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Therapeutic strategies targeting the LPS signaling and cytokines
Pathophysiology 16 (2009) 291–296
Review
Therapeutic strategies targeting the LPS signaling and cytokines Hua-Dong Wang, Da-Xiang Lu ∗ , Ren-Bin Qi Department of Pathophysiology, School of Medicine, Jinan University, Guangzhou 510632, China
Abstract Lipopolysaccharide (LPS) has been recognized as a major player in the pathogenesis of sepsis and neutralization of LPS or inhibition of its signal transduction mechanism is promising new treatment strategy in preclinical experiments. However, these therapeutic approaches have been shown unsuccessful in clinical trials. LPS activates Toll-like receptor 4 (TLR4) and induces pro-inflammatory and anti-inflammatory responses, the altered innate and adaptive immune responses eventually lead to the immunosuppressive state. The future therapeutic efforts in sepsis should focus on the immunosuppressive state. In this article, we will outline the current data on therapeutic strategies targeting LPS, TLR4 and single cytokine in sepsis and discuss the experimental and clinical evaluation of the immunomodulatory action of glycine and berberine. While we have demonstrated berberine in combination with yohimbine can modulate host immune responses in endotoxemia, it seems worthwhile to conduct clinical trials on the safe and efficacy of this new immunomodulatory therapy. © 2009 Elsevier Ireland Ltd. All rights reserved. Keywords: Lipopolysaccharide; Sepsis; Cytokines; Immunomodulation; Glycine; Berberine
Contents 1. 2. 3. 4.
5.
Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Targeting TLR4 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Targeting single cytokine . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Immunomodulatory therapies targeting cytokines . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4.1. Immunomodulatory effects of glycine . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4.2. Immunomodulatory effects of berberine . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Conclusion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Acknowledgments . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
1. Introduction Despite improvements in patient management, sepsis remains a leading cause of deaths worldwide in the intensive care unit, with an increasing incidence in recent years [1–3]. Lipopolysaccharide (LPS) or endotoxin is a principal component of the Gram-negative bacterial outer membrane, its role in sepsis pathogenesis was recognized in the 1960s [4]. Although endotoxin is usually associated with Gram-negative bacterial infection, intermediate ∗
Corresponding author. E-mail address: [email protected] (D.-X. Lu).
0928-4680/$ – see front matter © 2009 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.pathophys.2009.02.006
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level of endotoxin in Gram-positive bacteria infection was shown. Marshall et al. found that 57.2% of patients had either intermediate or high endotoxin levels on the first day of admission to the intensive care unit [5]. Many studies also demonstrated that treatment with some classes of beta-lactam antibiotics led to markedly increased endotoxin release in the systemic Gram-negative infection [6], and administration of LPS to experimental animals and humans results in a systemic inflammatory response, which partially mimics the feature of early sepsis [7,8]. These data suggest that the potential of therapeutic intervention targeting LPS in sepsis is promising. Therefore, a series of new antilipopolysaccharide treatment strategies for sepsis have
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developed for more than 20 years, including antiendotoxin antibodies, lipid A (harboring the LPS biological activity) antagonists, polymixin B, extracorporeal endotoxin absorber, bactericidal/permeability-increasing protein, cathelicidins, limulus antilipopolysaccharide factor and lactoferrin. These new therapeutic agents have demonstrated efficacy in animal research, however, numerous attempts to neutralize LPS in clinical trials with septic patients have failed to be effective [9,10]. LPS is a potent activator of the innate immune system. In mammalian cells, there are three kinds of receptors for LPS, that is, CD14-MD2 (myeloid differentiation protein2)-TLR4 (Toll-like receptor 4), CD11/CD18 molecules and scavenger receptors for lipid molecules. It is demonstrated that TLR4 is an important receptor for LPS. Once LPS binds to the TLR4, TLR4 undergoes oligomerization and recruits specific signaling adaptors, such as myeloid differentiation primary response gene 88 (MyD88) and Toll-interleukin-1 receptor (TIR) domain-containing adaptor inducing IFN- (TRIF), to its cytoplasmic TIR domains, and then activate MyD88-dependent and -independent signaling pathways. This signaling cascade ultimately activates the transcription factor NF-B, mitogen-activated protein kinases (MAPKs) and interferon-response factors, and then leads to a progressive production of cytokines and other inflammatory mediators, including interleukin-1 (IL-1), IL-6, IL-12, tumor necrosis factor (TNF)-␣, interferon (IFN)-␥, IFN and nitric oxide [10,11]. At the same time, LPS also induces the production of anti-inflammatory mediators, such as IL10, IL-4, transforming growth factor  and IL-1 receptor antagonist (IL-1Ra). Balance between the pro-inflammatory and anti-inflammatory play an important role in controlling LPS-induced immune activity. LPS-provoked strong pro-inflammatory responses can result in circulatory failure, coagulation disorder, organ dysfunction and even death [12]. Apparently, the current findings on LPS signaling suggest that therapeutic approaches to target at LPS signaling molecules and cytokines might be very appealing. On the other hand, inappropriate anti-inflammatory response results in the immunosuppressive state, immunomodulatory approaches are also important in the treatment of sepsis. This review will outline the current data on the therapeutic strategies targeting LPS signaling and cytokines and their potential use in sepsis, and discuss the experimental and clinical evaluation of the imunomodulatory action of glycine and berberine in sepsis.
2. Targeting TLR4 TLR4 is the important signaling receptor for LPS in mammals. LPS activates TLR4 and in turn induces excessive cytokine production, which is harmful to the host and can even be fatal. The current studies on the TLR4 function have raised the possibility of developing new drug targets to treat sepsis. E5564 and TAK-242 are the most impor-
tant TLR4 antagonists developed so far, they can bind to TLR4-MD2 complex, the structure and function of which are described in detail in the review by Leon et al. [13]. Accumulating experimental evidence indicates E5564 can inhibit LPS-induced production of TNF-␣ and other cytokines in a dose-dependent manner in animal macrophages and human myeloid cells as well as animal or human whole blood. In TLR4/MD2-transfected cells, E5564 can block the activation of the transcription factor NF-B by LPS [14,17]. In vivo, E5564 inhibits the production of LPS-induced cytokines and blocks LPS or bacterial-induced lethality in primed animals [14], although effective dosing of E5564 in rats depends on the timing of treatment and the route of LPS challenge [16]. Furthermore, Lynn et al. found that E5564 dose-dependently ameliorated or blocked all of the effects of LPS in healthy volunteers with experimental endotoxemia as measured by elevated temperature, C-reactive protein levels, white blood cell count, and cytokine levels such as TNF-␣ and IL-6 [15]. Similar to the E5564, another TLR4 antagonist, TAK242, has also been demonstrated to suppress LPS-provoked inflammation in animal and human cells [13]. These data indicate that the intervention of TLR4 may be a potential therapeutic approach to treating the sepsis. However, blocking TLR may cause the inappropriate immune responses such as immunological tolerance [19], the risks and benefits of this intervention in clinical practice remain to be further investigated [14]. Recently, Bennett-Guerrero et al. demonstrated that the administration of E5564 was not associated with overt toxicity in cardiac surgical patients. E5564 did not appear to confer any clear benefit to elective cardiac surgical patients [18].
3. Targeting single cytokine TNF-␣, an important early mediator of the innate inflammatory response, plays a major role in the pathogenesis of the septic shock syndrome. TNF exerts a range of beneficial and injurious actions that may ultimately cause organ dysfunction and death [20]. Intraarterial infusion of recombinant human TNF-␣ in dogs reproduced systemic inflammatory response and mortality found in the human septic shock [21]. When patients with the sepsis syndrome have detectable levels of circulating TNF-␣, there is a positive correlation between mortality and increased circulating TNF-␣ levels in patients with Gram-positive and -negative sepsis [22]. Furthermore, pretreatment with antibody against TNF-␣ prevents multiple organ failure and lethality in animal models of endotoxemia and bacteremia [22]. These observations indicate that blocking the effects of TNF-␣ may be a potential new therapeutic approach for the treatment of sepsis. The biologic effects of TNF-a are mediated by two different receptors (TNFR), TNFR1 or the p55 receptor and TNFR2 or the p75 receptor, respectively. Until now, a large number of approaches to neutralizing TNF in preclinical models has been investigated, including TNF-␣ knockout
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mice, TNFR1 or TNFR2 knockout mice, antibody to TNF, soluble TNFR, recombinant TNF-binding protein 1, TNFR fusion proteins and TNF siRNA [23]. However, this research shows that the effects of blocking TNF-␣ are quite variable. It has been demonstrated that a massive release of TNF-␣ is harmful to the host, but TNF-␣ itself also supports the host antibacterial defense. For example, inhibition of TNF-␣ lead to decreased bacterial clearance and increased mortality in some animal models of infection [23]. Our group showed that rhynchophylline pretreatment increased markedly the survival rate of LPS-challenged mice, decreased the release of TNF-␣, but significantly increased mortality of mice subjected to cecal ligation and puncture [24]. Recently, Lorente and Marshall analyzed the current data from 143 published reports in order to evaluate the consequences of blocking TNF in preclinical models of sepsis, and found that neutralization of TNF-␣ was beneficial in endotoxemia, or after systemic challenge with Gram-negative organisms, Staphylococcus aureus, or Group B streptococci, while neutralization was detrimental in infections caused by Streptococcus pneumoniae, or intracellular pathogens. The treatment was more efficacious when delivered before infectious challenge, and the beneficial effects of TNF blocking in systemic inflammation occur at the cost of impaired antimicrobial defenses [23]. In addition, other anti-cytokine strategies such as IL-1 receptor antagonist (IL-1Ra) were investigated in experimental or clinical trials. To date, there is no convincing evidence indicating that these anti-cytokine strategies are effective in the treatment for human sepsis [12,22].
4. Immunomodulatory therapies targeting cytokines 4.1. Immunomodulatory effects of glycine In recent years, evidence has accumulated in favor of the anti-inflammatory and immunomodulatory effects of glycine, some researchers focused on exploring the protective effects of glycine against endotoxemia. In 1996, our laboratory demonstrated that glycine inhibited the pyrogenicity of endotoxin markedly and might be a very promising endotoxin antagonist [25], at the same time, Ikejima et al. investigated the effects of a glycine-containing diet on mortality and liver injury provoked by intravenous injection of endotoxin in Sprague–Dawley rats. Fifty percent of the rats fed the control diet died within 24 h after LPS challenge, whereas feeding the rats with glycine totally prevented mortality and markedly reduced an LPS-induced increase in serum transaminase and TNF levels, hepatic necrosis, and lung injury [26]. These findings were further supported by results obtained in a mouse endotoxemia model. Mice were challenged with an intraperitoneal injection of d-Gal and LPS. The intervention group also received an intraperitoneal injection of glycine 24 h before and just after LPS challenge. Results showed that glycine significantly reduced the serum
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levels of liver enzymes and TNF-␣, the histologic necroinflammation score and the mortality rate in LPS-challenged mice. In addition, serum IL-10 levels in the glycine-treated mice were elevated. In vitro experiments, in lymphocytes isolated from either normal or glycine-pretreated mice, also demonstrated a significant inhibition of LPS-induced TNF-␣ secretion and an increase in IL-10 response after glycine treatment [27]. Similarly, in monocytes and a whole blood assay, glycine decreased LPS-induced TNF-␣ and IL-1 production and specific mRNA expression and increased IL-10 expression [28,29,33]. Furthermore, glycine significantly decreased CD11b/CD18 expression and E. coli phagocytosis, but did not modulate the expression of HLA-DR and CD64 on monocytes [29]. These results suggest that glycine can modulate pro-inflammatory and anti-inflammatory responses in an endotoxemia animal model. Except for the protection against LPS-induced liver injury, glycine has a beneficial effect on other organ function. In the isolated rat heart, Qi et al. found that glycine protected myocardial cells from the damage induced by endotoxin via inhibiting free radical production [30]. These data beg the question of how glycine impacts cytokine and other inflammatory mediator production during endotoxemia. An early suggestion was that the mechanism of glycine against endotoxin may be attributed to its positive amino terminal which may combine with the lipid A of endotoxin and interfere with its structural integrity [31]. However, this study did not consider the effect of pH on endotoxin. Our group and other studies have demonstrated that glycine not only inhibits the LPS-binding protein mRNA expression in the liver of LPS-challenged mice, but also TLR4 mRNA expression and DNA-binding activity of nuclear factor-kappa B in LPS-stimulated Kupffer cells [32,33], suggesting glycine affects the LPS-TLR4 signal pathway. In the central nervous system, glycine is an inhibitory neurotransmitter. It is well known that activation of the glycine-gated chloride channel is responsible for the mechanism of glycine action. Glycine binds to the glycine receptor (GlyR) in the postsynaptic neuronal membranes and leads to increases in chloride conductance. Recent studies provide pharmacological and molecular evidence for GlyR in nonneuronal cells, including splenic and alveolar macrophages, Kupffer cells, neutrophils, vascular endothelial cells, kidney cortical membrane fractions and renal proximal tubules and even cardiomyocytes [51]. In macrophages, Kupffer cells and neutrophils, GlyR has similar molecular and pharmacological properties to the channel expressed in the central nervous system. Glycine, in a dose-dependent manner, blunted the increase in intracellular calcium concentration and production of toxic radicals and cytokines induced by LPS, these effects of glycine were both strychnine sensitive and chloride dependent. Glycine also caused an influx of radiolabeled chloride [34–37]. These data strongly support that glycine exerts an immunomodulatory action via stimulating glycinegated chloride channel.
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Current evidence as mentioned above suggests that glycine has a potential to be used as an additional immunomodulatory agent in the early phase of sepsis. Yang et al. demonstrated that administration of glycine 1 h after cecal ligation and puncture attenuated liver injury, improved liver function and decreased mortality in experimental sepsis [38]. However, another study did not support the use of glycine in septic patients. In this study, Croner et al. investigated the effect of a glycine-enriched infusion on hepatic microcirculatory disturbances and mortality in a rat model of sepsis, although pretreatment with glycine reduced the hepatic inflammatory response and damage, there was no beneficial effect of intravenous administration of glycine after the onset of sepsis [39]. Thus, clinical use of glycine as an immunomodulatory agent in sepsis remains to be elucidated. 4.2. Immunomodulatory effects of berberine Berberine is an isoquinoline derivative alkaloid isolated from many Chinese medicinal herbs, and has many pharmacological actions, including inhibition of inflammation [43] and immunomodulation [45,46]. Fukuda et al. reported that berberine inhibited cyclooxygenase-2 expression via regulating activator protein 1 transcription factor [40], suggesting it may affect LPS-induced cytokine and inflammatory mediator production. This hypothesis is supported by the results from our laboratory. Neutral sulfate berberine can inhibit LPSinduced TNF-␣ production in primary cultured neonatal rat cardiomyocytes [41]. Moreover, pretreatment with 50 mg/kg neutral sulfate berberine once a day for 5 days significantly decreased the mortality and attenuated tissue injury in LPSchallenged mice. LPS induced a marked increase in plasma levels of TNF-␣, IFN-␥, IL-12, IL-10, and nitric oxide (NO). Pretreatment with berberine significantly reduced plasma TNF-␣, IFN-␥, and NO levels, but did not decrease IL-12 levels in mice exposed to LPS. On the contrary, pretreatment with berberine augmented IL-10 secretion provoked by LPS [42]. These findings indicate that pretreatment with berberine improves survival in endotoxemic mice by inhibiting pro-inflammatory mediator production and upregulating of IL-10 release. More recently, we further demonstrated that pretreatment with berberine decreased TNF production and mortality in mice challenged with LPS, which were enhanced by yohimbine, an alpha2-adrenoceptor antagonist. Berberine combined with yohimbine also improved survival in a mouse cecal ligation and puncture model [44]. In addition, pretreatment with berberine in combination with yohimbine reduced serum TNF-␣, and NO contents, increased serum IL-10, IL-6, IL-1 and IFN-␥ production, but did not decrease IL-12 p40 levels in LPS-treated mice, and significantly reduced mortality in a mouse endotoxemia model (unpublished data). Therefore, berberine, in combination with yohimbine modulates host immune responses during endotoxemia, may be a potential therapeutic agent in patients with sepsis.
5. Conclusion Today, sepsis remains a deadly disease. Although LPS has been recognized as a major player in the pathogenesis of sepsis and its signal pathway has been intensively investigated for many years, the treatment strategies targeting LPS, TLR4 and single cytokine, which are promising in preclinical studies, have failed to demonstrate efficacy in clinical practice. Some researchers believe that the failure of these approaches is partially attributed to these mediators being past their peak activity when patients are treated and late pro-inflammatory mediator, high-mobility group box-1 (HMGB1), may be an important therapeutic target. However, HMGB1 as a therapeutic target in septic patients also has more questions than answers [47]. In fact, most patients with sepsis will survive the early stage of the disease in the presence of modern intensive care medicine, and are subjected to an immunosuppressive state. Sepsis-induced immunosuppression renders the septic patients susceptible to secondary infections. Moreover, septic patients are not a homogenous group. Some patients may need suppression of inflammation, other patients may need to restore their ability to respond to pathogen-associated molecular patterns via enhancing their immune system [48,49]. Thus, it is very important to identify reliable markers of sepsis-induced immunoparalysis and develop better immunomodulatory strategies. Recently, immunostimulation using IFN-␥ or granulocyte-macrophage colony stimulating factor has been demonstrated to reverse the immunosuppression and show a beneficial effect in septic patients [48,50]. Our laboratory has demonstrated that berberine, in combination with yohimbine, modulates host immune responses during endotoxemia, especially inhibiting TNF-␣, and nitric oxide release and increasing serum IL-10, IL-6 and IFN-␥ production. It seems well worth it to conduct clinical trials on the safety and efficacy of this immunomodulatory therapy.
Acknowledgments This study was supported by the National Natural Science Foundation of China (grant no. 30670826) and grant (207140) from Ministry of Education of the People’s Republic of China.
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H.-D. Wang et al. / Pathophysiology 16 (2009) 291–296 survival in endotoxemic mice, Acta Pharmacol. Sin. 27 (2006) 1199–1205. C.L. Kuo, C.W. Chi, T.Y. Liu, The anti-inflammatory potential of berberine in vitro and in vivo, Cancer Lett. 203 (2004) 127–137. H.Q. Zhang, H.D. Wang, D.X. Lu, Berberine inhibits cytosolic phospholipase A2 and protects against LPS-induced lung injury and lethality independent of the alpha2-adrenergic receptor in mice, Shock 29 (2008) 617–622. B.Y. Kang, S.W. Chung, D. Cho, T.S. Kim, Involvement of p38 mitogen-activated protein kinase in the induction of interleukin-12 p40 production in mouse macrophages by berberine, a benzodioxoloquinolizine alkaloid, Biochem. Pharmacol. 63 (2002) 1901–1910. T.S. Kim, B.Y. Kang, D. Cho, S.H. Kim, Induction of interleukin-12 production in mouse macrophages by berberine, a benzodioxoloquinolizine alkaloid, deviates CD4+ T cells from a Th2 to a Th1 response, Immunology 109 (2003) 407–414.
[47] G.W. Waterer, High-mobility group box 1 (HMGB1) as a potential therapeutic target in sepsis-more questions than answers, Crit. Care Med. 35 (2007) 1205–1206. [48] J.C. Schefold, D. Hasper, H.D. Volk, P. Reinke, Sepsis: time has come to focus on the later stages, Med. Hypotheses 71 (2008) 203– 208. [49] T.S. Wang, J.C. Deng, Molecular and cellular aspects of sepsis-induced immunosuppression, J. Mol. Med. 86 (2008) 495–506. [50] J.J. Presneill, T. Harris, A.G. Stewart, J.F. Cade, J.W. Wilson, A randomized phase II trial of granulocyte-macrophage colony-stimulating factor therapy in severe sepsis with respiratory dysfunction, Am. J. Respir. Crit. Care Med. 166 (2002) 138–143. [51] R.B. Qi, J.Y. Zhang, D.X. Lu, H.D. Wang, H.H. Wang, C.J. Li, Glycine receptors contribute to cytoprotection of glycine in myocardial cells, Chin. Med. J. (Engl.) 120 (2007) 915–921.
Review
Therapeutic strategies targeting the LPS signaling and cytokines Hua-Dong Wang, Da-Xiang Lu ∗ , Ren-Bin Qi Department of Pathophysiology, School of Medicine, Jinan University, Guangzhou 510632, China
Abstract Lipopolysaccharide (LPS) has been recognized as a major player in the pathogenesis of sepsis and neutralization of LPS or inhibition of its signal transduction mechanism is promising new treatment strategy in preclinical experiments. However, these therapeutic approaches have been shown unsuccessful in clinical trials. LPS activates Toll-like receptor 4 (TLR4) and induces pro-inflammatory and anti-inflammatory responses, the altered innate and adaptive immune responses eventually lead to the immunosuppressive state. The future therapeutic efforts in sepsis should focus on the immunosuppressive state. In this article, we will outline the current data on therapeutic strategies targeting LPS, TLR4 and single cytokine in sepsis and discuss the experimental and clinical evaluation of the immunomodulatory action of glycine and berberine. While we have demonstrated berberine in combination with yohimbine can modulate host immune responses in endotoxemia, it seems worthwhile to conduct clinical trials on the safe and efficacy of this new immunomodulatory therapy. © 2009 Elsevier Ireland Ltd. All rights reserved. Keywords: Lipopolysaccharide; Sepsis; Cytokines; Immunomodulation; Glycine; Berberine
Contents 1. 2. 3. 4.
5.
Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Targeting TLR4 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Targeting single cytokine . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Immunomodulatory therapies targeting cytokines . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4.1. Immunomodulatory effects of glycine . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4.2. Immunomodulatory effects of berberine . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Conclusion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Acknowledgments . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
1. Introduction Despite improvements in patient management, sepsis remains a leading cause of deaths worldwide in the intensive care unit, with an increasing incidence in recent years [1–3]. Lipopolysaccharide (LPS) or endotoxin is a principal component of the Gram-negative bacterial outer membrane, its role in sepsis pathogenesis was recognized in the 1960s [4]. Although endotoxin is usually associated with Gram-negative bacterial infection, intermediate ∗
Corresponding author. E-mail address: [email protected] (D.-X. Lu).
0928-4680/$ – see front matter © 2009 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.pathophys.2009.02.006
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level of endotoxin in Gram-positive bacteria infection was shown. Marshall et al. found that 57.2% of patients had either intermediate or high endotoxin levels on the first day of admission to the intensive care unit [5]. Many studies also demonstrated that treatment with some classes of beta-lactam antibiotics led to markedly increased endotoxin release in the systemic Gram-negative infection [6], and administration of LPS to experimental animals and humans results in a systemic inflammatory response, which partially mimics the feature of early sepsis [7,8]. These data suggest that the potential of therapeutic intervention targeting LPS in sepsis is promising. Therefore, a series of new antilipopolysaccharide treatment strategies for sepsis have
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developed for more than 20 years, including antiendotoxin antibodies, lipid A (harboring the LPS biological activity) antagonists, polymixin B, extracorporeal endotoxin absorber, bactericidal/permeability-increasing protein, cathelicidins, limulus antilipopolysaccharide factor and lactoferrin. These new therapeutic agents have demonstrated efficacy in animal research, however, numerous attempts to neutralize LPS in clinical trials with septic patients have failed to be effective [9,10]. LPS is a potent activator of the innate immune system. In mammalian cells, there are three kinds of receptors for LPS, that is, CD14-MD2 (myeloid differentiation protein2)-TLR4 (Toll-like receptor 4), CD11/CD18 molecules and scavenger receptors for lipid molecules. It is demonstrated that TLR4 is an important receptor for LPS. Once LPS binds to the TLR4, TLR4 undergoes oligomerization and recruits specific signaling adaptors, such as myeloid differentiation primary response gene 88 (MyD88) and Toll-interleukin-1 receptor (TIR) domain-containing adaptor inducing IFN- (TRIF), to its cytoplasmic TIR domains, and then activate MyD88-dependent and -independent signaling pathways. This signaling cascade ultimately activates the transcription factor NF-B, mitogen-activated protein kinases (MAPKs) and interferon-response factors, and then leads to a progressive production of cytokines and other inflammatory mediators, including interleukin-1 (IL-1), IL-6, IL-12, tumor necrosis factor (TNF)-␣, interferon (IFN)-␥, IFN and nitric oxide [10,11]. At the same time, LPS also induces the production of anti-inflammatory mediators, such as IL10, IL-4, transforming growth factor  and IL-1 receptor antagonist (IL-1Ra). Balance between the pro-inflammatory and anti-inflammatory play an important role in controlling LPS-induced immune activity. LPS-provoked strong pro-inflammatory responses can result in circulatory failure, coagulation disorder, organ dysfunction and even death [12]. Apparently, the current findings on LPS signaling suggest that therapeutic approaches to target at LPS signaling molecules and cytokines might be very appealing. On the other hand, inappropriate anti-inflammatory response results in the immunosuppressive state, immunomodulatory approaches are also important in the treatment of sepsis. This review will outline the current data on the therapeutic strategies targeting LPS signaling and cytokines and their potential use in sepsis, and discuss the experimental and clinical evaluation of the imunomodulatory action of glycine and berberine in sepsis.
2. Targeting TLR4 TLR4 is the important signaling receptor for LPS in mammals. LPS activates TLR4 and in turn induces excessive cytokine production, which is harmful to the host and can even be fatal. The current studies on the TLR4 function have raised the possibility of developing new drug targets to treat sepsis. E5564 and TAK-242 are the most impor-
tant TLR4 antagonists developed so far, they can bind to TLR4-MD2 complex, the structure and function of which are described in detail in the review by Leon et al. [13]. Accumulating experimental evidence indicates E5564 can inhibit LPS-induced production of TNF-␣ and other cytokines in a dose-dependent manner in animal macrophages and human myeloid cells as well as animal or human whole blood. In TLR4/MD2-transfected cells, E5564 can block the activation of the transcription factor NF-B by LPS [14,17]. In vivo, E5564 inhibits the production of LPS-induced cytokines and blocks LPS or bacterial-induced lethality in primed animals [14], although effective dosing of E5564 in rats depends on the timing of treatment and the route of LPS challenge [16]. Furthermore, Lynn et al. found that E5564 dose-dependently ameliorated or blocked all of the effects of LPS in healthy volunteers with experimental endotoxemia as measured by elevated temperature, C-reactive protein levels, white blood cell count, and cytokine levels such as TNF-␣ and IL-6 [15]. Similar to the E5564, another TLR4 antagonist, TAK242, has also been demonstrated to suppress LPS-provoked inflammation in animal and human cells [13]. These data indicate that the intervention of TLR4 may be a potential therapeutic approach to treating the sepsis. However, blocking TLR may cause the inappropriate immune responses such as immunological tolerance [19], the risks and benefits of this intervention in clinical practice remain to be further investigated [14]. Recently, Bennett-Guerrero et al. demonstrated that the administration of E5564 was not associated with overt toxicity in cardiac surgical patients. E5564 did not appear to confer any clear benefit to elective cardiac surgical patients [18].
3. Targeting single cytokine TNF-␣, an important early mediator of the innate inflammatory response, plays a major role in the pathogenesis of the septic shock syndrome. TNF exerts a range of beneficial and injurious actions that may ultimately cause organ dysfunction and death [20]. Intraarterial infusion of recombinant human TNF-␣ in dogs reproduced systemic inflammatory response and mortality found in the human septic shock [21]. When patients with the sepsis syndrome have detectable levels of circulating TNF-␣, there is a positive correlation between mortality and increased circulating TNF-␣ levels in patients with Gram-positive and -negative sepsis [22]. Furthermore, pretreatment with antibody against TNF-␣ prevents multiple organ failure and lethality in animal models of endotoxemia and bacteremia [22]. These observations indicate that blocking the effects of TNF-␣ may be a potential new therapeutic approach for the treatment of sepsis. The biologic effects of TNF-a are mediated by two different receptors (TNFR), TNFR1 or the p55 receptor and TNFR2 or the p75 receptor, respectively. Until now, a large number of approaches to neutralizing TNF in preclinical models has been investigated, including TNF-␣ knockout
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mice, TNFR1 or TNFR2 knockout mice, antibody to TNF, soluble TNFR, recombinant TNF-binding protein 1, TNFR fusion proteins and TNF siRNA [23]. However, this research shows that the effects of blocking TNF-␣ are quite variable. It has been demonstrated that a massive release of TNF-␣ is harmful to the host, but TNF-␣ itself also supports the host antibacterial defense. For example, inhibition of TNF-␣ lead to decreased bacterial clearance and increased mortality in some animal models of infection [23]. Our group showed that rhynchophylline pretreatment increased markedly the survival rate of LPS-challenged mice, decreased the release of TNF-␣, but significantly increased mortality of mice subjected to cecal ligation and puncture [24]. Recently, Lorente and Marshall analyzed the current data from 143 published reports in order to evaluate the consequences of blocking TNF in preclinical models of sepsis, and found that neutralization of TNF-␣ was beneficial in endotoxemia, or after systemic challenge with Gram-negative organisms, Staphylococcus aureus, or Group B streptococci, while neutralization was detrimental in infections caused by Streptococcus pneumoniae, or intracellular pathogens. The treatment was more efficacious when delivered before infectious challenge, and the beneficial effects of TNF blocking in systemic inflammation occur at the cost of impaired antimicrobial defenses [23]. In addition, other anti-cytokine strategies such as IL-1 receptor antagonist (IL-1Ra) were investigated in experimental or clinical trials. To date, there is no convincing evidence indicating that these anti-cytokine strategies are effective in the treatment for human sepsis [12,22].
4. Immunomodulatory therapies targeting cytokines 4.1. Immunomodulatory effects of glycine In recent years, evidence has accumulated in favor of the anti-inflammatory and immunomodulatory effects of glycine, some researchers focused on exploring the protective effects of glycine against endotoxemia. In 1996, our laboratory demonstrated that glycine inhibited the pyrogenicity of endotoxin markedly and might be a very promising endotoxin antagonist [25], at the same time, Ikejima et al. investigated the effects of a glycine-containing diet on mortality and liver injury provoked by intravenous injection of endotoxin in Sprague–Dawley rats. Fifty percent of the rats fed the control diet died within 24 h after LPS challenge, whereas feeding the rats with glycine totally prevented mortality and markedly reduced an LPS-induced increase in serum transaminase and TNF levels, hepatic necrosis, and lung injury [26]. These findings were further supported by results obtained in a mouse endotoxemia model. Mice were challenged with an intraperitoneal injection of d-Gal and LPS. The intervention group also received an intraperitoneal injection of glycine 24 h before and just after LPS challenge. Results showed that glycine significantly reduced the serum
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levels of liver enzymes and TNF-␣, the histologic necroinflammation score and the mortality rate in LPS-challenged mice. In addition, serum IL-10 levels in the glycine-treated mice were elevated. In vitro experiments, in lymphocytes isolated from either normal or glycine-pretreated mice, also demonstrated a significant inhibition of LPS-induced TNF-␣ secretion and an increase in IL-10 response after glycine treatment [27]. Similarly, in monocytes and a whole blood assay, glycine decreased LPS-induced TNF-␣ and IL-1 production and specific mRNA expression and increased IL-10 expression [28,29,33]. Furthermore, glycine significantly decreased CD11b/CD18 expression and E. coli phagocytosis, but did not modulate the expression of HLA-DR and CD64 on monocytes [29]. These results suggest that glycine can modulate pro-inflammatory and anti-inflammatory responses in an endotoxemia animal model. Except for the protection against LPS-induced liver injury, glycine has a beneficial effect on other organ function. In the isolated rat heart, Qi et al. found that glycine protected myocardial cells from the damage induced by endotoxin via inhibiting free radical production [30]. These data beg the question of how glycine impacts cytokine and other inflammatory mediator production during endotoxemia. An early suggestion was that the mechanism of glycine against endotoxin may be attributed to its positive amino terminal which may combine with the lipid A of endotoxin and interfere with its structural integrity [31]. However, this study did not consider the effect of pH on endotoxin. Our group and other studies have demonstrated that glycine not only inhibits the LPS-binding protein mRNA expression in the liver of LPS-challenged mice, but also TLR4 mRNA expression and DNA-binding activity of nuclear factor-kappa B in LPS-stimulated Kupffer cells [32,33], suggesting glycine affects the LPS-TLR4 signal pathway. In the central nervous system, glycine is an inhibitory neurotransmitter. It is well known that activation of the glycine-gated chloride channel is responsible for the mechanism of glycine action. Glycine binds to the glycine receptor (GlyR) in the postsynaptic neuronal membranes and leads to increases in chloride conductance. Recent studies provide pharmacological and molecular evidence for GlyR in nonneuronal cells, including splenic and alveolar macrophages, Kupffer cells, neutrophils, vascular endothelial cells, kidney cortical membrane fractions and renal proximal tubules and even cardiomyocytes [51]. In macrophages, Kupffer cells and neutrophils, GlyR has similar molecular and pharmacological properties to the channel expressed in the central nervous system. Glycine, in a dose-dependent manner, blunted the increase in intracellular calcium concentration and production of toxic radicals and cytokines induced by LPS, these effects of glycine were both strychnine sensitive and chloride dependent. Glycine also caused an influx of radiolabeled chloride [34–37]. These data strongly support that glycine exerts an immunomodulatory action via stimulating glycinegated chloride channel.
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Current evidence as mentioned above suggests that glycine has a potential to be used as an additional immunomodulatory agent in the early phase of sepsis. Yang et al. demonstrated that administration of glycine 1 h after cecal ligation and puncture attenuated liver injury, improved liver function and decreased mortality in experimental sepsis [38]. However, another study did not support the use of glycine in septic patients. In this study, Croner et al. investigated the effect of a glycine-enriched infusion on hepatic microcirculatory disturbances and mortality in a rat model of sepsis, although pretreatment with glycine reduced the hepatic inflammatory response and damage, there was no beneficial effect of intravenous administration of glycine after the onset of sepsis [39]. Thus, clinical use of glycine as an immunomodulatory agent in sepsis remains to be elucidated. 4.2. Immunomodulatory effects of berberine Berberine is an isoquinoline derivative alkaloid isolated from many Chinese medicinal herbs, and has many pharmacological actions, including inhibition of inflammation [43] and immunomodulation [45,46]. Fukuda et al. reported that berberine inhibited cyclooxygenase-2 expression via regulating activator protein 1 transcription factor [40], suggesting it may affect LPS-induced cytokine and inflammatory mediator production. This hypothesis is supported by the results from our laboratory. Neutral sulfate berberine can inhibit LPSinduced TNF-␣ production in primary cultured neonatal rat cardiomyocytes [41]. Moreover, pretreatment with 50 mg/kg neutral sulfate berberine once a day for 5 days significantly decreased the mortality and attenuated tissue injury in LPSchallenged mice. LPS induced a marked increase in plasma levels of TNF-␣, IFN-␥, IL-12, IL-10, and nitric oxide (NO). Pretreatment with berberine significantly reduced plasma TNF-␣, IFN-␥, and NO levels, but did not decrease IL-12 levels in mice exposed to LPS. On the contrary, pretreatment with berberine augmented IL-10 secretion provoked by LPS [42]. These findings indicate that pretreatment with berberine improves survival in endotoxemic mice by inhibiting pro-inflammatory mediator production and upregulating of IL-10 release. More recently, we further demonstrated that pretreatment with berberine decreased TNF production and mortality in mice challenged with LPS, which were enhanced by yohimbine, an alpha2-adrenoceptor antagonist. Berberine combined with yohimbine also improved survival in a mouse cecal ligation and puncture model [44]. In addition, pretreatment with berberine in combination with yohimbine reduced serum TNF-␣, and NO contents, increased serum IL-10, IL-6, IL-1 and IFN-␥ production, but did not decrease IL-12 p40 levels in LPS-treated mice, and significantly reduced mortality in a mouse endotoxemia model (unpublished data). Therefore, berberine, in combination with yohimbine modulates host immune responses during endotoxemia, may be a potential therapeutic agent in patients with sepsis.
5. Conclusion Today, sepsis remains a deadly disease. Although LPS has been recognized as a major player in the pathogenesis of sepsis and its signal pathway has been intensively investigated for many years, the treatment strategies targeting LPS, TLR4 and single cytokine, which are promising in preclinical studies, have failed to demonstrate efficacy in clinical practice. Some researchers believe that the failure of these approaches is partially attributed to these mediators being past their peak activity when patients are treated and late pro-inflammatory mediator, high-mobility group box-1 (HMGB1), may be an important therapeutic target. However, HMGB1 as a therapeutic target in septic patients also has more questions than answers [47]. In fact, most patients with sepsis will survive the early stage of the disease in the presence of modern intensive care medicine, and are subjected to an immunosuppressive state. Sepsis-induced immunosuppression renders the septic patients susceptible to secondary infections. Moreover, septic patients are not a homogenous group. Some patients may need suppression of inflammation, other patients may need to restore their ability to respond to pathogen-associated molecular patterns via enhancing their immune system [48,49]. Thus, it is very important to identify reliable markers of sepsis-induced immunoparalysis and develop better immunomodulatory strategies. Recently, immunostimulation using IFN-␥ or granulocyte-macrophage colony stimulating factor has been demonstrated to reverse the immunosuppression and show a beneficial effect in septic patients [48,50]. Our laboratory has demonstrated that berberine, in combination with yohimbine, modulates host immune responses during endotoxemia, especially inhibiting TNF-␣, and nitric oxide release and increasing serum IL-10, IL-6 and IFN-␥ production. It seems well worth it to conduct clinical trials on the safety and efficacy of this immunomodulatory therapy.
Acknowledgments This study was supported by the National Natural Science Foundation of China (grant no. 30670826) and grant (207140) from Ministry of Education of the People’s Republic of China.
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