A Novel Allosteric Inhibitor of Macrophage Migration Inhibitory Factor (MIF)

Macrophage migration inhibitory factor (MIF) is a catalytic cytokine and an upstream mediator of the inflammatory pathway. MIF has broad regulatory properties, dysregulation of which has been implicated in the pathology of multiple immunological diseases. Inhibition of MIF activity with small molecules has proven beneficial in a number of disease models. Known small molecule MIF inhibitors typically bind in the tautomerase site of the MIF trimer, often covalently modifying the catalytic proline. Allosteric MIF inhibitors, particularly those that associate with the protein by noncovalent interactions, could reveal novel ways to block MIF activity for therapeutic benefit and serve as chemical probes to elucidate the structural basis for the diverse regulatory properties of MIF. In this study, we report the identification and functional characterization of a novel allosteric MIF inhibitor. Identified from a high throughput screening effort, this sulfonated azo compound termed p425 strongly inhibited the ability of MIF to tautomerize 4-hydroxyphenyl pyruvate. Furthermore, p425 blocked the interaction of MIF with its receptor, CD74, and interfered with the pro-inflammatory activities of the cytokine. Structural studies revealed a unique mode of binding for p425, with a single molecule of the inhibitor occupying the interface of two MIF trimers. The inhibitor binds MIF mainly on the protein surface through hydrophobic interactions that are stabilized by hydrogen bonding with four highly specific residues from three different monomers. The mode of p425 binding reveals a unique way to block the activity of the cytokine for potential therapeutic benefit in MIF-associated diseases.     

Macrophage Migration Inhibitory Factor (MIF) Enzymatic Activity and Lung Cancer

The cytokine macrophage migration inhibitory factor (MIF) possesses unique tautomerase enzymatic activity, which contributes to the biological functional activity of MIF. In this study, we investigated the effects of blocking the hydrophobic active site of the tautomerase activity of MIF in the pathogenesis of lung cancer. To address this, we initially established a Lewis lung carcinoma (LLC) murine model in Mif-KO and wild-type (WT) mice and compared tumor growth in a knock-in mouse model expressing a mutant MIF lacking enzymatic activity (Mif ^P1G). Primary tumor growth was significantly attenuated in both Mif-KO and Mif ^P1G mice compared with WT mice. We subsequently undertook a structure-based, virtual screen to identify putative small molecular weight inhibitors specific for the tautomerase enzymatic active site of MIF. From primary and secondary screens, the inhibitor SCD-19 was identified, which significantly attenuated the tautomerase enzymatic activity of MIF in vitro and in biological functional screens. In the LLC murine model, SCD-19, given intraperitoneally at the time of tumor inoculation, was found to significantly reduce primary tumor volume by 90% (p < 0.001) compared with the control treatment. To better replicate the human disease scenario, SCD-19 was given when the tumor was palpable (at d 7 after tumor inoculation) and, again, treatment was found to significantly reduce tumor volume by 81% (p < 0.001) compared with the control treatment. In this report, we identify a novel inhibitor that blocks the hydrophobic pocket of MIF, which houses its specific tautomerase enzymatic activity, and demonstrate that targeting this unique active site significantly attenuates lung cancer growth in in vitro and in vivo systems.     

Expression and Function of Macrophage Migration Inhibitory Factor (MIF) in Melioidosis

Background

Macrophage migration inhibitory factor (MIF) has emerged as a pivotal mediator of innate immunity and has been shown to be an important effector molecule in severe sepsis. Melioidosis, caused by Burkholderia pseudomallei, is an important cause of community-acquired sepsis in Southeast-Asia. We aimed to characterize the expression and function of MIF in melioidosis.

Methodology and Principal Findings

MIF expression was determined in leukocytes and plasma from 34 melioidosis patients and 32 controls, and in mice infected with B. pseudomallei. MIF function was investigated in experimental murine melioidosis using anti-MIF antibodies and recombinant MIF. Patients demonstrated markedly increased MIF mRNA leukocyte and MIF plasma concentrations. Elevated MIF concentrations were associated with mortality. Mice inoculated intranasally with B. pseudomallei displayed a robust increase in pulmonary and systemic MIF expression. Anti-MIF treated mice showed lower bacterial loads in their lungs upon infection with a low inoculum. Conversely, mice treated with recombinant MIF displayed a modestly impaired clearance of B. pseudomallei. MIF exerted no direct effects on bacterial outgrowth or phagocytosis of B. pseudomallei.

Conclusions

MIF concentrations are markedly elevated during clinical melioidosis and correlate with patients'' outcomes. In experimental melioidosis MIF impaired antibacterial defense.     

Role of Macrophage Migration Inhibitory Factor (MIF) in Pollen-Induced Allergic Conjunctivitis and Pollen Dermatitis in Mice

Pollen is a clinically important airborne allergen and one of the major causes of allergic conjunctivitis. A subpopulation of patients with atopic dermatitis (AD) are also known to have exacerbated skin eruptions on the face, especially around the eyelids, after contact with pollen. This pollen-induced skin reaction is now known as pollen dermatitis. Macrophage migration inhibitory factor (MIF) is a pluripotent cytokine that plays an essential role in allergic inflammation. Recent findings suggest that MIF is involved in several allergic disorders, including AD. In this study, MIF knockout (KO), MIF transgenic (Tg) and WT littermate mice were immunized with ragweed (RW) pollen or Japanese cedar (JC) pollen and challenged via eye drops. We observed that the numbers of conjunctiva- and eyelid-infiltrating eosinophils were significantly increased in RW and JC pollen-sensitized MIF Tg compared with WT mice or MIF KO mice. The mRNA expression levels of eotaxin, interleukin (IL)-5 and IL-13 were increased in pollen-sensitized eyelid skin sites of MIF Tg mice. An in vitro analysis revealed that high eotaxin expression was induced in dermal fibroblasts by MIF combined with stimulation of IL-4 or IL-13. This eotaxin expression was inhibited by the treatment with CD74 siRNA in fibroblasts. These findings indicate that MIF can induce eosinophil accumulation in the conjunctiva and eyelid dermis exposed to pollen. Therefore, targeted inhibition of MIF might result as a new option to control pollen-induced allergic conjunctivitis and pollen dermatitis.     

Identification and Characterization of Novel Classes of Macrophage Migration Inhibitory Factor (MIF) Inhibitors with Distinct Mechanisms of Action

Macrophage migration inhibitory factor (MIF), a proinflammatory cytokine, is considered an attractive therapeutic target in multiple inflammatory and autoimmune disorders. In addition to its known biologic activities, MIF can also function as a tautomerase. Several small molecules have been reported to be effective inhibitors of MIF tautomerase activity in vitro. Herein we employed a robust activity-based assay to identify different classes of novel inhibitors of the catalytic and biological activities of MIF. Several novel chemical classes of inhibitors of the catalytic activity of MIF with IC₅₀ values in the range of 0.2–15.5 μm were identified and validated. The interaction site and mechanism of action of these inhibitors were defined using structure-activity studies and a battery of biochemical and biophysical methods. MIF inhibitors emerging from these studies could be divided into three categories based on their mechanism of action: 1) molecules that covalently modify the catalytic site at the N-terminal proline residue, Pro¹; 2) a novel class of catalytic site inhibitors; and finally 3) molecules that disrupt the trimeric structure of MIF. Importantly, all inhibitors demonstrated total inhibition of MIF-mediated glucocorticoid overriding and AKT phosphorylation, whereas ebselen, a trimer-disrupting inhibitor, additionally acted as a potent hyperagonist in MIF-mediated chemotactic migration. The identification of biologically active compounds with known toxicity, pharmacokinetic properties, and biological activities in vivo should accelerate the development of clinically relevant MIF inhibitors. Furthermore, the diversity of chemical structures and mechanisms of action of our inhibitors makes them ideal mechanistic probes for elucidating the structure-function relationships of MIF and to further determine the role of the oligomerization state and catalytic activity of MIF in regulating the function(s) of MIF in health and disease.     

Structural and functional characterization of a secreted hookworm Macrophage Migration Inhibitory Factor (MIF) that interacts with the human MIF receptor CD74

Hookworms, parasitic nematodes that infect nearly one billion people worldwide, are a major cause of anemia and malnutrition. We hypothesize that hookworms actively manipulate the host immune response through the production of specific molecules designed to facilitate infection by larval stages and adult worm survival within the intestine. A full-length cDNA encoding a secreted orthologue of the human cytokine, Macrophage Migration Inhibitory Factor (MIF) has been cloned from the hookworm Ancylostoma ceylanicum. Elucidation of the three-dimensional crystal structure of recombinant AceMIF (rAceMIF) revealed an overall structural homology with significant differences in the tautomerase sites of the human and hookworm proteins. The relative bioactivities of human and hookworm MIF proteins were compared using in vitro assays of tautomerase activity, macrophage migration, and binding to MIF receptor CD74. The activity of rAceMIF was not inhibited by the ligand ISO-1, which was previously determined to be an inhibitor of the catalytic site of human MIF. These data define unique immunological, structural, and functional characteristics of AceMIF, thereby establishing the potential for selectively inhibiting the hookworm cytokine as a means of reducing parasite survival and disease pathogenesis.     

Macrophage Migration Inhibitory Factor (MIF) and Thyroid Hormone Alterations in Antineutrophil Cytoplasmic Antibody (ANCA)-Associated Vasculitis (AAV)

Macrophage migration inhibitory factor (MIF) is a proinflammatory cytokine known to be released from lymphocytes, macrophages and endothelial cells and also in animal models shown to be inducible with glucocorticoids (GC). In contrast, thyroxine seems to antagonize MIF activity. To investigate whether MIF is increased in active antineutrophil cytoplasmic antibody (ANCA)-associated vasculitis (AAV) and possible correlations with GC dosing and thyroid hormone levels, 27 consecutive patients with active AAV were studied and followed prospectively. Disease activity was assessed using Birmingham Vasculitis Activity Score 2003 (BVAS) at baseline and at follow-up at 3 and 6 months, along with MIF, thyroid hormones free triiodothyronine (fT3) and free thyroxine (fT4), C-reactive protein (CRP) and creatinine. MIF was elevated significantly at baseline compared with follow-up at 3 and 6 months (8,618 pg/mL versus 5,696 and 6,212 respectively; P < 0.002) but did not correlate to CRP, GC dose, creatinine or organ involvement. fT3 was depressed significantly at baseline compared with follow-up (1.99 pg/mL versus 2.31 and 2.67 respectively; P = 0.01) and correlated inversely to the BVAS score at baseline. We found a significant correlation between the MIF/fT4 ratio at baseline versus MIF/fT4 ratio at 6 months (ρ = 0.52, P < 0.005) and a trend between the baseline MIF/fT3 ratio versus MIF/fT3 ratio at 6 months (ρ = 0.39, P = 0.05). These results suggest a possible role for MIF and thyroid status in AAV. Further studies could reveal whether the association between AAV and thyroid hormone levels in the context of elevated MIF may present a link as well as a target of treatment.     

Elevated Serum Levels of Macrophage Migration Inhibitory Factor Are Associated with Progressive Chronic Cardiomyopathy in Patients with Chagas Disease

Clinical symptoms of chronic Chagas disease occur in around 30% of the individuals infected with Trypanosoma cruzi and are characterized by heart inflammation and dysfunction. The pathogenesis of chronic chagasic cardiomyopathy (CCC) is not completely understood yet, partially because disease evolution depends on complex host-parasite interactions. Macrophage migration inhibitory factor (MIF) is a pleiotropic proinflammatory cytokine that promotes numerous pathophysiological processes. In the current study, we investigated the link between MIF and CCC progression.Immunohistochemical analysis demonstrated MIF overexpression in the hearts from chronically T. cruzi-infected mice, particularly those showing intense inflammatory infiltration. We also found that MIF exogenously added to parasite-infected murine macrophage cultures is capable of enhancing the production of TNF-α and reactive oxygen species, both with pathogenic roles in CCC. Thus, the integrated action of MIF and other cytokines and chemokines may account for leukocyte influx to the infected myocardium, accompanied by enhanced local production of multiple inflammatory mediators. We further examined by ELISA the level of MIF in the sera from chronic indeterminate and cardiomyopathic chagasic patients, and healthy subjects. CCC patients displayed significantly higher MIF concentrations than those recorded in asymptomatic T. cruzi-infected and uninfected individuals. Interestingly, increased MIF levels were associated with severe progressive Chagas heart disease, in correlation with elevated serum concentration of high sensitivity C-reactive protein and also with several echocardiographic indicators of left ventricular dysfunction, one of the hallmarks of CCC. Our present findings represent the first evidence that enhanced MIF production is associated with progressive cardiac impairment in chronic human infection with T. cruzi, strengthening the relationship between inflammatory response and parasite-driven pathology. These observations contribute to unravel the elements involved in the pathogenesis of CCC and may also be helpful for the design of novel therapies aimed to control long-term morbidity in chagasic patients.     

Macrophage Migration Inhibitory Factor Inhibits the Migration of Cartilage End Plate-Derived Stem Cells by Reacting with CD74

Background

Macrophage migration inhibitory factor (MIF) is a multifunctional cytokine that regulates inflammatory reactions and the pathophysiology of many inflammatory diseases. Intervertebral disc (IVD) degeneration is characterized by an inflammatory reaction, but the potential role of MIF in IVD degeneration has not been determined. Recent studies have shown that MIF and its receptor, CD74, are involved in regulating the migration of human mesenchymal stem cells (MSCs); Thus, MIF might impair the ability of mesenchymal stem cells (MSCs) to home to injured tissues. Our previous studies indicated that cartilage endplate (CEP)-derived stem cells (CESCs) as a type of MSCs exist in human degenerate IVDs. Here, we investigate the role of MIF in regulating the migration of CESCs.

Methods and Findings

CESCs were isolated and identified. We have shown that MIF was distributed in human degenerate IVD tissues and was subject to regulation by the pro-inflammatory cytokine TNF-α. Furthermore, in vitro cell migration assays revealed that nucleus pulposus (NP) cells inhibited the migration of CESCs in a number-dependent manner, and ELISA assays revealed that the amount of MIF in conditioned medium (CM) was significantly increased as a function of increasing cell number. Additionally, recombinant human MIF (r-MIF) inhibited the migration of CESCs in a dose-dependent manner. CESCs migration was restored when an antagonist of MIF, (S, R)-3(4-hydroxyphenyl)-4, 5-dihydro-5-isoxazole acetic acid methyl ester (ISO-1), was added. Finally, a CD74 activating antibody (CD74Ab) was used to examine the effect of CD74 on CESCs motility and inhibited the migration of CESCs in a dose-dependent manner.

Conclusions

We have identified and characterized a novel regulatory mechanism governing cell migration during IVD degeneration. The results will benefit understanding of another possible mechanism for IVD degeneration, and might provide a new method to repair degenerate IVD by enhancing CESCs migration to degenerated NP tissues to exert their regenerative effects.     

调控巨噬细胞迁移抑制因子基因表达的影响因素

巨噬细胞迁移抑制因子(macrophage migration inhibitory factor, MIF)是一种广泛表达的多效性细胞因子,参与多种炎症和免疫疾病的过程并在其中发挥重要作用,是许多疾病的生物标志物或治疗靶点。MIF基因在系统发育中高度保守,在其启动子区有多种不同转录因子的特定结合位点,借此调节MIF的表达。MIF在细胞内外均发挥作用,且MIF是组成型表达。因此,研究调控MIF基因表达和刺激MIF分泌的相关因素具有重要意义。本文通过对MIF基因和MIF启动子上的结合位点的简述,对影响MIF基因表达的相关因素进行总结和归类。根据与MIF基因结合的方式,可分为：(1)与MIF基因启动子特定位点结合,改变转录活性;(2)与MIF CATT5-8微卫星重复序列结合,改变高表达MIF等位基因;(3)非编码RNA调控MIF表达;(4)影响MIF分泌的相关因素。通过对这4类调控MIF基因表达的相关因素的综述,进而认识MIF基因表达的调控机制和影响因素,以期对其治疗相关疾病提供理论基础。     

Negative Regulation of AMP-activated Protein Kinase (AMPK) Activity by Macrophage Migration Inhibitory Factor (MIF) Family Members in Non-small Cell Lung Carcinomas

AMP-activated protein kinase (AMPK) is a nutrient- and metabolic stress-sensing enzyme activated by the tumor suppressor kinase, LKB1. Because macrophage migration inhibitory factor (MIF) and its functional homolog, d-dopachrome tautomerase (d-DT), have protumorigenic functions in non-small cell lung carcinomas (NSCLCs) but have AMPK-activating properties in nonmalignant cell types, we set out to investigate this apparent paradox. Our data now suggest that, in contrast to MIF and d-DTs AMPK-activating properties in nontransformed cells, MIF and d-DT act cooperatively to inhibit steady-state phosphorylation and activation of AMPK in LKB1 wild type and LKB1 mutant human NSCLC cell lines. Our data further indicate that MIF and d-DT, acting through their shared cell surface receptor, CD74, antagonize NSCLC AMPK activation by maintaining glucose uptake, ATP production, and redox balance, resulting in reduced Ca²⁺/calmodulin-dependent kinase kinase β-dependent AMPK activation. Combined, these studies indicate that MIF and d-DT cooperate to inhibit AMPK activation in an LKB1-independent manner.     

The Dexamethasone-induced Inhibition of Proliferation, Migration, and Invasion in Glioma Cell Lines Is Antagonized by Macrophage Migration Inhibitory Factor (MIF) and Can Be Enhanced by Specific MIF Inhibitors

Glioblastomas (GBMs) are the most frequent and malignant brain tumors in adults. Glucocorticoids (GCs) are routinely used in the treatment of GBMs for their capacity to reduce the tumor-associated edema. Few in vitro studies have suggested that GCs inhibit the migration and invasion of GBM cells through the induction of MAPK phosphatase 1 (MKP-1). Macrophage migration inhibitory factor (MIF), an endogenous GC antagonist is up-regulated in GBMs. Recently, MIF has been involved in tumor growth and migration/invasion and specific MIF inhibitors have been developed on their capacity to block its enzymatic tautomerase activity site. In this study, we characterized several glioma cell lines for their MIF production. U373 MG cells were selected for their very low endogenous levels of MIF. We showed that dexamethasone inhibits the migration and invasion of U373 MG cells, through a glucocorticoid receptor (GR)- dependent inhibition of the ERK1/2 MAPK pathway. Oppositely, we found that exogenous MIF increases U373 MG migration and invasion through the stimulation of the ERK1/2 MAP kinase pathway and that this activation is CD74 independent. Finally, we used the Hs 683 glioma cells that are resistant to GCs and produce high levels of endogenous MIF, and showed that the specific MIF inhibitor ISO-1 could restore dexamethasone sensitivity in these cells. Collectively, our results indicate an intricate pathway between MIF expression and GC resistance. They suggest that MIF inhibitors could increase the response of GBMs to corticotherapy.     

Homotrimeric Macrophage Migration Inhibitory Factor (MIF) Drives Inflammatory Responses in the Corneal Epithelium by Promoting Caveolin-rich Platform Assembly in Response to Infection

Acute inflammation that arises during Pseudomonas aeruginosa-induced ocular infection can trigger tissue damage resulting in long term impairment of visual function, suggesting that the appropriate treatment strategy should include the use of anti-inflammatory agents in addition to antibiotics. We recently identified a potential target for modulation during ocular infection, macrophage migration inhibitory factor (MIF). MIF deficiency protected mice from inflammatory-mediated corneal damage resulting from acute bacterial keratitis. To gain a better understanding of the molecular mechanisms of MIF activity, we analyzed the oligomeric states and functional properties of MIF during infection. We found that in human primary corneal cells infected with P. aeruginosa, MIF is primarily in a homotrimeric state. Homotrimeric MIF levels correlated with the severity of infection in the corneas of infected mice, suggesting that the MIF homotrimers were the functionally active form of MIF. During infection, human primary corneal cells released more IL-8 when treated with recombinant, locked MIF trimers than when treated with lower MIF oligomers. MIF promoted P. aeruginosa–induced IL-8 responses via the formation of caveolin-1-rich “signaling hubs” in the corneal cells that led to elevated MAPK p42/p44 activation and sustained inflammatory signaling. These findings suggest that inhibiting homotrimerization of MIF or the functional activities of MIF homotrimers could have therapeutic benefits during ocular inflammation.     

巨噬细胞迁移抑制因子拮抗剂对子宫内膜异位症的影响

目的：探讨巨噬细胞迁移抑制因子拮抗剂（ISO-1）对子宫内膜异位症的影响。方法：以裸鼠为研究对象,构建子宫内膜异位元症动物模型,应用巨噬细胞迁移抑制因子拮抗剂进行干预,观察子宫内膜异位症小鼠的成活率和体重变化;采用RT-PCR检测基质金属蛋白酶-2（MMP-2）,基质金属蛋白酶抑制剂-2（TIMP-2）,血管内皮细胞生长因子（VEGF）,TNF-αmRNA的表达,ELISA检测TNF-α蛋白的表达。结果：ISO-1对子宫内膜异位症小鼠的存活率无明显影响,但可增加其体重（P〈0.05）。ISO-1减少子宫内膜异位症小鼠受损组织中MMP-2、VEGF、TNF-α的表达（P〈0.05）,但对TIMP-2的表达无明显影响。结论：巨噬细胞迁移抑制因子被特异性阻断后,可明显抑制受损组织的重构、血管生成和炎症,最终影响子宫内膜异位症的组织生长及进一步恶化,这可能是临床治疗子宫内膜异位症的新策略。     

Identification of Macrophage Migration Inhibitory Factor Isoforms in Bovine Brain

In the course of the study of the primary structures and molecular mechanisms of action of immunologically active compounds of the nervous system we have isolated from the soluble fraction of total bovine brain two heat-stable proteins. The purification procedure was mainly based on DEAE-Servacel ion-exchange chromatography and reversed-phase HPLC. The proteins were identified by the N-terminal Edman microsequence analysis and database searching as macrophage migration inhibitory factor (MIF). The N-terminal sequences for MIF1 and MIF2 were found to be identical. According to mass spectral analysis, the molecular masses for MIF1 and MIF2 were determined respectively as 12,369.21 and 12,299.7 Da. In addition, we have also isolated a third peptide having the same N-terminal sequence and Mr 9,496.2 that seems to be a proteolytic fragment of MIF. Using p-hydroxyphenylpyruvate as a substrate, we have not revealed tautomerase activity of either MIF1 or MIF2. As both the immunologic and enzymatic activities were reported to be expressed by the oligomeric structure of MIF, we suggest that the present study may give additional information on MIF in terms of structural properties of this protein. A comparatively simple purification procedure is presented that may be widely used for simultaneous isolation in one run of MIF isoforms.     

终末期肾病患者合并胃食管反流病的临床研究进展

胃食管反流病(GERD)是最常见的食管疾病之一。多项研究表明,终末期肾病(ERSD)患者GERD的患病率高于普通人群。目前对于ERSD患者特别是血液透析患者GERD的症状特点及严重程度的研究较少。ESRD患者常并发或伴发糖尿病、高血压等,而糖尿病神经病变可影响胃排空功能,钙拮抗剂和硝酸酯类药物可影响LES舒张功能。透析相关淀粉样变通过影响食管蠕动、食管下段括约肌张力和胃排空影响GERD的发生。ERSD患者中,相当比例的患者全身状况不佳,行胃镜风险较高,常常应用标准化量表或质子泵抑制剂诊断试验评估患者症状性GERD患病情况。ESRD及透析患者GERD的知晓率仍较低,部分患者自行服用碳酸氢钠等非一线药物控制症状。理论上对于ESRD及透析患者伴随的GERD进行早期诊断和治疗可能提高患者生活质量,并减少水钠摄入,改善血压及透析间期体重增加,降低心血管事件风险,具体的临床获益仍有待进一步研究证实。     

Charge Heterogeneity of Bovine Brain Macrophage Migration Inhibitory Factor

Macrophage migration inhibitory factor (MIF) is known as a ubiquitous pluripotent cytokine originally identified for its capacity to inhibit the random migration of macrophages in vitro. It is recognized as an important regulator of the immunological, neuroendocrine and enzymatic processes. MIF is widely expressed in brain, but its role in the nervous system is not yet understood. In the course of the study of the primary structure of bovine brain MIF we have previously identified a number of MIF-related proteins having identical N-terminal sequences. In this paper we report the results of isoelectric focusing of MIF isolated to a homogeneous state from bovine brain that revealed MIF charge heterogeneity. We have detected isoelectric forms of MIF with pI values of 6.9, 7.0, 7.3, and 7.8. The diverse actions of MIF within the immuno-neuroendocrine system is suggested to be a result of its occurrence in different isoforms and oligomerization states.