Murine salmonellosis studied by confocal microscopy: Salmonella typhimurium resides intracellularly inside macrophages and exerts a cytotoxic effect on phagocytes in vivo

Abstract

Salmonella typhimurium is considered a facultative intracellular pathogen, but its intracellular location in vivo has not been demonstrated conclusively. Here we describe the development of a new method to study the course of the histopathological processes associated with murine salmonellosis using confocal laser scanning microscopy of immunostained sections of mouse liver. Confocal microscopy of 30-micron-thick sections was used to detect bacteria after injection of approximately 100 CFU of S. typhimurium SL1344 intravenously into BALB/c mice, allowing salmonellosis to be studied in the murine model using more realistic small infectious doses. The appearance of bacteria in the mouse liver coincided in time and location with the infiltration of neutrophils in inflammatory foci. At later stages of disease the bacteria colocalized with macrophages and resided intracellularly inside these macrophages. Bacteria were cytotoxic for phagocytic cells, and apoptotic nuclei were detected immunofluorescently, whether phagocytes harbored intracellular bacteria or not. These data argue that Salmonella resides intracellularly inside macrophages in the liver and triggers cell death of phagocytes, processes which are involved in disease. This method is also applicable to other virulence models to examine infections at a cellular and subcellular level in vivo.

Figure 1

Infection kinetics. Numbers of bacteria recovered from homogenized liver (white) and spleen (black) at different days after infection following an intravenous dose of 115 CFU SL 1344. The mice died at days 6–7. The data represent the average number of bacteria recovered from the organs of four mice per time point, and the error bars indicate the standard deviation.

Figure 4

Neutrophils are present in the foci at an early stage of infection, while macrophages dominate the tissue at later stages. Mouse liver sections labeled with antineutrophil-FITC (purple), CD18-Cy5 (red), and anti-Salmonella–TxR (green/light blue/yellow) in (a) uninfected liver, (b) liver infected with 65 CFU SL1344 at day 3 after infection, and (c) the same dose at day 5 after infection showing that neutrophils extravasate into the liver as an early response, but disappear at a later stage of infection when the foci consists of macrophages. The bacteria colocalize to the phagocytes, not to the hepatocytes. Each image is a projection of an 8-μm z-stack collected through the ×20 objective on a Bio-Rad MRC-600 confocal microscope. Bar, 50 μm.

Figure 5

S. typhimurium are located inside macrophages at late stages of infection. (a) Mouse liver infected with 65 CFU SL1344 at day 5 after infection labeled with anti-Salmonella–Cy3 (orange) and CD18-Cy5 (green). The image is a projection of a 30-μm z-stack collected through the ×60 objective on a Bio-Rad MRC-600 confocal microscope. The boxed macrophage is analyzed further in b–d. Bar, 25 μm. (b) NIH Image was used to project the side view (yz) and the bottom view (xz) of the boxed macrophage (represented by the xy view), which together demonstrate the intracellular location of the bacteria. (c) A three-dimensional reconstitution of a liver macrophage at day 5 after infection (the same cell as in b) using volume rendering. (d) A tilted, 1.8-μm section of the macrophage in c shows the intracellular location of three bacteria (red). Other bacteria present in b were also intracellularly located, but in a different section of the macrophage.

Figure 6

S. typhimurium are cytotoxic for mouse liver phagocytes. Mouse liver sections labeled with anti-digoxigenin-FITC (green), anti- Salmonella–TxR (pink), and CD18-Cy5 (blue) in (a) uninfected liver, (b) liver infected with 65 CFU SL1344 at day 3 after infection, and (c) the same dose at day 5 after infection. The FITC labeling shows the cytotoxic effect of the bacteria on the phagocytes, while a single apoptotic cell in the uninfected tissue is indicated by the arrow in a. The images are projections of 8-μm z-stacks collected through the ×20 objective. Bar, 50 μm. (d) The same tissue and labeling as in c but the image is a projection of a 32-μm z-stack collected through the ×60 objective. Bar, 25 μm. (e) As d but focused on a single apoptotic cell with intracellular bacteria. Images in a–c were collected on a Bio-Rad MRC-600 confocal microscope, d–e were collected on a Bio-Rad 1024 confocal microscope.