Host-Adaptation of Francisella tularensis Alters the Bacterium's Surface-Carbohydrates to Hinder Effectors of Innate and Adaptive Immunity

The gram-negative bacterium Francisella tularensis survives in arthropods, fresh water amoeba, and mammals with both intracellular and extracellular phases and could reasonably be expected to express distinct phenotypes in these environments. The presence of a capsule on this bacterium has been controversial with some groups finding such a structure while other groups report that no capsule could be identified. Previously we reported in vitro culture conditions for this bacterium which, in contrast to typical methods, yielded a bacterial phenotype that mimics that of the bacterium's mammalian, extracellular phase.SDS-PAGE and carbohydrate analysis of differentially-cultivated F. tularensis LVS revealed that bacteria displaying the host-adapted phenotype produce both longer polymers of LPS O-antigen (OAg) and additional HMW carbohydrates/glycoproteins that are reduced/absent in non-host-adapted bacteria. Analysis of wildtype and OAg-mutant bacteria indicated that the induced changes in surface carbohydrates involved both OAg and non-OAg species. To assess the impact of these HMW carbohydrates on the access of outer membrane constituents to antibody we used differentially-cultivated bacteria in vitro to immunoprecipitate antibodies directed against outer membrane moieties. We observed that the surface-carbohydrates induced during host-adaptation shield many outer membrane antigens from binding by antibody. Similar assays with normal mouse serum indicate that the induced HMW carbohydrates also impede complement deposition. Using an in vitro macrophage infection assay, we find that the bacterial HMW carbohydrate impedes TLR2-dependent, pro-inflammatory cytokine production by macrophages. Lastly we show that upon host-adaptation, the human-virulent strain, F. tularensis SchuS4 also induces capsule production with the effect of reducing macrophage-activation and accelerating tularemia pathogenesis in mice.F. tularensis undergoes host-adaptation which includes production of multiple capsular materials. These capsules impede recognition of bacterial outer membrane constituents by antibody, complement, and Toll-Like Receptor 2. These changes in the host-pathogen interface have profound implications for pathogenesis and vaccine development

Targeting of a Fixed Bacterial Immunogen to Fc Receptors Reverses the Anti-Inflammatory Properties of the Gram-Negative Bacterium, Francisella tularensis, during the Early Stages of Infection.

Production of pro-inflammatory cytokines by innate immune cells at the early stages of bacterial infection is important for host protection against the pathogen. Many intracellular bacteria, including Francisella tularensis, the agent of tularemia, utilize the anti-inflammatory cytokine IL-10, to evade the host immune response. It is well established that IL-10 has the ability to inhibit robust antigen presentation by dendritic cells and macrophages, thus suppressing the generation of protective immunity. The pathogenesis of F. tularensis is not fully understood, and research has failed to develop an effective vaccine to this date. In the current study, we hypothesized that F. tularensis polarizes antigen presenting cells during the early stages of infection towards an anti-inflammatory status characterized by increased synthesis of IL-10 and decreased production of IL-12p70 and TNF-α in an IFN-ɣ-dependent fashion. In addition, F. tularensis drives an alternative activation of alveolar macrophages within the first 48 hours post-infection, thus allowing the bacterium to avoid protective immunity. Furthermore, we demonstrate that targeting inactivated F. tularensis (iFt) to Fcγ receptors (FcɣRs) via intranasal immunization with mAb-iFt complexes, a proven vaccine strategy in our laboratories, reverses the anti-inflammatory effects of the bacterium on macrophages by down-regulating production of IL-10. More specifically, we observed that targeting of iFt to FcγRs enhances the classical activation of macrophages not only within the respiratory mucosa, but also systemically, at the early stages of infection. These results provide important insight for further understanding the protective immune mechanisms generated when targeting immunogens to Fc receptors

Anti-inflammatory effect of <i>F</i>. <i>tularensis</i> LPS on mouse PECs is IFN-γ and IL-10 dependent.

<p>PECs from naïve and IL-10 genetically deficient C57BL/6 mice were obtained and resuspended in cell culture media. PECs were cultured in a 96-well plate at 2 x 10⁵ cells/well with either <i>Ft</i>-LPS or <i>E</i>. <i>coli</i>-LPS at 1 ng/mL in the presence or absence of recombinant IFN-γ at 100 U/ml. Cells cultured with PBS were used as a control. The cytokine production was measured using BD Biosciences Cytometric Bead Array (CBA) following vendor instructions. Results are representative of three independent experiments. (*) P-value < 0.1, (**) P-value < 0.05, bars represent SD.</p

FcγR targeting drives polarization of mouse macrophages towards the AM1 phenotype at the early stages of LVS infection.

<p>Lungs of immunized mice were harvested 24, 48 and 96 hours post-infection. For cell surface marker staining, cells were fluorescently labeled with antibodies against CD11b, F4/80, MHC class II, B7.1, B7.2, CCR7, or their corresponding isotype controls were added. Cells were then analyzed by flow cytometry on an LSRII flow cytometer (BD Biosciences). Results are representative of three independent experiments. (*) P-value < 0.1; (**) P-value < 0.05; bars represent the SD.</p

<i>tularensis</i> LPS contributes to the anti-inflammatory properties of <i>F</i>. <i>tularensis</i> LVS during the early stages of infection.

<p><b><i>F</i>.</b> PECs from C57BL/6 mice were obtained and cultured in a 96-well plate at 2 x 10⁵ cells/well in the presence or absence of either <i>Ft</i>-LPS or <i>E</i>. <i>coli</i>-LPS at 1 ng/mL, 10 ng/mL and 20 ng/ml. Cells incubated with PBS were used as a control. Levels of IL-12p70, TNF-α and IL-10 were measured using BD Biosciences Cytometric Bead Array (CBA) following vendor instructions. Results are representative of three independent experiments. (*) P-value < 0.1; (**) P-value < 0.05; bars represent SD.</p