Virulent Francisella tularensis destabilize host mRNA to rapidly suppress inflammation.

Highly virulent bacterial pathogens have evolved rapid means to suppress host inflammatory responses by unknown mechanisms. Here, we use virulent Francisella tularensis, the cause of lethal tularemia in humans, as a model to elucidate these mechanisms. We show that following infection of murine macrophages F. tularensis rapidly and selectively destabilizes mRNA containing adenylate-uridylate-rich elements that encode for cytokines and chemokines important in controlling bacterial infection. Degradation of host mRNA encoding interleukin (IL)-1ß, IL-6 and CXCL1 did not require viable bacteria or de novo protein synthesis, but did require escape of intracellular organisms from endocytic vesicles into the host cytosol. The specific targeting of host mRNA encoding inflammatory cytokines and chemokines for decay by a bacterial pathogen has not been previously reported. Thus, our findings represent a novel strategy by which a highly virulent pathogen modulates host inflammatory responses critical to the evasion of innate immunity.

IFN-ß mediates suppression of IL-12p40 in human dendritic cells following infection with virulent Francisella tularensis.

Active suppression of inflammation is a strategy used by many viral and bacterial pathogens, including virulent strains of the bacterium Francisella tularensis, to enable colonization and infection in susceptible hosts. In this study, we demonstrated that virulent F. tularensis strain SchuS4 selectively inhibits production of IL-12p40 in primary human cells via induction of IFN-ß. In contrast to the attenuated live vaccine strain, infection of human dendritic cells with virulent SchuS4 failed to induce production of many cytokines associated with inflammation (e.g., TNF-α and IL-12p40). Furthermore, SchuS4 actively suppressed secretion of these cytokines. Assessment of changes in the expression of host genes associated with suppression of inflammatory responses revealed that SchuS4, but not live vaccine strain, induced IFN-ß following infection of human dendritic cells. Phagocytosis of SchuS4 and endosomal acidification were required for induction of IFN-ß. Further, using a defined mutant of SchuS4, we demonstrated that the presence of bacteria in the cytosol was required, but not sufficient, for induction of IFN-ß. Surprisingly, unlike previous reports, induction of IFN-ß by F. tularensis was not required for activation of the inflammasome, was not associated with exacerbation of inflammatory responses, and did not control SchuS4 replication when added exogenously. Rather, IFN-ß selectively suppressed the ability of SchuS4-infected dendritic cells to produce IL-12p40. Together, these data demonstrated a novel mechanism by which virulent bacteria, in contrast to attenuated strains, modulate human cells to cause disease.

The presence of CD14 overcomes evasion of innate immune responses by virulent Francisella tularensis in human dendritic cells in vitro and pulmonary cells in vivo.

Francisella tularensis is a Gram-negative bacterium that causes acute, lethal disease following inhalation. We have previously shown that viable F. tularensis fails to stimulate secretion of proinflammatory cytokines following infection of human dendritic cells (hDC) in vitro and pulmonary cells in vivo. Here we demonstrate that the presence of the CD14 receptor is critical for detection of virulent F. tularensis strain SchuS4 by dendritic cells, monocytes, and pulmonary cells. Addition of soluble CD14 (sCD14) to hDC restored cytokine production following infection with strain SchuS4. In contrast, addition of anti-CD14 to monocyte cultures inhibited the ability of these cells to respond to strain SchuS4. Addition of CD14 or blocking CD14 following SchuS4 infection in dendritic cells and monocytes, respectively, was not due to alterations in phagocytosis or replication of the bacterium in these cells. Administration of sCD14 in vivo also restored cytokine production following infection with strain SchuS4, as assessed by increased concentrations of tumor necrosis factor alpha (TNF-alpha), interleukin-1beta (IL-1beta), IL-12p70, and IL-6 in the lungs of mice receiving sCD14 compared to mock-treated controls. In contrast to homogenous cultures of monocytes or dendritic cells infected in vitro, mice treated with sCD14 in vivo also exhibited controlled bacterial replication and dissemination compared to mock-treated controls. Interestingly, animals that lacked CD14 were not more susceptible or resistant to pulmonary infection with SchuS4. Together, these data support the hypothesis that the absence or low abundance of CD14 on hDC and in the lung contributes to evasion of innate immunity by virulent F. tularensis. However, CD14 is not required for development of inflammation during the last 24 to 48 h of SchuS4 infection. Thus, the presence of this receptor may aid in control of virulent F. tularensis infections at early, but not late, stages of infection.

Direct and indirect impairment of human dendritic cell function by virulent Francisella tularensis Schu S4.

The gram-negative, facultative intracellular bacterium Francisella tularensis causes acute, lethal pneumonic disease following infection with only 10 CFU. The mechanisms used by the bacterium to accomplish this in humans are unknown. Here, we demonstrate that virulent, type A F. tularensis strain Schu S4 efficiently infects and replicates in human myeloid dendritic cells (DCs). Despite exponential replication over time, Schu S4 failed to stimulate transforming growth factor beta, interleukin-10 (IL-10), IL-6, IL-1beta, IL-12, tumor necrosis factor alpha, alpha interferon (IFN-alpha), and IFN-beta throughout the course of infection. Schu S4 also suppressed the ability of directly infected DCs to respond to different Toll-like receptor agonists. Furthermore, we also observed functional inhibition of uninfected bystander cells. This inhibition was mediated, in part, by a heat-stable bacterial component. Lipopolysaccharide (LPS) from Schu S4 was present in Schu S4-conditioned medium. However, Schu S4 LPS was weakly inflammatory and failed to induce suppression of DCs at concentrations below 10 microg/ml, and depletion of Schu S4 LPS did not significantly alleviate the inhibitory effect of Schu S4-conditioned medium in uninfected human DCs. Together, these data show that type A F. tularensis interferes with the ability of a central cell type of the immune system, DCs, to alert the host of infection both intra- and extracellularly. This suggests that immune dysregulation by F. tularensis operates on a broader and more comprehensive scale than previously appreciated.

Prolonged costimulation is required for naive T cell activation.

Costimulation by members of the B7 family of molecules is critical for the activation of naive CD4+ T cells. While prolonged TCR signaling is necessary for T cell activation, the duration of costimulatory signals required has not been established. In this study, murine bone marrow-derived dendritic cells (DC) and naïve CD4+ T cells were used to determine the temporal costimulatory requirements for naive T cell activation. By blocking CD80/CD86 costimulation at various time points during DC-T cell interaction and using the CFSE technique to assess the dynamics of T cell proliferation, we found that prolonged costimulation was required for naive T cells to enter and progress through the cell cycle over a wide range of peptide concentrations. Prolonged costimulation was also important for IL-2 production and CD25/CD69 expression by naive T cells. Video microscopy demonstrated that DC and naive T cells formed stable conjugates that persisted for more than 6 h. Thus, persistent CD80/CD86 signaling during prolonged interactions with DC allows naive T cells to enter the cell cycle and programs the daughter cells to undergo subsequent divisions.