More tea for septic patients? - Green tea may reduce endotoxin-induced release of high mobility group box 1 (HMGB1) and other pro-inflammatory cytokines.

Summary

Despite recent advances in antibiotic therapy and intensive care, sepsis remains widespread problems in critically ill patients. The high mortality of sepsis is in part mediated by bacterial endotoxin, which stimulates macrophages/monocytes to sequentially release early (e.g., TNF, IL-1, and IFN-γ) and late (e.g., HMGB1) pro-inflammatory cytokines. In light of our recent discovery of HMGB1 as a late mediator of lethal systemic inflammation, and the observation that green tea (Camellia sinensis) dose-dependently attenuated bacterial endotoxin-induced HMGB1 release, we propose that regular tea intake might decrease the incidence of and mortality rates from lethal endotoxemia and sepsis.

Introduction

In 1347, a mysterious plague spread across the entire continent of Europe, taking the lives of 20 million people, wiping out approximately one third of the European population in the mere three years [1]. More than six hundred years have passed, yet our world continues to be plagued by infectious diseases. Being touted as modern day’s equivalent of the bubonic plague [1], sepsis refers to an overwhelming systemic inflammatory response to infection, and is defined by signs of organ dysfunction that include abnormalities in body temperature, heart rate, respiratory rate, and leukocyte counts. Despite recent advances in antibiotic therapy and intensive care, sepsis is still the most common cause of death in the intensive care units, claiming approximately 225,000 victims annually in the U.S. alone. Initiated by an infection, the pathogenesis of sepsis is attributable, at least in part, to dys-regulated systemic inflammatory responses characterized by excessive accumulation of various proinflammatory cytokines [2–5].

In response to bacterial toxins (e.g., lipopolysaccharide, LPS), macrophages/monocytes release reactive nitrogen intermediates (e.g., nitric oxide) [6], and various proinflammatory cytokines such as tumor necrosis factor (TNF) [7], interleukin (IL)-1 [8], interferon (IFN)-γ [9], and macrophage migration inhibitory factor (MIF) [10], which individually, or in combination, contribute to the pathogenesis of lethal endotoxemia or sepsis. For instance, neutralizing antibodies against TNF, the first cytokine elaborated in inflammatory cascade, reduces lethality in an animal model of endotoxemic/bacteremic shock [7]. However, the early release of TNF makes it difficult to target therapeutically in a clinical setting [7], prompting the search for late proinflammatory cytokines that may offer a wider therapeutic window for the treatment of lethal systemic inflammatory diseases.

HMGB1 as a late mediator of lethal endotoxemia and sepsis

We have discovered that a ubiquitous protein, high mobility group box 1 (HMGB1), is released by activated macrophages/monocytes [11–13], and functions as a late mediator of lethal endotoxemia and sepsis [11;14–16]. First, circulating HMGB1 levels are elevated in a delayed fashion (after 16–32 hours) in endotoxemic and septic animals [11;14], and in patients with sepsis [11]. Second, administration of recombinant HMGB1 to mice recapitulates many clinical signs of sepsis, including fever [17], allodynia [18], derangement of intestinal barrier function [19], lung injury [20], and lethal multiple organ failure [11]. Third, administration of anti-HMGB1 antibodies or inhibitors (e.g., ethyl pyruvate, nicotine, or stearoyl lysophosphatidylcholine) significantly protects mice against LPS-induced acute lung injury [20;21], and lethal endotoxemia [1;11;15;22;23]. Notably, these anti-HMGB1 reagents are also capable of rescuing animals from lethal experimental sepsis even when the first doses are given 24 hours after onset of sepsis [14;15;24], confirming a pathogenic role of HMGB1 in lethal experimental sepsis. Therefore, agents proven clinically safe, and yet still capable of attenuating HMGB1 release may hold potential in the prevention and treatment of inflammatory diseases.

Tea and human health

Tea has been brewed from the leaves of the Camellia sinensis plant for almost fifty centuries. In Maintaining Health by Drinking Tea, Eisai, the “Father of Tea” in Japan, called tea a “miraculous medicine for the maintenance of health.” Today, tea is cultivated in 30 different countries around the world, and its daily consumption ( 120 ml) is second only to water [25–28]. Although health benefits have been attributed to tea consumption since the beginning of its history, scientific investigations of this beverage and its constituents has been underway for less than three decades.

Epidemiological surveys have suggested a close association between green tea consumption and human longevity [29]. Indeed, tea contains abundant polyphenols that may be protective against chronic illness such as cardiovascular disease and cancer [25;26;28]. These beneficial effects have been well demonstrated in animal studies, but have subsequently confirmed only in a limited number of human studies. Confounding factors are multiple, and may include the insufficiency in the dosage of tea consumed by humans (as opposed to the dosage required to demonstrate the beneficial effects in animal models), as well as other lifestyle-related factors in different populations.

In addition, a number of pre-clinical animal studies have suggested some anti-inflammatory activities of green tea in models of collagen-induced arthritis [30] and pulmonary inflammation [31]. For example, polyphenolic fraction of green tea has been shown to markedly reduce the expression of pro-inflammatory cytokines such TNF and IFN-γ in joints of animals with collagen-induced arthritis [30]. However, it was previously unknown if green tea can attenuate bacterial endotoxin-induced release of late mediators of lethal endotoxemia and sepsis.

More tea for septic patients?

Accordingly, we examined the effects of natural Lipton (R) Green Tea (without artificial colors or additives) on bacterial endotoxin-induced HMGB1 release. As indicated in Figure 1, the Lipton green tea dose-dependently inhibited endotoxin-induced release of nitric oxide and HMGB1 (Fig. 1). At a dose as low as 10 μl/ml (equivalent of 75 mls /person, assuming a total body weight of 75 kg, and blood volume of 7,500 mls), green tea almost completely abolished endotoxin-induced HMGB1 release. Even at concentrations that almost completely abrogated HMGB1 release, green tea did not exhibit any cytotoxicity to macrophage cultures, because cell viability, as assessed by trypan blue exclusion, was not reduced [⁹²%, for control; versus 91%, in the presence of LPS + tea (10 μl/ml)]. In light of the capacity of green tea to attenuate endotoxin-induced release of nitric oxide and HMGB1, as well as their pathogenic roles in lethal systemic inflammation [11;15;32–35], we propose that green tea might be beneficial for patients with systemic inflammation (such as endotoxemia and sepsis). Therefore, regular tea intake might provide an approach to decrease the incidence of and mortality from lethal endotoxemia and sepsis.

Figure 1. Green tea dose-dependently suppressed endotoxin-induced HMGB1 release.

Murine macrophage-like RAW 264.7 cells were obtained from the American Type Culture Collection (ATCC, Rockville, MD) and cultured in DMEM medium (Gibco BRL, Grand Island, NY) supplemented with 10% fetal bovine serum and 2 mM glutamine. At 80–90% confluence, RAW 264.7 cells were washed twice with, and subsequently cultured in, serum-free DMEM medium before stimulation with bacterial endotoxin (lipopolysaccharide, LPS, E. coli 0111:B4, Sigma-Aldrich) alone, or in the presence of the green tea at indicated concentrations. Natural Lipton (R) Green Tea (3.2 gram/bag, Lipton, Englewood Cliffs, NJ, USA) was extracted in 100 ml hot water (100º C) for 10 minutes, and the aqueous extract was sterilized by filtration (through 0.22 μm filter) before adding to cell cultures. At 16 hours after stimulation, levels of nitric oxide and HMGB1 in the culture medium were determined by Griess reaction and Western blot, respectively [12]. Shown in the bar graph of panel A are levels of nitric oxide (expressed as mean ± SD of three experiments). Student’s t-test was performed and a P < 0.05 was considered significant (*). Shown in the bar graph of panel B are relative HMGB1 levels expressed as the optical band intensity (arbitrary units, AU). Shown in the lower portion of Panel B is a representative Western blot.

This hypothesis can be tested in pre-clinical animal, and clinical human studies. For instance, if green tea protects animals against lethal endotoxemia and experimental sepsis, it will support the above hypothesis. Similarly, if ingestion of green tea reduces mortality rate of patients with lethal sepsis, it will further support tea’s protective effects against lethal endotoxemia and sepsis. However, caution should be excised while using high doses of tea for disease prevention, because ingestion of large amount of caffeine by patients with intestinal or stomach ailments (e.g., ulcer) or kidney problems may cause dehydration, which may worsen the existing problems. Therefore, further studies are needed to elucidate the biologic activities of tea and to determine the optimal amount of tea consumption for possible health-beneficial effects.