Multiplex Immunofluorescence and Multispectral Imaging as a tool to evaluate host directed therapy against tuberculosis

<bold>Introduction:</bold> Mtb-containing centrally necrotizing granuloma with a fibrous capsule provide a therapeutic challenge in tuberculosis treatment in adults and children due to poor penetration of antibiotics. To improve treatment success in complicated or drug resistant TB and optimize drug dosages HDT is a promising approach. It aims to modify host cell factors, reduce inflammation and balance immune reactivity at sites of pathology. The aim of this study was to establish a workflow to evaluate potential HDTs regarding their effect on lung pathology. <bold>Methods:</bold> We used interleukin-13-overexpressing (IL-13tg) mice as a model. They form, contrary to wildtype mice, human-like pulmonary granulomas after infection with Mtb. In addition to conventional immunohistochemistry, multiplex immunofluorescence (mIF) allowing staining for multiple targets in one slide, was established. Combined with multispectral imaging, mIF provides a reliable algorithm-based analysis with a limited number of samples, enabling separation of tissue subtypes, measurement of cell counts and detection of cell-to-cell interactions. <bold>Results:</bold> IL-13tg mice (n=51) were infected with Mtb (H37Rv) and sacrificed at ten different time points between day 42 and 106 post infection. Comparison of algorithm-based tissue segmentation in sequential slides showed a correlation between conventional IHC and immunofluorescence staining (R=0,79, p< 2.2e-16). Mtb detection with a MPT64 antibody also correlated in both staining methods (R=0,95, p<1.2e-14). Discussion: Our advanced tissue-analysis in combination with the IL-13tg mouse model offers an improved evaluation of novel anti-TB drugs in the preclinical stage.