Hemophagocytic macrophages harbor Salmonella enterica during persistent infection

Abstract

Salmonella enterica subspecies can establish persistent, systemic infections in mammals, including human typhoid fever. Persistent S. enterica disease is characterized by an initial acute infection that develops into an asymptomatic chronic infection. During both the acute and persistent stages, the bacteria generally reside within professional phagocytes, usually macrophages. It is unclear how salmonellae can survive within macrophages, cells that evolved, in part, to destroy pathogens. Evidence is presented that during the establishment of persistent murine infection, macrophages that contain S. enterica serotype Typhimurium are hemophagocytic. Hemophagocytic macrophages are characterized by the ingestion of non-apoptotic cells of the hematopoietic lineage and are a clinical marker of typhoid fever as well as certain other infectious and genetic diseases. Cell culture assays were developed to evaluate bacterial survival in hemophagocytic macrophages. S. Typhimurium preferentially replicated in macrophages that pre-phagocytosed viable cells, but the bacteria were killed in macrophages that pre-phagocytosed beads or dead cells. These data suggest that during persistent infection hemophagocytic macrophages may provide S. Typhimurium with a survival niche.

Figure 1. S. Typhimurium Reside in Macrophages That Appear to Have Multiple Nuclei

Confocal fluorescence microscopy of 50-μm-thick liver sections from a 1-wk-infected Slc11a1 (Nramp1) wild-type mouse. S. Typhimurium (O-antigen, arrows) are red, macrophages (F4–80 and MOMA-2) are blue, DNA (DAPI) is gray, and phalloidin is green. (A) Low power image, scale bar is 40 μm. (B–H) Montage of 4-μm optical sections through the boxed region of (A). Scale bar is 20 μm. (I and J) Enlarged images showing actin rings (arrowheads) around nuclei within the multinucleate macrophage. The endogenous macrophage nucleus is visible in (I) and is labeled with an N. The video from which (A–J) were derived (Video S1) is available online.

Figure 2. S. Typhimurium-Infected Macrophages Containing Phagocytosed Neutrophils and T Cells

Confocal fluorescence microscopy of 50-μm-thick liver sections from 1-wk-infected Slc11a1 wild-type mice. (A–C) S. Typhimurium (O-antigen, arrows) are red, macrophages (F4–80 and MOMA-2) are blue, DNA (DAPI) is gray, phalloidin is green, and neutrophils (Gr-1/Ly-6G/RB6-8C5) are pink (arrowheads). (A) Collapsed image from a 40-μm Z-stack. Scale bar is 20 μm. (B and C) Sections from (A) that are 4 μm apart. The video from which (A–C) were derived (Video S2) is available online. (D–G) T cells within multinucleate macrophages. Macrophages (F4–80 and MOMA-2) are blue (D, G, and H), T cells (CD3_zeta) are red (D, G, arrowheads), DAPI is gray (E, G), actin-bound phalloidin is green (F, G). (G) Is a composite of (D, E, and F). Scale bars are 16 μm. (H) An image from a different mouse stained and labeled as described for (D–G). Scale bar is 8 μm. A video showing a T cell inside of a macrophage is available online (Video S3).

Figure 3. Infected Tissues Contain Few Terminal Deoxynucleotidyl Transferase (TdT)-Positive Nuclei

Nick-end (TdT) labeling (green, arrows) and DAPI staining (gray) of a liver inflammatory lesion, 1 wk post-infection. (A) Scale bar is 40 μm. (B) Close-up of box in (A). Scale bar is 8 μm.

Figure 4. Infected Tissues Have Significant Inflammatory Lesions at 1 wk and 3 wk but Contain Few Caspase-3-Positive Cells

Light microscopy of hematoxylin (purple), eosin (pink), and caspase-3 (brown, arrows) stained liver thin sections over an infection time course. Enlarged images of the boxed regions on the left (200-μm scale bars) are shown on the right (50-μm scale bars). Arrowheads show inflammatory lesions: (A–B) 4 d post-infection; (C–D) 1 wk; (E–F) 3 wk; and (G–H) 8 wk.

Figure 5. Activated Bone Marrow–Derived Macrophages (BMDMs) Phagocytose Live Host Cells

Confocal fluorescence microscopy. BMDMs are blue (F4–80 and MOMA-2), human T cells (Jurkats) are green (CMFDA-stained), and DNA is gray (DAPI). (A–C) 30 min after the addition of Jurkats to BMDMs. (A) Unactivated BMDMs show little association with Jurkat cells, many of which were washed away. Scale bar is 40 μm (B and C). Activated macrophages phagocytose Jurkat cells; arrow denotes engulfed cell, arrowheads show partially engulfed cells. Scale bars are 40 μm (B) and 8 μm (C). (D–F) 42 h after mock-infection (D), or S. Typhimurium-infection (O-antigen, red, arrows, E and F). Scale bars are 20 μm (D), 40 μm (E), and 16 μm (F).

Figure 6. S. Typhimurium Preferentially Survive within BMDMs That Phagocytosed Live Host Cells

Gentamicin protection assay analyzed by plating for colony-forming units (CFU). BMDMs were incubated with media alone (white bars), beads (light gray bars), apoptotic (dark gray bars), or live (black bars) cells (Jurkats) and then infected with S. Typhimurium. (A) Bacterial invasion, as determined 2 h post-infection. (B–C) S. Typhimurium survival expressed as percent of invasion (A). (B) 18 h post-infection and (C) 42 h post-infection.

Figure 7. S. Typhimurium Preferentially Survive within Individual BMDMs That Phagocytosed Live Cells

Gentamicin protection assays analyzed by fluorescence microscopy. Histograms of activated BMDMs that did (white bars) or did not (black bars) phagocytose live T cells. BMDMs were subsequently infected with S. Typhimurium. The x-axis indicates the number of bacteria (>6, 6, 5, 4, 3, 2, 1, or 0) per BMDM at 42 h post-infection. Experiments were performed with human (A) or mouse T cells (B). Student's t-test p-values are shown (**p < 0.01, * p < 0.05).

Figure 8. S. Typhimurium Preferentially Survive within Mouse Macrophage-Like Tissue Culture Cells That Phagocytosed Live Cells

Gentamicin protection assays analyzed by fluorescence microscopy. Histograms of activated RAW264.7 (Slc11a1^G169D) cells that did (white bars) or did not (black bars) phagocytose live T cells. RAW264.7 cells were subsequently infected with S. Typhimurium. The x-axis indicates the number of bacteria (>16, 11–15, 6–10, 1–5, or 0) per RAW264.7 at 42 h post-infection. Experiments were performed with human (A) or mouse T cells (B). Student's t test p-values are shown (**p < 0.01, * p < 0.05).