An In Vitro Model for the Lepromatous Leprosy Granuloma: Fate ofMycobacterium lepraefrom Target Macrophages after Interaction with Normal and Activated Effector Macrophages

Abstract

The lepromatous leprosy granuloma is a dynamic entity requiring a steady influx of macrophages (Mφ) for its maintenance. We have developed an in vitro model to study the fate of Mycobacterium leprae in a LL lesion, with and without immunotherapeutic intervention. Target cells, consisting of granuloma Mφ harvested from the footpads of M. leprae-infected athymic nu/nu mice, were cocultured with normal or IFN-γ-activated (ACT) effector Mφ. The bacilli were recovered and assessed for viability by radiorespirometry. M. leprae recovered from target Mφ possessed high metabolic activity, indicating a viable state in this uncultivable organism. M. leprae recovered from target Mφ incubated with normal effector Mφ exhibited significantly higher metabolism. In contrast, bacilli recovered from target Mφ cocultured with ACT effector Μφ displayed a markedly decreased metabolic activity. Inhibition by ACT Mφ required an E:T ratio of at least 5:1, a coculture incubation period of 3–5 days, and the production of reactive nitrogen intermediates, but not reactive oxygen intermediates. Neither IFN-γ nor TNF-α were required during the cocultivation period. However, cell-to-cell contact between the target and effector Mφ was necessary for augmentation of M. leprae metabolism by normal effector Mφ as well as for inhibition of M. leprae by ACT effector Mφ. Conventional fluorescence microscopy and confocal fluorescence microscopy revealed that the bacilli from the target Mφ were acquired by the effector Mφ. Thus, the state of Mφ infiltrating the granuloma may markedly affect the viability of M. leprae residing in Mφ in the lepromatous lesion.

Lymphotoxin-α and TNF Have Essential but Independent Roles in the Evolution of the Granulomatous Response in Experimental Leprosy

“…This is not unreasonable as a single macrophage can accommodate more than 100 viable M. leprae and remain physiologically normal and functional. 47,48 Furthermore, in immunocompetent mouse FP as well as in some KO mouse models, which on M. leprae infection develop an enlarged FP with a strong macrophage influx (eg, IFN␥ Ϫ/Ϫ , NOS2 Ϫ/Ϫ ), a large percentage of these cells are, in fact, uninfected (unpublished data).…”

The Continuing Challenges of Leprosy

“…The M is the primary host cell for M. leprae. In the absence of an effective adaptive immune response, these relatively nontoxic bacilli can multiply in M to over 100 organisms per cell (145). In vitro, M. leprae can be maintained in a metabolically active state for weeks in M supplemented with IL-10 and cultured at 33°C (122).…”

“…Methods have recently been developed to isolate granuloma M from the footpads of M. leprae-infected mice (145,373). This model, which enables the study of M from the actual site of infection in experimental leprosy, has allowed determination of culture characteristics, cytokine production, and cell surface phenotypic markers by flow cytometry of these granuloma-derived cells.…”

Insights from animal models on the immunogenetics of leprosy: a review

“…Studies in neonatally thymectomized or congenitally athymic mice and rats (Rees 1966, Colston & Hilson 1976, Dawson et al 1983, Chehl et al 1983), T900r mice (Ebenezer et al 2002), as well as later studies in SCID mice (Yogi et al 1991, Azouaou et al 1993, established the significance of lymphocytes, especially T cells, in host defence in leprosy. In athymic nu/nu mice, multiplication of M. leprae in the foot pad can reach 1 x 10 10 or more bacilli with the infected tissue composed primarily of macrophages heavily laden with the bacilli (Hagge et al 2004). In this regard, athymic nu/nu mice show characteristics of LL disease.…”