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

Introduction Mycobacterium tuberculosis (Mtb)-containing centrally necrotizing granuloma with a fibrous capsule provide a therapeutic challenge in tuberculosis (TB) treatment in adults and children due to poor penetration of antibiotics. To improve treatment success in complicated or drug resistant TB and to optimize drug dosages, host-directed therapy (HDT) as an adjunct to antibiotic treatment is a promising approach. It aims to modify host cell factors, modulate inflammation and balance immune reactivity at sites of infection within granulomatous lesions. The aim of this study was to establish a workflow to evaluate potential HDTs regarding their effect on lung pathology.

Materials and Methods We used interleukin-13-overexpressing (IL-13tg) mice as a model. These animals form, contrary to wildtype mice, human-like pulmonary granulomas after infection with Mtb. To evaluate alterations in histopathology during infection and treatment, multiplex immunofluorescence (mIF) allowing staining for multiple targets in one slide, was established in addition to conventional immunohistochemistry (IHC). 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.

Results 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). Furthermore, visual control revealed plausible cell and bacteria distribution in all tissue subtypes.

Discussion Our advanced tissue-analysis in combination with the IL-13tg mouse model offers an improved evaluation of novel anti-TB drugs and HDTs in the preclinical stage. Further studies are needed for verification in adults and children. The advanced tissue-analysis might potentially be applicable in other IL13-associated diseases such as asthma.