Selective modulation of autophagy, innate immunity, and adaptive immunity by small molecules

Macroautophagy (hereafter referred to as autophagy) is an evolutionarily conserved catabolic process that directs cytoplasmic proteins and organelles to the lysosome, where they are degraded for subsequent recycling. Autophagy plays important roles in normal development and differentiation, and helps maintain homeostasis in the face of nutrient and other cellular stresses. Autophagy has also been implicated in a wide variety of diseases, including cancer, neurodegenerative disease, susceptibility to pathogens, and inflammatory bowel disease.(1) Consistent with this broad disease involvement, autophagy (or individual autophagy proteins) exerts functional connections to many other cell processes, including host responses to intracellular bacteria, inflammatory signaling, and innate and adaptive immunity.(2, 3) For instance, autophagy leads to the killing of intracytoplasmic group A Streptococcus in an Atg5-dependent fashion,(4) and induction of autophagy decreases intracellular survival of M. tuberculosis. (5) Individual autophagy proteins (such as Atg5) can also mediate host defense against intracellular pathogens (such as L. Monocytogenes and the protozoan T. Gondii) in an autophagy-independent manner, highlighting that autophagy proteins may contribute to host defense through multiple mechanisms.(6) Autophagy also modulates inflammation and adaptive immunity. In myeloid cells, activation of the NLRP3 (NOD-like receptor family pyrin containing 3) inflammasome complex leads to caspase-1-mediated release of cytokines such as IL-1β, IL-18, and IL-33. Murine macrophages lacking the essential autophagy gene Atg16L1 produce increased amounts of IL-1β upon stimulation with the TLR4 ligand lipopolysaccharide (LPS).(7) Autophagy proteins also contribute to antigen presentation to CD4 and CD8 T cells. For instance, in dendritic cells, basal autophagy, as well as autophagy induced by the intracellular pattern recognition receptor NOD2, stimulates MHC class II presentation to CD4+ T cells, and antigen-specific CD4+ T cell proliferation.(8, 9) The pathways that mediate autophagy (and connect autophagy with inflammation and innate and adaptive immunity) likely involve a network of hundreds of proteins. Multiple steps of autophagy (including nucleation and elongation of an isolation membrane, fusion of the edges of the isolation membrane to form an autophagosome containing the cytoplasmic cargo, and fusion of the autophagosome with a lysosome to form an autolysosome) are mediated by multi-protein complexes and subject to regulatory inputs. A proteomic study of basal autophagy identified a network of 751 protein/protein interactions among over 400 proteins.(10) Genome-wide siRNA screens for modulators of basal or viral autophagy also support the notion of an autophagy network involving hundreds of proteins.(11, 12) To complement these genetic screens, and to better define the functional connections associated with autophagy, host response to pathogens, inflammation, and immunity, we undertook a chemical biology approach using FDA-approved drugs and bioactive compounds with predominantly known targets and mechanisms. We identified small molecules that induce basal autophagic flux, using image-based quantitation of autophagosomes and autolysosomes. Autophagic inducers were further characterized for their ability to reduce production of IL-1β by bone marrow-derived macrophages; enhance localization of the intracellular bacterium S. Typhimurium to autophagosomes in a manner that correlates with subsequent killing; and modulate differentiation of naive T-cells into Treg, TH17 and TH1 subsets. We find that compounds partition themselves into a variety of classes with distinct, selective activity profiles across these assays. Small molecules were further examined in macrophages from mice bearing a conditional knockout of the essential autophagy gene Atg16L1, where their ability to inhibit IL-1β suggests their potential utility in a variety of disease models.