Clofazimine Modulates the Expression of Lipid Metabolism Proteins in <em>Mycobacterium leprae-</em>Infected Macrophages

<div><p><em>Mycobacterium leprae (M. leprae)</em> lives and replicates within macrophages in a foamy, lipid-laden phagosome. The lipids provide essential nutrition for the mycobacteria, and <em>M. leprae</em> infection modulates expression of important host proteins related to lipid metabolism. Thus, <em>M. leprae</em> 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 <em>M. leprae</em>. 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 <em>in vitro</em>, 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 <em>M. leprae</em>-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 <em>M. leprae</em> and treated with clofazimine. Lipid droplets accumulated by <em>M. leprae</em>-infection were significantly decreased 48 h after clofazimine treatment. Such effects were not evident in cells without <em>M. leprae</em> infection. In clinical samples, ADRP expression was decreased and HSL expression was increased after treatment. These results suggest that clofazimine modulates lipid metabolism in <em>M. leprae</em>-infected macrophages by modulating the expression of ADRP and HSL. It also induces IFN production in <em>M. leprae</em>-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.</p> </div

Clofazimine counteracts <i>M. leprae</i> to modulate ADRP and HSL expression levels.

<p>THP-1 cells were cultured in six-well plates and infected with <i>M. leprae</i> (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 <i>M. leprae</i> 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.</p

Expression of ADRP and HSL is modulated by clofazimine in THP-1 cells infected with <i>M. leprae</i>.

<p>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 <i>M. leprae</i> 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.</p

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

<p>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.</p

Clofazimine increases mRNA expression of IFN-β and IFN-γ in <i>M. leprae</i>-infected THP-1 cells.

<p>THP-1 cells were cultured in six-well plates with or without 2.0 µg/ml clofazimine in the presence or absence of <i>M. leprae</i> 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.</p

Paleopathological Evidence and Detection of <i>Mycobacterium leprae</i> DNA from Archaeological Skeletal Remains of <i>Nabe-kaburi</i> (Head-Covered with Iron Pots) Burials in Japan

<div><p>The <i>Nabe-kaburi</i> is a unique burial method, the purpose of which is shrouded in mystery. The burials were performed during the 15^th to 18^th centuries in eastern Japan, and involved covering the heads of the deceased with iron pots or mortars. The identification of leprosy-specific osteological lesions among some of the excavated remains has led to the suggestion that <i>Nabe-kaburi</i> burials were a reflection of the social stigma against certain infectious diseases, such as leprosy, tuberculosis or syphilis. However, molecular evidence for the presence of disease has been lacking. The goal of this study was to detect <i>Mycobacterium leprae</i> (<i>M. leprae</i>) DNA in archaeological human skeletal remains from <i>Nabe-kaburi</i> burials. The paleopathological data from three <i>Nabe-kaburi</i> burials were re-evaluated before small samples were taken from affected and control areas. DNA was extracted and used as a template to target the <i>M. leprae</i>-specific DNA using a combination of whole genome amplification, PCR analysis and DNA sequencing. <i>M. leprae</i> DNA fragments were detected in the two sets of skeletal remains that had also shown paleopathological evidence of leprosy. These findings provide definitive evidence that some of the <i>Nabe-kaburi</i> burials were performed for people affected by leprosy. Demonstration of the presence of <i>M. leprae</i> DNA, combined with archeological and anthropological examinations, will aid in solving the mystery of why <i>Nabe-kaburi</i> burials were performed in medieval Japan.</p></div

Location of the <i>Tawara-ga-yatsu</i> and the <i>Usukubo</i> sites in Japan.

<p>Shaded area denotes the <i>Kanto</i> area. Inset illustrates the locations in relation to Tokyo.</p

Chronological and geographical distribution of <i>Nabe-kaburi</i> burials in Japan.

a<p>Shaded area in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0088356#pone-0088356-g001" target="_blank">Figure 1</a>.</p

Skeletal samples analyzed for the presence of <i>M. leprae</i> DNA.

<p>N.R.: No remarkable change.</p

Macroscopic view of the osteological lesions in the skeletal remains of TK5.

<p>(A) Frontal view of the skull with no paleopathological signs. The area from the glabella to the nasion and inion was in plane. No periostitis-caused osteological lesions were observed in the nasal bone or on the edges of the nasal aperture. (B) View of diaphysis of the femur. The arrow indicates extensive hypertrophy in the lower bone.</p