HMGB1 is a therapeutic target for sterile inflammation and infection

Abstract

A key question in immunology concerns how sterile injury activates innate immunity to mediate damaging inflammation in the absence of foreign invaders. The discovery that HMGB1, a ubiquitous nuclear protein, mediates the activation of innate immune responses led directly to the understanding that HMGB1 plays a critical role at the intersection of the host inflammatory response to sterile and infectious threat. HMGB1 is actively released by stimulation of the innate immune system with exogenous pathogen-derived molecules and is passively released by ischemia or cell injury in the absence of invasion. Established molecular mechanisms of HMGB1 binding and signaling through TLR4 reveal signaling pathways that mediate cytokine release and tissue damage. Experimental strategies that selectively target HMGB1 and TLR4 effectively reverse and prevent activation of innate immunity and significantly attenuate damage in diverse models of sterile and infection-induced threat.

Figure 1

Structure of human HMGB1, a 25-kDa protein of 215 amino acids. Note that 20% of the residues are lysines and that the protein is organized in three domains made up by two positively charged DNA-binding structures (A and B box) and a negatively charged acidic tail composed of 30 glutamic and aspartic acids, exclusively. The A and B boxes are helical structures, partly covered by the tail, which is folded over the protein. There are two nuclear emigration signals in the proximal part of the A and B boxes, respectively, that can bind to the nuclear exportin CRM1. There are also two nuclear-localization signals, as indicated in the figure. The primary HMGB1 sequence is 98.5% identical in all mammals, and two of the three substitutions occur in the repetitive carboxyl terminus with switches of aspartic and glutamic acids. Truncation of the full-length HMGB1 demonstrates that the extracellular cytokine activity resides within the B box. This activity can be competitively inhibited by truncated A box protein. The cysteine in position 106 in the B box is indispensable for its cytokine role, given that oxidation or selective mutation of this residue abolishes the activity of HMGB1 signaling to activate cytokine release.

Figure 2

HMGB1 is released during infection by activating innate immunity and during sterile injury as a passive phenomenon. Exposure to pathogens activates the highly conserved innate immune response, which triggers the release of HMGB1 from monocytes, macrophages, and other cells at the frontline of host defense. HMGB1 shuttles from the nucleus to the cytosol, where it accumulates in intracellular vesicles prior to secretion. This process can take up to 8 h to complete. The passive release of HMGB1 in cells undergoing necrotic cell death is much faster because HMGB1 is loosely attached to nuclear DNA in living cells. HMGB1 binding to chromatin increases during programmed cell death, causing some of it to be retained, but ingestion of apoptotic bodies by macrophages stimulates significant release of HMGB1 by the macrophages (not illustrated). Thus, infection, necrosis, and apoptosis can all lead to elevated HMGB1 levels. In low levels, HMGB1 mediates sickness behavior and antibacterial activities that contribute to inflammatory responses that can resolve. The release of larger amounts of HMGB1, however, is associated with the development of epithelial barrier failure, organ dysfunction, and even death.

Figure 3

The images illustrate intracellular HMGB1 localization in macrophages (a) before and (b) after 24-h LPS activation. Intracellular HMGB1, visualized with green immunofluorescent FITC staining, is predominately localized in the nucleus in resting macrophages (exemplified with white arrows in a). Following activation by exposure to LPS for 24 h, nuclear HMGB1 is phosphorylated, acetylated, and actively transported from the nucleus to the cytoplasm. Some nuclei even empty their HMGB1 contents (white arrows in b).

Figure 4

HMGB1 signals by binding to TLR4 to activate macrophage/monocyte cytokine release (left) and by binding to RAGE to modulate endothelial and tumor cell function (right). In monocytes/macrophages, HMGB1 binds TLR4 in the context of MD2 through a mechanism that requires cysteine in position 106. The addition of dithiothreitol (DTT) to HMGB1 denatures this interaction. HMGB1-TLR4 signaling activates MyD88-dependent nuclear translocation of NF-κB, which upregulates the expression of cytokine and other inflammatory mediators. In endothelial cells and other somatic cells, e.g., tumors and smooth muscle, HMGB1 interacts with RAGE. This signaling, which is not sensitive to DTT, is transduced by mechanisms that are incompletely known but culminate on Cdc42 and Rac. Other binding partners may be required, but RAGE signaling has been implicated in cell growth, differentiation, migration, and expression of cell surface proteins. HMGB1 interaction with CD24 and Siglec-10 can mediate a signal that inhibits activation of NF-κB and prevents cytokine release mediated by HMGB1-TLR4 signaling.

Figure 5

HMGB1 is an early mediator in sterile injury and a late mediator in infection. Infection activates innate immune cells to produce HMGB1, which occurs after a significant lag, placing it downstream of an early TNF response. During ischemia and other forms of sterile cell injury, HMGB1 is released as an early mediator that in turn activates the later release of TNF and other cytokines.