[Molecular mechanisms for intracellular parasitisation and exclusion in macrophage infected with Mycobacterium leprae].

It was previously demonstrated that TLR2 and CORO1A (TACO, Coronin 1, p57) localize phagosome membrane of macrophage. However, the functional relationship between TLR2 and CORO1A was not known. We show here that there is a functional counteraction between TLR2 and CORO1A.

Localization of CORO1A in the macrophages containing Mycobacterium leprae.

Mycobacteria have acquired an intracellular lifestyle within the macrophage, which is best exemplified by the enlarged infected histiocytes seen in lepromatous leprosy. To survive within the cell, mycobacteria must escape intracellular bactericidal mechanisms. In a study of Mycobacterium bovis Bacille Calmette-Guérin (M. bovis BCG) infection, it was shown that the host protein, CORO1A, also known as tryptophan aspartate-containing coat protein (TACO), accumulates on the phagosomal membrane, resulting in inhibition of phagosome-lysosome fusion, and thus augmenting intracellular survival. In this study, we show that CORO1A strongly localizes on the membrane of phagosomes that contain Mycobacterium leprae (M. leprae), where Toll-like receptor 2 was also visualized by immunostaining. When cultured macrophages were infected with M. leprae, CORO1A recruitment from the plasma membrane to the phagosomal membrane was observed. Moderate to strong CORO1A retention was observed in late lesions that contained foamy histiocytes, in which M. leprae were difficult to detect by acid-fast staining. These results suggest that components accumulating within the phagosome rather than viable bacilli are responsible for the retention of CORO1A, and that there is also a bactericidal mechanism in the macrophage that might counter the effects of CORO1A.

Role of the polypeptide region of a 33kDa mycobacterial lipoprotein for efficient IL-12 production.

Mycobacterium leprae lipoprotein, LpK, induced IL-12 production from human monocytes. To determine the components essential for cytokine production and the relative role of lipidation in the activation process, we produced lipidated and non-lipidated truncated forms of LpK. While 0.5nM of lipidated LpK-a having N-terminal 60 amino acids of LpK produced more than 700pg/ml IL-12 p40, the non-lipidated LpK-b having the same amino acids as that of LpK-a required more than 20nM of the protein to produce an equivalent dose of cytokine. Truncated protein having the C-terminal 192 amino acids of LpK did not induce any cytokine production. Fifty nanomolar of the synthetic lipopeptide of LpK produced only about 200pg/ml IL-12. Among the truncated LpK, only LpK-a and lipopeptide stimulated NF-kB-dependent reporter activity in TLR-2 transfectant. However, when monocytes were stimulated with lipopeptide in the presence of non-lipidated protein, they produced IL-12 synergistically. Therefore, both peptide regions of LpK and lipid residues are necessary for efficient IL-12 production.

Effect of hsp65 DNA vaccination carrying immunostimulatory DNA sequences (CpG motifs) against Mycobacterium leprae multiplication in mice.

A DNA expressing hsp65 of Mycobacterium leprae (pACB/hsp65) was constructed by using a vector containing immunostimulatory DNA sequences (pACB). At 12 weeks post-immunization, spleen cells from BALB/cA mice immunized with pACB/hsp65, produced a significantly higher amount of IFN-gamma than mice immunized with pACB in the absence of any in vitro stimulation, and further enhanced its production upon secondary in vitro stimulation with M. leprae lysate and hsp65. On the other hand, while production of IL-12 was observed in mice immunized with pACB/hsp65 12 weeks before, the cytokine production was inhibited by in vitro secondary stimulation with M. leprae or hsp65. At 18 weeks post-immunization, the production of both IFN-gamma and IL-12 was apparently down-regulated, but that of IL-10 was up-regulated. IL-10 seemed to suppress the IFN-gamma and IL-12 productions, because their production was recovered by neutralization of IL-10 with anti-IL-10 mAb. Furthermore, when the efficiency of pACB/hsp65 as a vaccine against M. leprae was evaluated in vivo, the mice immunized with pACB/hsp65 suppressed the multiplication of subsequently challenged M. leprae. These results suggest that a DNA containing M. leprae-derived hsp65 and immunostimulatory sequences might be a potent vaccine candidate against M. leprae infection.

Effect of hsp65 DNA vaccination carrying immunostimulatory DNA sequences (CpG Motifs) against mycobacterium leprae multiplication in mice

A DNA expressing hsp65 of Mycobacterium leprae (pACB/hsp65) was constructed by using a vector containing immunostimulatory DNA sequences (pACB). At 12 weeks post-immunization, spleen cells from BALB/cA mice immunized with pACB/hsp65, produced a significantly higher amount of IFN-gamma than mice immunized with pACB in the absence of any in vitro stimulation, and further enhanced its production upon secondary in vitro stimulation with M. leprae lysate and hsp65. On the other hand, while production of IL-12 was observed in mice immunized with pACB/hsp65 12 weeks before, the cytokine production was inhibited by in vitro secondary stimulation with M. leprae or hsp65. At 18 weeks post-immunization, the production of both IFN-gamma and IL-12 was apparently down-regulated, but that of IL-10 was up-regulated. IL-10 seemed to suppress the IFN-gamma and IL-12 productions, because their production was recovered by neutralization of IL-10 with anti-IL-10 mAb. Furthermore, when the efficiency of pACB/hsp65 as a vaccine against M. leprae was evaluated in vivo, the mice immunized with pACB/hsp65 suppressed the multiplication of subsequently challenged M. leprae. These results suggest that a DNA containing M. leprae-derived hsp65 and immunostimulatory sequences might be a potent vaccine candidate against M. leprae infection.