Abstract
Using a novel tissue-clearing method, we aimed to visualize the three-dimensional (3D) distribution of immune cells within Mycobacterium tuberculosis (Mtb)-infected mice lungs. Ethyl cinnamate-based tissue clearing of Mtb-infected mice lungs was performed to obtain transparent lung samples, which were then imaged using a light sheet fluorescence microscope. Using the 3D images, we performed quantitative analysis of the immune cell population within multiple granulomas. In addition, to compare the data from the tissue clearing method, we performed histopathological and immunofluorescence analyses, and flow cytometry. We then created 3D images of the Mtb-infected lung that successfully demonstrated the distribution of blood vessels, immune cells, and granulomas. Since the immune cells within a granuloma could be separately selected and counted, the immune cell population within a specific lesion could be quantified. In addition, macroscopic analysis, e.g., the size or shape of a granuloma, as well as microscopic analysis could be performed as intact lung samples were used. The use of the tissue clearing method in infected lungs could be a novel modality for understanding the role of the immune system in the pathogenesis of tuberculosis.
Highlights
Tuberculosis (TB), a pulmonary disease caused by a group of infectious agents named Mycobacterium tuberculosis (Mtb) complex, remains one of the major health concerns worldwide
These findings indicate that the role of neutrophils in the immune response of TB remains to be clarified and studying their functions at different stages of the disease may provide a deeper understanding of the pathogenesis
Flow cytometric studies were performed in order to quantify three types of immune cells: neutrophils, alveolar macrophages, and infiltrating macrophages, in both naïve and Mtb-infected mice lungs (Figure 1)
Summary
Tuberculosis (TB), a pulmonary disease caused by a group of infectious agents named Mycobacterium tuberculosis (Mtb) complex, remains one of the major health concerns worldwide. Novel therapeutic approaches for TB are required In light of this need for a new treatment, several researches have attempted to elucidate the role of the innate immune system in the pathogenesis of the disease. As Mtb enter the alveoli, alveolar macrophages phagocytose the bacteria and subsequently produce various cytokines, recruiting monocytes from the blood and leading to mobilization of monocyte derived macrophages. Data from patients suggest that higher neutrophil count is associated with worse prognosis in well-established TB (Barnes et al, 1988) To summarize, these findings indicate that the role of neutrophils in the immune response of TB remains to be clarified and studying their functions at different stages of the disease may provide a deeper understanding of the pathogenesis
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