Complement system in zebrafish

Zebrafish is recently emerging as a model species for the study of immunology and human diseases. Complement system is the humoral backbone of the innate immune defense, and our knowledge as such in zebrafish has dramatically increased in the recent years. This review summarizes the current research progress of zebrafish complement system. The global searching for complement components in genome database, together with published data, has unveiled the existence of all the orthologues of mammalian complement components identified thus far, including the complement regulatory proteins and complement receptors, in zebrafish. Interestingly, zebrafish complement components also display some distinctive features, such as prominent levels of extrahepatic expression and isotypic diversity of the complement components. Future studies should focus on the following issues that would be of special importance for understanding the physiological role of complement components in zebrafish: conclusive identification of complement genes, especially those with isotypic diversity; analysis and elucidation of function and mechanism of complement components; modulation of innate and adaptive immune response by complement system; and unconventional roles of complement-triggered pathways.     

A common human TLR1 polymorphism regulates the innate immune response to lipopeptides

Toll-like receptors (TLR) are critical mediators of the immune response to pathogens and human polymorphisms in this gene family regulate inflammatory pathways and are associated with susceptibility to infection. Lipopeptides are present in a wide variety of microbes and stimulate immune responses through TLR1/2 or TLR2/6 heterodimers. It is not currently known whether polymorphisms in TLR1 regulate the innate immune response. We stimulated human whole blood with triacylated lipopeptide, a ligand for TLR1/2 heterodimers, and found substantial inter-individual variation in the immune response. We sequenced the coding region of TLR1 and found a non-synonymous polymorphism, I602S (base pair T1805G), that regulated signalling. In comparison to TLR1_602S, the 602I variant mediated substantially greater basal and lipopeptide-induced NF-kappaB signalling in transfected HEK293 cells. These signalling differences among TLR1 variants were also found with stimulation by extracts of Mycobacterium tuberculosis. Furthermore, individuals with the 602II genotype produced substantially more IL-6 than those with the 602SS variant in a lipopeptide-stimulated whole-blood cytokine assay. Together, these observations demonstrate that variation in the inflammatory response to bacterial lipopeptides is regulated by a common TLR1 transmembrane domain polymorphism that could potentially impact the innate immune response and clinical susceptibility to a wide spectrum of pathogens.     

How Toll-like receptors signal: what we know and what we don't know

Signal transduction pathways activated by Toll-like receptors (TLRs) have continued to be a major focus of research for investigators interested in the initiation of innate immune responses and the induction of pro-inflammatory cytokines and type I interferons during infection. Biochemical details of the major signalling pathways have now been obtained, and the specific signalling pathways activated by different TLRs are being elucidated. New insights into the activation of IRF family members, notably IRF3, IRF5 and IRF7, have been obtained, and interesting spatiotemporal aspects of signalling by MyD88 leading to IRF7 activation revealed. Adapters in TLR signalling are targets for inhibition, both by endogenous regulators and by virally derived proteins. Selective targeting of pathways by anti-inflammatory glucocorticoids also indicates the potential for modulating TLR signalling therapeutically.     

Opposing effects of Toll-like receptor stimulation induce autoimmunity or tolerance

Toll-like receptor (TLR) activation by pathogens can induce the activation of diverse cell populations of the immune system and, therefore, can initiate or augment protective T-helper 1 immune responses. However, on a susceptible genetic background, TLR stimulation can also induce autoimmunity. The relative contribution of either microbe-derived or endogenous antigens, such as single-stranded RNA and unmethylated DNA, to TLR stimulation and the development of specific autoimmune diseases are still debated. Here, we review the different possibilities. Furthermore, tolerance induction by TLRs, which originally had been postulated to be protective by limiting excessive inflammation and, thus, preventing septic shock, has come into focus as a mechanism to control autoimmunity by inhibiting dendritic-cell maturation. In some murine models of systemic lupus erythematosus, TLR9 deficiency results in a shift from anti-nucleosome to TLR7-dependent anti-ribonucleoprotein IgG2a and IgG2b autoantibodies, and enhanced disease progression and mortality. Thus, not only can TLR signalling induce autoimmunity, but TLR(9) stimulation might also regulate tolerance.     

Radioresistant cells expressing TLR5 control the respiratory epithelium's innate immune responses to flagellin

Bacterial products (such as endotoxins and flagellin) trigger innate immune responses through TLRs. Flagellin‐induced signalling involves TLR5 and MyD88 and, according to some reports, TLR4. Whereas epithelial and dendritic cells are stimulated by flagellin in vitro, the cell contribution to the in vivo response is still unclear. Here, we studied the respective roles of radioresistant and radiosensitive cells in flagellin‐induced airway inflammation in mice. We found that i.n. delivery of flagellin elicits a transient change in respiratory function and an acute, pro‐inflammatory response in the lungs, characterized by TLR5‐ and MyD88‐dependent chemokine secretion and neutrophil recruitment. In contrast, TLR4, CD14 and TRIF were not essential for flagellin‐mediated responses, indicating that TLR4 does not cooperate with TLR5 in the lungs. Respiratory function, chemokine secretion and airway infiltration by neutrophils were dependent on radioresistant, TLR5‐expressing cells. Furthermore, lung haematopoietic cells also responded to flagellin by activating TNF‐α production. We suggest that the radioresistant lung epithelial cells are essential for initiating early, TLR5‐dependent signalling in response to flagellin and thus triggering the lung's innate immune responses.     

模式生物斑马鱼在免疫学研究中的应用

斑马鱼是目前新兴的免疫学研究模式生物。基因突变技术、细胞移植技术、显微注射技术等已经在斑马鱼上得以应用。荧光标记使斑马鱼成为可以跟踪研究免疫细胞的活体动物模型。PCR与芯片技术有助于寻找重要的免疫相关基因。胚胎培养与移植技术为建立斑马鱼病原体感染模型提供了技术保障。结合荧光标记,斑马鱼模型可以用来筛选免疫增强药物和免疫抑制剂。目前已经建立了细菌病毒感染斑马鱼模型、白血病斑马鱼模型、免疫基因筛选斑马鱼模型与药物筛选斑马鱼模型。研究斑马鱼具有很重要的医学与生物学价值。     

TLR signaling in B-cell development and activation

Expression of Toll-like receptors (TLRs) in B cells provides a cell-intrinsic mechanism for innate signals regulating adaptive immune responses. In combination with other signaling pathways in B cells, including through the B-cell receptor (BCR), TLR signaling plays multiple roles in B-cell differentiation and activation. The outcome of TLR signaling in B cells is largely context-dependent, which partly explains discrepancies among in vitro and in vivo studies, or studies using different immunogens. We focus on recent findings on how B-cell-intrinsic TLR signaling regulates antibody responses, including germinal center formation and autoantibody production in autoimmune disease models. In addition, TLR signaling also acts on the precursors of B cells, which could influence the immune response of animals by shaping the composition of the immune system. With TLR signaling modulating immune responses at these different levels, much more needs to be understood before we can depict the complete functions of innate signaling in host defense.     

Defense of zebrafish embryos against Streptococcus pneumoniae infection is dependent on the phagocytic activity of leukocytes

Severe community acquired pneumonia caused by Streptococcus pneumoniae is the most common cause of death from infection in developing countries. Serotype specific conjugate vaccines have decreased the incidence of invasive infections, but at the same time, disease due to non-vaccine serotypes have increased. New insights into host immune mechanisms against pneumococcus may provide better treatment and prevention strategies. Zebrafish is an attractive vertebrate model for studying host immune responses and infection biology. Here we show that an intravenous challenge with pneumococcus infects zebrafish embryos leading to death in a dose dependent manner. Survival rates correlate with the bacterial burden in the embryos. The production of proinflammatory cytokines is induced in zebrafish after pneumococcal exposure. Importantly, morpholino treated embryos lacking either myeloid cells or the ability to phagocytose bacteria have lowered survival rates compared to wild type embryos after pneumococcal challenge. These data suggest that the survival of zebrafish embryos upon intravenous infection with S. pneumoniae is dependent on the clearance of the bacteria by phagocytosing cells. Additionally, we demonstrate that mutant pneumococci lacking known virulence factors are attenuated in the zebrafish model. Our data demonstrate that zebrafish embryos can be used for study innate immune responses as well as virulence determinants in pneumococcal infections.     

Complement and Toll-like receptors: key regulators of adaptive immune responses

The innate immune system provides sophisticated defense mechanisms to protect complex macroorganisms from the attack of microorganisms. Among those, the complement system and Toll-like receptors are of paramount importance to discriminate between infectious non-self and non-infectious self and to provide critical danger signals instructing adaptive immune responses. Here, we will discuss recent advances in our understanding of the mechanisms underlying complement and TLR-mediated regulation of adaptive immunity. We will focus on the regulation of T cell immunity and discuss recent findings on the cross-talk between complement receptor and TLR signaling pathways. Such cross-talk is likely to affect the outcome of infections with intracellular pathogens, as well as the initiation and maintenance of aberrant immune responses leading to autoimmunity and atopy.     

Insights into the toll‐like receptors in sexually transmitted infections

Toll‐like receptors (TLRs) are like soldiers of an innate immune system, which protects vital biological processes against invading pathogens. TLR signalling pathways help in the removal of pathogens and mediate well‐established inflammatory processes. However, these processes may also aid in the development or augmentation of an infection or an autoimmune disease. Recent studies have delineated TLR polymorphism's role in the loss of function, making hosts more resistant or vulnerable to the development of an infection. In this review, we have discussed the association of TLRs with sexually transmitted infections (STIs), especially to the pathogen‐specific ligands. We have also assessed the impact on TLR downstream signalling and the maintenance of cellular homeostasis during immune responses. Besides, we have discussed the role of TLRs single nucleotide polymorphisms in various STIs. Since TLRs are known to play a part in defence mechanisms and in aiding infections therefore, a thorough understanding of TLRs structure and molecular mechanisms is required to explain how they can influence the outcome of an STI. Such a strategy may lead to the development of novel and useful immunotherapeutic approaches to control pathogen progression and prevent transmission.     

Complement fragments C3a and C5a: The salt and pepper of the immune response

The influence of complement on B‐cell responses has been known for many years, but the notion that T‐cell recognition, expansion and differentiation are complement dependent has only recently gained impetus. DC, and to a lesser extent T cells, produce a range of complement components necessary for complement activation, and these cells also express receptors that detect complement‐activation products such as C3a and C5a (anaphylatoxins). In the absence of C3a‐receptor (C3aR) signalling, DC lose their capacity to induce potent Th1 responses against alloantigen and also favour the emergence of Treg. A study in this issue of the European Journal of Immunology not only spotlights the importance of C5aR signalling in DC interaction with T cells, but also shows how cooperation with other signalling pathways determines the outcome of T‐cell activation. Remove C5aR from the equation, TLR2‐stimulated DC induce naive CD4⁺ Th cells to undergo differentiation not only mainly to Th17 cells but also to Treg, via a TGF‐β‐dependent pathway. Thus, anaphylatoxins in conjunction with other danger signalling pathways modify the function of DC in antigen presentation and help to shape the primary immune response. Future work will need to address the impact of anaphylatoxins on protective immunity in vivo and determine the wider implications of anaphylatoxins for allo‐ and autoimmunity.     

Toll‐like receptor 6 mediated inflammatory and functional responses of zinc oxide nanoparticles primed macrophages

Macrophages are among the most sensitive immune cells because of their phagocytic activity and are prone to become dysfunctional or not able to perform properly if nanoparticle load increases. We have previously reported that zinc oxide nanoparticles (ZNPs) induce inflammatory responses in macrophages that contribute to their death. Recognition of ZNPs by pattern recognition receptors such as toll‐like receptors (TLRs) might be a factor in the initiation of these responses in macrophages. Therefore, in this study we explored the role played by TLR6 and mitogen‐activated protein kinase (MAPKs) pathways in the inflammatory responses of macrophages during ZNPs exposure. ZNPs‐activated macrophages showed enhanced expression of activation and maturation markers (CD1d, MHC‐II, CD86 and CD71). Among various TLRs screened, TLR6 emerged as the most potent activator for ZNPs‐induced inflammatory responses. Downstream signalling proteins myeloid differentiation 88, interleukin‐1 receptor associated kinase and tumour necrosis factor receptor‐associated factor were also enhanced. On inhibiting MAPKs pathways individually, the inflammatory responses such as interleukin‐1β, interleukin‐6, tumour necrosis factor‐α, cyclooxygenase‐2 and inducible nitric oxide synthase were suppressed. TLR6 silencing significantly inhibited the pro‐inflammatory cytokine levels, reactive nitrogen species generation and inducible nitric oxide synthase expression. Also, inhibition of MAPKs in the absence of TLR6 signalling validated the link between TLR6 and MAPKs in ZNPs‐induced inflammatory responses. TLR6 was found to be co‐localized with autophagosomes. Macrophages lacking TLR6 inhibited the autophagosome marker protein‐microtubule‐associated protein1 light chain 3‐isoform II formation and phagocytosis. These results demonstrate that inflammatory responses caused by ZNPs‐activated macrophages strongly depend on TLR6‐mediated MAPK signalling.     

Dysregulation of human Toll-like receptor function in aging

Studies addressing immunosenescence in the immune system have expanded to focus on the innate as well as the adaptive responses. In particular, aging results in alterations in the function of Toll-like receptors (TLRs), the first described pattern recognition receptor family of the innate immune system. Recent studies have begun to elucidate the consequences of aging on TLR function in human cohorts and add to existing findings performed in animal models. In general, these studies show that human TLR function is impaired in the context of aging, and in addition there is evidence for inappropriate persistence of TLR activation in specific systems. These findings are consistent with an overarching theme of age-associated dysregulation of TLR signaling that likely contributes to the increased morbidity and mortality from infectious diseases found in geriatric patients.     

Dynamic balance of pro- and anti-inflammatory signals controls disease and limits pathology

Immune responses to pathogens are complex and not well understood in many diseases, and this is especially true for infections by persistent pathogens. One mechanism that allows for long-term control of infection while also preventing an over-zealous inflammatory response from causing extensive tissue damage is for the immune system to balance pro- and anti-inflammatory cells and signals. This balance is dynamic and the immune system responds to cues from both host and pathogen, maintaining a steady state across multiple scales through continuous feedback. Identifying the signals, cells, cytokines, and other immune response factors that mediate this balance over time has been difficult using traditional research strategies. Computational modeling studies based on data from traditional systems can identify how this balance contributes to immunity. Here we provide evidence from both experimental and mathematical/computational studies to support the concept of a dynamic balance operating during persistent and other infection scenarios. We focus mainly on tuberculosis, currently the leading cause of death due to infectious disease in the world, and also provide evidence for other infections. A better understanding of the dynamically balanced immune response can help shape treatment strategies that utilize both drugs and host-directed therapies.     

牙龈卟啉单胞菌免疫逃避机制研究进展

天然免疫即固有免疫是机体抵御微生物侵袭的第一道防线。牙龈卟啉单胞菌是牙周、尤其是慢性牙周病病变区或活动部位最主要的优势菌。牙龈卟啉单胞菌菌体表面有多种粘附素并能分泌大量的毒力因子,使牙龈卟啉单胞菌能逃避天然免疫系统的防御,破坏补体系统和TLR对牙龈卟啉单胞菌的识别,并且削弱两者之间相互作用的信号通路。本文就牙龈卟啉单胞菌与机体天然免疫中补体的相互作用,如何逃避TLRs的识别,以及如何削弱两者之间相互作用的信号通路进行了概述。     

Zebrafish mutants: behavioral genetic studies of visual system defects.

Zebrafish are a promising model for behavioral and genetic studies of vertebrate visual system development and retinal degeneration. In the past few years, numerous studies on zebrafish vision have been published. While most of the studies focus on the molecular and cellular characterization of mutations that disrupt zebrafish visual system structure in early development, others examine the mechanisms that underlie inherited visual system disorders in adults. Behavioral assays, along with morphologic and electrophysiological methods, are powerful tools for functional analyses of zebrafish visual development and performance.     

Evidence for a widespread brain stem escape network in larval zebrafish.

Zebrafish escape behaviors, which typically consist of a C bend, a counter-turn, and a bout of rapid swimming, are initiated by firing of the Mauthner cell and two segmental homologs. However, after laser-ablation of the Mauthner cell and its homologs, escape-like behaviors still occur, albeit at a much longer latency. This might suggest that additional neurons contribute to this behavior. We therefore recorded the activity of other descending neurons in the brain stem using confocal imaging of cells retrogradely labeled with fluorescent calcium indicators. A large majority of identified descending neurons present in the larval zebrafish, including both ipsilaterally and contralaterally projecting reticulospinal neurons, as well as neurons from the nucleus of the medial longitudinal fasciculus, showed short-latency calcium responses after gentle taps to the head of the larva-a stimulus that reliably evokes an escape behavior. Previous studies had associated such in vivo calcium responses with the firing of action potentials, and because all responding cells have axons projecting into to spinal cord, this suggests that these cells are relaying escape-related information to spinal cord. Other identified neurons failed to show consistent calcium responses to escape-eliciting stimuli. In conjunction with previous lesion studies, these results indicate that the neural control systems for turning and swimming behaviors are widely distributed in the larval zebrafish brain stem. The degree of robustness or redundancy of this system has implications for the descending control of vertebrate locomotion.     

The contribution of direct TLR signaling to T cell responses

It is well established that Toll-like receptors (TLRs) play a critical role in the generation of innate immune responses and thereby also play an important, indirect role in the initiation of subsequent adaptive T cell responses. However, T cells also express certain TLRs, and we have focused on the physiological importance of direct TLR signaling in T cells. TLRs can function as co-stimulatory receptors that complement TCR-induced signals to enhance effector T cell proliferation, survival and cytokine production. We also found that TLR signaling pathways in T cells are required for the effective clonal expansion of antigen-specific T cells during infection in vivo. Thus, the importance of TLRs in T cell-mediated immunity reflects both T cell-extrinsic and T cell-intrinsic components, which warrants a reconsideration of the dogma that restricts germ-line encoded pattern recognition to cells of the innate immune system.     

Functional characterization of naturally occurring genetic variants in the human TLR1-2-6 gene family

Toll-like receptors (TLRs) are considered an essential component of the innate immune system, initiating inflammatory responses following infection of the host. Humans have 10 functional TLRs, differing in their subcellular distributions and the microbial agonists they sense. The phylogenetically conserved TLR1-2-6 family is unique in that TLR1 and TLR6 form heterodimers with TLR2 to mediate signalling in response to agonists. Epidemiological genetic studies have identified several TLR variants that appear to influence susceptibility to infectious diseases, but the functional consequences of which remain largely unknown. Here, we assessed the functional impact of the TLR1-2-6 variants with altered amino acid sequences segregating naturally in the human population. We used an NF-κB reporter assay in TLR-transfected human embryonic kidney 293T cells stimulated with the corresponding TLR agonists. We found that among the 41 naturally occurring variants with amino acid alterations identified in the TLR1-2-6 family, 14 of them (five TLR1, four TLR2, and five TLR6 variants) displayed marked impairment of NF-κB activation. Most of these variants are present at very low population frequencies and are population-specific. These observations suggest that rare, nonsynonymous TLR mutations are likely to have deleterious effects on immune responses and may therefore contribute to complex susceptibility to infection at the population level.