Elsevier

Brain, Behavior, and Immunity

Volume 59, January 2017, Pages 322-332
Brain, Behavior, and Immunity

Full-length Article
Ds-HMGB1 and fr-HMGB induce depressive behavior through neuroinflammation in contrast to nonoxid-HMGB1

https://doi.org/10.1016/j.bbi.2016.09.017Get rights and content

Highlights

  • Both ds-HMGB1 and fr-HMGB1 induce depressive behavior.

  • The depressive behavior induced by ds-HMGB1 is related to TLR4 activation and a subsequent TNF-α increase.

  • Fr-HMGB1 evokes depressive behavior through TLR4, RAGE and CXCR4, which are related to TNF-α induction.

  • Nonoxid-HMGB1 cannot induce depressive behavior or a central TNF-α increase.

Abstract

High mobility group box 1 (HMGB1) has been implicated as a key factor in several neuroinflammatory conditions. Our previous study suggested that the release of central HMGB1 acts as a late-phase mediator in lipopolysaccharide (LPS)-induced depression. Recent findings indicate that the redox state of HMGB1 is a critical determinant of its immunomodulatory properties. Here, we aimed to investigate the potential mechanisms that link the redox states of HMGB1 to depression in mice. Distinct redox forms of recombinant HMGB1 (rHMGB1) were used that included fully reduced HMGB (fr-HMGB1), which acted as a chemokine, and disulfide-HMGB1 (ds-HMGB1), which possessed cytokine activity. Fr-HMGB1 in vivo was partially oxidized into ds-HMGB1; thus, the mutant protein non-oxidizable chemokine-HMGB (nonoxid-HMGB1) was applied. Concurrent with depressive behavior induced by four-week stress exposure, the HMGB1 concentrations in the serum and cerebral cortex substantially increased. Therefore, a single dose of rHMGB1 (200 ng/5 μl/mice) or vehicle was administered to mice via intracerebroventricular (i.c.v.) injection. The receptor inhibitors of TLR4/RAGE/CXCR4 (TAK-242/FPS-ZM1/AMD3100) (3 mg/kg) were intraperitoneally injected 30 min prior to rHMGB1 treatment. Depressive-like behavior was measured 20 h post i.c.v. injection. Administration of fr-HMGB1 prolonged the immobility duration in the tail suspension test (TST) and decreased sucrose preference. In addition to depressive behavior, the hippocampal TNF-α protein slightly increased. These depressive behaviors and upregulation of hippocampal TNF-α were alleviated or abrogated by pretreatment with the inhibitors AMD3100, FPS-ZM1, and TAK-242. Alternatively, nonoxid-HMGB1 failed to induce TNF-α protein or prolong the immobility duration. As expected, ds-HMGB1 administration substantially upregulated hippocampal TNF-α protein, increased the immobility time in the TST and decreased sucrose preference. Moreover, both glycyrrhizin and TAK-242 improved ds-HMGB1-induced depressive behavior. Furthermore, TAK-242 significantly blocked the upregulation of hippocampal TNF-α protein and protected hippocampal myelin basic protein from ds-HMGB1-induced reduction. These drugs had no effect on the total or central distance in the open field test. Collectively, this initial experiment demonstrates the role and receptor mechanisms of HMGB1 under different redox states on the induction of depressive-like behavior. Both ds-HMGB1 and fr-HMGB1 may induce depressive-like behavior in vivo mainly via neuroinflammatory response activation.

Introduction

Depression is a devastating mental disorder characterized by sadness, anhedonia, low-esteem, fatigue, disturbed sleep or appetite and poor concentration. Approximately one million individuals die from suicide each year (refer to url: http://www.who.int/mediacentre/factsheets/fs369/en/). Reports from the World Health Organization indicate that depression is a major contributor to the overall global burden of disease. In recent years, an inflammatory hypothesis has been proposed, and a substantial number of studies have indicated that immune system activation plays an important role in the pathogenesis of depression (Dantzer, 2012, Dantzer et al., 2008, Raison et al., 2006, Schiepers et al., 2005). Increased expression of proinflammatory cytokines, chemokines and soluble adhesion molecules in the peripheral blood and cerebrospinal fluid (CSF) have been demonstrated in patients with major depressive disorder (Miller and Raison, 2016, Monje et al., 2003). Further studies provide strong evidence that exogenous administration of several cytokines causes depressive symptoms in humans (McDonald et al., 1987).

High mobility group box 1 (HMGB1) is an alarmin protein discovered decades ago (Goodwin et al., 1973). It comprises a late-phase proinflammatory cytokine that links stress and inflammation, particularly in priming neuroinflammation (Balosso et al., 2014, Frank et al., 2015, Weber et al., 2015). As a key factor, HMGB1 is implicated in the mediation of neuroinflammatory processes in several pathophysiological conditions, including seizure and ischemia (Frank et al., 2016). Increasing evidence has indicated that HMGB1 is widely involved in the pathogenesis of several immune-related diseases, emotional and cognitive dysfunctions and neurological diseases (Bae, 2012, Bianchi and Manfredi, 2007, Takata et al., 2012). Moreover, many preclinical studies are attempting to alleviate the symptoms of diseases using blocking agents or neutralizing antibodies against HMGB1 (Musumeci et al., 2014, Nogueira-Machado and de Oliveira Volpe, 2012). However, HMGB1 did not receive attention in the field of depression until our first report on an LPS-induced acute depression model last year (Wu et al., 2015). Early phase proinflammatory cytokines have been the focus in the field of depression (Miller and Raison, 2016, Raison et al., 2006, Schiepers et al., 2005); thus, the inflammatory hypothesis was proposed.

As a ubiquitous nonhistone chromosomal protein, HMGB1 serves as a damage-associated molecular pattern (DAMP) to stimulate the innate immune system by itself or as part of complexes with cytokines, exogenous and endogenous molecules (Andersson and Tracey, 2011, Harris et al., 2012). HMGB1 is mainly located in the nucleus under physiological conditions and acts as a DNA chaperone engaged in many DNA activity associated events (Kang et al., 2014). Noticeably, when subjected to various stressors, biologically active HMGB1 may be transported to the cytosol, further excreted into the extracellular space and cause a series of reactions (Kang et al., 2014). Extracellular HMGB1 may function as a chemotactic or proinflammatory mediator through interactions with different receptors, such as toll-like receptors (TLRs), including TLR4 and TLR2, and receptor for advanced glycation end products (RAGE) (Balosso et al., 2014, Mazarati et al., 2011). In addition to direct interactions with receptors, HMGB1 may form complexes with inflammation-inducing agents, such as interleukin (IL)-1β and lipopolysaccharide (LPS), and dramatically amplifies inflammatory responses (Hreggvidsdottir et al., 2009).

Recent work has demonstrated that the biological activity of extracellular HMGB1 depends on the redox states of cysteines at positions 23, 45, and 106 within a protein (Venereau et al., 2012). Different redox forms of HMGB1 interact with different receptors in different phases of the pathophysiological process. There are three forms of HMGB1 depending on the redox states, namely, fully reduced HMGB1 (fr-HMGB1), disulfide HMGB1 (ds-HMGB1) and fully oxidized HMGB1 (ox-HMGB1). Fr-HMGB1 represents the status in which cysteines at positions C23, C45, and C106 are all reduced, thereby remaining in a thiol state. It functions as a chemotactic factor via interactions with the RAGE receptor and potentiates chemotactic activity via interactions with CXCR4 and the formation of a heterocomplex with CXCL12 (http://www.genecards.org/cgi-bin/carddisp.pl?gene=CXCL12) chemokine. In contrast, ds-HMGB1 exerts proinflammatory effects via binding to TLRs when cysteines at positions C23 and C45 become oxidized, whereas the cysteine at position C106 remains in a reduced thiol state (Frank et al., 2015, Yang et al., 2012). Ds-HMGB1 induces cytokine production through TLR4 signaling. When all three cysteines in HMGB1 are terminally oxidized, namely, ox-HMGB1, it is considered non-active. Ox-HMGB1 is not associated with an in vivo function (Agalave and Svensson, 2014, Kang et al., 2014, Tang et al., 2016, Yang et al., 2012). However, few studies have investigated the potential mechanism that links redox states of HMGB1 to neuroinflammatory processes. Balosso and colleagues demonstrated that ds-HMGB1 potentiated kainate-induced seizures (Balosso et al., 2014). Liesz and colleagues demonstrated that cerebral ischemia induced the release of fr-HMGB1 from necrotic brain lesions and ds-HMGB1 was released into serum (Liesz et al., 2015). Frank et al. demonstrated that ds-HMGB1 induced a primed immunophenotype in the central nervous system, which may result in an exacerbated neuroinflammatory response following exposure to a subsequent proinflammatory stimulus (Frank et al., 2015).

Our previous study suggested that the active release of central HMGB1 was involved in LPS-induced depressive-like behavior (Wu et al., 2015). After our first report, Cheng et al. recently proposed consistent results in acute stress induced depression (Cheng et al., 2016). However, it is not clear whether this finding is related to the redox states of HMGB1. In the present study, we aim to investigate the role and potential mechanisms of HMGB1 under different redox states on the induction of depressive behavior. First, we further confirmed the upregulation of central HMGB1 in a chronic stress induced depression model. On this basis, we subsequently implemented various molecular forms of HMGB1 through i.c.v. injection to investigate their potential effects on depressive-like behavior. Finally, primary receptor mechanisms were investigated.

Section snippets

Reagents

Recombinant human HMGB1/HMG-1 (expressed in E. coli, purity ⩾95%, endotoxin level <0.10 EU/μg protein, Cat#1690-HMB-050) was obtained from R&D SYSTEMS (R&D SYSTEMS, Minneapolis, USA). Fr-HMGB1 (produced in E.coli, LPS <0.4 ng/mg protein, Cat#HM-116) and Non-oxidizable chemokine-HMGB1 (non-oxid HMGB1, produced in E.coli, LPS <0.4 ng/mg protein, Cat#HM-132) were obtained from HMGBiotech (HMGBiotech, Milano, Italy). TAK-242 (C15H17CIFNO4S, Cat#HY-11109), FPS-ZM1 (C20H22CINO, Cat#HY-19370), and

Serum and central HMGB1 increased with depressive-like behavior

Chronic stress comprises a fundamental factor in the provocation of major depression disorder (Gambarana et al., 2001, Yan et al., 2010). Our previous work demonstrated the role of HMGB1 in LPS induced depressive-like behavior (Wu et al., 2015). To further confirm this finding, the HMGB1 level in the CUMS depression model was measured. Following the 4-week CUMS exposure, the SPT and TST were performed to assess the depressive status of the mice. The serum and cortex HMGB1 proteins were measured

Discussion

We first identified and established the important role of actively released HMGB1 during the depression process in our previous studies (Wu et al., 2015). The present study further investigated the redox states and receptor mechanism of HMGB1 closely related to the development of depressive-like behavior. The results demonstrate that both ds-HMGB1 and oxidizable fr-HMGB1 may induce depressive-like behavior through an upregulation of central TNF-α, whereas a non-oxidizable form does not induce

Conclusions

In conclusion, our findings suggest both ds-HMGB1 and fr-HMGB1, in contrast to nonoxidable HMGB1, induce depressive-like behavior mainly via neuroinflammatory response activation. Ds-HMGB1 induced the development of depressive-like behavior through a TLR4 mediated TNF-α increase and MBP reduction. Fr-HMGB1 may be converted into ds-HMGB1 in vivo to produce proinflammatory cytokines and subsequently evoke additional inflammatory mediators. These series of neuroinflammatory responses contribute to

Acknowledgments

This work was supported by the Natural Science Foundation of China (NSFC, NO.81171124 and 81571169) and the Military Medical Research Foundation (NO.2013JS13 and NO.13CXZ050).

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    Yong-Jie Lian and Hong Gong contributed equally to this work.

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