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Salmonella typhimurium persists within macrophages in the mesenteric lymph nodes of chronically infected Nramp1+/+ mice and can be reactivated by IFNgamma neutralization.

Monack DM, Bouley DM, Falkow S - J. Exp. Med. (2004)

Bottom Line: Host-adapted strains of Salmonella are capable of establishing a persistent infection in their host often in the absence of clinical disease.Here, we show that S. typhimurium can persist for as long as 1 yr in the mesenteric lymph nodes (MLNs) of 129sv Nramp1(+)(/)(+) (Slc11a1(+)(/)(+)) mice despite the presence of high levels of anti-S. typhimurium antibody.Finally, chronically infected mice treated with an interferon-gamma neutralizing antibody exhibited symptoms of acute systemic infection, with evidence of high levels of bacterial replication in most tissues and high levels of fecal shedding.

View Article: PubMed Central - PubMed

Affiliation: Department of Microbiology and Immunology, Stanford University School of Medicine, Stanford University, Stanford, CA 94305, USA. dmonack@leland.stanford.edu

ABSTRACT
Host-adapted strains of Salmonella are capable of establishing a persistent infection in their host often in the absence of clinical disease. The mouse model of Salmonella infection has primarily been used as a model for the acute systemic disease. Therefore, the sites of long-term S. typhimurium persistence in the mouse are not known nor are the mechanisms of persistent infection clearly understood. Here, we show that S. typhimurium can persist for as long as 1 yr in the mesenteric lymph nodes (MLNs) of 129sv Nramp1(+)(/)(+) (Slc11a1(+)(/)(+)) mice despite the presence of high levels of anti-S. typhimurium antibody. Tissues from 129sv mice colonized for 60 d contain numerous inflammatory foci and lesions with features resembling S. typhi granulomas. Tissues from mice infected for 365 d have very few organized inflammatory lesions, but the bacteria continue to persist within macrophages in the MLN and the animals generally remain disease-free. Finally, chronically infected mice treated with an interferon-gamma neutralizing antibody exhibited symptoms of acute systemic infection, with evidence of high levels of bacterial replication in most tissues and high levels of fecal shedding. Thus, interferon-gamma, which may affect the level of macrophage activation, plays an essential role in the control of the persistent S. typhimurium infection in mice.

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S. typhimurium persist inside of macrophages within the MLN of chronically infected mice. (A) MLN sections were labeled with anti-Salmonella antibody (green), antineutrophil antibody, Gr-1 (red), and an anti–mouse IgG antibody (blue) that nonspecifically stains other immune cells. (A, D, and E) The image is a projection of a 20-μm z-stack collected through the 60× objective on a confocal microscope (model MRC-600; Bio-Rad Laboratories). Arrow points to bacterium. Bar, 10 μm. (B) MLN sections were labeled with anti-Salmonella antibody (green), antimacrophage antibody, MOMA-2 (red), and ToTo-3 to stain all nuclei (blue). The image is a projection of a 15-μm z-stack collected through the 40× objective on a confocal microscope. (C) Volocity 2.0 was used to project the side view (yz) and bottom view (xz) of the infected macrophage in B (represented by the xy view), which together demonstrate the intracellular location of the bacterium. Bar, 5 μm. (D) Three-dimensional projection of macrophage in C. (E) MLN sections were labeled with anti-Salmonella antibody (green), antimacrophage antibody (red), and B220 (blue), the anti–B lymphocyte antibody. Bar, 10 μm.
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fig6: S. typhimurium persist inside of macrophages within the MLN of chronically infected mice. (A) MLN sections were labeled with anti-Salmonella antibody (green), antineutrophil antibody, Gr-1 (red), and an anti–mouse IgG antibody (blue) that nonspecifically stains other immune cells. (A, D, and E) The image is a projection of a 20-μm z-stack collected through the 60× objective on a confocal microscope (model MRC-600; Bio-Rad Laboratories). Arrow points to bacterium. Bar, 10 μm. (B) MLN sections were labeled with anti-Salmonella antibody (green), antimacrophage antibody, MOMA-2 (red), and ToTo-3 to stain all nuclei (blue). The image is a projection of a 15-μm z-stack collected through the 40× objective on a confocal microscope. (C) Volocity 2.0 was used to project the side view (yz) and bottom view (xz) of the infected macrophage in B (represented by the xy view), which together demonstrate the intracellular location of the bacterium. Bar, 5 μm. (D) Three-dimensional projection of macrophage in C. (E) MLN sections were labeled with anti-Salmonella antibody (green), antimacrophage antibody (red), and B220 (blue), the anti–B lymphocyte antibody. Bar, 10 μm.

Mentions: To determine the host cells that S. typhimurium are associated with in persistently infected MLN, we stained frozen sections with several antibodies to host immune cells. Initially, we stained sections with an antibody that recognized neutrophils, Gr-1, an anti-Salmonella antibody, and an antibody that nonspecifically stains the host cells. We found that bacteria did not colocalize with neutrophils; rather, they resided within large host cells that were surrounded by neutrophils (Fig. 6 A). This finding and the results of our histological staining led us to examine the remaining sections with an antibody that stains the cytoplasm of all macrophages, MOMA-2. The bacteria were mainly found colocalized with MOMA-2+ macrophages in macrophage-rich areas of the lymph nodes within the subcapsular sinus and outer cortex (Fig. 6 B). Further analysis of the z-stacks obtained on the confocal microscope revealed that bacteria were inside these macrophages, as seen in the xz, yz, and three-dimensional projections (Fig. 6, C–E). To determine the frequency with which S. typhimurium were located inside macrophages, we examined every bacterium found within all sections and scored whether it was inside or outside a MOMA-2+ host cell. 82% of the bacteria were clearly intracellular, whereas the remaining bacteria were either extracellular or their location was unclear (Table II). We noted at the same time the number of bacteria residing within the infected host cell and found that the mean number was between three and four bacteria per cell (Table II). We further characterized the host immune cells in the foci of bacterially infected macrophages and found that in addition to macrophages, the other immune cells in the vicinity of infected macrophages were predominantly B lymphocytes (Fig. 6 E). Thus, S. typhimurium persist intracellularly within macrophages of the MLN of 129sv mice.


Salmonella typhimurium persists within macrophages in the mesenteric lymph nodes of chronically infected Nramp1+/+ mice and can be reactivated by IFNgamma neutralization.

Monack DM, Bouley DM, Falkow S - J. Exp. Med. (2004)

S. typhimurium persist inside of macrophages within the MLN of chronically infected mice. (A) MLN sections were labeled with anti-Salmonella antibody (green), antineutrophil antibody, Gr-1 (red), and an anti–mouse IgG antibody (blue) that nonspecifically stains other immune cells. (A, D, and E) The image is a projection of a 20-μm z-stack collected through the 60× objective on a confocal microscope (model MRC-600; Bio-Rad Laboratories). Arrow points to bacterium. Bar, 10 μm. (B) MLN sections were labeled with anti-Salmonella antibody (green), antimacrophage antibody, MOMA-2 (red), and ToTo-3 to stain all nuclei (blue). The image is a projection of a 15-μm z-stack collected through the 40× objective on a confocal microscope. (C) Volocity 2.0 was used to project the side view (yz) and bottom view (xz) of the infected macrophage in B (represented by the xy view), which together demonstrate the intracellular location of the bacterium. Bar, 5 μm. (D) Three-dimensional projection of macrophage in C. (E) MLN sections were labeled with anti-Salmonella antibody (green), antimacrophage antibody (red), and B220 (blue), the anti–B lymphocyte antibody. Bar, 10 μm.
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fig6: S. typhimurium persist inside of macrophages within the MLN of chronically infected mice. (A) MLN sections were labeled with anti-Salmonella antibody (green), antineutrophil antibody, Gr-1 (red), and an anti–mouse IgG antibody (blue) that nonspecifically stains other immune cells. (A, D, and E) The image is a projection of a 20-μm z-stack collected through the 60× objective on a confocal microscope (model MRC-600; Bio-Rad Laboratories). Arrow points to bacterium. Bar, 10 μm. (B) MLN sections were labeled with anti-Salmonella antibody (green), antimacrophage antibody, MOMA-2 (red), and ToTo-3 to stain all nuclei (blue). The image is a projection of a 15-μm z-stack collected through the 40× objective on a confocal microscope. (C) Volocity 2.0 was used to project the side view (yz) and bottom view (xz) of the infected macrophage in B (represented by the xy view), which together demonstrate the intracellular location of the bacterium. Bar, 5 μm. (D) Three-dimensional projection of macrophage in C. (E) MLN sections were labeled with anti-Salmonella antibody (green), antimacrophage antibody (red), and B220 (blue), the anti–B lymphocyte antibody. Bar, 10 μm.
Mentions: To determine the host cells that S. typhimurium are associated with in persistently infected MLN, we stained frozen sections with several antibodies to host immune cells. Initially, we stained sections with an antibody that recognized neutrophils, Gr-1, an anti-Salmonella antibody, and an antibody that nonspecifically stains the host cells. We found that bacteria did not colocalize with neutrophils; rather, they resided within large host cells that were surrounded by neutrophils (Fig. 6 A). This finding and the results of our histological staining led us to examine the remaining sections with an antibody that stains the cytoplasm of all macrophages, MOMA-2. The bacteria were mainly found colocalized with MOMA-2+ macrophages in macrophage-rich areas of the lymph nodes within the subcapsular sinus and outer cortex (Fig. 6 B). Further analysis of the z-stacks obtained on the confocal microscope revealed that bacteria were inside these macrophages, as seen in the xz, yz, and three-dimensional projections (Fig. 6, C–E). To determine the frequency with which S. typhimurium were located inside macrophages, we examined every bacterium found within all sections and scored whether it was inside or outside a MOMA-2+ host cell. 82% of the bacteria were clearly intracellular, whereas the remaining bacteria were either extracellular or their location was unclear (Table II). We noted at the same time the number of bacteria residing within the infected host cell and found that the mean number was between three and four bacteria per cell (Table II). We further characterized the host immune cells in the foci of bacterially infected macrophages and found that in addition to macrophages, the other immune cells in the vicinity of infected macrophages were predominantly B lymphocytes (Fig. 6 E). Thus, S. typhimurium persist intracellularly within macrophages of the MLN of 129sv mice.

Bottom Line: Host-adapted strains of Salmonella are capable of establishing a persistent infection in their host often in the absence of clinical disease.Here, we show that S. typhimurium can persist for as long as 1 yr in the mesenteric lymph nodes (MLNs) of 129sv Nramp1(+)(/)(+) (Slc11a1(+)(/)(+)) mice despite the presence of high levels of anti-S. typhimurium antibody.Finally, chronically infected mice treated with an interferon-gamma neutralizing antibody exhibited symptoms of acute systemic infection, with evidence of high levels of bacterial replication in most tissues and high levels of fecal shedding.

View Article: PubMed Central - PubMed

Affiliation: Department of Microbiology and Immunology, Stanford University School of Medicine, Stanford University, Stanford, CA 94305, USA. dmonack@leland.stanford.edu

ABSTRACT
Host-adapted strains of Salmonella are capable of establishing a persistent infection in their host often in the absence of clinical disease. The mouse model of Salmonella infection has primarily been used as a model for the acute systemic disease. Therefore, the sites of long-term S. typhimurium persistence in the mouse are not known nor are the mechanisms of persistent infection clearly understood. Here, we show that S. typhimurium can persist for as long as 1 yr in the mesenteric lymph nodes (MLNs) of 129sv Nramp1(+)(/)(+) (Slc11a1(+)(/)(+)) mice despite the presence of high levels of anti-S. typhimurium antibody. Tissues from 129sv mice colonized for 60 d contain numerous inflammatory foci and lesions with features resembling S. typhi granulomas. Tissues from mice infected for 365 d have very few organized inflammatory lesions, but the bacteria continue to persist within macrophages in the MLN and the animals generally remain disease-free. Finally, chronically infected mice treated with an interferon-gamma neutralizing antibody exhibited symptoms of acute systemic infection, with evidence of high levels of bacterial replication in most tissues and high levels of fecal shedding. Thus, interferon-gamma, which may affect the level of macrophage activation, plays an essential role in the control of the persistent S. typhimurium infection in mice.

Show MeSH
Related in: MedlinePlus