Early activation of NK cells after lung infection with the intracellular bacterium, Francisella tularensis LVS

Francisella tularensis is a gram-negative intracellular bacterium that has been classified as a Category A biothreat because of its ability to induce deadly pneumonic tularemia when inhaled. In the present study, an experimental model of F. tularensis LVS intranasal infection was used to study the immune cells involved in cytokine secretion in the lungs after infection. Dramatic increases in the numbers of cells secreting IFN-gamma were observed 72 h after intranasal infection of BALB/c and C57BL/6 mice with sublethal (1000 CFU) or lethal (10,000 CFU) doses of F. tularensis LVS and the cells primarily responsible for this IFN-gamma expression were identified as CD11b+ DX5+ NK cells. The findings were further confirmed in C57BL/6 mice showing that cells responsible for IFN-gamma secretion in the lungs were CD11b+ DX5+ NK1.1+. NK cell depletion studies showed a decrease in the percentage of IFN-gamma secreting cells, due not only to a diminished proportion of IFN-gamma secreting NK cells, but also to a reduced percentage of T cells secreting IFN-gamma. The results indicate that IFN-gamma is secreted in response to respiratory infection with F. tularensis LVS, and that NK cells are the early responders responsible for IFN-gamma secretion.     

Orchestration of the protective immune response to intracellular bacteria: Francisella tularensis as a model organism

Abstract Francisella tularensis is used as a model organism in studies of mechanisms behind the induction of a protective T-cell response in the mammalian host. Protective immunity is associated with a CD4 and CD8 T-cell response towards a mosaic of proteins of F. tularensis and due to HLA restriction, each individual selects her own mosaic. No single protein has so far been shown to be immunodominant. Only live F. tularensis affords effective host protection. Subcellular antigen preparations induce only a marginal protective response even when combined with potent adjuvants such as immunostimulating complexes (ISCOMs). In mice, intradermal injection of live F. tularensis but not of killed bacteria results in an early cytokine expression in the infected liver, including interleukin-12, tumor necrosis factor-α, and interferon-γ. This cytokine response seems to be a prerequisite for effective priming of T cells to an array of proteins of F. tularensis to occur.     

Proteomics analysis of the Francisella tularensis LVS response to iron restriction: induction of the F. tularensis pathogenicity island proteins IglABC

Francisella tularensis is a highly virulent, facultative intracellular pathogen that causes tularemia in humans and animals. Although it is one of the most infectious bacterial pathogens, little is known about its virulence mechanisms. In this study, the response of F. tularensis live vaccine strain to iron depletion, which simulates the environment within the host, was investigated. In order to detect alterations in protein synthesis, metabolic labeling, followed by 2D-PAGE analysis was used. Globally, 141 protein spots were detected whose levels were significantly altered in the iron-restricted medium. About 65% of the spots were successfully identified using mass spectrometric approaches. Importantly, among the proteins produced at an increased level during iron-limited growth, three proteins were found encoded by the igl operon, located in the F. tularensis pathogenicity island I (FPI). Of these, the IglC and IglA proteins were previously reported to be necessary for full virulence of F. tularensis. These results, obtained at the proteome level, support and confirm recently published data showing that the igl operon genes are transcribed in response to iron limitation.     

Sensitivity of Francisella tularensis to ultrapure water and deoxycholate: implications for bacterial intracellular growth assay in macrophages

The ability of Francisella tularensis to replicate in macrophages is critical for its pathogenesis, therefore intracellular growth assays are important tools for assessing virulence. We show that two lysis solutions commonly used in these assays, deionized water and deoxycholate in PBS, lead to highly inaccurate measurements of intracellular bacterial survival.     

Prophylactic and therapeutic use of antibodies for protection against respiratory infection with Francisella tularensis

The role of Abs in protection against respiratory infection with the intracellular bacterium Francisella tularensis is not clear. To investigate the ability of Abs to clear bacteria from the lungs and prevent systemic spread, immune serum was passively administered i.p. to naive mice before intranasal F. tularensis live vaccine strain infection. It was found that immune serum treatment provided 100% protection against lethal challenge while normal serum or Ig-depleted immune serum provided no protection. Protective efficacy was correlated with increased clearance of bacteria from the lung and required expression of FcgammaR on phagocytes, including macrophages and neutrophils. However, complement was not required for protection. In vitro experiments demonstrated that macrophages were more readily infected by Ab-opsonized bacteria but became highly efficient in killing upon activation by IFN-gamma. Consistent with this finding, in vivo Ab-mediated protection was found to be dependent upon IFN-gamma. SCID mice were not protected by passive Ab transfer, suggesting that T cells but not NK cells serve as the primary source for IFN-gamma. These data suggest that a critical interaction of humoral and cellular immune responses is necessary to provide sterilizing immunity against F. tularensis. Of considerable interest was the finding that serum Abs were capable of conferring protection against lethal respiratory tularemia when given 24-48 h postexposure. Thus, this study provides the first evidence for the therapeutic use of Abs in Francisella-infected individuals.     

Utilization of Fc receptors as a mucosal vaccine strategy against an intracellular bacterium, Francisella tularensis

Numerous studies have demonstrated that targeting Ag to Fc receptors (FcR) on APCs can enhance humoral and cellular immunity. However, studies are lacking that examine both the use of FcR-targeting in generating immune protection against infectious agents and the use of FcRs in the induction of mucosal immunity. Francisella tularensis is a category A intracellular mucosal pathogen. Thus, intense efforts are underway to develop a vaccine against this organism. We hypothesized that protection against mucosal infection with F. tularensis would be significantly enhanced by targeting inactivated F. tularensis live vaccine strain (iFt) to FcRs at mucosal sites, via intranasal immunization with mAb-iFt complexes. These studies demonstrate for the first time that: 1) FcR-targeted immunogen enhances immunogen-specific IgA production and protection against subsequent infection in an IgA-dependent manner, 2) FcgammaR and neonatal FcR are crucial to this protection, and 3) inactivated F. tularensis, when targeted to FcRs, enhances protection against the highly virulent SchuS4 strain of F. tularensis, a category A biothreat agent. In summary, these studies show for the first time the use of FcRs as a highly effective vaccination strategy against a highly virulent mucosal intracellular pathogen.     

Immunologic consequences of Francisella tularensis live vaccine strain infection: role of the innate immune response in infection and immunity

Francisella tularensis (Ft), a Gram-negative intracellular bacterium, is the etiologic agent of tularemia. Although attenuated for humans, i.p. infection of mice with <10 Ft live vaccine strain (LVS) organisms causes lethal infection that resembles human tularemia, whereas the LD50 for an intradermal infection is >10(6) organisms. To examine the immunological consequences of Ft LVS infection on the innate immune response, the inflammatory responses of mice infected i.p. or intradermally were compared. Mice infected i.p. displayed greater bacterial burden and increased expression of proinflammatory genes, particularly in the liver. In contrast to most LPS, highly purified Ft LVS LPS (10 microg/ml) was found to be only minimally stimulatory in primary murine macrophages and in HEK293T cells transiently transfected with TLR4/MD-2/CD14, whereas live Ft LVS bacteria were highly stimulatory for macrophages and TLR2-expressing HEK293T cells. Despite the poor stimulatory activity of Ft LVS LPS in vitro, administration of 100 ng of Ft LVS LPS 2 days before Ft LVS challenge severely limited both bacterial burden and cytokine mRNA and protein expression in the absence of detectable Ab at the time of bacterial challenge, yet these mice developed a robust IgM Ab response within 2 days of infection and survived. These data suggest that prior administration of Ft LVS LPS protects the host by diminishing bacterial burden and blunting an otherwise overwhelming inflammatory response, while priming the adaptive immune response for development of a strong Ab response.     

TLR4-dependent activation of inflammatory cytokine response in macrophages by Francisella elongation factor Tu

The bacterial determinants of pulmonary Francisella induced inflammatory responses and their interaction with host components are not clearly defined. In this study, proteomic and immunoblot analyses showed presence of a cytoplasmic protein elongation factor Tu (EF-Tu) in the membrane fractions of virulent Francisella novicida, LVS and SchuS4, but not in an attenuated F. novicida mutant. EF-Tu was immunodominant in mice vaccinated and protected from virulent F. novicida. Moreover, recombinant EF-Tu induced macrophages to produce inflammatory cytokines in a TLR4 dependent manner. This study shows immune stimulatory properties of a cytoplasmic protein EF-Tu expressed on the membrane of virulent Francisella strains.     

Myeloid differentiation factor-88 (MyD88) is essential for control of primary in vivo Francisella tularensis LVS infection, but not for control of intra-macrophage bacterial replication

The means by which Francisella tularensis, the causative agent of tularemia, are recognized by mammalian immune systems are poorly understood. Here we wished to explore the contribution of the MyD88/Toll-like receptor signaling pathway in initiating murine responses to F. tularensis Live Vaccine Strain (LVS). MyD88 knockout (KO) mice, but not TLR2-, TLR4- or TLR9-deficient mice, rapidly succumbed following in vivo bacterial infection via the intradermal route even with a very low dose of LVS (5 x 10(1)) that was 100,000-fold less than the LD(50) of normal wild-type (WT) mice. By day 5 after LVS infection, bacterial organ burdens were 5-6 logs higher in MyD88 knockout mice; further, unlike infected WT mice, levels of interferon-gamma in the sera of LVS-infected MyD88 KO were undetectable. An in vitro culture system was used to assess the ability of bone marrow macrophages derived from either KO or WT mice to support bacterial growth, or to control intracellular bacterial replication when co-cultured with immune lymphocytes. In this assay, bacterial replication was similar in macrophages derived from either WT or any of the TLR KO mice. Bacterial growth was controlled in co-cultures containing macrophages from MyD88 KO mice or TLR KO mice as well as in co-cultures containing immune WT splenic lymphocytes and WT macrophages. Further, MyD88-deficient LVS-immune splenocytes controlled intracellular growth comparably to those from normal mice. Thus MyD88 is essential for innate host resistance to LVS infection, but is not required for macrophage control of intracellular bacterial growth.     

Mutations in Francisella tularensis, decreasing the virulence of these bacteria and leading to a change in the immune response upon infection of experimental animals with them]

The research was aimed at isolation of Francisella tularensis mutants possessing the decreased virulence for experimental animals and mediating the changes in the animal immune response. A number of spontaneous and induced mutants of the American and European subtypes of Francisella tularensis were selected for antibiotics resistance or detergent sensitivity. All the obtained mutants have the decreased virulence and differ in their ability to induce the protective antitularemia immunity or ability to induce the humoral immune response in the laboratory animals. The dimeric immunoprecipitation in gel as well as immunoblotting have shown the mutations decreasing the virulence to cause the loss by bacteria of a number of antigenic structures (in case the virulence is completely lost) or changes in antigenic structure resulting in inability of bacteria to induce the humoral immune response when immunizing the laboratory animals. The latter occurs in partially virulent mutants of the vaccine mutant type. The concomitant changes in virulence, ability to cause protective immunity or humoral immune response of the mutants is discussed.     

Identification of a novel Francisella tularensis factor required for intramacrophage survival and subversion of innate immune response

Francisella tularensis, the causative agent of tularemia, is one of the deadliest agents of biological warfare and bioterrorism. Extremely high virulence of this bacterium is associated with its ability to dampen or subvert host innate immune response. The objectives of this study were to identify factors and understand the mechanisms of host innate immune evasion by F. tularensis. We identified and explored the pathogenic role of a mutant interrupted at gene locus FTL_0325, which encodes an OmpA-like protein. Our results establish a pathogenic role of FTL_0325 and its ortholog FTT0831c in the virulent F. tularensis SchuS4 strain in intramacrophage survival and suppression of proinflammatory cytokine responses. This study provides mechanistic evidence that the suppressive effects on innate immune responses are due specifically to these proteins and that FTL_0325 and FTT0831c mediate immune subversion by interfering with NF-κB signaling. Furthermore, FTT0831c inhibits NF-κB activity primarily by preventing the nuclear translocation of p65 subunit. Collectively, this study reports a novel F. tularensis factor that is required for innate immune subversion caused by this deadly bacterium.     

Generation of a convalescent model of virulent Francisella tularensis infection for assessment of host requirements for survival of tularemia

Francisella tularensis is a facultative intracellular bacterium and the causative agent of tularemia. Development of novel vaccines and therapeutics for tularemia has been hampered by the lack of understanding of which immune components are required to survive infection. Defining these requirements for protection against virulent F. tularensis, such as strain SchuS4, has been difficult since experimentally infected animals typically die within 5 days after exposure to as few as 10 bacteria. Such a short mean time to death typically precludes development, and therefore assessment, of immune responses directed against virulent F. tularensis. To enable identification of the components of the immune system that are required for survival of virulent F. tularensis, we developed a convalescent model of tularemia in C57Bl/6 mice using low dose antibiotic therapy in which the host immune response is ultimately responsible for clearance of the bacterium. Using this model we demonstrate αβTCR(+) cells, γδTCR(+) cells, and B cells are necessary to survive primary SchuS4 infection. Analysis of mice deficient in specific soluble mediators shows that IL-12p40 and IL-12p35 are essential for survival of SchuS4 infection. We also show that IFN-γ is required for survival of SchuS4 infection since mice lacking IFN-γR succumb to disease during the course of antibiotic therapy. Finally, we found that both CD4(+) and CD8(+) cells are the primary producers of IFN-γand that γδTCR(+) cells and NK cells make a minimal contribution toward production of this cytokine throughout infection. Together these data provide a novel model that identifies key cells and cytokines required for survival or exacerbation of infection with virulent F. tularensis and provides evidence that this model will be a useful tool for better understanding the dynamics of tularemia infection.     

Construction of a reporter plasmid for screening in vivo promoter activity in Francisella tularensis

Francisella tularensis is a facultative intracellular bacterium that survives and multiplies inside macrophages. Here we constructed a new promoter probe plasmid denoted pKK214 by introduction of a promoter-less chloramphenicol acetyltransferase (cat) gene into the shuttle vector pKK202. A promoter library was created in F. tularensis strain LVS by cloning random chromosomal DNA fragments into pKK214. Approximately 15% of the recombinant bacteria showed chloramphenicol resistance in vitro. The promoter library was also used to infect macrophages in the presence of chloramphenicol and after two cycles of infection the library contained essentially only chloramphenicol resistance clones which shows that pKK214 can be used to monitor F. tularensis genes that are expressed during infection.     

Akt/Protein kinase B modulates macrophage inflammatory response to Francisella infection and confers a survival advantage in mice

The Gram-negative bacterium Francisella novicida infects primarily monocytes/macrophages and is highly virulent in mice. Macrophages respond by producing inflammatory cytokines that confer immunity against the infection. However, the molecular details of host cell response to Francisella infection are poorly understood. In this study, we demonstrate that F. novicida infection of murine macrophages induces the activation of Akt. Inhibition of Akt significantly decreases proinflammatory cytokine production in infected macrophages, whereas production of the anti-inflammatory cytokine IL-10 is enhanced. Analysis of the mechanism of Akt influence on cytokine response demonstrated that Akt promotes NF-kappaB activation. We have extended these findings to show that Akt activation may be regulated by bacterial genes associated with phagosomal escape. Infection with mglA mutants of F. novicida elicited sustained activation of Akt in comparison to cells infected with wild-type F. novicida. Concomitantly, there was significantly higher proinflammatory cytokine production and lower IL-10 production in cells infected with the mglA mutant. Finally, transgenic animals expressing constitutively active Akt displayed a survival advantage over their wild-type littermates when challenged with lethal doses of F. novicida. Together, these observations indicate that Akt promotes proinflammatory cytokine production by F. novicida-infected macrophages through its influence on NF-kappaB, thereby contributing to immunity against F. novicida infection.     

Inhibition of hyaluronan synthesis reduced inflammatory response in mouse synovial fibroblasts subjected to collagen-induced arthritis

Hyaluronan (HA) fragments are able to induce inflammation by stimulating both CD44 and toll-like receptor 4 (TLR-4). CD44 and TLR-4 activation stimulates the liberation of NF-kB and pro-inflammatory cytokine responses. The aim of this study was to investigate the effects of hyaluronidase (HYAL) treatment, which depolymerises HA into small fragments, and of the addition of specific hyaluronan synthases-1, 2, and 3 small interference RNA (HASs siRNA), which silence HASs activity, on normal mouse synovial fibroblasts (NSF) and on rheumatoid arthritis synovial fibroblasts (RASF) obtained from mice subjected to collagen induced arthritis (CIA). The addition of HYAL to NSF and/or RASF significantly increased the TLR-4, CD44 and NF-kB activity, as well as the pro-inflammatory cytokines, interleukin-1beta (IL-1beta), tumor necrosis factor-alpha (TNF-alpha), interleukin-6 (IL-6), and interleukin-33 (IL-33) in both groups, but to a greater extent in RASF. The addition to NSF and/or RASF of the HASs siRNA, which block HASs activity and therefore the availability of HA substrate for HYAL, was able to reduce HYAL effects in both NSF and RASF. Finally, the HA evaluation confirmed the increment of HA at low molecular weight after HYAL treatment.     

Transcriptional profiling of Francisella tularensis infected peripheral blood mononuclear cells: a predictive tool for tularemia

In this study, we analyzed temporal gene expression patterns in human peripheral blood mononuclear cells (PBMCs) infected with the Francisella tularensis live vaccine strain from 1 to 24 h utilizing a whole human Affymetrix gene chip. We found that a considerable number of induced genes had similar expression patterns and functions as reported previously for gene expression profiling in patients with ulceroglandular tularemia. Among the six uniquely regulated genes reported for tularemia patients as being part of the alarm signal gene cluster, five, namely caspase 1, PSME2, TAP-1, GBP1, and GCH1, were induced in vitro. We also detected four out of the seven potential biomarkers reported in tularemia patients, namely TNFAIP6 at 4 h and STAT1, TNFSF10, and SECTM1 at 16 and 24 h. These observations underscore the value of using microarray expression profiling as an in vitro tool to identify potential biomarkers for human infection and disease. Our results indicate the potential involvement of several host pathways/processes in Francisella infection, notably those involved in calcium, zinc ion binding, PPAR signaling, and lipid metabolism, which further refines the current knowledge of F. tularensis infection and its effects on the human host. Ultimately, this study provides support for utilizing in vitro microarray gene expression profiling in human PBMCs to identify biomarkers of infection and predict in vivo immune responses to infectious agents.     

Rapid onset of ICAM-1 expression is a marker of effective macrophages activation during infection of <Emphasis Type="Italic">Francisella tularensis</Emphasis> LVS in vitro

Francisella tularensis is capable to modulate immunobiological activities of the host cells. We focused on the expression of ICAM-1 (CD54) on J774.2 mouse macrophage cell line infected by F. tularensis live vaccine strain (LVS) in vitro as a putative marker of subsequent elimination of infection. J774.2 cell line cells were infected by F. tularensis LVS strain (multiplicity of infection, 1:100). Cell cultures were stimulated either 3 h before infection or 3 h after infection by either lipopolysaccharide (LPS) or interferon γ (IFN-γ). The expression of ICAM-1 was determined by flow cytometry 6 h after infection. The intensity of ICAM-1 expression after 6 h of J774.2 macrophage cells infection by F. tularensis is very sensitive indicator of the effective macrophages stimulation resulting in the elimination of F. tularensis infection. The mean fluorescence intensity MFI = 49.8 is set-up by our experiments as a reliable threshold of the effective elimination of F. tularensis experimental infection with 83.3% sensitivity and 96.7% specificity, respectively. Simultaneous stimulation of J774.2 macrophage cells by LPS and IFN-γ was essential to elicit the elimination of F. tularensis infection. The ICAM-1 expression determined by flow cytometry can be considered to be highly sensitive and specific approach to predict elimination of F. tularensis infection by J774.2 macrophages.