ABSTRACT 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 Δ purMCD , 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 Δ fevR 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 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.

647 Objectives Alkaline protease (AP) reduces the progress of TNF-α-induced mouse inflammatory bowel disease. FDG-PET provides accurate, non-invasive visualization of inflammation. We investigated whether FDG-PET imaging is useful to detect therapeutic response of alkaline protease in acute inflammatory bowel disease (IBD). Methods Rectal administration of the hapten reagent oxazolone dissolved in ethanol induces a severe IBD in C56BL/6 mice. The mice are divided into three groups: Control, Oxa, and Oxa+AP administered mice. After 4 hours of oxazolone administration, FDG-PET images were acquired using small animal PET/X-ray scanner. PET data were acquired from whole body approximately 60 min after intravenous administration of 50 μCi of 18F-FDG. The mice were sacrificed and the colon was removed. Ex vivo PET images were acquired. To determine whether AP blocks TNF-α activity in this acute inflammation induced mice, samples of their intestines were collected. And then TNF-α immunostaining of intestines was carried out. Results Oxazolone treated mouse model showed swelled intestine and darker color than the other group. Immunohistochemistry assay showed less TNF-α staining in villous epithelium of group Oxa+AP than of group Oxa. FDG uptake increased 2.0 times of control when oxazolone had treated (p=0.016). However, FDG uptake increased 1.4 times of control when oxazolone and AP administered together (p=0.001). Conclusions Pretreatment of alkaline protease reduced not only TNF-α expression but also FDG uptake. FDG-PET shows therapeutic effect of AP in mouse IBD.

To determine predictors of conversion from percutaneous (PEVAR) to an open approach (OEVAR) during groin access of endovascular aortic aneurysm repair (EVAR). A retrospective review of all EVAR patients from January 2009 through June 2011. Target vessels were classified as mild (<1/3), moderate (1/3 to ½) or severe (>½) according to circumference of calcification. We investigated 400 access sites for 200 patients who underwent EVAR. Averages were age 72.8 + 9.0, vessel size 9.6 + 1.8, sheath size 17.1 + 3.0, BMI 27.6 + 5.3, and eGFR 68.5 + 24.2%. Co-morbidities included dyslipidemia (64.5%), and hypertension (42.0%). There were 132 OEVAR (66 patients), 2 mixed (OEVAR and PEVAR for 1 patient) and 266 (133 patients) PEVAR approaches. Use of PEVAR increased over time [45.5% (2009), 77.8% (2010) and up to 88.5% (2011); P = .001] while conversion decreased [24.3% (2009), 8.7% (2010) and 4.3% (2011); P = .001]. Length of stay was significantly shorter for PEVAR (3.0 + 4.6 vs 6.1 + 9.1 days; P = .013). For the 266 PEVAR approaches, 32 (32/266 = 12.0%) had to be converted. After multivariate analysis, it was found that severely calcified arteries were most predictive of conversion (odds ratio (OR), 36.4; P < .001). Year of procedure (2010; OR, 0.17; P = .001), (2011; OR, 0.20; P = .049), female gender (OR, 3.1; P = .017), moderately calcified arteries (OR, 2.5; P = .085) and age (OR, 2.3 (per decade); P = .002) were all also significant. Vessel and sheath size along with BMI were non-significant. PEVAR was found to be safe, reliable and feasible. Several factors including vessel calcification, age and female gender should be considered before PEVAR intervention.

ABSTRACT 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.

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.

ABSTRACTHost 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) ofFrancisella tularensiswithin the mouse liver are the formation of granulomas and the production of gamma interferon (IFN-γ), we have asked what role IFN-γ plays in hepatic granuloma formation and function.Francisellaantigens were predominantly localized within granulomas of the livers of mice infected withF. tularensisLVS 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-γ-deficient mice did not form normal numbers of hepatic granulomas and showed widely disseminatedFrancisellaantigens within the liver. The incidence of cell death within hepatic granulomas also decreased significantly in the absence of IFN-γ. Inducible NO synthase (iNOS) expression was restricted to the granulomas of wild-type mice but was not seen for IFN-γ-deficient mice. Cell death within granulomas was also significantly decreased for iNOS-deficient mice. The predominant IFN-γ-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-γ and hepatic NK cells that are activated duringF. tularensisLVS infections regulate hepatic granuloma formation, the spatial containment of infection, the expression of iNOS, and the induction of cell death within the liver.

Historically colostrum and milk have been thought to confer immunity on the neonate only by virtue of their immunoglobulin content. Recently we have ohserved that colostrum also contains viable T lymphocytes capable of expressing cell-mediated immunity in vitro and have employed techniques of lymphocyte culture to elucidate the local nature of mammary tissue immunity at the T-cell level. The results indicate that the activity of colostral lymphocytes appears not to represent the total immunological experience of the mother but that they may contain reactive clones beneficial for the suckling. Colostral immunity appears to depend upon sensitizing events within the intestine and respiratory tract, followed by the migration of lymphoid precursors to the breast, suggesting a relationship between the expression of immunity at various secretory surfaces. The immunologic benefits afforded a newborn by suckling traditionally have been thought to be mediated exclusively by soluble milk proteins known as immunoglobulins. The primary vehicle for passive immunization of the neonate is either peripherally-synthesized immunoglobulin IgG antibody (often of a particular subclass) or secretory IgA produced locally within the mammary gland. Elucidation of the origin of colostral antibody has depended, in part, on techniques that allow for the identification of particular subpopulations of lymphoReceived November 10, 1976. 1 The cost of this study was defrayed in part by USPHS Grants AI-10678 and AI-42531 and a grant from the Ross Laboratories. cytes capable of synthesizing milk-borne immunoglobulins. In addition to providing exciting data about the origin of lymphoid cells in the parenchyma and alveolar epithelium of the mammary gland, these experiments also have determined that the mammary exosecretion, as with other exocrine secretions, contains a natural component of viable lymphocytes, the immunologic characterization of which has provided further insight into the local nature of secretory immunity at mucosal surfaces. This article will attempt to elucidate the cellular basis and origin of local secretory immunity, with special reference to the mammary gland and its secretions. In so doing, we will present data supporting the concept that immunity mediated by T lymphocytes also can be local in nature and suggest a conceptual framework that links secretory immunity (mediated by T or B lymphocytes) at distant mucosal surfaces. GUT-ASSOCIATED LYMPHOID TISSUE AS A MODEL FOR LOCAL IMMUNITY It has been known for approximately 50 yr that immunity to certain enteric pathogens correlates more with concentration of coproantibodies than with concentration of circulating serum antibody (11, 24). From these early observations the concept of local immunity has developed. Following the presentation of an antigen to a particular microenvironment, generally a mucosal surface, an immune response is elicited that is restricted anatomically to the site of immune induction and/or to a distant mucosal surface but is not manifested systemically. The classical example is the mammalian intestine, in which intraluminal antigen enters the gut-associated lymphoid tissue (GALT) and immunocompetent ceils are mobilized by a process that restricts immune effector functions to the intestinal mucosa (22, 40, 51, 87), often

ABSTRACTThe facultative intracellular bacteriumFrancisella tularensisis capable of causing systemic infections in various hosts, including mice and humans. The liver is a major secondary site ofF. tularensisinfection, 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-γ), 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-γ 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-γ induction. Neutralizing antibodies to either IL-12 or IL-18 inhibited IFN-γ production in vitro, and mice deficient in the p35 subunit of IL-12 failed to show IFN-γ responses to bacterial challenge either in vitro or in vivo. Clinical isolates of highly virulent type AFrancisella tularensissubsp.tularensisorganisms were comparable to the live attenuated vaccine strain ofFrancisella tularensissubsp.holarcticain their ability to induce IL-12 and IFN-γ expression. These findings demonstrate that cells capable of mounting IFN-γ responses toF. tularensisare resident within the livers of uninfected mice and depend on coactivation by IL-12 and IL-18 for optimum responses.

ABSTRACT The production of gamma interferon (IFN-γ) 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-γ expression. The present study was undertaken to determine whether or not macrophages (Mφ) or dendritic cells (DC) provide coactivating signals for the liver IFN-γ 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 Mφ and DC significantly augmented the IFN-γ 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-γ production in response to viable F. tularensis organisms. Conversely, liver lymphocytes from TLR2-deficient mice cocultured with wild-type accessory cells produced IFN-γ at levels comparable to those for wild-type hepatic lymphocytes. These findings indicate that TLR2 controls hepatic lymphocyte IFN-γ responses to F. tularensis by regulating DC IL-12 production. While Mφ also coinduced hepatic IFN-γ production in response to F. tularensis, they did so in a fashion less dependent on TLR2.

Enzyme replacement therapy is a growing clinical intervention primarily directed at correcting enzyme deficiencies, including the impaired production of digestive enzymes. We have asked whether microbial proteases have any potential therapeutic value in the treatment of inflammatory conditions whose pathogenesis involves peptide inflammatory mediators. The proteases of Aspergillus oryzae were selected due to their routine use in the processing foods. Pretreating mice with a partially purified mixture of A. oryzae proteases protected them against TNF-α-dependent lethality in an endotoxemia model. Pretreated mice showed decreased concentrations of detectable TNF-α, following challenge with bacterial endotoxin. Treating TNF- α with this protease preparation partially degraded the cytokine and blocked its ability to induce nitric oxide production by mouse myoblast cells in vitro. Three proteases were purified from this preparation by ion exchange chromatography and found by amino-terminal sequencing to be alkaline protease (AP), deuterolysin (DE) and 26 kDa protease. Purified AP inactivated mouse TNF-α. Treating TNF- α in vitro with AP, also destroyed its ability to induce activated caspase-3 in intestinal epithelial cells in vivo. Injecting with 1 μg AP by the i.p. route as much as 15 min prior to challenge with 5 ng TNF-α blocked the activation of intestinal caspase-3. These results suggest that A. oryzae AP has anti-inflammatory activity that may have therapeutic value in treating TNF-α-dependent inflammatory bowel diseases.

ABSTRACT 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-γ) 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-γ in a mouse bacterial peritonitis model and determine the effects of oxidative stress on their activation. The liver contained large numbers of IFN-γ-expressing lymphocytes following challenge with viable E. coli bacteria. The surface phenotypes of IFN-γ-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-γ production by NK cells, conventional T cells, and CD1d-restricted NK T cells in response to E. coli challenge. However, IFN-γ expression by a subset of NK T cells, which did not bind α-galactosylceramide-CD1d tetramers, was resistant to the inhibitory effects of tissue oxidative stress. Stress-resistant IFN-γ-expressing cells were also predominantly CD8+ and bore γδ T-cell antigen receptors. The residual IFN-γ 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.

Morrison, D. C.; Zhang, G.; Shen, J.; Huo, K.; Li, Y; Dhar, A.; Xue, Q.; Parmely, M. J.; Qureshi, N.; Papasian, C. J.; Van Way, C. III; Gao, J. J. Author Information

Oxidative stress occurs in animals challenged with bacterial endotoxin and can affect the expression of important host inflammatory genes. However, much less is known about the effects of oxidative stress on responses to gram-negative bacteria. The current study compared the effects of redox imbalance on hepatic responses of mice to Escherichia coli bacteria versus purified endotoxic lipopolysaccharide (LPS). Oxidative stress induced by glutathione depletion virtually eliminated hepatic tumor necrosis factor alpha responses to both E. coli and LPS. Inducible NO synthase (iNOS) and intercellular adhesion molecule-1 (ICAM-1) expression was also markedly inhibited by glutathione depletion in LPS challenged mice, but was unaffected in E. coli-infected animals. Three findings suggested that gamma interferon (IFN-gamma) production explained the differences between LPS and bacterial challenge. Glutathione depletion completely inhibited the IFN-gamma response to LPS, but only partially inhibited IFN-gamma production in infected mice. Exogenous IFN-gamma restored iNOS and ICAM-1 responses to LPS in stressed mice. Conversely, IFN-gamma deficient, glutathione depleted mice showed a marked decrease in iNOS and ICAM-1 expression when challenged with E. coli. These findings indicate that both the nature of the microbial challenge and the production of IFN-gamma can be important in determining the effects of redox imbalance during gram-negative bacterial infections.

Adenyl carbocyclic nucleosides have potent anti-inflammatory effects on a number of cell types. Notable in this regard is their ability to inhibit the production of tumor necrosis factor-alpha (TNF-alpha) by mouse macrophages that have been activated with bacterial lipopolysaccharide (LPS). Because the transcriptional activation of the mouse TNF-alpha gene is highly dependent on kappaB enhancers, the present study determined whether the synthetic carbocyclic nucleoside 9-[(1S,3R)-cis-cyclopentan-3-ol]adenine (cPA) inhibited LPS-induced nuclear factor-kappaB (NF-kappaB) activation in these cells. Stimulation of either mouse peritoneal macrophages or RAW 264. 7 macrophage-like cells with LPS led to the appearance of four distinct kappaB-binding nucleoprotein complexes detected by gel mobility shift assays. Cells treated with 100 microM cPA showed significantly reduced levels of NF-kappaB activation as evidenced by measurements of nucleoprotein kappaB-binding activity and diminished kappaB-dependent transcriptional activation. However, both the LPS-induced degradation of the cytoplasmic NF-kappaB inhibitor IkappaBalpha and the nuclear translocation of the NF-kappaB p50, p65, and c-Rel peptides were unaffected by treatment of the cells with the nucleoside. These findings suggest that certain adenyl carbocyclic nucleosides inhibit the activation of NF-kappaB/Rel complexes by a novel mechanism that results in an inhibition of their DNA-binding activities, without blocking their dissociation from IkappaBalpha or their nuclear translocation.

Endotoxemia is accompanied by significant changes in the reductive-oxidative (redox) balance of critical target organs. Redox stress has been shown to regulate the expression of proinflammatory genes that are induced by endotoxic lipopolysaccharide (LPS) in vitro; however, much less is known about the effects of redox imbalance on LPS-induced gene expression in vivo. To assess the effects of redox stress on inflammatory responses in endotoxemia, mice were treated with either diethyl maleate (DEM), a glutathione-depleting agent, or buthionine sulfoximine (BSO), an inhibitor of glutathione synthesis, and challenged with LPS. While serum tumor necrosis alpha (TNF-alpha) responses and the appearance of TNF-alpha-positive Kupffer cells in the liver were virtually eliminated by DEM or BSO treatment, the expression of both CD14 and inducible NO synthase (iNOS) by Kupffer cells was unaffected by glutathione depletion. By contrast, LPS-induced hepatocyte and hepatic sinusoidal endothelial cell iNOS expression was significantly inhibited in DEM-treated mice. Hepatocyte iNOS induced by recombinant mouse TNF-alpha was also inhibited by DEM treatment. These results indicate that the effects of oxidative stress in this organ are cell type specific and suggest that both the production and the action of TNF-alpha are substantially influenced by the redox state of the liver during endotoxemia.