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

Abstract

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.

Fig 1. SW-10 cells express S100 and NGFR p75, but neither MBP nor MPZ.

SW-10 cells were immunostained with antibodies against S100, NGFR p75, MBP, or MPZ. Nuclei were counterstained for 15 min with 10 μM Hoechst 33342 (Sigma-Aldrich Co. Ltd). Scale bar: 10 μm.

Fig 2. SW-10 cells have phagocytic activity.

The phagocytic activity of SW-10 cells was determined using a CytoSelect 96-well phagocytosis assay kit (Cell BioLabs). Cells were pre-treated with cytochalasin D, an inhibitor of phagocytosis and actin polymerization, at the designated concentration for 1h at 37°C, and then incubated with Zymosan particles at a 100:1 ratio for 2 h. Significance was calculated via one-way ANOVA. *P <0.05 versus control cells with only Zymosan uptake. Cyto D: cytochalasin D.

Fig 3. SW-10 cells are infected with M. leprae.

(A and B) SW-10 cells were incubated with M. leprae at the MOI of 10:1, 20:1, 50:1 and 100:1 for 6 h at 37°C. After extracellular M. leprae were washed away, M. leprae were stained with Auromine O. The percentage of M. leprae-infected cells and the number of M. leprae in a cell were determined in the oil immersion field of a light microscope. Significance was calculated via one-way ANOVA. *P <0.05 versus cells were incubated with M. leprae at the MOI of 100:1. (C) SW-10 cells were incubated with M. leprae at the MOI of 100:1 for 6 h at 37°C. After extracellular M. leprae were washed away, M. leprae were stained with Auramine O. Nuclei were counterstained for 15 min with 10 μM Hoechst 33342 (Sigma-Aldrich Co. Ltd). Scale bar: 10μm.

Fig 4. M.leprae infection does not induce apoptosis in SW-10 cells.

(A) SW-10 cells were incubated with M. leprae at the MOI of 100:1 for 6 h at 37°C. After extracellular M. leprae were washed away, the cells were again incubated for another 48 h. M. leprae were stained with Auramine O and the cells were immunostained with antibodies against active caspase-3. (B) SW-10 cells were treated with 1 μM staurosporine for 2 h. The cells were then immunostained with antibodies against active caspase-3. Nuclei were counterstained for 15 min with 10 μM Hoechst 33342 (Sigma-Aldrich Co. Ltd). Scale bar: 10μm.

Fig 5. Live M. leprae induces the formation of LDs in SW-10 cells.

SW-10 cells were incubated with either live or dead M.leprae, or latex beads (3.0 μm and Polysciences) at a MOI of 100:1 for 6 h at 37°C. After the extracellular M. leprae were washed away, the cells were again incubated either for another 48 h (A) or for the indicated times (B). The formation of LDs was examined by transmission electron microscopy (A). The expression of ADRP was determined by western blot analysis (B). Similar results were observed in three independent experiments. Scale bar: 2 μm.

Fig 6. Treatment with celecoxib or C-75 inhibits M.leprae-induced ADRP expression in SW-10 cells.

(A) Cells were pre-treated either with 20 μΜ celecoxib, an inhibitor of COX-2, or with 157 μΜ C-75, an inhibitor of FAS, for 1 h, and were then incubated with live M. leprae or latex beads (3.0 μm, Polysciences) at a MOI of 100:1 for 6 h at 37°C. The expression of ADRP was determined by western blot analysis. (B and C) The effect of celecoxib and C-75 on the phagocytic activity of SW-10 cells was determined using the CytoSelect 96-well phagocytosis assay kit (Cell BioLabs). Cells were pre-treated with celecoxib or C-75, and were then incubated with Zymosan particles at a 100:1 ratio for 2 h. Significance was calculated via one-way ANOVA. *P <0.05 versus control cells with only Zymosan uptake.

Fig 7. Treatment with celecoxib or C-75 reverses impairment of the fusion of live M.leprae-containing phagosomes with lysosomes in SW-10 cells.

(A and B) Cells were pre-treated either with 20 μΜ celecoxib, an inhibitor of COX-2, or with 157 μΜ C-75, an inhibitor of FAS, for 1 h, and were then incubated with either live or dead M. leprae at a MOI of 100:1 for 6 h at 37°C. After the extracellular M. leprae were washed away, the cells were again incubated for another 48 h and then were stained with Auramine O and a red-fluorescent dye for labeling and tracking acidic organelles, LysoTracker Red (Molecular probes). The maturation level of the phagosomes containing live M. leprae was determined by measuring the co-localization level of Auramine O-labeled M.leprae with Lyso Tracker Red (Molecular probes). Nuclei were counterstained for 15 min with 10 μM Hoechst 33342 (Sigma-Aldrich Co. Ltd). *P <0.05 and **P <0.01 between the indicated groups (B). Scale bar: 10 μm.

Fig 8. Treatment with celecoxib or C-75 reduces the ATP content of M.leprae in SW-10 cells.

Cells were pre-treated with 20 μΜ celecoxib, an inhibitor of COX-2, or 157 μΜ C-75, an inhibitor of FAS, for 1 h, and then incubated with live M. leprae at the MOI of 100:1 for 6 h at 37°C. After extracellular M. leprae were washed away, the cells were again incubated for another 72 h. The M. leprae-infected SW-10 cells were lysed with 0.1 N NaOH for 5 min. The amount of ATP was quantified using the BacTiter-Glo Microbial Cell Viability Assay kit (Promega), according to the manufacturer’s instructions. *P <0.05 between the indicated groups.