Multiple regulatory and effector roles of autophagy in immunity

Abstract

Autophagy is a cytoplasmic homeostasis pathway, enabling cells to digest their own cytosol, remove toxic protein aggregates, and eliminate defective or surplus organelles. A plenitude of studies has now expanded roles of autophagy to both effector and regulatory functions in innate and adaptive immunity. In its role of an immunological effector, autophagy plays many parts: (i) In its most primeval manifestation, it captures and digests intracellular microbes, (ii) it is an antimicrobial output of Toll-like receptor (TLR) response to pathogen associated molecular patterns (PAMP), and (iii) it is an effector of Th1-Th2 polarization in resistance or susceptibility to intracellular pathogens. As a regulator of immunity, autophagy plays a multitude of functions: (i) It acts as a topological inversion device servicing both innate and adaptive immunity by delivering cytosolic antigens to the lumen of MHC II compartments and cytosolic PAMPs to endosomal TLRs, (ii) it is crucial in T cell repertoire selection in the thymus and control of central tolerance, (iii) it plays a role in T and B cell homeostasis, and (iv) it is of significance for inflammatory pathology. A properly functioning autophagy helps prevent autoimmunity and assists in clearing pathogens. When aberrant, it contributes to human inflammatory disorders such as Crohn's disease.

Figure 1. Autophagy regulation and execution stages

Shown are the execution stages of autophagy, controlled by an upstream signaling cascade centered around the Ser/Thr kinase Tor, a metabolic regulator of autophagy, and a cascade of Atg factors, starting with Atg1. 1. Initiation. Upstream signaling brings about complex changes in autophagic initiation machinery leading to the formation of membranous precursors of autophagosome. (a) Signaling: nutrient or growth factor starvation signals via Ser/Thr kinase Tor; immunological stimuli (e.g. TLR activation with PAMPs) act through protein complexes containing MyD88 and Beclin 1 (Atg6), a key regulator of autophagy. (b) Beclin 1 is in an inhibitory complex with Bcl-2. Following stimulation, JNK-1 phosphorylates Bcl-2 (Bcl 2-P_i) releasing Beclin 1 from the inhibitory complex with Bcl-2. (c) Activated Beclin 1, in a tri-partite complex with type III phosphatidylinositol 3-kinase hVPS34 and Atg14, cooperates with other Atg factors to initiate autophagosome formation. (d) Atg16L1 (a genetic risk locus for inflammatory Crohn’s disease) is in a noncovalent complex with the Atg5-Atg12 protein conjugate Atg16L1 marks the site of phosphatidylinositol 3-phosphate (PI3P) -dependent initiation of autophagosome formation and sets off phases e, f and g (shown only in elongation phase for clarity). Autophagic isolation membrane (phagophore) wraps around the cytoplasmic target (cytosol, protein aggregates, mitochondria, peroxisomes, microbes, etc.). 2. Elongation. Phagophore enlarges and closes to form a double membrane organelle termed the autophagosome. (e,f) Atg16L1/Atg5-Atg12 (e) acts as an E3 enzyme to stimulates conversion of LC3 I into the LC3 II form (f) via a protein-protein and protein-lipid conjugation cascade (not shown) LC3 I has a free C-terminal Gly residue, whereas LC3 II C-terminal Gly is lipidated with phosphatidylethanolamine (PE) leading to membrane association. This stage is pre-proteolytic and the lumen is not yet acidified. (g) Atg4 reverses LC3 II into LC3 I by removing PE. Atg4 is sensitive to reactive oxygen species (ROS) released from mitochondria and may be one of the signals promoting initiation+elongation stages. 3. Maturation. Autophagosome matures into autolysosome, where the captured cargo is degraded. (h) The maturation phase is controlled by another Beclin 1-interacting factor, UVRAG. A second tri-partite complex with Beclin 1 and hVPS34 has been postulated (indicated by parentheses), containing UVRAG (Vps38) as a subunit in place of the initiation-specific factor Atg14; however, published evidence suggests that UVRAG acts independent of Beclin 1 at this stage. UVRAG interacts with HOPS which acts as a tethering and Rab gunanine nucleotide exchange factor (GEF). (i) HOPS with Vps39 stimulates activation of Rab7 by loading Rab7 with GTP via the Rab7 Vps39 (GEF) associated with HOPS complexes. Rab7 is a small GTPase controlling trafficking and identity of late endosomal/lysosomal compartments. (j) Maturation occurs through fusion with late endosomal/lysosomal organelles or delivery of trafficking intermediates carrying H⁺ ATPase components and lysosomal hydrolytic enzymes. The maturing autophagosome becomes acidified and is converted into a degradative organelle (autolysosome) with single delimiting membrane (the inner of the two membranes is dissolved) containing degraded material including internal membranes originating form the captured cytoplasmic material.

Figure 2. Signaling systems controlling autophagy

Green: positive regulators or agonists of autophagy induction; often, these are negative regulators of Tor. Red: activators of Tor, often acting as antagonists of autophagy. Tor is at the headquarters of the signaling center that decides on whether cells make or reduce biomass. Autophagy is a major mechanism for bulk reduction of cellular biomass. Subscripts: (n), nuclear; (c) or c, cytosolic. Updated and modified from Deretic, V., Current Opinion in Immunology, 2006, 18:375–382.

Figure 3. Autophagy role in Crohn’s disease

A Normal ileal crypt of Lieberkühn (CL) and vilus (V); AØ, autophagosome; E, enterocyte; E.c., adherent-invasive E. coli; G, Goblet cell; SCZ, stem cell zone; P, Paneth cell; TJ, tight junction. B. Dotted arrow, microbial translocation. 1. Possible roles of autophagy in Crohn’s disease. Autophagy may affect central tolerance by influencing negative and positive T cell selection in the thymus (for details, see text, section on autophagy in thymic selection). IRGM function in autophagy and Crohn’s disease is not known at present (indicated by a question mark). 2–4. Reported effects of Atg16L1 mutations: 2, reduced autophagy of invasive bacteria (shown using expression of Atg16L1*300A Crohn’s disease risk allele and infection of epithelial cells in vitro); 3, increased IL-1β activation (shown with ATG161ΔCCD knockout mice); 4, fewer granules or granule contents diffuse in the cytosol of Paneth cells (shown in ATG16L1^HM hypomorphic mice expressing 30% of ATG16L1 wild type levels). Modified with permission from Deretic et al., Dev Cell 2008, 15:641–642.