Clofazimine modulates the expression of lipid metabolism proteins in Mycobacterium leprae-infected macrophages

Abstract

Mycobacterium leprae (M. leprae) lives and replicates within macrophages in a foamy, lipid-laden phagosome. The lipids provide essential nutrition for the mycobacteria, and M. leprae infection modulates expression of important host proteins related to lipid metabolism. Thus, M. leprae infection increases the expression of adipophilin/adipose differentiation-related protein (ADRP) and decreases hormone-sensitive lipase (HSL), facilitating the accumulation and maintenance of lipid-rich environments suitable for the intracellular survival of M. leprae. HSL levels are not detectable in skin smear specimens taken from leprosy patients, but re-appear shortly after multidrug therapy (MDT). This study examined the effect of MDT components on host lipid metabolism in vitro, and the outcome of rifampicin, dapsone and clofazimine treatment on ADRP and HSL expression in THP-1 cells. Clofazimine attenuated the mRNA and protein levels of ADRP in M. leprae-infected cells, while those of HSL were increased. Rifampicin and dapsone did not show any significant effects on ADRP and HSL expression levels. A transient increase of interferon (IFN)-β and IFN-γ mRNA was also observed in cells infected with M. leprae and treated with clofazimine. Lipid droplets accumulated by M. leprae-infection were significantly decreased 48 h after clofazimine treatment. Such effects were not evident in cells without M. leprae infection. In clinical samples, ADRP expression was decreased and HSL expression was increased after treatment. These results suggest that clofazimine modulates lipid metabolism in M. leprae-infected macrophages by modulating the expression of ADRP and HSL. It also induces IFN production in M. leprae-infected cells. The resultant decrease in lipid accumulation, increase in lipolysis, and activation of innate immunity may be some of the key actions of clofazimine.

Figure 1. Expression of ADRP and HSL is modulated by clofazimine in THP-1 cells infected with M. leprae.

THP-1 cells were cultured in six-well plates with culture medium containing either 8.0 µg/ml rifampicin, 5.0 µg/ml dapsone or 2.0 µg/ml clofazimine with M. leprae infection (MOI = 10). After incubating for 24 h, total RNA was purified and RT-PCR analysis of ADRP, HSL and β-actin was performed. Representative results from three independent experiments are shown.

Figure 2. Only M. leprae-infected THP-1 cells are susceptible to clofazimine.

THP-1 cells were cultured in six-well plates with or without 2.0 µg/ml clofazimine in the presence or absence of M. leprae infection (MOI = 10). After incubating for the indicated period of time, total RNA and total cellular protein were purified and RT-PCR and Western blot analyses of ADRP, HSL and β-actin were performed. Representative results from three independent experiments are shown.

Figure 3. Clofazimine counteracts M. leprae to modulate ADRP and HSL expression levels.

THP-1 cells were cultured in six-well plates and infected with M. leprae (MOI = 10) for 24 h. Clofazimine (2.0 µg/ml) was added and incubation continued another 24 and 48 h (48 and 72 h from M. leprae infection). Total RNA and total cellular protein were purified and RT-PCR and Western blot analyses of ADRP, HSL and β-actin were performed. Representative results from three independent experiments are shown.

Figure 4. Clofazimine increases mRNA expression of IFN-β and IFN-γ in M. leprae-infected THP-1 cells.

THP-1 cells were cultured in six-well plates with or without 2.0 µg/ml clofazimine in the presence or absence of M. leprae infection (MOI = 10). After incubating for the indicated period of time, total RNA was purified and RT-PCR analysis of IFN-β (A) and IFN-γ (B) was performed. Representative results from three independent experiments are shown.

Figure 5. Clofazimine decreases cellular lipid accumulation in M. leprae-infected THP-1 cells.

THP-1 cells were grown on glass coverslips in 24-well plates. Cells with no treatment (A), infected with M. leprae (MOI = 10) (B), and infected with M. leprae (MOI = 10) and treated with clofazimine (2.0 µg/ml) (C) were cultured for 48 h. Oil-red-O staining followed by brief hematoxylin counter staining was performed and observed under a microscope. Representative results from three independent experiments are shown. Bars = 10 µm.

Figure 6. Detection of ADRP and HSL mRNA in slit-skin smear samples from leprosy patients.

Total RNA was isolated from slit-skin smear specimens taken from ten BL and four LL patients (A) or from one patient before and after treatment (B). Total RNA was purified and RT-PCR analysis of ADRP, HSL and β-actin was performed. Representative results from three independent experiments are shown.

Figure 7. Immunostaining of ADRP and HSL proteins in skin biopsy specimens before and after treatment.

Sections of skin biopsy specimens taken from one patient before (A and C) and after (B and D) treatment were subjected to immunostaining of ADRP (A and B) and HSL (C and D), followed by acid-fast staining for M. leprae and hematoxylin counterstaining. Arrows indicate phagosome membrane that contains M. leprae. Representative results from three independent experiments are shown. Bars = 20 µm.