Aspergillus fumigatus is an opportunistic fungal pathogen that can cause life-threatening invasive lung infections in immunodeficient patients. The cellular and molecular processes of infection during onset, establishment, and progression of A. fumigatus infections are highly complex and depend on both fungal attributes and the immune status of the host. Therefore, preclinical animal models are of paramount importance to investigate and gain better insight into the infection process. Yet, despite their extensive use, commonly employed murine models of invasive pulmonary aspergillosis are not well understood due to analytical limitations. Here, we present quantitative light sheet fluorescence microscopy (LSFM) to describe fungal growth and the local immune response in whole lungs at cellular resolution within its anatomical context. We analyzed three very common murine models of pulmonary aspergillosis based on immunosuppression with corticosteroids, chemotherapy-induced leukopenia, or myeloablative irradiation. LSFM uncovered distinct architectures of fungal growth and degrees of tissue invasion in each model. Furthermore, LSFM revealed the spatial distribution, interaction, and activation of two key immune cell populations in antifungal defense: alveolar macrophages and polymorphonuclear neutrophils. Interestingly, the patterns of fungal growth correlated with the detected effects of the immunosuppressive regimens on the local immune cell populations. Moreover, LSFM demonstrates that the commonly used intranasal route of spore administration did not result in complete intra-alveolar deposition, as about 80% of fungal growth occurred outside the alveolar space. Hence, characterization by LSFM is more rigorous than by previously used methods employing murine models of invasive pulmonary aspergillosis and pinpoints their strengths and limitations. IMPORTANCE The use of animal models of infection is essential to advance our understanding of the complex host-pathogen interactions that take place during Aspergillus fumigatus lung infections. As in the case of humans, mice need to suffer an immune imbalance in order to become susceptible to invasive pulmonary aspergillosis (IPA), the most serious infection caused by A. fumigatus. There are several immunosuppressive regimens that are routinely used to investigate fungal growth and/or immune responses in murine models of invasive pulmonary aspergillosis. However, the precise consequences of the use of each immunosuppressive model for the local immune populations and for fungal growth are not completely understood. Here, to pin down the scenarios involving commonly used IPA models, we employed light sheet fluorescence microscopy (LSFM) to analyze whole lungs at cellular resolution. Our results will be valuable to optimize and refine animal models to maximize their use in future research.

Activating mutations in the human KIT receptor is known to drive severe hematopoietic disorders and tumor formation spanning various entities. The most common mutation is the substitution of aspartic acid at position 816 to valine (D816V), rendering the receptor constitutively active independent of ligand binding. As the role of the KIT receptor in placental signaling cascades is poorly understood, we analyzed the impact of KITD816V expression on placental development using a humanized mouse model. Placentas from KITD816V animals present with a grossly changed morphology, displaying a reduction in labyrinth and spongiotrophoblast layer and an increase in the Parietal Trophoblast Giant Cell (P-TGC) layer. Elevated differentiation to P-TGCs was accompanied with reduced differentiation to other Trophoblast Giant Cell (TGC) subtypes and by severe decrease in proliferation. The embryos display growth retardation and die in utero. KITD816V-trophoblast stem cells (TSC) differentiate much faster compared to wild type (WT) controls. In undifferentiated KITD816V-TSCs, levels of Phosphorylated Extracellular-signal Regulated Kinase (P-ERK) and Phosphorylated Protein Kinase B (P-AKT) are comparable to wildtype cultures differentiating for 3–6 days. Accordingly, P-TGC markers Placental Lactogen 1 (PL1) and Proliferin (PLF) are upregulated as well. The results reveal that KIT signaling orchestrates the fine-tuned differentiation of the placenta, with special emphasis on P-TGC differentiation. Appropriate control of KIT receptor action is therefore essential for placental development and nourishment of the embryo.

The regulation of immune cell migration throughout the body is essential to warrant immunosurveillance and to maintain immune homeostasis. Marking and tracking of these cells has proven important to study mechanisms of immune cell trafficking and cell interaction in vivo. Photoconversion is a well-suited technique for intravital application because it enables contactless time- and location-specific marking of cells in the tissue without surgically manipulating the microenvironment of the cells in question. However, in dividing cells the converted fluorescent protein may decline quickly. Here, we provide a detailed description of the photoconversion technique and its applicability to tracking highly proliferating T cells from the priming site of T cell activation to peripheral target organs of effector function in a preclinical model. Dendra2+ T cells were photoconverted in the Peyer’s patches during the initiation phase of acute graft-versus-host disease (GvHD) and tracked through the mesenteric lymph nodes and the peripheral blood to the small intestine with flow cytometry and intravital two-photon microscopy. Photoconverted alloreactive T cells preserved the full proliferative capacity, homing, and migration of alloreactive T cells in the intestinal lamina propria. We conclusively proved that photoconversion of highly proliferative alloreactive T cells in the Peyer’s patches is an effective tool to study trafficking of alloreactive T cells under physiologic conditions and to GvHD target tissues. This technique can also be applied to the study of immune cell tracking under inflammatory and non-inflammatory conditions.

Acute graft versus host disease (aGVHD) remains a major complication in patients undergoing allogeneic hematopoietic stem cell transplantation (allo-HSCT), the only curative treatment for many malignant hematologic diseases. After initial priming in secondary lymphoid organs, alloreactive donor T cells efficiently migrate to the intestinal tract, liver and skin. We observed that alloreactive effector T cells infiltrating and attacking the lamina propria of the small and large intestines closely interact with intestinal myeloid cells of host origin. Here we asked whether these intimate interactions regulate alloreactive effector T cell responses and how they impact intestinal aGvHD. To address these questions, we employed non-invasive bioluminescence imaging, fluorescence and confocal microscopy, clinical and histopathologic scoring, flow cytometry and single cell RNA sequencing in murine models of myeloablative, MHC-mismatched allo-HSCT. In the intestinal lamina propria, we observed that allogeneic T cells closely interacted with CD11b+CD11c+CD103- radio-resistant host type hematopoietic myeloid antigen presenting cells. Selective depletion of intestinal CD11chi or CX3CR1+CD11chi host cells 3 or 8 days after allo-HSCT accelerated alloreactive T cell infiltration, increased T cell mediated inflammatory cytokine production and exacerbated tissue damage resulting in hyperacute lethal aGvHD. These results suggested a strong immunoregulatory effect of these intestinal host-type myeloid cells. Single cell RNA-Seq analysis and flow cytometry (e.g. MHC II, CD11c, F4/80, CD26, CD64, CCR2, CX3CR1), lineage reporter- and defined knockout mice determined these cells as non-classical monocyte derived macrophages as the development and differentiation of these cells did not depend on Flt3, Zbtb46, and CCR2 but rather on CSF-1R and CX3CR1. Adoptive transfer, bone marrow chimeras and parabiosis experiments revealed that these non-classical monocyte derived macrophages differentiated from non-circulating non-classical monocytic precursors. Finally, PD-L1 expression on these intestinal host non-classical monocyte derived macrophages but not on stroma or other hematopoietic cells regulated alloreactive T cell responses in the intestinal tract. Based on these findings we postulate that a specialized and persistent subpopulation of host non-classical monocyte derived macrophages can potently suppress alloreactive T cells in the lamina propria of the intestinal tract. Fostering the differentiation and function of these tissue resident cells may represent an attractive therapeutic strategy to prevent intestinal aGvHD. Disclosures No relevant conflicts of interest to declare.

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