L-leucyl-L-leucine methyl ester does not release cysteine cathepsins to the cytosol but inactivates them in transiently permeabilized lysosomes.

L-leucyl-L-leucine methyl ester (LLOMe) induces apoptosis, which is thought to be mediated by release of lysosomal cysteine cathepsins from permeabilized lysosomes into the cytosol. Here, we demonstrated in HeLa cells that apoptotic as well as sub-apoptotic concentrations of LLOMe caused rapid and complete lysosomal membrane permeabilization (LMP), as evidenced by loss of the proton gradient and release into the cytosol of internalized lysosomal markers below a relative molecular mass of 10,000. However, there was no evidence for the release of cysteine cathepsins B and L into the cytosol; rather they remained within lysosomes, where they were rapidly inactivated and degraded. LLOMe-induced adverse effects, including LMP, loss of cysteine cathepsin activity, caspase activation and cell death could be reduced by inhibition of cathepsin C, but not by inhibiting cathepsins B and L. When incubated with sub-apoptotic LLOMe concentrations, lysosomes transiently lost protons but annealed and re-acidified within hours. Full lysosomal function required new protein synthesis of cysteine cathepsins and other hydrolyses. Our data argue against the release of lysosomal enzymes into the cytosol and their proposed proteolytic signaling during LLOMe-induced apoptosis.

Translocation of nanoparticles and Mycobacterium marinum across the intestinal epithelium in zebrafish and the role of the mucosal immune system.

Nano- and microparticles are promising carrier systems for oral delivery of drugs or vaccines, particularly in fish aquaculture. However, the mechanisms of uptake, trans-epithelial transport and immune response to nano/micrometer sized particles, or microorganisms such as bacteria are poorly understood in fish. Here, adult zebrafish were used to study the uptake of different nano- and microparticles and the pathogenic bacteria Mycobacterium marinum in the intestine, and their interactions with epithelial cells and the mucosal immune system. Fluorescent particles or bacteria were delivered directly into the adult zebrafish intestine by oral intubation and their localization was imaged in intestine, liver and spleen sections. Zebrafish do not appear to have M-cells, but both nanoparticles and bacteria were rapidly taken up in the intestine and transported to the liver and spleen. In each tissue, both bacteria and particles largely localized to leukocytes, presumably macrophages.

Layer-by-layer nanocoating of live Bacille-Calmette-Guérin mycobacteria with poly(I:C) and chitosan enhances pro-inflammatory activation and bactericidal capacity in murine macrophages.

Tuberculosis (TB) is a major disease burden globally causing more than 1.5 million deaths per year. The attenuated live vaccine strain Bacille Calmette-Guérin (BCG), although providing protection against childhood TB, is largely ineffective against adult pulmonary TB. A major aim therefore is to increase the potency of the BCG vaccine to generate stronger and more sustained immunity against TB. Here, we investigated the use of layer-by-layer (LbL) nanocoating of the surface of live BCG with several layers of polyinosinic-polycytidylic acid (poly(I:C)), a strong inducer of cell-mediated immunity, and the biodegradable polysaccharide chitosan to enhance BCG immunogenicity. Nanocoating of live BCG did not affect bacterial viability or growth in vitro but induced killing of the BCG in infected mouse bone marrow-derived macrophages and enhanced macrophage production of pro-inflammatory cytokines and expression of surface co-stimulatory molecules relative to uncoated BCG. In addition, poly(I:C) surface-coated BCG, but not BCG alone or together with soluble poly(I:C), induced high production of nitric oxide (NO) and IL-12. These results argue that BCG and surface absorbed poly(I:C) act in a synergistic manner to elicit pro-inflammatory macrophage activation. In conclusion, nanocoating of live BCG with the immunostimulatory agent poly(I:C) may be an appropriate strategy to enhance and modulate host responses to the BCG vaccine.