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Tracing conidial fate and measuring host cell antifungal activity using a reporter of microbial viability in the lung.

ABSTRACT: Fluorescence can be harnessed to monitor microbial fate and to investigate functional outcomes of individual microbial cell-host cell encounters at portals of entry in native tissue environments. We illustrate this concept by introducing fluorescent Aspergillus reporter (FLARE) conidia that simultaneously report phagocytic uptake and fungal viability during cellular interactions with the murine respiratory innate immune system. Our studies using FLARE conidia reveal stepwise and cell-type-specific requirements for CARD9 and Syk, transducers of C-type lectin receptor and integrin signals, in neutrophil recruitment, conidial uptake, and conidial killing in the lung. By achieving single-event resolution in defined leukocyte populations, the FLARE method enables host cell profiling on the basis of pathogen uptake and killing and may be extended to other pathogens in diverse model host organisms to query molecular, cellular, and pharmacologic mechanisms that shape host-microbe interactions.

SUBMITTER: Jhingran A

PROVIDER: S-EPMC3712646 | biostudies-literature | 2012 Dec

REPOSITORIES: biostudies-literature

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Tracing conidial fate and measuring host cell antifungal activity using a reporter of microbial viability in the lung.

Jhingran Anupam A Mar Katrina B KB Kumasaka Debra K DK Knoblaugh Sue E SE Ngo Lisa Y LY Segal Brahm H BH Iwakura Yoichiro Y Lowell Clifford A CA Hamerman Jessica A JA Lin Xin X Hohl Tobias M TM

Cell reports 20121129 6

Fluorescence can be harnessed to monitor microbial fate and to investigate functional outcomes of individual microbial cell-host cell encounters at portals of entry in native tissue environments. We illustrate this concept by introducing fluorescent Aspergillus reporter (FLARE) conidia that simultaneously report phagocytic uptake and fungal viability during cellular interactions with the murine respiratory innate immune system. Our studies using FLARE conidia reveal stepwise and cell-type-specif ...[more]

PMID: 23200858

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Project description:Idiopathic pulmonary fibrosis (IPF) is a progressive and devastating interstitial lung disease. The origin of myofibroblasts is still to be elucidated and the existence of epithelial-mesenchymal transition (EMT) in IPF remains controversial. Hence, it is important to clarify the origin of fibroblasts by improving modeling and labeling methods and analyzing the differentiation pathway of alveolar epithelial cells (AEC) in pulmonary fibrosis with cell tracking technology. In the present study, adult transgenic mice with SPC-rtTA +/- /tetO 7 -CMV-Cre +/- /mTmG +/- were induced with doxycycline for 15 days. The gene knockout phenomenon occurred in type II AECs in the lung and the reporter gene cell membrane-localized enhanced green fluorescence protein (mEGFP) was expressed in the cell membrane. The expression of Cre recombinase and SPC was analyzed using immunohistochemical (IHC) staining to detect the labeling efficiency. A repetitive intraperitoneal bleomycin-induced pulmonary fibrosis model was established, and the mice were sacrificed on day 28. The co-localization of mEGFP and mesenchymal markers α-smooth muscle actin (α-SMA) and S100 calcium binding protein A4 (S100A4) were detected by multiple IHC staining. The results revealed that Cre was expressed in the airway and AECs in the lung tissue of adult transgenic mice with SPC-rtTA +/- /tetO 7 -CMV-Cre +/- /mTmG +/- induced by doxycycline; the labeling efficiency in the peripheral lung tissue was 63.27±7.51%. mEGFP was expressed on the membrane of type II AECs and their differentiated form of type I AECs. Expression of mEGFP was mainly observed in the fibrotic region in bleomycin-induced pulmonary fibrosis; 1.94±0.08% of α-SMA-positive cells were mEGFP-positive and 9.68±2.06% of S100A4-positive cells were mEGFP-positive in bleomycin-induced pulmonary fibrosis. In conclusion, the present results suggested that while EMT contributes to the pathogenesis of pulmonary fibrosis, it is not the major causative factor of this condition.

Dataset Information

Tracing conidial fate and measuring host cell antifungal activity using a reporter of microbial viability in the lung.

Publications

Tracing conidial fate and measuring host cell antifungal activity using a reporter of microbial viability in the lung.

Similar Datasets

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