Novel inhibitors of macrophage migration inhibitory factor prevent cytokine-induced beta cell death

doi:10.1016/j.ejphar.2014.06.009

European Journal of Pharmacology

Volume 740, 5 October 2014, Pages 683-689

https://doi.org/10.1016/j.ejphar.2014.06.009 Get rights and content

Abstract

Macrophage migration inhibitory factor is a multifunctional cytokine involved in the regulation of immune processes and also in apoptosis induction. Elevated MIF expression is detrimental for insulin-producing beta cells and MIF inhibition protected beta cells from several cytotoxic insults such as inflammatory cytokines, high fatty acids or high glucose concentrations. Therefore, the aim of this study was to investigate two newly synthesized small molecule MIF inhibitors (K664-1 and K647-1) and to compare them with previously established effects of the prototypical MIF inhibitor, ISO-1. Our results indicate that K664-1 and K647-1 are 160- and 40-fold more effective in inhibition of MIF׳s tautomerase activity than ISO-1. Also, new inhibitors confer beta cell protection from cytokine-triggered apoptosis at significantly lower concentrations than ISO-1. Although all three MIF inhibitors inhibit caspase 3 activity, K664-1 and K647-1 suppress pro-apoptotic BAX protein expression and up-regulate anti-apoptotic Bcl-2 mRNA. Finally, all three MIF inhibitors operate through blockade of nitric oxide production stimulated by cytokines. In conclusion, two novel MIF inhibitors are more potent than ISO-1 and operate through inhibition of the mitochondria-related apoptotic pathway. We propose that these compounds represent a unique class of anti-MIF antagonists that should be further tested for therapeutic use.

Introduction

Type 1diabetes is an autoimmune disease characterized by a local inflammatory reaction in and around the pancreatic islets, followed by progressive destruction of insulin producing beta cells that leads to hyperglycemia. Until now, a number of pathological stimuli have been identified to induce beta cell apoptosis: reactive oxidative and nitrogen species, perforins and granzymes, Fas ligand and most importantly pro-inflammatory cytokines such as interferon-γ (IFN-γ), interleukin-1β (IL-1β) and tumor necrosis factor α (TNF-α) (Padgett et al., 2013). Type 2 diabetes is characterized by insulin resistance in peripheral tissues that manifests as hyperglycemia when beta cells fail to regulate insulin production due to their demise mediated by elevated nutrients and cytokines. All mentioned cytotoxic stimuli that contribute to beta cell loss identified in type 1 and type 2 diabetes converge on mitochondria to lead the beta cell to apoptosis (Szabadkai and Duchen, 2009). Recently, another cytokine, macrophage migration inhibitory factor (MIF) has been identified as an important factor for beta cell survival and MIF inhibition plays a crucial role in blocking a mitochondria-mediated apoptotic process. MIF absence or blockade ensures protection of beta cells from exposure to either pro-inflammatory cytokines (Stojanovic et al., 2012a) or elevated nutrients (palmitic acid or glucose) in vitro (Stojanovic et al., 2012b).

in vivo experiments proved that MIF is crucial for development of type 1 diabetes (Bojunga et al., 2003) and that MIF inhibition by neutralizing antibodies (Cvetkovic et al., 2005) or complete MIF absence (MIF knock-out) ameliorates initiation and/or progression of type 1 diabetes (Stosic-Grujicic et al., 2008). These data indicate that the inhibition of MIF could be exploited as a therapeutic strategy (Stosic-Grujicic et al., 2009, Greven et al., 2010). Having in mind that the usage of neutralizing antibodies for therapy could result in side-effects and is expensive, several pharmacologically designed MIF inhibitors have been synthetized and have shown promising results. One of the best characterized and also most well-known MIF inhibitors is (S,R)-3-(4-hydroxyphenyl)-4,5-dihydro-5-isoxazole acetic acid methyl ester (ISO-1) (reviewed in Al-Abed et al., 2011). It was shown that ISO-1 has beneficial effects in experimental colitis (Shah et al., 2008), experimental allergic neuritis (Nicoletti et al., 2005) and in endotoxemia (Al-Abed et al., 2005). In MIF׳s homotrimeric structure a hydrophobic pocket region formed between each adjacent subunit plays an important role in its biological function, as evidenced by the fact that ISO-1 binding of this region decreases downstream MIF signaling, MIF biological activities and clinical outcomes that are associated with MIF (Al-Abed et al., 2011).

Since we have previously shown that in vivo MIF inhibition by ISO-1 ameliorates streptozotocin induced type 1 diabetes development in C57BL/6 mice (Cvetkovic et al., 2005) and that in vitro ISO-1 protects insulinoma cells or pancreatic islets from pro-inflammatory cytokines (Stojanovic et al., 2008) the aim of this study was to compare the activity of two novel MIF inhibitors with ISO-1 and to investigate, in more detail, the mechanism of their action.

Section snippets

Materials and methods

Unless stated otherwise, all chemicals were from Sigma-Aldrich (St Louis, MO, USA) and all plastics used in the experiments were from Sardstedt (Numbrecht, Germany).

Novel MIF inhibitors K664-1 and K647-1 are potent inhibitors of MIF enzymatic activity

The concentration required to inhibit 50% of the control tautomerase activity (IC-50) was determined by established methods and compared for the two new compounds versus the established MIF inhibitor, ISO-1. The IC-50′s of K647-1 (0.41×0.01 μM) and K664-1 (0.11±0.03 μM) were approximately 40 and 160x more potent than ISO-1 (Table 1).

K664-1 and K647-1 inhibit the recognition of MIF

Interaction of ISO-1 with recombinant MIF in vitro down-regulates immunorecognition of MIF during ELISA (Hegde et al., 2012). Therefore, we first sought to

Discussion

In the present study, we demonstrate that the two newly synthetized compounds, K664-1 and K647-1, are potent inhibitors of MIF tautomerase activity and that they rescue beta cells from cytokine-driven apoptosis. This protection is provided at lower concentrations compared to ISO-1. The new MIF inhibitors likely operate through inhibition of BAX-mediated apoptosis, unlike ISO-1. However, all three inhibitors prevent caspase-3 activation and NO production.

MIF is an important regulator of

Acknowledgments

We would like to thank Melita Vidakovic, PhD, from the Institute for Biological Research “Sinisa Stankovic”, University of Belgrade, Belgrade, Serbia for the kind gift of RINm5f cell culture. This work was supported by Ministry of Education, Science and Technological Development, Republic of Serbia (Project no. 173013).

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This issue will be discussed further in the next section. Intriguingly, both genetic MIF deletion and MIF inhibition with different MIF inhibitors prevented cytokine-induced beta cell death through down-regulation of BAX or caspase 3-related pathways in vitro [61,62]. This means that in the circumstances of MIF overproduction (under the influence of cytokines or palmitic acid), MIF acquires detrimental influence on beta cells.
Pro-inflammatory nature of macrophage migration inhibitory factor (MIF) has been generally related to the propagation of inflammatory and autoimmune diseases. But this molecule possesses many other peculiar functions, unrelated to the immune system, among which is its supportive role in the post-translational modifications of insulin. In this way MIF enables proper insulin conformation within the pancreatic beta cell and its full activity. The inherent or acquired changes in MIF expression might therefore lead to different insulin processing and initiation of autoimmunity. The relation between MIF and insulin does not stop at this point; these two molecules continue to interact during pathological states characterized by inflammation and insulin resistance. In this context, MIF indirectly and negatively influences insulin action by boosting inflammatory environment and disabling target cells to respond to insulin. On the other side, insulin might interfere with MIF action as well, acting as an anti-inflammatory mediator. Therefore, the proper interaction between MIF and insulin is crucial for maintaining homeostasis, while anti-inflammatory therapies based on the systemic MIF blockage may disturb this balance. This review covers MIF-insulin relationship in the physiological and pathological conditions and discusses the approaches for MIF inhibition and their net effect specifically considering possible impact on insulin misfolding and the possible misinterpretation of previous results due to the discovery of MIF functional homolog D-dopachrome tautomerase.
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MIF upregulated and knock-down PD cells model was successful established using lentivirus (P < 0.001; Fig. 2D) and siRNA (P < 0.01; Fig. 2E), respectively, for use in subsequent experiments. According to the above results, ISO-1, an inhibitor of MIF (Vujicic et al., 2014) (Fig. 2F), was added in order to verify the role of MIF in the MPP + SH-SY5Y cell model. As shown in Fig. 2G, the expression of MIF was significantly downregulated in the MPP + SH-SY5Y cell model upon ISO-1 addition (P < 0.001).
The influence of neuroinflammation in the development and progression of Parkinson's disease (PD) remains unknown. Macrophage migration inhibitory factor (MIF) is a multipotent and key cytokine involved in the pathogenesis of acute and chronic inflammatory and immune disorders. The aim of this study was to investigate the neuroprotective effects mediated by MIF in PD. Cellular apoptosis was measured by RT-qPCR analysis, fluorescence-activated cell sorting (FACS) analysis and western blotting. JC-1 staining was used to analyze the mitochondrial membrane potential (MMP). The formation of autophagosomes was detected by western blot analysis. Autophagic flux was assessed by tandem mRFP-GFP-LC3 fluorescence microscopy. Expression of MIF, cleaved-PARP and LC3B-II was increased significantly in both acute and chronic PD animal models. MIF was positively associated with IL-10 (P < 0.001), but inversely with TNF-α (P < 0.05). The PD cells (1 mM MPP + treated group) showed an increase in early-apoptotic cells by FACS. Upregulating MIF expression resulted in a lower concentration of cleaved-PARP than the control group (P < 0.001). The MMP was higher in the MIF upregulated group than in the MIF knockdown group (P < 0.001). Upregulating MIF expression resulted in a higher concentration of LC3B-II than the control group (P < 0.001). Finally, LC3 puncta were markedly increased in the MIF upregulated group and in the MIF + MPP + group. This study indicates that MIF mediates a neuroprotective effect via suppressing inflammatory responses, inhibiting apoptosis and inducing autophagy in PD.
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Fenofibrate attenuates fatty acid-induced islet β-cell dysfunction and apoptosis via inhibiting the NF-κB/MIF dependent inflammatory pathway
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Fatty acid-induced lipotoxicity and macrophage migration inhibitory factor (MIF) affect pancreatic β-cell function, and may promote the development of diabetes mellitus. However, the association of lipotoxicity with MIF and the effect of Fenofibrate on β-cell function remain unknown.
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¹: Authors equally contributed.

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Immunopharmacology and inflammationNovel inhibitors of macrophage migration inhibitory factor prevent cytokine-induced beta cell death

Abstract

Introduction

Section snippets

Materials and methods

Novel MIF inhibitors K664-1 and K647-1 are potent inhibitors of MIF enzymatic activity

K664-1 and K647-1 inhibit the recognition of MIF

Discussion

Acknowledgments

J. Biol. Chem.

Cytokine

Anal. Biochem.

Biochem. Biophys. Res. Commun.

J. Biol. Chem.

J. Immunol. Methods

J. Neuroimmunol.

Autoimmun. Rev.

MIF as a disease target: ISO-1 as a proof-of-concept therapeutic

Future Med. Chem.

Thyroxine is a potential endogenous antagonist of macrophage migration inhibitory factor (MIF) activity

Proc. Natl. Acad. Sci. U. S. A.