Autophagy and pattern recognition receptors in innate immunity

Abstract

Autophagy is a physiologically and immunologically controlled intracellular homeostatic pathway that sequesters and degrades cytoplasmic targets including macromolecular aggregates, cellular organelles such as mitochondria, and whole microbes or their products. Recent advances show that autophagy plays a role in innate immunity in several ways: (i) direct elimination of intracellular microbes by digestion in autolysosomes, (ii) delivery of cytosolic microbial products to pattern recognition receptors (PRRs) in a process referred to as topological inversion, and (iii) as an anti-microbial effector of Toll-like receptors and other PRR signaling. Autophagy eliminates pathogens in vitro and in vivo but, when aberrant due to mutations, contributes to human inflammatory disorders such as Crohn's disease. In this review, we examine these relationships and propose that autophagy is one of the most ancient innate immune defenses that has possibly evolved at the time of alpha-protobacteria-pre-eukaryote relationships, leading up to modern eukaryotic cell-mitochondrial symbiosis, and that during the metazoan evolution, additional layers of immunological regulation have been superimposed and integrated with this primordial innate immunity mechanism.

Fig. 1. Autophagy pathway: signaling and execution stages of autophagy

The Tor signaling cascade controls autophagy execution. During starvation or other physiological conditions, Tor is inhibited. This is possibly dependent on the type III PI3K. When Tor is inhibited, Atg1 and hVPS34 complexed with Beclin 1 (Atg6) lead to activation of downstream Atg factors (Atg1) and set in motion the autophagy execution phases: 1. Initiation, formation of phagophore (isolation membrane). 2. Elongation, growth of the phagophore and its closure to generate a double membrane autophagosome. 3. Maturation of the double membrane autophagosomes into autolysosomes. The membrane elongation and shape are controlled by two protein (and lipid) conjugation systems, akin in activation and conjugation cascade principle to the ubiquitination system but distinct from it. Atg12 is initially conjugated to Atg7, which acts as an E1-activating enzyme, and then gets transferred to the E2-like conjugating enzyme Atg10. This intermediate presents Atg12 to be conjugated to a Lys residue in Atg5. Atg5 is essential for autophagy in the mouse. The Atg5–12 conjugate, stabilized in a noncovalent complex with Atg16, acts as an E3 enzyme for the second conjugation system, and triggers oligomerization on the outside membrane of the growing phagophore, and also enhances conversion of LC3-I (Atg8) into its C-terminally lipidated (with PE) form LC3-II. Upon autophagosome closure, sealing the typical double membrane organelle, Atg5–12/16 and LC3 (delipidated by Atg4) dissociate from the outer autophagosomal membrane and get recycled. The LC3 associated with the lumenal membrane remains trapped in the autophagosome and is degraded during maturation into the autolysosome, which involves fusion of autophagosomes with late endosomal (LE), multivesicular bodies endosomes (MVB) and lysosomal organelles (Lys), dissolution of internal membrane, and conversion of the maturing autophagic organelles into autolysosomes. hVPS34, interacting with UVRAG and HOPS, plays a role in maturation stages of autophagy. Depicted are autophagosomes capturing an intracellular organelle (mitochondria) or microbes such as intraphagosomal bacteria or bacteria released into the cytosol.

Fig. 2. Th1 and Th2 cytokines activate and inhibit autophagy, respectively

See text for description. Note that in the mouse, IFN-γ activates expression of immunity related GTPase Irgm1 (LRG-47) to induce autophagy, while in human cells, the IRG factor IRGM is expressed independent of IFN-γ but is required for IFN-γ-induced autophagy.

Fig. 3. Autophagy as a topological inversion device feeding immune processes

For MHC class II-restricted endogenous antigen presentation, cytosolic proteins are captured by autophagosomes and are delivered to intracellular antigen-processing and loading compartments to meet the lumenally-oriented MHC II molecules. Note that the topological inversion occurs by sequestration of cytosolic proteins into the autophagosome, where they now are facing the lumen of endomembranous compartments, which puts them on the same side of the membrane as MHC II groove. For delivery of microbial products, e.g. PAMPs, a similar process sequesters them into autophagosomal lumen and follows the steps as above to deliver PAMP to lumenally oriented TLRs.

Fig. 4. Autophagy is a downstream effector of TLR signaling

LPS induces autophagy in macrophages by activating the TLR4/TRIF pathway. Zymosan induces autophagy and additionally may induce LC3 translocation to the phagosome (directly or mediated by rapid fusion with LC3-positive autophagic organelles). TLR3 (or RIG-I/MDA5) may activate autophagy upon recognition of double-stranded RNA (dsRNA). TLR7 (and TLR8, not shown) induce autophagy. In a viral infection, viral RNA is recognized by TLR7 (endocytic ssRNA) or by RIG-I (short dsRNA or 3′-triphosphate ssRNA). In macrophages, TLR7 ligands induce autophagy, activating the TLR7/MyD88 pathway. In most cells, but not in pDCs, the viral ligands detected by RIG-I induce the IPS-1 pathway (IPS-1 is associated with mitochondria). As a potential negative feedback, Atg5–Atg12 conjugate inhibits this pathway. In pDCs, constitutive autophagy delivers TLR7 ligands from the cytosol to the compartments containing TLR7.

Fig. 5. Model of interactions between autophagy and immune regulatory systems presented as an evolutionary timeline