Shock/Sepsis/Trauma/Critical care
Endogenous HMGB1 is required in endotoxin tolerance

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Abstract

Background

High-mobility group box 1 protein (HMGB1), a downstream inflammatory response modifier in sepsis and endotoxemia, alters endotoxin tolerance by affecting cellular hyporesponsiveness and tumor necrosis factor α and interleukin 1 production.

Objective

Endogenous HMGB1 signaling mechanisms during low-dose lipopolysaccharide (LPS)-induced endotoxin tolerance were investigated.

Methods

BALB/c mice were preconditioned with either 0.1 mL low-dose LPS (0.2 mg/kg) or phosphate-buffered saline (PBS) (control) followed by treatment with three consecutive injections of anti-HMGB1, IgY (an nonspecific antibody), or PBS, at 2, 12, and 22 h, respectively, Mice were then subjected to 0.1 mL high-dose LPS (10 mg/kg) or PBS at 24 h. Serum and hepatic tissue samples were obtained 1 or 3 h after final treatments. Signaling mechanisms were further investigated in the serum and hepatic tissues of mice preconditioned with 0.1 mL HMGB1 (1 mg/kg), low-dose LPS (0.2 mg/kg), or PBS for 1 h, and then high-dose LPS treatment for 3 h.

Results

The signaling mechanisms involved in low-dose LPS preconditioning required enhanced endogenous HMGB1 expression and secretion. Neutralizing endogenous HMGB1 with anti-HMGB1 antibodies following low-dose LPS preconditioning altered endotoxin tolerance by increasing serum tumor necrosis factor α, reducing hepatic interleukin-1R-associated kinase M expression, and partially restoring nuclear factor κB in vivo. The translocation from nucleus to cytoplasm of endogenous HMGB1 in RAW264.7 cells was also observed during low-dose LPS–induced endotoxin tolerance.

Conclusions

Increased interleukin-1R-associated kinase M and decreased nuclear factor κB activity in endotoxin tolerance is associated with endogenous HMGB1 expression after low-dose LPS preconditioning. These findings provide a basis for a better mechanistic understanding and the development of safer clinical therapeutics utilizing induced endotoxin tolerance.

Introduction

Lipopolysaccharide (LPS), a bacterial endotoxin, is a potent inflammatory agent capable of stimulating an immune response by altering macrophage production of proinflammatory cytokines, proteases, eicosinoids, and reactive oxygen species [1]. As early at 1946, low-dose endotoxin administration was reported to improve patient tolerance during subsequent endotoxin exposure [2]. This process, termed endotoxin tolerance, is characterized by a transient state of cellular hyporesponsiveness and decreased production of proinflammatory cytokines tumor necrosis factor α (TNF-α) and interleukin 1 [3]. Clinically, this state is a key contributor to both immunosuppression and mortality in sepsis patients [4], leading to broad clinical applications of endotoxin tolerance therapies using low-dose LPS to decrease infection severity, ischemic damage, and reperfusion injury [5]. However, detrimental outcomes, such as diminished responsiveness to repeat bacterial challenge and nosocomial infections, have also been reported [5]. In order to increase the safety and clinical applicability of induced endotoxin tolerance therapies, it is necessary to more precisely determine the cell-signaling and mechanistic pathways associated with LPS tolerance.

Numerous studies have explored the mechanistic relationships between endotoxins and the endogenous ligands responsible for tissue damage following infection, or “alarmins,” including high-mobility group box 1 (HMGB1) [6]. Most notably, preconditioning with recombinant HMGB1, a protein generally found in the nucleus that promotes DNA bending by binding to nucleosomes [7], has been reported to mediate damage caused by inflammatory responses following ischemic reperfusion in the liver. Furthermore, this phenomenon may be associated with upregulation of the negative signaling protein interleukin-1R-associated kinase M (IRAK-M) [8]. Aneja et al. [9] demonstrated in vivo that preconditioning with recombinant HMGB1 induced LPS tolerance in mice. In septic patients, several studies have confirmed reduced monocyte and macrophage capacity for proinflammatory cytokine release following further LPS stimulation by impaired intracellular signaling pathway activation, particularly activation of the nuclear factor κB (NF-κB) pathway [10], [11], [12]. In studies of induced tolerance to bacterial lipoteichoic acid and LPS, recombinant HMGB1 has been shown to decrease degradation of inhibitor of NF-κB nuclear factor of kappa light polypeptide gene enhancer in B-cell inhibitor alpha (IκBα) and the DNA binding to NF-κB [9], [13]. The mechanism of endogenous HMGB1 induction of LPS tolerance, however, remains widely explored and debated in current research.

HMGB1 plays a key role in the intracellular signaling pathways involved in the development of inflammation [14]. The activity of HMGB1 varies according to the redox states of cysteine residues involved in Toll-like receptor 4 bindings, potentially forming immunostimulatory complexes with cytokines and other endogenous factors [15]. Contrary to its proinflammatory functions, some evidence also suggests that HMGB1 has regenerative effects that can initiate tissue repair [16]. HMGB1 is also a key player in immune state changes, mediating the shift from inflammation to immunosuppression through regulation of T cells through Toll-like receptor 4 [17]. Harris et al. [18] demonstrated the effects of high level HMGB1 in vivo in rheumatoid arthritis, myositis, and systemic lupus erythematosus patients and animal models, revealing that extranuclear expression of HMGB1 is increased and blockade of such HMGB1 expression attenuates inflammation.

Although the ability of exogenous HMGB1 to induce endotoxin tolerance to LPS is well documented [8], [9], [13], the complex effects of low-dose LPS preconditioning on immune signaling mechanisms are not fully understood. Therefore, we assume that endogenous HMGB1 was required in endotoxin tolerance with low-dose LPS preconditioning. Thus, the aim of the present study is to clarify the signaling mechanisms involved in endogenous HMGB1 during the development of endotoxin tolerance following preconditioning with low-dose LPS. More importantly, the molecular basis for switching between immune states of inflammation and immunosuppression is explored, potentially providing a basis for design of safer induced endotoxin tolerance therapies.

Section snippets

Animal subjects

A total of 150 male BALB/c mice 8–12 wk old weighing 20–25 g were purchased from the XiangYa Medical Experimental Animal Center (Changsha, Hu’nan, China). All animal subjects were housed in a laminar-flow, temperature-controlled, pathogen-free environment at 22°C ± 2°C and a 12-h light/dark cycle at the Experimental Animal Laboratory of the Third XiangYa Hospital of Central South University (Changsha, Hu’nan, China). Mice were provided with ad libitum access to food and unfiltered tap water.

Endogenous HMGB1 expression enhancement by low-dose LPS preconditioning

In model 1, low-dose LPS preconditioning for 24 h before lethal-dose LPS injection induced hepatic HMGB1 expression. Mice pretreated with low-dose LPS before PBS injection also expressed high levels of HMBG1. However, no significant variation in HMGB1 expression was observed after lethal-dose LPS injection alone at early time points (before 3 h) (P > 0.05) (Fig. 2A). The release of HMGB1 was also confirmed in serum (Fig. 2B), demonstrating that serum HMGB1 levels after low-dose LPS

Discussion

Endogenous HMGB1 expression is necessary for establishing tolerance to the Gram-negative bacterial endotoxin LPS, as demonstrated by increased IRAK-M expression and decreased NF-κB activity. Although the recombinant HMGB1 preconditioning has been previously reported [8], [9], [13], the current study presents a novel assessment of the signaling mechanisms involved in this preconditioning process. These findings provide a basis for the development and enhancement of induced LPS tolerance

Acknowledgment

This work was supported by Hunan Provincial Innovation Foundation For Postgraduate (No. CX2012B103) and the project supported by the Specialized Research Fund for the Doctoral Program of Higher Education of China (Grant No. 20090162110020).

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