Delayed presence of alternatively activated macrophages during a Francisella tularensis infection.

Francisella tularensis is an intracellular bacterium that has the ability to multiply within the macrophage. The phenotype of a macrophage can determine whether the infection is cleared or the host succumbs to disease. Previously published data has suggested that F. tularensis LVS actively induces the alternative phenotype as a survival mechanism. In these studies we demonstrate that this is not the case for the more virulent strain of F. tularensis SCHU-S4. During an intranasal mouse model of infection, immuno-histochemistry identified that iNOS positive ("classical") macrophages are present at 72 h post-infection and remain high (supported by CCL-5 release) in numbers. In contrast, arginase/FIZZ-1 positive ("alternative") cells appear later and in low numbers during the development of the disease tularemia.

[Features of resistance formation to beta-lactam antibiotics in Francisella tularensis subsp. mediasiatica].

AIM: Determination of penicillin resistance features in tularemia causative agents of the mediasiatica subspecies, stability evaluation of differences in strains of various taxa and development of a rapid method of F. tularensis intraspecies differentiation. MATERIALS AND METHODS: Beta-lactamase activity was determined in 30 strains of Francisella genus bacteria by quantitative iodometric method. RESULTS: All the strains regardless of subspecies membership were characterized by high resistance to beta-lactam antibiotics, and bacteria of the mediasiatica subspecies in contrast to other francisella did not synthesize beta-lactamase. Our attempts to induce beta-lactamase activity in vitro and in vivo in strains of this subspecies did not succeed. A method of intraspecies differentiation of F. tularensis by nitorcefin disks is proposed based on the distinctive feature. CONCLUSION: A high level of F. tularensis subsp. mediasiatica resistance to beta-lactams in vitro and their inefficiency during therapy of experimental tularemia due to a beta-lactamase negative strain suggests that F. tularensis beta-lactamase is not the leading factor in formation of native penicillin resistance of tularemia causative agent.

Glycogen synthase kinase-3 is an early determinant in the differentiation of pathogenic Th17 cells.

CD4(+) T cells are critical for host defense but are also major drivers of immune-mediated diseases. The classical view of Th1 and Th2 subtypes of CD4(+) T cells was recently revised by the identification of the Th17 lineage of CD4(+) T cells that produce IL-17, which have been found to be critical in the pathogenesis of autoimmune and other diseases. Mechanisms controlling the differentiation of Th17 cells have been well described, but few feasible targets for therapeutically reducing Th17 cells are known. The generation of Th17 cells requires IL-6 and activation of STAT3. During polarization of CD4(+) T cells to Th17 cells, we found that inhibition of glycogen synthase kinase-3 (GSK3) blocked IL-6 production, STAT3 activation, and polarization to Th17 cells. Polarization of CD4(+) T cells to Th17 cells increased by 10-fold the expression of GSK3ß protein levels in Th17 cells, whereas GSK3ß was unaltered in regulatory T cells. Diminishing GSK3 activity either pharmacologically or molecularly blocked Th17 cell production, and increasing GSK3 activity promoted polarization to Th17 cells. In vivo inhibition of GSK3 in mice depleted constitutive Th17 cells in intestinal mucosa, blocked Th17 cell generation in the lung after Francisella tularensis infection, and inhibited the increase in spinal cord Th17 cells and disease symptoms in the experimental autoimmune encephalomyelitis mouse model of multiple sclerosis. These findings identify GSK3 as a critical mediator of Th17 cell production and indicate that GSK3 inhibitors provide a potential therapeutic intervention to control Th17-mediated diseases.

Multiple mechanisms of NADPH oxidase inhibition by type A and type B Francisella tularensis.

Ft is a facultative intracellular pathogen that infects many cell types, including neutrophils. In previous work, we demonstrated that the type B Ft strain LVS disrupts NADPH oxidase activity throughout human neutrophils, but how this is achieved is incompletely defined. Here, we used several type A and type B strains to demonstrate that Ft-mediated NADPH oxidase inhibition is more complex than appreciated previously. We confirm that phagosomes containing Ft opsonized with AS exclude flavocytochrome b(558) and extend previous results to show that soluble phox proteins were also affected, as indicated by diminished phosphorylation of p47(phox) and other PKC substrates. However, a different mechanism accounts for the ability of Ft to inhibit neutrophil activation by formyl peptides, Staphylococcus aureus, OpZ, and phorbol esters. In this case, enzyme targeting and assembly were normal, and impaired superoxide production was characterized by sustained membrane accumulation of dysfunctional NADPH oxidase complexes. A similar post-assembly inhibition mechanism also diminished the ability of anti-Ft IS to confer neutrophil activation and bacterial killing, consistent with the limited role for antibodies in host defense during tularemia. Studies of mutants that we generated in the type A Ft strain Schu S4 demonstrate that the regulatory factor fevR is essential for NADPH oxidase inhibition, whereas iglI and iglJ, candidate secretion system effectors, and the acid phosphatase acpA are not. As Ft uses multiple mechanisms to block neutrophil NADPH oxidase activity, our data strongly suggest that this is a central aspect of virulence.

Francisella tularensis induces extensive caspase-3 activation and apoptotic cell death in the tissues of infected mice.

Although Francisella tularensis subsp. tularensis is known to cause extensive tissue necrosis, the pathogenesis of tissue injury has not been elucidated. To characterize cell death in tularemia, C57BL/6 mice were challenged by the intranasal route with type A F. tularensis, and the pathological changes in infected tissues were characterized over the next 4 days. At 3 days postinfection, well-organized inflammatory infiltrates developed in the spleen and liver following the spread of infection from the lungs. By the next day, extensive cell death, characterized by the presence of pyknotic cells containing double-strand DNA breaks, was apparent throughout these inflammatory foci. Cell death was not mediated by activated caspase-1, as has been reported for cells infected with other Francisella subspecies. Mouse macrophages and dendritic cells that had been stimulated with type A F. tularensis did not release interleukin-18 in vitro, a response that requires the activation of procaspase-1. Dying cells within type A F. tularensis-infected tissues expressed activated caspase-3 but very little activated caspase-1. When caspase-1-deficient mice were challenged with type A F. tularensis, pathological changes, including extensive cell death, were similar to those seen in infected wild-type mice. In contrast, type A F. tularensis-infected caspase-3-deficient mice showed much less death among their F4/80+ spleen cells than did infected wild-type mice, and they retained the ability to express tumor necrosis factor alpha and inducible NO synthase. These findings suggest that type A F. tularensis induces caspase-3-dependent macrophage apoptosis, resulting in the loss of potentially important innate immune responses to the pathogen.

Glycogen synthase kinase-3beta (GSK3beta) inhibition suppresses the inflammatory response to Francisella infection and protects against tularemia in mice.

Francisella tularensis, the causative agent of tularemia, is currently considered a category A bioterrorism agent due to its high virulence. Infection with F. tularensis results in an inflammatory response that plays an important role in the pathogenesis of the disease; however, the cellular mechanisms regulating this response are poorly understood. Glycogen synthase kinase-3beta (GSK3beta) is a serine/threonine protein kinase that has recently emerged as a key regulatory switch in the modulation of the inflammatory response. In this study, we investigated the effect of GSK3beta inhibition in regulating F. tularensis LVS-induced inflammatory responses. F. tularensis LVS infection of murine peritoneal macrophages induced a TLR2 dependent phosphorylation of GSK3beta. Inhibition of GSK3beta resulted in a significant decrease in the production of pro-inflammatory cytokine IL-6, IL-12p40 and TNF-alpha, as well as a significant increase in the production of the anti-inflammatory cytokine IL-10. GSK3beta regulated the F. tularensis LVS-induced cytokine response by differentially affecting the activation of transcription factors NF-kappaB and CREB. Inhibition of GSK3beta by lithium in vivo suppressed the inflammatory response in mice infected with F. tularensis LVS and conferred a survival advantage. In addition, we show that the production of IFN-gamma contributed to the development of tularemia and to the fatal outcome of the infected animals, depending on the timing and the relative level of the IFN-gamma produced. IFN-gamma potentiated F. tularensis LVS-induced cytokine production by increasing GSK3beta activity and the nuclear translocation of NF-kappaB. Taken together, these results demonstrate a regulatory function of GSK3beta in modulating inflammatory responses that can be detrimental to the host during an F. tularensis LVS infection, and suggest that inhibition of GSK3beta may represent a novel therapeutic approach in the treatment of tularemia.

Matrix metalloproteinase 9 activity enhances host susceptibility to pulmonary infection with type A and B strains of Francisella tularensis.

A striking feature of pulmonary infection with the Gram-negative intracellular bacterium Francisella tularensis, a category A biological threat agent, is an intense accumulation of inflammatory cells, particularly neutrophils and macrophages, at sites of bacterial replication. Given the essential role played by host matrix metalloproteinases (MMPs) in modulating leukocyte recruitment and the potentially indiscriminate destructive capacity of these cells, we investigated whether MMP-9, an important member of this protease family released by neutrophils and activated macrophages, plays a role in the pathogenesis of respiratory tularemia. We found that F. tularensis induced expression of MMP-9 in FVB/NJ mice and that the action of this protease is associated with higher bacterial burdens in pulmonary and extrapulmonary tissues, development of more extensive histopathology predominated by neutrophils, and increased morbidity and mortality compared with mice lacking MMP-9 (MMP-9(-/-)). Moreover, MMP-9(-/-) mice were able to resolve infection with either the virulence-attenuated type B (live vaccine strain) or the highly virulent type A (SchuS4) strain of F. tularensis. Disease resolution was accompanied by diminished leukocyte recruitment and reductions in both bacterial burden and proinflammatory cytokine production. Notably, neutrophilic infiltrates were significantly reduced in MMP-9(-/-) mice, owing perhaps to limited release of Pro-Gly-Pro, a potent neutrophil chemotactic tripeptide released from extracellular matrix through the action of MMP-9. Collectively, these results suggest that MMP-9 activity plays a central role in modulating the clinical course and severity of respiratory tularemia and identifies MMPs as novel targets for therapeutic intervention as a means of modulating neutrophil recruitment.

[The activity of the phagocytic bactericidal systems in intact guinea pigs and in guinea pigs immunized against tularemia]. / Aktivnost' bakteritsidnykh sistem fagotsitov u intaktnykh i immunizirovannykh protiv tuliaremii morskikh svinok.

The activities of NADP-oxidase and glucose-6-phosphate dehydrogenase in mononuclear phagocytes and polymorphonuclear leukocytes (PML) during the phagocytosis of Francisella tularensis and the influence of immunization of on the activity of oxygen-dependent metabolism were studied. The phagocytosis of F.tularensis was found to be accompanied by a rise in the activity of NADP.H-oxidase and glucose-6-phosphate dehydrogenase. In the process of immunogenesis the rearrangement of the enzymatic system ensuring the antimicrobial action of phagocytes occurred. Macrophages exhibited higher enzymatic activity than PML.

Effects of Streptococcus pneumoniae, Salmonella typhimurium and Francisella tularensis infections on oxidative, glycolytic and lysosomal enzyme activity in red and white skeletal muscle in the rat.

Since opinions differ as to whether the oxidative and glycolytic capabilities of skeletal muscle are altered in acute infection, enzyme activities in oxidative, glycolytic and degradative (acid hydrolases) pathways and total protein and DNA were determined in skeletal muscle of rats infected with Streptococcus pneumoniae, Salmonella typhimurium or Francisella tularensis. Studies were performed separately in red (slow twitch) and white (fast twitch) muscle tissue because these fibers function during different types of exercise. In the salmonella- and tularemia-infected rats, the intramitochondrially located oxidative enzymes of muscle were decreased to 56-83% of controls whereas the glycolytic enzyme situated in the cytosol showed an earlier and more pronounced loss of activity, 30-75% of controls. In the pneumococcal infection, only reduced glycolytic activity was significant. DNA concentrations were unchanged in any infection. Reductions during tularemia were statistically correlated with whole-cell protein degradation, while that of the glycolytic enzyme was parallelled by activation of lysosomal enzymes. Red and white muscle tissues responded similarly, in contrast to several other pathologic states that involve a catabolic component of muscle with a predominant response (or damage) in one or the other fiber type.

[Cytochemical changes in the peripheral blood leukocytes of guinea pigs inoculated against plague, tularemia and anthrax]. / Tsitokhimicheskie izmeneniia v leikotsitakh perifericheskoi krovi u morskikh svinok, privitykh protiv chumy, tuliaremii i sibirskoi iazvy.

The immunization of guinea pigs with trivaccine and monovaccines against plaque, tularemia and anthrax induces a decrease in the activity of acidic phosphatase in lymphocytes, as well as a decrease in the number of lymphocytes containing this enzyme. A decrease in the activity of alkaline phosphatase and peroxidase had been found to occur in neutrophil leukocytes. Besides, neutrophil leukocytes have shown an increase in the activity of acidic phosphatase and nonspecific esterases. The study based on the evaluation of the activity of the above-mentioned enzymes in lymphocytes and neutrophils has not revealed the predominant influence exercised by any of the antigens, different in their nature and used separately or in the form of a combined preparation, on immunogenesis.

Relationship of serum beta-glucuronidase and lysozyme to pathogenesis of tularemia in immune and nonimmune rats.

A temporal study is reported of the febrile responses, tissue bacterial contents, and serum concentration of the lysosomal enzymes, beta-glucuronidase and lysozyme, in nonimmune rats inoculated with virulent or attenuated strains of Francisella tularensis, and in immune rats challenged with either a high or low dose of virulent organisms. The level of serum beta-glucuronidase appears to be an indicator of hepatocyte damage, whereas serum lysozyme correlates with the appearance, frequency, and severity of pyogranulomatous lesions. Survival of nonimmune rats after a challenge with either virulent or attenuated organisms appears to depend on a balance between dose of bacterial inoculum, celerity of irreversible pathologic events, and the ability of the reticuloendothelial and immune systems to collaboratively mount a response to limit or prevent dissemination of the infection. In immune rats, infection of parenchymal hepatic cells does not occur after a low dose (10-4) virulent challenge. Infection of parenchymal hepatic cells, however, does occur in immunized rats when the challenge dose is sufficiently large (10-8) so as to overcome the capacity of the reticuloendothelial to clear opsonized organisms.

Effect of infection and endotoxicosis on plasma lactate dehydrogenase isozymes in white rats.

Vertical slab electrophoresis in polyacrylamide gels was used to monitor changes in lactate dehydrogenase (LDH) isozymes in plasma of white rats during bacterial infection and endotoxin poisoning. Peritoneal infection with Francisella tularensis and Salmonella typhimurium and administration of S. typhimurium endotoxin stimulated significant increases in plasma LDH-5. Rates of change in enzyme activity after infection were directly related to size of infecting dose and type of agent employed. Infection with 1 median lethal dose of F. tularensis stimulated both an early, temporary and a prolonged, secondary elevation in LDH-5 activity, whereas salmonellosis, endotoxicosis, killed cells, and latex particles elicited only an initial response of short duration. Changes observed in plasma LDH-5 after exposure to these agents suggest that, as a result of phagocytosis or cell damage, peritoneal leukocytes contribute to early increases in plasma enzyme activity, whereas extensive liver involvement is responsible for high secondary LDH-5 levels during progressive tularemic infection.