Macrophage transplantation rescues RNASET2-deficient leukodystrophy by replacing deficient microglia in a zebrafish model.

RNASET2-deficient leukodystrophy is a rare infantile white matter disorder mimicking a viral infection and resulting in severe psychomotor impairments. Despite its severity, there is little understanding of cellular mechanisms of pathogenesis and no treatments. Recent research using the rnaset2 mutant zebrafish model has suggested that microglia may be the drivers of the neuropathology, due to their failure to digest apoptotic debris during neurodevelopment. Therefore, we developed a strategy for microglial replacement through transplantation of adult whole kidney marrow-derived macrophages into embryonic hosts. Using live imaging, we revealed that transplant-derived macrophages can engraft within host brains and express microglia-specific markers, suggesting the adoption of a microglial phenotype. Tissue-clearing strategies revealed the persistence of transplanted cells in host brains beyond embryonic stages. We demonstrated that transplanted cells clear apoptotic cells within the brain, as well as rescue overactivation of the antiviral response otherwise seen in mutant larvae. RNA sequencing at the point of peak transplant-derived cell engraftment confirms that transplantation can reduce the brain-wide immune response and particularly, the antiviral response, in rnaset2-deficient brains. Crucially, this reduction in neuroinflammation resulted in behavioral rescue-restoring rnaset2 mutant motor activity to wild-type (WT) levels in embryonic and juvenile stages. Together, these findings demonstrate the role of microglia as the cellular drivers of neuropathology in rnaset2 mutants and that macrophage transplantation is a viable strategy for microglial replacement in the zebrafish. Therefore, microglia-targeted interventions may have therapeutic benefits in RNASET2-deficient leukodystrophy.

What can we learn about fish neutrophil and macrophage response to immune challenge from studies in zebrafish.

Fish rely, to a high degree, on the innate immune system to protect them against the constant exposure to potential pathogenic invasion from the surrounding water during homeostasis and injury. Zebrafish larvae have emerged as an outstanding model organism for immunity. The cellular component of zebrafish innate immunity is similar to the mammalian innate immune system and has a high degree of sophistication due to the needs of living in an aquatic environment from early embryonic stages of life. Innate immune cells (leukocytes), including neutrophils and macrophages, have major roles in protecting zebrafish against pathogens, as well as being essential for proper wound healing and regeneration. Zebrafish larvae are visually transparent, with unprecedented in vivo microscopy opportunities that, in combination with transgenic immune reporter lines, have permitted visualisation of the functions of these cells when zebrafish are exposed to bacterial, viral and parasitic infections, as well as during injury and healing. Recent findings indicate that leukocytes are even more complex than previously anticipated and are essential for inflammation, infection control, and subsequent wound healing and regeneration.

Experimental evolution of Staphylococcus aureus in macrophages: dissection of a conditional adaptive trait promoting intracellular survival.

Staphylococcus aureus is a major pathogen associated with important diseases in humans and animals. Macrophages are a key component of the innate immune response to S. aureus infection and play a major role in disease outcomes. To investigate the adaptive evolution of S. aureus in response to macrophages, we developed an experimental infection assay. S. aureus strains representing major human epidemic clones were passaged many times in a macrophage cell line, accumulating mutations in an array of genomic loci. Phenotypic analysis revealed the emergence of a lineage exhibiting increased survival in macrophages and human blood, and resistance to vancomycin. The evolved lineage exhibited a previously undescribed small colony variant (SCV) phenotype characterized by hyper-pigmentation, which resulted from a missense mutation in rsbW. Notably, the novel SCV was a conditional adaptive trait that was unstable in nutrient-replete conditions in vitro, rapidly converting from hyper-pigmented SCV to a non-pigmented large colony variant via spontaneous sigB deletion events. Importantly, we identified similar deletions in the genome sequences of a limited number of clinical S. aureus isolates from public databases, indicating that related events may occur during clinical infection. Experimental infection of zebrafish did not reveal a difference in virulence between parent and novel SCV but demonstrated an in vivo fitness cost for the compensatory sigB deletion events. Taken together, we report an experimental evolutionary approach for investigating bacterial innate immune cell interactions, revealing a conditional adaptation that promotes S. aureus survival in macrophages and resistance to vancomycin. IMPORTANCE: Staphylococcus aureus is an important human bacterial pathogen. The host response to S. aureus involves the production of innate immune cells such as macrophages which are important for fighting infection. Here we report a new model of experimental evolution for studying how S. aureus can evade killing by macrophages. We identified a novel adaptive phenotype that promotes survival in macrophages and blood and resistance to antibiotics. The phenotype is lost rapidly upon growth in nutrient-rich conditions via disruption of the alternative sigma factor sigB, revealing a conditional niche-specific fitness advantage. Genomic analysis of clinical isolates suggests similar adaptations may occur during human infections. Our model may be used broadly to identify adaptations of S. aureus to the innate immune response.