Role of Nods in bacterial infection

Research into intracellular sensing of microbial products is an up and coming field in innate immunity. Nod1 and Nod2 are members of the rapidly expanding family of NACHT domain-containing proteins involved in intracellular recognition of bacterial products. Nods proteins are involved in the cytosolic detection of peptidoglycan motifs of bacteria, recognized through the LRR domain. The role of the NACHT-LRR system of detection in innate immune responses is highlighted at the mucosal barrier, where most of the membranous Toll like receptors (TLRs) are not expressed, or with pathogens that have devised ways to escape TLR sensing. For a given pathogen, the sum of the pathways induced by the recognition of the different "pathogen associated molecular patterns" (PAMPs) by the different pattern recognition receptors (PRRs) trigger and shape the subsequent innate and adaptive immune responses. Knowledge gathered during the last decade on PRR and their agonists, and recent studies on bacterial infections provide new insights into the immune response and the pathogenesis of human infectious diseases.     

Innate immune response in insects: recognition of bacterial peptidoglycan and amplification of its recognition signal

The major cell wall components of bacteria are lipopolysaccharide, peptidoglycan, and teichoic acid. These molecules are known to trigger strong innate immune responses in the host. The molecular mechanisms by which the host recognizes the peptidoglycan of Gram-positive bacteria and amplifies this peptidoglycan recognition signals to mount an immune response remain largely unclear. Recent, elegant genetic and biochemical studies are revealing details of the molecular recognition mechanism and the signalling pathways triggered by bacterial peptidoglycan. Here we review recent progress in elucidating the molecular details of peptidoglycan recognition and its signalling pathways in insects. We also attempt to evaluate the importance of this issue for understanding innate immunity.     

Peptidoglycan recognition protein (PGRP)-LE and PGRP-LC act synergistically in Drosophila immunity

In innate immunity, pattern recognition molecules recognize cell wall components of microorganisms and activate subsequent immune responses, such as the induction of antimicrobial peptides and melanization in Drosophila. The diaminopimelic acid (DAP)-type peptidoglycan potently activates imd-dependent induction of antibacterial peptides. Peptidoglycan recognition protein (PGRP) family members act as pattern recognition molecules. PGRP-LC loss-of-function mutations affect the imd-dependent induction of antibacterial peptides and resistance to Gram-negative bacteria, whereas PGRP-LE binds to the DAP-type peptidoglycan, and a gain-of-function mutation induces constitutive activation of both the imd pathway and melanization. Here, we generated PGRP-LE null mutants and report that PGRP-LE functions synergistically with PGRP-LC in producing resistance to Escherichia coli and Bacillus megaterium infections, which have the DAP-type peptidoglycan. Consistent with this, PGRP-LE acts both upstream and in parallel with PGRP-LC in the imd pathway, and is required for infection-dependent activation of melanization in Drosophila. A role for PGRP-LE in the epithelial induction of antimicrobial peptides is also suggested.     

Induction of autophagy via innate bacterial recognition

Macroautophagy (referred to hereafter as autophagy) functions not only in self-digestion, but also in the killing and degradation of infectious pathogens in in vitro-cultured cells. Based on genetic manipulations of both the host, Drosophila and pathogen, Listeria monocytogenes, we recently reported that L. monocytogenes-induced autophagy is dependent on the recognition of the pathogen by the Drosophila pattern recognition protein, PGRP-LE. Autophagy and PGRP-LE are crucial for inhibition of the intracellular growth of bacteria in hemocytes, the target cells of L. monocytogenes infection in vivo. The importance of autophagy in the resistance of Drosophila against L. monocytogenes is further indicated in in vivo survival experiments. The signaling pathway(s) that induces autophagy by PGRP-LE is independent of the known immune signaling pathways, suggesting that another unidentified pathway(s) is involved. The results of the present study demonstrate that the induction of autophagy, as an innate immune response targeting intracellular pathogens, is activated by intracellular sensors through unidentified pathways.     

Innate immunity to mycobacteria: vitamin D and autophagy

Autophagy is an ancient mechanism of protein degradation and a novel antimicrobial strategy. With respect to host defences against mycobacteria, autophagy plays a crucial role in antimycobacterial resistance, and contributes to immune surveillance of intracellular pathogens and vaccine efficacy. Vitamin D3 contributes to host immune responses against Mycobacterium tuberculosis through LL‐37/hCAP‐18, which is the only cathelicidin identified to date in humans. In this review, we discuss recent advances in our understanding of host immune strategies against mycobacteria, including vitamin D‐mediated innate immunity and autophagy activation. This review also addresses our current understanding regarding the autophagy connection to principal innate machinery, such as ubiquitin‐ or inflammasome‐involved pathways. Integrated dialog between autophagy and innate immunity may contribute to adequate host immune defences against mycobacterial infection.     

Intracellular innate resistance to bacterial pathogens

Mammalian innate immunity stimulates antigen-specific immune responses and acts to control infection prior to the onset of adaptive immunity. Some bacterial pathogens replicate within the host cell and are therefore sheltered from some protective aspects of innate immunity such as complement. Here we focus on mechanisms of innate intracellular resistance encountered by bacterial pathogens and how some bacteria can evade destruction by the innate immune system. Major strategies of intracellular antibacterial defence include pathogen compartmentalization and iron limitation. Compartmentalization of pathogens within the host endocytic pathway is critical for generating high local concentrations of antimicrobial molecules, such as reactive oxygen species, and regulating concentrations of divalent cations that are essential for microbial growth. Cytosolic sensing, autophagy, sequestration of essential nutrients and membrane attack by antimicrobial peptides are also discussed.     

Mammalian peptidoglycan recognition protein TagL inhibits Listeria monocytogenes invasion into epithelial cells

Peptidoglycan recognition proteins are a family of evolutionary conserved proteins that play a basic role in the innate immunity of insects, but their role in the immunity of mammals remains unclear. To elucidate its functions, a mouse member of the peptidoglycan recognition proteins family, TagL, was stably expressed in colon adenocarcinoma HT29 cells, and its effect on the invasion and intracellular growth of the enteroinvasive pathogenic bacterium Listeria monocytogenes was assessed. The expression of TagL substantially impaired bacterial invasion and early intracellular growth. The observed effects were partly caused by a loss of viability by intraphagosomal bacteria. Efficient phagosome escaping but not efficient invasion helped bacteria to overplay TagL.     

Eating the strangers within: host control of intracellular bacteria via xenophagy

Many bacterial pathogens rely on an intracellular cycle to ensure their proliferation within infected hosts, through their ability to avoid or circumvent host bactericidal pathways. Recent evidence supports an increasingly important role for the autophagy pathway in innate immune defences against intracellular pathogens, as a mechanism of capture of either cytosol-adapted or vacuolar bacteria that redirect them to the lysosomal compartment for killing. Antibacterial autophagy, also referred to as xenophagy, involves selective recognition of intracellular bacteria and their targeting to the autophagic machinery for degradation. Here we review recent advances in our molecular understanding of these processes, and in how bacteria have adapted to avoid xenophagy or even take advantage of this innate immune process.     

Analysis of signal-dependent changes in the proteome of Drosophila blood cells during an immune response

Innate immunity is based on the recognition of cell-surface molecules of infecting agents. Microbial substances, such as peptidoglycan, lipopolysaccharide, and beta-1,3-glucans, produce functional responses in Drosophila hemocytes that contribute to innate immunity. We have used two-dimensional gel electrophoresis and MS to resolve lipopolysaccharide-induced changes in the protein profile of a Drosophila hemocytic cell line. We identified 24 intracellular proteins that were up- or down-regulated, or modified, in response to immune challenge. Several proteins with predicted immune functions, including lysosomal proteases, actin-binding/remodeling proteins, as well as proteins involved in cellular responses to oxidative stress, were affected by the immune assault. Intriguingly, a number of the proteins identified in this study have recently been implicated in phagocytosis in higher vertebrates. We suggest that phagocytosis is activated in Drosophila hemocytes by the presence of microbial substances, and that this activation constitutes an evolutionarily conserved arm of innate immunity. In addition, a number of proteins involved in calcium-regulated signaling, mRNA processing, and nuclear transport were affected, consistent with a possible role in reprogramming of gene expression. In conclusion, the present proteome analysis identified many proteins previously not linked to innate immunity, demonstrating that differential protein profiling of Drosophila hemocytes is a valuable tool for identification of new players in immune-related cellular processes.     

The perplexing role of autophagy in plant innate immune responses

Autophagy is a major intracellular process for the degradation of cytosolic macromolecules and organelles in the lysosomes or vacuoles for the purposes of regulating cellular homeostasis and protein and organelle quality control. In complex metazoan organisms, autophagy is highly engaged during the immune responses through interfaces either directly with intracellular pathogens or indirectly with immune signalling molecules. Studies over the last decade or so have also revealed a number of important ways in which autophagy shapes plant innate immune responses. First, autophagy promotes defence‐associated hypersensitive cell death induced by avirulent or related pathogens, but restricts unnecessary or disease‐associated spread of cell death. This elaborate regulation of plant host cell death by autophagy is critical during plant immune responses to the types of plant pathogens that induce cell death, which include avirulent biotrophic pathogens and necrotrophic pathogens. Second, autophagy modulates defence responses regulated by salicylic acid and jasmonic acid, thereby influencing plant basal resistance to both biotrophic and necrotrophic pathogens. Third, there is an emerging role of autophagy in virus‐induced RNA silencing, either as an antiviral collaborator for targeted degradation of viral RNA silencing suppressors or an accomplice of viral RNA silencing suppressors for targeted degradation of key components of plant cellular RNA silencing machinery. In this review, we summarize this important progress and discuss the potential significance of the perplexing role of autophagy in plant innate immunity.     

Positive and negative regulation of the Drosophila immune response

Insects mount a robust innate immune response against a wide array of microbial pathogens. The hallmark of the Drosophila humoral immune response is the rapid production of antimicrobial peptides in the fat body and their release into the circulation. Two recognition and signaling cascades regulate expression of these antimicrobial peptide genes. The Toll pathway is activated by fungal and many Gram-positive bacterial infections, whereas the immune deficiency (IMD) pathway responds to Gram-negative bacteria. Recent work has shown that the intensity and duration of the Drosophila immune response is tightly regulated. As in mammals, hyperactivated immune responses are detrimental, and the proper down-modulation of immunity is critical for protective immunity and health. In order to keep the immune response properly modulated, the Toll and IMD pathways are controlled at multiple levels by a series of negative regulators. In this review, we focus on recent advances identifying and characterizing the negative regulators of these pathways.     

Peptidoglycan recognition proteins of the innate immune system

Peptidoglycan (PGN) is the major component of bacterial cell walls and one of the main microbial products recognized by the innate immune system. PGN recognition is mediated by several families of pattern recognition molecules, including Toll-like receptors, nucleotide-binding oligomerization domain-containing proteins, and peptidoglycan recognition proteins (PGRPs). However, only the interaction of PGN with PGRPs, which are highly conserved from insects to mammals, has so far been characterized at the molecular level. Here, we describe recent structural studies of PGRPs that reveal the basis for PGN recognition and provide insights into the signal transduction and antibacterial activities of these innate immune proteins.     

Autophagy and toll-like receptors: a new link in cancer cells

In addition to its clean-up function, autophagy is considered as an innate immunity mechanism due to its role in the removal of intracellular pathogens. Toll-like receptors (TLRs) are crucial components of innate immunity involved in the recognition of a diverse array of microbial products. Recent works demonstrated that different pathogen-associated molecular patterns (PAMPs) such as lipopolysaccharide (LPS) and single-strand RNA are able to induce autophagy via different TLRs in immune cells. In a recent report, we showed that bacterial CpG motifs, another PAMP, can induce autophagy in rodent and human tumor cell lines and that this process is TLR9-dependent. In addition, an increase in the number of autophagosomes can also be observed in vivo after the intratumoral injection of CpG motifs. These results extend the link between TLRs and autophagy to non-immune tumor cells and may be relevant for cancer treatment and more generally for gene therapy approaches in TLR9-positive tissues. In this addendum, we discuss the potential mechanisms and the consequences of the CpG-induced autophagy in tumor cells.     

昆虫免疫识别与病原物免疫逃避机理研究进展

昆虫在长期进化过程中形成复杂的天然免疫系统,病原识别是启动下游免疫反应的第一步,这一过程主要是由不同的模式识别蛋白来完成的。目前发现并鉴定的昆虫模式识别蛋白主要包括肽聚糖识别蛋白、类免疫球蛋白、β-1,3-葡聚糖结合蛋白、C型凝集素及具多功能的载脂蛋白等,不同的蛋白种类具有不同的结构、功能及识别对象。与昆虫免疫识别相对应的是,不同昆虫病原物在进化过程中发展出不同策略的免疫逃避能力,以战胜宿主免疫而致病或最终杀死昆虫。本文就昆虫免疫过程中不同模式识别蛋白的结合对象、结构与功能,以及逐渐兴起的病原物通过分子伪装等进行免疫逃避的研究进展进行了综述。并在此基础上,作者就昆虫免疫与昆虫病理研究的发展方向进行了展望,认为只有当两方面研究相结合时,才能更好地揭示昆虫宿主与病原物之间免疫与抗免疫的动态相互作用过程。     

Immunoamphisomes in dendritic cells amplify TLR signaling and enhance exogenous antigen presentation on MHC-II

Autophagy, a specialized lysosomal degradation pathway, has proven to be a potent cell-autonomous defense mechanism against a range of intracellular microbes. In addition, autophagy emerged recently as a critical regulator of innate and adaptive immune responses. Links between autophagy and innate immunity are being progressively unveiled. For instance, several TLR (Toll-Like Receptor) agonists upregulate autophagy flux in immune cell types such as DC (dendritic cells) or macrophages. Conversely, and perhaps surprisingly, is the observation that TLR7-mediated responses might depend on autophagy in plasmacytoid DC, thus suggesting a more complex link between TLR-dependent responses and autophagy. Recently, the demonstration that NOD2 increases autophagy suggests that innate immune responses initiated via a broad range of pathogen recognition receptors can regulate autophagy. In addition to its involvement in innate immune responses, autophagy regulates adaptive immune responses via both MHC class I and class II molecules depending on the cellular context and the nature of the antigen.     

Structure and function of Toll-like receptor proteins

Beginning in 1997 with the identification of the first human homologue of the Drosophila protein Toll, a family of related molecules have been identified in both humans and other mammals. These Toll-like receptor (TLR) proteins appear to represent a conserved family of innate immune recognition receptors. TLR proteins share extended homology with receptors for the cytokines interleukin 1 (IL-1) and interleukin 18 (IL-18). These receptors are coupled to a signaling pathway that is conserved in mammals, insects, and plants, resulting in cellular activation, thereby stimulating innate immune defenses. A variety of bacterial and fungal products have been identified that serve as TLR ligands, and more recent studies have identified the first endogenous protein ligands for TLR proteins. While TLR signaling is likely to be a key feature of innate immune responses, these proteins may also regulate homeostasis via interaction with endogenous protein ligands.     

Autophagy in CD4+ T-cell immunity and tolerance

Autophagy is a homeostatic process that enables eukaryotic cells to deliver cytoplasmic constituents for lysosomal degradation, to recycle nutrients and to survive during starvation. In addition to these primordial functions, autophagy has emerged as a key mechanism in orchestrating innate and adaptive immune responses to intracellular pathogens. Autophagy restricts viral infections as well as replication of intracellular bacteria and parasites and delivers pathogenic determinants for TLR stimulation and for MHC class II presentation to the adaptive immune system. Apart from its role in defense against pathogens, autophagy-mediated presentation of self-antigens in the steady state could have a crucial role in the induction and maintenance of CD4(+) T-cell tolerance. This review describes the mechanisms by which the immune system utilizes autophagic degradation of cytoplasmic material to regulate adaptive immune responses.     

Nod1 mediates cytoplasmic sensing of combinations of extracellular bacteria

During mucosal colonization, epithelial cells are concurrently exposed to numerous microbial species. Epithelial cytokine production is an early component of innate immunity and contributes to mucosal defence. We have previously demonstrated a synergistic response of respiratory epithelial cells to costimulation by two human pathogens, Streptococcus pneumoniae and Haemophilus influenzae. Here we define a molecular mechanism for the synergistic activation of epithelial signalling during polymicrobial colonization. H. influenzae peptidoglycan synergizes with the pore-forming toxin pneumolysin from S. pneumoniae. Radiolabelled peptidoglycan enters epithelial cells more efficiently in the presence of pneumolysin, consistent with peptidoglycan gaining access to the cytoplasm via toxin pores. Other pore-forming toxins (including anthrolysin O from Bacillus anthracis and Staphylococcus aureus alpha-toxin) can substitute for pneumolysin in the generation of synergistic responses. Consistent with a requirement for pore formation, S. pneumoniae expressing pneumolysin but not an isogenic mutant expressing a non-pore-forming toxoid prime epithelial responses. Nod1, a host cytoplasmic peptidoglycan-recognition molecule, is crucial to the epithelial response. Taken together, these findings demonstrate a role for cytosolic recognition of peptidoglycan in the setting of polymicrobial epithelial stimulation. We conclude that combinations of extracellular organisms can activate innate immune pathways previously considered to be reserved for the detection of intracellular microorganisms.