内毒素耐受的机制研究

内毒素是革兰阴性菌细胞壁的成分,能够激发机体的免疫反应。当细菌释放大量的内毒素到血液,即可引起内毒素血症,内毒素血症可以伴随多种疾病出现,引起致死性感染性休克,循环功能衰竭,其病死率极高。内毒素耐受是指机体接受小剂量内毒素刺激后对后续内毒素刺激的反应性降低,表现为促炎因子释放减少而抗炎因子释放增加,机体发热,缺氧,低血压,休克的症状减轻。内毒素耐受的发生机制极其复杂,受机体内多种因素的调节,但目前尚无明确的结论。近年来,有关其机制的研究有许多报道,其中,对内毒素耐受的信号机制的研究最为广泛,大量的研究表明内毒素的主要受体,细胞内的信号蛋白,负调控因子以及转录因子可能在内毒素耐受的发生过程中起重要作用。也有报道表明免疫细胞的凋亡,染色体修饰和基因重排以及小RNA的参与可能诱导内毒素耐受的发生。本文从细胞、分子水平对内毒素耐受的发生机制进行综述,拟对炎症性疾病如内毒素血症的预防和治疗提供理论依据。     

Immune Responsive Gene 1 (IRG1) Promotes Endotoxin Tolerance by Increasing A20 Expression in Macrophages through Reactive Oxygen Species

Sepsis-associated immunosuppression (SAIS) is regarded as one of main causes for the death of septic patients at the late stage because of the decreased innate immunity with a more opportunistic infection. LPS-tolerized macrophages, which are re-challenged by LPS after prior exposure to LPS, are regarded as the common model of hypo-responsiveness for SAIS. However, the molecular mechanisms of endotoxin tolerance and SAIS remain to be fully elucidated. In addition, negative regulation of the Toll-like receptor (TLR)-triggered innate inflammatory response needs further investigation. Here we show that expression of immune responsive gene 1 (IRG1) was highly up-regulated in the peripheral blood mononuclear cells of septic patients and in LPS-tolerized mouse macrophages. IRG1 significantly suppressed TLR-triggered production of proinflammatory cytokines TNF-α, IL-6, and IFN-β in LPS-tolerized macrophages, with the elevated expression of reactive oxygen species (ROS) and A20. Moreover, ROS enhanced A20 expression by increasing the H3K4me3 modification of histone on the A20 promoter domain, and supplement of the ROS abrogated the IRG1 knockdown function in breaking endotoxin tolerance by increasing A20 expression. Our results demonstrate that inducible IRG1 promotes endotoxin tolerance by increasing A20 expression through ROS, indicating a new molecular mechanism regulating hypoinflammation of sepsis and endotoxin tolerance.     

Recent advances in endotoxin tolerance

Endotoxin tolerance is defined as a reduced capacity of a cell to respond endotoxin (lipopolysaccharide, LPS) challenge after an initial encounter with endotoxin in advance. The body becomes tolerant to subsequent challenge with a lethal dose of endotoxin and cytokines release and cell/tissue damage induced by inflammatory reaction are significantly reduced in the state of endotoxin tolerance. The main characteristics of endotoxin tolerance are downregulation of inflammatory mediators such as tumor necrosis factor α (TNF-α), interleukin-1β (IL-1β), and C-X-C motif chemokine 10 (CXCL10) and upregulation of anti-inflammatory cytokines such as IL-10 and transforming growth factor β (TGF-β). Therefore, endotoxin tolerance is often regarded as the regulatory mechanism of the host against excessive inflammation. Endotoxin tolerance is a complex pathophysiological process and involved in multiple cellular signal pathways, receptor alterations, and biological molecules. However, the exact mechanism remains elusive up to date. To better understand the underlying cellular and molecular mechanisms of endotoxin tolerance, it is crucial to investigate the comprehensive cellular signal pathways, signaling proteins, cell surface molecules, proinflammatory and anti-inflammatory cytokines, and other mediators. Endotoxin tolerance plays an important role in reducing the mortality of sepsis, endotoxin shock, and other endotoxin-related diseases. Recent reports indicated that endotoxin tolerance is also related to other diseases such as cystic fibrosis, acute coronary syndrome, liver ischemia-reperfusion injury, and cancer. The aim of this review is to discuss the recent advances in endotoxin tolerance mainly based on the cellular and molecular mechanisms by outline the current state of the knowledge of the involvement of the toll-like receptor 4 (TLR4) signaling pathways, negative regulate factor, microRNAs, apoptosis, chromatin modification, and gene reprogramming of immune cells in endotoxin tolerance. We hope to provide a new idea and scientific basis for the rational treatment of endotoxin-related diseases such as endotoxemia, sepsis, and endotoxin shock clinically.     

IL-12 suppression during experimental endotoxin tolerance: dendritic cell loss and macrophage hyporesponsiveness

Endotoxin tolerance, the transient, secondary down-regulation of a subset of endotoxin-driven responses after exposure to bacterial products, is thought to be an adaptive response providing protection from pathological hyperactivation of the innate immune system during bacterial infection. However, although protecting from the development of sepsis, endotoxin tolerance also can lead to fatal blunting of immunological responses to subsequent infections in survivors of septic shock. Despite considerable experimental effort aimed at characterizing the molecular mechanisms responsible for a variety of endotoxin tolerance-related phenomena, no consensus has been achieved yet. IL-12 is a macrophage- and dendritic cell (DC)-derived cytokine that plays a key role in pathological responses to endotoxin as well as in the induction of protective responses to pathogens. It recently has been shown that IL-12 production is suppressed in endotoxin tolerance, providing a likely partial mechanism for the increased risk of secondary infections in sepsis survivors. We examined the development of IL-12 suppression during endotoxin tolerance in mice. Decreased IL-12 production in vivo is clearly multifactorial, involving both loss of CD11c(high) DCs as well as alterations in the responsiveness of macrophages and remaining splenic DCs. We find no demonstrable mechanistic role for B or T lymphocytes, the soluble mediators IL-10, TNF-alpha, IFN-alphabeta, or nitric oxide, or the NF-kappaB family members p50, p52, or RelB.     

IRAK-M is a negative regulator of Toll-like receptor signaling

Toll-like receptors (TLRs) detect microorganisms and protect multicellular organisms from infection. TLRs transduce their signals through MyD88 and the serine/threonine kinase IRAK. The IRAK family consists of two active kinases, IRAK and IRAK-4, and two inactive kinases, IRAK-2 and IRAK-M. IRAK-M expression is restricted to monocytes/macrophages, whereas other IRAKs are ubiquitous. We show here that IRAK-M is induced upon TLR stimulation and negatively regulates TLR signaling. IRAK-M prevented dissociation of IRAK and IRAK-4 from MyD88 and formation of IRAK-TRAF6 complexes. IRAK-M(-/-) cells exhibited increased cytokine production upon TLR/IL-1 stimulation and bacterial challenge, and IRAK-M(-/-) mice showed increased inflammatory responses to bacterial infection. Endotoxin tolerance, a protection mechanism against endotoxin shock, was significantly reduced in IRAK-M(-/-) cells. Thus, IRAK-M regulates TLR signaling and innate immune homeostasis.     

IL-1 receptor-associated kinase modulates host responsiveness to endotoxin

Endotoxin triggers many of the inflammatory, hemodynamic, and hematological derangements of Gram-negative septic shock. Recent genetic studies in mice have identified the Toll-like receptor 4 as the transmembrane endotoxin signal transducer. The IL-1 intracellular signaling pathway has been implicated in Toll-like receptor signal transduction. LPS-induced activation of the IL-1 receptor-associated kinase (IRAK), and the influence of IRAK on intracellular signaling and cellular responses to endotoxin has not been explored in relevant innate immune cells. We demonstrate that LPS activates IRAK in murine macrophages. IRAK-deficient macrophages, in contrast, are resistant to LPS. Deletion of IRAK disrupts several endotoxin-triggered signaling cascades. Furthermore, macrophages lacking IRAK exhibit impaired LPS-stimulated TNF-alpha production, and IRAK-deficient mice withstand the lethal effects of LPS. These findings, coupled with the critical role for IRAK in IL-1 and IL-18 signal transduction, demonstrate the importance of this kinase and the IL-1/Toll signaling cassette in sensing and responding to Gram-negative infection.     

Regulation of endotoxin-induced proinflammatory activation in human coronary artery cells: expression of functional membrane-bound CD14 by human coronary artery smooth muscle cells

Low-level endotoxemia has been identified as a powerful risk factor for atherosclerosis. However, little is known about the mechanisms that regulate endotoxin responsiveness in vascular cells. We conducted experiments to compare the relative responses of human coronary artery endothelial cells (HCAEC) and smooth muscle cells (HCASMC) to very low levels of endotoxin, and to elucidate the mechanisms that regulate endotoxin responsiveness in vascular cells. Endotoxin (10-fold higher in magnitude at >10-fold lower threshold concentrations (10-30 pg/ml) compared with HCAEC. This remarkable sensitivity of HCASMC to very low endotoxin concentrations, comparable to that found in circulating monocytes, was not due to differential expression of TLR4, which was detected in HCAEC, HCASMC, and intact coronary arteries. Surprisingly, membrane-bound CD14 was detected in seven different lines of HCASMC, conferring responsiveness to endotoxin and to lipoteichoic acid, a product of Gram-positive bacteria, in these cells. These results suggest that the low levels of endotoxin associated with increased risk for atherosclerosis are sufficient to produce inflammatory responses in coronary artery cells. Because CD14 recognizes a diverse array of inflammatory mediators and functions as a pattern recognition molecule in inflammatory cells, expression of membrane-bound CD14 in HCASMC implies a potentially broader role for these cells in transducing innate immune responses in the vasculature.     

Multiple mechanisms of reduced major histocompatibility complex class II expression in endotoxin tolerance

Patients after polytrauma, burns, or septic shock frequently develop a life-threatening immunodeficiency. This state is associated with specific functional alterations of monocytic cells. We previously proposed endotoxin tolerance, the monocyte state after acute response to lipopolysaccharide, as a respective model system. One major feature in both the clinical situation and the in vitro model is the dramatic down-regulation of monocyte major histocompatibility complex (MHC) class II surface expression, which is associated with impaired antigen presentation capacity. This study focused on the mechanisms behind reduced MHC class II expression in endotoxin tolerance. Endotoxin priming provoked a decrease of monocyte intracellular MHC class II. It also led to a reduced expression of the chaperonic invariant chain and to an inhibited synthesis of the major lysosomal enzyme for final cleavage of the invariant chain going along with a relative accumulation of p10. The expression of HLA-DM necessary for loading MHC class II with antigenic peptide was also decreased. Additionally, reduced export of MHC class II alphabeta complexes to the cell surface was observed. The down-regulation of HLA-DR, invariant chain, and HLA-DM was regulated at the mRNA level and may be the consequence of reduced class II transactivator expression observed in this study. The simultaneous interference at different regulatory levels may explain the uniquely strong and long lasting MHC class II down-modulating effect of endotoxin priming compared with transforming growth factor-beta and interleukin-10. These results not only contribute to a better understanding of experimental endotoxin tolerance but may also give rise to new therapeutics for temporary immunodeficiency and, conversely, for MHC class II-dependent diseases such as autoimmunity and transplant rejection.     

The role of tuberous sclerosis complex 1 in regulating innate immunity

The mechanisms that control TLR-induced responses, including endotoxin tolerance, have been not well understood. The tuberous sclerosis complex 1 (TSC1) is a tumor suppressor that inhibits the mammalian target of rapamycin (mTOR). We show in this study that deficiency of TSC1 results in enhanced activation of not only mTOR complex 1 (mTORC1), but also JNK1/2, following LPS stimulation in macrophages. TSC1-deficient macrophages produce elevated proinflammatory cytokines and NO in response to multiple TLR ligands. Such enhanced TLR-induced responses can be inhibited by reducing mTORC1 and JNK1/2 activities with chemical inhibitors or small hairpin RNA, suggesting that TSC1 negatively controls TLR responses through both mTORC1 and JNK1/2. The impact of TSC1 deficiency appeared not limited to TLRs, as NOD- and RIG-I/MDA-5-induced innate responses were also altered in TSC1-deficient macrophages. Furthermore, TSC1 deficiency appears to cause impaired induction of endotoxin tolerance in vitro and in vivo, which is correlated with increased JNK1/2 activation and can be reversed by JNK1/2 inhibition. Our results reveal a critical role of TSC1 in regulating innate immunity by negative control of mTORC1 and JNK1/2 activation.     

Flt3 ligand treatment reverses endotoxin tolerance-related immunoparalysis

Endotoxin tolerance, the secondary blunting of a subset of microbial product-driven responses, is presumed to provide protection from pathological hyperactivation of the innate immune system during infection. However, endotoxin tolerance can itself be harmful. A significant percentage of sepsis survivors exhibit the phenotype of systemic endotoxin tolerance, a state termed immunoparalysis. Similar immune hyporeactivity, associated with an elevated risk of succumbing to bacterial superinfection, is also seen in the aftermath of major trauma, surgery, and burns. We recently demonstrated that in vivo endotoxin tolerance in murine models involves dendritic cell loss as well as alterations in the responsiveness of macrophages and remaining dendritic cells. Furthermore, the kinetics of recovery from immunoparalysis-associated inhibition of proinflammatory and immunoregulatory cytokine production directly parallels the kinetics of dendritic cell repopulation in these models. Given this, we examined whether recovery from immunoparalysis could be accelerated therapeutically with flt3 ligand, a growth factor that stimulates the differentiation and mobilization of dendritic cells. Notably, administration of flt3 ligand rapidly reverses immunoparalysis in vivo, accelerating and amplifying repopulation of tissues with proinflammatory and immunoregulatory cytokine-producing dendritic cells.     

Regulation of cyclooxygenase 2 expression by agonists of PPAR nuclear receptors in the model of endotoxin tolerance in astrocytes

Endotoxin tolerance (ET) represents a state of an altered immune response induced by multiple stimulations of a cell, a tissue, or an organism with lipopolysaccharide. Characteristics of ET include downregulation of induction of proinflammatory genes (TNFα, IL6, and others) and enhancement of induction of antiinflammatory genes (IL10, TGFβ). ET generally has protective functions; nevertheless, it might result in a state of innate immune deficiency and cause negative outcomes. A current issue is the search for the mechanisms controlling the level of inflammation in the course of endotoxin tolerance. In this work, we investigated the change in cyclooxygenase 2 (Cox2) expression in the model of endotoxin tolerance in astrocytes and analyzed the possibility of regulating this process applying nuclear receptor PPAR agonists. Our results indicate that: 1) endotoxin tolerance can be induced in astrocytes and results in TNFα and Cox2 mRNA induction decrease upon secondary stimulation; 2) tolerance is revealed on the level of TNFα release and Cox2 protein expression; 3) PPAR agonists GW7647, L-165041, and rosiglitazone control Cox2 mRNA expression levels under conditions of endotoxin tolerance. In particular, rosiglitazone (a PPARγ agonist) induces Cox2 mRNA expression, while GW7647 (a PPARα agonist) and L-165041 (a PPARβ agonist) suppress the expression. Our results demonstrate that Cox2 can be upand downregulated during endotoxin tolerance in astrocytes, and PPAR agonists might be effective for controlling this target under conditions of multiple proinflammatory stimulations of brain tissues with endotoxin.     

The Early Induction of Suppressor of Cytokine Signaling 1 and the Downregulation of Toll-like Receptors 7 and 9 Induce Tolerance in Costimulated Macrophages

Toll-like receptors (TLR) 7 and 9 transduce a cellular signal through the MyD88-dependent pathway and induce the production of inflammatory mediators against microbial nucleotide components. The repeated stimulation of TLR4 leads to endotoxin tolerance, but the molecular mechanisms of tolerance induced through the costimulation of individual TLR has not yet been established, although endosomal TLRs share signaling pathways with TLR4. In the present study, mouse macrophages were simultaneously stimulated with the TLR7 agonist, gardiquimod (GDQ), and the TLR9 agonist, CpG ODN 1826, to examine the mechanism and effector functions of macrophage tolerance. Compared with individual stimulation, the costimulation of both TLRs reduced the secretion of TNF-α and IL-6 through the delayed activation of the NF-κB pathway; notably, IL-10 remained unchanged in costimulated macrophages. This tolerance reflected the early induction of suppressor of cytokine signaling-1 (SOCS-1), according to the detection of elevated TNF-α secretion and restored NF-κB signaling in response to the siRNA-mediated abrogation of SOCS-1 signaling. In addition, the restimulation of each TLRs using the same ligand significantly reduced the expression of both TLRs in endosomes. These findings revealed that the costimulation of TLR7 and TLR9 induced macrophage tolerance via SOCS-1, and the restimulation of each receptor or both TLR7 and TLR9 downregulated TLR expression through a negative feedback mechanisms that protects the host from excessive inflammatory responses. Moreover, the insufficient and impaired immune response in chronic viral infection might also reflect the repeated and simultaneous stimulation of those endosomal TLRs.     

Vascular cells contribute to atherosclerosis by cytokine- and innate-immunity-related inflammatory mechanisms

Cardiovascular diseases are the human diseases with the highest death rate and atherosclerosis is one of the major underlying causes of cardiovascular diseases. Inflammatory and innate immune mechanisms, employing monocytes, innate receptors, innate cytokines, or chemokines are suggested to be involved in atherogenesis. Among the inflammatory pathways the cytokines are central players. Plasma levels of cytokines and related proteins, such as CRP, have been investigated in cardiovascular patients, tissue mRNA expression was analyzed and correlations to vascular diseases established. Consistent with these findings the generation of cytokine-deficient animals has provided direct evidence for a role of cytokines in atherosclerosis. In vitro cell culture experiments further support the suggestion that cytokines and other innate mechanisms contribute to atherogenesis. Among the initiation pathways of atherogenesis are innate mechanisms, such as toll-like-receptors (TLRs), including the endotoxin receptor TLR4. On the other hand, innate cytokines, such as IL-1 or TNF, or even autoimmune triggers may activate the cells. Cytokines potently activate multiple functions relevant to maintain or spoil homeostasis within the vessel wall. Vascular cells, not least smooth muscle cells, can actively contribute to the inflammatory cytokine-dependent network in the blood vessel wall by: (i) production of cytokines; (ii) response to these potent cell activators; and (iii) cytokine-mediated interaction with invading cells, such as monocytes, T-cells, or mast cells. Activation of these pathways results in accumulation of cells and increased LDL- and ECM-deposition which may serve as an 'immunovascular memory' resulting in an ever-growing response to subsequent invasions. Thus, vascular cells may potently contribute to the inflammatory pathways involved in development and acceleration of atherosclerosis.     

Characterization of interleukin-1 receptor-associated kinase in normal and endotoxin-tolerant cells

Interleukin-1 receptor-associated kinase (IRAK), a signal transducer for interleukin-1, has also been suggested to participate in the Toll-like receptor-mediated innate immune response to bacterial endotoxin lipopolysaccharide (LPS). Using the human promonocytic THP-1 cell line, we demonstrated that the endogenous IRAK is quickly activated in response to bacterial LPS stimulation, as measured by its in vitro kinase activity toward myelin basic protein. LPS also triggers the association of IRAK with MyD88, the adaptor protein linking IRAK to the Toll-like receptor/interleukin-1beta receptor intracellular domain. Macrophage cells with prolonged LPS treatment become tolerant to additional dose of LPS and no longer express inflammatory cytokines. Endotoxin tolerance is a common phenomenon observed in blood from sepsis patients. We observed for the first time that the quantity of IRAK is greatly reduced in LPS-tolerant THP-1 cells, and its activity no longer responds to further LPS challenge. In addition, IRAK does not associate with MyD88 in the tolerant cells. Furthermore, application of AG126, a putative tyrosine kinase inhibitor, can substantially alleviate the LPS-induced cytokine gene expression and can also decrease IRAK level and activity. Our study indicates that IRAK is essential for LPS-mediated signaling and that cells may develop endotoxin tolerance by down-regulating IRAK.     

The biology of endotoxin

Endotoxin (lipopolysaccharide, LPS) is the major component of the outer leaflet of Gram-negative bacteria and has profound immunostimulatory and inflammatory capacity. The septic shock syndrome caused by endotoxin still has an unacceptably high mortality rate and, owing to increasing numbers of resistant strains, remains an ongoing threat throughout the world. However, the past years have provided new insights especially into the receptors of the innate immune system that are involved into the recognition of LPS and the initial signal transduction pathways that are engaged after the primary recognition on the cell surface. The knowledge about the molecular basis for the responses to endotoxin may eventually lead to the development of new drugs to fight the fatal effects of bacterial infections.     

Arachidonic acid turnover in peritoneal macrophages is altered in endotoxin-tolerant rats

Peritoneal macrophages from endotoxin-tolerant rats have been found to exhibit depressed metabolism of arachidonic acid (AA) to prostaglandins and thromboxane in response to endotoxin. The effect of endotoxin tolerance on AA turnover in peritoneal macrophages was investigated by measuring [14C]AA incorporation and release from membrane phospholipids. Endotoxin tolerance did not affect the amount of [14C]AA incorporated into macrophages (30 min-24 h). However, the temporal incorporation of [14C]AA into individual phospholipid pools (15 min-24 h) was altered. In endotoxin-tolerant macrophages, [14C]AA incorporation into phosphatidylcholine (PC) (2, 4, 24 h) and phosphatidylethanolamine (PE) (8 h) was increased, while the incorporation into phosphatidylserine (PS) (2-24 h) was reduced (P less than 0.005) compared to control macrophages. There was no change in [14C]AA incorporation into phosphatidylinositol (PI). Following 2 or 24 h of incorporation of [14C]AA, macrophages were incubated (3 h) with endotoxin (50 micrograms/ml) or A23187 (1 microM), and [14C]AA release was measured. Endotoxin-tolerant macrophages released decreased (P less than 0.05) amounts of [14C]AA in response to both endotoxin and the calcium ionophore A23187 compared to controls. Control macrophages in response to endotoxin released [14C]AA from PC, PI and PE. In contrast, tolerant cells released [14C]AA only from PC (P less than 0.05). A23187 released [14C]AA from all four pools in the control cells, but only from PC and PE in the tolerant cells. These data demonstrate that endotoxin tolerance alters the uptake and release of AA from specific macrophage phospholipid pools. These results suggest that changes in AA turnover and/or storage are associated with endotoxin tolerance.     

Innate immune mechanisms of colitis and colitis-associated colorectal cancer

The innate immune system provides first-line defences in response to invading microorganisms and endogenous danger signals by triggering robust inflammatory and antimicrobial responses. However, innate immune sensing of commensal microorganisms in the intestinal tract does not lead to chronic intestinal inflammation in healthy individuals, reflecting the intricacy of the regulatory mechanisms that tame the inflammatory response in the gut. Recent findings suggest that innate immune responses to commensal microorganisms, although once considered to be harmful, are necessary for intestinal homeostasis and immune tolerance. This Review discusses recent findings that identify a crucial role for innate immune effector molecules in protection against colitis and colitis-associated colorectal cancer and the therapeutic implications that ensue.     

Inhalation of (1-->3)-beta-D-glucan causes airway eosinophilia

BACKGROUND: Moulds are present in a variety of environments and aerosols of fungal spores are generated when mouldy materials are handled. Molds contain (1-->3)-beta-D-glucan, a polyglucose which is present in the cell wall of fungi, certain bacteria and plants. AIM: This study was undertaken to investigate the cellular inflammatory response in the lung after inhalation of (1-->3)-beta-D-glucan and bacterial endotoxin. METHODS: Guinea pigs were exposed daily to an aerosol of pure (1-->3)-beta-D-glucan and pure endotoxin for five weeks. Lung lavage and lung interstitial cell preparations were done and the inflammatory cells counted. Histological sections were prepared from the trachea. RESULTS: There was an increase in eosinophil numbers in lung lavage, lung interstitium, and the airway epithelium of animals exposed to (1-->3)-beta-D-glucan. In animals simultaneously exposed to endotoxin, there was no increase in eosinophils. In the lung interstitium, (1-->3)-beta-D-glucan exposure caused an increase in lymphocytes, which was not found after endotoxin exposure. Endotoxin exposure caused an increase in neutrophils and macrophages in lung lavage, which was not found after (1-->3)-beta-D-glucan exposure. CONCLUSIONS: The results support previous findings that (1-->3)-beta-D-glucan causes a different response in the airways as compared to endotoxin. Endotoxin modulated the increase in eosinophils caused by (1-->3)-beta-D-glucan exposure, suggesting a complex interaction between the microbial cell wall components.