pH-dependent Internalization of Muramyl Peptides from Early Endosomes Enables Nod1 and Nod2 Signaling

Nod1 and Nod2 are members of the Nod-like receptor family that detect intracellular bacterial peptidoglycan-derived muramyl peptides. The biological effects of muramyl peptides have been described for over three decades, but the mechanism underlying their internalization to the cytosol remains unclear. Using the human epithelial cell line HEK293T as a model system, we demonstrate here that Nod1-activating ligands entered cells through endocytosis, most likely by the clathrin-coated pit pathway, as internalization was dynamin-dependent but not inhibited by methyl-β-cyclodextrin. In the endocytic pathway, the cytosolic internalization of Nod1 ligands was pH-dependent, occurred prior to the acidification mediated by the vacuolar ATPase, and was optimal at pH ranging from 5.5 to 6. Similarly, the Nod2 ligand MDP was internalized into host cytosol through a similar pathway with optimal pH for internalization ranging from 5.5 to 6.5. Moreover, Nod1-activating muramyl peptides likely required processing by endosomal enzymes, prior to transport into the cytosol, suggesting the existence of a sterically gated endosomal transporter for Nod1 ligands. In support for this, we identified a role for SLC15A4, an oligopeptide transporter expressed in early endosomes, in Nod1-dependent NF-κB signaling. Interestingly, SLC15A4 expression was also up-regulated in colonic biopsies from patients with inflammatory bowel disease, a disorder associated with mutations in Nod1 and Nod2. Together, our results shed light on the mechanisms by which muramyl peptides get access to the host cytosol, where they are detected by Nod1 and Nod2, and might have implications for the understanding of human diseases, such as inflammatory bowel disease.Innate immunity relies on the detection of conserved microbial- or danger-associated molecular patterns (MAMPs or DAMPs),² by pattern-recognition molecules. In mammals, several families of pattern-recognition molecules have been recently identified, including the transmembrane Toll-like receptors (TLRs), cytosolic Nod-like receptors (NLRs), and RIG-I-like receptors (1). NLR proteins include Nod1 and Nod2, which trigger pro-inflammatory pathways such as NF-κB and mitogen-activated protein kinases, in response to bacterial peptidoglycan (2), and NLRPs (also known as Nalps), such as NLRP1 and NLRP3, which induce the activation of caspase-1 inflammasomes in response to various MAMPs and DAMPs (3).In the case of TLRs, there is accumulating evidence that the subcellular localization and the function of these pattern-recognition molecules is tightly associated, at multiple levels, with endocytosis and phagocytosis, which represent evolutionary conserved mechanisms for the internalization of small (<0.5 μm) and large (>0.5 μm) particles, respectively. Indeed, whereas some TLRs are expressed at the plasma membrane, others (such as TLR3, -7, and -9) are found predominantly associated with the endoplasmic reticulum and endosomal compartments, where they detect their respective microbial-derived nucleic acid MAMPs (4). In particular, TLR9 has been shown to move from the endoplasmic reticulum to CpG DNA-containing endosomes, concurrent with the accumulation of MyD88, thus showing that endosomes represent the physiological location where TLR9-dependent signaling arises (5). In addition, studies on TLR4 have demonstrated that lipopolysaccharide (LPS) is endocytosed by a receptor-mediated mechanism dependent on dynamin and clathrin and co-localized with TLR4 on early/sorting endosomes (6). In the case of this TLR, it is believed that endosomal trafficking is associated with termination of the MyD88-dependent pro-inflammatory signal (6). In contrast, TLR4 in early endosomes has been shown recently to engage TRAM and TRIF adaptors, resulting in the ignition of type I interferon signaling in response to LPS (7). Therefore, the nature of the cellular response to LPS is dependent upon the subcellular localization of TLR4, thus reinforcing the importance of the interplay between TLR signaling and endosomal trafficking.A number of studies have also linked TLR signaling with phagosome maturation. Although it remains controversial whether TLR-dependent signaling actually drives phagosomal maturation (8, 9), it is clear that the processing of engulfed microbes within phagosomes regulates the availability of MAMPs within this compartment. Accordingly, Herskovits et al. have recently demonstrated that, in interferon Γ-activated macrophages, the degradation of Listeria monocytogenes in the phagolysosome generates bacterial molecules, which could specifically trigger type I interferon responses through a Nod2-dependent pathway (10). This interesting observation suggests that innate immune signaling and microbial degradation within the phagolysosome are processes that are intimately linked. It also provides support to the concept that Nod-dependent signaling is associated with intracellular vesicular trafficking.Nod1 and Nod2 both detect specific structures from bacterial peptidoglycan (11). Whereas Nod2 detects muramyl dipeptide (MDP) (12, 13), a motif found in almost all bacteria, Nod1 specifically senses diaminopimelic acid (DAP)-containing muramyl peptides (14, 15). In particular, human Nod1 preferentially detects N-acetylmuramyl-l-Ala-d-Glu-mesoDAP (M-Tri-DAP) (16), and the minimal motif for Nod1-dependent sensing is the dipeptide d-Glu-mesoDAP (iE-DAP) (11, 14). Interestingly, long before the identification of Nod1 and Nod2 as sensors of muramyl peptides and bacterial peptidoglycan, the biological activities of these bacterial-derived molecules had been studied extensively (17, 18). It is well documented that these muramyl peptides trigger a multitude of immune responses, such as the induction of cytokines/chemokines, the production of nitric oxide and reactive oxygen species, and the clearance of microbes by phagocytic cells (17, 18). A considerable literature also demonstrated that these muramyl peptides synergize with MAMPs detected by TLRs, such as LPS (19). Although the identification of Nod1 and Nod2 as sensors of muramyl peptides has provided an acceleration in this field of investigation, it also brought the question of how such microbial molecules could get access to the host cytosol, where Nod1 and Nod2 reside. Interestingly, research aiming at improving the biological activities of these muramyl peptides demonstrated early on that the addition of lipophilic groups to these molecules enhanced their activity considerably, suggesting that their internalization was likely a key factor in determining their efficiency (20–23).The mechanisms by which muramyl peptides get access to the host cytosol remain unclear. This question is of fundamental importance for our understanding of Nod-dependent signaling and potentially holds broad therapeutic implications. Indeed, mutations in Nod1 and Nod2 have been associated with inflammatory bowel disease (IBD) in humans (24). In particular, Nod2 has been identified as the first susceptibility gene for Crohn''s disease (25, 26).In this report, we used the HEK293T epithelial cell line to study the mechanism of internalization of Nod1 ligands. We demonstrated that these peptidoglycan-derived molecules enter cells by endocytosis, and that the composition of the Nod1-activating molecules dramatically affected their intrinsic uptake capacity. Our data also suggested that this internalization was mediated by clathrin-dependent endocytosis, because internalization of Nod1 ligands required dynamin and was independent from caveolae. Further, we showed that, within endosomes, the internalization of Nod1 ligands was critically dependent on pH, and was optimal at pH ranging from 5.5 to 6, which are characteristic of early endosomes. Accordingly, internalization of Nod1-activating molecules did not require the action of the vacuolar ATPase (V-ATPase) complex. We also provide evidence that the Nod2 ligand MDP enters cells through a similar endocytic process. Our results also show that the internalization of Nod1 ligands is a process that is sterically gated, and likely requires the action of hydrolytic endosomal enzymes prior to transport into the cytosol, thus suggesting the existence of one or several specific transporters for Nod1 ligands in early endosomes. Using knockdown assays, we identified SLC15A4 as a putative transporter for Nod1 ligands in early endosomes. SLC15A4 expression was found to be significantly up-regulated in tissue biopsies from IBD patients, therefore highlighting a potential role for the modulation of peptidoglycan access to the cytosol in IBD etiology. Together, our results uncover the mechanism by which Nod ligands traffic into cells and get access to the cytosol where they are detected by Nod1 and Nod2. Our observations also highlight the previously unappreciated link between endosomal acidification/maturation and Nod-dependent signaling.