Antimicrobial Actions of the Nadph Phagocyte Oxidase and Inducible Nitric Oxide Synthase in Experimental Salmonellosis. I. Effects on Microbial Killing by Activated Peritoneal Macrophages in Vitro

Abstract

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.

Toll-Like Receptor 4 Dependence of Innate and Adaptive Immunity to Salmonella: Importance of the Kupffer Cell Network

“…In fact, elicited macrophages from C3H/ HeN and C3H/HeJ mice produced similar quantities of hydrogen peroxide and superoxide 1 h after infection with Salmonella as measured by HRP-dependent oxidation of phenol red and cytochrome c reduction (data not shown). However, TLR4-deficient macrophages were substantially impaired in their ability to restrict Salmonella growth at 20 h after infection, an action previously shown to be dependent on iNOS (13). Stimulation of TLR4-deficient macrophages with IFN-␥ 20 h before Salmonella ingestion was able to completely restore the antibacterial activity of these cells at 20 h (Fig.…”

“…inoculation of 1 mg/ml sodium periodate, as described (13). The peritoneal exudate cells were resuspended in RPMI 1640 supplemented with 10% heat-inactivated FCS (Gemini Bio-Products, Calabassas, CA), 1 mM sodium pyruvate, 10 mM HEPES, and 2 mM L-glutamine (all from Sigma-Aldrich, St. Louis, MO).…”

“…Amplification of GAPDH was used as an internal control. Production of reactive nitrogen species by Salmonella-infected macrophages was determined by spectrophotometric detection of nitrite at 550 nm using the Griess reagent (0.5% sulfanilimide and 0.05% N-1-naphthylethylenediamide hydrochloride in 2.5% acetic acid) (13).…”

Stimulation of Toll-Like Receptor 4 by Lipopolysaccharide During Cellular Invasion by Live Salmonella typhimurium Is a Critical But Not Exclusive Event Leading to Macrophage Responses

“…Mice that lack iNOS are more susceptible to salmonellae and are unable to limit bacterial growth in the later stages of Salmonella infection (41,42). Experiments with macrophages from these animals indicate that iNOS may contribute to both early and late limitation of bacterial growth by enhancing the early oxygen radical killing of S. typhimurium and by a later NO-dependent bacteristatic effect (43). We show in this study that live S. typhimurium infection stimulates the production of NO from macrophages by an LPS-and TLR-4-dependent mechanism.…”

Survival of Salmonella serovar Typhimurium inside porcine monocytes is associated with complement binding and suppression of the production of reactive oxygen species

“…Possibly, intracellular trafficking and thus survival of the Salmonella bacteria might be influenced by entry in the host cell through complement receptor binding (Ishibashi and Arai, 1996). The production of ROS by host macrophages is an important first defence mechanism (Vazquez Torres et al, 2000), which Salmonella must circumvent in order to survive intracellularly inside the host cell. The Salmonella Typhimurium strain was able to suppress the production of ROS in porcine monocytes.…”