Limited mycobacterial infection of the liver as a consequence of its microanatomical structure causing restriction of mycobacterial growth to professional phagocytes

Abstract

Among sites of extrapulmonary growth of Mycobacterium tuberculosis, the liver is the least infected. Our data suggest that this is due to the complete restriction of mycobacterial growth to liver macrophages. Unlike in organs more persistently seeded by M. tuberculosis, in the liver the bacteria do not infect cell types other than professional phagocytes.

FIG. 1

M. tuberculosis titers in vivo. C57BL/6 mice were infected intravenously with 10⁴ CFU of M. tuberculosis strain Erdman. Five weeks postinfection, mice were treated with isoniazide (INH; 0.5 g/liter) in the drinking water for 4 weeks. Log CFU were determined in liver (A), spleen (B), and lung (C) at the indicated time points. Shown are values for four individual mice per group (solid circles) and the means for four mice (line) per time point and group for all three organs. The detection limit of the assay is indicated by the broken line. Hatched area, time interval of isoniazide treatment. n.d., first time point CFU not determined for lung. Shown is one representative experiment of two similar ones.

FIG. 2

Confocal analysis of liver cell infection in vitro. TIB-75 mouse hepatocyte and J774A.1 mouse macrophage cell lines were infected with rBCG-GFP at an MOI of 10 for 1 h, resulting in an infection rate of 5 to 10% of the cells and analyzed after 1 h (A and C) and 24 h (B and D), respectively. For 24 h of infection, cultures were supplemented after 1 h of incubation with 25 mg of gentamicin sulfate per liter. Prior to analysis, cells were fixed with 4% PFA and stained with phalloidin-TRITC in order to visualize actin. The rBCG-GFP were localized within the cells by confocal laser microscopy. Shown are projections of eight confocal laser scans. Original magnification, ×1,000. Shown is one representative experiment of two similar ones.

FIG. 3

Confocal analysis of liver cell infection in vivo. C57BL/6 mice were either left untreated (A to E) or treated with 1.5 mg (F to J) of clodronate liposomes intraperitoneally at day −3. At day 0, mice were infected intravenously with 10⁶ CFU of rBCG-GFP. At 15 min and 2 days postinfection, liver sections were stained with the rat MAb F4/80 and the secondary PAb goat anti-rat alkaline phosphatase (A and F) or PAb goat anti-rat Cy3.18 (B to E and G to J) in order to detect liver macrophages. Clusters of rBCG-GFP were detected inside the liver macrophages by confocal laser microscopy. Original magnification, ×200 (A, B, D, F, G, and I) and ×630 (C, E, H, and J). Shown is one representative experiment of two similar ones.