Fluorescent microscopy.

<p>The representative picture was chosen to show the difference in the numbers of A20 cells infected with <b>(A)</b> unopsonized <i>F</i>. <i>tularensis</i> LVS/GFP bacteria, <b>(B)</b><i>F</i>. <i>tularensis</i> LVS/GFP opsonized with murine fresh serum, and <b>(C)</b> bacteria opsonized with immune sera. A20 cells in total volume 0.5 mL (1 x 10⁶ cells per well) were infected with <i>F</i>. <i>tularensis</i> 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.</p

Blocking of FcγR.

<p>Peritoneal cells were incubated with the antibody against CD16/32 (dFcRg). Thereafter, cells were infected with <i>F</i>. <i>tularensis</i> LVS/GFP (GFP), <i>F</i>. <i>tularensis</i> LVS/GFP opsonized with antibodies (GFP+Ab), and <i>F</i>. <i>tularensis</i> 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 <i>t</i>-test was used to test for significant differences between GFP and GFP+Ab and between GFP+Ab and dFcRg+GFP+Ab+C (*** <i>P</i> < 0.001, ** <i>P</i> < 0.01). Results shown from one experiment are representative of three independent experiments</p

Entry of <i>Francisella tularensis</i> into Murine B Cells: The Role of B Cell Receptors and Complement Receptors

<div><p><i>Francisella tularensis</i>, 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 <i>F</i>. <i>tularensis</i> into B cells within <i>in vivo</i> and <i>in vitro</i> infection models. Here, we present data showing that <i>Francisella tularensis</i> subsp. <i>holarctica</i> strain LVS significantly infects individual subsets of murine peritoneal B cells early after infection. Depending on a given B cell subset, uptake of <i>Francisella</i> into B cells is mediated by B cell receptors (BCRs) with or without complement receptor CR1/2. However, <i>F</i>. <i>tularensis</i> strain FSC200 Δ<i>iglC</i> and Δ<i>ftdsbA</i> deletion mutants are defective in the ability to enter B cells. Once internalized into B cells, <i>F</i>. <i>tularensis</i> 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 <i>F</i>. <i>tularensis</i> 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 <i>F</i>. <i>tularensis</i>.</p></div

Deletion mutant <i>F</i>. <i>tularensis</i> strains failed to enter the A20 cells.

<p>A20 cells were infected with wild type <i>F</i>. <i>tularensis</i> FSC200 (FSC200), with deletion mutant <i>F</i>. <i>tularensis</i> FSC200 Δ<i>ftdsbA</i> (FSC200 ΔftdsbA), and with deletion mutant <i>F</i>. <i>tularensis</i> FSC200 Δ<i>iglC</i> (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-<i>F</i>. <i>tularensis sera</i> and goat anti-rabbit secondary antibody conjugated with Alexa Fluor 488 to visualize <i>F</i>. <i>tularensis</i>. Error bars indicate SD around the means of samples processed in triplicate. Two-tailed <i>t</i>-test was used to test for significant differences between FSC200 and FSC200 ΔftdsbA and FSC200 ΔiglC. (*** <i>P</i> < 0.001). Results shown from one experiment are representative of three independent experiments.</p

Disturbance of lipid rafts.

<p>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 <b>(A)</b><i>F</i>. <i>tularensis</i> LVS/GFP or <b>(B)</b> opsonized <i>F</i>. <i>tularensis</i> 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 <i>t</i>-test was used to test for significant differences between untreated B cells and cyclodextrin- or filipin-treated cells (*** <i>P</i> < 0.001). Results shown from one experiment are representative of three independent experiments.</p

<i>F</i>. <i>tularensis</i> infecting subsets of B cells <i>in vitro</i>.

<p>Subsets of B cells were infected for 3 h with unopsonized <i>F</i>. <i>tularensis</i> LVS/GFP (GFP), <i>F</i>. <i>tularensis</i> 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 <i>t</i>-test was used to test for significant differences between GFP and GFP+C and GFP+Ab (*** <i>P</i> < 0.001, ** <i>P</i> < 0.01, * <i>P</i> < 0.05). Results shown from one experiment are representative of three independent experiments.</p

Blocking of BCR receptor.

<p>Peritoneal cells were incubated with the blocking antibody anti-IgM (BCR). Thereafter, the cells were infected for 3 h with <b>(A)</b> unopsonized <i>F</i>. <i>tularensis</i> LVS/GFP (GFP), <b>(B)</b><i>F</i>. <i>tularensis</i> LVS/GFP opsonized with complement (GFP+C), and <b>(C)</b><i>F</i>. <i>tularensis</i> 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 <i>t</i>-test was used to test for significant differences between untreated cells and cells with blocked BCR (*** <i>P</i> < 0.001, ** <i>P</i> < 0.01). Results shown from one experiment are representative of three independent experiments.</p

Intracellular trafficking.

<p>A20 mouse B cell line (1 x 10⁶ per well in total volume 0.5 mL) was infected with <i>F</i>. <i>tularensis</i> 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 <i>F</i>. <i>tularensis</i> LVS by fluorescent microscopy. Error bars indicate SD around the means of samples obtained from three independent experiments.</p

Blocking of CRs.

<p>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 <i>F</i>. <i>tularensis</i> LVS/GFP (GFP) or <i>F</i>. <i>tularensis</i> 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 <i>t</i>-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 (*** <i>P</i> < 0.001). Results shown from one experiment are representative of three independent experiments.</p