Despite significant advances in intensive care therapy and antibiotics, severe sepsis accounts for 9% of all deaths in the United States annually. The pathological sequelae of sepsis are characterized by a systemic inflammatory response, but experimental therapeutics that target specific early inflammatory mediators [tumor necrosis factor (TNF) and IL-1␤] have not proven efficacious in the clinic. We recently identified high mobility group box 1 (HMGB1) as a late mediator of endotoxin-induced lethality that exhibits significantly delayed kinetics relative to TNF and IL-1␤. Here, we report that serum HMGB1 levels are increased significantly in a standardized model of murine sepsis, beginning 18 h after surgical induction of peritonitis. Specific inhibition of HMGB1 activity [with either anti-HMGB1 antibody (600 g per mouse) or the DNAbinding A box (600 g per mouse)] beginning as late as 24 h after surgical induction of peritonitis significantly increased survival (nonimmune IgG-treated controls ‫؍‬ 28% vs. anti-HMGB1 antibody group ‫؍‬ 72%, P < 0.03; GST control protein ‫؍‬ 28% vs. A box ‫؍‬ 68%, P < 0.03). Animals treated with either HMGB1 antagonist were protected against the development of organ injury, as evidenced by improved levels of serum creatinine and blood urea nitrogen. These observations demonstrate that specific inhibition of endogenous HMGB1 therapeutically reverses lethality of established sepsis indicating that HMGB1 inhibitors can be administered in a clinically relevant time frame.

Toll-like receptor 2 (TLR2) agonists induce a subset of TLR4-inducible proinflammatory genes, which suggests the use of differential signaling pathways. Murine macrophages stimulated with the TLR4 agonist Escherichia coli lipopolysaccharide (LPS), but not with TLR2 agonists, induced phosphorylation of signal transducer and activator of transcription 1alpha (STAT1alpha) and STAT1beta, which was blocked by antibodies to interferon beta (IFN-beta) but not IFN-alpha. All TLR2 agonists poorly induced IFN-beta, which is encoded by an immediate early LPS-inducible gene. Thus, the failure of TLR2 agonists to induce STAT1-dependent genes resulted, in part, from their inability to express IFN-beta. TLR4-induced IFN-beta mRNA was MyD88- and PKR (double-stranded RNA-dependent protein kinase)-independent, but TIRAP (Toll-interleukin 1 receptor domain-containing adapter protein)-dependent. Together, these findings provide the first mechanistic basis for differential patterns of gene expression activated by TLR4 and TLR2 agonists.

In response to bacterial endotoxin (e.g., LPS) or endogenous proinflammatory cytokines (e.g., TNF and IL-1beta), innate immune cells release HMGB1, a late cytokine mediator of lethal endotoxemia and sepsis. The delayed kinetics of HMGB1 release makes it an attractive therapeutic target with a wider window of opportunity for the treatment of lethal systemic inflammation. However, the receptor(s) responsible for HMGB1-mediated production of proinflammatory cytokines has not been well characterized. Here we demonstrate that in human whole blood, neutralizing antibodies against Toll-like receptor 4 (TLR4, but not TLR2 or receptor for advanced glycation end product) dose-dependently attenuate HMGB1-induced IL-8 release. Similarly, in primary human macrophages, HMGB1-induced TNF release is dose-dependently inhibited by anti-TLR4 antibodies. In primary macrophages from knockout mice, HMGB1 activates significantly less TNF release in cells obtained from MyD88 and TLR4 knockout mice as compared with cells from TLR2 knockout and wild-type controls. However, in human embryonic kidney 293 cells transfected with TLR2 or TLR4, HMGB1 effectively induces IL-8 release only from TLR2 overexpressing cells. Consistently, anti-TLR2 antibodies dose-dependently attenuate HMGB1-induced IL-8 release in human embryonic kidney/TLR2-expressing cells and markedly reduce HMGB1 cell surface binding on murine macrophage-like RAW 264.7 cells. Taken together, our data suggest that there is a differential usage of TLR2 and TLR4 in HMGB1 signaling in primary cells and in established cell lines, adding complexity to studies of HMGB1 signaling which was not previously expected.

Upon tissue injury, high m.w. hyaluronan (HA), a ubiquitously distributed extracellular matrix component, is broken down into lower m.w. (LMW) fragments, which in turn activate an innate immune response. In doing so, LMW HA acts as an endogenous danger signal alerting the immune system of a breach in tissue integrity. In this report, we demonstrate that LMW HA activates the innate immune response via TLR-2 in a MyD88-, IL-1R-associated kinase-, TNFR-associated factor-6-, protein kinase Cζ-, and NF-κB-dependent pathway. Furthermore, we show that intact high m.w. HA can inhibit TLR-2 signaling. Finally, we demonstrate that LMW HA can act as an adjuvant promoting Ag-specific T cell responses in vivo in wild-type but not TLR-2null mice.