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Nod1 Detects a Unique Muropeptide from Gram-Negative Bacterial Peptidoglycan
Abstract
Although the role of Toll-like receptors in extracellular bacterial sensing has been investigated intensively, intracellular detection of bacteria through Nod molecules remains largely uncharacterized. Here, we show that human Nod1 specifically detects a unique diaminopimelate-containing N-acetylglucosamine-N-acetylmuramic acid (GlcNAc-MurNAc) tripeptide motif found in Gram-negative bacterial peptidoglycan, resulting in activation of the transcription factor NF-kappaB pathway. Moreover, we show that in epithelial cells (which represent the first line of defense against invasive pathogens), Nod1is indispensable for intracellular Gram-negative bacterial sensing.
... We have recently described a role for NOD1 ligand (NOD1L) in the maintenance of hematopoietic precursor pools in BM 19 . To further address the role of NOD1 in hematopoiesis, we compared the cellularity of lymphoid organs under steady-state conditions in and two independently generated strains of NOD1-deficient mice 22,23 . ...
... Both of the NOD1-deficient lines we utilized have been employed separately in numerous studies to assess NOD1 involvement. Neither is responsive to NOD1L stimulation due to disruption of exons 2 and 3 which encode the CARD domain 22,23 . Unexpectedly, we found that only one line completely lacks Nod1 messenger RNA expression, referred to here as NOD1 −/− (ref. ...
... Unexpectedly, we found that only one line completely lacks Nod1 messenger RNA expression, referred to here as NOD1 −/− (ref. 23), while the other line expresses a transcript corresponding to the NOD plus LRR regions but not the CARD domain and, thus, is referred to here as ΔCARD NOD1 (ref. 22) (Extended Data Fig. 1a-d). ...
Aberrant differentiation of progenitor cells in the hematopoietic system is known to severely impact host immune responsiveness. Here we demonstrate that NOD1, a cytosolic innate sensor of bacterial peptidoglycan, also functions in murine hematopoietic cells as a major regulator of both the generation and differentiation of lymphoid progenitors as well as peripheral T lymphocyte homeostasis. We further show that NOD1 mediates these functions by facilitating STAT5 signaling downstream of hematopoietic cytokines. In steady-state, loss of NOD1 resulted in a modest but significant decrease in numbers of mature T, B and natural killer cells. During systemic protozoan infection this defect was markedly enhanced, leading to host mortality. Lack of functional NOD1 also impaired T cell-dependent anti-tumor immunity while preventing colitis. These findings reveal that, in addition to its classical role as a bacterial ligand receptor, NOD1 plays an important function in regulating adaptive immunity through interaction with a major host cytokine signaling pathway.
... A limited number of Gram-negative bacteria, including the human pathogens N. gonorrhoeae, N. meningitidis, and Bordetella pertussis release sufficient amounts of proinflammatory PG fragments to stimulate production of proinflammatory cytokines in tissue explants (18)(19)(20)(21). NOD1 and NOD2 are immune receptors found in host cells that are activated by small PG fragments (22,23). ...
... PG dimers that are digested by host lysozyme yield PG monomers with reducing ends, and these molecules are NOD2 agonists (41). The tripeptide PG monomers and the free tripeptides are human NOD1 agonists, while the tetrapeptide PG monomers and free tetrapeptides are mouse NOD1 agonists (22,42). Small amounts of dipeptide PG monomers are released and serve as NOD2 agonists (43). ...
Neisseria gonorrhoeae, a human restricted pathogen, releases inflammatory peptidoglycan (PG) fragments that contribute to the pathophysiology of pelvic inflammatory disease. The genus Neisseria is also home to multiple species of human- or animal-associated Neisseria that form part of the normal microbiota. Here we characterized PG release from the human-associated nonpathogenic species Neisseria lactamica and Neisseria mucosa and animal-associated Neisseria from macaques and wild mice. An N. mucosa strain and an N. lactamica strain were found to release limited amounts of the proinflammatory monomeric PG fragments. However, a single amino acid difference in the PG fragment permease AmpG resulted in increased PG fragment release in a second N. lactamica strain examined. Neisseria isolated from macaques also showed substantial release of PG monomers. The mouse colonizer Neisseria musculi exhibited PG fragment release similar to that seen in N. gonorrhoeae with PG monomers being the predominant fragments released. All the human-associated species were able to stimulate NOD1 and NOD2 responses. N. musculi was a poor inducer of mouse NOD1, but ldcA mutation increased this response. The ability to genetically manipulate N. musculi and examine effects of different PG fragments or differing amounts of PG fragments during mouse colonization will lead to a better understanding of the roles of PG in Neisseria infections. Overall, we found that only some nonpathogenic Neisseria have diminished release of proinflammatory PG fragments, and there are differences even within a species as to types and amounts of PG fragments released.
... It has previously been reported that N. gonorrhoeae and its outer membrane vesicles (OMVs) are able to initiate cytosolic nucleotide-binding oligomerization domain 1 (NOD1) responses in epithelial cells, resulting in the activation of NF-κB and innate immune responses [17,18]. Human NOD1 senses the peptidoglycan diaminopimelate-containing GlcNAc-MurNAc tripeptide motif (GM-TriDAP) found in Gram-negative bacteria [19,20]. N. gonorrhoeae releases large quantities of GM-TriDAP, which is mediated by dedicated peptidoglycan processing and secretion proteins, and explains the strong inflammatory response observed during a gonococcal infection [21][22][23][24]. ...
... Gonococcal type IV pili are sequence and phase variable [34][35][36], with pilus-negative strains being more invasive [37,38]. We showed that invading and intracellular N. gonorrhoeae is recognized by the NOD1 cytoplasmic pattern recognition receptor that detects the peptidoglycan fragment GM-TriDAP [19]. It has previously been shown for S. flexneri and L. monocytogenes that their detection by NOD1/NOD2 results in the recruitment of ATG16L1 to the entry site for further autophagy targeting [25]. ...
Neisseria gonorrhoeae establishes tight interactions with mucosal epithelia through activity of its type IV pilus, while pilus retraction forces activate autophagic responses toward invading gonococci. Here we studied pilus-independent epithelial cell responses and showed that pilus-negative gonococci residing in early and late endosomes are detected and targeted by nucleotide-binding oligomerization domain 1 (NOD1). NOD1 subsequently forms a complex with immunity-related guanosine triphosphatase M (IRGM) and autophagy-related 16–like 1 (ATG16L1) to activate autophagy and recruit microtubule-associated protein light chain 3 (LC3) to the intracellular bacteria. IRGM furthermore directly recruits syntaxin 17 (STX17), which is able to form tethering complexes with the lysosome. Importantly, IRGM-STX17 interactions are enhanced by LC3 but were still observed at lower levels in an LC3 knockout cell line. These findings demonstrate key roles for NOD1 and IRGM in the sensing of intracellular N gonorrhoeae and subsequent directing of the bacterium to the lysosome for degradation.
... NOD1 and NOD2 have been intensively studied in the gut and are responsible for recognition of bacterial cell wall peptidoglycan (PGN). NOD1 senses the meso-diaminopimelic type of PGN, which is most commonly found in Gram-negative bacteria (Girardin et al., 2003a;Girardin et al., 2003b). NOD2 has a broader sensing spectrum, recognizing the muramyl dipeptide N-acetylmuramyl-L-alanyl-D-glutamate, which is common to both Gram-negative and Gram-positive bacteria (Girardin et al., 2003b;Girardin et al., 2003c). ...
... Thus, the alpha-protein kinase-1 (ALPK1) is primarily involved in the detection of freely replicating cytosolic bacteria and elicits a robust NF-κB response following activation of the peptidoglycan sensor NOD1 (Gaudet et al., 2017). NOD1 is known to mediate an initial transient burst of NF-κB activation during bacterial invasion (Girardin et al., 2003a). The ALPK1-TIFA-mediated pathogen recognition system is thought to play a role in supporting a sustained inflammatory response during the later stages of bacterial infection after the initial transient NOD1-mediated NF-κB activation. ...
Intestinal epithelial cells (IECs) perform several physiological and metabolic functions at the epithelial barrier. IECs also play an important role in defining the overall immune functions at the mucosal region. Pattern recognition receptors (PRRs) on the cell surface and in other cellular compartments enable them to sense the presence of microbes and microbial products in the intestinal lumen. IECs are thus at the crossroads of mediating a bidirectional interaction between the microbial population and the immune cells present at the intestinal mucosa. This communication between the microbial population, the IECs and the underlying immune cells has a profound impact on the overall health of the host. In this review, we focus on the various PRRs present in different cellular compartments of IECs and discuss the recent developments in the understanding of their role in microbial recognition. Microbial recognition and signaling at the epithelial barrier have implications in the maintenance of intestinal homeostasis, epithelial barrier function, maintenance of commensals, and the overall tolerogenic function of PRRs in the gut mucosa. We also highlight the role of an aberrant microbial sensing at the epithelial barrier in the pathogenesis of inflammatory bowel disease (IBD) and the development of colorectal cancer.
... A sa partie carboxy-terminale, le domaine riche en Leucine (LRR) assure la détection des ligands spécifiques du récepteur NOD1 et NOD2(215,216). Le récepteur NOD1 reconnaît une fragment de motif de peptidoglycane bactérien présent dans la plupart des bactéries gram négatives et dans quelques bactéries gram positives comme Bacillus subtilis ou Listeria monocytogenes(217,218). La plus petite fraction de peptidoglycane reconnue par Nod1 est le γ-D-Glutamyl-meso-diaminopimelic acid(iE-DAP) provenant d'une structure contenant le N-acetyl glucosamine-N-acetyl muramic acid (GlcNAc-MurNAc (217,218). ...
... Other associations have been demonstrated with GWAS 217 I n r e v i e w studies but consistently replicated (64). The postulated pathogenesis of COPD includes protease-anti- 218 protease imbalance, recruitment of inflammatory cells, peri-bronchiolar fibrosis and oxidative stress. 219 ...
L’asthme est une maladie inflammatoire chronique des voies aériennesresponsable d’une morbi-mortalité significative et définie par une association entre dessymptômes cliniques et une obstruction bronchique variable. Les caractéristiques cardinalesde l’asthme sont une hyper-réactivité des voies aériennes, une inflammation bronchiqueassociée à une modification de la structure des bronches dont une hyperproduction demucus.L’ensemble de ces caractéristiques aboutit à de nombreuses présentations cliniques,appelées des phénotypes asthmatiques, qui sont la conséquence de multiples mécanismesphysiopathologiques distincts. Le phénotype asthmatique le plus fréquent est l’asthmeallergique dont les acariens représentent l’un des principaux pneumallergènes.L’hétérogénéité de cette pathologie requiert une approche globale incluant des travauxfondamentaux et une approche clinique translationnelle. Ces deux approches ont étédéveloppées dans cette thèse.Dans la première partie, l’implication d’un récepteur de l’immunité innée, lerécepteur Nod1, dans l’asthme allergique aux acariens a été investiguée. Ce récepteurreconnaît des fragments de peptidoglycanes bactériens et participe à la réponse immune. Ilest également impliqué dans la régulation du microbiote digestif. Chez les souris Nod1-/-, lesparamètres allergiques asthmatiques induits par les acariens sont réduits comparativementaux souris sauvages. Cette atténuation de la réponse allergique asthmatique n’est pas liée àune modification de la flore digestive des souris Nod1-/-. En revanche, les extraits d’acarienscontiennent une flore microbienne, principalement composée de bacille gram négatif,susceptible d’activer directement le récepteur Nod1 au niveau de l’épithélium des voiesrespiratoires. Cette activation participe à l’exacerbation de la réponse allergique asthmatiqueinduite aux acariens et offre une nouvelle perspective thérapeutique dans le traitement del’asthme allergique aux acariens.La deuxième partie de ce travail est consacrée à l’impact fonctionnel de bouchons demucus présents dans les voies aériennes de patients asthmatiques en fonction de leur statuttabagique en utilisant des outils précédemment développés. La présence de bouchons demucus est fréquente quel que soit le statut tabagique des asthmatiques. Une corrélationinverse a été retrouvée entre le nombre de segments pulmonaires présentant au moins unbouchon de mucus et l’obstruction des voies aériennes. Cette caractéristique clinique estcorrélée au pourcentage de polynucléaires éosinophiles dans les expectorations. Chez lespatients ayant un antécédent de tabagisme, la présence d’occlusions des voies aériennesest associée au pourcentage de neutrophiles. Les bouchons de mucus apparaissent commeun marqueur de sévérité de l’asthme et sont corrélés à différents types d’inflammationbronchique selon le statut tabagique.Cette thèse a contribué à faire avancer les connaissances fondamentales et clinicofonctionellesdans l’asthme. Ces résultats invitent à poursuivre les investigations dans cesdifférents domaines.
... Depending on the particular CLR-ligand partners, the interaction may lead to cellular immunosuppression or an inflammatory response 34 . NLRs, RLRs, ALRs, and cGAS are all present in the cytosol, but recognize different ligands: NLRs sense intracellular infection and stress, and the first NLRs that were described, NOD1 and NOD2, bind structures from bacterial peptidoglycans [35][36][37] . RLRs recognize dsRNA and are important in defence against viral infections 38,39 . ...
IL-12p70 is a crucial cytokine for T helper 1 (Th1) polarization and the generation of type 1 immunity that are required to fight cancer and pathogens. Therefore, strategies to optimize the production of IL-12p70 by human dendritic cells (DCs) may significantly improve the efficacy of vaccines and immunotherapies. However, the rules governing the production of IL-12p70 remain obscure. Here, we stimulated pattern recognition receptors (PRRs) representing all five families of PRRs, to evaluate their ability to elicit high production of IL-12p70 by monocyte-derived DCs. We used ten well-characterized agonists and stimulated DCs in vitro with either single agonists or 27 different combinations. We found that poly(I:C), which engages the RNA-sensing PRRs TLR3 and MDA5, and LPS which stimulates TLR4, were the only agonists that could elicit notable IL-12p70 production when used as single ligands. We identified six different combinations of PRR agonists, all containing either the TLR3/MDA5 agonist poly(I:C) or the TLR7/8 agonist R848, that could synergize to elicit high production of IL-12p70 by human DCs. Five of the six combinations also triggered high production of the antiviral and antitumor cytokine IFNβ. Overall, the tested PRR ligands could be divided into three groups depending on whether they triggered production of both IL-12p70 and IFNβ, only one of the two, or neither. Thus, combinations of PRR agonists were found to increase the production of IL-12p70 by human DCs in a synergistic manner, and we identified six PRR agonist combinations that may represent strong adjuvant candidates, in particular for therapeutic cancer vaccines.
... Gonococcal OMVs are endocytosed by epithelial cells in a dynamin-dependent manner ( Supplementary Fig. 2), and purified OMVs from both strain ATCC 49226 and contemporary clinical isolate ZJXSH86 (Fig. 1d) subsequently activated autophagy fluxes (Fig. 1e, f). Human NOD1 detects bacterial GlcNAc-MurNAc tripeptide motif (GM-Tri DAP )-containing peptidoglycan fragments 26 , while a gonococcal ΔldcA mutant is unable to generate and secrete GM-Tri DAP peptidoglycan and therefore escapes NOD1 sensing 9,27 and NOD1dependent autophagy targeting 9,15,16 . Gonococcal OMVs obtained from the ΔldcA mutant still activated autophagy, albeit at lower levels then OMVs from the WT strain (Fig. 1g, h), but OMVs from the ΔldcA mutant largely escaped direct autophagy targeting based on minimal colocalization with LC3 puncta (Fig. 1g). ...
The bacterial pathogen Neisseria gonorrhoeae is able to invade epithelial cells and survive intracellularly. During this process, it secretes outer membrane vesicles (OMVs), however, the mechanistic details for interactions between gonococcal OMVs and epithelial cells and their impact on intracellular survival are currently not established. Here, we show that gonococcal OMVs induce epithelial cell mitophagy to reduce mitochondrial secretion of reactive oxygen species (ROS) and enhance intracellular survival. We demonstrate that OMVs deliver PorB to mitochondria to dissipate the mitochondrial membrane potential, resulting in mitophagy induction through a conventional PINK1 and OPTN/NDP52 mechanism. Furthermore, PorB directly recruits the E3 ubiquitin ligase RNF213, which decorates PorB lysine residue 171 with K63-linked polyubiquitin to induce mitophagy in a p62-dependent manner. These results demonstrate a mechanism in which polyubiquitination of a bacterial virulence factor that targets mitochondria directs mitophagy processes to this organelle to prevent its secretion of deleterious ROS.
... Surprisingly, the pre-treatment with a synthetic anhydro-PGN effectively suppressed the LPS-induced proinflammatory cytokine expressions in murine macrophage RAW 264.7 cells in vitro. As Bifidobacterium anhydro-PGNs are non-agnostic to the canonical NOD1 and NOD2 immune receptors, (91)(92)(93)(94) the underlying mechanisms of their anti-inflammatory roles are yet to be elucidated. Importantly, our work suggests the anti-inflammatory effects of Bifidobacterium spp. as probiotics likely are attributed to such abundant anhydro-PGNs. ...
Peptidoglycan is an essential exoskeletal polymer present across all bacteria. The gut microbiota-derived peptidoglycan fragments (PGNs) are increasingly recognized as key effector molecules that impact host biology, offering attractive yet untapped potential to combat microbiome-associated diseases in humans. Unfortunately, comprehensive peptidoglycan profiling of gut bacteria has been hampered by the lack of a robust and automated analysis workflow. Currently, PGN identification still relies on manual deconvolutions of acquired tandem mass spectrometry (MS/MS) data, which are highly laborious and inconsistent. Recognizing the unique sugar and amino acid makeup of bacterial peptidoglycan and guided by the experimental MS/MS fragmentation patterns of known PGNs, we developed a computational tool PGN_MS2 that reliably simulates MS/MS spectra of PGNs. Integrating PGN_MS2 into the customizable in silico PGN database, we built an open-access PGN MS library of predicted MS/MS spectra for all molecules in the user-defined in silico PGN search space. With this library, automated searching and spectral matching can be used to identify PGN. We then performed comprehensive peptidoglycan profiling for several gut bacteria species, revealing distinct PGN structural features that may be implicated in microbiota-host crosstalk. Strikingly, the probiotic Bifidobacterium spp. has an exceedingly high proportion of anhydro-PGNs, which exhibit anti-inflammatory effects in vitro. We further identified MltG and RfpB homologs in Bifidobacterium as lytic transglycosylases (LTs), which demonstrate distinct substrate preferences to produce anhydro-PGNs. Overall, our novel PGN_MS2 prediction tool contributes to the robust and automated peptidoglycan analysis workflow, advancing efforts to elucidate the structures and functions of gut microbiota-derived PGNs in the host.
... NOD1 and NOD2 are functionally well described. NOD1 detects γ-D-glutamyl-meso- diaminopimelic acid (iE-DAP), and NOD2 recognizes muropeptides from bacterial peptidoglycan [102][103][104] . NLR signaling can induce the expression of costimulatory and coinhibitory molecules. ...
Our bodies are inhabited by trillions of microorganisms. The host immune system constantly interacts with the microbiota in barrier organs, including the intestines. Over decades, numerous studies have shown that our mucosal immune system is dynamically shaped by a variety of microbiota-derived signals. Elucidating the mediators of these interactions is an important step for understanding how the microbiota is linked to mucosal immune homeostasis and gut-associated diseases. Interestingly, the efficacy of cancer immunotherapies that manipulate costimulatory and coinhibitory pathways has been correlated with the gut microbiota. Moreover, adverse effects of these therapies in the gut are linked to dysregulation of the intestinal immune system. These findings suggest that costimulatory pathways in the immune system might serve as a bridge between the host immune system and the gut microbiota. Here, we review mechanisms by which commensal microorganisms signal immune cells and their potential impact on costimulation. We highlight how costimulatory pathways modulate the mucosal immune system through not only classical antigen-presenting cells but also innate lymphocytes, which are highly enriched in barrier organs. Finally, we discuss the adverse effects of immune checkpoint inhibitors in the gut and the possible relationship with the gut microbiota.
... Nucleotide-binding oligomerization domain-containing 1 and 2 (NOD1 and NOD2) proteins are intracellular pattern recognition receptors sensing bacterial infections and contributing to gastrointestinal homeostasis (Philpott et al, 2014;Kayama Hisako, 2020;Trindade & Chen, 2020). Receptor activation by bacterial peptidoglycan breakdown products, γ-D-Glu-mdiaminopimelic acid (iE-DAP) or muramyl-dipeptide (MDP), respectively (Chamaillard et al, 2003;Girardin et al, 2003Girardin et al, , 2016Inohara et al, 2003), triggers production of NF-κB and MAPK cascade activation. This results in transcriptional up-regulation and release of pro-inflammatory cytokines and antimicrobial peptides (Ferrand & Ferrero, 2013;Boyle et al, 2014;Philpott et al, 2014;Trindade & Chen, 2020). ...
RIPK2 is an essential adaptor for NOD signalling and its kinase domain is a drug target for NOD-related diseases, such as inflammatory bowel disease. However, recent work indicates that the phosphorylation activity of RIPK2 is dispensable for signalling and that inhibitors of both RIPK2 activity and RIPK2 ubiquitination prevent the essential interaction between RIPK2 and the BIR2 domain of XIAP, the key RIPK2 ubiquitin E3 ligase. Moreover, XIAP BIR2 antagonists also block this interaction. To reveal the molecular mechanisms involved, we combined native mass spectrometry, NMR, and cryo-electron microscopy to determine the structure of the RIPK2 kinase BIR2 domain complex and validated the interface with in cellulo assays. The structure shows that BIR2 binds across the RIPK2 kinase antiparallel dimer and provides an explanation for both inhibitory mechanisms. It also highlights why phosphorylation of the kinase activation loop is dispensable for signalling while revealing the structural role of RIPK2–K209 residue in the RIPK2–XIAP BIR2 interaction. Our results clarify the features of the RIPK2 conformation essential for its role as a scaffold protein for ubiquitination.
... Their structure is characterized by a central nucleotide-binding and oligomerization domain (NOD or NACHT) and C-terminal leucine-rich repeats (LRRs) as PAMP binding sites [188]. The NOD1 receptor senses gram-negative peptidoglycan [189,190], whereas the NOD2 receptor is a general sensor of bacterial peptidoglycan [191]. ...
Chronic airway inflammation is the cornerstone on which bronchial asthma arises, and in turn, chronic inflammation arises from a complex interplay between environmental factors such as allergens and pathogens and immune cells as well as structural cells constituting the airway mucosa. Airway epithelial cells (AECs) are at the center of these processes. On the one hand, they represent the borderline separating the body from its environment in order to keep inner homeostasis. The airway epithelium forms a multi-tiered, self-cleaning barrier that involves an unstirred, discontinuous mucous layer, the dense and rigid mesh of the glycocalyx, and the cellular layer itself, consisting of multiple, densely interconnected cell types. On the other hand, the airway epithelium represents an immunologically highly active tissue once its barrier has been penetrated: AECs play a pivotal role in releasing protective immunoglobulin A. They express a broad spectrum of pattern recognition receptors, enabling them to react to environmental stressors that overcome the mucosal barrier. By releasing alarmins—proinflammatory and regulatory cytokines—AECs play an active role in the formation, strategic orientation, and control of the subsequent defense reaction. Consequently, the airway epithelium is of vital importance to chronic inflammatory diseases, such as asthma.
... The activated RIPK2 later induces its autophosphorylation and lysine-63 (K63)-linked polyubiquitin, which promotes mitogen-activated protein (MAP) and the NF-κB signaling pathway. Transcription of multiple inflammatory factors is then activated [3]. Dysregulation of the NF-κB signaling pathway is associated with various diseases like inflammatory diseases [4], severe pulmonary sarcoidosis [5] and multiple sclerosis. ...
Receptor-interacting protein kinase 2 (RIPK2) belongs to the receptor-interacting protein family (RIPs), which is mainly distributed in the cytoplasm. RIPK2 is widely expressed in human tissues, and its mRNA level is highly expressed in the spleen, leukocytes, placenta, testis, and heart. RIPK2 is a dual-specificity kinase with multiple domains, which can interact with tumor necrosis factor receptor (TNFR), and participate in the Toll-like receptor (TLR) and nucleotide-binding oligomerization domain (NOD) signaling pathways. It is considered as a vital adapter molecule involved in the innate immunity, adaptive immunity, and apoptosis. Functionally, RIPK2 and its targeted small molecules are of great significance in inflammatory responses, autoimmune diseases and tumors. The present study reviews the molecule structure and biological functions of RIPK2, and its correlation between human diseases. In addition, we focus on the structure-activity relationship of small molecule inhibitors of RIPK2 and their therapeutic potential in human diseases.
... 7 Their recognition by PRRs belonging to various classes of proteins, such as nod-like receptors (NLRs) and peptidoglycan recognition proteins (PGRPs) in animals and lysine-motif (Lys-M) in plants, is necessary to mobilize host antibacterial defenses, but also to support developmental processes or activate behavior, to name a few. [8][9][10][11][12][13] For the past few decades, Drosophila melanogaster has served as a model for studying aspects of innate immunity that might otherwise be obscured by the actions of the adaptive immune response. [14][15][16][17] Previous work reveals that PGN sensing by PGRP proteins explains many of the interactions that take place between flies and bacteria. ...
Interactions between prokaryotes and eukaryotes require a dialogue between MAMPs and PRRs. In Drosophila, bacterial peptidoglycan is detected by PGRP receptors. While the components of the signaling cascades activated upon PGN/PGRP interactions are well characterized, little is known about the subcellular events that translate these early signaling steps into target gene transcription. Using a Drosophila enteric infection model, we show that gut-associated bacteria can induce the formation of intracellular PGRP-LE aggregates which colocalized with the early endosome marker Rab5. Combining microscopic and RNA-seq analysis, we demonstrate that RNAi inactivation of the endocytosis pathway in the Drosophila gut affects the expression of essential regulators of the NF-κB response leading not only to a disruption of the immune response locally in the gut but also at the systemic level. This work sheds new light on the involvement of the endocytosis pathway in the control of the gut response to intestinal bacterial infection.
... Specifically, NOD1 and NOD2 recognize the major components of PG D-glutamyl-mesodiaminopimelic acid (iE-DAP) and muramyl dipeptide (MDP), respectively (145,146). Their assembly recruits RIPK2 through characterized by a caspase-recruitment domain (CARD)-CARD interactions, leading to the formation of the TAB1/TAK1 complex (147). TAK1 becomes activated and then stimulates the downstream IKK complex, resulting in IKK phosphorylation, activation of the NF-kB signaling pathway, and the secretion of IL-6 and IL-8 (148). ...
Outer membrane vesicles (OMVs) are spherical, bilayered, and nanosized membrane vesicles that are secreted from gram-negative bacteria. OMVs play a pivotal role in delivering lipopolysaccharide, proteins and other virulence factors to target cells. Multiple studies have found that OMVs participate in various inflammatory diseases, including periodontal disease, gastrointestinal inflammation, pulmonary inflammation and sepsis, by triggering pattern recognition receptors, activating inflammasomes and inducing mitochondrial dysfunction. OMVs also affect inflammation in distant organs or tissues via long-distance cargo transport in various diseases, including atherosclerosis and Alzheimer’s disease. In this review, we primarily summarize the role of OMVs in inflammatory diseases, describe the mechanism through which OMVs participate in inflammatory signal cascades, and discuss the effects of OMVs on pathogenic processes in distant organs or tissues with the aim of providing novel insights into the role and mechanism of OMVs in inflammatory diseases and the prevention and treatment of OMV-mediated inflammatory diseases.
... PAMPs sensation by PRRs activates intracellular signal transduction pathways triggering stormy reactions such as antimicrobial activity and inflammatory response to eliminate the pathogenic microorganism (217). (237,238). PG delivered by the H. pylori CagT4SS is sensed by NOD1 in the cytoplasm of epithelial cells and causes the activation of NF-κB signalling and upregulation of pro-inflammatory immune responses (55). It was shown that the immunomodulatory glycoprotein olfactomedin 4 (OLFM4) targets NOD1 and NOD2 in H. pylori-infected cells (239). ...
Helicobacter pylori ( H. pylori ) is a worldwide spread bacterium, co-evolving with humans for at least 100 000 years. Despite the uncertainty about the mode of H. pylori transmission, the development of intra-gastric and extra-gastric diseases is attributed to this bacterium. The morphological transformation and production of heterogenic virulence factors enable H. pylori to overcome the harsh stomach environment. Using numerous potent disease-associated virulence factors makes H. pylori a prominent pathogenic bacterium. These bacterial determinants are adhesins (e.g., blood group antigen-binding adhesin (BabA)/sialic acid-binding adhesin (SabA)), enzymes (e.g., urease), toxins (e.g., vacuolating cytotoxin A (VacA)), and effector proteins (e.g., cytotoxin-associated gene A (CagA)) involved in colonisation, immune evasion, and disease induction. H. pylori not only cleverly evades the immune system but also robustly induces immune responses. This insidious bacterium employs various strategies to evade human innate and adaptive immune responses, leading to a life-long infection. Owing to the alteration of surface molecules, innate immune receptors couldn't recognise this bacterium; moreover, modulation of effector T cells subverts adaptive immune response. Most of the infected humans are asymptomatic and only a few of them present severe clinical outcomes. Therefore, the identification of virulence factors will pave the way for the prediction of infection severity and the development of an effective vaccine. H. pylori virulence factors are hereby comprehensively reviewed and the bacterium evasion from the immune response is properly discussed.
... NOD1 and NOD2 are cytosolic PRRs that sense bacterial PGN fragmentsto initiate pro-inflammatory responses [200]. The minimal ligand for NOD1 is γ-D-glutamyl-mesodiaminopimelic acid (iE-DAP), which is part of the cell wall of Gram-negative and certain Gram-positive bacteria [201,202], while NOD2 recognizes muramyl dipeptide (MDP), which is present in both bacterial categories [203,204]. Upon activation, NOD1 and NOD2 homodimerize and recruit RIPK2 (RIP2), which initiates inflammation via NF-κB activation and the induction of adaptive immune responses to defend against intracellular bacteria. ...
Obesity and its associated metabolic morbidities have been and still are on the rise, posing a major challenge to health care systems worldwide. It has become evident over the last decades that a low-grade inflammatory response, primarily proceeding from the adipose tissue (AT), essentially contributes to adiposity-associated comorbidities, most prominently insulin resistance (IR), atherosclerosis and liver diseases. In mouse models, the release of pro-inflammatory cytokines such as TNF-alpha (TNF-α) and interleukin (IL)-1β and the imprinting of immune cells to a pro-inflammatory phenotype in AT play an important role. However, the underlying genetic and molecular determinants are not yet understood in detail. Recent evidence demonstrates that nucleotide-binding and oligomerization domain (NOD)-like receptor (NLR) family proteins, a group of cytosolic pattern recognition receptors (PRR), contribute to the development and control of obesity and obesity-associated inflammatory responses. In this article, we review the current state of research on the role of NLR proteins in obesity and discuss the possible mechanisms leading to and the outcomes of NLR activation in the obesity-associated morbidities IR, type 2 diabetes mellitus (T2DM), atherosclerosis and non-alcoholic fatty liver disease (NAFLD) and discuss emerging ideas about possibilities for NLR-based therapeutic interventions of metabolic diseases.
... Two well-studied NLR proteins are NOD1 and NOD2. The NOD1 recognizes molecules containing D-Glu-mDAP (205), whereas NOD2 are vital for the regulation of NAM-D-Ala-D-Glu unit of the molecules (206). Recognition of muropeptide from Lactobacilli by NOD2 can induce anti-inflammatory properties and protect mice from colitis development (94). ...
... Treatment of cell cultures with 2′,3′-cyclic GMP-AMP (cGAMP) or poly(I:C) was performed as previously described 53,54 . For cGAMP treatment, cells were incubated in digitonin permeabilization buffer (50 mM Article HEPES pH 7.3, 100 mM KCl, 3 mM MgCl 2 , 0.1 mM DTT, 85 mM sucrose, 0.2% BSA, and 1 μg ml −1 digitonin) for 30 min, then fresh medium was added with 0.5 μg ml −1 or 4 μg ml −1 cGAMP (Invivogen, tlrl-nacga23) or no additions. ...
Metastasis frequently develops from disseminated cancer cells that remain dormant after the apparently successful treatment of a primary tumour. These cells fluctuate between an immune-evasive quiescent state and a proliferative state liable to immune-mediated elimination1–6. Little is known about the clearing of reawakened metastatic cells and how this process could be therapeutically activated to eliminate residual disease in patients. Here we use models of indolent lung adenocarcinoma metastasis to identify cancer cell-intrinsic determinants of immune reactivity during exit from dormancy. Genetic screens of tumour-intrinsic immune regulators identified the stimulator of interferon genes (STING) pathway as a suppressor of metastatic outbreak. STING activity increases in metastatic progenitors that re-enter the cell cycle and is dampened by hypermethylation of the STING promoter and enhancer in breakthrough metastases or by chromatin repression in cells re-entering dormancy in response to TGFβ. STING expression in cancer cells derived from spontaneous metastases suppresses their outgrowth. Systemic treatment of mice with STING agonists eliminates dormant metastasis and prevents spontaneous outbreaks in a T cell- and natural killer cell-dependent manner—these effects require cancer cell STING function. Thus, STING provides a checkpoint against the progression of dormant metastasis and a therapeutically actionable strategy for the prevention of disease relapse.
... NLR has been demonstrated to be crucial in the congenital immunological illness inflammatory bowel disease. NOD1 and NOD2 intracellular protein families are Nod molecules [30], and NOD1 is primarily found in Ellan-negative bacteria [31]. As gram-negative bacteria, Non-O1 V. cholerae also causes zoonotic acute diarrheal infectious illness. ...
Non-O1 Vibrio cholerae, a member of the Vibrio family, could cause gastrointestinal infection of Macrobrachium rosenbergii and result in significant economic losses. However, few studies on microRNA immunity related to non-O1 V. cholerae infection of M. rosenbergii. The aim of this study was to elucidate the mechanism of miRNA in the potential immune response of M. rosenbergii. to non-O1 V. cholerae MSVC-GY01 infection by transcriptome sequencing. Following quality screening, the control group received 10,616,712 clean reads, whereas the infected group received 9,727,616. The miRNA sequences in the two samples are extremely consistent and have a length of roughly 23 nt. In all, 871 known miRNAs were discovered, with 279 differentially expressed miRNAs (DEMs). Meanwhile, 62 novel miRNAs were predicted, including 43 DEMs. In order to understand the immune-related biological functions of DEMs, target genes were predicted. Pathway function annotation analysis showed that non-O1 V. cholerae affected the NOD-like receptor signaling pathway, RIG-I-like receptor signaling pathway, and Toll-like receptor signaling pathway, suggesting that miRNAs in the hepatopancreas play a key role in immune responses to pattern recognition receptors. Twelve DEMs were randomly selected for Quantitative Real-time PCR (qRT-PCR). Overall, the expression trends of qRT-PCR were consistent with the sequencing results. These findings corroborate the immunomodulatory function of miRNA in M. rosenbergii against non-O1 V. cholerae infection and provide guidance for the prevention and treatment of related illnesses.
... Peptidoglycan (PGN) is the conserved structure of Gramnegative bacterial which could be recognized by nucleotidebinding oligomerization domain-1 (NOD1) (72). Once activated by PGN, NOD1 could further activate the downstream NF-κB (72) and MAPK (73) pathway through interacting with a caspase activated and recruitment domain (CARD) and its adaptor protein, the receptor-interacting protein 2 (RIP2) (74)(75)(76). ...
Colorectal cancer (CRC) is one of the most prevalent and life-threatening cancer types with limited therapeutic options worldwide. Gut microbiota has been recognized as the pivotal determinant in maintaining gastrointestinal (GI) tract homeostasis, while dysbiosis of gut microbiota contributes to CRC development. Recently, the beneficial role of postbiotics, a new concept in describing microorganism derived substances, in CRC has been uncovered by various studies. However, a comprehensive characterization of the molecular identity, mechanism of action, or routes of administration of postbiotics, particularly their role in CRC, is still lacking. In this review, we outline the current state of research toward the beneficial effects of gut microbiota derived postbiotics against CRC, which will represent the key elements of future precision-medicine approaches in the development of novel therapeutic strategies targeting gut microbiota to improve treatment outcomes in CRC.
... In addition, PGN fragments stimulate inflammatory responses via NOD1 and NOD2, which are cytoplasmic proteins that sense bacterial byproducts. It was reported that PGN-derived, gram-positive bacteria stimulate NOD2 [206][207][208], while gram-negative PGN is a potent stimulator of NOD1 and a weak stimulator of NOD2 [206,207,209]. Although NOD1 is expressed in most tissues, NOD2 is especially expressed in immune cells, such as macrophages [210,211]. ...
Periodontitis is the sixth most common chronic inflammatory disease, destroying the tissues supporting the teeth. There are three distinct stages in periodontitis: infection, inflammation, and tissue destruction, where each stage has its own characteristics and hence its line of treatment. Illuminating the underlying mechanisms of alveolar bone loss is vital in the treatment of periodontitis to allow for subsequent reconstruction of the periodontium. Bone cells, including osteoclasts, osteoblasts, and bone marrow stromal cells, classically were thought to control bone destruction in periodontitis. Lately, osteocytes were found to assist in inflammation-related bone remodeling besides being able to initiate physiological bone remodeling. Furthermore, mesenchymal stem cells (MSCs) either transplanted or homed exhibit highly immunosuppressive properties, such as preventing monocytes/hematopoietic precursor differentiation and downregulating excessive release of inflammatory cytokines. In the early stages of bone regeneration, an acute inflammatory response is critical for the recruitment of MSCs, controlling their migration, and their differentiation. Later during bone remodeling, the interaction and balance between proinflammatory and anti-inflammatory cytokines could regulate MSC properties, resulting in either bone formation or bone resorption. This narrative review elaborates on the important interactions between inflammatory stimuli during periodontal diseases, bone cells, MSCs, and subsequent bone regeneration or bone resorption. Understanding these concepts will open up new possibilities for promoting bone regeneration and hindering bone loss caused by periodontal diseases.
... NODs are cytosolic pattern recognition molecules that bind to PGN, a component of cell walls common to Gram-positive and Gram-negative bacteria. NOD1 typically responds to a unique muropeptide motif called γ-D-glutamyl-meso-diaminopimelic acid (iE-DAP) which is present in Gram-negative bacterial PGN and PGN of particular Grampositive bacteria such as Bacillus subtilis, while NOD2 recognized muramyl dipeptide (MDP) and mediates the response to both Gram-positive and Gram-negative PGN [31,32]. Synergized with TLRs, activation of NODs leads to cytokine and antimicrobial peptides (AMPs) production via distinct molecular mechanisms [33]. ...
Introduction
Innate immunity serves as the frontline to combat invading pathogens. Oral microbiota is the total collection of microorganisms colonized within the oral cavity. By recognizing the resident microorganisms through pattern recognition receptors, innate immunity is capable of interacting with oral microbiota and maintaining homeostasis. Dysregulation of interaction may lead to the pathogenesis of several oral diseases. Decoding the crosstalk between oral microbiota and innate immunity may be contributory to developing novel therapies for preventing and treating oral diseases.
Areas covered
This article reviewed pattern recognition receptors in the recognition of oral microbiota, the reciprocal interaction between innate immunity and oral microbiota, and discussed how the dysregulation of this relationship lead to the pathogenesis and development of oral diseases.
Expert opinion
Many studies have been conducted to illustrate the relationship between oral microbiota and innate immunity and its role in the occurrence of different oral diseases. The impact and mechanisms of innate immune cells on oral microbiota and the mechanisms of dysbiotic microbiota in altering innate immunity are still needed to be investigated. Altering the oral microbiota might be a possible solution for treating and preventing oral diseases.
... NLRs are important PRRs that sense bacterial products in the cytoplasm, and they play crucial roles in the recognition of bacterial or viral invasion in eukaryotic cells (47,99). Key members of the NLR pathways include the NLR family members nucleotide oligomerization domain 1 and 2 (NOD1 and NOD2), receptor interacting serine/threonine kinase 2 (RIPK2), NEMO, IKKs (IKKa and IKKb), and p50/p65. ...
Nuclear factor-kappa B (NF-κB) pathways have a close relationship with many diseases, especially in terms of the regulation of inflammation and the immune response. Non-coding RNAs (ncRNAs) are a heterogeneous subset of endogenous RNAs that directly affect cellular function in the absence of proteins or peptide products; these include microRNAs (miRNAs), long noncoding RNAs (lncRNAs), circular RNAs (circRNAs), etc. Studies on the roles of ncRNAs in targeting the NF-κB pathways in aquatic animals are scarce. A few research studies have confirmed detailed regulatory mechanisms among ncRNAs and the NF-κB pathways in aquatic animals. This comprehensive review is presented concerning ncRNAs targeting the NF-κB pathway in aquatic animals and provides new insights into NF-κB pathways regulatory mechanisms of aquatic animals. The review discusses new possibilities for developing non-coding-RNA-based antiviral applications in fisheries.
Mechanisms by which Mycobacterium tuberculosis (Mtb) evades pathogen recognition receptor activation during infection may offer insights for the development of improved tuberculosis (TB) vaccines. Whilst Mtb elicits NOD-2 activation through host recognition of its peptidoglycan-derived muramyl dipeptide (MDP), it masks the endogenous NOD-1 ligand through amidation of glutamate at the second position in peptidoglycan side-chains. As the current BCG vaccine is derived from pathogenic mycobacteria, a similar situation prevails. To alleviate this masking ability and to potentially improve efficacy of the BCG vaccine, we used CRISPRi to inhibit expression of the essential enzyme pair, MurT-GatD, implicated in amidation of peptidoglycan side-chains. We demonstrate that depletion of these enzymes results in reduced growth, cell wall defects, increased susceptibility to antibiotics, altered spatial localization of new peptidoglycan and increased NOD-1 expression in macrophages. In cell culture experiments, training of a human monocyte cell line with this recombinant BCG yielded improved control of Mtb growth. In the murine model of TB infection, we demonstrate that depletion of MurT-GatD in BCG, which is expected to unmask the D-glutamate diaminopimelate (iE-DAP) NOD-1 ligand, yields superior prevention of TB disease compared to the standard BCG vaccine. In vitro and in vivo experiments in this study demonstrate the feasibility of gene regulation platforms such as CRISPRi to alter antigen presentation in BCG in a bespoke manner that tunes immunity towards more effective protection against TB disease.
Inflammatory bowel disease (IBD) is an idiopathic, chronic condition characterized by episodes of inflammation in the gastrointestinal tract. The nuclear factor κB (NF-κB) system describes a family of dimeric transcription factors. Canonical NF-κB signaling is stimulated by and enhances inflammation, whereas noncanonical NF-κB signaling contributes to immune organogenesis. Dysregulation of NF-κB factors drives various inflammatory pathologies, including IBD. Signals from many immune sensors activate NF-κB subunits in the intestine, which maintain an equilibrium between local microbiota and host responses. Genetic association studies of patients with IBD and preclinical mouse models confirm the importance of the NF-κB system in host defense in the gut. Other studies have investigated the roles of these factors in intestinal barrier function and in inflammatory gut pathologies associated with IBD. NF-κB signaling modulates innate and adaptive immune responses and the production of immunoregulatory proteins, anti-inflammatory cytokines, antimicrobial peptides, and other tolerogenic factors in the intestine. Furthermore, genetic studies have revealed critical cell type–specific roles for NF-κB proteins in intestinal immune homeostasis, inflammation, and restitution that contribute to the etiopathology of IBD-associated manifestations. Here, we summarize our knowledge of the roles of these NF-κB pathways, which are activated in different intestinal cell types by specific ligands, and their cross-talk, in fueling aberrant intestinal inflammation. We argue that an in-depth understanding of aberrant immune signaling mechanisms may hold the key to identifying predictive or prognostic biomarkers and developing better therapeutics against inflammatory gut pathologies.
Peptidoglycan is an essential exoskeletal polymer across all bacteria. Gut microbiota-derived peptidoglycan fragments (PGNs) are increasingly recognized as key effector molecules that impact host biology. However, the current peptidoglycan analysis workflow relies on laborious manual identification from tandem mass spectrometry (MS/MS) data, impeding the discovery of novel bioactive PGNs in the gut microbiota. In this work, we built a computational tool PGN_MS2 that reliably simulates MS/MS spectra of PGNs and integrated it into the user-defined MS library of in silico PGN search space, facilitating automated PGN identification. Empowered by PGN_MS2, we comprehensively profiled gut bacterial peptidoglycan composition. Strikingly, the probiotic Bifidobacterium spp. manifests an abundant amount of the 1,6-anhydro-MurNAc moiety that is distinct from Gram-positive bacteria. In addition to biochemical characterization of three putative lytic transglycosylases (LTs) that are responsible for anhydro-PGN production in Bifidobacterium, we established that these 1,6-anhydro-PGNs exhibit potent anti-inflammatory activity in vitro, offering novel insights into Bifidobacterium-derived PGNs as molecular signals in gut microbiota-host crosstalk.
Pattern recognition receptors (PRRs) represent a re-emerging class of therapeutic targets for vaccine adjuvants, inflammatory diseases and cancer. In this review article, we summarize exciting developments in discovery and characterization of small molecule PRR modulators, focusing on Toll-like receptors (TLRs), NOD-like receptors (NLRs) and the cGAS-STING pathway. We also highlight PRRs that are currently lacking small molecule modulators and opportunities for chemical biology and therapeutic discovery.
The research identified a list of molecules involved in the mechanisms of innate immunity in cattle and the recognition of bacterial pathogens. The current list of molecular receptors has expanded to include TLR receptors and the recently defined NOD-like receptors (NLRs): NOD, NALP, NAIP, and IPAF. TLR molecules are designed to transmit a ligand-binding signal on the cell surface or endosome and activate specific molecules of bacterial origin in the cytosol, such as peptidoglycans, RNA, toxins and flagellins. The obtained data on the molecular structure of TLR and NLR receptors indicate their anti-inflammatory role, mediated by signals through nuclear transcription factor κB and activation of caspase-1 in the inflammasome. It has been shown that the role of immunosensors of extracellular and intracellular perception of bacteria in regulating inflammation is synergistic. Mutations in TLR and NOD receptors are associated with autoimmune inflammatory syndromes. This review examines the body's ways of recognising intracellular pathogens, describes the problem of their mimicry from the animal immune system, and the molecular mechanisms of such interactions. Variants of molecular interactions of innate immune receptors with peptidoglycans, bacterial DNA and toxins, cell wall compartments, and bacterial flagellin receptors are also considered. This study aimed to analyse the current understanding of the genetic and molecular structure of the immune response to bacterial environmental factors and the mechanisms and characteristics of the reaction of the animal body.
Introduction. Hepatitis C is a liver disease with high chronicity, the cause of cirrhosis and hepatocarcinoma. The main obstacle to controlling hepatitis C is the lack of vaccines.
The aim of the work was to compare the immunogenic activity of nonstructural recombinant proteins NS3, NS4 and NS5B of hepatitis C virus (HCV) as components of a subunit candidate vaccine and to analyze the adjuvant properties of two available commercial drugs, polymuramil and pyrogenalum.
Materials and methods. BALB/c, DBA/2J and C57BL/6 mice were immunized with nonstructural proteins without adjuvants or with polymuramyl (NOD1 and NOD2 agonist) and pyrogenalum (TLR-4 agonist). The activity of antibodies was determined in ELISA, the cellular response by antigen-specific lymphocyte proliferation and by production of IFN- in vitro.
Results. Recombinant proteins showed different immunogenicity. NS4 induced antibodies more efficiently than NS3 and NS5B. Significant differences were found in the immune response of three inbred lines mice: the level of IFN- in BALB/c and DBA/2J mice induced by NS5B protein was 30 times higher than in C57Bl/6 mice. In contrast, the induction of antibodies in BALB/c mice was lower than in C57Bl/6 and DBA/2J. Polymuramil did not increase the humoral response to NS5B and enhanced the cellular response only in C57BL/6 mice. The combined use of polymuramil with pyrogenalum significantly increased both the humoral and cellular response of mice to all recombinant HCV proteins.
Conclusion. Different immunogenic properties and different functions of recombinant non-structural HCV proteins indicate the feasibility of their combined inclusion in subunit vaccines. It was established for the first time that immunization with HCV proteins with a complex adjuvant (polymuramyl + pyrogenalum) has a synergistic effect, significantly exceeding the effect of each of them separately.
Methanogenic archaea (methanogens) represent a diverse group of microorganisms that inhabit various environmental and host-associated microbiomes. These organisms play an essential role in global carbon cycling given their ability to produce methane, a potent greenhouse gas, as a by-product of their energy production. Recent advances in culture-independent and -dependent studies have highlighted an increased prevalence of methanogens in the host-associated microbiome of diverse animal species. Moreover, there is increasing evidence that methanogens, and/or the methane they produce, may play a substantial role in human health and disease. This review addresses the expanding host-range and the emerging view of host-specific adaptations in methanogen biology and ecology, and the implications for host health and disease.
Parasitic protozoans such as Trypanosoma and Plasmodium cause many of the most prevalent and dreadful diseases in humans. The survival of these parasitic protozoans relies on the successful evasion of the host organism immune system. These include invasion and replication with host cells, as well as alterations of their surface proteins. In some cases, products from parasitic protozoans are responsible for immunosuppression observed in the host organism and it is associated with antigenic mimicry, which is usually linked to diseases induced by these parasites. Other immune responses to protozoans includes activation of T cells and inflammatory cascade of host organism. The evasion strategies used by parasitic protozoans differ depending on the stage of their life cycle, the route of entry into host organisms and the environment in which they exist. Therefore, this chapter highlights the various immune response mechanisms to parasitic protozoans.
Vaccines comprising innovative adjuvants are rapidly reaching advanced translational stages, such as the authorized nanotechnology adjuvants in mRNA vaccines against COVID-19 worldwide, offering new strategies to effectively combat diseases threatening human health. Adjuvants are vital ingredients in vaccines, which can augment the degree, extensiveness, and longevity of antigen specific immune response. The advances in the modulation of physicochemical properties of nanoplatforms elevate the capability of adjuvants in initiating the innate immune system and adaptive immunity, offering immense potential for developing vaccines against hard-to-target infectious diseases and cancer. In this review, we provide an essential introduction of the basic principles of prophylactic and therapeutic vaccination, key roles of adjuvants in augmenting and shaping immunity to achieve desired outcomes and effectiveness, and the physiochemical properties and action mechanisms of clinically approved adjuvants for humans. We particularly focus on the preclinical and clinical progress of highly immunogenic emerging nanotechnology adjuvants formulated in vaccines for cancer treatment or infectious disease prevention. We deliberate on how the immune system can sense and respond to the physicochemical cues (e.g., chirality, deformability, solubility, topology, and chemical structures) of nanotechnology adjuvants incorporated in the vaccines. Finally, we propose possible strategies to accelerate the clinical implementation of nanotechnology adjuvanted vaccines, such as in-depth elucidation of nano-immuno interactions, antigen identification and optimization by the deployment of high-dimensional multiomics analysis approaches, encouraging close collaborations among scientists from different scientific disciplines and aggressive exploration of novel nanotechnologies.
The oral organism Tannerella forsythia is auxotrophic for peptidoglycan amino sugar N-acetylmuramic acid (MurNAc). It survives in the oral cavity by scavenging MurNAc- and MurNAc-linked peptidoglycan fragments (muropeptides) secreted by co-habiting bacteria such as Fusobacterium nucleatum with which it forms synergistic biofilms. Muropeptides, MurNAc-l-Ala-d-isoGln (MDP, muramyl dipeptide) and d-γ-glutamyl-meso-DAP (iE-DAP dipeptide), are strong immunostimulatory molecules that activate nucleotide oligomerization domain (NOD)-like innate immune receptors and induce the expression of inflammatory cytokines and antimicrobial peptides. In this study, we utilized an in vitro T. forsythia-F. nucleatum co-culture model to determine if T. forsythia can selectively scavenge NOD ligands from the environment and impact NOD-mediated inflammation. The results showed that NOD-stimulatory molecules were secreted by F. nucleatum in the spent culture broth, which subsequently induced cytokine and antimicrobial peptide expression in oral epithelial cells. In the spent broth from T. forsythia-F. nucleatum co-cultures, the NOD-stimulatory activity was significantly reduced. These data indicated that F. nucleatum releases NOD2-stimulatory muropeptides in the environment, and T. forsythia can effectively scavenge the muropeptides released by co-habiting bacteria to dampen NOD-mediated host responses. This proof-of-principle study demonstrated that peptidoglycan scavenging by T. forsythia can impact the innate immunity of oral epithelium by dampening NOD activation.
Neospora caninum, an intracellular protozoan parasite, causes neosporosis resulting in major losses in the livestock industry worldwide. However, no effective drugs or vaccines have been developed to control neosporosis. An in-depth study on the immune response against N. caninum could help to search for effective approaches to prevent and treat neosporosis. The host unfolded protein response (UPR) functions as a double-edged sword in several protozoan parasite infections, either to initiate immune responses or to help parasite survival. In this study, the roles of the UPR in N. caninum infection in vitro and in vivo were explored, and the mechanism of the UPR in resistance to N. caninum infection was analyzed. The results revealed that N. caninum triggered the UPR in mouse macrophages, such as the activation of the IRE1 and PERK branches, but not the ATF6 branch. Inhibition of the IRE1α-XBP1s branch increased the N. caninum number both in vitro and in vivo, while inhibition of the PERK branch did not affect the parasite number. Furthermore, inhibition of the IRE1α-XBP1s branch reduced the production of cytokines by inhibiting NOD2 signalling and its downstream NF-κB and MAPK pathways. Taken together, the results of this study suggest that the UPR is involved in the resistance of N. caninum infection via the IRE1α-XBP1s branch by regulating NOD2 and its downstream NF-κB and MAPK pathways to induce the production of inflammatory cytokines, which provides a new perspective for the research and development of anti-N. caninum drugs.
Nucleotide-binding oligomerization domain-containing protein 1 and 2 (NOD 1/2) are important cytosolic pattern recognition receptors that initiate host immune response. The dysregulation of NOD signaling is highly associated with inflammatory bowel disease (IBD) that needs novel treatment options. Receptor-interacting protein kinase 2 (RIPK2) is a critical mediator of NOD signaling and considered a promising therapeutic target for IBD treatment. However, there are currently no RIPK2 inhibitors available for clinical use. Here, we report the discovery and characterization of Zharp2-1 as a novel and potent RIPK2 inhibitor that effectively blocks RIPK2 kinase function and NOD-mediated NF-κB/MAPK activation in both human and mouse cell lines. Zharp2-1 exhibits significantly superior solubility compared to the non-prodrug form of the advanced RIPK2 inhibitor prodrug GSK2983559. The improved solubility combined with favorable in vitro metabolic stability translated to excellent in vivo pharmacokinetic profiles for Zharp2-1. In addition, Zharp2-1 demonstrates better effects than GSK2983559 in inhibiting the muramyl dipeptide (MDP)-induced production of pro-inflammatory cytokines in human peripheral blood mononuclear cells (PBMCs) and MDP-induced peritonitis in mice. Furthermore, Zharp2-1 markedly reduces Listeria monocytogenes infection-induced cytokines release in both human and mouse cells. Importantly, Zharp2-1 significantly ameliorates DNBS-induced colitis in rats and suppressed pro-inflammatory cytokine release in intestinal specimens from IBD patients. Collectively, our findings indicate that Zharp2-1 is a promising RIPK2 inhibitor with the potential to be further developed for IBD therapy.
The gut microbiota plays a key role in host health and disease, particularly through their interactions with the immune system. Intestinal homeostasis is dependent on the symbiotic relationships between the host and the diverse gut microbiota, which is influenced by the highly co-evolved immune-microbiota interactions. The first step of the interaction between the host and the gut microbiota is the sensing of the gut microbes by the host immune system. In this review, we describe the cells of the host immune system and the proteins that sense the components and metabolites of the gut microbes. We further highlight the essential roles of pattern recognition receptors (PRRs), the G protein coupled receptors (GPCRs), aryl hydrocarbon receptor (AHR) and the nuclear receptors expressed in the intestinal epithelial cells (IECs) and the intestine-resident immune cells. We also discuss the mechanisms by which the disruption of microbial sensing because of genetic or environmental factors causes human diseases such as the inflammatory bowel disease (IBD).
The characterization of microbiota mechanisms in health and disease has reinvigorated pattern recognition receptors as prominent targets for immunotherapy. Notably, our recent studies on Enterococcus species revealed peptidoglycan remodeling and activation of NOD2 as key mechanisms for microbiota enhancement of immune checkpoint inhibitor therapy. Inspired by this work and other studies of NOD2 activation, we performed in silico ligand screening and developed N-arylpyrazole dipeptides as novel NOD2 agonists. Importantly, our N-arylpyrazole NOD2 agonist is enantiomer-specific and effective at promoting immune checkpoint inhibitor therapy and requires NOD2 for activity in vivo. Given the significant functions of NOD2 in innate and adaptive immunity, these next-generation agonists afford new therapeutic leads and adjuvants for a variety of NOD2-responsive diseases.
Bacterial cell wall peptidoglycan is composed of innate immune ligands and, due to its important structural role, also regulates access to many other innate immune ligands contained within the bacteria. There is a growing body of literature demonstrating how innate immune recognition impacts the metabolic functions of immune cells and how metabolic changes are not only important to inflammatory responses but are often essential. Peptidoglycan is primarily sensed in the context of the whole bacteria during lysosomal degradation; consequently, the innate immune receptors for peptidoglycan are primarily intracellular cytosolic innate immune sensors. However, during bacterial growth, peptidoglycan fragments are shed and can be found in the bloodstream of humans and mice, not only during infection but also derived from the abundant bacterial component of the gut microbiota. These peptidoglycan fragments influence cells throughout the body and are important for regulating inflammation and whole-body metabolic function. Therefore, it is important to understand how peptidoglycan-induced signals in innate immune cells and cells throughout the body interact to regulate how the body responds to both pathogenic and nonpathogenic bacteria. This mini-review will highlight key research regarding how cellular metabolism shifts in response to peptidoglycan and how systemic peptidoglycan sensing impacts whole-body metabolic function.
Interactions between gut microbiome and host immune system are fundamental to maintaining the intestinal mucosal barrier and homeostasis. At the host-gut microbiome interface, cell wall-derived molecules from gut commensal bacteria have been reported to play a pivotal role in training and remodeling host immune responses. In this article, we review gut bacterial cell wall-derived molecules with characterized chemical structures, including peptidoglycan and lipid-related molecules that impact host health and disease processes via regulating innate and adaptive immunity. Also, we aim to discuss the structures, immune responses and underlying mechanisms of these immunogenic molecules. Based on current advances, we propose cell wall-derived components as important sources of medicinal agents for the treatment of infection and immune diseases.
The mammalian NLR gene family was first reported over 20 years ago, although several genes that were later grouped into the family were already known at that time. Although it is widely known that NLRs include inflammasome receptors and/or sensors that promote the maturation of caspase 1, IL-1β, IL-18 and gasdermin D to drive inflammation and cell death, the other functions of NLR family members are less well appreciated by the scientific community. Examples include MHC class II transactivator (CIITA), a master transcriptional activator of MHC class II genes, which was the first mammalian NBD-LRR-containing protein to be identified, and NLRC5, which regulates the expression of MHC class I genes. Other NLRs govern key inflammatory signalling pathways or interferon responses, and several NLR family members serve as negative regulators of innate immune responses. Multiple NLRs regulate the balance of cell death, cell survival, autophagy, mitophagy and even cellular metabolism. Perhaps the least discussed group of NLRs are those with functions in the mammalian reproductive system. The focus of this Review is to provide a synopsis of the NLR family, including both the intensively studied and the underappreciated members. We focus on the function, structure and disease relevance of NLRs and highlight issues that have received less attention in the NLR field. We hope this may serve as an impetus for future research on the conventional and non-conventional roles of NLRs within and beyond the immune system.
Receptor-interacting serine/threonine kinase 2 (RIPK2) is a vital immunomodulator that plays critical roles in nucleotide-binding oligomerization domain 1 (NOD1), NOD2, and Toll-like receptors (TLRs) signaling. Stimulated NOD1 and NOD2 interact with RIPK2 and lead to the activation of nuclear factor kappa B (NF-κB) and mitogen-activated protein kinases (MAPK), followed by the production of pro-inflammatory cytokines such as TNF-α, IL-6, and IL-12/23. Defects in NOD/RIPK2 signaling are associated with numerous inflammatory diseases, including asthma, sarcoidosis, inflammatory bowel disease (Crohn's disease and ulcerative colitis), multiple sclerosis, and Blau syndrome. As RIPK2 is a crucial element of innate immunity, small molecules regulating RIPK2 functions are attractive to establish novel immunotherapies. The increased interest in developing RIPK2 inhibitors has led to the clinical investigations of novel drug candidates. In this review, we attempt to summarize recent advances in the development of RIPK2 inhibitors and degraders.
Many clinically important enteric pathogens initiate disease by invading and passing through the intestinal epithelium, a
process accompanied by increased epithelial expression of proinflammatory cytokines. To further define the role intestinal
epithelial cells play in initiating and modulating the host response to infection with invasive bacteria, hybrid selection
on high density cDNA arrays was used to characterize the mRNA expression profile of ∼4,300 genes in human intestinal epithelial
cells after infection with the prototypic invasive bacteria,Salmonella. Selected findings were further evaluated by reverse transcription-polymerase chain reaction, Northern blot analysis, and
protein assays. Epithelial infection withSalmonella significantly up-regulated mRNA expression of a relatively small fraction of all genes tested. Of these, several cytokines
(granulocyte colony-stimulating factor, inhibin A, Epstein-Barr virus-induced gene 3, interleukin-8, macrophage inflammatory
protein-2α), kinases (TKT, Eck, HEK), transcription factors (interferon regulatory factor-1), and HLA class I were the most
prominent. Furthermore, the transcription factor NF-κB is shown to be important for inducible mRNA expression for a broad
group of genes tested. These findings expand the repertoire of known epithelial cell responses to infection with an invasive
enteric pathogen. The results also show that evaluation of mRNA expression profiles by cDNA array analysis is a powerful approach
to characterizing and understanding host-pathogen interactions.
Epithelial cells are refractory to extracellular lipopolysaccharide (LPS), yet when presented inside the cell, it is capable of initiating an inflammatory response. Using invasive Shigella flexneri to deliver LPS into the cytosol, we examined how this factor, once intracellular, activates both NF-kappaB and c-Jun N-terminal kinase (JNK). Surprisingly, the mode of activation is distinct from that induced by toll-like receptors (TLRs), which mediate LPS responsiveness from the outside-in. Instead, our findings demonstrate that this response is mediated by a cytosolic, plant disease resistance-like protein called CARD4/Nod1. Biochemical studies reveal enhanced oligomerization of CARD4 upon S. flexneri infection, an event necessary for NF-kappaB induction. Dominant-negative versions of CARD4 block activation of NF-kappaB and JNK by S. flexneri as well as microinjected LPS. Finally, we showed that invasive S. flexneri triggers the formation of a transient complex involving CARD4, RICK and the IKK complex. This study demonstrates that in addition to the extracellular LPS sensing system mediated by TLRs, mammalian cells also possess a cytoplasmic means of LPS detection via a molecule that is related to plant disease-resistance proteins.
Apaf-1 and Nod1 are members of a protein family, each of which contains a caspase recruitment domain (CARD) linked to a nucleotide-binding
domain, which regulate apoptosis and/or NF-κB activation. Nod2, a third member of the family, was identified. Nod2 is composed
of two N-terminal CARDs, a nucleotide-binding domain, and multiple C-terminal leucine-rich repeats. Although Nod1 and Apaf-1
were broadly expressed in tissues, the expression of Nod2 was highly restricted to monocytes. Nod2 induced nuclear factor
κB (NF-κB) activation, which required IKKγ and was inhibited by dominant negative mutants of IκBα, IKKα, IKKβ, and IKKγ. Nod2
interacted with the serine-threonine kinase RICK via a homophilic CARD-CARD interaction. Furthermore, NF-κB activity induced
by Nod2 correlated with its ability to interact with RICK and was specifically inhibited by a truncated mutant form of RICK
containing its CARD. The identification of Nod2 defines a subfamily of Apaf-1-like proteins that function through RICK to
activate a NF-κB signaling pathway.
All clinical isolates of methicillin-resistant Staphylococcus aureus contain an extra penicillin binding protein (PBP) 2A in addition to four PBPs present in all staphylococcal strains. This extra PBP is thought to be a transpeptidase essential for the continued cell wall synthesis and growth in the presence of beta-lactam antibiotics. As an approach of testing this hypothesis we compared the muropeptide composition of cell walls of a highly methicillin-resistant S. aureus strain containing PBP2A and its isogenic Tn551 derivative with reduced methicillin resistance, which contained no PBP2A because of the insertional inactivation of the PBP2A gene. Purified cell walls were hydrolyzed into muropeptides which were subsequently resolved by reversed-phase high-performance liquid chromatography and identified by chemical and mass spectrometric analysis. The peptidoglycan composition of the two strains were identical. Both peptidoglycans were highly cross-linked mainly through pentaglycine cross-bridges, although other, chemically distinct peptide cross-bridges were also present including mono-, tri-, and tetraglycine; alanine; and alanyl-tetraglycine. Our experiments provided no experimental data for a unique transpeptidase activity associated with PBP2A.
The cytokine-induced activation cascade of NF-kappaB in mammals and the activation of the morphogen dorsal in Drosophila embryos show striking structural and functional similarities (Toll/IL-1, Cactus/I-kappaB, and dorsal/NF-kappaB). Here we demonstrate that these parallels extend to the immune response of Drosophila. In particular, the intracellular components of the dorsoventral signaling pathway (except for dorsal) and the extracellular Toll ligand, spätzle, control expression of the antifungal peptide gene drosomycin in adults. We also show that mutations in the Toll signaling pathway dramatically reduce survival after fungal infection. Antibacterial genes are induced either by a distinct pathway involving the immune deficiency gene (imd) or by combined activation of both imd and dorsoventral pathways.
The nematode CED-4 protein and its human homolog Apaf-1 play a central role in apoptosis by functioning as direct activators of death-inducing caspases. A novel human CED-4/Apaf-1 family member called CARD4 was identified that has a domain structure strikingly similar to the cytoplasmic, receptor-like proteins that mediate disease resistance in plants. CARD4 interacted with the serine-threonine kinase RICK and potently induced NF-kappaB activity through TRAF-6 and NIK signaling molecules. In addition, coexpression of CARD4 augmented caspase-9-induced apoptosis. Thus, CARD4 coordinates downstream NF-kappaB and apoptotic signaling pathways and may be a component of the host innate immune response.
Nod1 is an Apaf-1-like molecule composed of a caspase-recruitment domain (CARD), nucleotide-binding domain, and leucine-rich
repeats that associates with the CARD-containing kinase RICK and activates nuclear factor κB (NF-κB). We show that self-association
of Nod1 mediates proximity of RICK and the interaction of RICK with the γ subunit of the IκB kinase (IKKγ). Similarly, the
RICK-related kinase RIP associated via its intermediate region with IKKγ. A mutant form of IKKγ deficient in binding to IKKα
and IKKβ inhibited NF-κB activation induced by RICK or RIP. Enforced oligomerization of RICK or RIP as well as of IKKγ, IKKα,
or IKKβ was sufficient for induction of NF-κB activation. Thus, the proximity of RICK, RIP, and IKK complexes may play an
important role for NF-κB activation during Nod1 oligomerization or trimerization of the tumor necrosis factor α receptor.
Crohn's disease is a chronic inflammatory disorder of the gastrointestinal tract, which is thought to result from the effect of environmental factors in a genetically predisposed host. A gene location in the pericentromeric region of chromosome 16, IBD1, that contributes to susceptibility to Crohn's disease has been established through multiple linkage studies, but the specific gene(s) has not been identified. NOD2, a gene that encodes a protein with homology to plant disease resistance gene products is located in the peak region of linkage on chromosome 16 (ref. 7). Here we show, by using the transmission disequilibium test and case-control analysis, that a frameshift mutation caused by a cytosine insertion, 3020insC, which is expected to encode a truncated NOD2 protein, is associated with Crohn's disease. Wild-type NOD2 activates nuclear factor NF-kappaB, making it responsive to bacterial lipopolysaccharides; however, this induction was deficient in mutant NOD2. These results implicate NOD2 in susceptibility to Crohn's disease, and suggest a link between an innate immune response to bacterial components and development of disease.
Crohn's disease and ulcerative colitis, the two main types of chronic inflammatory bowel disease, are multifactorial conditions of unknown aetiology. A susceptibility locus for Crohn's disease has been mapped to chromosome 16. Here we have used a positional-cloning strategy, based on linkage analysis followed by linkage disequilibrium mapping, to identify three independent associations for Crohn's disease: a frameshift variant and two missense variants of NOD2, encoding a member of the Apaf-1/Ced-4 superfamily of apoptosis regulators that is expressed in monocytes. These NOD2 variants alter the structure of either the leucine-rich repeat domain of the protein or the adjacent region. NOD2 activates nuclear factor NF-kB; this activating function is regulated by the carboxy-terminal leucine-rich repeat domain, which has an inhibitory role and also acts as an intracellular receptor for components of microbial pathogens. These observations suggest that the NOD2 gene product confers susceptibility to Crohn's disease by altering the recognition of these components and/or by over-activating NF-kB in monocytes, thus documenting a molecular model for the pathogenic mechanism of Crohn's disease that can now be further investigated.
Components of microbial cell walls are potent activators of innate immune responses in animals. For example, the mammalian TLR4 signaling pathway is activated by bacterial lipopolysaccharide and is required for resistance to infection by Gram-negative bacteria. Other components of microbial surfaces, such as peptidoglycan, are also potent activators of innate immune responses, but less is known about how those components activate host defense. Here we show that a peptidoglycan recognition protein, PGRP-LC, is absolutely required for the induction of antibacterial peptide genes in response to infection in Drosophila and acts by controlling activation of the NF-kappaB family transcription factor Relish.
The immune system consists of two evolutionarily different but closely related responses, innate immunity and adaptive immunity. Each of these responses has characteristic receptors-Toll-like receptors (TLRs) for innate immunity and antigen-specific receptors for adaptive immunity. Here we show that the caspase recruitment domain (CARD)-containing serine/threonine kinase Rip2 (also known as RICK, CARDIAK, CCK and Ripk2) transduces signals from receptors of both immune responses. Rip2 was recruited to TLR2 signalling complexes after ligand stimulation. Moreover, cytokine production in Rip2-deficient cells was reduced on stimulation of TLRs with lipopolysaccharide, peptidoglycan and double-stranded RNA, but not with bacterial DNA, indicating that Rip2 is downstream of TLR2/3/4 but not TLR9. Rip2-deficient cells were also hyporesponsive to signalling through interleukin (IL)-1 and IL-18 receptors, and deficient for signalling through Nod proteins-molecules also implicated in the innate immune response. Furthermore, Rip2-deficient T cells showed severely reduced NF-kappaB activation, IL-2 production and proliferation on T-cell-receptor (TCR) engagement, and impaired differentiation to T-helper subtype 1 (TH1) cells, indicating that Rip2 is required for optimal TCR signalling and T-cell differentiation. Rip2 is therefore a signal transducer and integrator of signals for both the innate and adaptive immune systems.
The recognition and phagocytosis of microbes by macrophages is a principal aspect of innate immunity that is conserved from insects to humans. Drosophila melanogaster has circulating macrophages that phagocytose microbes similarly to mammalian macrophages, suggesting that insect macrophages can be used as a model to study cell-mediated innate immunity. We devised a double-stranded RNA interference-based screen in macrophage-like Drosophila S2 cells, and have defined 34 gene products involved in phagocytosis. These include proteins that participate in haemocyte development, vesicle transport, actin cytoskeleton regulation and a cell surface receptor. This receptor, Peptidoglycan recognition protein LC (PGRP-LC), is involved in phagocytosis of Gram-negative but not Gram-positive bacteria. Drosophila humoral immunity also distinguishes between Gram-negative and Gram-positive bacteria through the Imd and Toll pathways, respectively; however, a receptor for the Imd pathway has not been identified. Here we show that PGRP-LC is important for antibacterial peptide synthesis induced by Escherichia coli both in vitro and in vivo. Furthermore, totem mutants, which fail to express PGRP-LC, are susceptible to Gram-negative (E. coli), but not Gram-positive, bacterial infection. Our results demonstrate that PGRP-LC is an essential component for recognition and signalling of Gram-negative bacteria. Furthermore, this functional genomic approach is likely to have applications beyond phagocytosis.
The antimicrobial defence of Drosophila relies largely on the challenge-induced synthesis of an array of potent antimicrobial peptides by the fat body. The defence against Gram-positive bacteria and natural fungal infections is mediated by the Toll signalling pathway, whereas defence against Gram-negative bacteria is dependent on the Immune deficiency (IMD) pathway. Loss-of-function mutations in either pathway reduce the resistance to corresponding infections. The link between microbial infections and activation of these two pathways has remained elusive. The Toll pathway is activated by Gram-positive bacteria through a circulating Peptidoglycan recognition protein (PGRP-SA). PGRPs appear to be highly conserved from insects to mammals, and the Drosophila genome contains 13 members. Here we report a mutation in a gene coding for a putative transmembrane protein, PGRP-LC, which reduces survival to Gram-negative sepsis but has no effect on the response to Gram-positive bacteria or natural fungal infections. By genetic epistasis, we demonstrate that PGRP-LC acts upstream of the imd gene. The data on PGRP-SA with respect to the response to Gram-positive infections, together with the present report, indicate that the PGRP family has a principal role in sensing microbial infections in Drosophila.
Toll-like receptor 2 (TLR2)-mediated cell activation induced by commercial preparations of LPS was recently shown to arise from impurities whose identities are not known. In this work, we determined the molecules responsible for TLR2-mediated cell activation in LPS derived from Escherichia coli K-12 strain LCD25. When LCD25 LPS was phenol extracted, two proteins capable of TLR2-mediated cell activation were purified and identified as E. coli lipoproteins. We cloned, expressed, and purified these two lipoproteins, Lip19 and Lip12. Lip19 or Lip12 activated TNF-alpha production from RAW264.7 and THP-1 cells in a TLR2-dependent manner. However, neither Lip19 nor Lip12 activated HUVECs, which lack endogenous TLR2. Additionally, IkappaB kinase beta and c-Jun N-terminal kinase 1 activation in THP-1 cells induced by Lip19 or Lip12 was observed. TLR2 activation by Lip19 and Lip12 in HEK293 cells was blocked by inhibitory anti-TLR2 mAbs. The unlipidated mutants, Lip19-C19S and Lip12-C21S, in which the NH(2)-terminal cysteine was substituted by serine, lost their ability to activate TLR2-transfected HEK 293 cells. Taken together, these results demonstrate that two lipoproteins constitute the major contaminants responsible for TLR2-mediated cell activation in E. coli LCD25 LPS.
NOD2, a protein associated with susceptibility to Crohn's disease, confers responsiveness to bacterial preparations of lipopolysaccharide and peptidoglycan, but the precise moiety recognized remains elusive. Biochemical and functional analyses identified muramyl dipeptide (MurNAc-L-Ala-D-isoGln) derived from peptidoglycan as the essential structure in bacteria recognized by NOD2. Replacement of L-Ala for D-Ala or D-isoGln for L-isoGln eliminated the ability of muramyl dipeptide to stimulate NOD2, indicating stereoselective recognition. Muramyl dipeptide was recognized by NOD2 but not by TLR2 or co-expression of TLR2 with TLR1 or TLR6. NOD2 mutants associated with susceptibility to Crohn's disease were deficient in their recognition of muramyl dipeptide. Notably, peripheral blood mononuclear cells from individuals homozygous for the major disease-associated L1007fsinsC NOD2 mutation responded to lipopolysaccharide but not to synthetic muramyl dipeptide. Thus, NOD2 mediates the host response to bacterial muropeptides derived from peptidoglycan, an activity that is important for protection against Crohn's disease. Because muramyl dipeptide is the essential structure of peptidoglycan required for adjuvant activity, these results also have implications for understanding adjuvant function and effective vaccine development.
Nod2 activates the NF-κB pathway following intracellular stimulation by bacterial products. Recently, mutations inNod2 have been shown to be associated with Crohn's disease, suggesting a role for bacteria-host interactions in the etiology of
this disorder. We show here that Nod2 is a general sensor of peptidoglycan through the recognition of muramyl dipeptide (MDP),
the minimal bioactive peptidoglycan motif common to all bacteria. Moreover, the 3020insC frameshift mutation, the most frequent
Nod2 variant associated with Crohn's disease patients, fully abrogates Nod2-dependent detection of peptidoglycan and MDP.
Together, these results impact on the understanding of Crohn's disease development. Additionally, the characterization of
Nod2 as the first pathogen-recognition molecule that detects MDP will help to unravel the well known biological activities
of this immunomodulatory compound.
About 80 different muropeptides, the subunits which comprise the polymer murein of Escherichia coli, were resolved by high-performance liquid chromatography. The muropeptides were released from isolated murein by complete digestion with muramidase from Chalaropsis spec. The separation method is based on reversed phase chromatography of the sodium borohydride-reduced compounds using ODS (C18) columns and a linear gradient elution with sodium phosphate buffer and methanol as organic modifier. The effect of temperature, pH, ionic strength, and the steepness of the gradient and of different support materials on the separation of the muropeptides was investigated. The new method represents a major improvement over previous methods with respect to resolution, sensitivity, and speed. Analytical as well as preparative separations can be realized. Quantitative analysis of murein composition is achieved by a linear gradient from 50 mM sodium phosphate, pH 4.31, to 75 mM sodium phosphate, pH 4.95, containing 15% methanol for 135 min on a 250 X 4.6 mm 3-micron Hypersil ODS column at 55 degrees C using a flow rate of 0.5 ml/min. With uv detection at 205 nm about 20 micrograms of murein per analysis is sufficient. The detection limit per compound is about 5 ng. A method for the evaluation of the analytical data allowing a convenient comparison of different muropeptide pattern is described.
The gene encoding a 102 kDa autolysin has been cloned from an expression library of Listeria monocytogenes EGD genomic DNA, using a direct screening protocol. The encoded protein has two domains, an N-terminal enzymic domain showing a high level of homology to the amidase domain of the major autolysin (atl) of Staphylococcus aureus, and a C-terminal, putative cell-wall-binding domain containing four imperfect direct repeats. In order to examine the role of the enzyme, the autolysin-encoding gene was insertionally inactivated by site-specific integration of a temperature sensitive plasmid. The enzyme accounts for 66% of the total lytic enzyme activity when L. monocytogenes walls are used as substrate and several of the major autolytic bands are missing on renaturing gels when compared to the wild-type. The enzyme does not appear to be directly involved in cell separation but has a role in motility. Characterization of the recombinant enzyme expressed in Escherichia coli has revealed it to be an amidase and to be able to hydrolyse a range of peptidoglycan substrates.
Ced-4 and Apaf-1 belong to a major class of apoptosis regulators that contain caspase-recruitment (CARD) and nucleotide-binding oligomerization domains. Nod1, a protein with an NH2-terminal CARD-linked to a nucleotide-binding domain and a COOH-terminal segment with multiple leucine-rich repeats, was identified. Nod-1 was found to bind to multiple caspases with long prodomains, but specifically activated caspase-9 and promoted caspase-9-induced apoptosis. As reported for Apaf-1, Nod1 required both the CARD and P-loop for function. Unlike Apaf-1, Nod1 induced activation of nuclear factor-kappa-B (NF-kappaB) and bound RICK, a CARD-containing kinase that also induces NF-kappaB activation. Nod1 mutants inhibited NF-kappaB activity induced by RICK, but not that resulting from tumor necrosis factor-alpha stimulation. Thus, Nod1 is a leucine-rich repeat-containing Apaf-1-like molecule that can regulate both apoptosis and NF-kappaB activation pathways.
MyD88 is a general adaptor protein that plays an important role in the Toll/IL-1 receptor family signalings. Recently, Toll-like receptors 2 and 4 (TLR2 and TLR4) have been suggested to be the signaling receptors for lipopolysaccharide (LPS). In this study, we demonstrate that MyD88 knockout mice lack the ability to respond to LPS as measured by shock response, B cell proliferative response, and secretion of cytokines by macrophages and embryonic fibroblasts. However, activation of neither NF-kappaB nor the mitogen-activated protein (MAP) kinase family is abolished in MyD88 knockout mice. These findings demonstrate that signaling via MyD88 is essential for LPS response, but the inability of MyD88 knockout mice to induce LPS-dependent gene expression cannot simply be attributed to lack of the activation of MAP kinases and NF-kappaB.
The immune response to microbial pathogens is initiated by recognition of specific pathogen components by host cells both at the cell surface and in the cytosol. While the response triggered by pathogen products at the surface of immune cells is well characterized, that initiated in the cytosol is poorly understood. Nod1 is a member of a growing family of intracellular proteins with structural homology to apoptosis regulators Apaf-1/Ced-4 and a class of plant disease-resistant gene products. Here we show that bacterial lipopolysaccharides, but not other pathogen components tested, induced TLR4- and MyD88-independent NF-kappaB activation in human embryonic kidney 293T cells expressing trace amounts of Nod1. Nod2, another Nod family member, also conferred responsiveness to bacterial components but with a response pattern different from that observed with Nod1. As it was reported for plant disease-resistant R proteins, the leucine-rich repeats of Nod1 and Nod2 were required for lipopolysaccharide-induced NF-kappaB activation. A lipopolysaccharide binding activity could be specifically coimmunopurified with Nod1 from cytosolic extracts. These observations suggest that Nod1 and Nod2 are mammalian counterparts of plant disease-resistant gene products that may function as cytosolic receptors for pathogen components derived from invading bacteria.
The lytic transglycosylases are a class of autolysins which cleave the bacterial cell wall heteropolymer peptidoglycan (murein) to facilitate its biosynthesis and turnover. A search of the National Center for Biotechnology Information (NCBI) databases using the primary sequences of the six characterized lytic transglycosylases of Escherichia coli, a membrane-bound form of the enzyme from Pseudomonas aeruginosa, and the endolysins of lambda bacteriophage permitted the identification of a total of 127 known and hypothetical enzymes from a wide variety of bacteria and bacteriophage. These amino acid sequences have been arranged into four families based on alignments, and consensus motifs have been identified. Family 1 represents a superfamily comprising 86 sequences which are subdivided into five (1A--1E) subfamilies.
The innate immune system recognizes pathogen-associated molecular patterns (PAMPs) that are expressed on infectious agents, but not on the host. Toll-like receptors (TLRs) recognize PAMPs and mediate the production of cytokines necessary for the development of effective immunity. Flagellin, a principal component of bacterial flagella, is a virulence factor that is recognized by the innate immune system in organisms as diverse as flies, plants and mammals. Here we report that mammalian TLR5 recognizes bacterial flagellin from both Gram-positive and Gram-negative bacteria, and that activation of the receptor mobilizes the nuclear factor NF-kappaB and stimulates tumour necrosis factor-alpha production. TLR5-stimulating activity was purified from Listeria monocytogenes culture supernatants and identified as flagellin by tandem mass spectrometry. Expression of L. monocytogenes flagellin in non-flagellated Escherichia coli conferred on the bacterium the ability to activate TLR5, whereas deletion of the flagellin genes from Salmonella typhimurium abrogated TLR5-stimulating activity. All known TLRs signal through the adaptor protein MyD88. Mice challenged with bacterial flagellin rapidly produced systemic interleukin-6, whereas MyD88-null mice did not respond to flagellin. Our data suggest that TLR5, a member of the evolutionarily conserved Toll-like receptor family, has evolved to permit mammals specifically to detect flagellated bacterial pathogens.
Recognition of pathogens is mediated by a set of germline-encoded receptors that are referred to as pattern-recognition receptors (PRRs). These receptors recognize conserved molecular patterns (pathogen-associated molecular patterns), which are shared by large groups of microorganisms. Toll-like receptors (TLRs) function as the PRRs in mammals and play an essential role in the recognition of microbial components. The TLRs may also recognize endogenous ligands induced during the inflammatory response. Similar cytoplasmic domains allow TLRs to use the same signaling molecules used by the interleukin 1 receptors (IL-1Rs): these include MyD88, IL-1R--associated protein kinase and tumor necrosis factor receptor--activated factor 6. However, evidence is accumulating that the signaling pathways associated with each TLR are not identical and may, therefore, result in different biological responses.
Microbial infection activates two distinct intracellular signalling cascades in the immune-responsive fat body of Drosophila. Gram-positive bacteria and fungi predominantly induce the Toll signalling pathway, whereas Gram-negative bacteria activate the Imd pathway. Loss-of-function mutants in either pathway reduce the resistance to corresponding infections. Genetic screens have identified a range of genes involved in these intracellular signalling cascades, but how they are activated by microbial infection is largely unknown. Activation of the transmembrane receptor Toll requires a proteolytically cleaved form of an extracellular cytokine-like polypeptide, Spätzle, suggesting that Toll does not itself function as a bona fide recognition receptor of microbial patterns. This is in apparent contrast with the mammalian Toll-like receptors and raises the question of which host molecules actually recognize microbial patterns to activate Toll through Spätzle. Here we present a mutation that blocks Toll activation by Gram-positive bacteria and significantly decreases resistance to this type of infection. The mutation semmelweis (seml) inactivates the gene encoding a peptidoglycan recognition protein (PGRP-SA). Interestingly, seml does not affect Toll activation by fungal infection, indicating the existence of a distinct recognition system for fungi to activate the Toll pathway.
Host defences to microorganisms rely on a coordinated interplay between the innate and adaptive responses of immunity. Infection with intracellular bacteria triggers an immediate innate response requiring macrophages, neutrophils and natural killer cells, whereas subsequent activation of an adaptive response through development of T-helper subtype 1 cells (TH1) proceeds during persistent infection. To understand the physiological role of receptor-interacting protein 2 (Rip2), also known as RICK and CARDIAK, we generated mice with a targeted disruption of the gene coding for Rip2. Here we show that Rip2-deficient mice exhibit a profoundly decreased ability to defend against infection by the intracellular pathogen Listeria monocytogenes. Rip2-deficient macrophages infected with L. monocytogenes or treated with lipopolysaccharide (LPS) have decreased activation of NF-kappaB, whereas dominant negative Rip2 inhibited NF-kappaB activation mediated by Toll-like receptor 4 and Nod1. In vivo, Rip2-deficient mice were resistant to the lethal effects of LPS-induced endotoxic shock. Furthermore, Rip2 deficiency results in impaired interferon-gamma production in both TH1 and natural killer cells, attributed in part to defective interleukin-12-induced Stat4 activation. Our data reflect requirements for Rip2 in multiple pathways regulating immune and inflammatory responses.
Toll-like receptors have a crucial role in the detection of microbial infection in mammals and insects. In mammals, these receptors have evolved to recognize conserved products unique to microbial metabolism. This specificity allows the Toll proteins to detect the presence of infection and to induce activation of inflammatory and antimicrobial innate immune responses. Recognition of microbial products by Toll-like receptors expressed on dendritic cells triggers functional maturation of dendritic cells and leads to initiation of antigen-specific adaptive immune responses.
The Drosophila immune system discriminates between different classes of infectious microbes and responds with pathogen-specific defense reactions through selective activation of the Toll and the immune deficiency (Imd) signaling pathways. The Toll pathway mediates most defenses against Gram-positive bacteria and fungi, whereas the Imd pathway is required to resist infection by Gram-negative bacteria. The bacterial components recognized by these pathways remain to be defined. Here we report that Gram-negative diaminopimelic acid-type peptidoglycan is the most potent inducer of the Imd pathway and that the Toll pathway is predominantly activated by Gram-positive lysine-type peptidoglycan. Thus, the ability of Drosophila to discriminate between Gram-positive and Gram-negative bacteria relies on the recognition of specific forms of peptidoglycan.