Dendritic cells inhibit the progression of Listeria monocytogenes intracellular infection by retaining bacteria in major histocompatibility complex class II-rich phagosomes and by limiting cytosolic growth

Abstract

Dendritic cells (DC) provide a suboptimal niche for the growth of Listeria monocytogenes, a facultative intracellular bacterial pathogen of immunocompromised and pregnant hosts. This is due in part to a failure of large numbers of bacteria to escape to the cytosol, an essential step in the intracellular life cycle that is mediated by listeriolysin O (LLO). Here, we demonstrate that wild-type bacteria that failed to enter the cytosol of bone marrow-derived DC were retained in a LAMP2+ compartment. An isogenic L. monocytogenes strain that produces an LLO protein with reduced pore-forming activity had a severe escape and growth phenotype in DC. Few mutant bacteria entered the cytosol in the first 2 h and were instead found in LAMP2+, major histocompatibility complex class II+ (MHC-II+) H2-DM vesicles characteristic of MHC-II antigen loading compartments (MIIC). In contrast, the mutant had a minor phenotype in bone marrow-derived macrophages (BMM) despite the reduced LLO activity. In the first hour, DC phagosomes acidified to a pH that was, on average, half a point higher than that of BMM phagosomes. Unlike BMM, L. monocytogenes growth in DC was minimal after 5 h, and consequently, DC remained viable and matured late in infection. Taken together, the data are consistent with a model in which phagosomal maturation events associated with the acquisition of MHC-II molecules present a suboptimal environment for L. monocytogenes escape to the DC cytosol, possibly by limiting the activity of LLO. This, in combination with an undefined mechanism that controls bacterial growth late in infection, promotes DC survival during the critical maturation response.

FIG. 1.

DC retain significant numbers of L. monocytogenes cells in a LAMP2⁺ compartment. DC were infected for 30 min with wild-type L. monocytogenes 10403S, washed, and chased for 1 to 2.5 h. Cells were fixed and permeabilized, and three-color staining for total bacteria, LAMP2, and actin was performed. The three-color confocal images, depicting a 0.5-μm optical section through a representative field of DC infected for 3 h, were split, and the LAMP2 and actin images were converted to grayscale. LAMP2 and actin images overlaid with bacteria (green) are also shown. Cumulative results indicating the percentage of bacteria colocalized with LAMP2 (mean ± standard deviation) from seven (DC) and six experiments (BMM) are shown. Significance was determined by unpaired Student's t test.

FIG. 2.

Differential growth of wild-type (10403S) and LLO G486D mutants in DC and BMM. (a) DC and BMM were infected with 10403S (open symbols) or the LLO G486D mutant (closed symbols), and intracellular growth was measured for 12 h as described in Materials and Methods. Cumulative results from multiple experiments are shown; points represent the means ± standard deviations from 9 to 12 coverslips per time point for DC (squares) and 6 to 9 coverslips per time point for BMM (circles). To normalize for the number of CFU at 2 h, the G486D mutant was added at a 3-fold-higher MOI than that for wild-type bacteria for both DC and BMM. (b) Additional coverslips were processed for fluorescence microscopy at 1.5 to 3.0 h postinfection, and individual bacteria were scored as cytosolic or noncytosolic based on staining for total bacteria and for actin. The percentage of cytosolic bacteria (phalloidin⁺ bacteria/total bacteria) and the fold decrease for the mutant compared to the level for the WT is shown for each cell type. The results are from three (BMM) or seven (DC) independent experiments. The total numbers of bacteria analyzed were the following: for DC, n = 640 (WT) and 741 (G486D); for BMM, n = 1,109 (WT) and 551 (G486D).

FIG. 3.

L. monocytogenes is retained in an MHC-II-rich DC compartment. (a) Uninfected DC were fixed, permeabilized, and stained for MHC-II (red) and the MHC-II accessory molecule H2-DM (green). Two distinct staining patterns are shown, that of immature DC (day 7 adherent cells) and that of mature DC (day 7 nonadherent cells immobilized on poly-l-lysine-coated coverslips). In the former, which is representative of DC used in the current studies, MHC-II and H2-DM colocalize in intracellular vesicles. (b) DC were infected with the LLO G486D strain for a total of 1 to 3 h. Cells were stained for total L. monocytogenes and H2-DM and scored for the colocalization of bacteria with H2-DM by confocal microscopy. The top panel shows a representative z-stacked image (1-μm optical sections, 8 μm thick) with bacteria colocalized (arrows) and not colocalized (arrow heads) with H2-DM. The bottom panel depicts four optical sections through the same cell. The percentage of bacteria colocalized with H2-DM (n = 277; three independent experiments) and LAMP2 (n = 177; two independent experiments) is shown in the graph. DIC, differential interference contrast. (c) Optical section (0.5 μm) through a DC infected with WT L. monocytogenes for 3 h. The first two panels show the colocalization of H2-DM (green) with MHC-II (red) in intracellular vesicles, and the second two panels show the colocalization of L. monocytogenes (magenta) with both markers (yellow).

FIG. 4.

L. monocytogenes-containing DC compartment acidifies by 1 h. (a) DC were pretreated for 30 min with BafA₁ (0.5 μM), infected for 30 min with CFSE-labeled LLO G486D, washed, and chased in the presence of BafA₁ for an additional 30 min. Intracellular CFSE fluorescence in the presence and absence of BafA₁ was measured by flow cytometry. The MFI of the CFSE⁺ gate is shown. Histograms represent fluorescence intensity in the absence (solid line) and presence (dotted line) of BafA₁. The shaded histogram represents cells infected with unlabeled bacteria. Data represent one of three experiments with similar results. (b) DC were infected with unlabeled LLO G486D as described above and chased in the presence of LTR (1 μM) (red). Cells were fixed, permeabilized, and stained for total L. monocytogenes using an Alexfluor 488-conjugated secondary antibody (green). Bacteria that colocalized with LTR were scored by confocal microscopy. The single-channel images (converted to grayscale) depict a 0.5-μm optical section through a representative field. The arrow indicates the one bacterium in the field that does not colocalize with LTR. A total of 259 bacteria were scored in three independent experiments (n = 84, 114, and 61), and the results are expressed as percent bacteria localized to LTR⁺ vesicles (% LTR⁺).

FIG. 5.

Quantitative analysis of DC phagosomal pH in the first hour of infection. (a) DC were infected with dual-labeled LLO G486D (MOI, 10) for 10 min, washed, and chased for 35 min (total infection time, 45 min). Cells were harvested and exposed to media of defined pH in the presence (open circles) or absence (closed circles) of ionophores as described in Materials and Methods. CFSE and AF647 intracellular fluorescence were measured by flow cytometry after gating on the predominant infected cell population. The ratio of CFSE/AF647 mean fluorescence intensity (MFI) was plotted as a function of pH. (b) Cumulative results for DC (10 independent determinations) and BMM (infected as per DC; 6 independent determinations). The line denotes the mean pH for each group of experiments. Significance was determined by unpaired Student's t test.

FIG. 6.

Controlled growth of L. monocytogenes in DC preserves cellular integrity and leads to maturation at late stages of infection. (a) Cells were infected with wild-type L. monocytogenes per the growth experiment described for Fig. 2a, and supernatants were collected at the indicated time points for the measurement of LDH activity. Points represent the means ± standard deviations of triplicate samples, and data from one of two experiments with similar results is shown. (b) DC and BMM from the experiment depicted in panel a were fixed and stained at 24 h with Diff-Quik, illustrating cytotoxic effects and high bacterial load in BMM specifically. (c) L. monocytogenes infection (MOI, 0.3; 10 μg/ml gentamicin was present for the duration of the experiment) induced DC to mature by 24 h. Infected cells were harvested at 1.5 and 24 h after infection and analyzed by flow cytometry for CD86 expression. The gray-shaded histogram indicates the uninfected DC control. L. monocytogenes infection (red) and CD86 expression (green) also were evaluated at the same time points by immunofluorescence microscopy.