CLARITY and PACT-based imaging of adult zebrafish and mouse for whole-animal analysis of infections

Abstract

Visualization of infection and the associated host response has been challenging in adult vertebrates. Due to their transparency, zebrafish larvae have been used to directly observe infection in vivo, but as larvae have not yet developed a functional adaptive immune system. Thus, the study of many aspects of vertebrate infection requires dissection of adult organs or ex vivo isolation of immune cells. Cells involved in adaptive immunity mature later and thus have been difficult to access optically in intact animals. Recently, CLARITY and PACT 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 mouse lungs enables imaging of mycobacterial granulomas deep within tissue to a depth of more than 1 mm. Using established transgenic lines, we are able to image normal and pathogenic structures and their surrounding host context at high resolution. We identify the three-dimensional organization of granuloma-associated angiogenesis, an important feature of mycobacterial infection, and characterize the induction of the cytokine TNF within the granuloma using an established fluorescent reporter line. We observe 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.