CD11b+Gr-1low cells that accumulate in M.leprae-induced granulomas of the footpad skin of nude mice have the characteristics of monocytic-myeloid-derived suppressor cells.

CD11b+Gr-1low cells that are increased in the lungs of a Mycobacterium (M) tuberculosis-infection mouse model have the characteristics of monocytic (M)-myeloid-derived suppressor cells (MDSCs) and harbor M.tuberculosis. Interestingly, a high number of M-MDSCs have also been observed in skin lesions of patients with lepromatous leprosy. We hypothesized that CD11b+Gr-1low cells might be involved in the pathogenesis of leprosy, as they are in tuberculosis. In the current study, we investigated the issue of whether CD11b+Gr-1low cells accumulate in Mycobacterium (M) leprae-induced granulomas of the footpad skin of nude mice. Our results show that CD11b+Gr-1low cells began to accumulate in the 7-month-old M.leprae-induced granulomas and were replaced by other leukocytes, including CD11b+Gr-1high over time during M.leprae infections. CD11b + Gr-1low cells expressed the surface markers of M-MDSC, Ly6Chigh and Ly6Glow. In addition, CD11b+Gr-1low cells have the nuclei of a mononuclear cell type and expressed higher levels of arginase 1 (Arg1) and inducible NO synthetase (iNOS). Furthermore, they showed a higher infection rate by M.leprae. Taken together, our results indicate that the inoculation with M.leprae induced an accumulation of CD11b + Gr-1low at a relatively early stage, 7-month-old M.leprae-induced granulomas, and that CD11b+Gr-1low have the characteristics of M-MDSC and may act as a reservoir for M.leprae.

M. leprae interacts with the human epidermal keratinocytes, neonatal (HEKn) via the binding of laminin-5 with α-dystroglycan, integrin-ß1, or -ß4.

Although Mycobacterium leprae (M.leprae) is usually found in macrophages and nerves of the dermis of patients with multibacillary leprosy, it is also present in all layers of the epidermis, basal, suprabasal, prickle cells, and keratin layers. However, the mechanism by which M.leprae invades the dermis remains unknown, whereas the underlying mechanism by which M.leprae invades peripheral nerves, especially Schwann cells, is well defined. M. leprae binds to the α-dystroglycan (DG) of Schwann cells via the interaction of α-DG and laminin (LN) -α2 in the basal lamina, thus permitting it to become attached to and invade peripheral nerves. In the current study, we investigated the issue of how M.leprae is phagocytosed by human epidermal keratinocytes, neonatal (HEKn). LN-5 is the predominant form of laminin in the epidermis and allows the epidermis to be stably attached to the dermis via its interaction with α/ß-DG as well as integrins that are produced by keratinocytes. We therefore focused on the role of LN-5 when M. leprae is internalized by HEKn cells. Our results show that M.leprae preferentially binds to LN-5-coated slides and this binding to LN-5 enhances its binding to HEKn cells. The findings also show that pre-treatment with an antibody against α-DG, integrin-ß1, or -ß4 inhibited the binding of LN-5-coated M.leprae to HEKn cells. These results suggest that M. leprae binds to keratinocytes by taking advantage of the interaction of LN-5 in the basal lamina of the epidermis and a surface receptor of keratinocytes, such as α-DG, integrin-ß1, or -ß4.

The formation of lipid droplets favors intracellular Mycobacterium leprae survival in SW-10, non-myelinating Schwann cells.

Leprosy is a chronic infectious disease that is caused by the obligate intracellular pathogen Mycobacterium leprae (M.leprae), which is the leading cause of all non-traumatic peripheral neuropathies worldwide. Although both myelinating and non-myelinating Schwann cells are infected by M.leprae in patients with lepromatous leprosy, M.leprae preferentially invades the non-myelinating Schwann cells. However, the effect of M.leprae infection on non-myelinating Schwann cells has not been elucidated. Lipid droplets (LDs) are found in M.leprae-infected Schwann cells in the nerve biopsies of lepromatous leprosy patients. M.leprae-induced LD formation favors intracellular M.leprae survival in primary Schwann cells and in a myelinating Schwann cell line referred to as ST88-14. In the current study, we initially characterized SW-10 cells and investigated the effects of LDs on M.leprae-infected SW-10 cells, which are non-myelinating Schwann cells. SW-10 cells express S100, a marker for cells from the neural crest, and NGFR p75, a marker for immature or non-myelinating Schwann cells. SW-10 cells, however, do not express myelin basic protein (MBP), a marker for myelinating Schwann cells, and myelin protein zero (MPZ), a marker for precursor, immature, or myelinating Schwann cells, all of which suggests that SW-10 cells are non-myelinating Schwann cells. In addition, SW-10 cells have phagocytic activity and can be infected with M. leprae. Infection with M. leprae induces the formation of LDs. Furthermore, inhibiting the formation of M. leprae-induced LD enhances the maturation of phagosomes containing live M.leprae and decreases the ATP content in the M. leprae found in SW-10 cells. These facts suggest that LD formation by M. leprae favors intracellular M. leprae survival in SW-10 cells, which leads to the logical conclusion that M.leprae-infected SW-10 cells can be a new model for investigating the interaction of M.leprae with non-myelinating Schwann cells.

Palmitate induces RIP1-dependent necrosis in RAW 264.7 cells.

OBJECTIVE: The kinase receptor-interacting protein (RIP) 1, a serine/threonine protein kinase, is a key signaling molecule for necrosis. The possible involvement of RIP1 in palmitate-induced macrophage death and its underlying molecular mechanism was investigated in this study. METHODS: Cell viability was measured by an MTT reduction assay. The type of cell death was determined by staining with annexin V, propidium iodide (PI) and the APOPercentage dye, and by examining cell morphology using transmission electron microscopy. The down-regulation of RIP1 was performed by siRNA transfection. Intracellular reactive oxygen species (ROS) were measured by staining with H(2)DCF-DA. RESULTS: Palmitate largely induced necrosis in RAW 264.7 cells, whereas C2-ceramide induced apoptosis. Palmitate-induced necrosis was inhibited by Necrostatin-1, an inhibitor of RIP1, and by RIP1 siRNA transfection, whereas ordinary cell death was not inhibited by z-VAD-fmk. In addition, the presence of palmitate caused a significant increase in intracellular ROS levels compared to control cells. Pre-treatment with Tempol, a cell permeable ROS scavenger, and MnTBAP, an inhibitor of mitochondrial oxidative stress, protected cells from palmitate-induced cell death. Furthermore, the down-regulation of RIP1 by siRNA transfection significantly decreased palmitate-induced ROS generation compared to control cells. CONCLUSION: The findings reported herein indicate that palmitate induces necrotic cell death via RIP1-dependent ROS generation in RAW 264.7 cells. These findings may provide a new mechanism that explains the link between elevated levels of free fatty acids (FFAs) and macrophage death.

Mycobacterial infections in coal workers' pneumoconiosis patients in South Korea.

Coal workers' pneumoconiosis (CWP) is the most common occupational disease in South Korea and is an important factor in the development of infections with Mycobacterium tuberculosis (MTB) and non-tuberculous mycobacteria (NTM). In the current study, we identified mycobacterial species that cause pulmonary infections in CWP patients, using rpoB DNA-PCR-restriction analysis. Among the 129 CWP patients studied, 35 (27.1%) were diagnosed as having mycobacterial infections. Among these, the proportion of NTM infections (21/35, 60.0%) was higher than that for MTB infections (14/35, 40.0%). Of the 21 NTM strains, the most common was M. intracellulare (6/21, 28.6%), followed by M. avium (5/21, 23.8%). We also compared the characteristics of CWP patients between the MTB and NTM infection groups. A higher proportion of CWP patients with NTM infections compared with those with MTB infections had a history of having been involved in rock work (38.1% vs 21.4%), and had complicated CWP (85.7% vs 35.7%) and a past history of TB treatment (61.9% vs 50.0%). We also discovered 3 MTB mutants that are resistant to first-line anti-TB drugs, in CWP patients. These results demonstrate the features of pulmonary mycobacterial infections with a predominance of NTM in CWP patients in South Korea.

Rifampicin Inhibits the LPS-induced Expression of Toll-like Receptor 2 via the Suppression of NF-kappaB DNA-binding Activity in RAW 264.7 Cells.

Rifampicin is a macrocyclic antibiotic which is used extensively for treatment against Mycobacterium tuberculosis and other mycobacterial infections. Recently, a number of studies have focused on the immune-regulatory effects of rifampicin. Therefore, we hypothesized that rifampicin may influence the TLR2 expression in LPS-activated RAW 264.7 cells. In this study, we determined that rifampicin suppresses LPS-induced TLR2 mRNA expression. The down-regulation of TLR2 expression coincided with decreased production of TNF-alpha. Since NF-kappaB is a major transcription factor that regulates genes for TLR2 and TNF-alpha, we examined the effect of rifampicin on the LPS-induced NF-kappaB activation. Rifampicin inhibited NF-kappaB DNA-binding activity in LPS-activated RAW 264.7 cells, while it did not affect IKKalpha/beta activity. However, rifampicin slightly inhibited the nuclear translocation of NF-kappaB p65. In addition, rifampicin increased physical interaction between pregnane X receptor, a receptor for rifampicin, and NF-kappaB p65, suggesting pregnane X receptor interferes with NF-kappaB binding to DNA. Taken together, our results demonstrate that rifampicin inhibits LPS-induced TLR2 expression, at least in part, via the suppression of NF-kappaB DNA-binding activity in RAW 264.7 cells. Thus, the present results suggest that the rifampicin-mediated inhibition of TLR2 via the suppression of NF-kappaB DNA-binding activity may be a novel mechanism of the immune-suppressive effects of rifampicin.