PUBLICATION
An explant technique for high-resolution imaging and manipulation of mycobacterial granulomas
- Authors
- Cronan, M.R., Matty, M.A., Rosenberg, A.F., Blanc, L., Pyle, C.J., Espenschied, S.T., Rawls, J.F., Dartois, V., Tobin, D.M.
- ID
- ZDB-PUB-181207-12
- Date
- 2018
- Source
- Nature Methods 15: 1098-1107 (Journal)
- Registered Authors
- Cronan, Mark, Espenschied, Scott "Ted", Matty, Molly, Rawls, John F., Rosenberg, Allison, Tobin, David
- Keywords
- none
- MeSH Terms
-
- Animals
- Animals, Genetically Modified
- Granuloma/drug therapy
- Granuloma/microbiology
- Granuloma/pathology*
- Image Processing, Computer-Assisted/methods*
- Imaging, Three-Dimensional/methods*
- Indoles/pharmacology
- Macrophages/drug effects
- Macrophages/microbiology
- Macrophages/pathology*
- Maleimides/pharmacology
- Mycobacterium tuberculosis/drug effects
- Protein Kinase Inhibitors/pharmacology
- Tuberculosis/drug therapy
- Tuberculosis/microbiology
- Tuberculosis/pathology*
- Zebrafish
- PubMed
- 30504889 Full text @ Nat. Methods
Citation
Cronan, M.R., Matty, M.A., Rosenberg, A.F., Blanc, L., Pyle, C.J., Espenschied, S.T., Rawls, J.F., Dartois, V., Tobin, D.M. (2018) An explant technique for high-resolution imaging and manipulation of mycobacterial granulomas. Nature Methods. 15:1098-1107.
Abstract
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.
Genes / Markers
Expression
Phenotype
Mutations / Transgenics
Human Disease / Model
Sequence Targeting Reagents
Fish
Orthology
Engineered Foreign Genes
Mapping