Toll-like receptor 2 is required for inflammatory responses to Francisella tularensis LVS

Francisella tularensis, a gram-negative bacterium, is the etiologic agent of tularemia and has recently been classified as a category A bioterrorism agent. Infections with F. tularensis result in an inflammatory response that plays an important role in the pathogenesis of the disease; however, the cellular mechanisms mediating this response have not been completely elucidated. In the present study, we determined the role of Toll-like receptors (TLRs) in mediating inflammatory responses to F. tularensis LVS, and the role of NF-kappaB in regulating these responses. Stimulation of bone marrow-derived dendritic cells from C57BL/6 wild-type (wt) and TLR4-/- but not TLR2-/- mice, with live F. tularensis LVS elicited a dose-dependent increase in the production of tumor necrosis factor alpha. F. tularensis LVS also induced in a dose-dependent manner an up-regulation in the expression of the costimulatory molecules CD80 and CD86 and of CD40 and the major histocompatibility complex class II molecules on dendritic cells from wt and TLR4-/- but not TLR2-/- mice. TLR6, not TLR1, was shown to be involved in mediating the inflammatory response to F. tularensis LVS, indicating that the functional heterodimer is TLR2/TLR6. Stimulation of dendritic cells with F. tularensis resulted in the activation of NF-kappaB, which resulted in a differential effect on the production of pro- and anti-inflammatory cytokines. Taken together, our results demonstrate the role of TLR2/TLR6 in the host's inflammatory response to F. tularensis LVS in vitro and the regulatory function of NF-kappaB in modulating the inflammatory response.     

Toll-like receptor 2 controls the gamma interferon response to Francisella tularensis by mouse liver lymphocytes

The production of gamma interferon (IFN-gamma) is a key step in the protective innate immune response to Francisella tularensis. Natural killer cells and T cells in the liver are important sources of this cytokine during primary F. tularensis infections, and interleukin-12 (IL-12) appears to be an essential coactivating cytokine for hepatic IFN-gamma expression. The present study was undertaken to determine whether or not macrophages (Mphi) or dendritic cells (DC) provide coactivating signals for the liver IFN-gamma response in vitro, whether IL-12 mediates these effects, and whether Toll-like receptor (TLR) signaling is essential to induce this costimulatory activity. Both bone marrow-derived Mphi and DC significantly augmented the IFN-gamma response of F. tularensis-challenged liver lymphocytes in vitro. While both cell types produced IL-12p40 in response to F. tularensis challenge, only DC secreted large quantities of IL-12p70. DC from both IL-12p35-deficient and TLR2-deficient mice failed to produce IL-12p70 and did not costimulate liver lymphocytes for IFN-gamma production in response to viable F. tularensis organisms. Conversely, liver lymphocytes from TLR2-deficient mice cocultured with wild-type accessory cells produced IFN-gamma at levels comparable to those for wild-type hepatic lymphocytes. These findings indicate that TLR2 controls hepatic lymphocyte IFN-gamma responses to F. tularensis by regulating DC IL-12 production. While Mphi also coinduced hepatic IFN-gamma production in response to F. tularensis, they did so in a fashion less dependent on TLR2.     

Toll-like receptor 2 is required for control of pulmonary infection with Francisella tularensis

Toll-like receptor 2 (TLR2) deficiency enhances murine susceptibility to infection by Francisella tularensis as indicated by accelerated mortality, higher bacterial burden, and greater histopathology. Analysis of pulmonary cytokine levels revealed that TLR2 deficiency results in significantly lower levels of tumor necrosis factor alpha and interleukin-6 but increased amounts of gamma interferon and monocyte chemoattractant protein 1. This pattern of cytokine production may contribute to the exaggerated pathogenesis seen in TLR2-/- mice. Collectively, these findings suggest that TLR2 plays an important role in tempering the host response to pneumonic tularemia.     

Immunization with the recombinant PorB outer membrane protein induces a bactericidal immune response against Neisseria meningitidis

Infections with Neisseria meningitidis are characterized by life-threatening meningitis and septicemia. The meningococcal porin proteins from serogroup B meningococci have been identified as candidates for inclusion in vaccines to prevent such infections. In this study, we investigated the vaccine potential of the PorB porin protein free of other meningococcal components. The porB gene from a strain of Neisseria meningitidis expressing the class 3 outer membrane porin protein (PorB3) was cloned into the pRSETB vector, and the protein was expressed at high levels in a heterologous host Escherichia coli. The recombinant protein was purified to homogeneity by affinity chromatography and used for immunization after incorporation into liposomes and into micelles composed either of zwitterionic detergent or nondetergent sulfobetaine. The immunogenicity of these preparations was compared to recombinant PorB protein adsorbed to Al(OH)(3) adjuvant as a control. Although sera raised against the protein adsorbed to Al(OH)(3) reacted with the purified recombinant protein, sera raised against liposomes and micelles showed greater activity with native protein, as measured by enzyme immunoassay with outer membranes and by whole-cell immunofluorescence. Reactivity with native protein was considerably enhanced by incorporation of the adjuvant monophosphoryl lipid A into the liposome or micelle preparations. Recognition of the native protein was in a serotype-specific manner and was associated with the ability of the antisera to promote high levels of serotype-specific complement-mediated killing of meningococci. These results demonstrate that the PorB protein should be considered as a component of a vaccine designed to prevent serogroup B meningococcal infection.     

Molecular immunology of experimental primary tularemia in mice infected by respiratory or intradermal routes with type A Francisella tularensis

The type A subspecies of Francisella tularensis is a highly virulent facultative intracellular bacterial pathogen, and a potential biological weapon. Recently, there has been renewed interest in developing new vaccines and therapeutics against this bacterium. Natural cases of disease, tularemia, caused by the type A subspecies are very rare. Therefore, the United States Food and Drug Administration will rely on the so-called Animal Rule for efficacy testing of anti-Francisella medicines. This requires the human disease to be modeled in one or more animal species in which the pathogenicity of the agent is reasonably well understood. Mice are natural hosts for F. tularensis, and might be able to satisfy this requirement. Tularemia pathogenesis appears to be primarily due to the host inflammatory response which is poorly understood at the molecular level. Additionally, the extent to which this response varies depending on host and pathogen genetic background, or by pathogen challenge route or dose is unknown. Therefore, the present study examined sera and infected tissues from C57BL/6 and BALB/c mice challenged by natural intradermal (ID) and respiratory routes with one of two distinct type A strains of the pathogen for cytokine and chemokine responses that might help to explain the morbidity associated with tularemia. The results show that the molecular immune response was mostly similar regardless of the variables examined. For instance, mRNA for the proinflammatory cytokine IL-6, and chemokines KC, and IP-10 was consistently upregulated at all sites of infection. Upregulation of mRNA for several other cytokines and chemokines occurred in a more tissue restricted manner. For instance, IFN-γ was highly upregulated in the skin of BALB/c, but not C57BL/6 mice after ID inoculation of the pathogen, whilst IL-10 mRNA upregulation was only consistently seen in the skin and lungs.     

Production and characterization of monoclonal antibodies directed against the lipopolysaccharide of Francisella tularensis.

Two monoclonal antibodies (FT14 and FT2F11) directed against the lipopolysaccharide (LPS) of Francisella tularensis were produced for use in tests to detect the organism in environmental samples and clinical specimens. The specificity of the antibodies was determined by enzyme-linked immunosorbent assay (ELISA) and immunoblotting. Both antibodies detected LPS from F. tularensis by ELISA, but only one antibody, FT14, was serologically active in an immunoblot. Treatment of the LPS with detergents prior to ELISA eliminated its binding to FT2F11 but not FT14. Qualitatively, both antibodies detected 10 different strains of F. tularensis by ELISA, but quantitatively, FT14 gave a detectable reaction with 10(3) organisms, whereas FT2F11 was able to detect only 10(5) organisms. FT14 did not cross-react with LPS from a range of other gram-negative species of bacteria, whereas FT2F11 cross-reacted against Vibrio cholerae LPS. Neither antibody showed cross-reactions when entire gram-negative organisms were used as antigens. In a competition ELISA, the two monoclonal antibodies were shown to compete for different epitopes. FT14 was strongly inhibited by purified O side chain from F. tularensis LPS, but FT2F11 was only weakly inhibited. It was inferred from those results that FT14 is directed against the O side chain and that FT2F11 is directed against the core.     

Toll-like receptor 2-mediated signaling requirements for Francisella tularensis live vaccine strain infection of murine macrophages

Francisella tularensis, an aerobic, non-spore-forming, gram-negative coccobacillus, is the causative agent of tularemia. We reported previously that F. tularensis live vaccine strain (LVS) elicited strong, dose-dependent NF-kappaB reporter activity in Toll-like receptor 2 (TLR2)-expressing HEK293T cells and proinflammatory gene expression in primary murine macrophages. Herein, we report that F. tularensis LVS-induced murine macrophage proinflammatory cytokine gene and protein expression are overwhelmingly TLR2 dependent, as evidenced by the abrogated responses of TLR2(-/-) macrophages. F. tularensis LVS infection also increased expression of TLR2 both in vitro, in mouse macrophages, and in vivo, in livers from F. tularensis LVS-infected mice. Colocalization of intracellular F. tularensis LVS, TLR2, and MyD88 was visualized by confocal microscopy. Signaling was abrogated if the F. tularensis LVS organisms were heat or formalin killed or treated with chloramphenicol, indicating that the TLR2 agonist activity is dependent on new bacterial protein synthesis. F. tularensis LVS replicates in macrophages; however, bacterial replication was not required for TLR2 signaling because LVSDeltaguaA, an F. tularensis LVS guanine auxotroph that fails to replicate in the absence of exogenous guanine, activated NF-kappaB in TLR2-transfected HEK293T cells and induced cytokine expression in wild-type macrophages comparably to wild-type F. tularensis LVS. Collectively, these data indicate that the primary macrophage response to F. tularensis LVS is overwhelmingly TLR2 dependent, requires de novo bacterial protein synthesis, and is independent of intracellular F. tularensis replication.     

Toll-like receptor 4 (TLR4) does not confer a resistance advantage on mice against low-dose aerosol infection with virulent type A Francisella tularensis

Francisella tularensis, the causative agent of tularemia, is a gram-negative facultative intracellular bacterium. Toll-like receptor (TLR) 4 is considered to be critical for inducing host innate immunity against many gram-negative bacteria including many respiratory pathogens. To determine the role of TLR4 in host defense against airborne F. tularensis infection, TLR4-defective C3H/HeJ (TLR4(d)) or wild-type C3H/HeOuJ (WT) mice were challenged by low-dose aerosol with type A F. tularensis, and the course of the infection and host responses were compared at day 2 and 4 post-inoculation (dpi). At dpi 2, bacterial burdens in the lungs were similar between TLR4(d) and WT mice, but TLR4(d) mice surprisingly harbored approximately 10-fold fewer bacteria in their spleens and livers. However, the bacterial burdens at dpi 4, the mortality and median time to irreversible moribundity were indistinguishable between the two mouse strains. In addition, the inflammatory responses to the infection, as reflected by the cytokine levels and leukocyte influx in the bronchoalveolar lavage fluid and histopathological analysis, were similar between both mouse strains. Additionally, as with C3H mice, we found no difference in either the median time to death or the survival rate between TLR4-deleted C57BL/10ScNJ mice and WT C57BL/10 mice. Combined, these data suggest that TLR4 does not contribute to resistance of mice to airborne type A F. tularensis infection.     

Basis for the failure of Francisella tularensis lipopolysaccharide to prime human polymorphonuclear leukocytes

Francisella tularensis is the intracellular gram-negative coccobacillus that causes tularemia, and its virulence and infectiousness make it a potential agent of bioterrorism. Previous studies using mononuclear leukocytes have shown that the lipopolysaccharide (LPS) of F. tularensis is neither a typical proinflammatory endotoxin nor an endotoxin antagonist. This inertness suggests that F. tularensis LPS does not bind host LPS-sensing molecules such as LPS-binding protein (LBP). Using priming of the polymorphonuclear leukocyte (PMN) oxidase as a measure of endotoxicity, we found that F. tularensis live vaccine strain LPS did not behave like either a classic endotoxin or an endotoxin antagonist in human PMNs, even when the concentration of LBP was limiting. Furthermore, F. tularensis LPS did not compete with a radiolabeled lipooligosaccharide from Neisseria meningitidis for binding to LBP or to the closely related PMN granule protein, bactericidal/permeability-increasing protein. Our results suggest that the inertness of F. tularensis LPS and the resistance of F. tularensis to oxygen-independent PMN killing may result from the inability of F. tularensis LPS to be recognized by these important LPS-sensing molecules of the innate immune system.     

Experimental murine tularemia caused by Francisella tularensis, live vaccine strain: a model of acquired cellular resistance

We have established a model of experimentally-induced tularemia in mice, using the live vaccine strain of Francisella tularensis. A sublethal, intravenous inoculation of this organism caused in C57BL/6 strain mice an acute infection which lasted approximately 12 days. The clearance of Francisella from the bloodstream was shown to be complete by 5.5 hours postinfection. At this time, approximately twice as many bacteria were isolated from the spleen as from the liver. Mice which had recovered from a primary infection demonstrated a significant resistance to re-infection with autologous Francisella, a memory which persisted for at least 15 weeks. Resistance to experimental tularemia could be passively transferred from infected mice to naive mice by means of non-adherent spleen cells. Cells capable of adoptive transfer of resistance were present at a maximal concentration 7 days following infection, and persisted in significant numbers within the spleen cell population for at least 20 days after infection. Treatment of mice with serum from recovered animals caused a decrease in resistance when measured in the livers, and an increase in resistance when measured in the spleens. Suppression of T cell-mediated immunity during infection by treatment with cyclosporin A resulted in a dramatic increase in the tissue bacterial counts. Cyclosporin A-induced suppression of antitularemic resistance was first noted 2-3 days following infection and remained apparent for at least 8 days. The results of these experiments demonstrate that resistance to experimental murine tularemia is mediated predominantly by a cell-mediated mechanism. This mechanism involves T cells which become activated as early as 2-3 days following infection. Experimental, non-lethal infection with Francisella tularensis is thus an excellent model for investigating the mechanisms of acquired cellular immunity.     

Neisseria meningitidis lipooligosaccharide structure-dependent activation of the macrophage CD14/Toll-like receptor 4 pathway

Meningococcal lipopoly(oligo)saccharide (LOS) is a major inflammatory mediator of fulminant meningococcal sepsis and meningitis. Highly purified wild-type meningococcal LOS and LOS from genetically defined mutants of Neisseria meningitidis that contained specific mutations in LOS biosynthesis pathways were used to confirm that meningococcal LOS activation of macrophages was CD14/Toll-like receptor 4 (TLR4)-MD-2 dependent and to elucidate the LOS structural requirement for TLR4 activation. Expression of TLR4 but not TLR2 was required, and antibodies to both TLR4 and CD14 blocked meningococcal LOS activation of macrophages. Meningococcal LOS alpha or beta chain oligosaccharide structure did not influence CD14/TLR4-MD-2 activation. However, meningococcal lipid A, expressed by meningococci with defects in 3-deoxy-D-manno-octulosonic acid (KDO) biosynthesis or transfer, resulted in an approximately 10-fold (P < 0.0001) reduction in biologic activity compared to KDO2-containing meningococcal LOS. Removal of KDO2 from LOS by acid hydrolysis also dramatically attenuated cellular responses. Competitive inhibition assays showed similar binding of glycosylated and unglycosylated lipid A to CD14/TLR4-MD-2. A decrease in the number of lipid A phosphate head groups or penta-acylated meningococcal LOS modestly attenuated biologic activity. Meningococcal endotoxin is a potent agonist of the macrophage CD14/TLR4-MD-2 receptor, helping explain the fulminant presentation of meningococcal sepsis and meningitis. KDO2 linked to meningococcal lipid A was structurally required for maximal activation of the human macrophage TLR4 pathway and indicates an important role for KDO-lipid A in endotoxin biologic activity.     

The ability of CpG oligonucleotides to protect mice against Francisella tularensis live vaccine strain but not fully virulent F. tularensis subspecies holarctica is reflected in cell-based assays

CpG DNA is a potent activator of the innate immune system. Here the protective effects of CpG DNA are assessed against the facultative intracellular pathogen Francisella tularensis. Dosing of mice with CpG DNA provided protection against disease caused by F. tularensis subsp. holarctica live vaccine strain (LVS) but did not protect against the fully virulent F. tularensis subsp holarctica strain HN63. Similarly, in vitro studies in J774A murine macrophage-like cells demonstrated that stimulation with CpG DNA enables control of intracellular replication of LVS but not HN63. These data confirm findings that CpG DNA may have limited efficacy in providing protection against fully virulent strains of F. tularensis and also suggest that in vitro assays may be useful for the evaluation of novel treatments for virulent F. tularensis.     

Cross-reactive polyclonal antibodies to the inner core of lipopolysaccharide from Neisseria meningitidis

Sera from mice immunized with native or detergent-extracted outer membrane vesicles derived from lipopolysaccharide (LPS) mutant 44/76(Mu-4) of Neisseria meningitidis were analyzed for antibodies to LPS. The carbohydrate portion of 44/76(Mu-4) LPS consists of the complete inner core, Glc beta 1-->4[GlcNAc alpha 1-->2Hep alpha 1-->3]Hep alpha 1-->5KDO[4-->2 alpha KDO]. Immunoblot analysis revealed that some sera contained antibodies to wild-type LPS which has a fully extended carbohydrate chain of immunotype L3,7, as well as to the homologous LPS. Sera reacted only weakly to LPS from 44/76(Mu-3), which lacks the terminal glucose of the inner core. No binding to more truncated LPS was observed. Consequently, the cross-reactive epitopes are expressed mainly by the complete inner core. Dephosphorylation of wild-type LPS abolished antibody binding to LPS in all but one serum. Thus, at least two specificities of cross-reactive antibodies exist: one is dependent on phosphoethanolamine groups in LPS, and one is not. Detection of these cross-reactive antibodies strongly supports the notion that epitopes expressed by meningococcal LPS inner core are also accessible to antibodies when the carbohydrate chain is fully extended. Also, these inner core epitopes are sufficiently immunogenic to induce antibody levels detectable in polyclonal antibody responses. Meningococci can escape being killed by antibodies to LPS that bind only to a specific LPS variant, by altering the carbohydrate chain length. Cross-reactive antibodies may prevent such escape. Therefore, inner core LPS structures may be important antigens in future vaccines against meningococcal disease.     

Competitive enzyme immunoassay for antibodies to a 43,000-molecular-weight Francisella tularensis outer membrane protein for the diagnosis of tularemia.

Antibodies against a 43,000-molecular-weight Francisella tularensis outer membrane (OM) protein (43K protein) were measured in paired serum specimens from 23 patients with tularemia and matched against antibodies in sera from 25 patients with nontularemic infectious diseases and from 25 blood donors. Antibodies were measured by a competition enzyme-linked immunosorbent assay which tested the ability of human serum to compete with rabbit anti-43K protein antibodies for its binding to the F. tularensis OM coat. The sera from nontularemic patients and from blood donors, in dilutions of 1:16 and 1:64, respectively, showed no or very low levels of antibodies. All of the tularemia patients showed positive tests with the first, the second, or both of the serum specimens examined. For instance, with serum diluted 1:64, each of the serum specimens showed a sensitivity of 95.7% and a specificity of 96%. When used for antibody competition in Western blotting (immunoblotting), the rabbit anti-43K selectivity blocked the binding of human serum antibodies to the 43,000-molecular-weight protein. This protein was immunoaccessible in Formalin-killed F. tularensis. These data indicate an important role of the 43,000-molecular-weight OM protein in the immunobiology of tularemia and emphasize its potential usefulness as an antigen in serodiagnostic tests.     

Immunization of mice with an attenuated Salmonella typhimurium strain expressing a membrane protein of Francisella tularensis. A model for identification of bacterial determinants relevant to the host defence against tularemia

A 17-kilodalton (kDA) protein of the facultative intracellular bacterium Francisella tularensis is one of several membrane proteins that induce an in vitro response in T cells from F. tularensis-primed humans. A DNA fragment containing two genes, one of which encodes the 17-kDa protein, was cloned into an attenuated Salmonella typhimurium strain. Mice orally immunized with the recombinant S. typhimurium strain showed lower viable counts in livers and spleens after challenge with F. tularensis LVS (live vaccine strain) than did animals immunized with the non-recombinant strain. Cyclosporin A neutralized the protective effect of the recombinant S. typhimurium strain.     

Conjugation of meningococcal lipopolysaccharide R-type oligosaccharides to tetanus toxoid as route to a potential vaccine against group B Neisseria meningitidis.

Oligosaccharides were obtained by the mild acid hydrolysis of the lipopolysaccharides from a number of different strains of Neisseria meningitidis, serotypes L2, L3, L4, L5, and L10. The dephosphorylated oligosaccharides were conjugated to tetanus toxoid as their 2-(4-isothiocyanatophenyl)-ethylamine derivatives, which resulted in the incorporation of from 18 to 38 oligosaccharides per molecule of tetanus toxoid. When injected in rabbits, the conjugates produced oligosaccharide-specific antibodies which were predominantly serologically specific but which also exhibited some cross-reactivity. These serological results can be attributed to regions of structural dissimilarity and similarity within the oligosaccharides. The oligosaccharide-specific antibodies were also lipopolysaccharide serotype specific, thus indicating that the oligosaccharides are the determinants associated with this serotype specificity. Consistent with the serological results, the conjugate antisera were bactericidal for the homologous serotype meningococcal organisms and in some cases for heterologous serotype organisms.     

Comparative analysis of antibodies to Francisella tularensis antigens during the acute phase of tularemia and eight years later.

Approximately 8 years after treatment for tularemia, 14 of 22 (63.6%) individuals tested still had a positive microagglutination test for Francisella tularensis antibodies. An enzyme-linked immunosorbent assay for anti-F. tularensis outer membrane antibodies was positive for 55% (immunoglobulin A [IgA]), 95% (IgG), and 27% (IgM) of the late-phase sera, but with antibody levels significantly reduced from those in the acute-phase sera. IgG and IgA antibody levels in the late-phase sera showed significant correlation with levels in the acute-phase sera. The IgG/IgM ratio calculation discriminated between acute-phase and persistent antibodies for most sera, but Western blot (immunoblot) patterns did not. Immunoblotting indicated that the F. tularensis lipopolysaccharide is a major target for antibodies in both groups of sera. Our results substantiate the need for caution in the interpretation of positive serological test results for tularemia, which could result from disease occurring years earlier.     

Disruption of the C5a receptor gene fails to protect against experimental allergic encephalomyelitis

Activation of the complement system generates the anaphylatoxic peptide C5a, which elicits a broad range of inflammatory activities. The biological activities of C5a are mediated through its binding to the widely expressed C5a receptor (C5aR), a G-protein-coupled seven transmembrane domain receptor. In experimental autoimmune encephalomyelitis (EAE), an animal model for multiple sclerosis, the C5aR is expressed on monocytes/macrophages, reactive astrocytes and T cells infiltrating the central nervous system (CNS). To investigate the role of the C5aR in this T cell-driven autoimmune model, we induced EAE in C5aR-deficient mice (C5aR(-/-)) and wild-type mice using a myelin oligodendrocyte glycoprotein (MOG) peptide as the immunogen. We found that C5aR(-/-) mice were fully susceptible to MOG-induced EAE with no difference in disease onset or severity in C5aR(-/-) mice compared to control mice. Cellular infiltrates (macrophages and T cells) were similar in the spinal cords of both animal groups and splenic T cells from C5aR(-/-) mice and control mice responded identically to MOG in T cell proliferation assays. Ribonuclease protection assays demonstrated no significant differences in pro-inflammatory gene expression between receptor-deficient and sufficient mice. These results indicate that the C5aR is not an essential mediator in the induction and progression of EAE.     

Inability of the Francisella tularensis lipopolysaccharide to mimic or to antagonize the induction of cell activation by endotoxins.

We studied the ability of the lipopolysaccharide (LPS) extracted from a vaccine strain of Francisella tularensis (LPS-Ft) to mimic LPSs from other gram-negative bacteria for activation of various murine cell types or to antagonize the effects of other LPSs. We found that activation of macrophages for the production of tumor necrosis factor alpha and NO, of pre-B lymphocytes for the expression of surface immunoglobulins, and of bone marrow cells for the expression of LPS-binding sites was either undetectable with LPS-Ft or required concentrations 100 to 1,000 times higher than for standard LPSs. Preexposure of macrophages to LPS-Ft also failed to trigger down-regulation of tumor necrosis factor alpha (desensitization) or up-regulation of NO responses to an endotoxin challenge. In contrast to other atypical LPSs, LPS-Ft was also unable to antagonize any of the endotoxin-induced cellular responses mentioned above, suggesting that this LPS does not interact with LPS receptors.