Francisella and Antibodies

Immune responses to intracellular pathogens depend largely upon the activation of T helper type 1-dependent mechanisms. The contribution of B cells to establishing protective immunity has long been underestimated. Francisella tularensis, including a number of subspecies, provides a suitable model for the study of immune responses against intracellular bacterial pathogens. We previously demonstrated that Francisella infects B cells and activates B-cell subtypes to produce a number of cytokines and express the activation markers. Recently, we documented the early production of natural antibodies as a consequence of Francisella infection in mice. Here, we summarize current knowledge on the innate and acquired humoral immune responses initiated by Francisella infection and their relationships with the immune defense systems

Innate Immune Recognition: Implications for the Interaction of Francisella tularensis with the Host Immune System

The intracellular bacterial pathogen Francisella tularensis causes serious infectious disease in humans and animals. Moreover, F. tularensis, a highly infectious pathogen, poses a major concern for the public as a bacterium classified under Category A of bioterrorism agents. Unfortunately, research has so far failed to develop effective vaccines, due in part to the fact that the pathogenesis of intracellular bacteria is not fully understood and in part to gaps in our understanding of innate immune recognition processes leading to the induction of adaptive immune response. Recent evidence supports the concept that immune response to external stimuli in the form of bacteria is guided by the primary interaction of the bacterium with the host cell. Based on data from different Francisella models, we present here the basic paradigms of the emerging innate immune recognition concept. According to this concept, the type of cell and its receptor(s) that initially interact with the target constitute the first signaling window; the signals produced in the course of primary interaction of the target with a reacting cell act in a paracrine manner; and the innate immune recognition process as a whole consists in a series of signaling windows modulating adaptive immune response. Finally, the host, in the strict sense, is the interacting cell

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

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

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

<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

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

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

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