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Sepsis is a complex immune disorder with a mortality rate of 20-50% and currently has no therapeutic interventions. It is thus critical to identify and characterize molecules/factors responsible for its development. We have recently shown that pulmonary infection with Francisella results in sepsis development. As extensive cell death is a prominent feature of sepsis, we hypothesized that host endogenous molecules called alarmins released from dead or dying host cells cause a hyperinflammatory response culminating in sepsis development. In the current study we investigated the role of galectin-3, a mammalian β-galactoside binding lectin, as an alarmin in sepsis development during F. novicida infection. We observed an upregulated expression and extracellular release of galectin-3 in the lungs of mice undergoing lethal pulmonary infection with virulent strain of F. novicida but not in those infected with a non-lethal, attenuated strain of the bacteria. In comparison with their wild-type C57Bl/6 counterparts, F. novicida infected galectin-3 deficient (galectin-3(-/-)) mice demonstrated significantly reduced leukocyte infiltration, particularly neutrophils in their lungs. They also exhibited a marked decrease in inflammatory cytokines, vascular injury markers, and neutrophil-associated inflammatory mediators. Concomitantly, in-vitro pre-treatment of primary neutrophils and macrophages with recombinant galectin-3 augmented F. novicida-induced activation of these cells. Correlating with the reduced inflammatory response, F. novicida infected galectin-3(-/-) mice exhibited improved lung architecture with reduced cell death and improved survival over wild-type mice, despite similar bacterial burden. Collectively, these findings suggest that galectin-3 functions as an alarmin by augmenting the inflammatory response in sepsis development during pulmonary F. novicida infection.

The virulence mechanisms of Francisella tularensis, the causative agent of severe pneumonia in humans and a CDC category A bioterrorism agent, are not fully defined. As sepsis is the leading cause of mortality associated with respiratory infections, we determined whether, in the absence of any known bacterial toxins, a deregulated host response resulting in sepsis syndrome is associated with lethality of respiratory infection with the virulent human Type A strain SchuS4 of F. tularensis. The C57BL/6 mice infected intranasally with a lethal dose of SchuS4 exhibited high bacterial burden in systemic organs and blood indicative of bacteremia. In correlation, infected mice displayed severe tissue pathology and associated cell death in lungs, liver and spleen. Consistent with our studies with murine model strain Francisella novicida, infection with SchuS4 caused an initial delay in upregulation of inflammatory mediators followed by development of severe sepsis characterized by exaggerated cytokine release, upregulation of cardiovascular injury markers and sepsis mediator alarmins S100A9 and HMGB1. This study shows that pulmonary tularemia caused by the Type A strain of F. tularensis results in a deregulated host response leading to severe sepsis and likely represents the major cause of mortality associated with this virulent pathogen., (Copyright © 2011 Elsevier Ltd. All rights reserved.)

Aging is a complex phenomenon that has been shown to affect many organ systems including the innate and adaptive immune systems. The current study was designed to examine the potential effect of immunosenescence on the pulmonary immune response using a Francisella tularensis live vaccine strain (LVS) inhalation infection model. F. tularensis is a Gram-negative intracellular pathogen that can cause a severe pneumonia. In this study both young (8-12 week old) and aged (20-24 month old) mice were infected intranasally with LVS. Lung tissues from young and aged mice were used to assess pathology, recruitment of immune cell types and cytokine expression levels at various times post infection. Bacterial burdens were also assessed. Interestingly, the lungs of aged animals harbored fewer organisms at early time points of infection (day 1, day 3) compared with their younger counterparts. In addition, only aged animals displayed small perivascular aggregates at these early time points that appeared mostly mononuclear in nature. However, the kinetics of infiltrating polymorphonuclear neutrophils (PMNs) and increased cytokine levels measured in the bronchial alveolar lavage fluid (BALF) were delayed in infected aged animals relative to young infected animals with neutrophils appearing at day 5 post infection (PI) in the aged animals as opposed to day 3 PI in the young infected animals. Also evident were alterations in the ratios of mononuclear to PMNs at distinct post infection times. The above evidence indicates that aged mice elicit an altered immune response in the lung to respiratory F. tularensis LVS infections compared to their younger counterparts., (Copyright (c) 2009 Elsevier Inc. All rights reserved.)

Background: Francisella tularensis is the causative agent of tularemia and is classified as a Category A select agent. Recent studies have implicated TLR2 as a critical element in the host protective response to F. tularensis infection, but questions remain about whether TLR2 signaling dominates the response in all circumstances and with all species of Francisella and whether F. tularensis PAMPs are predominantly recognized by TLR2/TLR1 or TLR2/TLR6. To address these questions, we have explored the role of Toll-like receptors (TLRs) in the host response to infections with F. tularensis Live Vaccine Strain (LVS) and F. tularensis subspecies (subsp.) novicida in vivo., Methodology/principal Findings: C57BL/6 (B6) control mice and TLR- or MyD88-deficient mice were infected intranasally (i.n.) or intradermally (i.d.) with F. tularensis LVS or with F. tularensis subsp. novicida. B6 mice survived >21 days following infection with LVS by both routes and survival of TLR1(-/-), TLR4(-/-), and TLR6(-/-) mice infected i.n. with LVS was equivalent to controls. Survival of TLR2(-/-) and MyD88(-/-) mice, however, was significantly reduced compared to B6 mice, regardless of the route of infection or the subspecies of F. tularensis. TLR2(-/-) and MyD88(-/-) mice also showed increased bacterial burdens in lungs, liver, and spleen compared to controls following i.n. infection. Primary macrophages from MyD88(-/-) and TLR2(-/-) mice were significantly impaired in the ability to secrete TNF and other pro-inflammatory cytokines upon ex vivo infection with LVS. TNF expression was also impaired in vivo as demonstrated by analysis of bronchoalveolar lavage fluid and by in situ immunofluorescent staining., Conclusions/significance: We conclude from these studies that TLR2 and MyD88, but not TLR4, play critical roles in the innate immune response to F. tularensis infection regardless of the route of infection or the subspecies. Moreover, signaling through TLR2 does not depend exclusively on TLR1 or TLR6 during F. tularensis LVS infection.

Francisella tularensis is the causative agent of zoonotic tularemia, a severe pneumonia in humans, and Francisella novicida causes a similarly severe tularemia in mice upon inhalation. The correlates of protective immunity, as well as the virulence mechanisms of this deadly pathogen, are not well understood. In the present study, we compared the host immune responses of lethally infected and vaccinated mice to highlight the host determinants of protection from this disease. Intranasal infection with an attenuated mutant (Mut) of F. novicida lacking a 58-kDa hypothetical protein protected C57BL/6 mice from a subsequent challenge with the fully virulent wild-type strain U112 via the same route. The protection conferred by Mut vaccination was associated with reduced bacterial burdens in systemic organs, as well as the absence of bacteremia. Also, there was reduced lung pathology and associated cell death in the lungs of vaccinated mice. Both vaccinated and nonvaccinated mice displayed an initial 2-day delay in upregulation of signature inflammatory mediators after challenge. Whereas the nonvaccinated mice developed severe sepsis characterized by hypercytokinemia and T-cell depletion, the vaccinated mice displayed moderated cytokine induction and contained increased numbers of alphabeta T cells. The recall response in vaccinated mice consisted of a characteristic Th1-type response in terms of cytokines, as well as antibody isotypes. Our results show that a regulated Th1 type of cell-mediated and humoral immunity in the absence of severe sepsis is associated with protection from respiratory tularemia, whereas a deregulated host response leading to severe sepsis contributes to mortality.

The bacterial or host determinants of lethality associated with respiratory Francisella infections are currently unknown. No exo- or endotoxins that contribute to the severity of this disease have been identified. However, a deregulated host immune response upon infection is characterized by an initial 36- to 48-h delay followed by a rapid and excessive inflammatory response prior to death at 72-120 h. Here, we extend these findings by comparing host immune responses between sublethal and lethal respiratory infections of mice with an attenuated transposon mutant (Mut) of F. novicida (F.n.) strain U112 (sublethal) versus the wild-type (WT) strain (lethal). Infection with WT bacteria, but not the Mut, was characterized by sustained bacteremia and systemic dissemination of the pathogen with temporal increases in bacterial burdens in liver and spleen. Severe pathology with large foci of infiltrates associated with extensive tissue damage was evident in WT-infected lungs, and Mut-infected mice displayed much reduced pathology with intact lung architecture. Similar to other experimental models of severe sepsis, WT- but not the Mut-infected mice exhibited a robust increase in numbers of Gr1+ and CD11b+ cells, while displaying a significant depletion of alphabeta T cells. Further, a dramatic up-regulation of multiple cytokines and chemokines was observed only in lethal WT infection. In addition, an earlier and larger increased expression of S100A9, a known mediator of sepsis, was observed in WT-infected mice. Taken together, these results show that a hyperinflammatory host immune response, culminating in severe sepsis, is responsible for the lethal outcome of respiratory tularemia.

Respiratory Francisella infections induce a delayed innate immune response followed by a severe sepsis like condition. In this study, mice infected intranasally with Francisella novicida showed a depletion of alphabeta T cells in lungs while exhibiting large accumulations of other leukocytes correlating with disease severity. The depleted T cells were predominantly CD4(+). The alphabeta T cells in infected mice showed significantly higher levels of Annexin V binding than those in mock control mice suggesting increased apoptosis of T cells. These results suggest that lack of transition from an innate to adaptive host response is associated with lethality of respiratory tularemia.

Background: Francisella tularensis subsp. tularensis is classified as a Category A bioweapon that is capable of establishing a lethal infection in humans upon inhalation of very few organisms. However, the virulence mechanisms of this organism are not well characterized. Francisella tularensis subsp. novicida, which is an equally virulent subspecies in mice, was used in concert with a microPET scanner to better understand its temporal dissemination in vivo upon intranasal infection and how such dissemination compares with other routes of infection. Adult mice were inoculated intranasally with F. tularensis subsp. novicida radiolabeled with 64Cu and imaged by microPET at 0.25, 2 and 20 hours post-infection., Results: 64Cu labeled F. tularensis subsp. novicida administered intranasally or intratracheally were visualized in the respiratory tract and stomach at 0.25 hours post infection. By 20 hours, there was significant tropism to the lung compared with other tissues. In contrast, the images of radiolabeled F. tularensis subsp. novicida when administered intragastrically, intradermally, intraperitoneally and intravenouslly were more generally limited to the gastrointestinal system, site of inoculation, liver and spleen respectively. MicroPET images correlated with the biodistribution of isotope and bacterial burdens in analyzed tissues., Conclusion: Our findings suggest that Francisella has a differential tissue tropism depending on the route of entry and that the virulence of Francisella by the pulmonary route is associated with a rapid bacteremia and an early preferential tropism to the lung. In addition, the use of the microPET device allowed us to identify the cecum as a novel site of colonization of Francisella tularensis subsp. novicida in mice.

Francisella tularensis is a highly virulent facultative intracellular bacterium and is considered a potential biological warfare agent. Inhalation tularemia can lead to the development of bronchopneumonia, which is frequently fatal without medical intervention. Treatment strategies that directly target the respiratory mucosa may extend the efficacy of therapy, particularly for the medical management of acute aerosol exposure. To this end, we describe an intranasal (i.n.) strategy for the treatment of pulmonary Francisella infection in mice that uses a combinatorial approach with the conventional antibiotic gentamicin and interleukin 12 (IL-12). The i.n. administration of IL-12 alone promoted bacterial clearance and extended the time to death but did not prevent mortality against lethal pulmonary challenge with Francisella tularensis subsp. novicida. However, i.n. treatment with gentamicin and IL-12 therapeutically at 8 and 24 h after challenge markedly enhanced the rate of survival (70 to 100%) against pulmonary infection compared to the rates of survival for animals treated with antibiotic alone (17%) or IL-12 alone (0%). A delay in combinatorial therapy over a span of 4 days progressively decreased the efficacy of this treatment regimen. This combinatorial treatment was shown to be highly dependent upon the induction of endogenous gamma interferon and may also involve the activation of natural killer cells. Together, these findings suggest that IL-12 may be a potent adjunct for chemotherapy to enhance drug effectiveness against pulmonary Francisella infection.