Complement C3 as a Prompt for Human Macrophage Death during Infection with Francisella tularensis Strain SCHU S4.

Tularemia is caused by the Gram-negative bacterial pathogen Francisella tularensis Infection of macrophages and their subsequent death are believed to play important roles in the progression of disease. Because complement is a particularly effective opsonin for Francisella, we asked whether complement-dependent uptake of F. tularensis strain SCHU S4 affects the survival of primary human macrophages during infection. Complement component C3 was found to be an essential opsonin in human serum not only for greatly increased uptake of SCHU S4 but also for the induction of macrophage death. Single-cell analysis also revealed that macrophage death did not require a high intracellular bacterial burden. In the presence of C3, macrophage death was observed at 24 h postinfection in a quarter of the macrophages that contained only 1 to 5 bacterial cells. Macrophages infected in the absence of C3 rarely underwent cell death, even when they contained large numbers of bacteria. The need for C3, but not extensive replication of the pathogen, was confirmed by infections with SCHU S4 ΔpurMCD, a mutant capable of phagosome escape but of only limited cytosolic replication. C3-dependent Francisella uptake alone was insufficient to induce macrophage death, as evidenced by the failure of the phagosome escape-deficient mutant SCHU S4 ΔfevR to induce cell death despite opsonization with C3. Together, these findings indicate that recognition of C3-opsonized F. tularensis, but not extensive cytosolic replication, plays an important role in regulating macrophage viability during intracellular infections with type A F. tularensis.

Francisella tularensis Confronts the Complement System.

Francisella tularensis has developed a number of effective evasion strategies to counteract host immune defenses, not the least of which is its ability to interact with the complement system to its own advantage. Following exposure of the bacterium to fresh human serum, complement is activated and C3b and iC3b can be found covalently attached to the bacterial surface. However, the lipopolysaccharide and capsule of the F. tularensis cell wall prevent complement-mediated lysis and endow the bacterium with serum resistance. Opsonization of F. tularensis with C3 greatly increases its uptake by human neutrophils, dendritic cells and macrophages. Uptake occurs by an unusual looping morphology in human macrophages. Complement receptor 3 is thought to play an important role in opsonophagocytosis by human macrophages, and signaling through this receptor can antagonize Toll-like receptor 2-initiated macrophage activation. Complement C3 also determines the survival of infected human macrophages and perhaps other cell types. C3-opsonization of F. tularensis subsp. tularensis strain SCHU S4 results in greatly increased death of infected human macrophages, which requires more than complement receptor engagement and is independent of the intracellular replication by the pathogen. Given its entry into the cytosol of host cells, F. tularensis has the potential for a number of other complement-mediated interactions. Studies on the uptake C3-opsonized adenovirus have suggested the existence of a C3 sensing system that initiates cellular responses to cytosolic C3b present on invading microbes. Here we propose that C3 peptides enter the cytosol of human macrophages following phagosome escape of F. tularensis and are recognized as intruding molecular patterns that signal host cell death. With the discovery of new roles for intracellular C3, a better understanding of tularemia pathogenesis is likely to emerge.

Programmed cell death and the pathogenesis of tissue injury induced by type A Francisella tularensis.

Francisella tularensis is a highly virulent bacterial species that causes various forms of tularemia in humans. The urgency in understanding the pathogenesis of these diseases has stimulated unprecedented interest in this bacterial species over the past few years. Recent findings underscore a number of important distinctions between the Francisella ssp. and emphasize the importance of using type A F. tularensis strains when characterizing pathophysiological responses that are relevant to the lethal forms of human disease. This review focuses on the mediators of cell death induction in infected tissues and the implications of these processes on the pathophysiological changes observed in various host species.

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.

NK cells and gamma interferon coordinate the formation and function of hepatic granulomas in mice infected with the Francisella tularensis live vaccine strain.

Host innate immune responses to many intracellular pathogens include the formation of inflammatory granulomas that are thought to provide a physical barrier between the microbe and host. Because two common features of infections with the live vaccine strain (LVS) of Francisella tularensis within the mouse liver are the formation of granulomas and the production of gamma interferon (IFN-gamma), we have asked what role IFN-gamma plays in hepatic granuloma formation and function. Francisella antigens were predominantly localized within granulomas of the livers of mice infected with F. tularensis LVS 4 days postinfection. Hepatic granulomas also contained large numbers of dying cells, some of which coexpressed the F4/80 macrophage antigen and activated caspase-3. IFN-gamma-deficient mice did not form normal numbers of hepatic granulomas and showed widely disseminated Francisella antigens within the liver. The incidence of cell death within hepatic granulomas also decreased significantly in the absence of IFN-gamma. Inducible NO synthase (iNOS) expression was restricted to the granulomas of wild-type mice but was not seen for IFN-gamma-deficient mice. Cell death within granulomas was also significantly decreased for iNOS-deficient mice. The predominant IFN-gamma-expressing cells in the liver were NK cells. Depleting NK cells resulted in the expression of bacterial antigens and iNOS outside the granulomas and the appearance of extensive hepatic focal necrosis. These findings indicate that IFN-gamma and hepatic NK cells that are activated during F. tularensis LVS infections regulate hepatic granuloma formation, the spatial containment of infection, the expression of iNOS, and the induction of cell death within the liver.

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.

Coactivating signals for the hepatic lymphocyte gamma interferon response to Francisella tularensis.

The facultative intracellular bacterium Francisella tularensis is capable of causing systemic infections in various hosts, including mice and humans. The liver is a major secondary site of F. tularensis infection, but hepatic immune responses to the pathogen remain poorly defined. Immune protection against the pathogen is thought to depend on the cytokine gamma interferon (IFN-gamma), but the cellular basis for this response has not been characterized. Here we report that natural killer cells from the livers of naïve uninfected mice produced IFN-gamma when challenged with live bacteria in vitro and that the responses were greatly increased by coactivation of the cells with either recombinant interleukin-12 (IL-12) or IL-18. Moreover, the two cytokines had strong synergistic effects on IFN-gamma induction. Neutralizing antibodies to either IL-12 or IL-18 inhibited IFN-gamma production in vitro, and mice deficient in the p35 subunit of IL-12 failed to show IFN-gamma responses to bacterial challenge either in vitro or in vivo. Clinical isolates of highly virulent type A Francisella tularensis subsp. tularensis organisms were comparable to the live attenuated vaccine strain of Francisella tularensis subsp. holarctica in their ability to induce IL-12 and IFN-gamma expression. These findings demonstrate that cells capable of mounting IFN-gamma responses to F. tularensis are resident within the livers of uninfected mice and depend on coactivation by IL-12 and IL-18 for optimum responses.

Gamma interferon production by hepatic NK T cells during Escherichia coli infection is resistant to the inhibitory effects of oxidative stress.

The reductive-oxidative status of tissues regulates the expression of many inflammatory genes that are induced during gram-negative bacterial infections. The cytokine gamma interferon (IFN-gamma) is a potent stimulus for host inflammatory gene expression, and oxidative stress has been shown to inhibit its production in mice challenged with Escherichia coli bacteria. The objective of the present study was to characterize the cells that produced IFN-gamma in a mouse bacterial peritonitis model and determine the effects of oxidative stress on their activation. The liver contained large numbers of IFN-gamma-expressing lymphocytes following challenge with viable E. coli bacteria. The surface phenotypes of IFN-gamma-expressing hepatic lymphocytes were those of natural killer (NK) cells (NK1.1(+) CD3(-)), conventional T cells (NK1.1(-) CD3(+)), and NK T cells (NK1.1(+) CD3(+)). Treating mice with diethyl maleate to deplete tissue thiols significantly impaired IFN-gamma production by NK cells, conventional T cells, and CD1d-restricted NK T cells in response to E. coli challenge. However, IFN-gamma expression by a subset of NK T cells, which did not bind alpha-galactosylceramide-CD1d tetramers, was resistant to the inhibitory effects of tissue oxidative stress. Stress-resistant IFN-gamma-expressing cells were also predominantly CD8(+) and bore gamma delta T-cell antigen receptors. The residual IFN-gamma response by NK T cells may explain previous reports of hepatic gene expression following gram-negative bacterial challenge in thiol-depleted mice. The finding also demonstrates that innate immune cells differ significantly in their responses to altered tissue redox status.