Induction of antimicrobial pathways during early-phase immune response to Salmonella spp. in murine macrophages: gamma interferon (IFN-gamma) and upregulation of IFN-gamma receptor alpha expression are required for NADPH phagocytic oxidase gp91-stimulated oxidative burst and control of virulent Salmonella spp

Abstract

The effect of gamma interferon (IFN-gamma) on elevation of reactive oxygen species and the viability of virulent wild-type and avirulent mutants of Salmonella enterica serovar Typhimurium and S. enterica serovar Infantis was studied in a murine macrophage cell line (J774.2 cells). S. enterica serovar Typhimurium 14028 phoP and a rough lipopolysaccharide mutant of S. enterica serovar Infantis 1326/28 (phi(r)) (avirulent mutants) induced NADPH phagocytic oxidase gp91 (gp91(phox)) activity and a significant (P < 0.05) elevation of reactive oxygen species within 12 h without coculture with IFN-gamma. This coincided with reduced survival of S. enterica serovar Typhimurium14028 phoP or stasis of S. enterica serovar Infantis phi(r). Fluorometric studies indicated that expression of IFN-gamma on infected J774.2 cells was not significantly (P > 0.05) elevated. However, studies with the virulent S. enterica serovar Typhimurium strains showed that a comparable level of control of bacterial numbers could only be achieved by coculture with IFN-gamma. This coincided with significant upregulation of IFN-gamma receptor alpha expression on the surface of J774.2 cells and was completely abolished by N-acetyl-L-cysteine captopril (an inhibitor of reactive oxygen species). Delay in reactive oxygen species induction due to a requirement for IFN-gamma and upregulation of IFN-gamma receptor alpha in macrophages infected with virulent salmonellae may result in greater dissemination of virulent salmonellae in host tissue.

FIG. 1.

Survival of Salmonella spp. and induction of respiratory burst (ROS) in J774.2 monolayers cocultured with IFN-γ from 2 to 24 h postinfection. ▪, wild-type S. enterica serovar Typhimurium 14028; ♦, S. enterica serovar Typhimurium 14028 phoP; •, S. enterica serovar Typhimurium F98; ▪, S. enterica serovar Infantis 1326/28 φ^r. (A, C, E, and G) Salmonella survival; (B, D, F, and H) oxidative burst. (A and B) Zero IFN-γ; (C and D) cocultured with IFN-γ (10 U/ml), (E and F) cocultured with IFN-γ (100 U/ml); (G and H) cocultured with IFN-γ (1,000 U/ml). Black bars, 2 h postinfection; dark gray bars, 7 h postinfection; light gray bars, 12 h postinfection; open bars, 24 h postinfection. *, significant (P = 0.05) increase from control values at the same time point. All points represent a mean of three replicate experiments performed on at least five different occasions. MOI was 10 in all experiments.

FIG. 2.

Effect of coculture with N-acetyl-l-cysteine captopril (ACC) and IFN-γ on Salmonella survival and ROS induction in J774.2 cells. (A) J774 cells infected with salmonellae (MOI = 10) and cocultured with ACC (10 mM) and IFN-γ (1,000 U/ml) from 2 to 24 h postinfection. Black bars, 2 h postinfection; dark gray bars, 7 h postinfection; light gray bars, 12 h postinfection; open bars, 24 h postinfection. (B) Survival of salmonellae in J774 cells cocultured with ACC (10 mM) and IFN-γ (1,000 U/ml). ⋄, wild-type 14028 with ACC; □, 14028 phoP mutant with ACC; ♦, wild-type 14028 without ACC; ▪, 14028 phoP mutant without ACC. (C) Effect of increasing IFN-γ concentration on the induction of ROS in J774 cells. Black bars, 2 h postinfection; dark gray bars, 7 h postinfection; light gray bars, 12 h postinfection; open bars, 24 h postinfection. *, significant (P = 0.05) increase above the control (zero IFN-γ) at the same time point. Each point represents a mean of three replicate experiments repeated on at least five separate occasions at an MOI of 10.

FIG.3.

Confocal laser scanning microscope images of gp91^phox activity in J774.2 macrophages infected with salmonellae and cocultured with or without IFN-γ (1,000 U/ml). (A) Uninfected control, 2 h in culture; (B) uninfected control, 12 h in culture; (C) 14028 phoP mutant, 12 h postinfection, without IFN-γ; (D) 14028 phoP, 12 h postinfection, with IFN-γ; (E) high-power image of 14028 phoP mutant-infected cell, 12 h postinfection, without IFN-γ (image taken 4 μm below cell surface); (F) wild-type 14028, 12 h postinfection, without IFN-γ; (G) wild-type 14028, 12 h postinfection, with IFN-γ; (H) high-power image of wild-type 14028, 12 h postinfection, without IFN-γ (4 μm below cell surface); (I) IFN-γ only, 2 h in culture; (J) IFN-γ only, 12 h in culture. Yellow-green shows gp91^phox activity. Open arrow shows gp91^phox localization on cell membrane; solid arrow shows gp91^phox cytoplasmic localization. Solid arrows in E and H show bacteria in cells. Scale bars, 10 μm.

FIG. 4.

Fluorometric microsphere analysis of IFN-γRα surface expression on J774.2 cells following Salmonella infection (MOI, 10) and cocultured with or without IFN-γ (1,000 U/ml) from 2 to 12 h postinfection. (A) Cocultured without IFN-γ; (B) cocultured with IFN-γ. ▪, wild-type 14028; ▴, 14028 phoP mutant; ♦, uninfected control; □, IFN-γ only; +, microspheres coated with bovine serum albumin; •, cultured cells not exposed to any treatment (autofluorescence). *, significant (P = 0.05) increase above the uninfected control value. Each experiment represents a mean of three replicates repeated on at least five separate occasions.

FIG. 5.

Survival of salmonellae in hydrogen peroxide. (A) Wild-type 14028; (B) S. enterica serovar Typhimurium 14028 phoP; (C) S. enterica serovar Typhimurium F98; (D) S. enterica serovar Infantis 1326/28 φ^r. Each experiment was repeated three times on three separate occasions.