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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-β. 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-β following infection of human dendritic cells. Phagocytosis of SchuS4 and endosomal acidification were required for induction of IFN-β. 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-β. Surprisingly, unlike previous reports, induction of IFN-β 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-β 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.

An inadequate innate immune response appears to contribute to the virulence of Francisella tularensis following pulmonary infection. Studies in mice suggest that this poor response results from suppression of proinflammatory cytokine production early during infection, but the mechanisms involved are not understood. PI3K is known to regulate proinflammatory cytokine expression, but its exact role (positive versus negative) is controversial. We sought to clarify the role of PI3K in regulating proinflammatory signaling and cytokine production during infection with F. tularensis live vaccine strain (LVS). In this study, we demonstrate that the induction of TNF and IL-6 expression by LVS in mouse bone marrow-derived macrophages was markedly enhanced when PI3K activity was inhibited by either of the well-known chemical inhibitors, wortmannin or LY294002. The enhanced cytokine expression was accompanied by enhanced activation of p38 MAPK and ERK1/2, both of which were critical for LVS-induced expression of TNF and IL-6. LVS-induced MAPK activation and cytokine production were TLR2- and MyD88- dependent. PI3K/Akt activation was MyD88-dependent, but was surprisingly TLR2-independent. LVS infection also rapidly induced MAPK phosphatase-1 (MKP-1) expression; PI3K and TLR2 signaling were required. Peak levels of MKP-1 correlated closely with the decline in p38 MAPK and ERK1/2 phosphorylation. These data suggest that infection by LVS restrains the TLR2-triggered proinflammatory response via parallel activation of PI3K, leading to enhanced MKP-1 expression, accelerated deactivation of MAPKs, and suppression of proinflammatory cytokine production. This TLR2-independent inhibitory pathway may be an important mechanism by which Francisella suppresses the host's innate immune response.

N-Formylated (N-f-met) peptides derived from proteins of the intracellular bacterium Listeria monocytogenes generate a protective, H2-M3-restricted CD8 T cell response in C57BL/6 mice. N-f-met peptide-specific CTL were generated in vitro when mice previously immunized with gp96 isolated from donor mice infected with L. monocytogenes were stimulated with these peptides. No significant peptide-specific CTL activity was observed in mice immunized with gp96 from uninfected animals. Masses corresponding to one N-f-met peptide were found by matrix-assisted laser desorption/ionization-mass spectrometry on gp96 isolated from C57BL/6 mice infected with L. monocytogenes, but not on gp96 from noninfected mice. Therefore, bacterial N-f-met peptides from intracellular bacteria can bind to gp96 in the infected host, and gp96 loaded with these peptides can generate N-f-met-peptide-specific CTL. We assume a unique role of gp96 in Ag processing through the H2-M3 pathway.

Anti-pneumococcal antibody-producing lymphocytes from a patient who was immunized with Pneumovax (a polysaccharide mixture from 14 different pneumococci types) were selected and immortalized with Epstein Barr virus (EBV). The cells of the emerging cloned line were shown on a single cell level assay to secrete an IgM, kappa anti-pneumococci antibody. This human monoclonal antibody bound specifically to polysaccharides of pneumococci type 8, but did not bind to any of the other 13 pneumococci types that were present in the Pneumovax mixture. In competition experiments, inhibition of binding occurred with polysaccharide type 8 and Pneumovax only. This diploid cell line has actively secreted the specific antibody (2.8 micrograms/10(6) cells/ml) now for more than 9 mo. In patients, increased anti-pneumococcal antibody titers generated by active immunization with Pneumovax improves resistance to the corresponding bacterial infection. Production in vitro of human monoclonal antibodies directed toward bacterial and viral antigens enables a new possible approach for passive immune protection.