| Autor: |
Cronan MR; Department of Molecular Genetics and Microbiology, Duke University School of Medicine, Durham, NC, USA. mark.cronan@duke.edu., Matty MA; Department of Molecular Genetics and Microbiology, Duke University School of Medicine, Durham, NC, USA., Rosenberg AF; Department of Molecular Genetics and Microbiology, Duke University School of Medicine, Durham, NC, USA., Blanc L; Public Health Research Institute, New Jersey Medical School, Rutgers, The State University of New Jersey, Newark, NJ, USA., Pyle CJ; Department of Molecular Genetics and Microbiology, Duke University School of Medicine, Durham, NC, USA., Espenschied ST; Department of Molecular Genetics and Microbiology, Duke University School of Medicine, Durham, NC, USA., Rawls JF; Department of Molecular Genetics and Microbiology, Duke University School of Medicine, Durham, NC, USA., Dartois V; Public Health Research Institute, New Jersey Medical School, Rutgers, The State University of New Jersey, Newark, NJ, USA.; Department of Medicine, New Jersey Medical School, Rutgers, The State University of New Jersey, Newark, NJ, USA., Tobin DM; Department of Molecular Genetics and Microbiology, Duke University School of Medicine, Durham, NC, USA. david.tobin@duke.edu.; Department of Immunology, Duke University School of Medicine, Durham, NC, USA. david.tobin@duke.edu. |
| Abstrakt: |
A central and critical structure in tuberculosis, the mycobacterial granuloma consists of highly organized immune cells, including macrophages that drive granuloma formation through a characteristic epithelioid transformation. Difficulties in imaging within intact animals and caveats associated with in vitro assembly models have severely limited the study and experimental manipulation of mature granulomas. Here we describe a new ex vivo culture technique, wherein mature, fully organized zebrafish granulomas are microdissected and maintained in three-dimensional (3D) culture. This approach enables high-resolution microscopy of granuloma macrophage dynamics, including epithelioid macrophage motility and granuloma consolidation, while retaining key bacterial and host characteristics. Using mass spectrometry, we find active production of key phosphotidylinositol species identified previously in human granulomas. We also describe a method to transfect isolated granulomas, enabling genetic manipulation, and provide proof-of-concept for host-directed small-molecule screens, identifying protein kinase C (PKC) signaling as an important regulator of granuloma macrophage organization. |
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