Dynamic changes in Mycobacterium tuberculosis-induced granulomas: developing new tools

Abstract

Abstract Continuous cell replacement has been demonstrated in a transplantation model of BCG infected liver, however, the Mtb lung granuloma has yet to be studied as lung tissue is not amenable to granuloma transplantation. To demonstrate the plasticity of Mtb lung granulomas, we treated mice 6 weeks post infection with chemokine inhibitors for 7 days. Treated lungs showed a strong, significant reduction in granuloma size, which suggests continuous cell replacement and indicates that Mtb granulomas are actively maintained during infection. To examine cell fate in more detail, we utilized STOCK Tg(CAG-KikGR)33Hadj/J mice to photo-tag lung granulomas in situ. Following exposure to 405 nm light KikGR Red (583/593 nm) photoconverted lung regions or cells could be differentiated from unexposed Green (507/517 nm) systemic cells. These mice were aerosol infected with 200 CFU Mtb H37Rv. Portions of infected lungs were photoconverted 4 wpi, harvested up to 7 days post-exposure, and examined, along with mediastinal lymph nodes, by confocal microscopy. Areas of photoconversion were successfully identified in infected lungs. F4/80+ macrophages and CD4+ T-cells were stained and screened for red versus green fluorescence. Unconverted cells of both types were found, demonstrating the utility of this technique for detecting influx into the granuloma. Efflux of cells from the granuloma was also confirmed by the detection of photoconverted cells within the unconverted MLNs. While further optimization is necessary to use this model for quantitative analysis, these results implicate continuous cell recruitment into granulomatous lesions. Elucidating cell traffic to granulomas with this technique can lead to new therapies for many granulomatous diseases.