Autophagy gets the ‘NOD’ to enhance bacterial handling and antigen presentation

Astudy by Cooney et al.1 in January issue of Nature Medicine, shows that nucleotide-binding oligermization domain-containing-2 (NOD2) induces autophagy in human dendritic cells (DCs), influencing bacterial killing and enhancing presentation of bacterial antigens. Another report by Travassos et al.,2 appears almost at the same time in Nature Immunology, shows that both NOD1 and NOD2 induce autophagy to enhance bacterial killing. These studies carry additional significance because of the known association of NOD2 and autophagy with Crohn's disease. Host defense to microbial invaders is triggered by the recognition of pathogen products sensed by pattern recognition receptors (PRRs) located both on the cell surface, as well as in the cytoplasm. In addition to sensing microbial products, PRRs can detect endogenous products released from damaged cells,3 referred to as danger signals or danger-associated molecular patterns. Recognition by PRRs of the invading pathogens or danger signals, initiates both innate and adaptive immune responses. The most studied PRRs are the Toll-like receptors (TLRs). Recently, it has been reported that TLR signaling pathway is a sensor for the induction of autophagy4,5 and that autophagy is important for pathogen removal and for adaptive immunity through major histocompatibility complex (MHC) class II antigen presentation.6,7 Several other classes of PRRs have been implicated in innate defenses. These include the C-type lectin receptors, the RIG-like helicases, cytosolic DNA sensors and members of the NOD-like receptor (NLR) family.8 The NLRs are a large family of cytosolic sensors, comprising about 23 members in humans. This family includes NOD proteins, which are intracellular PRRs that sense peptidoglycan moieties released from the bacterial cell wall and recruit receptor-interacting protein 2 (RIP2) to activate NF-κB (Figure 1). The NLR family also includes members of NALP inflammasome, a mutiprotein complex activated by a number of microbial products and endogenous stimuli, involved in caspase 1 activation. One important function of the NLRs is to regulate the production of the pro-inflammatory cytokines interleukin (IL)-1β, IL-18 and IL-33. Production of IL-1β occurs by a two-step process. The first step is synthesis of pro-IL-1β, which is partly by the activation of NF-κB. The second step is the cleavage of pro-IL-1β by the inflammasome (Figure 1). Nucleotide-binding oligermization domain-containing-1 is expressed in non-myeloid cells such as epithelial cells and fibroblasts, whereas NOD2 is expressed in intestinal epithelial paneth cells and in myeloid cells such as monocytes, macrophages and DCs. Polymorphisms in the ligand-recognition domain of NOD2 are associated with Crohn's disease, a chronic inflammatory disease of the human intestine. Additional studies have linked two autophagy-related genes to Crohn's disease. These are autophagy gene (ATG)16L1 and immunity-related GTPase family M.9,10 Mice deficient in ATG16L1 exhibit hyperactivation of the inflammasome and increased susceptibility to a colitis model of Crohn's disease.11 NOD2 stimulation has been shown to be involved in an antigen-specific immune response with Th2 polarization.12 However, it is not clear how bacterial detection by NOD2 is linked to its role in antigen presentation. Here, the study by Cooney et al.,1 provides a new insight into this rapidly evolving field. The investigators found that NOD2 activation by its bacterial ligand muramylidpeptide (MDP) induced autophagy in human DCs and that NOD2-mediated autophagy is required for antigen presentation. Autophagy induction had been previously linked to the activation of TLRs in primary mouse and human macrophages.4,5,6 In this study, induction of autophagy by MDP in DCs was as robust as that induced by stimulation of TLR4 by LPS or TLR1-2 by PAM3CYS4. Thus this study links another PRR to autophagy activation. MDP can also activate the NALP3 inflammasome (Figure 1). The investigators, however, found that autophagy induction by MDP did not require the inflammasome. Whether NOD-induced autophagy is mediated by RIP2 remains questionable. In this study, RIP2 deficiency seemed to interfere with MDP-induced autophagy. However, in the study by Travassos et al.,2 investigators found that both NOD1 and NOD2 can induce autophagy in epithelial cells and macrophages, respectively, but neither RIP2 nor NF-κB was required for autophagy induction. Instead, they found that NOD proteins interact with ATG16L1 and recruit it to the plasma membrane at the site of bacterial entry. One experimental difference between the two studies is that the RIP2-deficient cells used in Cooney's studies were DCs, whereas Travassos et al.2 used mouse embryonic fibroblasts. In addition, one limitation of the studies by Cooney et al.1 is that they did not test if NF-κB was indeed required for NOD2 induction of autophagy in DCs. The studies by Cooney et al.,1 however, added new dimension to their findings by focusing on an additional function of autophagy, namely antigen presentation. Recent studies have shown an important role for autophagy in antigen presentation of MHC class II-peptides by DCs.7 This observation has been exploited in vivo for vaccine therapy.6 Cooney's et al.,1 found that in DCs, antigen presentation of bacterial peptides was significantly reduced in cells deficient in NOD2 or ATG16L, suggesting that NOD2-induced autophagy promotes NOD2-mediated MHC class II antigen presentation. The most revealing insights in Cooney's study are derived from experiments done on DCs obtained from patients with Crohn's disease. The authors carefully selected patients with quiescent disease and who were free of immunotherapy for more than 12 months. DCs expressing the Crohn's disease NOD2 variant had reduced autophagic response and antigen presentation in response to MDP. These data provide strong evidence in support of the authors' conclusion of the role of NOD2 in autophagy induction and antigen presentation. However, it is not clear whether the wild-type NOD2 DCs were obtained from Crohn's disease patients not expressing the NOD2 or ATG16L mutations, or from normal subjects. It is interesting to note that DCs expressing the Crohn's disease variant ATG16LT300A showed a defect in the autophagic response and in antigen presentation after treatment with the NOD2 agonist MDP, but not after exposure to the TLR1-2 agonist PAM3CYS4. The most straightforward explanation of this finding is that ATG16L is involved in NOD2 mediated autophagy and thus these results provide indirect support for NOD2 interacting with ATG16L as shown in the study by Tarvassos et al.2 Additional experiments using DCs expressing Crohn's disease NOD2 and ATG16L variants showed defective bacterial handling. It is important to note that induction of autophagy with rapamycin corrected the bacterial handling defect in Crohn's disease DCs. The studies by Cooney et al.,1 and Travassos et al.,2 provide a biological explanation for the association of the genetic variants of NOD2 and ATG16L with Crohn's disease. Defects in autophagy induction in response to pathogens could explain the bacterial persistence associated with the disease. Several questions remain to be answered. How do NOD proteins induce autophagy? Is this induction mTOR-dependent? Clarification of the role of RIP2 and NF-κB in this pathway is also needed. It is important to note that TLR-mediated autophagy was still induced in NOD2-Atg16L1 variant expressing DCs, and that functional autophagy induction in these cells was achieved by rapamycin. This indicates that the conventional autophagy machinery is functional in Crohn's disease DCs. Therefore, potential future therapy aiming at regulating bacterial-induced autophagy could be a useful therapy in this disease. Autophagy modulation has been recently shown to regulate inflammasome activation.10,11 Similar to LPS, MDP here is shown to stimulate both autophagy and the inflammasome. The relationship between autophagy and the inflammasome is poorly understood. It is possible that induction of autophagy functions as an anti-inflammatory defense by providing protection against inflammasome activation. Hence, the balance between PRR-induced autophagy and inflammasome activation may be important in determining the inflammatory state. Thus in Crohn's disease associated with variants in NOD2 or ATG16L1, autophagy deficiency is associated with a constitutively active inflammasome. Future work is needed to dissect the evolving interwoven relationship between PRRs, including NOD proteins, autophagy and the inflammosome. This triad is emerging as the control axis of innate and adaptive immunity, as well as of cell survival and death. Model of NOD2-mediated pro-inflammatory response and antigen-specific CD4+ T-cell response in human dendritic cells. The activation of NOD2 by intracellular pathogens induces two independent pathways. The binding of MDP, a degradation product of peptidoglycans, to NOD2, initiates the recruitment of RIP2, which results in NF-κB activation and proIL-1β synthesis. MDP is also able to stimulate NALP3 inflammasome, which is a multiprotein complex containing NALP3, ASC and caspase 1, activated caspase 1 induce cleavage of proIL-1β and secretion of mature IL-1β. The activation of NOD2 by intracellular pathogens also induces the formation of autophagosome, which is mediated by Atg5, Atg7 and Atg16L1. Intracellular pathogens are engulfed by autophagosomes, degraded by lysosomes and their peptides get loaded on MHC class II in MIIC, and then migrate to cell surface for presentation to CD4+ T cells.