The cellular niche of Listeria monocytogenes infection changes rapidly in the spleen

Abstract

The spleen is an important organ for the host response to systemic bacterial infections. Many cell types and cell surface receptors have been shown to play role in the capture and control of bacteria, yet these are often studied individually and a coherent picture has yet to emerge of how various phagocytes collaborate to control bacterial infection. We analyzed the cellular distribution of Listeria monocytogenes (LM) in situ during the early phase of infection. Using an immunohistochemistry approach, five distinct phagocyte populations contained LM after i.v. challenge and accounted for roughly all bacterial signal in tissue sections. Our analysis showed that LM was initially captured by a wide range of phagocytes in the marginal zone, where the growth of LM appeared to be controlled. The cellular distribution of LM within phagocyte populations changed rapidly during the first few hours, decreasing in marginal zone macrophages and transiently increasing in CD11c(+) DC. After 4-6 h LM was transported to the periarteriolar lymphoid sheath where the infective foci developed and LM grew exponentially.

Figure 1

Histological characterization of resident phagocytes in the spleen Cryosections from normal BALB/c mice were stained with different combinations of host cell markers to demonstrate staining specificity. (A) Anti-F4/80 (blue), anti-MOMA-1 (green) and anti-MARCO (red). (B) Anti-CD11b (blue), MOMA-1 (green), and CD11c (red). (C) Anti-CD11b (blue), anti-F4/80 (green), and anti-CD11c (red). (D) Anti-ER-TR9 (green) and anti-MARCO (red). Nearly all ER-TR9⁺ cells also express MARCO (yellow), suggesting that they are a subset of the MARCO⁺ MZM. (E) Anti-Gr-1 (green) and anti-CD11b (red). Approximately, 95% of CD11b⁺ cells are small round and co-stain with Gr-1. (F) CD11b⁺ cells do not stain with F4/80, anti-F4/80 (green) and anti-CD11b (red). Scale bar = 200 μm in (A–C) and 100 μm in (D–F). Images are representative of 3 independent experiments. WP: white pulp. BC: bridging channel.

Figure 2

Figure 2 LM is broadly distributed in splenocytes immediately after i.v. infection. (A) Infected host cells in stained cryosections 30 min following infection with 10⁶ EGD strain of LM. LM (green) and host cell markers (red). Scale bar = 10 μm. Images are representative of 3 independent experiments. (B) LM colocalization with host cell types. Bars represent mean + SD of at least 30 images per each cell type. (C) spleens at 30 min post infection with 2 × 10⁸ LM. Cryosections were stained with phalloidin-Alexa Fluor 350, anti-LM (green) and indicated host cell markers (red). Yellow represents colocalization between LM and stained host-cells. Blue lines represent the edge of the MZ determined by phalloidin staining. Scale bar = 50 μm. (D), colocalization analysis of (D) 2 × 10⁸ LM, (E) 7.2 × 10⁸ fluorescent 1.0 μm beads, and (F) 200 μg of 70 kD FITC-conjugated dextran with splenic phagocytes 30 min after i.v. injection. Bars represent mean + SD of 10 images per cell type. (G) 129/SvEv, IFNAR and IFNGR mice were infected intravenously with 10⁷ CFU of LM EGD strain and spleen colony counts were determined at 30 and 240 minutes post-infection. MZ: marginal zone. RP: red pulp, WP: white pulp.

Figure 3

LM signal decreases in MZM 0.5– 6h post-infection. (A) The colocalization coefficients of LM and host cell markers over time. Scatter plots show mean values for individual images and pooled means. (B) Spleen sections from mice infected with 2 × 10⁸ LM at 30 min, 2 h and 6 h. Cryosections were stained with phallodin-Alexa Fluor 350, anti-LM (green) and anti-SIGNR1 (red). Colocalization between LM and host-cells appears yellow in the image. Blue lines highlight the MZ edge drawn based on phalloidin staining. Scale bar = 100 μm. Images are representative of 10 images per time point. (C) Colocalization of LM in ER-TR9⁺ cells and, (D) covariance of LM and ER-TR9 signal (Pearson’s correlation), at increasing times after infection. Each dot is the mean value of a single image and pooled means are shown. (E), LM signal in pixels per image at 30 min, 2 h and 6 h post infection (2 × 10⁸ LM). Bars show mean + SD of 50 images per time point. (F) CFU per spleen 30 min, 2 h and 6 h post infection (2 × 10⁸ LM). Bars show mean + SD, 5 mice per group. (G) Host cell signal expressed in pixels per image at increasing times after infection. Graph shows mean of 12 images per group. All data are representative of 3 independent experiments with similar results. MZ: marginal zone. RP: red pulp, WP: white pulp.

Figure 4

Host cell distribution of LM 12 h and 24 h post infection. LM colocalization with splenic phagocytes 12 h (A) and 24 h (B) after infection. Bars represent mean + SD of 10 images per cell type. Bar graphs are representative of 3 independent experiments. LM disappears rapidly from MARCO⁺ MZM, but increases transiently in CD11c⁺ cells.