Taken together, our results suggest a previously undescribed positive feedback loop between cancer cells and macrophages mediated by MAPK4 silencing that facilitates gastric cancer liver metastasis.

Abstract 10387: Macrophage Migration Inhibitory Factor Maintains Mitochondrial Integrity in Aging During Ischemic Insults

Introduction: The expression and secretion of cardiac MIF (macrophage migration inhibitory factor) are downregulated in aging. A small molecule MIF agonist, MIF20 can increase the binding affinity of MIF with CD74 receptor to modulate AMPK signaling. However, the pharmacological mechanisms by which MIF20 triggers MIF-AMPK signaling cascade to benefit heart in response to ischemia and reperfusion (I/R) stress remain unknown. Hypothesis: MIF20 rescues an impaired MIF-AMPK signaling in aging through augmenting the interaction between MIF and CD74 receptor to maintain mitochondrial integrity of aged heart under ischemic stress. Methods: Young (3-5 months) and aged (21-24 months) C57BL/6J mice were subjected to 45 min of ligation of LAD (left anterior descending coronary artery) and injection of MIF20 (0.15 μg/kg, i.v.) 5 min before 24 hr of reperfusion with release of LAD. Echocardiography measures cardiac functions, and TTC/Evans blue staining for determination of myocardial infarction size. Seahorse XF Analyzer for mitochondrial respiration functions of isolated cardiomyocytes. Results: The echocardiography data showed that I/R caused cardiac systolic dysfunctions but not effect on cardiac diastolic functions of both young and aged C57BL/6J mice. There were no differences between male and female groups. Administration of MIF20 significantly ameliorated I/R-induced cardiac dysfunctions in both young and aged mice, while more improvement observed in aged versus young mice. MIF20 treatment reduced myocardial infarct size caused by I/R in both young and aged mice, but the reduction extent of aged hearts was higher than that of young hearts (p<0.05). Mitochondrial respiration rates of isolated cardiomyocytes as indicted by ATP production were impaired by I/R versus sham operations in both young and aged hearts. Intriguingly, MIF20 treatment significantly rescued the impaired mitochondrial functions caused by I/R in both young and aged cardiomyocytes. Moreover, the mitochondrial functions of young and aged cardiomyocytes were improved to a similar level under I/R with MIF20. Conclusions: MIF agonist MIF20 rescues the impaired energy metabolism in aged heart. The mitochondrial integrity under I/R by MIF20 could limit ischemic insults in aging.

Blockage of CD72 reduces B cell proliferation in immune thrombocytopenic purpura, involving interleukin 1 and macrophage migration inhibitory factor secretion

ABSTRACT Aim This study aims to explore the expression and role of CD72 in B lymphocytes in immune thrombocytopenic purpura (ITP). Methods The expression level of CD72 in B lymphocytes was detected by flow cytometry in 18 ITP patients and 19 controls of healthy donor or iron-deficiency anemia patients. B cell proliferation was determined by 5-bromo-2′-deoxyuridine incorporation (BrdU) in the culture of 17 ITP patients’ and 11 controls’ peripheral mononuclear cells (PMNCs). The secretion levels of antibodies against human platelet antigens (HPA), as well as B cell proliferation-related cytokine interleukin 1(IL-1) and macrophage migration inhibitory factor (MIF) in culture supernatants were measured by ELISA. Results CD72 was significantly increased in B cells of newly diagnosed or persistent ITP compared with ITP in remission. B cell proliferation in culture with CD72 antibody addition was significantly decreased both in ITP patients and in controls compared with isotype antibody addition. CD72 antibody did not significantly alter HPA antibody level in ITP patients. CD72 antibody increased IL-1 and MIF levels in ITP patients’ cell culture supernatant but not in controls. Conclusion CD72 expression elevation accompanies the active status of ITP. In vitro addition of CD72 antibody has a negative impact on B cell proliferation. The function of CD72 in B cell proliferation in ITP may be related to IL-1 and MIF secretion.

Abstract 2113: Suppression of macrophage migration inhibitory factor (MIF) impairs tumor growth and overcomes immunotherapy resistance in KEAP1-deficient NSCLC tumors

Abstract Background: KEAP1, which regulates the degradation of the antioxidant transcription factor NRF2, is the third most commonly mutated tumor suppressor in lung adenocarcinoma (LUAD). Recent reports have provided clinical evidence that mutations in STK11/LKB1 and KEAP1 are strongly associated with immune checkpoint blockade resistance in LUAD, particularly those harboring KRAS mutations. Nevertheless, the specific mechanisms by which loss of KEAP1 impacts anti-tumor immunity in KRAS mutant tumors remains to be determined. Methods: KRAS-mutant (K) and LKB1 (KL), and/or KEAP1-deficient (KK and KLK) murine tumor models were profiled using single-cell RNA sequencing (scRNA-seq) and multiplex staining, and response to anti-PD1 treatment was assessed. Clinical samples from the MD Anderson ICON study (a cohort of 148 resected tumors from early-stage lung cancer patients) and TCGA lung cohorts were used to validate pre-clinical findings. Results: While K tumors were sensitive to anti-PD1 treatment, KEAP1-deficient isogenic tumors (KK; KLK) were refractory. KEAP1-deficient tumors were found to exhibit low immune cell infiltration and an enrichment of cancer associated fibroblasts (CAFs) and endothelial cells. scRNA-seq analysis indicated that KEAP1-deficient tumors had reduced T cell infiltration, in particular, CD8 and NK T cells, decreased B cell populations, and a marked change in M2 macrophage polarization as compared to KEAP1-proficient tumors. Multiplex analysis of CD3 and F4/80 markers confirmed these findings. In the TCGA lung cancer cohort, CD8B expression was dramatically decreased while MIF (macrophage migration inhibitory factor) was upregulated in KK tumors as compared to K LUAD tumors, and expression of KEAP1 inversely correlated with CD163, ARG2 and IL10, which are mainly secreted by macrophages. KEAP1-deficient pre-clinical tumor models showed a significant upregulation of MIF expression and secretion. CRISPR-Cas9 deletion of MIF dramatically impaired in vivo tumor growth, and enhanced T cell cytotoxic effects, anti-tumor immune response and anti-PD1 treatment in KK and KLK tumor models. Conclusions: These findings indicate that loss of KEAP1, alone or in combination with STK11/LKB1 alterations, contributes to an immunosuppressed tumor immune microenvironment. These changes appear to be mediated at least in part through MIF upregulation, providing a potential therapeutic strategy for overcoming KEAP1-dependent resistance to immunotherapy. Citation Format: Ana Galan-Cobo, Yu Qian, Fuduan Peng, Daniel James McGrail, Sonia Patel, Fahao Zhang, Xiang Zhang, Ferdinandos Skoulidis, Edwin Parra, Minghao Dang, Saxon Rodriguez, Alexandre Reuben, Ignacio Wistuba, Linghua Wang, John Victor Heymach. Suppression of macrophage migration inhibitory factor (MIF) impairs tumor growth and overcomes immunotherapy resistance in KEAP1-deficient NSCLC tumors [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 2113.

Chloroquine Induces ROS-mediated Macrophage Migration Inhibitory Factor Secretion and Epithelial to Mesenchymal Transition in ER-positive Breast Cancer Cell Lines.

Breast cancer (BC) is the leading cause of cancer-related death in women in the world. Since tumor cells employ autophagy as a survival pathway, it has been proposed that autophagy inhibition could be beneficial for cancer treatment. There are several onging clinical trials where autophagy is being inhibited (using chloroquine, CQ or hydroxychloroquine, HCQ) along with chemotherapy with promising results. However, there is also in vitro evidence in which autophagy inhibition can induce epithelial to mesenchymal transition (EMT) in cancer cells, indicating that, at least in some cases, this strategy could be detrimental for cancer patients. In this study, we found that the genetic inhibition of autophagy primed cells for EMT by inducing a decrease in E-cadherin protein levels, while CQ treatment decreased E-cadherin levels, induced morphological changes related to EMT, increased EMT-related transcription factor (EMT-TF) expression and migration in estrogen receptor positive (ER +) BC cell lines. Importantly, CQ treatment increased intracellular reactive oxygen species (ROS) which induced the secretion of macrophage migration inhibitory factor (MIF), a pro-inflammatory cytokine related to malignancy. Both ROS production and MIF secretion were responsible for the mesenchymal morphology and increased migratory capacity induced by CQ. Our results indicate that CQ treatment increased malignancy by inducing ROS production, MIF secretion and EMT and suggest that autophagy inhibition in ER + BC patients might have detrimental effects. Our data indicates that a careful selection of patients should be performed in order to determine who will benefit the most from autophagy inhibition with available pharmacological agents for the treatment of breast cancer.

The Role of Macrophage Migration Inhibitory Factor in Adipose-Derived Stem Cells Under Hypoxia.

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-1β 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-1β under hypoxia and normoxia, and neutralizing MIF-antibodies diminished TNF-α and IL-1β 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-1β.

Autophagy inhibition in breast cancer cells induces ROS-mediated MIF expression and M1 macrophage polarization

Autophagy can function as a survival mechanism for cancer cells and therefore, its inhibition is currently being explored as a therapy for different cancer types. For breast cancer, triple negative breast cancer (TNBC) is the subtype most sensitive to the inhibition of autophagy; but its inhibition has also been shown to promote ROS-dependent secretion of macrophage migration inhibitory factor (MIF), a pro-tumorigenic cytokine. In this work, we explore the role of MIF in breast cancer, the mechanism by which autophagy inhibition promotes MIF secretion and its effects on neighboring cancer cell signaling and macrophage polarization. We analyzed MIF mRNA expression levels in tumors from breast cancer patients from different subtypes and found that Luminal B, HER2 and Basal subtypes, which are associated to high proliferation, displayed high MIF levels. However, MIF expression had no prognostic relevance in any breast cancer subtype. In addition, we found that autophagy inhibition in 66cl4 TNBC cells increased intracellular Reactive Oxygen Species (ROS) levels, which increased MIF expression and secretion. MIF secreted from 66cl4 TNBC cells induced the activation of MIF-regulated pathways in syngeneic cell lines, increasing Akt phosphorylation in 4T1 cells and ERK phosphorylation in 67NR cells. Regarding MIF/ chemokine receptors, higher levels of CD74 and CXCR2 were found in TNBC tumor cell lines when compared to non-tumorigenic cells and CXCR7 was elevated in the highly metastatic 4T1 cell line. Finally, secreted MIF from autophagy deficient 66cl4 cells induced macrophage polarization towards the M1 subtype. Together, our results indicate an important role for the inhibition of autophagy in the regulation of ROS-mediated MIF gene expression and secretion, with paracrine effects on cancer cell signaling and pro-inflammatory repercussions in macrophage M1 polarization. This data should be considered when considering the inhibition of autophagy as a therapy for different types of 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".

IGF-1 affects the development of myocarditis in LDL-R knockout mice by inhibiting peritoneal infiltration of macrophages.

Infiltration of the inflammatory cells, such as macrophages and myocardial necrosis, are involved in myocarditis. Insulin-like growth factor (IGF-1) exerts a variety of biological effects. However, the role of IGF-1 in myocarditis remains unclear. LDL-R knockout mice were randomly divided into the control group, myocarditis group, and IGF-1 siRNA group. Real Time-Polymerase Chain Reaction (PCR) and Western blot were used to measure IGF-1 expression in myocardial tissue. The myocardial tissue changes were analyzed by HE staining. The total cholesterol (TC) and low-density lipoprotein cholesterol (LDL) in each group were detected. Flow cytometry was used to analyze the number of macrophages. The secretion of TNF-α and INF-γ and macrophage migration inhibitory factor (MIF) were detected by enzyme-linked immunosorbent assay (ELISA). Compared with the control group, IGF-1 expression, TC, and LDL in myocarditis group was significantly increased, along with decreased heart rate (HR), left ventricular end-systolic diameter (LVEDs), left ventricular end-diastolic diameter (LVEDd), and left ventricular mass index (LVMI). In addition, inflammatory cell infiltration, fibrosis, and macrophages number in the peritoneum were increased. Moreover, the secretion of TNF-α, INF-γ, and MIF was also significantly increased (p<0.05). However, IGF-1 siRNA treatment inhibited IGF-1 expression and reversed the changes in the myocarditis group with statistically significant differences compared with the myocarditis group (p<0.05). IGF-1 expression is increased in myocarditis. The downregulation of IGF-1 expression inhibits macrophages infiltration, reduces the expression of MIF and inflammatory factors, and improves myocarditis injury.

Roles of Macrophage Migration Inhibitory Factor in Dengue Pathogenesis: From Pathogenic Factor to Therapeutic Target.

Dengue virus (DENV) infection is the most prevalent mosquito-borne viral infection and can lead to severe dengue hemorrhagic fever (DHF) and even life-threatening dengue shock syndrome (DSS). Although the cytokine storm has been revealed as a critical factor in dengue disease, the limited understanding of dengue immunopathogenesis hinders the development of effective treatments. Macrophage migration inhibitory factor (MIF) is a pleiotropic proinflammatory cytokine that mediates diverse immune responses, and the serum level of MIF positively correlates with disease severity in patients with dengue. MIF is involved in DENV replication and many pathological changes, such as vascular leakage, during DENV infection. In this paper, the pathogenic roles of MIF and the regulation of MIF secretion during DENV infection are reviewed. Furthermore, whether MIF is a potential therapeutic target against DENV infection is also discussed.

44-OR: PAR2-Regulated MIF Secretion from Non-Immune Cells in Adipose Tissue Is a Key Mechanism Involved in the Development of Metabolic Dysfunction

Macrophage migration inhibitory factor (MIF) is a cytokine that is increased in obesity and contributes to metabolic dysfunction. Our present study describes a novel mechanism by which fatty acids activate non-immune cells to increase MIF secretion in adipose tissue, initiating cross-talk between preadipocytes and adipocytes. High fatty acids specifically induce protease-activated receptor 2 (PAR2) activation in preadipocytes, which downregulates pref-1 expression and release. Preadipocyte factor 1 (Pref-1) has an autocrine-paracrine action to inhibit the release of MIF from both preadipocytes and adipocytes. Thus, the loss of Pref-1 secretion during high fat feeding is responsible in part for both increased MIF secretion in adipose tissue and the rise in plasma MIF levels that ensues. The physiological consequences are most evident in adipose tissue. Genetic deletion of Par2 confirms the importance of this mechanism in mice, protecting against high fat diet-induced metabolic dysfunction. Importantly, this novel mechanism is also operative in adipose tissue from obese human subjects. These results provide the scientific rationale to consider whether strategies to either block Par2 expression or augment Pref-1 secretion might improve metabolic health in obesity or type 2 diabetes. Disclosure Y. Huang: None. L. Chen: None. Y. Qi: None. D. Qi: None.

Autophagy Inhibition Induces the Secretion of Macrophage Migration Inhibitory Factor (MIF) with Autocrine and Paracrine Effects on the Promotion of Malignancy in Breast Cancer.

Breast cancer is the main cause of cancer-related death in women in the world. Because autophagy is a known survival pathway for cancer cells, its inhibition is currently being explored in clinical trials for treating several types of malignancies. In breast cancer, autophagy has been shown to be necessary for the survival of cancer cells from the triple negative subtype (TNBC), which has the worst prognosis among breast cancers and currently has limited therapeutic options. Autophagy has also been involved in the regulation of protein secretion and, of importance for this work, the inhibition of autophagy is known to promote the secretion of proinflammatory cytokines from distinct cell types. We found that the inhibition of autophagy in TNBC cell lines induced the secretion of the macrophage migration inhibitory factor (MIF), a pro-tumorigenic cytokine involved in breast cancer invasion and immunomodulation. MIF secretion was dependent on an increase in reactive oxygen species (ROS) induced by the inhibition of autophagy. Importantly, MIF secreted from autophagy-deficient cells increased the migration of cells not treated with autophagy inhibitors, indicating that autophagy inhibition in cancer cells promoted malignancy in neighboring cells through the release of secreted factors, and that a combinatorial approach should be evaluated for cancer therapy.

Abstract 4676: Cancer stem cell induces chemoresistance in breast cancer via macrophage migration inhibitory factor mediated activation of AKT pathway

Abstract Chemoresistance is one of the significant problems of cancer management. Cellular crosstalk in tumor microenvironment plays a vital role in regulating the chemoresistance in cancer. Cancer stem cell (CSC) is one of the key players in the regulation of chemoresistance in cancer, and they modulate the tumor microenvironment to enhance the resistance to anti-cancer therapy. However, the exact mechanism by which CSC can influence the cellular crosstalk in the tumor microenvironment is mostly unknown. Macrophage migration inhibitory factor (MIF) is attributed to inducing therapeutic resistance in several cancers. We previously found a close relation of MIF secretion with a group of polyploid giant cells in chemoresistant breast cancer. MIF secretion was increased in the media of the mammosphere of MDA MB 231 cell (231) as well as Doxorubicin (Dox) and 5-Fluorouracil (5FU) resistant 231 cells. MIF secretion also increased upon treating 231 with Dox and 5FU in a time-dependent manner. Addition of recombinant MIF in culture medium increased the resistance of 231 to chemotherapeutic agents, and a similar phenomenon was also observed upon treatment with conditioned medium from CSC. Chemoresistant breast cancer is enriched with the CSC population, and hence we hypothesized that the MIF was secreted from CSC populations and had a significant role in the regulation of chemoresistance in breast cancer. MIF expression was also increased in Chemoresistant breast cancer tissues compare to non-resistant ones. MIF also induced anti-apoptotic protein Bcl2 and Bcl-xl by downregulating Bax and Bad as well as contributed to the chemoresistant phenotype. Conditioned media from CSC and exogenous MIF were unable to induce chemoresistance in parental 231 cell line in the presence of 4-IPP, a MIF inhibitor. The anti-apoptotic effect of exogenous MIF was also reversed by the inhibitor. Thus we confirmed that MIF played a significant role in the regulation of resistant phenotype in breast cancer microenvironment. Upon further analysis of molecular pathways, we found phosphorylation of AKT corresponded with the MIF secretion in CSC as well as chemoresistant 231 cell lines. MIF was able to increase the level of phosphorylated AKT in 231 cell line in a dose-dependent manner. When chemoresistant 231 cells were treated with MIF inhibitor, the AKT phosphorylation was inhibited. Silencing the AKT pathway via siRNA reversed the sensitization of exogenous MIF and reduced the resistance of both parental 231 as well as resistant 231 cells. So, overall we conclude that MIF plays a crucial role in inducing chemoresistance in breast cancer by upregulating anti-apoptotic protein Bcl2 and Bcl-xl &amp; downregulating Bax and Bad through AKT phosphorylation. The MIF is mainly secreted by CSC population during self-renewal or stress caused by chemotherapy which activates anti-apoptotic pathways in tumor microenvironment. Citation Format: Subhayan Das, Moumita Kundu, Aditya Parekh, Deblina Bharadwaj, Mahitosh Mandal. Cancer stem cell induces chemoresistance in breast cancer via macrophage migration inhibitory factor mediated activation of AKT pathway [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 4676.

Autophagy mediates the secretion of macrophage migration inhibitory factor from cardiomyocytes upon serum-starvation.

Macrophage migration inhibitory factor (MIF) is an inflammatory cytokine. It is elevated early in the blood of acute myocardial infarction patients. However, it is unclear whether and how MIF is released. This study investigated the cellular source and mechanism of MIF release from hearts. An ischemia-mimic treatment induced the secretion of MIF from neonatal rat cardiomyocytes but not from fibroblasts. The treatment did not cause significant leakage of lactate dehydrogenase, suggesting that ischemia induced the MIF secretion without causing severe cell damage. Plasma samples from patients with acute chest pain at the emergency department were collected for the detection of MIF. MIF levels in patients with acute coronary syndrome (ACS) increased early, when cardiac injury markers were not yet elevated, suggesting that ischemia can induce MIF secretion before the occurrence of severe myocardial damage. Serum-starvation caused MIF secretion from rat cardiomyocytes and Langendorff-perfused rat hearts. The secretion was suppressed by the inhibition of autophagy by inhibitors or by silencing of Atg5. In conclusion, serum-starvation induces the secretion of MIF from cardiomyocytes via autophagy dependent pathway. Clarifying the mechanism of MIF secretion will be helpful for its application in the early diagnosis and treatment of ACS.

Involvement of HPV Infection in the Release of Macrophage Migration Inhibitory Factor in Head and Neck Squamous Cell Carcinoma.

Human papilloma virus (HPV) infection has been well-established as a risk factor in head and neck squamous cell carcinoma (HNSCC). The carcinogenic effect of HPV is mainly due to the E6 and E7 oncoproteins, which inhibit the functions of p53 and pRB, respectively. These oncoproteins could also play a role in the Warburg effect, thus favoring tumor immune escape. Here, we demonstrated that the pro-inflammatory cytokine macrophage migration inhibitory factor (MIF) is expressed at higher levels in HPV-negative patients than in HPV-positive patients. However, the secretion of MIF is higher in HPV-positive human HNSCC cell lines, than in HPV-negative cell lines. In-HPV positive cells, the half inhibitory concentration (IC50) of MIF inhibitor (4-iodo-6-phenylpyrimidine (4-IPP)) is higher than that in HPV-negative cells. This result was confirmed in vitro and in vivo by the use of murine SCCVII cell lines expressing either E6 or E7, or both E6 and E7. Finally, to examine the mechanism of MIF secretion, we conducted proton nuclear magnetic resonance (1H-NMR) experiments, and observed that lactate production is increased in both the intracellular and conditioned media of HPV-positive cells. In conclusion, our data suggest that the stimulation of enzymes participating in the Warburg effect by E6 and E7 oncoproteins increases lactate production and hypoxia inducible factor 1α (HIF-1α) expression, and finally induces MIF secretion.