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Project

Can high-resolution optical projection tomography of the interplay between cryptococci and macrophages unravel the enigmas in pulmonary and cerebral cryptococosis pathogenesis?

 

Scientific state-of-the-art & aims of this proposal:

Cryptococcus neoformans and C. gattii are encapsulated yeasts that can cause life-threatening disease in both immune-competent and immune-suppressed individuals (e.g. cancer therapy patients or patients with AIDS). Cryptococcosis affects the lung and may spread to the brain, manifesting itself by meningitis or pseudocystic brain lesions. It remains unknown why, how and when the cryptococci are able to cross an apparently intact blood-brain barrier (BBB). The role of the host immune cells in the course of the disease and in particular in the process of crossing the BBB is known to be important, but is still largely enigmatic. Imaging techniques are therefore indispensable to monitor dynamic processes in vivo, to define the relevant time frames for detailed cellular analysis and this for each animal individually.

The overall objective of our research is to dynamically monitor immune cells and cryptococci in individual animals in vivo to resolve their interplay during the course of the initial lung infection and eventually their interaction with the BBB. By using different, complementary imaging modalities, we aim to track the different types of cells involved and correlate this to the progression of the infection in a mouse model for pulmonary and cerebral cryptococcosis, thereby defining the key events in cryptococcosis pathogenesis, which is essential for its diagnosis and therapy. However, imaging techniques specific for fungal infections are currently very limited as fungi pose some particular challenges1,2. We have been able to successfully develop non-invasive imaging methods to visualize and quantify the overall cryptococcal lung disease progression, using lung MRI and micro-CT. However, a method to image fungal load in vivo is currently unavailable. Using GFP-positive C. gattii, we were able to image the cryptococci in the lung with intravital confocal fluorescence microscopy – however, the field-of-view (FOV) is very limited and there is no reference to the surrounding tissue context, essential for mapping the cryptococcal cell density and spatial progression across the entire lung, as there is evidence that these parameters change during infection, depending on the disease stage3,4. This is thought to be directly related to virulence and therefore most probably to important events during pathogenesis including cryptococcal invasion of the brain. However, not much is known about this phenomenon as currently no methods are available to investigate the cryptococcal cellular distribution in intact infected mouse lungs, in 3D and with cellular resolution.

We have the ambition to provide the missing link between whole-body imaging methods that visualize overall lung disease progress in 3D, and microscopic techniques that give information with high, cellular resolution in 2D and with a very small FOV. Optical projection tomography (OPT) is a relatively new optical imaging technology that was developed to bridge this imaging gap by providing high-resolution 3D imaging and quantification of morphological features in intact mouse organs5,6. It has been successfully applied in embryology and in diabetes research7-9, but its use for adult mouse lung and brain imaging has not been explored before. Nonetheless, OPT of Cryptococcus-infected lungs would be the perfect tool to give us the missing information on fungal load, to correlate disease parameters such as cryptococcal distribution and density with infection stages (as assessed by in vivo lung MRI and CT) and eventually, with the time of crossing the BBB and invasion of the brain. As OPT offers the possibility of multiplexing different fluorescent signals in an entire organ of the mouse7,10, we plan to combine OPT for GFP-expressing cryptococci with OPT looking at the spatial distribution and co-localization with fluorescently labeled macrophages. Macrophages are very important for cryptococcosis pathogenesis. This is illustrated by the fact that cryptococci can survive inside macrophages after being phagocytized11. This observation has led to the hypothesis that cryptococci might be shuttled over the BBB inside macrophages like a Trojan horse - but the exact mechanism is not known 12. To unravel this is one of our ultimate goals, by studying the interaction between cryptococci and macrophages during lung infection, hereby defining key events during pathogenesis of cryptococcosis.

We expect that the introduction of the here developed novel complementary imaging approaches will greatly push the cryptococcosis research forward as it will give us the tools to answer the still enigmatic key phases in the pathogenesis of cryptococcosis, thereby contributing to the identification of potential targets for therapy that may result in new diagnostic approaches. These imaging tools will have further applications in enabling rapid in vivo screening and ex vivo validation of different (pathogenic) strains, including mutant strains in order to identify key virulence factors and potential therapeutic targets in mouse models of infection. The research on other life-threatening fungal infections will benefit from the research we will conduct, as our approach will be readily or with minor adaptations translatable to other (fungal) infectious disease models, such as invasive aspergillosis.

Description of the research method:

As it is currently unknown if lungs of adult mice can be imaged with OPT, a first step will be a proof-of-concept for lung imaging with OPT of normal, adult mouse lungs, optimizing the sample preparation starting from the currently available protocols for optical clearing and making the sample compatible for OPT. Contrary to embryonic lungs, adult mouse lungs contain air which is detrimental for OPT imaging and one of the challenges that need to be tackled. Therefore, careful organ preparation by tying off the trachea after inflating the lungs with OPT-compatible liquid and adaptation of the sample processing steps (clearing, embedding, imaging, data processing) will be undertaken. Possible proof-of-principle (PPoP): OPT of healthy adult mouse lung. We have an ongoing collaboration with Prof. Sharpe’s lab, co-inventor of OPT, and a shared interest in validating OPT for lung and brain imaging. We agreed to collaborate on validating lung OPT and I am very welcome in their lab to share our mutual expertise for optimal feasibility.

A next challenge will be the immunostaining of a large organ such as the lungs, as reagents have to diffuse throughout the whole lung which would require specific optimization (such as tissue permeabilization) and long incubation times. For stainings using primary (anti-GFP) and secondary (Alexa488-labeled) antibodies, we will try an approach whereby the immunostaining reagents will be intratracheally applied to ensure optimal distribution and facilitate diffusion throughout the entire lung tissue while keeping the trachea closed (tied off) to avoid reagents leaking out of the lung and air from entering the lung. PPoP: OPT of crypto-infected adult mouse lung.

In a next step, we would cross-validate our established in vivo imaging protocols (MRI, CT) with high-resolution OPT of the 3D-distribution of fungal load in the intact lung during pulmonary cryptococcosis. Infected mice will be scanned at baseline and weekly up to 5 weeks post infection with MRI and CT to monitor overall disease progression, after every imaging time point sacrificing three mice for OPT. Signal intensities and lung parameters will be quantified and cross-correlated with OPT results, with parallel ‘traditional’ histology and fungal load quantification. PPoP: cross-validated OPT and in vivo MRI & CT, validated against standard histology and fungal load quantification.

A second OPT-imaging mode that is available and very promising to answer our research questions, is selective plane illumination microscopy (SPIM). This imaging mode works best with smaller samples (up to a few millimeters) such as individual cryptococcomas, but can resolve individual cells. The sample preparation protocol is essentially the same as for OPT apart from the smaller sample size. SPIM would be the perfect modality to test the hypothesis that different capsule sizes and therefore density of the cryptococcal distribution in cryptococcomas reflect virulence and correlate with the severity of infection, and possibly with cryptococci crossing the BBB. PPoP: time line of cryptococcal density over the course of the infection, with 3D cellular density within lung cryptococcomas, in order to be correlated with cryptococci detected in the brain (with non-invasive optical imaging approaches and/or OPT of brain (see further), or in the worst case, with standard histological techniques).

It is not known how and when cryptococci are able to cross an intact BBB to infect the brain, therefore we aim to image 3D cryptococcal distribution in the brain with OPT in order to help unraveling the process. As a first step, we would optimize the sample preparation for OPT of the intact mouse brain. PPoP: OPT of healthy adult mouse brain. Imaging cryptococci invading the brain with OPT involves immunostaining of an intact, large organ, which is again highly challenging. PPoP: OPT of cryptococci-infected intact adult mouse brain.

A next step would be to use OPT and SPIM to image the interplay between cryptococci and immune cells, i.e. host macrophages. They are known to be important for spread of the infection, but it is not known how and when this happens. Cryptococci can survive intracellularly after being phagocytized by macrophages. Therefore the hypothesis has been formulated that cryptococci are being shuttled over the BBB by macrophages, as in a Trojan horse. PPOP: It is our ultimate aim to verify this hypothesis by imaging this interaction. A challenging aspect here will be to identify the exact time frame during the course of infection when the cryptococci will cross the BBB. As the process of crossing the BBB probably involves few cells, it will be challenging to track the cells dynamically in vivo. Therefore, we expect that a complimentary approach involving multi-modality (MRI, CT and optical) imaging approach will be necessary to define the time frame, combining overall infection

imaging (MRI, CT) with in vivo BLI of cryptococci. We will engineer cryptococci to express luciferase, the reporter gene for BLI. This will hopefully enable us to quantify viable cryptococci in the lung during initial lung infection and to non-invasively track cryptococcal cells that are spreading from the lungs to other organs and to the brain. PPOP: BLI of luciferase-expressing cryptococci; established time profile for the spreading of cryptococcosis (theoretical time resolution of one day). A potential challenge is that the BLI signal intensity needs to be high enough to reach optimal sensitivity. If needed, we could use the Nanoluc® luciferase gene (higher BLI signal for lower intracellular substrate concentrations; the cDNA is available in the lab) or engineer cryptococci to express an extracellularly located Gaussia luciferase, as shown to improve BLI of other yeast infections by overcoming limitations regarding the intracellular uptake of the substrate13,14. BLI is particularly challenging for fungal pathogens2,15 as also illustrated by the limited applications of fungal BLI in the literature. However, experience in our and other laboratories has shown that these challenges can be overcome13,14.

In general, we will validate OPT as a generic cross-validation tool for our in vivo experiments. This will also form the basis for future development and validation of probes for in vivo fluorescence imaging, having also applications in particular for easier clinical diagnosis of fungal infections. We will also build up expertise for a possible future implementation of OPT at KU Leuven.

Proposed staffing: postdoc (myself, for protocol development, OPT imaging) working together with a full-time technician (mainly for performing established in vivo imaging (MRI, CT, optical), sample preparation and immunostaining, mouse model induction, fungal cell culture,… during one year. The financing would be used for a full-time technician, animals and housing, consumables such as 1° and 2° Abs, scanning costs incl. anesthesia and contrast agents, open access publication costs (detailed calculations have been made).

Date:1 Oct 2014 →  31 Mar 2016
Keywords:cerebral cryptococosis
Disciplines:Multimedia processing, Biological system engineering, Signal processing, Laboratory medicine, Palliative care and end-of-life care, Regenerative medicine, Other basic sciences, Other health sciences, Nursing, Other paramedical sciences, Other translational sciences, Other medical and health sciences