Endotoxin recognition: in fish or not in fish?

The interaction between pathogens and their multicellular hosts is initiated by activation of pathogen recognition receptors (PRRs). These receptors, that include most notably members of the toll-like receptor (TLR) family, recognize specific pathogen-associated molecular patterns (PAMPs). TLR4 is a central part of the receptor complex that is involved in the activation of the immune system by lipopolysaccharide (LPS) through the specific recognition of its endotoxic moiety (Lipid A). This is a critical event that is essential for the immune response to Gram-negative bacteria as well as the etiology of endotoxic shock. Interestingly, compared to mammals, fish are resistant to endotoxic shock. This in vivo resistance concurs with in vitro studies demonstrating significantly lowered sensitivity of fish leukocytes to LPS activation. Further, our in vitro analyses demonstrate that in trout mononuclear phagocytes, LPS fails to induce antiviral genes, an event that occurs downstream of TLR4 and is required for the development of endotoxic shock. Finally, an in silico approach that includes mining of different piscine genomic and EST databases, reveals the presence in fish of all of the major TLR signaling elements except for the molecules specifically involved in TLR4-mediated endotoxin recognition and signaling in mammals. Collectively, our analysis questions the existence of TLR4-mediated cellular responses to LPS in fish. We further speculate that other receptors, in particular beta-2 integrins, may play a primary role in the activation of piscine leukocytes by LPS.     

鱼类Toll样受体及其信号传导的研究进展

鱼类是脊椎动物中的一个重要类群, 在其生存与进化的过程中, 免疫系统担负着保护鱼类免受病原感染的重任, 其中Toll样受体家族等介导的先天性免疫是鱼类抗病免疫的第一道防线, 并在连接先天性免疫与获得性免疫反应中起着桥梁作用. 虽然从无脊椎动物到高等脊椎动物, Toll样受体家族内多数成员在蛋白质结构与功能上都较为保守, 但是鱼类作为最低等的脊椎动物, 在其进化过程中又形成了一些特有Toll样受体分子, 其剪接类型也更丰富; 鱼类Toll样受体家族介导的免疫识别、免疫信号传导、激活和调控方式与高等脊椎动物也不尽相同. 文章主要综述了鱼类Toll样受体的结构、种类、功能、多样性、免疫信号传导及其调控特点, 为深入了解鱼类的免疫反应奠定基础.       

无脊椎动物先天免疫模式识别受体研究进展

免疫系统的基本功能是“自己”与“非己”识别.对入侵物的识别是免疫防御的起始,最终引发效应物反应系统,包括吞噬作用、包被作用、激活蛋白酶级联反应和黑化作用以及诱导抗菌肽的合成等,从而清除或消灭入侵物.研究证明,这种“非己”识别是因为存在某些特异性的、可溶的或与细胞膜结合的模式识别受体,可以识别或结合微生物表面保守的、而在宿主中又不存在的病原相关分子模式.模式识别受体通过对病原相关分子的识别启动先天免疫防御.近年来这方面的研究进展很快,已经在无脊椎动物中确定了多种模式识别受体,包括肽聚糖识别蛋白、含硫酯键蛋白、革兰氏阴性菌结合蛋白、清除受体、C型凝集素、硫依赖型凝集素、Toll样受体和血素等,并对其性质和功能进行了研究.     

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.     

Endotoxin, toll-like receptor 4, and the afferent limb of innate immunity

Positional cloning work and subsequent biochemical analyses have revealed that Toll-like receptor 4 (Tlr4) transduces the lipopolysaccharide (LPS) signal, alerting the host to infection by Gram-negative bacteria. Moreover, it appears that the LPS sensing pathway is a solitary one: disruption of Tlr4 causes complete unresponsiveness to LPS. As several Tlr family members exist in vertebrates, it appears likely that the innate immune system defends the host by recognizing a small number of structurally conserved molecules that distinguish the microbial world from tissues of the host.     

Innate immune responses to LPS in mouse lung are suppressed and reversed by neutralization of GM-CSF via repression of TLR-4

The innate immune inflammatory response to lipopolysaccharide (LPS, an endotoxin) is essential for lung host defense against infection by gram-negative bacteria but is also implicated in the pathogenesis of some lung diseases. Studies on genetically altered mice implicate granulocyte-macrophage colony-stimulating factor (GM-CSF) in lung responses to LPS; however, the physiological effects of GM-CSF neutralization are poorly characterized. We performed detailed kinetic and dose-response analyses of the lung inflammation response to LPS in the presence of the specific GM-CSF-neutralizing antibody 22E9. LPS instilled into the lungs of BALB/c mice induced a dose-dependent inflammation comprised of intense neutrophilia, macrophage infiltration and proliferation, TNF-alpha and matrix metalloproteinase release, and macrophage inflammatory protein-2 induction. The neutralization of anti-GM-CSF in a dose-dependent fashion suppressed these inflammatory indexes by 85% when given before or after LPS or after repeat LPS challenges. Here we report for the first time that the physiological expression of Toll-like receptor-4 in lung is reduced by anti-GM-CSF. We observed that lower Toll-like receptor-4 expression correlated with a similar decline in peak TNF- levels in response to endotoxin. Consequently, sustained expression of key inflammatory mediators over 24 h was reduced. These data expand the understanding of the contribution of GM-CSF to innate immune responses in lung and suggest that blocking GM-CSF might benefit some lung diseases where LPS has been implicated in etiology.     

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.     

Overexpression of Toll-like receptor 4 amplifies the host response to lipopolysaccharide and provides a survival advantage in transgenic mice

Toll-like receptors are transmembrane proteins that are involved in the innate immune recognition of microbial constituents. Among them, Toll-like receptor 4 (Tlr4) is a crucial signal transducer for LPS, the major component of Gram-negative bacteria outer cell membrane. The contribution of Tlr4 to the host response to LPS and to infection with virulent Salmonella typhimurium was studied in four transgenic (Tg) strains including three overexpressing Tlr4. There was a good correlation between the level of Tlr4 mRNA expression and the sensitivity to LPS both in vitro and in vivo: Tg mice possessing the highest number of Tlr4 copies respond the most to LPS. Overexpression of Tlr4 by itself appears to have a survival advantage in Tg mice early during infection: animals possessing more than two copies of the gene survived longer and in a greater percentage to Salmonella infection. The beneficial effect of Tlr4 overexpression is greatly enhanced when the mice present a wild-type allele at natural resistance-associated macrophage protein 1, another critical innate immune gene involved in resistance to infection with SALMONELLA: Tlr4 and natural resistance-associated macrophage protein 1 exhibit functional epistatic interaction to improve the capacity of the host to control bacterial replication. However, this early improvement in disease resistance is not conducted later during infection, because mice overexpressing Tlr4 developed an excessive inflammatory response detrimental to the host.     

Innate immune defense of the sponge Suberites domuncula against bacteria involves a MyD88-dependent signaling pathway. Induction of a perforin-like molecule

Sponges (phylum Porifera) are the phylogenetically oldest metazoa; as filter feeders, they are abundantly exposed to marine microorganisms. Here we present data indicating that the demosponge Suberites domuncula is provided with a recognition system for gram-negative bacteria. The lipopolysaccharide (LPS)-interacting protein was identified as a receptor on the sponge cell surface, which recognizes the bacterial endotoxin LPS. The cDNA was isolated, and the protein (Mr 49,937) was expressed. During binding to LPS, the protein dimerizes and interacts with MyD88, which was also identified and cloned. The sponge MyD88 (Mr 28,441) is composed of two protein interaction domains, a Toll/interleukin-1 receptor domain (found in MyD88 and in Toll-like receptors) and a death domain (present in MyD88 and interleukin-1 receptor-associated kinase). Northern blot experiments and in situ hybridization studies showed that after LPS treatment, the level of the LPS-interacting protein remains unchanged, whereas MyD88 is strongly up-regulated. A perforin-like molecule (Mr 74,171), the macrophage-expressed protein, was identified as an executing molecule of this pathway. This gene is highly expressed after LPS treatment, especially at the surfaces of the animals. The recombinant protein possesses biological activity and eliminates gram-negative bacteria; it is inactive against gram-positive bacteria. These data indicate that S. domuncula is provided with an innate immune system against gram-negative bacteria; the ligand LPS (a pathogen-associated molecular pattern) is recognized by the pattern recognition receptor (LPS-interacting protein), which interacts with MyD88. A signal transduction is established, which results in an elevated expression of MyD88 as well as of the macrophage-expressed protein as an executing protein.     

Mass Spectrometry-based Structural Analysis and Systems Immunoproteomics Strategies for Deciphering the Host Response to Endotoxin

One cause of sepsis is systemic maladaptive immune response of the host to bacteria and specifically, to Gram-negative bacterial outer-membrane glycolipid lipopolysaccharide (LPS). On the host myeloid cell surface, proinflammatory LPS activates the innate immune system via Toll-like receptor-4/myeloid differentiation factor-2 complex. Intracellularly, LPS is also sensed by the noncanonical inflammasome through caspase-11 in mice and 4/5 in humans. The minimal functional determinant for innate immune activation is the membrane anchor of LPS called lipid A. Even subtle modifications to the lipid A scaffold can enable, diminish, or abolish immune activation. Bacteria are known to modify their LPS structure during environmental stress and infection of hosts to alter cellular immune phenotypes. In this review, we describe how mass spectrometry-based structural analysis of endotoxin helped uncover major determinations of molecular pathogenesis. Through characterization of LPS modifications, we now better understand resistance to antibiotics and cationic antimicrobial peptides, as well as how the environment impacts overall endotoxin structure. In addition, mass spectrometry-based systems immunoproteomics approaches can assist in elucidating the immune response against LPS. Many regulatory proteins have been characterized through proteomics and global/targeted analysis of protein modifications, enabling the discovery and characterization of novel endotoxin-mediated protein translational modifications.     

Synthesis of a dimeric monosaccharide lipid A mimic and its synergistic effect on the immunostimulatory activity of lipopolysaccharide

Bacterial endotoxin lipopolysaccharide (LPS) is a potent immune stimulant, with the recognition of LPS and its active principal lipid A mediated by the Toll-like receptor 4 (TLR4)/MD-2 receptor complex. Due to the broad downstream implications of TLR4-mediated signalling, TLR4 ligands show great potential for immunotherapeutic manipulations. In this paper a dimeric monosaccharide lipid A mimic (3) has been designed as a potential TLR4 ligand. The chemical synthesis and the preliminary biological studies are described. Compound 3 shows a significant synergistic effect on LPS-induced ICAM-1 expression in human monocytic THP-1 cells.     

Innate recognition of lipopolysaccharide by Toll-like receptor 4-MD-2

Toll-like receptors (TLRs) are pathogen recognition molecules that activate the immune system as part of the innate immune response. Microbial recognition by TLRs plays a crucial role in the host immune system's decision to respond or not to a particular microbial infection. Lipopolysaccharide (LPS), a membrane glycolipid of Gram-negative bacteria, exhibits strong immunostimulating activity among TLR ligands and has been studied in great detail. Recent studies have shown that cell surface TLR4-MD-2 physically interacts with LPS and triggers the release of an LPS signal, revealing a host-pathogen interaction mediated by TLR.     

Immune control of phagosomal bacteria by p47 GTPases

Sequestered from the action of complement, antibody and lytic peptides, phagosomal pathogens pose a unique problem for the innate immune system both in terms of detection and disposal. An immunologically induced 47-kDa (p47) GTPase family recruited to nascent phagosomes (PGs) has provided new insights into how vertebrates deal with facultative bacteria occupying a vacuolar niche. Research over the past 2 years in particular has identified several molecular determinants that underlie the membrane trafficking functions of LRG-47 and other p47 GTPases as part of a PG remodeling program. When coupled to signals issuing from pathogen-specific Toll-like receptors, the p47 proteins may constitute a novel sensory system enlisted by mammals, birds and fish to decode the language of immune recognition against this particular class of infectious agents.     

N-acetylcysteine improves in vitro the function of macrophages from mice with endotoxin-induced oxidative stress

Reactive oxygen species (ROS) and proinflammatory cytokines produced by immune cells cause the oxidative stress involved in septic shock induced by endotoxin. This oxidative stress can be controlled to a certain degree by antioxidants, which is specially important for a type of immune cell, i.e. the phagocyte, that uses ROS to kill microorganisms and needs antioxidants in order to support its functions. In a previous study we have observed changes in several functions of peritoneal macrophages from BALB/c mice with lethal endotoxic shock caused by intraperitoneal injection of Escherichia coli lipopolysaccharide (LPS) (100 mg/kg), which were associated with a high production of superoxide anion. N-acetylcysteine (NAC) is a thiolic antioxidant that improves the immune response, and we have observed that when administered intraperitoneally (150 mg/kg) at 30 min after LPS injection it counteracts the effects of LPS on macrophages and lymphocytes. In the present work, we have studied the in vitro effect of several concentrations of NAC (0.001, 0.01, 0.1, 1 and 2.5 mM) on the following functions: adherence to substrate, chemotaxis, ingestion of particles, ROS production and the release of tumor necrosis factor (TNFalpha) of peritoneal macrophages from BALB/c mice at 2, 4,12 and 24 h after LPS injection. The results show that the administration of NAC (especially at 0.1 mM) decreases raised adherence, ingestion, ROS production and TNFalpha levels in macrophages from animals injected with LPS, bringing these functions to values near those of control animals. These effects which seem to be linked to a modulation of NF-kappaB, suggest that the improvement of immune functions observed in previous work after injection of NAC to animals with endotoxic shock could be due to a direct action of this thiol antioxidant on immune cells.     

Ascorbic acid and N-acetylcysteine improve in vitro the function of lymphocytes from mice with endotoxin-induced oxidative stress.

Oxidative stress associated with reactive oxygen species (ROS) and cytokines produced by immune cells, which is involved in septic shock caused by endotoxin, can be controlled to a certain degree by antioxidants with free radical scavenging action. N-acetylcysteine (NAC) and ascorbic acid (AA) are ROS scavengers that improve the immune response, and modulate macrophage function in mice with endotoxin-caused oxidative stress. Therefore, we have investigated the in vitro effects of these antioxidants on the functions of lymphocytes from BALB/c mice with lethal endotoxic shock caused by intraperitoneal injection of E. coli lipopolysaccharide (LPS) (100 mg/kg). Adherence to tissues and chemotaxis (the earliest two functions of lymphocytes in the immune response), as well as ROS levels and TNF alpha production were determined in the presence or absence of NAC or AA (0.001, 0.01, 0.1, 1 and 2.5 mM) in lymphocytes from peritoneum, axillary nodes, spleen and thymus obtained at several times (2, 4, 12 and 24 hours) after LPS injection. Endotoxic shock decreases the chemotaxis of lymphocytes from all the above localizations and increases their adherence, TNF alpha and ROS production. These changes in lymphocyte function were counteracted by NAC and AA, bringing these functions to values near those of control animals. Our data suggest that lymphocytes are important targets of endotoxins contributing to oxidative stress by septic shock, and that antioxidants can preserve the function of lymphocytes, preventing the homeostatic disturbances caused by endotoxin.     

Toxoplasma gondii interferes with lipopolysaccharide-induced mitogen-activated protein kinase activation by mechanisms distinct from endotoxin tolerance

We show in this study that Toxoplasma gondii infection induces rapid activation of p38 mitogen-activated protein kinase (MAPK), extracellular signal-regulated kinase 1/2, and stress-activated protein kinase/c-Jun N-terminal kinase MAPK, followed promptly by their deactivation in mouse macrophages. Nevertheless, when infected cells were subsequently subjected to LPS triggering, MAPK activation was severely defective, in particular in the case of p38 MAPK, which is required for LPS-triggered TNF-alpha and IL-12 production. Similar effects occurred during endotoxin tolerance, but the phenomena were distinct. LPS pretriggering failed to activate the major p38 MAPK kinase, MAPK kinase 3/6. Toxoplasma infection, in contrast, resulted in sustained activation of this kinase. Furthermore, endotoxin pre-exposure blocked IkappaBalpha degradation upon subsequent LPS triggering, but this was not the case for Toxoplasma preinfection. Endotoxin-mediated down-regulation of the LPS receptor, Toll-like receptor 4, has been suggested as one possible mechanism contributing to tolerance, and we found in this study that LPS down-modulated Toll-like receptor 4 expression. In contrast, Toxoplasma infection induced up-regulation of this pattern recognition receptor. Our results show that T. gondii blocks LPS-triggered cytokine production in part through MAPK inactivation, and that this occurs through pathways distinct from endotoxin-induced tolerance.     

LPS, TLR4 and infectious disease diversity

Innate immune receptors recognize microorganism-specific motifs. One such receptor-ligand complex is formed between the mammalian Toll-like receptor 4 (TLR4)-MD2-CD14 complex and bacterial lipopolysaccharide (LPS). Recent research indicates that there is significant phylogenetic and individual diversity in TLR4-mediated responses. In addition, the diversity of LPS structures and the differential recognition of these structures by TLR4 have been associated with several bacterial diseases. This review will examine the hypothesis that the variability of bacterial ligands such as LPS and their innate immune receptors is an important factor in determining the outcome of infectious disease.     

Photoperiod alters macrophage responsiveness, but not expression of Toll-like receptors in Siberian hamsters

Defense against pathogens is a critical component of comparative and ecological biology. However, pathogen recognition, a process necessary for the facilitation of systemic immune response, remains understudied in a comparative context, yet could provide insight into how the immune system interacts with pathogens in variable environments. We examined pathogen recognition by macrophages in relation to an ecological variable, day length, in Siberian hamsters (Phodopus sungorus). Because peritoneal macrophages collected in long, summer-like day lengths are more responsive to a lipopolysaccharide (LPS) challenge compared to macrophages collected during short, winter-like day lengths, we hypothesized that these functional differences are mediated by variation in pathogen recognition, which occurs through binding to Toll-like receptors (TLRs). We predicted that expression of TLR2 and 4, the receptors that bind and respond specifically to LPS, would be upregulated in long vs. short days, and that expression of these receptors would reflect macrophage responsiveness to LPS. Macrophages collected during long days were again more responsive to LPS challenge compared to short-day macrophages; however, TLR2 and TLR4 expression was similar between photoperiods and were unrelated to our measure of macrophage responsiveness suggesting that other downstream intracellular mechanisms may be responsible for photoperiod-based variation in macrophage responsiveness in this species.     

Ascorbic acid and N-acetylcysteine improve in vitro the function of lymphocytes from mice with endotoxin-induced oxidative stress

Oxidative stress associated with reactive oxygen species (ROS) and cytokines produced by immune cells, which is involved in septic shock caused by endotoxin, can be controlled to a certain degree by antioxidants with free radical scavenging action. N-acetylcysteine (NAC) and ascorbic acid (AA) are ROS scavengers that improve the immune response, and modulate macrophage function in mice with endotoxin-caused oxidative stress. Therefore, we have investigated the in vitro effects of these antioxidants on the functions of lymphocytes from BALB/c mice with lethal endotoxic shock caused by intraperitoneal injection of E. coli lipopolysaccharide (LPS) (100 mg/kg). Adherence to tissues and chemotaxis (the earliest two functions of lymphocytes in the immune response), as well as ROS levels and TNFα production were determined in the presence or absence of NAC or AA (0.001, 0.01, 0.1, 1 and 2.5 mM) in lymphocytes from peritoneum, axillary nodes, spleen and thymus obtained at several times (2, 4, 12 and 24 hours) after LPS injection. Endotoxic shock decreases the chemotaxis of lymphocytes from all the above localizations and increases their adherence, TNFα and ROS production. These changes in lymphocyte function were counteracted by NAC and AA, bringing these functions to values near those of control animals. Our data suggest that lymphocytes are important targets of endotoxins contributing to oxidative stress by septic shock, and that antioxidants can preserve the function of lymphocytes, preventing the homeostatic disturbances caused by endotoxin.