In vivo assessment of differences in fungal cell density in cerebral cryptococcomas of mice infected with Cryptococcus neoformans or Cryptococcus gattii.

In cerebral cryptococcomas caused by Cryptococcus neoformans or Cryptococcus gattii, the density of fungal cells within lesions can contribute to the overall brain fungal burden. In cultures, cell density is inversely related to the size of the cryptococcal capsule, a dynamic polysaccharide layer surrounding the cell. Methods to investigate cell density or related capsule size within fungal lesions of a living host are currently unavailable, precluding in vivo studies on longitudinal changes. Here, we assessed whether intravital microscopy and quantitative magnetic resonance imaging techniques (diffusion MRI and MR relaxometry) would enable non-invasive investigation of fungal cell density in cerebral cryptococcomas in mice. We compared lesions caused by type strains C. neoformans H99 and C. gattii R265 and evaluated potential relations between observed imaging properties, fungal cell density, total cell and capsule size. The observed inverse correlation between apparent diffusion coefficient and cell density permitted longitudinal investigation of cell density changes. Using these imaging methods, we were able to study the multicellular organization and cell density within brain cryptococcomas in the intact host environment of living mice. Since the MRI techniques are also clinically available, the same approach could be used to assess fungal cell density in brain lesions of patients.

CryptoCEST: A promising tool for spatially resolved identification of fungal brain lesions and their differentiation from brain tumors with MRI.

Infectious brain lesions caused by the pathogenic fungi Cryptococcus neoformans and C. gattii, also referred to as cryptococcomas, could be diagnosed incorrectly as cystic brain tumors if only based on conventional magnetic resonance (MR) images. Previous MR spectroscopy (MRS) studies showed high local concentrations of the fungal disaccharide trehalose in cryptococcomas. The aim of this study was to detect and localize fungal brain lesions caused by Cryptococcus species based on Chemical Exchange Saturation Transfer (CEST) MR imaging of endogenous trehalose, and hereby to distinguish cryptococcomas from gliomas. In phantoms, trehalose and cryptococcal cells generated a concentration-dependent CEST contrast in the 0.2 - 2 ppm chemical shift range, similar to glucose, but approximately twice as strong. In vivo single voxel MRS of a murine cryptococcoma model confirmed the presence of trehalose in cryptococcomas, but mainly for lesions that were large enough compared to the size of the MRS voxel. With CEST MRI, combining the more specific CEST signal at 0.7 ppm with the higher signal-to-noise ratio signal at 4 ppm in the CryptoCEST contrast enabled localization and distinction of cryptococcomas from the normal brain and from gliomas, even for lesions smaller than 1 mm3. Thanks to the high endogenous concentration of the fungal biomarker trehalose in cryptococcal cells, the CryptoCEST contrast allowed identification of cryptococcomas with high spatial resolution and differentiation from gliomas in mice. Furthermore, the CryptoCEST contrast was tested to follow up antifungal treatment of cryptococcomas. Translation of this non-invasive method to the clinic holds potential for improving the differential diagnosis and follow-up of cryptococcal infections in the brain.

Trehalose as quantitative biomarker for in vivo diagnosis and treatment follow-up in cryptococcomas.

Brain lesions caused by Cryptococcus neoformans or C. gattii (cryptococcomas) are typically difficult to diagnose correctly and treat effectively, but rapid differential diagnosis and treatment initiation are crucial for good outcomes. In previous studies, cultured cryptococcal isolates and ex vivo lesion material contained high concentrations of the virulence factor and fungal metabolite trehalose. Here, we studied the in vivo metabolic profile of cryptococcomas in the brain using magnetic resonance spectroscopy (MRS) and assessed the relationship between trehalose concentration, fungal burden, and treatment response in order to validate its suitability as marker for early and noninvasive diagnosis and its potential to monitor treatment in vivo. We investigated the metabolites present in early and late stage cryptococcomas using in vivo 1H MRS in a murine model and evaluated changes in trehalose concentrations induced by disease progression and antifungal treatment. Animal data were compared to 1H and 13C MR spectra of Cryptococcus cultures and in vivo data from 2 patients with cryptococcomas in the brain. In vivo MRS allowed the noninvasive detection of high concentrations of trehalose in cryptococcomas and showed a comparable metabolic profile of cryptococcomas in the murine model and human cases. Trehalose concentrations correlated strongly with the fungal burden. Treatment studies in cultures and animal models showed that trehalose concentrations decrease following exposure to effective antifungal therapy. Although further cases need to be studied for clinical validation, this translational study indicates that the noninvasive MRS-based detection of trehalose is a promising marker for diagnosis and therapeutic follow-up of cryptococcomas.

The Added Value of Longitudinal Imaging for Preclinical In Vivo Efficacy Testing of Therapeutic Compounds against Cerebral Cryptococcosis.

Brain infections with Cryptococcus neoformans are associated with significant morbidity and mortality. Cryptococcosis typically presents as meningoencephalitis or fungal mass lesions called cryptococcomas. Despite frequent in vitro discoveries of promising novel antifungals, the clinical need for drugs that can more efficiently treat these brain infections remains. A crucial step in drug development is the evaluation of in vivo drug efficacy in animal models. This mainly relies on survival studies or postmortem analyses in large groups of animals, but these techniques only provide information on specific organs of interest at predefined time points. In this proof-of-concept study, we validated the use of noninvasive preclinical imaging to obtain longitudinal information on the therapeutic efficacy of amphotericin B or fluconazole monotherapy in meningoencephalitis and cryptococcoma mouse models. Bioluminescence imaging enabled the rapid in vitro and in vivo evaluation of drug efficacy, while complementary high-resolution anatomical information obtained by magnetic resonance imaging of the brain allowed a precise assessment of the extent of infection and lesion growth rates. We demonstrated a good correlation between both imaging readouts and the fungal burden in various organs. Moreover, we identified potential pitfalls associated with the interpretation of therapeutic efficacy based solely on postmortem studies, demonstrating the added value of this noninvasive dual imaging approach compared to standard mortality curves or fungal load endpoints. This novel preclinical imaging platform provides insights in the dynamic aspects of the therapeutic response and facilitates a more efficient and accurate translation of promising antifungal compounds from bench to bedside.

Longitudinal In Vivo Assessment of Host-Microbe Interactions in a Murine Model of Pulmonary Aspergillosis.

The fungus Aspergillus fumigatus is ubiquitous in nature and the most common cause of invasive pulmonary aspergillosis (IPA) in patients with a compromised immune system. The development of IPA in patients under immunosuppressive treatment or in patients with primary immunodeficiency demonstrates the importance of the host immune response in controlling aspergillosis. However, study of the host-microbe interaction has been hampered by the lack of tools for their non-invasive assessment. We developed a methodology to study the response of the host's immune system against IPA longitudinally in vivo by using fluorine-19 magnetic resonance imaging (19F MRI). We showed the advantage of a perfluorocarbon-based contrast agent for the in vivo labeling of macrophages and dendritic cells, permitting quantification of pulmonary inflammation in different murine IPA models. Our findings reveal the potential of 19F MRI for the assessment of rapid kinetics of innate immune response against IPA and the permissive niche generated through immunosuppression.

Sensitive bioluminescence imaging of fungal dissemination to the brain in mouse models of cryptococcosis.

Cryptococcus neoformans is a leading cause of fungal brain infection, but the mechanism of dissemination and dynamics of cerebral infection following pulmonary disease are poorly understood. To address these questions, non-invasive techniques that can study the dynamic processes of disease development and progression in living animal models or patients are required. As such, bioluminescence imaging (BLI) has emerged as a powerful tool to evaluate the spatial and temporal distribution of infection in living animals. We aimed to study the time profile of the dissemination of cryptococcosis from the lung to the brain in murine models by engineering the first bioluminescent C. neoformans KN99α strain, expressing a sequence-optimized red-shifted luciferase. The high pathogen specificity and sensitivity of BLI was complemented by the three-dimensional anatomical information from micro-computed tomography (µCT) of the lung and magnetic resonance imaging (MRI) of the brain. These non-invasive imaging techniques provided longitudinal readouts on the spatial and temporal distribution of infection following intravenous, intranasal or endotracheal routes of inoculation. Furthermore, the imaging results correlated strongly with the fungal load in the respective organs. By obtaining dynamic and quantitative information about the extent and timing of brain infections for individual animals, we found that dissemination to the brain after primary infection of the lung is likely a late-stage event with a timeframe that is variable between animals. This novel tool in Cryptococcus research can aid the identification of host and pathogen factors involved in this process, and supports development of novel preventive or therapeutic approaches.

A Multimodal Imaging Approach Enables In Vivo Assessment of Antifungal Treatment in a Mouse Model of Invasive Pulmonary Aspergillosis.

Aspergillus fumigatus causes life-threatening lung infections in immunocompromised patients. Mouse models are extensively used in research to assess the in vivo efficacies of antifungals. In recent years, there has been an increasing interest in the use of noninvasive imaging techniques to evaluate experimental infections. However, single imaging modalities have limitations concerning the type of information they can provide. In this study, magnetic resonance imaging and bioluminescence imaging were combined to obtain longitudinal information on the extent of developing lesions and fungal load in a leukopenic mouse model of invasive pulmonary aspergillosis (IPA). This multimodal imaging approach was used to assess changes occurring within lungs of infected mice receiving voriconazole treatment starting at different time points after infection. The results showed that IPA development depends on the inoculum size used to infect animals and that disease can be successfully prevented or treated by initiating intervention during early stages of infection. Furthermore, we demonstrated that a reduction in fungal load is not necessarily associated with the disappearance of lesions on anatomical lung images, especially when antifungal treatment coincides with immune recovery. In conclusion, multimodal imaging allows an investigation of different aspects of disease progression or recovery by providing complementary information on dynamic processes, which are highly useful for assessing the efficacy of (novel) therapeutic compounds in a time- and labor-efficient manner.

Bronchoscopic fibered confocal fluorescence microscopy for longitudinal in vivo assessment of pulmonary fungal infections in free-breathing mice.

Respiratory diseases, such as pulmonary infections, are an important cause of morbidity and mortality worldwide. Preclinical studies often require invasive techniques to evaluate the extent of infection. Fibered confocal fluorescence microscopy (FCFM) is an emerging optical imaging technique that allows for real-time detection of fluorescently labeled cells within live animals, thereby bridging the gap between in vivo whole-body imaging methods and traditional histological examinations. Previously, the use of FCFM in preclinical lung research was limited to endpoint observations due to the invasive procedures required to access lungs. Here, we introduce a bronchoscopic FCFM approach that enabled in vivo visualization and morphological characterisation of fungal cells within lungs of mice suffering from pulmonary Aspergillus or Cryptococcus infections. The minimally invasive character of this approach allowed longitudinal monitoring of infection in free-breathing animals, thereby providing both visual and quantitative information on infection progression. Both the sensitivity and specificity of this technique were high during advanced stages of infection, allowing clear distinction between infected and non-infected animals. In conclusion, our study demonstrates the potential of this novel bronchoscopic FCFM approach to study pulmonary diseases, which can lead to novel insights in disease pathogenesis by allowing longitudinal in vivo microscopic examinations of the lungs.

Nanoparticle-induced inflammation can increase tumor malignancy.

Nanomaterials, such as aluminum oxide, have been regarded with high biomedical promise as potential immune adjuvants in favor of their bulk counterparts. For pathophysiological conditions where elevated immune activity already occurs, the contribution of nanoparticle-activated immune reactions remains unclear. Here, we investigated the effect of spherical and wire-shaped aluminum oxide nanoparticles on primary splenocytes and observed a clear pro-inflammatory effect of both nanoparticles, mainly for the high aspect ratio nanowires. The nanoparticles resulted in a clear activation of NLRP3 inflammasome, and also secreted transforming growth factor ß. When cancer cells were exposed to these cytokines, this resulted in an increased level of epithelial-to-mesenchymal-transition, a hallmark for cancer metastasis, which did not occur when the cancer cells were directly exposed to the nanoparticles themselves. Using a syngeneic tumor model, the level of inflammation and degree of lung metastasis were significantly increased when the animals were exposed to the nanoparticles, particularly for the nanowires. This effect could be abrogated by treating the animals with inflammatory inhibitors. Collectively, these data indicate that the interaction of nanoparticles with immune cells can have secondary effects that may aggravate pathophysiological conditions, such as cancer malignancy, and conditions must be carefully selected to finely tune the induced aspecific inflammation into cancer-specific antitumor immunity. STATEMENT OF SIGNIFICANCE: Many different types of nanoparticles have been shown to possess immunomodulatory properties, depending on their physicochemical parameters. This can potentially be harnessed as a possible antitumor therapy. However, in the current work we show that inflammation elicited by nanomaterials can have grave effects in pathophysiological conditions, where non-specific inflammation was found to increase cancer cell mobility and tumor malignancy. These data show that immunomodulatory properties of nanomaterials must be carefully controlled to avoid any undesired side-effects.

Longitudinal, in vivo assessment of invasive pulmonary aspergillosis in mice by computed tomography and magnetic resonance imaging.

Invasive aspergillosis is an emerging threat to public health due to the increasing use of immune suppressive drugs and the emergence of resistance against antifungal drugs. To deal with this threat, research on experimental disease models provides insight into the pathogenesis of infections caused by susceptible and resistant Aspergillus strains and by assessing their response to antifungal drugs. However, standard techniques used to evaluate infection in a preclinical setting are severely limited by their invasive character, thereby precluding evaluation of disease extent and therapy effects in the same animal. To enable non-invasive, longitudinal monitoring of invasive pulmonary aspergillosis in mice, we optimized computed tomography (CT) and magnetic resonance imaging (MRI) techniques for daily follow-up of neutropenic BALB/c mice intranasally infected with A. fumigatus spores. Based on the images, lung parameters (signal intensity, lung tissue volume and total lung volume) were quantified to obtain objective information on disease onset, progression and extent for each animal individually. Fungal lung lesions present in infected animals were successfully visualized and quantified by both CT and MRI. By using an advanced MR pulse sequence with ultrashort echo times, pathological changes within the infected lung became visually and quantitatively detectable at earlier disease stages, thereby providing valuable information on disease onset and progression with high sensitivity. In conclusion, these non-invasive imaging techniques prove to be valuable tools for the longitudinal evaluation of dynamic disease-related changes and differences in disease severity in individual animals that might be readily applied for rapid and cost-efficient drug screening in preclinical models in vivo.

Longitudinal micro-CT provides biomarkers of lung disease that can be used to assess the effect of therapy in preclinical mouse models, and reveal compensatory changes in lung volume.

In vivo lung micro-computed tomography (micro-CT) is being increasingly embraced in pulmonary research because it provides longitudinal information on dynamic disease processes in a field in which ex vivo assessment of experimental disease models is still the gold standard. To optimize the quantitative monitoring of progression and therapy of lung diseases, we evaluated longitudinal changes in four different micro-CT-derived biomarkers [aerated lung volume, lung tissue (including lesions) volume, total lung volume and mean lung density], describing normal development, lung infections, inflammation, fibrosis and therapy. Free-breathing mice underwent micro-CT before and repeatedly after induction of lung disease (bleomycin-induced fibrosis, invasive pulmonary aspergillosis, pulmonary cryptococcosis) and therapy (imatinib). The four lung biomarkers were quantified. After the last time point, we performed pulmonary function tests and isolated the lungs for histology. None of the biomarkers remained stable during longitudinal follow-up of adult healthy mouse lungs, implying that biomarkers should be compared with age-matched controls upon intervention. Early inflammation and progressive fibrosis led to a substantial increase in total lung volume, which affects the interpretation of aerated lung volume, tissue volume and mean lung density measures. Upon treatment of fibrotic lung disease, the improvement in aerated lung volume and function was not accompanied by a normalization of the increased total lung volume. Significantly enlarged lungs were also present in models of rapidly and slowly progressing lung infections. The data suggest that total lung volume changes could partly reflect a compensatory mechanism that occurs during disease progression in mice. Our findings underscore the importance of quantifying total lung volume in addition to aerated lung or lesion volumes to accurately document growth and potential compensatory mechanisms in mouse models of lung disease, in order to fully describe and understand dynamic processes during lung disease onset, progression and therapy. This is highly relevant for the translation of therapy evaluation results from preclinical studies to human patients.

Longitudinal in vivo microcomputed tomography of mouse lungs: No evidence for radiotoxicity.

Before microcomputed tomography (micro-CT) can be exploited to its full potential for longitudinal monitoring of transgenic and experimental mouse models of lung diseases, radiotoxic side effects such as inflammation or fibrosis must be considered. We evaluated dose and potential radiotoxicity to the lungs for long-term respiratory-gated high-resolution micro-CT protocols. Free-breathing C57Bl/6 mice underwent four different retrospectively respiratory gated micro-CT imaging schedules of repeated scans during 5 or 12 wk, followed by ex vivo micro-CT and detailed histological and biochemical assessment of lung damage. Radiation exposure, dose, and absorbed dose were determined by ionization chamber, thermoluminescent dosimeter measurements and Monte Carlo calculations. Despite the relatively large radiation dose delivered per micro-CT acquisition, mice did not show any signs of radiation-induced lung damage or fibrosis when scanned weekly during 5 and up to 12 wk. Doubling the scanning frequency and once tripling the radiation dose as to mimic the instant repetition of a failed scan also stayed without detectable toxicity after 5 wk of scanning. Histological analyses confirmed the absence of radiotoxic damage to the lungs, thereby demonstrating that long-term monitoring of mouse lungs using high-resolution micro-CT is safe. This opens perspectives for longitudinal monitoring of (transgenic) mouse models of lung diseases and therapeutic response on an individual basis with high spatial and temporal resolution, without concerns for radiation toxicity that could potentially influence the readout of micro-CT-derived lung biomarkers. This work further supports the introduction of micro-CT for routine use in the preclinical pulmonary research field where postmortem histological approaches are still the gold standard.

Magnetic resonance imaging for noninvasive assessment of lung fibrosis onset and progression: cross-validation and comparison of different magnetic resonance imaging protocols with micro-computed tomography and histology in the bleomycin-induced mouse model.

OBJECTIVES: Bleomycin instillation is frequently used to model lung fibrosis, although the onset and severity of pathology varies highly between mice. This makes non-invasive fibrosis detection and quantification essential to obtain a comprehensive analysis of the disease course and to validate novel therapies. Magnetic resonance imaging (MRI) of lung disease progression and therapy may provide such a sensitive in vivo readout of lung fibrosis, bypassing radiotoxicity concerns (when using micro-CT [µCT]) and elaborate invasive end point measurements (histology). We aimed to optimize and evaluate 3 different lung MRI contrast and acquisition methods to visualize disease onset and progression in the bleomycin-induced mouse model of lung fibrosis using a small-animal MRI scanner. For validation, we compared the MRI results with established µCT and histological measures of lung fibrosis. MATERIALS AND METHODS: Free-breathing bleomycin-instilled and control mice were scanned in vivo with respiration-triggered conventional, ultrashort echo time and self-gated MRI pulse sequences (9.4 T) and µCT at baseline and weekly at days 7, 14, 21, and 28 after bleomycin instillation. After the last imaging time point, the mice were killed and the lungs were isolated for criterion standard histological analysis of lung fibrosis and quantification of lung collagen content for validation of the imaging results. The agreement between quantitative MRI and µCT data and standard measurements was analyzed by linear regression. RESULTS: All 3 MRI protocols were able to visualize and quantify lung pathology onset and progression in individual bleomycin-instilled mice. In vivo MRI results were in excellent agreement with in vivo µCT and criterion standard histological measures of lung fibrosis. Ultrashort echo time MRI appeared particularly useful for detecting early disease; self-gated MRI, for improved breathing motion handling. DISCUSSION: Magnetic resonance imaging sensitively visualizes and quantifies lung fibrosis in vivo, which makes it a noninvasive, translatable, safe, and potentially more versatile alternative to invasive methods or µCT, thereby stimulating pathogenesis and preclinical research.