Entry of Francisella tularensis into Murine B Cells: The Role of B Cell Receptors and Complement Receptors

Abstract

Francisella tularensis, the etiological agent of tularemia, is an intracellular pathogen that dominantly infects and proliferates inside phagocytic cells but can be seen also in non-phagocytic cells, including B cells. Although protective immunity is known to be almost exclusively associated with the type 1 pathway of cellular immunity, a significant role of B cells in immune responses already has been demonstrated. Whether their role is associated with antibody-dependent or antibody-independent B cell functions is not yet fully understood. The character of early events during B cell-pathogen interaction may determine the type of B cell response regulating the induction of adaptive immunity. We used fluorescence microscopy and flow cytometry to identify the basic requirements for the entry of F. tularensis into B cells within in vivo and in vitro infection models. Here, we present data showing that Francisella tularensis subsp. holarctica strain LVS significantly infects individual subsets of murine peritoneal B cells early after infection. Depending on a given B cell subset, uptake of Francisella into B cells is mediated by B cell receptors (BCRs) with or without complement receptor CR1/2. However, F. tularensis strain FSC200 ΔiglC and ΔftdsbA deletion mutants are defective in the ability to enter B cells. Once internalized into B cells, F. tularensis LVS intracellular trafficking occurs along the endosomal pathway, albeit without significant multiplication. The results strongly suggest that BCRs alone within the B-1a subset can ensure the internalization process while the BCRs on B-1b and B-2 cells need co-signaling from the co receptor containing CR1/2 to initiate F. tularensis engulfment. In this case, fluidity of the surface cell membrane is a prerequisite for the bacteria's internalization. The results substantially underline the functional heterogeneity of B cell subsets in relation to F. tularensis.

Fig 1. Fluorescent microscopy.

The representative picture was chosen to show the difference in the numbers of A20 cells infected with (A) unopsonized F. tularensis LVS/GFP bacteria, (B) F. tularensis LVS/GFP opsonized with murine fresh serum, and (C) bacteria opsonized with immune sera. A20 cells in total volume 0.5 mL (1 x 10⁶ cells per well) were infected with F. tularensis LVS/GFP at MOI 500 for 3 h. The cell nuclei were stained with DAPI. Note: The number of infected cells was counted using flow cytometry.

Fig 2. F. tularensis infecting A20 cells in vitro.

Cells from the A20 mouse B cell line (5 x 10⁵ per well in total volume 0.5 mL) were infected by F. tularensis LVS/GFP (GFP) opsonized with fresh uninactivated serum (GFP+C) from naïve mice and bacteria opsonized with heat-inactivated immune sera (GFP+Ab) at MOI 500. The cultures were then incubated at 36.8°C and 5% CO2 atmosphere for 3 h. Proportion of infected cells was determined by flow cytometry. Error bars indicate SD around the means of samples processed in triplicate. Two-tailed t-tests were used to test for significant differencess between GFP and GFP+C and GFP+iC and GFP+Ab (*** P < 0.001, ** P < 0.01). Results shown from one experiment are representative of three independent experiments.

Fig 3. F. tularensis infecting subsets of B cells in vitro.

Subsets of B cells were infected for 3 h with unopsonized F. tularensis LVS/GFP (GFP), F. tularensis LVS/GFP opsonized with fresh un-inactivated serum (GFP+C) from naïve mice, and bacteria opsonized with heat-inactivated immune sera (GFP+Ab). The proportions of infected CD19⁺ cells from all measured cells and of infected B-1a, B-1b, and B-2 cells from CD19⁺ cells were measured by flow cytometry. Error bars indicate SD around the means of samples processed in triplicate. Two-tailed t-test was used to test for significant differences between GFP and GFP+C and GFP+Ab (*** P < 0.001, ** P < 0.01, * P < 0.05). Results shown from one experiment are representative of three independent experiments.

Fig 4. Deletion mutant F. tularensis strains failed to enter the A20 cells.

A20 cells were infected with wild type F. tularensis FSC200 (FSC200), with deletion mutant F. tularensis FSC200 ΔftdsbA (FSC200 ΔftdsbA), and with deletion mutant F. tularensis FSC200 ΔiglC (FSC200 ΔiglC), respectively, at MOI 500. The infected cells were determined by florescent microscopy. The cells were stained with DAPI to visualize nuclei and with rabbit anti-F. tularensis sera and goat anti-rabbit secondary antibody conjugated with Alexa Fluor 488 to visualize F. tularensis. Error bars indicate SD around the means of samples processed in triplicate. Two-tailed t-test was used to test for significant differences between FSC200 and FSC200 ΔftdsbA and FSC200 ΔiglC. (*** P < 0.001). Results shown from one experiment are representative of three independent experiments.

Fig 5. Blocking of BCR receptor.

Peritoneal cells were incubated with the blocking antibody anti-IgM (BCR). Thereafter, the cells were infected for 3 h with (A) unopsonized F. tularensis LVS/GFP (GFP), (B) F. tularensis LVS/GFP opsonized with complement (GFP+C), and (C) F. tularensis LVS/GFP opsonized with antibodies (GFP+Ab). Entry into CD19⁺ cells (expressed as percentage of infected CD19⁺ from all CD19⁺ cells) and individual B cell subsets (expressed as percentage of infected B-1a from all B-1a cells, infected B-1b from all B-1b cells, and infected B-2 from all B-2 cells) was detected by flow cytometry. Error bars indicate SD around the means of samples processed in triplicate. Two-tailed t-test was used to test for significant differences between untreated cells and cells with blocked BCR (*** P < 0.001, ** P < 0.01). Results shown from one experiment are representative of three independent experiments.

Fig 6. Blocking of CRs.

Peritoneal cells were incubated with the antibodies against CD21/CD35 (CR1/2), CD11b (CR3), and CD11c (CR4). After blocking, the cells were infected for 3 h with either F. tularensis LVS/GFP (GFP) or F. tularensis LVS/GFP opsonized with complement (GFP+C) and the proportions of infected CD19⁺ cells were detected by flow cytometry. Error bars indicate SD around the means of samples processed in triplicate. Two-tailed t-test was used to test for significant differences against GFP. The significance of CR blocking effect was calculated between GFP+C and all groups with blocked CRs (*** P < 0.001). Results shown from one experiment are representative of three independent experiments.

Fig 7. Blocking of FcγR.

Peritoneal cells were incubated with the antibody against CD16/32 (dFcRg). Thereafter, cells were infected with F. tularensis LVS/GFP (GFP), F. tularensis LVS/GFP opsonized with antibodies (GFP+Ab), and F. tularensis LVS/GFP opsonized with murine fresh serum and antibodies (dFcRg+GFP+Ab+C) at MOI 500. Entry into all B cells (CD19⁺) and individual B cell subsets was detected 3 h after infection by flow cytometry. Error bars indicate SD around the means of samples processed in triplicate. Two-tailed t-test was used to test for significant differences between GFP and GFP+Ab and between GFP+Ab and dFcRg+GFP+Ab+C (*** P < 0.001, ** P < 0.01). Results shown from one experiment are representative of three independent experiments

Fig 8. Disturbance of lipid rafts.

For disturbing lipid rafts, the cholesterol-binding agent filipin or methyl-beta cyclodextrin (Cyclodex) was used. The peritoneal B cells were pretreated with 10 μg/mL filipin or 10 mM cyclodextrin and consequently infected with (A) F. tularensis LVS/GFP or (B) opsonized F. tularensis LVS/GFP with complement. Entry into all B cells (CD19⁺) and individual B cell subsets was detected by flow cytometry. Error bars indicate SD around the means of samples processed in triplicate. Two-tailed t-test was used to test for significant differences between untreated B cells and cyclodextrin- or filipin-treated cells (*** P < 0.001). Results shown from one experiment are representative of three independent experiments.

Fig 9. Intracellular trafficking.

A20 mouse B cell line (1 x 10⁶ per well in total volume 0.5 mL) was infected with F. tularensis LVS (MOI 500). Cells were infected for 5, 15 and 30 min, as well as 1 and 2 h. To identify intracellular trafficking, endosomal/lysosomal membrane markers EEA1, LAMP-1, and Cathepsin D were used for determining colocalization of these markers with F. tularensis LVS by fluorescent microscopy. Error bars indicate SD around the means of samples obtained from three independent experiments.