High mobility group box 1 secretion blockade results in the reduction of early pancreatic islet graft loss

https://doi.org/10.1016/j.bbrc.2019.05.003Get rights and content

Highlights

  • Isolated pancreatic islets secrete HMGB1 during normoxic and hypoxic incubation.

  • Inflachromene blocks HMGB1 secretion from isolated pancreatic islets.

  • Inflachromene lowers serum and graft HMGB1 levels in islet transplant recipients.

  • HMGB1 blockade increased in vitro and in vivo viability of pancreatic islets.

  • Inflachromene's mass-sparing effect cured diabetic mice with marginal-mass islets.

Abstract

Pancreatic islet transplantation has been known as the best cure for patients suffering from severe type 1 diabetes mellitus (T1DM). Despite meaningful advances in human allogeneic islet transplantation field, significant amounts of islet loss in early post-transplantation periods is still a big concern for clinicians. One of the major factors determining the fate of the islets is the danger-associated molecular patterns (DAMPs) secreted by activated immune cells or islets themselves under hypoxic stress. High mobility group box 1 (HMGB1) protein is one of the best characterized DAMP molecules associated with islets. HMGB1 is known to be passively released by transplanted murine islet cells after taking damages from cytokines, reactive oxygen species, and other DAMPS, and the released HMGB1 harms neighboring islet cells by interacting with receptors expressed on murine islets such as toll-like receptor 2 (TLR2) and TLR4, thereby forming a vicious cycle. Here, we show that a small molecule inhibitor inflachromene (ICM) was capable of blocking the secretion of HMGB1 from murine islet cells during the normoxic and hypoxic post-isolation period. Notably, the treatment of ICM during the islet isolation process resulted in decreased HMGB1 levels during the subsequent cell culture. ICM's in vivo efficacy was evaluated in murine syngeneic islet transplantation model, and it significantly reduced the serum and graft level of HMGB1. Ultimately, the intraperitoneal administration of ICM prevented the loss of marginal-mass islet grafts and reversed the diabetes in mice.

Introduction

Pancreatic islet transplantation has now become a routine procedure for T1DM patients with hypoglycemia unawareness, severe hypoglycemic episodes, and glycemic liability [1]. Despite scientific and technical advances so far, the loss of implanted islet mass in the early period of transplantation is still inevitable, thus requiring two to three donors to cure one diabetic patient [2]. Previous reports connected this misfortune to the hypoxic stress inflicted upon the islet graft and the vulnerability of the pancreatic islets to the stress [3]. Because of the low-oxygen tension of the implantation sites [4] and low revascularization rate of islet grafts [5], hypoxic stress against islets is unavoidable. Hence, reducing the hypoxia-related damage in the entire islet isolation and transplantation procedure would be critical for the successful engraftment of pancreatic islets.

It is well known that many DAMPs are released following cell death due to hypoxic damage, and they again accelerate the destruction of transplanted islets [6]. HMGB1 is a nuclear protein involved in chromatin stabilization and transcription process, but it has been also known to act as a DAMP when released to the extracellular environment at times of immune activation or cell death [7]. In fact, HMGB1 is highly associated with hypoxia-induced islet cell loss either through direct binding to the islets via TLR2 or TLR4 engagement [8]. Naturally, there were attempts to prevent the loss of pancreatic islet graft by blocking the secretion of HMGB1 [9] or neutralizing its effect [10]. Many of these efforts succeeded in protecting islet graft and reversed the diabetes in mice [[11], [12], [13]], which re-emphasizes the detrimental function of extracellular HMGB1 in islet transplantation settings.

In this study, we tested whether ICM, a small-molecule inhibitor of HMGB1 previously known to block the HMGB1 secretion in neuro-inflammatory cells with great potency and little toxicity [14], could work in the same manner on pancreatic islets in vitro and islet transplantation in vivo. We discovered that ICM could block the secretion of HMGB1 in isolated pancreatic islets, and showed that the HMGB1 blockade by ICM could spare the mass of islet grafts in diabetic mice recipients.

Section snippets

Animals

Female BALB/cAnHsd (BALB/c) mice at 8–12 weeks of age were purchased from Jackson Laboratories (Bar Harbor, ME) and maintained in the Seoul National University specific pathogen-free (SPF) animal facilities. All experimental procedures were conducted in accordance with the guidelines outlined in the Guide for the Care and Use of Laboratory Animals prepared by the Institute of Laboratory Animal Resources (NIH Publication No. 86–23, revised 2011) and published by the National Institute of Health.

ICM reduces the level of HMGB1 in islet cultures

The first step we took was to determine whether ICM could demonstrate the same potency to pancreatic islets as it did to the microglia. Since islets express the receptors for LPS [8,10], we sought to investigate the ICM's effect on LPS-treated MIN6 cells. Through ICC, we observed that ICM treatment reduced the expression of LPS-induced HMGB1 in MIN6 cells (Fig. 1A). Particularly, HMGB1 seemed to localize to the nuclear compartment of the cells after ICM treatment (white arrows). In fact, ICM

Discussion

Here, we tried to show that usage of ICM, a small-molecule inhibitor of HMGB1, has certain gains in murine islet transplantation. Recently, ICM was proven to be effective in the sepsis treatment [15], indicating that it could be used on the broader spectrum of HMGB1-secreting cells. We speculated that ICM could be used in pancreatic islet transplantation settings, where pancreatic islets act as a substantial reservoir of HMGB1 protein [20,23] and increased serum level of HMGB1 is negatively

Acknowledgements

This work was supported by a grant from the Korea Healthcare Technology R&D Project through the Korea Health Industry Development Institute and funding from the Ministry for Health and Welfare, Republic of Korea (Grant No. HI13C0954). This work also was supported by the Creative Research Initiative Grant (2014R1A3A2030423) and the Bio & Medical Technology Development Program (2012M3A9C4048780) through the National Research Foundation of Korea funded by the Ministry of Science & ICT, Korea.

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    Hyun-Je Kim's Current address is, Department of Dermatology and the Laboratory of Inflammatory Skin Diseases, Icahn School of Medicine at Mount Sinai, New York, NY, USA.

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