Automated quantitative assay of fibrosis characteristics in tuberculosis granulomas

Introduction: Granulomas, the pathological hallmark of Mycobacterium tuberculosis (Mtb) infection, are formed by different cell populations. Across various stages of tuberculosis conditions, most granulomas are classical caseous granulomas. They are composed of a necrotic center surrounded by multilayers of histocytes, with the outermost layer encircled by fibrosis. Although fibrosis characterizes the architecture of granulomas, little is known about the detailed parameters of fibrosis during this process. Methods: In this study, samples were collected from patients with tuberculosis (spanning 16 organ types), and Mtb-infected marmosets and fibrotic collagen were characterized by second harmonic generation (SHG)/two-photon excited fluorescence (TPEF) microscopy using a stain-free, fully automated analysis program. Results: Histopathological examination revealed that most granulomas share common features, including necrosis, solitary and compact structure, and especially the presence of multinuclear giant cells. Masson's trichrome staining showed that different granuloma types have varying degrees of fibrosis. SHG imaging uncovered a higher proportion (4%similar to 13%) of aggregated collagens than of disseminated type collagens (2%similar to 5%) in granulomas from matched tissues. Furthermore, most of the aggregated collagen presented as short and thick clusters (200 similar to 620 mu m), unlike the long and thick (200 similar to 300 mu m) disseminated collagens within the matched tissues. Matrix metalloproteinase-9, which is involved in fibrosis and granuloma formation, was strongly expressed in the granulomas in different tissues. Discussion: Our data illustrated that different tuberculosis granulomas have some degree of fibrosis in which collagen strings are short and thick. Moreover, this study revealed that the SHG imaging program could contribute to uncovering the fibrosis characteristics of tuberculosis granulomas.