CLARITY and PACT-based imaging of adult zebrafish and mouse for whole-animal analysis of infections.
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2015-12
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Visualization of infection and the associated host response has been challenging in adult vertebrates. Owing to their transparency, zebrafish larvae have been used to directly observe infection in vivo; however, such larvae have not yet developed a functional adaptive immune system. Cells involved in adaptive immunity mature later and have therefore been difficult to access optically in intact animals. Thus, the study of many aspects of vertebrate infection requires dissection of adult organs or ex vivo isolation of immune cells. Recently, CLARITY and PACT (passive clarity technique) methodologies have enabled clearing and direct visualization of dissected organs. Here, we show that these techniques can be applied to image host-pathogen interactions directly in whole animals. CLARITY and PACT-based clearing of whole adult zebrafish and Mycobacterium tuberculosis-infected mouse lungs enables imaging of mycobacterial granulomas deep within tissue to a depth of more than 1 mm. Using established transgenic lines, we were able to image normal and pathogenic structures and their surrounding host context at high resolution. We identified the three-dimensional organization of granuloma-associated angiogenesis, an important feature of mycobacterial infection, and characterized the induction of the cytokine tumor necrosis factor (TNF) within the granuloma using an established fluorescent reporter line. We observed heterogeneity in TNF induction within granuloma macrophages, consistent with an evolving view of the tuberculous granuloma as a non-uniform, heterogeneous structure. Broad application of this technique will enable new understanding of host-pathogen interactions in situ.
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Cronan, Mark R, Allison F Rosenberg, Stefan H Oehlers, Joseph W Saelens, Dana M Sisk, Kristen L Jurcic Smith, Sunhee Lee, David M Tobin, et al. (2015). CLARITY and PACT-based imaging of adult zebrafish and mouse for whole-animal analysis of infections. Dis Model Mech, 8(12). pp. 1643–1650. 10.1242/dmm.021394 Retrieved from https://hdl.handle.net/10161/12636.
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David M. Tobin
Tuberculosis: Mycobacterial Pathogenesis and Host Susceptibility
Tuberculosis kills 1.5 million people annually. Our laboratory aims to understand the intricate interplay between mycobacteria and their hosts using a combination of model organism genetics, human genetics, pharmacology and high-resolution microscopy. By identifying key pathways utilized by the infecting bacteria and the host innate immune system, we hope to discover new therapeutic targets and interventions to combat this enduringly destructive disease.
Using a Mycobacterium/zebrafish model, we have identified new host susceptibility loci for tuberculosis. Zebrafish are natural hosts to Mycobacterium marinum, the closest relative of the Mycobacterium tuberculosis complex. Because zebrafish embryos and larvae are optically transparent, we are able to visualize the complex details of mycobacterial pathogenesis in whole, live animals. The facile genetics of the zebrafish allow us to map and positionally clone affected host susceptibility genes. In addition, zebrafish larvae are remarkably permeable to small molecules, providing a platform for whole-animal pharmacological manipulation of specific host immune responses.
We have identified novel pathways that modulate susceptibility to tuberculosis. We have shown that genes identified in the zebrafish model are also important in human tuberculosis. We find robust associations of human variants in a specific eicosanoid pathway with susceptibility to both tuberculosis and leprosy.
We have active collaborations in both Vietnam and Guatemala. In Guatemala, we are working with the Clínica Familiar Luis Angel García and the Asociación de Salud Integral to support projects involving HIV-infected patients and to understand the dynamics of TB transmission in Central America.
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