Elsevier

Brain, Behavior, and Immunity

Volume 42, November 2014, Pages 169-177
Brain, Behavior, and Immunity

Identification of a functional interaction of HMGB1 with Receptor for Advanced Glycation End-products in a model of neuropathic pain

https://doi.org/10.1016/j.bbi.2014.06.199Get rights and content

Abstract

Recent studies indicate that the release of high mobility group box 1 (HMGB1) following nerve injury may play a central role in the pathogenesis of neuropathic pain. HMGB1 is known to influence cellular responses within the nervous system via two distinct receptor families; the Receptor for Advanced Glycation End-products (RAGE) and Toll-like receptors (TLRs). The degree to which HMGB1 activates a receptor is thought to be dependent upon the oxidative state of the ligand, resulting in the functional isoforms of all-thiol HMGB1 (at-HMGB1) acting through RAGE, and disufide HMGB1 (ds-HMGB1) interacting with TLR4. Though it is known that dorsal root ganglia (DRG) sensory neurons exposed to HMGB1 and TLR4 agonists can influence excitation, the degree to which at-HMGB1 signaling through neuronal RAGE contributes to neuropathic pain is unknown. Here we demonstrate that at-HMGB1 activation of nociceptive neurons is dependent on RAGE and not TLR4. To distinguish the possible role of RAGE on neuropathic pain, we characterized the changes in RAGE mRNA expression up to one month after tibial nerve injury (TNI). RAGE mRNA expression in lumbar dorsal root ganglion (DRG) is substantially increased by post-injury day (PID) 28 when compared with sham injured rodents. Protein expression at PID28 confirms this injury-induced event in the DRG. Moreover, a single exposure to monoclonal antibody to RAGE (RAGE Ab) failed to abrogate pain behavior at PID 7, 14 and 21. However, RAGE Ab administration produced reversal of mechanical hyperalgesia on PID28. Thus, at-HMGB1 activation through RAGE may be responsible for sensory neuron sensitization and mechanical hyperalgesia associated with chronic neuropathic pain states.

Introduction

Recent studies indicate inflammatory mediators released by nerve injury play a central role in the pathogenesis of chronic pain conditions (Calvo et al., 2012). Although poorly understood, a key feature of these inflammatory events is the presence of Danger Associated Molecular Patterns (DAMPs; alarmins) Bianchi, 2007. A DAMP of particular interest to the injured nervous system is high mobility group protein box-1 (HMGB1; previously known as amphoterin) (Andersson and Tracey, 2011). HMGB1, a nuclear protein that binds DNA and regulates gene expression is structurally composed of two tandem DNA-binding domains, Box A and B, and a highly acidic C-terminal tail composed of a string of aspartate and glutamate residues (Bianchi et al., 1992, Czura et al., 2001, Dumitriu et al., 2005, Giese et al., 1992). Originally described as a membrane-associated protein which regulates neurite outgrowth during development, it is now known that HMGB1 also plays a crucial role in the inflammatory responses associated with tissue injury, reparative responses and disease (Parkkinen et al., 1993, Hori et al., 1995, Ulloa and Tracey, 2005, Maroso et al., 2010, Zhang et al., 2011) and may contribute significantly to chronic neuropathic pain states (Feldman et al., 2012, Shibasaki et al., 2010).

The action of HMGB1 on different cell types is known to differ dramatically based on the oxidation state of the protein. When first released into the extracelluar space, HMGB1 is initially in the all-thiol state (at-HMGB1) and is thought to largely act on a member of the Ig superfamily, the Receptor for Advanced Glycation End-products (RAGE) Huttunen et al., 2002. There are also reports that at-HMGB1 can form a complex with CXCL12 and act through CXCR4 (Venereau et al., 2013). Once present in an oxidative environment, cysteines 23 and 46 of HMGB1 Box A form a sulfide bond, effectively producing the disulfide isoform of HMGB1 (ds-HMGB1). ds-HMGB1 appears to primarily act on the receptor toll-like receptor 4 (TLR4) in order to influence the production of inflammatory cytokines (Venereau et al., 2013, Yang et al., 2012). ds-HMGB1 can then be further reduced by sulfonation of cysteine 106 in the Box B domain of the ligand, resulting in an inert form (Kazama et al., 2008).

It has been suggested that release of HMGB1 from injured neurons can contribute to seizure activity associated with epilepsy; however, the receptor responsible for this pathological activity is still disputed. (Maroso et al., 2010, Iori et al., 2013). The actions of HMGB1 have also been implicated in both inflammatory and neuropathic pain conditions, though it is unclear as to whether the TLR4 or RAGE receptor is responsible (Feldman et al., 2012, Shibasaki et al., 2010, Chacur et al., 2001, O’Connor et al., 2003, Otoshi et al., 1976, Nakamura et al., 2013). Endotoxin-mediated TLR4 activation is known to directly increase neuronal excitation states in acutely dissociated nociceptive neurons (Hua et al., 1996, Diogenes et al., 2011, Ochoa-Cortes et al., 2010, Due et al., 2012) and administration of xenobiotic TLR4 agonists can produce tactile behavioral hypersensitivity in uninjured rodents (Due et al., 2012). Since the effects of HMGB1 could be mediated by either TLR4 and RAGE depending on the oxidation state of the protein, we set out to determine the contribution of at-HMGB1 on neuronal excitation of nociceptive neurons using a small molecule inhibitor of TLR4 (Bevan et al., 2010, Due et al., 2012) and a neutralizing antibody against RAGE, 11E6 (Guo et al., 2012, Strakhova et al., 2013). Moreover, as little direct evidence exists to support a direct role for RAGE in pain, additional studies examined tibial nerve injury (TNI)-induced RAGE expression in associated lumbar DRG and the ability of RAGE neutralizing antibody to reverse injury-induced behavioral hypersensitivity in the rat across time. Our data indicates that at-HMGB1 elicits neuronal excitation via RAGE, in acutely dissociated sensory neurons. In addition, the use of the neutralizing RAGE antibody reverses tactile pain hypersensitivity. This evidence, together with the increased expression of RAGE in the sensory ganglia, identifies a new potential therapeutic target which appears to contribute to pathological pain.

Section snippets

Animals

Pathogen-free, adult female and male Sprague–Dawley (S/D) rats (150–200 g; Harlan Laboratories, Madison, WI) were housed in temperature (23 ± 3 °C) and light (12-h light: 12-h dark cycle; lights on at 07:00 h) controlled rooms with standard rodent chow and autoclaved tap water available. Experiments were performed during the light cycle. Animals were randomly assigned to the treatment groups. All animal related experiments were approved by the Institutional Animal Care and Use Committee of Indiana

Neuronal RAGE-immunoreactivity colocalizes with TLR4, the isolectin IB4 and calcitonin-gene related peptide (CGRP) expression

We previously reported that the TLR4 receptor is localized to both peptidergic and non-peptidergic sensory neurons within the dorsal root ganglia (Due et al., 2012). We examined cells in vitro for co-expression of RAGE and TLR4, IB4 and CGRP. Dissociated L4 and L5 DRGs removed from naïve animals and cultured for 16–20 h, exhibited a near complete colocalization of TLR4 and RAGE in small and medium diameter neurons (Fig. 1 B, C) with little to no TLR4 or RAGE immunoreactivity present in

Discussion

Our study provides evidence that the ligand at-HMGB1 acting through RAGE is a critical regulator of nociceptive signaling and sensitization in vitro. Moreover, as ongoing pain following peripheral nerve injury is thought to be maintained in part by activity in sensitized primary afferent neurons and could be influenced by an injury-induced upregulation of de novo receptors (White et al., 2005), latent upregulation of RAGE in the DRG corresponds with the ability of a monoclonal antibody against

Acknowledgments

This work was supported by the Indiana Spinal Cord & Brain Injury Research Grant (FAW); NIH (DA026040 to FAW; DK100905 to RK and FAW); the National Scientist Development from the American Heart Association (SDG5280023 to RK); the Neurofibromatosis New Investigator Award from the DOD/CDMRP (NF1000099 to RK). Additional support for YMA as an Indiana CTSI Predoctoral trainee was provided by UL1 TR001107, NIH/NCATS (A. Shekhar, PI). The authors declare no conflicts of interest.

Dr. Khanna is

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      High mobility group box-1 (HMGB1) is a non-histone DNA-binding protein, which exists in the nucleus (Javaherian et al., 1978). HMGB1 is recognized as a late proinflammatory cytokine that triggers the release of numerous inflammatory mediators (Allette et al., 2014; Lotze and Tracey, 2005). Previous studies have shown that HMGB1 plays an important role in chronic pain (Wan et al., 2016; Zhan et al., 2018), including BCP (Tong et al., 2010).

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