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-μm-thick sections was used to detect bacteria after injection of ∼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.

Toll-like receptor (TLR) 4 is mainly found on cells of the myelopoietic lineage. It recognizes lipopolysaccharide (LPS) and mediates cellular activation and production of proinflammatory cytokines. Less is known about the distribution and role of TLR4 in epithelial cells that are continuously exposed to microbes and microbial products. Here we show that the murine small intestinal epithelial cell line m-ICcl2 is highly responsive to LPS and expresses both CD14 and TLR4. Transcription and surface membrane staining for CD14 were up-regulated upon LPS exposure. Surprisingly, TLR4 immunostaining revealed a strictly cytoplasmic paranuclear distribution. This paranuclear compartment could be identified as the Golgi apparatus. LPS added to the supernatant was internalized by m-ICcl2 cells and colocalized with TLR4. Continuous exposure to LPS led to a tolerant phenotype but did not alter TLR4 expression nor cellular distribution. Thus, intestinal epithelial cells might be able to provide the initial proinflammatory signal to attract professional immune cells to the side of infection. The cytoplasmic location of TLR4, which is identical to the final location of internalized LPS, further indicates an important role of cellular internalization and cytoplasmic traffic in the process of innate immune recognition.