Nanoparticles (NPs) enclosing antibiotics have provided promising therapy against Mycobacterium tuberculosis (Mtb) in different mammalian models. However, the NPs were not visualized in any of these animal studies. Here, we introduce the transparent zebrafish embryo as a system for noninvasive, simultaneous imaging of fluorescent NPs and the fish tuberculosis (TB) agent Mycobacterium marinum (Mm). The study was facilitated by the use of transgenic lines of macrophages, neutrophils, and endothelial cells expressing fluorescent markers readily visible in the live vertebrate. Intravenous injection of Mm led to phagocytosis by blood macrophages. These remained within the vasculature until 3 days postinfection where they started to extravasate and form aggregates of infected cells. Correlative light/electron microscopy revealed that these granuloma-like structures had significant access to the vasculature. Injection of NPs induced rapid uptake by both infected and uninfected macrophages, the latter being actively recruited to the site of infection, thereby providing an efficient targeting into granulomas. Rifampicin-loaded NPs significantly improved embryo survival and lowered bacterial load, as shown by quantitative fluorescence analysis. Our results argue that zebrafish embryos offer a powerful system for monitoring NPs in vivo and rationalize why NP therapy was so effective against Mtb in earlier studies; bacteria and NPs share the same cellular niche.

Graphical Abstract Highlights d Neuronal processing by microglia depends on Slc37a2mediated phagosomal shrinkage d Phagosomes fuse with the gastrosome, a unique compartment in the phagocytic pathway d Loss of Slc37a2 blocks phagosomal shrinkage, resulting in gastrosomal expansion d Gastrosomal expansion affects microglia phagocytosis and migration toward injuries SUMMARYPhagocytic immune cells such as microglia can engulf and process pathogens and dying cells with high efficiency while still maintaining their dynamic behavior and morphology. Effective intracellular processing of ingested cells is likely to be crucial for microglial function, but the underlying cellular mechanisms are poorly understood. Using both living fish embryos and mammalian macrophages, we show that processing depends on the shrinkage and packaging of phagosomes into a unique cellular compartment, the gastrosome, with distinct molecular and ultra-structural characteristics. Loss of the transporter Slc37a2 blocks phagosomal shrinkage, resulting in the expansion of the gastrosome and the dramatic bloating of the cell. This, in turn, affects the ability of microglia to phagocytose and migrate toward brain injuries. Thus, this work identifies a conserved crucial step in the phagocytic pathway of immune cells and provides a potential entry point for manipulating their behavior in development and disease.

Objective. Antinucleosome autoantibodies are pathogenic factors in lupus nephritis. Defects in apoptotic pathways may result in increased levels of apoptotic nucleosomes. The objectives of this study were 1) to determine whether low molecular weight oligonucleosomes are present in the kidneys of autoimmune (NZB ؋ NZW)F 1 mice, 2) to analyze whether the presence of glomerular membrane-associated TUNELpositive electron-dense structures reflect the existence of low molecular weight oligonucleosomes, and 3) to determine an eventual temporal relationship between glomerular electron-dense structures, oligonucleosomes, and proteinuria in these mice.Methods. DNA was isolated from mouse 111s34 hybridoma cells and from the kidneys of normal BALB/c mice in which apoptosis was induced by camptothecin and from the kidneys of (NZB ؋ NZW)F 1 mice at ages 4 weeks, 8 weeks, 20 weeks, and >26 weeks (nephritic mice). The DNA fragmentation pattern was determined with an Agilent bioanalyzer. An electron microscopybased TUNEL assay was performed to detect apoptotic chromatin in glomerular membranes, and immunoelectron microscopy was used to determine antibody binding. Transcription levels for nucleases associated with apoptosis and necrosis were determined by real-time polymerase chain reaction.Results. DNA from camptothecin-treated cell lines and BALB/c mouse kidneys, but not that from untreated (NZB ؋ NZW)F 1 mouse kidneys, demonstrated DNA cleavage consistent with apoptotic fragmentation. DNA from (NZB ؋ NZW)F 1 mice was devoid of apoptotic fragmentation, irrespective of the age of the mice, whereas TUNEL-positive chromatin particles were detected in glomerular membranes in nephritic mice. Renal DNase I transcription was reduced in nephritic mice. Nucleosomal DNA fragmentation in response to camptothecin exposure was highly reduced in (NZB ؋ NZW)F 1 mouse kidneys compared with that in their normal counterparts.Conclusion. The results of this study demonstrate that TUNEL-positive chromatin particles are deposited in the glomeruli of nephritic (NZB ؋ NZW)F 1 mice, due to reduced fragmentation and clearance of chromatin.

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