Inducible Lysosome Renitence in Macrophages.

Lysosomes are membrane-bounded intracellular compartments responsible for degradation of macromolecules and destruction of engulfed microbes. Extracellular molecules are taken up by pinocytosis in the case of soluble molecules and by phagocytosis in the case of particulates while cytoplasmic molecules are isolated by the process of autophagy. All of these pathways converge on delivery of contents to lysosomes where lysosomal acid-hydrolases degrade macromolecules to basic subunits for recycling. Damage to the lysosomal membrane spills acid-hydrolases and lysosome contents into the cytosol, which can lead to inflammasome activation and cell death. While lysosome damage has been studied for decades, previous assays for measuring lysosome damage were non-quantitative or insufficiently sensitive to detect small levels of damage. In this thesis I describe a novel, ratiometric, live-cell, fluorescence imaging method for measuring lysosome damage which allowed us to study mechanisms of damage and repair. Murine bone marrow-derived macrophage lysosomes were loaded with fluorescein-dextran and release from acidic lysosomes into the neutral cytosol was measured using the pH-sensitive fluorescence of fluorescein. Using this assay, we showed that lysosome damage following phagocytosis depends on the physical properties of the phagocytosed particle. Inflammasome assembly and IL-1β secretion correlated with the extent of lysosome damage. Importantly, pre-stimulation of macrophages with lipopolysaccharide resulted in reduced lysosome damage following silica particle phagocytosis. Peptidoglycan, interferon-γ, and tumor necrosis factor-α were observed to induce similar lysosome protection. Macrophage lysosomes were protected from different kinds of damage, implying that this induced effect is a general lysosome resistance to physical damage, which we call lysosome renitence. Lysosome renitence inhibited Listeria monocytogenes vacuolar escape in macrophages and lysosome damage resulting from Listeria infection, indicating that induced lysosome renitence is a novel activity which activated macrophages use to combat infection. The mechanisms responsible for lysosome renitence were examined. Luminal calcium and autophagy may be part of the lysosome repair mechanism. Lysosome renitence is a potentially important therapeutic target, as pharmacological induction of renitence without inflammation could bolster defenses against intracellular pathogens and irritant particulates.Ph.D.ImmunologyUniversity of Michigan, Horace H. Rackham School of Graduate Studieshttp://deepblue.lib.umich.edu/bitstream/2027.42/86543/1/davimich_1.pd