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J. Biochem. 2013;154(6):491–499 doi:10.1093/jb/mvt099 JB Review How do cells optimize luminal environments of endosomes/ lysosomes for efficient inflammatory responses? Received September 19, 2013; accepted October 23, 2013; published online October 31, 2013 Department of Molecular Immunology and Inflammation, Research Institute, National Center for Global Health and Medicine, 1-21-1 Toyama, Shinjuku-ku, Tokyo 162-8655, Japan *Noriko Toyama-Sorimachi, Department of Molecular Immunology and Inflammation, Research Institute, National Center for Global Health and Medicine, 1-21-1 Toyama, Shinjuku-ku, Tokyo 162-8655, Japan. Tel: þ81-3-3202-7181, Fax: þ81-3-3202-7364, email: [email protected] The endosome/lysosome compartments play pivotal roles in immune cell functions as signalling platforms. These intracellular compartments can efficiently restrict the localization of signalling complexes and temporally regulate signalling events to produce qualitatively different outcomes. Immune cells also exploit the endosome/lysosome system for signal transduction and intercellular communication to elicit immune responses. Antigen-presenting cells such as dendritic cells and macrophages take up pathogens by endocytosis and prepare antigens via the endosome/lysosome system. At the same time, pathogen-derived DNA and RNA are recognized by immune sensors at the endosome/ lysosome compartments, which transmit signals to induce immune responses. Recent studies revealed the importance of controlling the endosomal/lysosomal environment for eliciting efficient signalling events at the endosomes/lysosomes. Many factors including pH, membrane potential, amino acid concentrations and lipid composition are finely tuned at the endosome/lysosome compartments, and dysregulation of these factors greatly affect immune cell functions. Redox-related molecules and various types of transporters are involved in the control of endosomal/lysosomal environment and could be good therapeutic targets for treating autoimmune diseases. Keywords: dendritic cells/inflammation/signalling endosome/toll-like receptor/solute carrier protein15A4. Abbreviations: APC, antigen-presenting cells; BCR, B cell receptors; CpG-ODN, CpG oligo DNA; DC, dendritic cell; EGFR, epidermal growth factor receptor; ENU, N-ethyl-N-nitrosourea; ER, endoplasmic reticulum; GPCR, G protein-coupled receptor; IBD, inflammatory bowel disease; iE-DAP, g-Dglutamyl-meso-diaminopimelic acid; IFN-b, interferon-b; IL-6, interleukin-6; LCMV, lymphocytic choriomeningitis virus; LPS, lipopolysaccharide; MAPK, mitogen-activated protein kinase; MDP, muramyl dipeptide; MHC, major histocompatibility complex; NADPH, reduced nicotinamide adenine dinucleotide phosphate; NALP3, NACHT, LRR and PYD domains-containing protein 3; NF-kB, nuclear factor-kappa B; NOD, nucleotide-binding oligomerization domain; NOX2, NADPH oxidase 2; NGFR, nerve growth factor receptor; PGN, peptidoglycan; POT, proton-coupled oligopeptide transporters; SLC, solute carrier family; SLE, systemic lupus erythematosus; TIRAP, toll/interleukin-1 receptor domaincontaining adapter protein; TLR, Toll-like receptors; TNFa, tumor necrosis factor alpha; TRAM, translocation associated membrane protein; TRIF, TIRdomain-containing adapter-inducing interferon-b. Endocytosis is a universal cellular activity to uptake various materials including nutrition, pathogens and cell surface receptors into cells. Until a decade ago, endocytosis of cell surface receptors and the following lysosomal degradation process were thought to be important regulatory mechanisms for down-modulation and termination of signalling by cell surface receptors. Today, endosomes/lysosomes also sometimes play important roles as signalling platforms (13). In these cases, after a surface receptor binds to its ligand, the receptorligand complex is internalized and continuously promotes downstream signalling events via signalling complexes that are spatially restricted to particular compartments in the cell. Such ‘signalling endosomes’ can efficiently regulate the localization of signalling complexes via transportation and enable particular signalling activities to be sustained for long periods. Once activated, many receptors, such as epidermal growth factor receptor (EGFR), nerve growth factor receptor (NGFR) (TrkA), and G proteincoupled receptor (GPCR), use this system (2, 46). In parallel, a growing body of evidence has demonstrated that lysosomes, too, can function as important signalling platforms and are not only compartments used for degradation. Cellular nutritional conditions as well as the metabolic state are sensed at lysosomal compartments, and signals are transmitted at the lysosomal compartments to induce various gene transcriptions (79). Thus, the endosome/lysosome system plays key roles in various cellular functions. Immune cells also exploit the signal transduction capacity of the endosome/lysosome system for eliciting immune responses (10, 11). In particular, some immune cells, such as dendritic cells (DCs), macrophages, and B cells, take up various antigens, recognize danger-associated molecular patterns at the endosomes/ lysosomes, and transmit signals from these compartments to produce immune responses (12, 13). Because ß The Authors 2013. Published by Oxford University Press on behalf of the Japanese Biochemical Society. All rights reserved 491 Featured Article Toshihiko Kobayashi, Tsubasa Tanaka and Noriko Toyama-Sorimachi* T. Kobayashi et al. these compartments can restrict signals spatially and temporally to elicit qualitatively different outcomes, understanding the molecular bases of endosome/lysosome-dependent signalling events could be helpful for developing strategies to control immune cell functions. Recent studies have revealed that efficient signalling at these compartments is achieved by controlling the endosomal/lysosomal environment. In this review, we highlight the endosome/lysosome-dependent signalling events and the unique regulation of the endosomal/ lysosomal environment in immune cells. Endosomes/Lysosomes Spatiotemporally Sort TLR4 signalling Components to Induce Immune Responses Toll-like receptors (TLRs) are well-characterized innate immune sensors that play a key role in host defense (14). They recognize conserved microbial components at the cell surface, or in intracellular compartments such as endosomes/lysosomes, and induce immune responses (14, 15). At the early stage of inflammation, antigen-presenting cells (APCs), such as DCs and macrophages, that encounter microbes or their components are activated through TLRs to produce inflammatory and/or immunomodulatory cytokines, including tumor necrosis factor a (TNFa), interleukin-6 (IL-6) and type I interferon. At the same time, the TLR-mediated activation of these cells leads to a transcription of a panel of genes that are pivotal for the induction of acquired immune responses following antigen presentation. Importantly, depending on the type of TLRs triggered, APCs produce different cytokines and induce specific types of effector T cells that are suitable to eliminate the infecting microbes; that is, which TLRs are triggered determines the type of immune response elicited (16, 17). TLR4 recognizes lipopolysaccharide (LPS) on gramnegative bacteria at the cell surface and activates two qualitatively distinct signalling pathways, MyD88dependent and MyD88-independent, at the cell surface and endosomes, respectively (Fig. 1A) (18, 19). After LPS binds to TLR4 at the cell surface, the MyD88dependent pathway is activated through the adaptor proteins TIRAP (toll/interleukin-1 receptor domaincontaining adapter protein) and MyD88, which in turn activate the transcription factor NF-kB (nuclear factor-kappa B) and mitogen-activated protein kinase (MAPK), culminating in the production of pro-inflammatory cytokines such as IL-6 and TNFa. Subsequently, TLR4 is internalized, the adaptor proteins TRAM (translocation associated membrane protein) and TIR-domain-containing adapter-inducing interferon-b (TRIF) replace TIRAP and MyD88, and the MyD88-independent pathway is consequently initiated at the endosomes. Its activation results in the generation of interferon-b (IFN-b) and IL-10. The transition from the TIRAP-MyD88 pathway to the TRAM-TRIF pathway downstream of TLR4 is mediated by PI3 kinase-dependent phosphatidylinositol turnover. TIRAP binds PtdIns(4,5)P2 through its amino-terminal polybasic domain and is localized to 492 PtdIns(4,5)P2-rich plasma membrane regions. Conversion of PtdIns(4,5)P2 to PtdIns(3,4,5)P3 by PI3 kinase p110d causes the release of TIRAP into the cytoplasm, and then TRAM is recruited to TLR4. These sequential events at the distinct compartments are pivotal for the efficient initiation and termination of inflammatory responses, by shifting the cytokines released from pro-inflammatory to the antiinflammatory ones. Indeed, PI3Kd-mutant mice were more susceptible to endotoxic shock than wild-type controls because of sustained production of TNF, IL-6 and IL-12 but restrained production of IL-10 and IFN-b (20). Thus, the compartmentalization of signalling complexes at endosomes confers spatiotemporal properties on TLR4-mediated signalling and is an important mechanism for mounting an effective and harmless immune response. Endosome/Lysosome-Dependent Recognition of Nucleic Acids by TLRs and Its Importance in Immune Defense Some TLRs, including TLR3, 7 and 9, localize to endosomes, where they recognize nucleic acids and transmit signals to induce various cytokines (21, 22). The manner by which nucleic acids are recognized by these endosomal TLRs appears to enable selective recognition of ingested microbe-derived nucleic acids while avoiding undesirable responses to self-tissuederived nucleotides in the serum. TLR9 recognizes unmethylated CpG oligo DNA (CpG-ODN) at the endosomes and induces pro-inflammatory cytokines and type I IFN production, which is essential for antiviral immune responses (Fig. 1B) (23). In this process, endoplasmic reticulum (ER)-resident TLR9 is transported to endosomes, where it binds CpG-ODN, and then passes to specialized lysosome-related compartments (24). This TLR9 trafficking is crucial for generating functionally active TLR9, given that the limited proteolysis of TLR9 by lysosomal proteases such as cathepsins is necessary for CpG-ODN recognition (25, 26). These lysosomal proteases are most active under acidic conditions, and therefore the acidification of endosome/lysosome compartments during their maturation is important for triggering inflammatory responses. Intriguingly, the trafficking of TLR9/ CpG-ODN from endosomes to lysosomes is affected by the CpG-ODN structure, with different structures leading to different cellular responses (27). The precise molecular mechanism by which such traffickingcoupled signalling events are spatially and temporally regulated at the endosomal/lysosomal compartments is not fully understood. Endosomes/Lysosomes as Sites for BCR-TLR Collaboration and Autoimmune-Response Induction B cell receptors (BCRs) are composed of a membraneassociated form of immunoglobulin (Ig) that is noncovalently associated with a disulphide-linked heterodimer composed of two integral membrane proteins, Endosomal/lysosomal signalling and inflammation Fig. 1 Examples of endosome/lysosome-dependent signalling events in the immune system. (A) Model for endosome-dependent TLR4 signalling. At the plasma membrane, LPS-bound TLR4 binds to the sorting adaptor TIRAP, which is anchored with the plasma membrane lipid PI (4,5) P2. TIRAP then recruits the signalling adaptor MyD88, and MyD88-dependent signalling induces the production of proinflammatory cytokines including TNFa, IL-6 and IL-12. LPS induces recruitment of the PI3 kinase p110d to the TLR4-TIRAP-MyD88 complex, and p110d then decreases the amount of PI(4,5)P2, which probably causes TIRAP to be released from the membrane and promotes the switch to endosomal TRAM signalling. The released TIRAP and MyD88 are degraded through a proteasome-dependent process. This subcellular shuttling of TLR4 results in a cytokine shift from proinflammatory to anti-inflammatory. (B) Recognition of nucleic acids by TLR7 or TLR9 in endosomes. Some TLRs that sense nucleotides are localized to endosomes and transduce signals to elicit inflammatory responses. TLR7 and TLR9 recognize single-strand RNA and unmethylated CpG-ODN, respectively, when they enter endolysosomes. These TLRs are localized to the ER in the steady state and then relocate to endosomes in an AP3- and Unc93B-dependent manner. In endolysosomes, these TLRs are cleaved by cathepsins, which confers ligand-binding ability on them. The environmental control of the endolysosomes and the proper trafficking and subcellular localization of the TLRs are required to induce inflammatory responses. (C) Essential role of endocytosis in regulating the outcome of BCR signalling. The endocytosis and subcellular localization of the BCR following its activation are necessary for BCR-mediated kinase activation and the downstream gene transcription. BCRs are internalized after cross-linking with antigens, and they traffic from early endosomes to late endosomes (LAMP-1þ compartments). Lyn and Syk are phosphorylated at the cell surface. The phosphorylation of MAP kinases occurs at the endosomes, and the phosphorylation of Akt and its downstream targets, resulting from internalized BCRs, occurs at the late endosomes. Importantly, inhibiting BCR’s endocytosis alters the amount of kinase phosphorylation in the BCR signalling pathway and its outcome. CD79a and CD79b (28). After ligating with its antigen, the BCR initiates signalling at the plasma membrane, which involves the phosphorylation of BCR components by Src family kinases and the recruitment of a proximal kinase, Syk. The BCR is then internalized and induces the sequential phosphorylation of MAPKs and the kinase Akt (Fig. 1C) (29). The inhibition of BCR endocytosis dysregulates these phosphorylation events, leading to gene-transcription defects. Thus, proper trafficking of the BCR into endosomes is required for BCR signalling and appropriate immune responses. Furthermore, endosomal signalling via BCRs is a pivotal event for induction of autoimmune response. Autoantigens include self-DNA, histones, RNA or ribonucleoproteins, which are thought to be released from apoptotic cells. The endosomal localization of the BCRs allows them to engage synergistically with TLR9 in the presence of DNA-containing antigens; this process has been implicated in inducing the activation of autoimmune B cells (30). In fact, the simultaneous engagement of BCRs with TLR9 and their endosomal activation contributes to the production of DNAspecific autoantibodies in mouse models of lupus (30, 31). Thus, signalling endosomes also serve as a meeting place for multiple signalling pathways mediated by different immune sensors. Importance of Controlling the Endosome/Lysosome Environment in Inflammatory Cells Endosomes are gradually acidified by V-ATPase as they mature (32). As described earlier, the acidic environment of endosomes is important for the cathepsinmediated limited proteolysis of TLR9, which converts this receptor into its ligand-accessible form. In addition, the binding affinity of CpG-ODN to TLR9 itself is affected by the pH (33). At neutral pH, TLR9’s binding to CpG-DNA is weak, but lowering the pH leads to a strong TLR9-CpG-DNA interaction. Treatment with compounds that interfere with luminal acidification of endosomes/lysosomes, such as bafilomycin A1 (a specific inhibitor of the vacuolar proton pump V-ATPase) or chloroquine (a weak base that can partition into vesicles and increase the pH) impairs the CpG-DNA-driven signalling via TLR9 (3436). Thus, the optimization of luminal pH is an important regulatory mechanism for efficient signalling through TLR9. From this viewpoint, factors that control the ‘luminal’ pH in immune cells could be useful as targets for anti-inflammatory drugs to treat autoimmune diseases. In fact, chloroquine and its derivatives have already been used to effectively treat autoimmune diseases such as rheumatoid arthritis and systemic lupus 493 T. Kobayashi et al. erythematosus (SLE) (37, 38). The mechanism underlying chloroquine’s action in autoimmune diseases is not fully understood, but findings in a murine model of SLE suggest that it blocks the TLR9-dependent, chromatin-antibody complex-induced stimulation of self-reactive B cells (39, 40). Endosomes finally fuse to lysosomes, which contain numerous hydrolases for the degradation of proteins, lipids, and other biomolecules. In lysosomes, these molecules are completely degraded into their building blocks, which are recycled for biosynthesis of macromolecules. An impairment in the lysosomes’ degradative function leads to the accumulation of undegraded molecules, which causes various types of lysosomal storage diseases (41, 42). DCs, the most potent APCs, however, limit the lysosomes’ proteolytic activity for antigen presentation to T cells. DCs ingest protein-containing macromolecules and process them into short peptides, which they present as an antigen together with major histocompatibility complex (MHC) molecules, a critical event for the induction of the adaptive immune response (43). To generate peptides with a certain length and to maximize half-life of the peptides within lysosomes, DCs possess a unique mechanism for phagosome maturation. DCs maintain the phagosomal pH between 7 and 7.5 in the first few hours after phagocytosis (44). DCs recruit a component of nicotinamide adenine dinucleotide phosphate (NADPH) oxidase NOX2 to the phagosomes in Rab27a-dependent fashion (45), where oxygen radicals generated by the NADPH oxidase cause proton consumption and neutralize the acidic environment, counteracting the actions of the proton pump V-ATPase (44, 46). Because of this phagosomal alkalization machinery, the phagolysosomal pH still remained above pH 7 after fusing to the lysosomes. This makes it possible for peptides to be processed to the appropriate size for loading onto MHC molecules by preventing the complete degradation of peptides (44). This mechanism is particularly important for antigen processing for MHC class I, called antigen cross-presentation, in which exogenously ingested antigens are presented together with MHC class I to activate CD8þ cytotoxic T cells (47). The alkalized phagolysosomes should be finally acidified to hydrolyze contents. Recent study further improved our understanding on how the neutralized phagolysosomes are safely acidified to fulfil the obligation of lysosomes as degradative compartments (48). On the surface of phagosomes containing grampositive bacteria, caspase-1 activated via NACHT, LRR and PYD domains-containing protein 3 (NALP3) inflammasome accumulates and induces phagosomal acidification by negatively regulating NADPH oxidase NOX2 activity. An important observation is that phagosomes containing gram-negative bacteria did not recruit caspase-1 and appeared to be acidified via different machinery. This implies that the phagosomal environment is differentially controlled by bacterial species. Thus environmental regulation within phagosomes/phagolysosomes is mediated by multiple complex processes depending on different cargos. 494 A New Player in Endosomal/Lysosomal Environmental Control Immune cells possess a multilayered system for controlling the endosomal/lysosomal environment, involving not only the proton concentration but also the concentrations of other ions and substances (Fig. 2). We and another group recently discovered that the lysosome-resident amino acid transporter, solute carrier family 15A4 (SLC15A4), is critical for signal transduction by TLR7 and TLR9 (Fig. 3) (49, 50). Cellular transporters are currently categorized into two large groups: the SLC family, which functions independently of ATP, and the ATP-binding cassette family, which is ATP-dependent. SLC15A4 is a member of the SLC family, which includes roughly 300 genes to date, and is further divided into many subfamilies (51). SLC15A4 belongs to the SLC15 subfamily, which has five members, A1 to A5, and is well conserved across species (52, 53). These are so-called protoncoupled oligopeptide transporters (POTs) and carry amino acids and oligopeptides together with a proton as a common feature, but their functions and expression patterns differ. A1 and A2 are mainly expressed in the small intestine, liver and kidney, whereas A3 and A4 have low sequence similarity with A1 and A2 and are expressed in immune cells and the nervous system (the localization of A5 is currently unknown). Moreover, both A1 and A2 have low substrate specificity and the ability to carry 600 different di-peptides and 8,000 tri-peptides, whereas A3 and A4 transport only histidine and several histidine-containing di- or tri-peptides (Fig. 3A) (51). Because of their expression pattern and substrate specificity, A1 and A2 are thought to function in the absorption or reabsorption of digested nutrients. By contrast, A3 and A4 appear to have a role in immune regulation; however, the functional importance of these transporters was not investigated until recently. The transporter activity of A4 is pH dependent, with higher transport of histidine/oligopeptides and protons at pH 5.5 than pH 7.0 (Fig. 3B) (53). Therefore, the transporter activity of SLC15A4 is maximized in the acidic environment of lysosomes. Several studies have demonstrated an association between SLC15A4 function and certain inflammatory disorders, including diabetes and inflammatory bowel disease (IBD) (54, 55). Importantly, a recent genome-wide association study identified SLC15A4 as an SLE-susceptibility gene (56), suggesting a link between the function of SLC15A4 and autoimmune disease in humans. An attractive hypothesis emerging from these observations is that SLC15A4 contributes to the regulation of inflammatory responses by regulating lysosomal functions or environments. We tested this hypothesis using SLC15A4 knockout mice and found that SLC15A4-deficient DCs showed an impaired TLR9-mediated production of cytokines such as IFN-b, IL-12 and IL-15 (50). Consistent with this finding, SLC15A4-knockout mice exhibited a less severe form of colitis than wild-type mice. Beutler and his colleagues also demonstrated using N-ethyl-Nnitrosourea (ENU)-induced SLC15A4 mutant mice Endosomal/lysosomal signalling and inflammation Fig. 2 Tuning of the endosomal/lysosomal environment in immune cells. The endosomal/lysosomal compartments play pivotal roles in immune cell functions as signalling platforms. In addition to the TLRs and BCR mentioned in the text, other important receptor systems such as chemokine receptors and cytokine receptors use such compartments for their signalling. The ‘luminal’ environments of endosomes/lysosomes are strictly regulated by various transporters and redox-related molecules, such as V-ATPase, NOX2 and SLC15A4, to optimize endosome/lysosome-dependent signalling events. In addition to these players, phosphatidylinositol turnover is important for generating spatial and temporal properties of signalling molecules. The lysosomal system in immune cells is also finely tuned to efficiently generate antigen peptides. Phagosomal alkalization through NOX2-mediated reactive oxygen production limits the vesicles’ proteolytic capacity, thereby preserving peptides from complete degradation and making it possible to load peptides of the appropriate size onto MHC molecules. that SLC15A4 plays a crucial role in regulation of the functions of TLR7 as well as TLR9, which is necessary for recognizing single-stranded RNA (Fig. 3C). SLC15A4-mutant mice is highly susceptible to influenza virus infection (49). SLC15A4 is also required to protect mice from infection by mouse lymphocytic choriomeningitis virus, in which the DCs prime T cells in an SLC15A4-dependent manner to clear the virus (57). How does SLC15A4 contribute to TLR7- or TLR9mediated signalling? Considering that SLC15A4’s function is to export histidines/oligopeptides together with protons from these compartments to the cytosol, one possibility is that the loss of this transporter substantially affects the proton concentration of the endolysosome. Another possibility is that the loss of this transporter causes an abnormal histidine concentration in endolysosomes, eventually disrupting the histidine homeostasis. Histidine has an ionizable imidazole ring in its side chain and the ability, unique among amino acids, to function as an acidbase catalyst. The pKa value for the imidazole ring is 7.0. In fact, histidine’s buffering capacity is so high that it is used as a buffer in histidine-tryptophan-ketoglutarate solution, which is used for organ preservation. Therefore, in the course of endolysosomal acidification, too much histidines within endolysosomes may affect endolysosomal pH. This idea is supported by the observation that histidine administration in mice leads to the amelioration of inflammatory responses (50). We also showed that free histidine, but not alanine, in a certain concentration range can inhibit the enzymatic activities of cathepsins B and L, both of which are important for TLR9’s processing and activation in lysosomes (25, 26). Because the addition of exogenous histidine did not exacerbate the impaired TLR9-mediated cytokine production of DCs lacking SLC15A4, the endolysosomal histidine homeostasis of these cells may already have been disrupted. The precise mechanism for the suppression of inflammatory responses in the absence of this transporter needs to be clarified. Besides DCs, we revealed that B cells also express SLC15A4 and B cell’s SLC15A4 is crucial for autoimmune symptom (manuscript under revision). Nevertheless, the finding that disruption of a lysosome-resident transporter greatly affects lysosome-dependent immunological events revealed the importance of controlling the environment of endolysosome compartments for adequate immune response induction. Another important finding obtained from SLC15A4knockout mice is that SLC15A4 is responsible for transporting a certain type of microbial component from inside the lysosomes to the cytosol for initiating cytokine production and inflammatory responses (50). The bacterial cell wall peptidoglycan (PGN) contains unique 495 T. Kobayashi et al. Fig. 3 Roles of the lysosomal histidine/oligopeptide transporter, SLC15A4, in innate immunity. (A, B) SLC15A4 contains 12 transmembrane domains, with both the N- and C-termini facing the cytosol, and transports histidines/oligopeptides uphill using proton-motive force. The transporter activity of SLC15A4 is pH dependent. In mature lysosomes, histidines/oligopeptides derived from lysosomal degradation products are actively transported out by SLC15A4. (C) The ligand binding and signalling of TLR9 are strictly regulated by the lysosomal state and functions. Histidine in lysosomes might be maintained within a defined concentration, which contributes to the maximal functional efficiency of lysosomal components, including TLR9. SLC15A4 deficiency causes homeostasis failure of the lysosomal environment and disrupts TLR9 function. Furthermore, SLC15A4 is responsible for transporting the peptide glycan-like NOD1 ligand, tri-DAP, from lysosomes to the cytosol, and induces IL-1b production to eliminate bacterial invaders. Thus, this lysosomal transporter also functions as a receiving clerk for pathogenic components. structure such as g-D-glutamyl-meso-diaminopimelic acid (iE-DAP) or muramyl dipeptide (MDP). These components are recognized by cytoplasmic pathogen sensors to initiate the activation of NF-kB and MAP kinases and the induction of innate immune responses. Nucleotide-binding oligomerization domain (NOD) 1 and NOD2 are well characterized cytoplasmic sensors and recognize iE-DAP and MDP, respectively (58). The importance of these molecules is underscored by their genetic association with IBD (59). Although both NOD1 and NOD2 detect PGN components in the cytosol, how such microbially derived components reach the host cytosol is still unclear. SLC15A4-knockout mice exhibit a severe defect in NOD1-dependent IL-1b production in vivo, which is caused by the unresponsiveness of SLC15A4-deficient DCs and macrophages to triDAP (Fig. 3C) (50). Thus, this lysosomal transporter also functions as a receiving clerk for pathogenic components. Because the lysosomes are the site at which phagocytosed pathogens are digested, the transport of the digestion products by a pH-dependent lysosomal transporter seems to be a reasonable and safe way for the cell to monitor pathogen invasion. 496 Conclusion and Perspectives In summary, the endosome/lysosome compartments play pivotal roles in immune cell functions as signalling platforms. Their environments are strictly regulated as the occasion demands by various transporters and redox-related molecules such as V-ATPase, NOX2 and SLC15A4 to optimize endosome/lysosomedependent signalling events. The endosome/lysosome system is an attractive target for controlling immune diseases, although our understanding of it is still quite primitive. Although not discussed here in detail, certain immune cells, such as natural killer cells and cytotoxic T cells, develop specialized lysosome-related organelles, called secretory lysosomes, which contain cytotoxic components including granzymes and perforin and are essential for the cells’ cytotoxic function. Thus, the environmental control of secretory lysosomes is another interesting and important issue. Because lysosome functions are closely related to immune cell functions, further investigations of the endolysosome system should reveal novel regulatory Endosomal/lysosomal signalling and inflammation mechanisms for the immune system. We still have more to learn about which molecules control the signalling-competent endolysosomal environment, and how and when such molecules are recruited to and regulate compartments. To better understand the molecular bases of these processes, a comprehensive understanding of dynamic organization of proteins and lipid species at the endosomes/lysosomes is necessary. Funding This work was supported by the Funding Program for Next Generation World-Leading Researchers (Next Program) (for N.T.-S., LS134), grants-in-aid for Scientific Research from the Ministry of Education, Science, Sports and Culture of Japan (for N.T.-S., 21390123, for T.K., 25871165), and a grant from the National Center for Global Health and Medicine (for N.T.-S., 23S001). Conflict of Interest None declared. References 1. Platta, H.W. and Stenmark, H. (2011) Endocytosis and signaling. Curr. Opin. Cell Biol. 23, 393403 2. Murphy, J.E., Padilla, B.E., Hasdemir, B., Cottrell, G.S., and Bunnett, N.W. (2009) Endosomes: a legitimate platform for the signaling train. Proc. Natl. Acad. Sci. U S A. 106, 1761517622 3. Hupalowska, A. and Miaczynska, M. (2012) The new faces of endocytosis in signaling. Traffic 13, 918 4. Delcroix, J.D., Valletta, J.S., Wu, C., Hunt, S.J., Kowal, A.S., and Mobley, W.C. (2003) NGF signaling in sensory neurons: evidence that early endosomes carry NGF retrograde signals. Neuron 39, 6984 5. Wiley, H.S. and Burke, P.M. (2001) Regulation of receptor tyrosine kinase signaling by endocytic trafficking. Traffic 2, 1218 6. Irannejad, R., Tomshine, J.C., Tomshine, J.R., Chevalier, M., Mahoney, J.P., Steyaert, J., Rasmussen, S.G., Sunahara, R.K., El-Samad, H., Huang, B., and von Zastrow, M. (2013) Conformational biosensors reveal GPCR signalling from endosomes. Nature 495, 534538 7. Settembre, C., Zoncu, R., Medina, D.L., Vetrini, F., Erdin, S., Huynh, T., Ferron, M., Karsenty, G., Vellard, M.C., Facchinetti, V., Sabatini, D.M., and Ballabio, A. (2012) A lysosome-to-nucleus signalling mechanism senses and regulates the lysosome via mTOR and TFEB. EMBO J. 31, 10951108 8. Sancak, Y., Bar-Peled, L., Zoncu, R., Markhard, A.L., Nada, S., and Sabatini, D.M. (2010) Ragulator-Rag complex targets mTORC1 to the lysosomal surface and is necessary for its activation by amino acids. Cell 141, 290303 9. Cang, C., Zhou, Y., Navarro, B., Seo, Y.J., Aranda, K., Shi, L., Battaglia-Hsu, S., Nissim, I., Clapham, D.E., and Ren, D. (2013) mTOR regulates lysosomal ATP-sensitive two-pore Na(þ) channels to adapt to metabolic state. Cell 152, 778790 10. Watts, C. (2012) The endosome-lysosome pathway and information generation in the immune system. Biochim. Biophys. Acta 1824, 1421 11. Kagan, J.C. (2012) Signaling organelles of the innate immune system. Cell 151, 11681178 12. Niedergang, F. and Chavrier, P. (2004) Signaling and membrane dynamics during phagocytosis: many roads lead to the phagos(R)ome. Curr. Opin. Cell Biol. 16, 422428 13. Savina, A. and Amigorena, S. (2007) Phagocytosis and antigen presentation in dendritic cells. Immunol. Rev. 219, 143156 14. Kawai, T. and Akira, S. (2008) Toll-like receptor and RIGI-like receptor signaling. Ann. N Y Acad. Sci. 1143, 120 15. Gilliet, M., Cao, W., and Liu, Y.J. (2008) Plasmacytoid dendritic cells: sensing nucleic acids in viral infection and autoimmune diseases. Nat. Rev. Immunol. 8, 594606 16. Dabbagh, K. and Lewis, D.B. (2003) Toll-like receptors and T-helper-1/T-helper-2 responses. Curr. Opin. Infect. Dis. 16, 199204 17. Steinman, R.M. and Hemmi, H. (2006) Dendritic cells: translating innate to adaptive immunity. Curr. Top. Microbiol. Immunol. 311, 1758 18. Kagan, J.C., Su, T., Horng, T., Chow, A., Akira, S., and Medzhitov, R. (2008) TRAM couples endocytosis of Toll-like receptor 4 to the induction of interferon-beta. Nat. Immunol. 9, 361368 19. Tanimura, N., Saitoh, S., Matsumoto, F., AkashiTakamura, S., and Miyake, K. (2008) Roles for LPSdependent interaction and relocation of TLR4 and TRAM in TRIF-signaling. Biochem. Biophys. Res. Commun. 368, 9499 20. Aksoy, E., Taboubi, S., Torres, D., Delbauve, S., Hachani, A., Whitehead, M.A., Pearce, W.P., Berenjeno-Martin, I., Nock, G., Filloux, A., Beyaert, R., Flamand, V., and Vanhaesebroeck, B. (2012) The p110delta isoform of the kinase PI(3)K controls the subcellular compartmentalization of TLR4 signaling and protects from endotoxic shock. Nat. Immunol. 13, 10451054 21. Blasius, A.L. and Beutler, B. (2010) Intracellular toll-like receptors. Immunity 32, 305315 22. Blasius, A.L. and Beutler, B. (2010) Intracellular toll-like receptors. Immunity 32, 305315 23. Kumagai, Y., Takeuchi, O., and Akira, S. (2008) TLR9 as a key receptor for the recognition of DNA. Adv. Drug Deliv. Rev. 60, 795804 24. Latz, E., Schoenemeyer, A., Visintin, A., Fitzgerald, K.A., Monks, B.G., Knetter, C.F., Lien, E., Nilsen, N.J., Espevik, T., and Golenbock, D.T. (2004) TLR9 signals after translocating from the ER to CpG DNA in the lysosome. Nat. Immunol. 5, 190198 25. Matsumoto, F., Saitoh, S., Fukui, R., Kobayashi, T., Tanimura, N., Konno, K., Kusumoto, Y., AkashiTakamura, S., and Miyake, K. (2008) Cathepsins are required for Toll-like receptor 9 responses. Biochem. Biophys. Res. Commun. 367, 693699 26. Park, B., Brinkmann, M.M., Spooner, E., Lee, C.C., Kim, Y.M., and Ploegh, H.L. (2008) Proteolytic cleavage in an endolysosomal compartment is required for activation of Toll-like receptor 9. Nat. Immunol. 9, 14071414 27. Honda, K., Ohba, Y., Yanai, H., Negishi, H., Mizutani, T., Takaoka, A., Taya, C., and Taniguchi, T. (2005) Spatiotemporal regulation of MyD88-IRF-7 signalling for robust type-I interferon induction. Nature 434, 10351040 28. Hsueh, R.C. and Scheuermann, R.H. (2000) Tyrosine kinase activation in the decision between growth, differentiation, and death responses initiated from the B cell antigen receptor. Adv. Immunol. 75, 283316 29. Chaturvedi, A., Martz, R., Dorward, D., Waisberg, M., and Pierce, S.K. (2011) Endocytosed BCRs sequentially 497 T. Kobayashi et al. 30. 31. 32. 33. 34. 35. 36. 37. 38. 39. 40. 41. 42. 43. 44. 45. 498 regulate MAPK and Akt signaling pathways from intracellular compartments. Nat. Immunol. 12, 11191126 Chaturvedi, A., Dorward, D., and Pierce, S.K. (2008) The B cell receptor governs the subcellular location of Toll-like receptor 9 leading to hyperresponses to DNAcontaining antigens. Immunity 28, 799809 Rawlings, D.J., Schwartz, M.A., Jackson, S.W., and Meyer-Bahlburg, A. (2012) Integration of B cell responses through Toll-like receptors and antigen receptors. Nat. Rev. Immunol. 12, 282294 Mindell, J.A. (2012) Lysosomal acidification mechanisms. Annu. Rev. Physiol. 74, 6986 Rutz, M., Metzger, J., Gellert, T., Luppa, P., Lipford, G.B., Wagner, H., and Bauer, S. (2004) Toll-like receptor 9 binds single-stranded CpG-DNA in a sequence- and pH-dependent manner. Eur. J. Immunol. 34, 25412550 Hacker, H., Mischak, H., Miethke, T., Liptay, S., Schmid, R., Sparwasser, T., Heeg, K., Lipford, G.B., and Wagner, H. (1998) CpG-DNA-specific activation of antigen-presenting cells requires stress kinase activity and is preceded by non-specific endocytosis and endosomal maturation. EMBO J. 17, 62306240 Yi, A.K., Tuetken, R., Redford, T., Waldschmidt, M., Kirsch, J., and Krieg, A.M. (1998) CpG motifs in bacterial DNA activate leukocytes through the pH-dependent generation of reactive oxygen species. J. Immunol. 160, 47554761 Ahmad-Nejad, P., Hacker, H., Rutz, M., Bauer, S., Vabulas, R.M., and Wagner, H. (2002) Bacterial CpGDNA and lipopolysaccharides activate Toll-like receptors at distinct cellular compartments. Eur. J. Immunol. 32, 19581968 Katz, S.J. and Russell, A.S. (2011) Re-evaluation of antimalarials in treating rheumatic diseases: re-appreciation and insights into new mechanisms of action. Curr. Opin. Rheumatol. 23, 278281 Lee, S.J., Silverman, E., and Bargman, J.M. (2011) The role of antimalarial agents in the treatment of SLE and lupus nephritis. Nat. Rev. Nephrol. 7, 718729 Leadbetter, E.A., Rifkin, I.R., Hohlbaum, A.M., Beaudette, B.C., Shlomchik, M.J., and MarshakRothstein, A. (2002) Chromatin-IgG complexes activate B cells by dual engagement of IgM and Toll-like receptors. Nature 416, 603607 Viglianti, G.A., Lau, C.M., Hanley, T.M., Miko, B.A., Shlomchik, M.J., and Marshak-Rothstein, A. (2003) Activation of autoreactive B cells by CpG dsDNA. Immunity 19, 837847 Platt, F.M., Boland, B., and van der Spoel, A.C. (2012) The cell biology of disease: lysosomal storage disorders: the cellular impact of lysosomal dysfunction. J. Cell Biol 199, 723734 Schultz, M.L., Tecedor, L., Chang, M., and Davidson, B.L. (2011) Clarifying lysosomal storage diseases. Trends Neurosci. 34, 401410 Neefjes, J., Jongsma, M.L., Paul, P., and Bakke, O. (2011) Towards a systems understanding of MHC class I and MHC class II antigen presentation. Nat. Rev. Immunol. 11, 823836 Savina, A., Jancic, C., Hugues, S., Guermonprez, P., Vargas, P., Moura, I.C., Lennon-Dumenil, A.M., Seabra, M.C., Raposo, G., and Amigorena, S. (2006) NOX2 controls phagosomal pH to regulate antigen processing during crosspresentation by dendritic cells. Cell 126, 205218 Jancic, C., Savina, A., Wasmeier, C., Tolmachova, T., El-Benna, J., Dang, P.M., Pascolo, S., Gougerot- 46. 47. 48. 49. 50. 51. 52. 53. 54. 55. 56. Pocidalo, M.A., Raposo, G., Seabra, M.C., and Amigorena, S. (2007) Rab27a regulates phagosomal pH and NADPH oxidase recruitment to dendritic cell phagosomes. Nat. Cell Biol. 9, 367378 Savina, A., Peres, A., Cebrian, I., Carmo, N., Moita, C., Hacohen, N., Moita, L.F., and Amigorena, S. (2009) The small GTPase Rac2 controls phagosomal alkalinization and antigen crosspresentation selectively in CD8(þ) dendritic cells. Immunity 30, 544555 Ramachandra, L., Simmons, D., and Harding, C.V. (2009) MHC molecules and microbial antigen processing in phagosomes. Curr. Opin. Immunol. 21, 98104 Sokolovska, A., Becker, C.E., Ip, W.K., Rathinam, V.A., Brudner, M., Paquette, N., Tanne, A., Vanaja, S.K., Moore, K.J., Fitzgerald, K.A., Lacy-Hulbert, A., and Stuart, L.M. Activation of caspase-1 by the NLRP3 inflammasome regulates the NADPH oxidase NOX2 to control phagosome function. Nat. Immunol. 14, 543553 Blasius, A.L., Arnold, C.N., Georgel, P., Rutschmann, S., Xia, Y., Lin, P., Ross, C., Li, X., Smart, N.G., and Beutler, B. (2010) Slc15a4, AP-3, and HermanskyPudlak syndrome proteins are required for Toll-like receptor signaling in plasmacytoid dendritic cells. Proc. Natl. Acad. Sci. U S A. 107, 1997319978 Sasawatari, S., Okamura, T., Kasumi, E., TanakaFuruyama, K., Yanobu-Takanashi, R., Shirasawa, S., Kato, N., and Toyama-Sorimachi, N. (2011) The solute carrier family 15A4 regulates TLR9 and NOD1 functions in the innate immune system and promotes colitis in mice. Gastroenterology 140, 15131525 Yamashita, T., Shimada, S., Guo, W., Sato, K., Kohmura, E., Hayakawa, T., Takagi, T., and Tohyama, M. (1997) Cloning and functional expression of a brain peptide/histidine transporter. J. Biol. Chem. 272, 1020510211 Daniel, H. and Kottra, G. (2004) The proton oligopeptide cotransporter family SLC15 in physiology and pharmacology. Pflugers Arch. 447, 610618 Bhardwaj, R.K., Herrera-Ruiz, D., Eltoukhy, N., Saad, M., and Knipp, G.T. (2006) The functional evaluation of human peptide/histidine transporter 1 (hPHT1) in transiently transfected COS-7 cells. Eur. J. Pharm. Sci. 27, 533542 Takeuchi, F., Ochiai, Y., Serizawa, M., Yanai, K., Kuzuya, N., Kajio, H., Honjo, S., Takeda, N., Kaburagi, Y., Yasuda, K., Shirasawa, S., Sasazuki, T., and Kato, N. (2008) Search for type 2 diabetes susceptibility genes on chromosomes 1q, 3q and 12q. J. Hum. Genet. 53, 314324 Lee, J., Tattoli, I., Wojtal, K.A., Vavricka, S.R., Philpott, D.J., and Girardin, S.E. (2009) pH-dependent internalization of muramyl peptides from early endosomes enables Nod1 and Nod2 signaling. J. Biol. Chem. 284, 2381823829 Han, J.W., Zheng, H.F., Cui, Y., Sun, L.D., Ye, D.Q., Hu, Z., Xu, J.H., Cai, Z.M., Huang, W., Zhao, G.P., Xie, H.F., Fang, H., Lu, Q.J., Li, X.P., Pan, Y.F., Deng, D.Q., Zeng, F.Q., Ye, Z.Z., Zhang, X.Y., Wang, Q.W., Hao, F., Ma, L., Zuo, X.B., Zhou, F.S., Du, W.H., Cheng, Y.L., Yang, J.Q., Shen, S.K., Li, J., Sheng, Y.J., Zuo, X.X., Zhu, W.F., Gao, F., Zhang, P.L., Guo, Q., Li, B., Gao, M., Xiao, F.L., Quan, C., Zhang, C., Zhang, Z., Zhu, K.J., Li, Y., Hu, D.Y., Lu, W.S., Huang, J.L., Liu, S.X., Li, H., Ren, Y.Q., Wang, Z.X., Yang, C.J., Wang, P.G., Zhou, W.M., Lv, Y.M., Zhang, A.P., Zhang, S.Q., Lin, D., Low, H.Q., Shen, M., Zhai, Z.F., Wang, Y., Zhang, F.Y., Yang, S., Liu, J.J., Endosomal/lysosomal signalling and inflammation and Zhang, X.J. (2009) Genome-wide association study in a Chinese Han population identifies nine new susceptibility loci for systemic lupus erythematosus. Nat. Genet. 41, 12341237 57. Blasius, A.L., Krebs, P., Sullivan, B.M., Oldstone, M.B., and Popkin, D.L. (2012) Slc15a4, a gene required for pDC sensing of TLR ligands, is required to control persistent viral infection. PLoS Pathog. 8, e1002915 58. Franchi, L., Warner, N., Viani, K., and Nunez, G. (2009) Function of Nod-like receptors in microbial recognition and host defense. Immunol. Rev. 227, 106128 59. Hampe, J., Cuthbert, A., Croucher, P.J., Mirza, M.M., Mascheretti, S., Fisher, S., Frenzel, H., King, K., Hasselmeyer, A., MacPherson, A.J., Bridger, S., van Deventer, S., Forbes, A., Nikolaus, S., Lennard-Jones, J.E., Foelsch, U.R., Krawczak, M., Lewis, C., Schreiber, S., and Mathew, C.G. (2001) Association between insertion mutation in NOD2 gene and Crohn’s disease in German and British populations. Lancet 357, 19251928 499