NLR proteins: integral members of innate immunity and mediators of inflammatory diseases

Abstract

The innate immune system is the first line of defense against microorganisms and is conserved in plants and animals. The nucleotide-binding domain, leucine rich containing (NLR) protein family is a recent addition to the members of innate immunity effector molecules. These proteins are characterized by a central oligomerization domain, termed nucleotide-binding domain (NBD) and a protein interaction domain, leucine-rich repeats (LRRs) at the C terminus. It has been shown that NLR proteins are localized to the cytoplasm and recognize microbial products. To date, it is known that Nod1 and Nod2 detect bacterial cell wall components, whereas Ipaf and Naip detect bacterial flagellin, and NACHT/LRR/Pyrin 1 has been shown to detect anthrax lethal toxin. NLR proteins comprise a diverse protein family (over 20 in humans), indicating that NLRs have evolved to acquire specificity to various pathogenic microorganisms, thereby controlling host-pathogen interactions. Activation of NLR proteins results in inflammatory responses mediated by NF-kappaB, MAPK, or Caspase-1 activation, accompanied by subsequent secretion of proinflammatory cytokines. Mutations in several members of the NLR protein family have been linked to inflammatory diseases, suggesting these molecules play important roles in maintaining host-pathogen interactions and inflammatory responses. Therefore, understanding NLR signaling is important for the therapeutic intervention of various infectious and inflammatory diseases.

Figure 1. NLRs and relevant proteins

NLRs are classified into subfamilies by protein interaction domains such as CARD or pyrin domain. NACHT (NBD) and LRRs are domains common to all NLRs. Two major subfamilies are the CARD and Pyrin subfamilies. Although they are not NLRs, putative helper and adaptor proteins are also shown, including ASC (adaptor for NLRs), Nalp10 and PYRIN (negative regulator of Caspase-1 activation).

Figure 2. Nod1/Nod2 activating peptidoglycan moieties

Schematic diagram of the Gram negative bacterial cell wall consisting of an outer (OM) and inner membrane (IM) with a periplasmic space in between. Unique to Gram negative bacteria is the presence of lipopolysaccharide (LPS) (green) on the outer membrane. The periplasm contains the thin peptidoglycan layer made of N-acetylglucosamine (NAG) and N-acetylmuramic acid (NAM) sugars and short peptide chains creating a crystal lattice-like structure. The proposed natural ligand for Nod1 is GM-tripeptide containing the meso-DAP and muramyl dipeptide (MDP) for Nod2.

Figure 3. Resistance to live bacteria conferred by NLRs

Bacteria are internalized into the host cell by phagocytosis. Some bacteria survive in the phagosomes by suppressing host defenses and phagolysosomal fusion, while others have pathogenic mechanisms that allow them to escape into the cytosol. Bacteria that remain in the phagosomes may use their pathogenic secretion system generated pores to leak PAMPs into the cytosol for recognition by NLRs. Such a mechanism has been suggested for flagellin perception by Ipaf and Naip5. In addition, peptidoglycan moiety perception by Nod1 conferring resistance to extracellular Helicobacter pylori has been suggested to be mediated by that bacterium’s type IV secretion system [175]. NLRs such as Nod1, Nod2 and Nalp3 have been shown to confer resistance to other bacteria. Depicted here are some of the representative cases based on live infection studies (please see Table 2. for a complete list of ligands and live bacteria).

Figure 4. NLR signaling

Cellular signaling of NLRs and their activation model are depicted. A. There are two major NLR signaling pathways, Rip2- and ASC- dependent pathways. Nod1 and Nod2 detect active moieties in bacterial peptidolycan, GM-triDAP and MDP, respectively. Nod1 and Nod2 signal through a kinase, Rip2, which activates NF-κB and MAPKs, leading to the activation of immune response genes. In contrast to the Rip2 dependent pathway, the ASC dependent pathway results in the activation of Caspase-1. NLRs such as Nalp3, Nalp1, Ipaf and Naip activate Caspase-1 upon ligand recognition. Active Caspase-1 has dual roles: production of mature IL-1β by cleavages of proIL-1β and induction of programmed cell death of host cells, which may act as a host defense mechanism against pathogenic organisms. B. The current proposed model of NLR activation is analogous to the activation of Apaf-1, a critical mediator of apoptosis by the mitochondrial pathway. Without apoptotic stimuli, Apaf-1 holds an autoinhibited conformation by binding of WD40 domains to CARD. When Cytochrome-c released from mitochondria binds to the WD-40 domain (Ligand binding), this event changes the conformation of Apaf-1, allowing access of ATP to the NBD. ATP binding to the NBD of Apaf-1 further changes the confirmation of Apaf-1 (Nucleotide binding), generating “active” Apaf-1. Apaf-1 oligomerizes as a heptamer protein complex using NBD as an oligomerization domain (Oligomerization). The oligomerized Apaf-1 recruits the downstream effector, Caspase-9 which causes autocleavage. By analogy, ligand binding of Nod2 and Nalp3 may result in a conformational change, subsequent oligomerization and recruitment of downstream effectors. Nod2 may recruit the Rip2 kinase, which autophosphorylates and activates downstream signaling cascades. Nalp3 may recruit ASC adaptor and pro-Caspase-1, which activates self and cleaves downstream substrates such as pro-IL1β. C. Ligand binding and ATP catalysis result in the oligomerization via the NACHT domain. Effectors are recruited to protein-protein interaction domains, such as CARD or pyrin domain. The NBD is indicated by blue, LRRs are indicated by yellow and protein-protein interaction domains are indicated by red.

Figure 5. Proposed disease mechanisms caused by mutations in NOD2 and NALP3

(A) Upon recognition of muramyl dipeptide (MDP), NOD2 signals downstream (via Rip2) to activate NF-κB. (B) Loss-of-function mutations within the LRRs of NOD2 (indicated by arrows) prevent the recognition of MDP and subsequent NF-κB activation. Such mutations predispose toward the development of Crohn’s disease. (C) Gain-of-function mutations within the NACHT domain of NOD2 (indicated by arrows) result in constitutive NF-κB activation while mutations within the NACHT domain of NALP3 result in constitutive caspase-1 activation followed by increased IL-1β production. Gain-of-function mutations within the NACHT domain of NOD2 results in Blau Syndrome and EOS while mutations within the NACHT domain of NALP3 results in diseases such as MWS, FCU and NOMID.