Human Vitreous Humor Serves as an Effective Extracellular Matrix for an In Vitro Granuloma System to Study Mycobacterium tuberculosis Infection

Abstract

Background: Granuloma formation is the hallmark of Mycobacterium tuberculosis (Mtb) infection. This blockade by leukocytes prevents dissemination and develops a niche that provides the mycobacteria’s survival. The extracellular matrix (ECM) facilitates the differentiation and activation of macrophages that compose the granuloma. Current commercially available sources of ECMs do not provide effective models to study this complex pathophysiology. Human vitreous humor (hVH) has demonstrated to work as an effective ECM for fibroblast differentiation.

Methods: Here we describe the use of hVH as an ECM for an in vitro granuloma model upon Mtb infection. THP-1 human monocytes containing reporter plasmids for nuclear factor kappa-B (NF-kB) and interferon regulatory factor (IRF) were seeded onto hVH obtained from human cadavers. Cells were then cultured under three conditions: hVH only, hVH + vitamin D₃, and hVH + phorbol 12-myristate 13-acetate (PMA). After 3 days, macrophages were inoculated with Mtb. Formation, length, and area of aggregation were measured over time. Supernatants were also collected to measure the activity of NF-kB and IRF transcription factors.

Results: We observed that human monocyte-derived macrophages form granulomatous-like monocytic aggregate (GLMA) in hVH as of day 1 in all conditions, with peaks on days 3 and 6. Uninfected controls exhibited no aggregate formation. Significantly, NF-kB activation precedes IRF activation over time in cells infected with Mtb.

Conclusions: Our findings support the use of hVH as a viable ECM model to study Mtb granuloma formation and provide evidence of cell activation. Inclusion of other cell types into this system may better mimic human tissue phenomena and improve our understanding of the Mtb granuloma formation process.

Clin Infect Immun. 2022;7(2):49-57
doi: https://doi.org/10.14740/cii157