Abstract
ABSTRACTVisualization 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.
Highlights
Owing to the limited optical clarity of vertebrate tissue, analysis of host immune cell interactions with pathogenic organisms has generally focused on either ex vivo analysis by flow cytometry or traditional pathology techniques on thin sections
The authors show that CLARITY and PACT reduce or eliminate these pigments, allowing imaging to depths in excess of 1 mm in whole animals
Their analysis reveals considerable heterogeneity within the granuloma. The authors apply these techniques to Mycobacterium tuberculosis infection in mouse lungs, enabling threedimensional visualization of infection using a Biosafety Level 3 (BSL-3) pathogen
Summary
Owing to the limited optical clarity of vertebrate tissue, analysis of host immune cell interactions with pathogenic organisms has generally focused on either ex vivo analysis by flow cytometry or traditional pathology techniques on thin sections. These techniques provide only limited spatial information, resulting in the loss of much of the three-dimensional context of infection. The study of host-microbe interactions in the context of infection frequently involves techniques that provide limited spatial information and models that are unsuitable for visualizing the adaptive immune system. The techniques have generally been applied for imaging of dissected organs
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