Specialized epithelia known as M cells overlying the lymphoid follicles of Peyer's patches are important in the mucosal immune system, but also provide a portal of entry for pathogens such as Salmonella typhimurium, Mycobacterium bovis, Shigella flexneri, Yersinia enterocolitica and reoviruses. Penetration of intestinal M cells and epithelial cells by Salmonella typhimurium requires the invasion genes of Salmonella Pathogenicity Island 1 (SPI1). SPI1-deficient S. typhimurium strains gain access to the spleen following oral administration and cause lethal infection in mice without invading M cells or localizing in Peyer's patches, which indicates that Salmonella uses an alternative strategy to disseminate from the gastrointestinal tract. Here we report that Salmonella is transported from the gastrointestinal tract to the bloodstream by CD18-expressing phagocytes, and that CD18-deficient mice are resistant to dissemination of Salmonella to the liver and spleen after oral administration. This CD18-dependent pathway of extraintestinal dissemination may be important for the development of systemic immunity to gastrointestinal pathogens, because oral challenge with SPI1-deficient S. typhimurium elicits a specific systemic IgG humoral immune response, despite an inability to stimulate production of specific mucosal IgA.

The contribution of the NADPH phagocyte oxidase (phox) and inducible nitric oxide (NO) synthase (iNOS) to the antimicrobial activity of macrophages for Salmonella typhimurium was studied by using peritoneal phagocytes from C57BL/6, congenic gp91phox −/−, iNOS −/−, and doubly immunodeficient phox −/−iNOS −/− mice. The respiratory burst and NO radical (NO·) made distinct contributions to the anti-Salmonella activity of macrophages. NADPH oxidase–dependent killing is confined to the first few hours after phagocytosis, whereas iNOS contributes to both early and late phases of antibacterial activity. NO-derived species initially synergize with oxyradicals to kill S. typhimurium, and subsequently exert prolonged oxidase-independent bacteriostatic effects. Biochemical analyses show that early killing of Salmonella by macrophages coincides with an oxidative chemistry characterized by superoxide anion (O2·−), hydrogen peroxide (H2O2), and peroxynitrite (ONOO−) production. However, immunofluorescence microscopy and killing assays using the scavenger uric acid suggest that peroxynitrite is not responsible for macrophage killing of wild-type S. typhimurium. Rapid oxidative bacterial killing is followed by a sustained period of nitrosative chemistry that limits bacterial growth. Interferon γ appears to augment antibacterial activity predominantly by enhancing NO· production, although a small iNOS-independent effect was also observed. These findings demonstrate that macrophages kill Salmonella in a dynamic process that changes over time and requires the generation of both reactive oxidative and nitrosative species.

A type III protein secretion system encoded by Salmonella pathogenicity island 2 (SPI2) has been found to be required for virulence and survival within macrophages. Here, SPI2 was shown to allow Salmonella typhimurium to avoid NADPH oxidase-dependent killing by macrophages. The ability of SPI2-mutant bacteria to survive in macrophages and to cause lethal infection in mice was restored by abrogation of the NADPH oxidase-dependent respiratory burst. Ultrastructural and immunofluorescence microscopy demonstrated efficient localization of the NADPH oxidase in the proximity of vacuoles containing SPI2-mutant but not wild-type bacteria, suggesting that SPI2 interferes with trafficking of oxidase-containing vesicles to the phagosome.

The role of leptin was investigated in two models of T cell-mediated hepatitis: the administration of Con A or of Pseudomonas aeruginosa exotoxin A (PEA). In both models, leptin-deficient (ob͞ob) mice were protected from liver damage and showed lower induction of tumor necrosis factor (TNF) ␣ and IL-18 compared with their lean littermates. Neutralization of TNF-␣ reduced induction of IL-18 by either Con A (70% reduction) or PEA (40% reduction). Pretreatment of lean mice with either soluble TNF receptors or with an anti-IL-18 antiserum significantly reduced Con A-and PEA-induced liver damage. The simultaneous neutralization of TNF-␣ and IL-18 fully protected the mice against liver toxicity. However, neutralization of either IL-18 or TNF-␣ did not inhibit Con A-induced production of IFN-␥. Thymus atrophy and alterations in the number of circulating lymphocytes and monocytes were observed in ob͞ob mice. Exogenous leptin replacement restored the responsiveness of ob͞ob mice to Con A and normalized their lymphocyte and monocyte populations. These results demonstrate that leptin deficiency leads to reduced production of TNF-␣ and IL-18 associated with reduced T cell-mediated hepatotoxicity. In addition, both TNF-␣ and IL-18 appear to be essential mediators of T cell-mediated liver injury.