Protection Afforded by Fluoroquinolones in Animal Models of Respiratory Infections with Bacillus anthracis, Yersinia pestis, and Francisella tularensis.

Successful treatment of inhalation anthrax, pneumonic plague and tularemia can be achieved with fluoroquinolone antibiotics, such as ciprofloxacin and levofloxacin, and initiation of treatment is most effective when administered as soon as possible following exposure. Bacillus anthracis Ames, Yersinia pestis CO92, and Francisella tularensis SCHU S4 have equivalent susceptibility in vitro to ciprofloxacin and levofloxacin (minimal inhibitory concentration is 0.03 µg/ml); however, limited information is available regarding in vivo susceptibility of these infectious agents to the fluoroquinolone antibiotics in small animal models. Mice, guinea pig, and rabbit models have been developed to evaluate the protective efficacy of antibiotic therapy against these life-threatening infections. Our results indicated that doses of ciprofloxacin and levofloxacin required to protect mice against inhalation anthrax were approximately 18-fold higher than the doses of levofloxacin required to protect against pneumonic plague and tularemia. Further, the critical period following aerosol exposure of mice to either B. anthracis spores or Y. pestis was 24 h, while mice challenged with F. tularensis could be effectively protected when treatment was delayed for as long as 72 h postchallenge. In addition, it was apparent that prolonged antibiotic treatment was important in the effective treatment of inhalation anthrax in mice, but short-term treatment of mice with pneumonic plague or tularemia infections were usually successful. These results provide effective antibiotic dosages in mice, guinea pigs, and rabbits and lay the foundation for the development and evaluation of combinational treatment modalities.

Lack of demonstrable memory T cell responses in humans who have spontaneously recovered from leptospirosis in the Peruvian Amazon.

BACKGROUND. We tested the hypothesis that patients who have recovered from leptospirosis have peripheral blood memory T cells that are specific for Leptospira or Leptospira protein antigens. METHODS. Peripheral blood mononuclear cells (PBMCs) were obtained from patients who had recovered from leptospirosis, as well as from control individuals. PBMCs were assessed for in vitro proliferation, phenotyping, and cytokine production after stimulation with different strains of Leptospira, recombinant LipL32, or overlapping synthetic peptides of different outer membrane proteins. RESULTS. PBMCs from both control subjects and patients produced significant proliferative responses to all Leptospira strains. Proliferation from control PBMCs was significantly greater than responses produced by patient PBMCs. Select strains of Leptospira expanded both T cell receptor (TCR) alphabeta and TCRgammadelta T cells in both control subject and patient PBMCs. Patient and control subject PBMCs produced equivalent levels of tumor necrosis factor alpha and interferon gamma, but patient PBMCs produced significantly less interleukin 10 than did control subject PBMCs after stimulation by different strains of Leptospira. PBMCs from patients failed to respond to recombinant LipL32 or to any of the Leptospira peptides. CONCLUSION. Leptospira induced significant proliferative responses, TCRgammadelta T cell expansion, and cytokine production in both control subject and patient PBMCs. Patient PBMCs failed to recognize Leptospira protein antigens. Leptospirosis does not seem to generate memory T cells that can be activated by in vitro stimulation.

Tetraacylated lipopolysaccharide of Yersinia pestis can inhibit multiple Toll-like receptor-mediated signaling pathways in human dendritic cells.

BACKGROUND: Yersinia pestis, the causative agent of plague, showed a temperature-dependent change in lipid A composition, with a reduced degree of acylation when bacteria were grown at 37 degrees C (tetraacylated) versus ambient temperature (hexaacylated). METHODS: Human monocytes and monocyte-derived dendritic cells (DCs) were exposed to Y. pestis grown at 26 degrees C or 37 degrees C, to their corresponding lipopolysaccharides (LPS-26 degrees C or LPS-37 degrees C), and to ligands of different Toll-like receptors (TLRs), such as LPS from Escherichia coli (TLR4), lipoprotein (TLR2), polyinosinic-polycytidylic acid (poly-IC) (TLR9), and their combinations. Production of cytokines was measured, along with expression of surface markers of DC maturation. RESULTS: Y. pestis grown at 37 degrees C or LPS-37 degrees C induced much lower production of cytokines (such as tumor necrosis factor alpha and interleukins 1beta, 10, and 12) by DCs than did Y. pestis grown at 26 degrees C or LPS-26 degrees C. Expression of the surface markers HLA-DR, CD86, and CD40 by DCs was also reduced in response to treatment with LPS-37 degrees C compared with LPS-26 degrees C. Pretreatment of DCs with LPS-37 degrees C inhibited subsequent stimulation with LPS-26 degrees C, control LPS from E. coli, lipoprotein, or poly-IC. CONCLUSIONS: LPS-37 degrees C can inhibit stimulation of DCs not only via TLR4 signaling but also via TLR2 and TLR3. [corrected]

Comparative antimicrobial activity of granulysin against bacterial biothreat agents.

Granulysin is a cationic protein produced by human T cells and natural killer cells that can kill bacterial pathogens through disruption of microbial membrane integrity. Herein we demonstrate antimicrobial activity of the granulysin peptide derived from the active site against Bacillus anthracis, Yersinia pestis, Francisella tularensis, and Burkholderia mallei, and show pathogen-specific differences in granulysin peptide effects. The susceptibility of Y. pestis to granulysin is temperature dependent, being less susceptible when grown at the flea arthropod vector temperature (26°C) than when grown at human body temperature. These studies suggest that augmentation of granulysin expression by cytotoxic lymphocytes, or therapeutic application of granulysin peptides, could constitute important strategies for protection against select agent bacterial pathogens. Investigations of the microbial surface molecules that determine susceptibility to granulysin may identify important mechanisms that contribute to pathogenesis.

Levofloxacin rescues mice from lethal intra-nasal infections with virulent Francisella tularensis and induces immunity and production of protective antibody.

The ability to protect mice against respiratory infections with virulent Francisella tularensis has been problematic and the role of antibody-versus-cell-mediated immunity controversial. In this study, we tested the hypothesis that protective immunity can develop in mice that were given antibiotic therapy following infection via the respiratory tract with F. tularensis SCHU S4. We show that mice infected with a lethal dose of SCHU S4, via an intra-nasal challenge, could be protected with levofloxacin treatment. This protection was evident even when levofloxacin treatment was delayed 72h post-infection. At early time points after levofloxacin treatment, significant numbers of bacteria could be recovered from the lungs and spleens of mice, which was followed by a dramatic disappearance of bacteria from these tissues. Mice successfully treated with levofloxacin were later shown to be almost completely resistant to re-challenge with SCHU S4 by the intra-nasal route. Serum antibody appeared to play an important role in this immunity. Normal mice, when given sera from animals protected by levofloxacin treatment, were solidly protected from a lethal intra-nasal challenge with SCHU S4. The protective antiserum contained high titers of SCHU S4-specific IgG2a, indicating that a strong Th1 response was induced following levofloxacin treatment. Thus, this study describes a potentially valuable animal model for furthering our understanding of respiratory tularemia and provides suggestive evidence that antibody can protect against respiratory infections with virulent F. tularensis.

Subversion of complement activation at the bacterial surface promotes serum resistance and opsonophagocytosis of Francisella tularensis.

Francisella tularensis (Ft) is resistant to serum but requires complement factor C3-derived opsonins for uptake by phagocytic cells and subsequent intracellular growth. In this study, we show that C3 fragments, deposited on Ft, are detected by anti-C3d and -iC3b mAb and that the classical and the alternative pathways are involved in this event. This was demonstrated using C2-depleted sera and specific inhibitors of the classical-versus-alternative pathways of complement activation. Further, we demonstrate that factor C4b, which is crucial for the classical pathway, is deposited on the surface of Ft. In contrast, the C5b-C9 membrane attack complex (MAC) is not assembled on the surface of Ft, which may explain its resistance to complement killing. Deposition of C3 opsonins leads to enhanced phagocytosis by human immature dendritic cells (DC), which leads to intracellular survival, growth, and DC death. Finally, we show that factor H (fH) can bind to the surface of Ft. We believe our data suggest that important virulence factors for Ft are its ability to bind fH and inactivate C3b to iC3b, which culminates in opsonin-induced uptake for subsequent intracellular growth. C3b inactivation also leads to inefficient MAC assembly, which contributes to the ability of this bacterium to resist complement lysis.

Braun lipoprotein (Lpp) contributes to virulence of yersiniae: potential role of Lpp in inducing bubonic and pneumonic plague.

Yersinia pestis evolved from Y. pseudotuberculosis to become the causative agent of bubonic and pneumonic plague. We identified a homolog of the Salmonella enterica serovar Typhimurium lipoprotein (lpp) gene in Yersinia species and prepared lpp gene deletion mutants of Y. pseudotuberculosis YPIII, Y. pestis KIM/D27 (pigmentation locus minus), and Y. pestis CO92 with reduced virulence. Mice injected via the intraperitoneal route with 5 x 10(7) CFU of the Deltalpp KIM/D27 mutant survived a month, even though this would have constituted a lethal dose for the parental KIM/D27 strain. Subsequently, these Deltalpp KIM/D27-injected mice were solidly protected against an intranasally administered, highly virulent Y. pestis CO92 strain when it was given as five 50% lethal doses (LD(50)). In a parallel study with the pneumonic plague mouse model, after 72 h postinfection, the lungs of animals infected with wild-type (WT) Y. pestis CO92 and given a subinhibitory dose of levofloxacin had acute inflammation, edema, and masses of bacteria, while the lung tissue appeared essentially normal in mice inoculated with the Deltalpp mutant of CO92 and given the same dose of levofloxacin. Importantly, while WT Y. pestis CO92 could be detected in the bloodstreams and spleens of infected mice at 72 h postinfection, the Deltalpp mutant of CO92 could not be detected in those organs. Furthermore, the levels of cytokines/chemokines detected in the sera were significantly lower in animals infected with the Deltalpp mutant than in those infected with WT CO92. Additionally, the Deltalpp mutant was more rapidly killed by macrophages than was the WT CO92 strain. These data provided evidence that the Deltalpp mutants of yersiniae were significantly attenuated and could be useful tools in the development of new vaccines.

Immunological responses against Salmonella enterica serovar Typhimurium Braun lipoprotein and lipid A mutant strains in Swiss-Webster mice: potential use as live-attenuated vaccines.

We generated and characterized Salmonella enterica serovar Typhimurium mutants that were deleted for the genes encoding Braun lipoprotein (lpp) alone or in conjunction with the msbB gene, which codes for an enzyme required for the acylation of the lipid A moiety of lipopolysaccharide. Two copies of the lpp gene, designated as lppA and lppB, exist on the chromosome of S. Typhimurium. These mutants were highly attenuated in a mouse infection model and induced minimal histopathological changes in mouse organs compared to those seen in infection with wild-type (WT) S. Typhimurium. The lppB/msbB and the lppAB/msbB mutants were maximally attenuated, and hence further examined in this study for their ability to induce humoral and cellular immune responses. Importantly, infection of out-bred Swiss-Webster mice with the mutant S. Typhimurium generated superior T helper cell type 2 (Th2) responses compared to WT S. Typhimurium, as determined by measuring IgG subclasses and cytokines. WT S. Typhimurium induced higher levels of IgG2a in sera of infected mice, while the lppB/msbB and lppAB/msbB mutants mounted higher levels of IgG1 as determined by an enzyme-linked immunosorbent assay. Mice immunized with lppB/msbB and lppAB/msbB mutants rapidly cleared WT S. Typhimurium upon subsequent rechallenge, and naïve mice passively immunized with sera from animals infected with S. Typhimurium mutants were protected against subsequent challenge with WT S. Typhimurium. Splenic T cells produced higher levels of interferon-gamma following ex vivo exposure to WT S. Typhimurium, while splenic T cells infected with the above-mentioned two mutants evoked higher levels of interleukin-6. Further, mice infected with lppB/msbB and lppAB/msbB mutants showed much higher levels of splenic T cell activation as measured by CD44(+) expression on CD4(+) T cells by flow cytometry and by incorporation of (3)H-thymidine compared to mice that were infected with WT S. Typhimurium. We expect the lppB/msbB and lppAB/msbB mutants to be excellent live-attenuated vaccine candidates, because they induced minimal inflammatory responses and evoked stronger and specific antibody and cellular immune responses.

Critical role for serum opsonins and complement receptors CR3 (CD11b/CD18) and CR4 (CD11c/CD18) in phagocytosis of Francisella tularensis by human dendritic cells (DC): uptake of Francisella leads to activation of immature DC and intracellular survival of the bacteria.

Francisella tularensis is one of the most infectious human pathogens known. Although much has been learned about the immune response of mice using an attenuated live vaccine strain (LVS) derived from F. tularensis subspecies holarctica (Type B), little is known about the responses of human monocyte-derived immature dendritic cells (DC). Here, we show that optimal phagocytosis of LVS by DC is dependent on serum opsonization. We demonstrate that complement factor C3-derived opsonins and the major complement receptors expressed by DC, the integrins CR3 (CD11b/CD18) and CR4 (CD11c/CD18), play a critical role in this adhesion-mediated phagocytosis. LVS induced proinflammatory cytokine production and up-regulation of costimulatory surface proteins (CD40, CD86, and MHC Class II) on DC but resisted killing. Once taken up, LVS grew intracellularly, resulting in DC death. DC maturation and cytokine production were induced by direct contact/phagocytosis of LVS or interaction with soluble products of the bacteria, and enhanced activation was seen when LVS was pretreated with serum. Sonicated LVS and supernatants from LVS cultures were potent activators of DC, but LVS LPS failed to activate DC maturation or cytokine production. Serum-treated LVS rapidly induced (within 6 h) a number of cytokines including IL-10, a potent suppressor of macrophage functions and down-regulator of Th1-like responses and the Th1 response inducer IL-12. These results suggest that the simultaneous production of an activating (IL-12, IL-1beta, and TNF-alpha) and a suppressing (IL-10) cytokine profile could contribute to the immunopathogenesis of tularemia.

Leptospira interrogans activation of human peripheral blood mononuclear cells: preferential expansion of TCR gamma delta+ T cells vs TCR alpha beta+ T cells.

Innate and adaptive immune responses induced by leptospirosis have not been well characterized. In this study we show that in vitro exposure of naive human PBMC to Leptospira interrogans results in cell proliferation and the production of IFN-gamma, IL-12, and TNF-alpha. Cell proliferation was highest when using high numbers of Leptospira. Optimal cell proliferation occurred at 6-8 days, and the majority of cells contained in these cultures were gamma/delta T cells. These cultures showed a 10- to 50-fold expansion of gamma/delta T cells compared with the initial cellular input. Additionally, these cultures contained elevated numbers of NK cells. In contrast, exposure of PBMC to low numbers of Leptospira failed to induce gammadelta T cell or NK cell expansion, but induced significant alphabeta T cell expansion. Vgamma9/Vdelta2 were expressed on all gamma/delta T cells expanded by exposure of PBMC to Leptorspira: Leptospira stimulation of purified TCRgammadelta(+) T cells, obtained from 8-day cultures of Leptospira-stimulated PBMC, induced high levels of IFN-gamma production, but no cell proliferation, suggesting that such stimulation of gammadelta T cells did not depend on specialized accessory cells or Ag processing. Finally, in patients with acute leptospirosis, there was a significant (4- to 5-fold) increase in the number of peripheral blood TCRgammadelta(+) T cells. These results indicate that Leptospira can activate gammadelta T cells and alphabeta T cells and will guide further investigations into the roles of these T cell populations in host defense and/or the pathology of leptospirosis.

Binding of the hepatitis C virus envelope protein E2 to CD81 inhibits natural killer cell functions.

Infection with hepatitis C virus (HCV) is a leading cause of chronic liver disease worldwide. Little is known about how this virus is able to persist or whether this persistence might be because of its ability to alter the early innate immune response. The major HCV envelope protein E2 has been shown to bind to CD81. Thus, HCV binding to natural killer (NK) cells could result in the cross-linking of CD81. To explore this possibility, we investigated whether cross-linking CD81 on NK cells could alter NK cell function. CD81 cross-linking by monoclonal antibody (mAb) specific for CD81 or by immobilized E2 have been shown to result in costimulatory signals for human T cells. In this study, we show that CD81 cross-linking via immobilized E2 or mAbs specific for CD81 inhibits not only non major histocompatibility complex-restricted cytotoxicity mediated by NK cells but also interferon (IFN)-gamma production by NK cells after exposure to interleukin (IL)-2, IL-12, IL-15, or CD16 cross-linking. These results show that CD81 cross-linking mediates completely different signals in NK cells versus T cells. Importantly, these results suggest that one mechanism whereby HCV can alter host defenses and innate immunity is via the early inhibition of IFN-gamma production by NK cells.