Splenectomy inactivates the cholinergic antiinflammatory pathway during lethal endotoxemia and polymicrobial sepsis.

The innate immune system protects against infection and tissue injury through the specialized organs of the reticuloendothelial system, including the lungs, liver, and spleen. The central nervous system regulates innate immune responses via the vagus nerve, a mechanism termed the cholinergic antiinflammatory pathway. Vagus nerve stimulation inhibits proinflammatory cytokine production by signaling through the alpha7 nicotinic acetylcholine receptor subunit. Previously, the functional relationship between the cholinergic antiinflammatory pathway and the reticuloendothelial system was unknown. Here we show that vagus nerve stimulation fails to inhibit tumor necrosis factor (TNF) production in splenectomized animals during lethal endotoxemia. Selective lesioning of the common celiac nerve abolishes TNF suppression by vagus nerve stimulation, suggesting that the cholinergic pathway is functionally hard wired to the spleen via this branch of the vagus nerve. Administration of nicotine, an alpha7 agonist that mimics vagus nerve stimulation, increases proinflammatory cytokine production and lethality from polymicrobial sepsis in splenectomized mice, indicating that the spleen is critical to the protective response of the cholinergic pathway. These results reveal a specific, physiological connection between the nervous and innate immune systems that may be exploited through either electrical vagus nerve stimulation or administration of alpha7 agonists to inhibit proinflammatory cytokine production during infection and tissue injury.

Splenectomy protects against sepsis lethality and reduces serum HMGB1 levels.

High mobility group box 1 (HMGB1) is a critical mediator of lethal sepsis. Previously, we showed that apoptotic cells can activate macrophages to release HMGB1. During sepsis, apoptosis occurs primarily in lymphoid organs, including the spleen and thymus. Currently, it is unclear whether this accelerated lymphoid organ apoptosis contributes to systemic release of HMGB1 in sepsis. In this study, we report that splenectomy significantly reduces systemic HMGB1 release and improves survival in mice with polymicrobial sepsis. Treatment with a broad-spectrum caspase inhibitor reduces systemic lymphocyte apoptosis, suppresses circulating HMGB1 concentrations, and improves survival during polymicrobial sepsis, but fails to protect septic mice following splenectomy. These findings indicate that apoptosis in the spleen is essential to the pathogenesis of HMGB1-mediated sepsis lethality.

Spermine protects mice against lethal sepsis partly by attenuating surrogate inflammatory markers.

The pathogenesis of sepsis is partly attributable to dysregulated inflammatory response mediated by pathogen-associated molecular patterns (PAMPs) (for example, endotoxin) and damage-associated molecular patterns (DAMPs) (for example, high-mobility group box 1 [HMGB1]). An endogenous ubiquitous polyamine, spermine, inhibits endotoxin-induced cytokine release in vitro, but its capacities to attenuate sepsis- and HMGB1-induced inflammatory responses was previously unknown. We thus tested the hypothesis that spermine protects mice against lethal sepsis by attenuating sepsis-induced local and systemic inflammatory responses. Intraperitoneal (i.p.) administration of spermine (10 mg/kg, twice daily, for 3 d) conferred a significant protection against lethal sepsis. The protective effects were associated with a significant reduction in peritoneal and serum levels of several surrogate markers of sepsis (for example, Interleukin-6 [IL-6], keratinocyte-derived chemokine [KC], monocytes chemoattractant protein-1 [MCP-1], macrophage inflammatory protein-2 [MIP-2], tissue inhibitor of metalloproteinase-1 [TIMP-1], soluble tumor necrosis factor-alpha receptor I [sTNFRI], and soluble tumor necrosis factor-alpha receptor II [sTNFRII]) during a late stage of sepsis. In vitro, spermine effectively inhibited HMGB1-induced release of the above surrogate markers in peritoneal macrophages. Thus, spermine confers protection against lethal sepsis partly by attenuating sepsis- and HMGB1-induced inflammatory responses.

A major ingredient of green tea rescues mice from lethal sepsis partly by inhibiting HMGB1.

BACKGROUND: The pathogenesis of sepsis is mediated in part by bacterial endotoxin, which stimulates macrophages/monocytes to sequentially release early (e.g., TNF, IL-1, and IFN-gamma) and late (e.g., HMGB1) pro-inflammatory cytokines. Our recent discovery of HMGB1 as a late mediator of lethal sepsis has prompted investigation for development of new experimental therapeutics. We previously reported that green tea brewed from the leaves of the plant Camellia sinensis is effective in inhibiting endotoxin-induced HMGB1 release. METHODS AND FINDINGS: Here we demonstrate that its major component, (-)-epigallocatechin-3-gallate (EGCG), but not catechin or ethyl gallate, dose-dependently abrogated HMGB1 release in macrophage/monocyte cultures, even when given 2-6 hours post LPS stimulation. Intraperitoneal administration of EGCG protected mice against lethal endotoxemia, and rescued mice from lethal sepsis even when the first dose was given 24 hours after cecal ligation and puncture. The therapeutic effects were partly attributable to: 1) attenuation of systemic accumulation of proinflammatory mediator (e.g., HMGB1) and surrogate marker (e.g., IL-6 and KC) of lethal sepsis; and 2) suppression of HMGB1-mediated inflammatory responses by preventing clustering of exogenous HMGB1 on macrophage cell surface. CONCLUSIONS: Taken together, these data suggest a novel mechanism by which the major green tea component, EGCG, protects against lethal endotoxemia and sepsis.

A cardiovascular drug rescues mice from lethal sepsis by selectively attenuating a late-acting proinflammatory mediator, high mobility group box 1.

The pathogenesis of sepsis is mediated in part by bacterial endotoxin, which stimulates macrophages/monocytes to sequentially release early (e.g., TNF, IL-1, and IFN-gamma) and late (e.g., high mobility group box 1 (HMGB1) protein) proinflammatory cytokines. The recent discovery of HMGB1 as a late mediator of lethal sepsis has prompted investigation for development of new experimental therapeutics. We found that many steroidal drugs (such as dexamethasone and cortisone) and nonsteroidal anti-inflammatory drugs (such as aspirin, ibuprofen, and indomethacin) failed to influence endotoxin-induced HMGB1 release even at superpharmacological concentrations (up to 10-25 microM). However, several steroid-like pigments (tanshinone I, tanshinone IIA, and cryptotanshinone) of a popular Chinese herb, Danshen (Salvia miltiorrhiza), dose dependently attenuated endotoxin-induced HMGB1 release in macrophage/monocyte cultures. A water-soluble tanshinone IIA sodium sulfonate derivative (TSNIIA-SS), which has been widely used as a Chinese medicine for patients with cardiovascular disorders, selectively abrogated endotoxin-induced HMGB1 cytoplasmic translocation and release in a glucocorticoid receptor-independent manner. Administration of TSNIIA-SS significantly protected mice against lethal endotoxemia and rescued mice from lethal sepsis even when the first dose was given 24 h after the onset of sepsis. The therapeutic effects were partly attributable to attenuation of systemic accumulation of HMGB1 (but not TNF and NO) and improvement of cardiovascular physiologic parameters (e.g., decrease in total peripheral vascular resistance and increase in cardiac stroke volume) in septic animals. Taken together, these data re-enforce the pathogenic role of HMGB1 in lethal sepsis, and support a therapeutic potential for TSNIIA-SS in the treatment of human sepsis.

Transcutaneous vagus nerve stimulation reduces serum high mobility group box 1 levels and improves survival in murine sepsis.

OBJECTIVE: Electrical vagus nerve stimulation inhibits proinflammatory cytokine production and prevents shock during lethal systemic inflammation through an alpha7 nicotinic acetylcholine receptor (alpha7nAChR)-dependent pathway to the spleen, termed the cholinergic anti-inflammatory pathway. Pharmacologic alpha7nAChR agonists inhibit production of the critical proinflammatory mediator high mobility group box 1 (HMGB1) and rescue mice from lethal polymicrobial sepsis. Here we developed a method of transcutaneous mechanical vagus nerve stimulation and then investigated whether this therapy can protect mice against sepsis lethality. DESIGN: Prospective, randomized study. SETTING: Institute-based research laboratory. SUBJECTS: Male BALB/c mice. INTERVENTIONS: Mice received lipopolysaccharide to induce lethal endotoxemia or underwent cecal ligation and puncture to induce polymicrobial sepsis. Mice were then randomized to receive electrical, transcutaneous, or sham vagus nerve stimulation and were followed for survival or euthanized at predetermined time points for cytokine analysis. MEASUREMENTS AND MAIN RESULTS: Transcutaneous vagus nerve stimulation dose-dependently reduced systemic tumor necrosis factor levels during lethal endotoxemia. Treatment with transcutaneous vagus nerve stimulation inhibited HMGB1 levels and improved survival in mice with polymicrobial sepsis, even when administered 24 hrs after the onset of disease. CONCLUSIONS: Transcutaneous vagus nerve stimulation is an efficacious treatment for mice with lethal endotoxemia or polymicrobial sepsis.

The aqueous extract of a popular herbal nutrient supplement, Angelica sinensis, protects mice against lethal endotoxemia and sepsis.

Despite recent advances in antibiotic therapy and intensive care, sepsis remains a widespread problem in critically ill patients. The high mortality from sepsis is in part mediated by bacterial endotoxin, which stimulates macrophages/monocytes to sequentially release early (e.g., tumor necrosis factor, interleukin-1, and interferon-gamma) and late [e.g., high mobility group box 1 protein (HMGB1)] proinflammatory cytokines. Our discovery of HMGB1 as a late mediator of lethal systemic inflammation has initiated a new field of investigation for the development of experimental therapeutics. A popular Chinese herb, Angelica sinensis (also known as Dang Gui or Dong Quai) has been used traditionally for treating women with gynecological disorders (such as dysmenorrheal and hot flashes). Here we examined the effect of Angelica sinensis extract on endotoxin-induced HMGB1 release in vitro, and explored its therapeutic potential in animal models of lethal endotoxemia and sepsis [induced by cecal ligation and puncture (CLP)] in vivo. We demonstrated that a low-molecular-weight (<10 kDa) fraction of A. sinensis extract significantly attenuated endotoxin-induced HMGB1 release in part through interfering with its cytoplasmic translocation in macrophage cultures. Prophylactic administration of an aqueous extract of A. sinensis significantly attenuated systemic HMGB1 accumulation in vivo, and conferred a dose-dependent protection against lethal endotoxemia. Furthermore, delayed administration of A. sinensis extract beginning 24 h after CLP attenuated systemic HMGB1 accumulation, and significantly rescued mice from lethal sepsis. Taken together, these data suggest that A. sinensis contains water-soluble components that exert protective effects against lethal endotoxemia and experimental sepsis in part by attenuating systemic accumulation of a late proinflammatory cytokine, HMGB1.