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The World Health Organization estimates ~180,000 deaths occur annually from burn-related injuries. Many victims who survive the initial burn trauma succumb to bacterial infections that lead to sepsis during treatment. Although advancements in burn care continue to improve in high-income countries due to their burn centers and advanced research, low and middle-income countries continue to see high frequencies of burn injuries and burn-related deaths due to secondary infections. Bacterial-derived sepsis is the most life-threatening danger for people that survive burn injuries. Here we provide evidence for the first time that a subeschar seroma forms postburn even in the absence of infection in mice. The seroma fills with a volume estimated at 500 µL of fluid, 25% of the blood supply, free of red blood cells. The seroma fluid supports robust Pseudomonas aeruginosa (PA) growth and contains inflammatory cytokines and chemokines, which recruit immature neutrophils and monocytes to the seroma in the absence of endothelial breakdown. These immune cells fail to contain PA expansion and dissemination. This recruitment of monocytes and immature neutrophils may result in sequestering these critical immune cells away from other tissues during a pivotal time during bacterial dissemination, promoting PA-mediated sepsis.

Despite the dramatic increase in antimicrobial resistance, there is a dearth of antibiotics in development and few pharmaceutical companies working in the field. Further, any new antibiotics are likely to have a short shelf life. Ab-based interventions offer alternatives that are not likely to be circumvented by the widely prevalent antibiotic resistance genes. Bovine colostrum (BC)—the first milk after parturition, rich in nutrients and immune components—promotes gut integrity and modulates the gut microbiome. We developed a hyperimmune BC (HBC) enriched in Abs to a highly conserved LOS core region of Gram-negative bacteria by immunizing pregnant cows with a vaccine comprised of detoxified LOS from Escherichia coli O111 Rc (J5) mutant non-covalently complexed to group B meningococcal outer membrane protein (J5dLOS/OMP). This vaccine generated robust levels of anti-J5 LOS Ab in the colostrum. When given orally to neutropenic rats challenged orally with Pseudomonas aeruginosa, administration of HBC improved survival compared to non-immune rats, while both BC preparations improved survival compared to PBS controls. Elevated circulating endotoxin levels correlated with mortality. HBC and to a lesser extent non-immune BC reduced bacterial burden from the liver, lung, and spleen. We conclude that HBC and to a lesser extent BC may be effective supplements that improve outcome from lethal gut-derived disseminated infection and may reduce transmission of Gram-negative bacilli from the gastrointestinal tract.

BACKGROUND Aeromedical evacuation can expose traumatically injured patients to low pressure (hypobaria) and hypoxia. Here, we sought to assess the impact of hypobaria on inflammation, organ injury, and mortality in a mouse model of polytrauma. METHODS Eight to 12-week-old male C57BL/6J mice were subjected to sham or polytrauma consisting of bowel ischemia by superior mesenteric artery occlusion, hindlimb muscle crush, and tibia fracture. Two hours after injury, animals were randomized to undergo either 6 h of hypobaria or sea-level, room air conditions. At 8 or 24 h after injury, transthoracic echocardiography was performed. Acute kidney injury (AKI) biomarkers were measured by qRT-PCR. Plasma cytokine and endothelial injury markers were determined by enzyme-linked immunosorbent assay. RESULTS Eight hours after traumatic injury, mice exhibited a marked increase in plasma IL-6 (57 pg/mL vs. 1,216 pg/mL), AKI with increased Ngal and Kim-1, and endothelial injury as evidenced by significantly increased plasma hyaluronic acid (96 ng/mL vs.199 ng/mL), thrombomodulin (23.2 ng/mL vs. 58.9 ng/mL), syndecan-1 (0.99 ng/mL vs. 4.34 ng/mL), and E-selectin (38.6 ng/mL vs. 62.7 ng/mL). The trauma mice also developed cardiac dysfunction with decreased cardiac output and stroke volume at 8 h postinjury. Hypobaric exposure after polytrauma led to decreased ejection fraction (81.0% vs. 74.2%, P

Pseudomonas aeruginosa is a Gram-negative nosocomial pathogen and one of the most prevalent organisms isolated from burn wounds worldwide. Pseudomonas aeruginosa strain M2 (O5 serotype, type B flagella) is utilized for examining the murine model associated with burns. Pseudomonas aeruginosa M2 is similar in lethality to common laboratory P. aeruginosa strains when infecting CD-1 mice. Conversely, we recently showed that, relative to these strains, P. aeruginosa M2-infected mice are more susceptible to sepsis and demonstrate a 6-log reduction in LD50 from subcutaneous infection at the infection site directly after 10% total body surface area burn. To better understand this striking phenotypic difference from other P. aeruginosa strains employed in burn models, we sequenced the P. aeruginosa M2 genome. A total of 4,136,641 read pairs were obtained, providing an average genome coverage of 97.5X; subsequent assembly yielded a draft genome with 187 contigs comprising 6,360,304 bp with a G + C content of 66.45%. Genome-based phylogeny estimation of 92 P. aeruginosa strains placed P. aeruginosa M2 with P. aeruginosa-12-4-4(59), a nonairway clinical strain isolated from the blood culture of a burn patient. Phylogenomic analyses identified genes shared between P. aeruginosa M2 and P. aeruginosa 14, another strain exhibiting increased lethality in thermal tissues, as well as P. aeruginosa M2 unique genes with diverse functions like degradation of toxic aromatic compounds, iron scavenging, swarming motility and biofilm formation, defense against invasive DNA, and host assault. Predicted lateral gene transfers illuminate proteins heretofore uncharacterized for roles in P. aeruginosa biology. Our work yields a rich resource for assessing P. aeruginosa genes required for increased lethality in burn tissue seroma.

To gain insight into sialic acid biology and sialidase/neuraminidase (NEU) expression in mature human neutrophil (PMN)s, we studied NEU activity and expression in PMNs and the HL60 promyelocytic leukemic cell line, and changes that might occur in PMNs undergoing apoptosis and HL60 cells during their differentiation into PMN-like cells. Mature human PMNs contained NEU activity and expressed NEU2, but not NEU1, the NEU1 chaperone, protective protein/cathepsin A(PPCA), NEU3, and NEU4 proteins. In proapoptotic PMNs, NEU2 protein expression increased > 30.0-fold. Granulocyte colony-stimulating factor protected against NEU2 protein upregulation, PMN surface desialylation and apoptosis. In response to 3 distinct differentiating agents, dimethylformamide, dimethylsulfoxide, and retinoic acid, total NEU activity in differentiated HL60 (dHL60) cells was dramatically reduced compared to that of nondifferentiated cells. With differentiation, NEU1 protein levels decreased > 85%, PPCA and NEU2 proteins increased > 12.0-fold, and 3.0-fold, respectively, NEU3 remained unchanged, and NEU4 increased 1.7-fold by day 3, and then returned to baseline. In dHL60 cells, lectin blotting revealed decreased α2,3-linked and increased α2,6-linked sialylation. dHL60 cells displayed increased adhesion to and migration across human bone marrow-derived endothelium and increased bacterial phagocytosis. Therefore, myeloid apoptosis and differentiation provoke changes in NEU catalytic activity and protein expression, surface sialylation, and functional responsiveness.

Of the 486,000 burn injuries that required medical treatment in the United States in 2016, 40,000 people were hospitalized, with >3,000 fatalities. After burn injury, humans are at increased risk of sepsis and mortality from infections caused by Pseudomonas aeruginosa, an opportunistic pathogen. We hypothesize that systemic events were initiated from the burn that increased the host’s susceptibility to P. aeruginosa. A nonlethal 10% total body surface area (TBSA), full-thickness flame burn was performed in CD-1 mice without and with subsequent P. aeruginosa (strain M2) infection. The 50% lethal dose for subcutaneous infection with P. aeruginosa M2 at the burn site immediately after the burn decreased by 6 log, with mortality occurring between 18 and 26 h, compared with P. aeruginosa-infected mice without burn injury. Bacteria in distal organs were detected by 18 h, concurrent with the onset of clinical symptoms. Serum proinflammatory cytokines (interleukin-6 [IL-6], IL-1β, gamma interferon, and tumor necrosis factor alpha) and the anti-inflammatory cytokine IL-10 were first detected at 12 h postburn with infection and continued to increase until death. Directly after burn alone, serum levels of HMGB1, a danger-associated molecular pattern and TLR4 agonist, transiently increased to 50 ng/ml before returning to 20 ng/ml. Burn with P. aeruginosa infection increased serum HMGB1 concentrations >10-fold (250 ng/ml) at the time of death. This HMGB1-rich serum stimulated TLR4-mediated NF-κB activation in a TLR4 reporter cell line. Treatment of infected burned mice with P5779, a peptide inhibitor of HMGB1, increased the mean survival from 23 to 42 h (P

Soft-tissue bacterial infection can progress to severe sepsis and septic shock as a result of a disproportionate inflammatory response, characterized by an excessive release of cytokines and influx of immune cells. Reltecimod (previously known as AB103 or p2TA), a peptide derived from the T-cell receptor CD28, modulates the host immune response by targeting the co-stimulatory pathway, which is essential for the induction of multiple pro-inflammatory cytokines. Consequently, reltecimod has demonstrated beneficial effects against different bacterial infections, their exotoxins and endotoxins, and ionizing radiation. The dosing regimen of reltecimod was evaluated in three mouse models of infection. The effect of the number of reltecimod doses with respect to survival, cytokine/chemokine levels, and blood leukocyte profiles was assessed. Overall, mice treated with a single intravenous dose of reltecimod (5 mg/kg) at 1–2 h after infection showed significantly greater survival as compared with saline-treated controls. Mice treated with a second doses demonstrated improved survival compared with saline-treated controls. However, in all models of infection, administration of a single therapeutic dose of reltecimod was superior to two or multiple doses. Further examination showed that the single therapeutic dose of reltecimod was associated with an early (within 24 h) decrease in cytokine/chemokine levels and most circulating leukocyte subpopulations. A second dose of reltecimod did not improve these early positive effects and appeared to attenuate further changes. These results provided insight into the mechanism of action of reltecimod and established a basis for the dosing regimen utilized in clinical trials, where reltecimod is administered as a single dose.

We previously reported that flagellin-expressing Pseudomonas aeruginosa (Pa) provokes NEU1 sialidase-mediated MUC1 ectodomain (MUC1-ED) desialylation and MUC1-ED shedding from murine lungs in vivo. Here, we asked whether Pa in the lungs of patients with ventilator-associated pneumonia might also increase MUC1-ED shedding. The levels of MUC1-ED and Pa-expressed flagellin were dramatically elevated in bronchoalveolar lavage fluid (BALF) harvested from Pa-infected patients, and each flagellin level, in turn, predicted MUC1-ED shedding in the same patient. Desialylated MUC1-ED was only detected in BALF of Pa-infected patients. Clinical Pa strains increased MUC1-ED shedding from cultured human alveolar epithelia, and FlaA and FlaB flagellin-expressing strains provoked comparable levels of MUC1-ED shedding. A flagellin-deficient isogenic mutant generated dramatically reduced MUC1-ED shedding compared with the flagellin-expressing wild-type strain, and purified FlaA and FlaB recapitulated the effect of intact bacteria. Pa:MUC1-ED complexes were detected in the supernatants of alveolar epithelia exposed to wild-type Pa, but not to the flagellin-deficient Pa strain. Finally, human recombinant MUC1-ED dose-dependently disrupted multiple flagellin-driven processes, including Pa motility, Pa biofilm formation, and Pa adhesion to human alveolar epithelia, while enhancing human neutrophil-mediated Pa phagocytosis. Therefore, shed desialylated MUC1-ED functions as a novel flagellin-targeting, Pa-responsive decoy receptor that participates in the host response to Pa at the airway epithelial surface.

Klebsiella pneumoniae is a common cause of sepsis and is particularly associated with healthcare-associated infections. New strategies are needed to prevent or treat infections due to the emergence of multi-drug resistant K. pneumoniae. The goal of this study was to determine the diversity and distribution of O (lipopolysaccharide) and K (capsular polysaccharide) antigens on a large (>500) global collection of K. pneumoniae strains isolated from blood to inform vaccine development efforts. A total of 645 K. pneumoniae isolates were collected from the blood of patients in 13 countries during 2005-2017. Antibiotic susceptibility was determined using the Kirby-Bauer disk diffusion method. O antigen types including the presence of modified O galactan types were determined by PCR. K types were determined by multiplex PCR and wzi capsular typing. Sequence types of isolates were determined by multilocus sequence typing (MLST) targeting seven housekeeping genes. Among 591 isolates tested for antimicrobial resistance, we observed that 19.3% of isolates were non-susceptible to carbapenems and 62.1% of isolates were multidrug resistant (from as low as 16% in Sweden to 94% in Pakistan). Among 645 isolates, four serotypes, O1, O2, O3, and O5, accounted for 90.1% of K. pneumoniae strains. Serotype O1 was associated with multidrug resistance. Fifty percent of 199 tested O1 and O2 strains were gmlABC-positive, indicating the presence of the modified polysaccharide subunit D-galactan III. The most common K type was K2 by both multiplex PCR and wzi capsular typing. Of 39 strains tested by MLST, 36 strains were assigned to 26 known sequence types of which ST14, ST25, and ST258 were the most common. Given the limited number of O antigen types, diverse K antigen types and the high multidrug resistance, we believe that an O antigen-based vaccine would offer an excellent prophylactic strategy to prevent K. pneumoniae invasive infection. ispartof: FRONTIERS IN MICROBIOLOGY vol:11 ispartof: location:Switzerland status: published

Injured warfighters air evacuated to tertiary medical care facilities are subjected to many stresses that may promote the development of sepsis. In this study, we tested the hypothesis that exposure to "in-flight" hypobaria and/or hyperoxia within 24 hours after onset of intra-abdominal infection in rats accelerates the development and/or severity of sepsis and neurologic injury in survivors.Sprague-Dawley rats underwent cecal ligation/puncture (CLP) or sham procedures. Twenty-four hours later, rats were then placed in hypobaric chambers for 6 hours and assigned to normobaric conditions and maintained at either 21% or 100% O2, or under hypobaric conditions (pressure equivalent to an altitude of 8,000 ft) but maintained under either 28% or 100% O2. Two days after CLP or sham, blood samples were obtained for cytokine levels, and mitochondria were isolated from the brain and heart of a subset of animals for analysis of mitochondrial oxygen consumption. Animals were also evaluated for neuromotor impairment before and 15 days postsurgery.Among the 70 rats studied, 16.7% of CLP but none of the sham-treated rats died. All of the CLP but none of the sham rats had evidence of peritonitis at 2 days. Twenty percent (6 of 30) CLP rats undergoing hypobaria versus 12.5% (3 of 24) of CLP rats exposed to normobaria died (p = 0.715) while 12% (3 of 25) of CLP rats exposed to hyperoxia versus 20.7% (6 of 29) of CLP rats exposed to normoxia died (p = 0.48). The ratio of mitochondrial ATP-generating O2 consumption to resting respiration was higher in the CLP plus hypobaria under 100% compared with shams. The only difference in H2O2 production was observed in mitochondria from CLP rats exposed to hyperoxia under normobaric conditions. Composite neurologic scores obtained 15 days postinjury were lower than those at baseline for shams.We conclude that neither "in-flight" hyperoxia nor hypobaria exacerbate sepsis or neurologic injury.

Introduction: Klebsiella pneumoniae (KP) are a leading cause of healthcare-associated infections. The dramatic increase in microbial resistance to third-generation cephalosporin and carbapenem 'front line' antimicrobial agents and the paucity of new antimicrobials have left clinicians with few therapeutic options and resulted in increased morbidity and mortality. Vaccines may reduce the incidence of infections thereby reducing the necessity for antimicrobials and are not subject to antimicrobial resistance mechanisms. Areas covered: We review whole cell, subunit, capsular polysaccharide (CPS), O polysaccharide (OPS) and conjugate vaccines against KP infection, as well as alternative KP vaccine platforms. Expert opinion: Vaccine-induced antibodies to KP CPS have been protective in preclinical studies, but the number of CPS types (>77) makes vaccines against this virulence factor less feasible. Since four OPS serotypes account of ~80% of invasive KP infections and anti-OPS antibodies are also protective in preclinical studies, both OPS-based conjugate and multiple antigen presenting system (MAPS) vaccines are in active development. Vaccines based on other KP virulence factors, such as outer membrane proteins, type 3 fimbriae (MrkA) and siderophores are at earlier stages of development. Novel strategies for the clinical testing of KP vaccines need to be developed.

Pseudomonas aeruginosa (Pa) expresses an adhesin, flagellin, that engages the mucin 1 (MUC1) ectodomain (ED) expressed on airway epithelia, increasing association of MUC1-ED with neuraminidase 1 (NEU1) and MUC1-ED desialylation. The MUC1-ED desialylation unmasks both cryptic binding sites for Pa and a protease recognition site, permitting its proteolytic release as a hyperadhesive decoy receptor for Pa. We found here that intranasal administration of Pa strain K (PAK) to BALB/c mice increases MUC1-ED shedding into the bronchoalveolar compartment. MUC1-ED levels increased as early as 12 h, peaked at 24–48 h with a 7.8-fold increase, and decreased by 72 h. The a-type flagellin–expressing PAK strain and the b-type flagellin–expressing PAO1 strain stimulated comparable levels of MUC1-ED shedding. A flagellin-deficient PAK mutant provoked dramatically reduced MUC1-ED shedding compared with the WT strain, and purified flagellin recapitulated the WT effect. In lung tissues, Pa increased association of NEU1 and protective protein/cathepsin A with MUC1-ED in reciprocal co-immunoprecipitation assays and stimulated MUC1-ED desialylation. NEU1-selective sialidase inhibition protected against Pa-induced MUC1-ED desialylation and shedding. In Pa-challenged mice, MUC1-ED–enriched bronchoalveolar lavage fluid (BALF) inhibited flagellin binding and Pa adhesion to human airway epithelia by up to 44% and flagellin-driven motility by >30%. Finally, Pa co-administration with recombinant human MUC1-ED dramatically diminished lung and BALF bacterial burden, proinflammatory cytokine levels, and pulmonary leukostasis and increased 5-day survival from 0% to 75%. We conclude that Pa flagellin provokes NEU1-mediated airway shedding of MUC1-ED, which functions as a decoy receptor protecting against lethal Pa lung infection.

Klebsiella pneumoniae (KP) and Pseudomonas aeruginosa (PA) are important human pathogens that are associated with a range of infection types, including wound and disseminated infections. Treatment has been complicated by rising rates of antimicrobial resistance. Immunoprophylactic strategies are not constrained by antimicrobial resistance mechanisms. Vaccines against these organisms would be important public health tools, yet they are not available. KP surface O polysaccharides (OPS) are protective antigens in animal models of infection. Similarly, PA flagellin (Fla), the major subunit of the flagellar filament, is required for virulence and is a target of protective antibodies in animal models. We report herein the development of a combined KP and PA glycoconjugate vaccine comprised of the four most common KP OPS types associated with human infections (O1, O2, O3, O5), chemically linked to the two Fla types of PA (FlaA, FlaB). Conjugation of KP OPS to PA Fla enhanced anti-polysaccharide immune responses and produced a formulation that generated antibody titers to the four KP OPS types and both PA Fla antigens in rabbits. Passive transfer of vaccine-induced rabbit antisera reduced the bacterial burden and protected mice against fatal intravenous KP infection. Mice passively transferred with conjugate-induced antisera were also protected against PA infection after thermal injury with a FlaB-expressing isolate, but not a FlaA isolate. Taken together, these promising preclinical results provide important proof-of-concept for a broad spectrum human vaccine to prevent KP and PA infections.

Recent advances in lipopolysaccharide (LPS) biology have led to its use in drug discovery pipelines, including vaccine and vaccine adjuvant discovery. Desirable characteristics for LPS vaccine candidates include both the ability to produce a specific antibody titer in patients and a minimal host inflammatory response directed by the innate immune system. However, in-depth chemical characterization of most LPS extracts has not been performed; hence, biological activities of these extracts are unpredictable. Additionally, the most widely adopted workflow for LPS structure elucidation includes nonspecific chemical decomposition steps before analyses, making structures inferred and not necessarily biologically relevant. In this work, several different mass spectrometry workflows that have not been previously explored were employed to show proof-of-principle for top down LPS primary structure elucidation, specifically for a rough-type mutant (J5) E. coli-derived LPS component of a vaccine candidate. First, ion mobility filtered precursor ions were subjected to collision induced dissociation (CID) to define differences in native J5 LPS v. chemically detoxified J5 LPS (dLPS). Next, ultra-high mass resolving power, accurate mass spectrometry was employed for unequivocal precursor and product ion empirical formulae generation. Finally, MS3 analyses in an ion trap instrument showed that previous knowledge about dissociation of LPS components can be used to reconstruct and sequence LPS in a top down fashion. A structural rationale is also explained for differential inflammatory dose-response curves, in vitro, when HEK-Blue hTLR4 cells were administered increasing concentrations of native J5 LPS v. dLPS, which will be useful in future drug discovery efforts.

Background Blood alcohol is present in a third of trauma patients and has been associated with organ dysfunction. In both human studies and in animal models, it is clear that alcohol intoxication exerts immunomodulatory effects several hours to days after exposure, when blood alcohol is no longer detectable. The early immunomodulatory effects of alcohol while blood alcohol is still elevated are not well understood. Methods Human volunteers achieved binge alcohol intoxication after high-dose alcohol consumption. Blood was collected for analysis prior to alcohol ingestion, and 20 min, 2 h, and 5 h after alcohol ingestion. Flow cytometry was performed on isolated peripheral blood mononuclear cells, and cytokine generation in whole blood was measured by enzyme-linked immunosorbent assay (ELISA) after 24-h stimulation with lipopolysaccharide (LPS) and phytohemagglutinin-M (PHA) stimulation. Results An early pro-inflammatory state was evident at 20 min when blood alcohol levels were ∼130 mg/dL, which was characterized by an increase in total circulating leukocytes, monocytes, and natural killer cells. During this time, a transient increase in LPS-induced tumor necrosis factor (TNF)-α levels and enhanced LPS sensitivity occurred. At 2 and 5 h post-alcohol binge, an anti-inflammatory state was shown with reduced numbers of circulating monocytes and natural killer cells, attenuated LPS-induced interleukin (IL)-1β levels, and a trend toward increased interleukin (IL)-10 levels. Conclusions A single episode of binge alcohol intoxication exerted effects on the immune system that caused an early and transient pro-inflammatory state followed by an anti-inflammatory state.

We measured the antibody response in 10 victims of acute blunt trauma and penetrating trauma who were immunized against Klebsiella pneumoniae and Pseudomonas species within 72 hours of injury. The two vaccines, which were previously shown to be safe and immunogenic in uninjured humans, were a 24-valent K. pneumoniae capsular polysaccharide vaccine and an eight-valent Pseudomonas O-polysaccharide-toxin A conjugate vaccine. The patients were between 18 and 44 years of age, had Injury Severity Scores that ranged between 9 and 34, and did not have chronic infections or malignancies. On days 14 and 28 after immunization, all patients had a response of greater than fourfold to at least six of the nine Pseudomonas vaccine antigens. Half of the patients responded to eight of the nine antigens. Nine patients responded to at least 18 of 24 Klebsiella antigens, and seven patients responded to 22 of the 24 antigens. No important side effects were attributed to the vaccines. The results of this preliminary study indicate that active immunization against potential pathogens is possible in victims of acute trauma.

Inhalational anthrax, caused by the gram positive bacteria Bacillus anthracis, is a potentially fatal form of anthrax infection. It is initiated after inhaled spores are deposited in the lung, phagocytosed by immune cells, and subsequently transported to nearby lymph nodes. Intracellular spores that successfully germinate and become vegetative bacteria can lyse their host cell and contribute to bacterial outgrowth and toxin production. To better understand the early disease dynamics of the host-pathogen interaction, we develop a mathematical model of ordinary differential Equations and estimate parameters using available data. The model which consists of two subsystems is designed in accordance with an in vitro experimental protocol in which macrophages were challenged with varying doses of spores at spore-to-macrophage ratios of 1:1, 1:2, 1:10, 1:20. Initial modeling results suggested the need to consider two distinct subpopulations of anthrax bacteria: newly germinated bacteria which cannot replicate immediately and fully vegetative bacteria that can. Additional modeling results provide insights into possible reasons why macrophage-induced killing is more effective at the 1:20 ratio.

Background Translocation of gut-derived Gram-negative bacterial (GNB) lipopolysaccharide (LPS, or endotoxin) is a source of systemic inflammation that exacerbates HIV, cardiovascular and gastrointestinal diseases and malnutrition. The oral administration of bovine colostrum (BC) reduces endotoxemia in patients with impaired gut barrier function. Consequently, BC enriched in antibodies to LPS may ameliorate endotoxemia-related morbidities. We developed a detoxified J5 LPS/group B meningococcal outer membrane protein (J5dLPS/OMP) vaccine that induces antibodies against a highly conserved core region of LPS and protects against heterologous GNB infection. We now examine the ability of this vaccine to elicit anti-core endotoxin antibodies in BC. Methods Two cohorts of pregnant cows were immunized with this vaccine in combination with FICA (Cohort 1) or Emulsigen-D® (Cohort 2) adjuvants. Antibody responses to the J5 core LPS antigen were measured in both serum and colostrum and compared to antibody levels elicited by a commercially available veterinary vaccine (J5 Bacterin®) comprised of heat-killed Escherichia coli O111, J5 mutant bacteria, from which the J5 LPS was purified. Results The J5dLPS/OMP vaccine induced high titers of serum IgG antibody to J5 LPS in all seven cows. Both IgG and to a lesser extent IgA anti-J5 LPS antibodies were generated in the colostrum. The J5dLPS/OMP vaccine was significantly more immunogenic in mice than was the J5 Bacterin®. BC enriched in anti-J5 LPS antibody reduced circulating endotoxin levels in neutropenic rats, a model of “leaky gut”. Conclusion The J5dLPS/OMP vaccine elicits high titers of serum anti-endotoxin antibodies in cows that is passed to the colostrum. This BC enriched in anti-core LPS antibodies has the potential to reduce endotoxemia and ameliorate endotoxin-related systemic inflammation in patients with impaired gut barrier function. Since this vaccine is significantly more immunogenic than the J5 Bacterin® vaccine, this J5dLPS/OMP vaccine might prove to be more useful for veterinary indications as well.

Polysialic acid (polySia) is a unique linear homopolymer of α2,8-linked sialic acid that has been studied extensively as a posttranslational modification of neural cell adhesion molecule in the central nervous system. Only two proteins are known to be polysialylated in cells of the immune system: CD56 on human natural killer cells and murine bone marrow (BM) leukocytes, and neuropilin-2 (NRP-2) on dendritic cells (DCs). We tested the hypothesis that polySia expression is regulated during maturation and migration of leukocytes and plays a role in functional activity. Using wild-type and NCAM(-/-) mice, we show that BM neutrophils express only polysialylated CD56, whereas a subset of BM monocytes expresses polysialylated CD56 and/or another polysialylated protein(s). We demonstrate that polysialylated CD56 expression is progressively down-regulated in wild-type monocytes and monocyte-derived cells during migration from BM through peripheral blood to pulmonary and peritoneal sites of inflammation. Freshly isolated monocyte-derived peritoneal macrophages are devoid of polySia yet re-express polySia on NRP-2 and an additional protein(s) after maintenance in culture. Removal of polySia from these cells enhances phagocytosis of Klebsiella pneumoniae, suggesting that down-regulation of polySia on macrophages facilitates bacterial clearance. Using wild-type and NRP-2(-/-) mice, we demonstrate that NRP-2 and an additional protein(s) are polysialylated by ST8 SiaIV in BM-derived DCs. We conclude that polySia expression in monocyte-derived cells is dynamically regulated by ST8 SiaIV activity and by expression of carrier proteins during recruitment to sites of inflammation and influences cellular interactions with microbes, contributing to innate and adaptive immune responses.

Sialic acids on glycoconjugates play a pivotal role in many biological processes. In the airways, sialylated glycoproteins and glycolipids are strategically positioned on the plasma membranes of epithelia to regulate receptor-ligand, cell-cell, and host-pathogen interactions at the molecular level. We now demonstrate, for the first time, sialidase activity for ganglioside substrates in human airway epithelia. Of the four known mammalian sialidases, NEU3 has a substrate preference for gangliosides and is expressed at mRNA and protein levels at comparable abundance in epithelia derived from human trachea, bronchi, small airways, and alveoli. In small airway and alveolar epithelia, NEU3 protein was immunolocalized to the plasma membrane, cytosolic, and nuclear subcellular fractions. Small interfering RNA-induced silencing of NEU3 expression diminished sialidase activity for a ganglioside substrate by >70%. NEU3 immunostaining of intact human lung tissue could be localized to the superficial epithelia, including the ciliated brush border, as well as to nuclei. However, NEU3 was reduced in subepithelial tissues. These results indicate that human airway epithelia express catalytically active NEU3 sialidase.

The neutralization of toxins is considered essential for protection against lethal infection with Bacillus anthracis (BA), a select agent and bioterrorism threat. However, toxin-neutralizing activity alone would not be expected to provide sterile immunity. Therefore, we hypothesized that the development of an adaptive immune response against BA is required for bacterial clearance. We found that human monocyte-derived dendritic cells (hDCs) kill germinated BA bacilli, but not nongerminated BA spores. hDCs produce IL-1β, IL-6, IL-12, and IL-23, and these cytokines are differentially regulated by germination-proficient versus germination-deficient BA spores. Moreover, the IL-23 response to BA spores is regulated by IL-1R-mediated signaling. hDCs infected with germinating BA spores stimulated autologous CD4(+) T cells to secrete IL-17A and IFN-γ in a contact-dependent and antigen-specific manner. The T-cell response to BA spores was not recapitulated by hDCs infected with germination-deficient BA spores, implying that the germination of spores into replicating bacilli triggers the proinflammatory cytokine response in hDCs. Our results provide primary evidence that hDCs can generate a BA-specific Th17 response, and help elucidate the mechanisms involved. These novel findings suggest that the IL-23/Th17 axis is involved in the immune response to anthrax in humans.

We previously reported that removal of sialyl residues primed PBMCs to respond to bacterial LPS stimulation in vitro. Therefore, we speculated that prior desialylation can sensitize the host to generate an enhanced inflammatory response upon exposure to a TLR ligand, such as LPS, in a murine model of acute lung injury. Intratracheal instillation of neuraminidase (NA) 30 min prior to intratracheal administration of LPS increased polymorphonuclear leukocytes (PMNs) in the bronchoalveolar lavage fluid and the wet-to-dry lung weight ratio, a measure of pulmonary edema, compared with mice that received LPS alone. Administration of NA alone resulted in desialylation of bronchiolar and alveolar surfaces and induction of TNF-α, IL-1β, and chemokines in lung homogenates and bronchoalveolar lavage fluid; however, PMN recruitment in mice treated with NA alone did not differ from that of PBS-administered controls. NA pretreatment alone induced apoptosis and markedly enhanced LPS-induced endothelial apoptosis. Administration of recombinant Bcl-2, an antiapoptotic molecule, abolished the effect of NA treatment on LPS-induced PMN recruitment and pulmonary edema formation. We conclude that NA pretreatment potentiates LPS-induced lung injury through enhanced PMN recruitment, pulmonary edema formation, and endothelial and myeloid cell apoptosis. A similar “reprogramming” of immune responses with desialylation may occur during respiratory infection with NA-expressing microbes and contribute to severe lung injury.

Gram-negative bacterial (GNB) infections are a leading cause of serious infections both in hospitals and the community. The mortality remains high despite potent antimicrobials and modern supportive care. In the last decade invasive GNB have become increasingly resistant to commonly used antibiotics, and attempts to intervene with novel biological therapies have been unsuccessful. Earlier studies with antibodies directed against a highly conserved core region in the GNB lipopolysaccharide (LPS, or endotoxin) suggested that this approach may have therapeutic benefit, and led to the development of a subunit vaccine that has progressed to phase 1 clinical testing. Since only a few serogroups of GNB cause bacteremia, O-specific vaccines had been developed, but these were not deployed because of the availability of other therapeutic options at the time. Given the likelihood that new antibiotics will not be soon available, the development of vaccines and antibodies directed against endotoxin, both O and core an...

Infants born prematurely suffer the greatest incidence of and impact from sepsis among all age groups. Therapeutic interventions aimed at reducing morbidity and mortality in this vulnerable population have been unsuccessful. Interleukin (IL)-18 is a proinflammatory member of the IL-1 superfamily. Serum IL-18 concentrations in uninfected premature infants are increased as compared with healthy adults. We show that IL-18 in the setting of sepsis results in gut injury, a potentiation of the host’s inflammatory response, increased bacteremia, and mortality mediated by IL-1 receptor 1 (IL-1R1)–dependent IL-17A produced by γδT and myeloid cells. The discovery of this novel IL-18/IL-1R1/IL-17A axis brings new hope for therapeutic interventions that target downstream IL-17A and ultimately reduce the increased mortality from sepsis in this understudied population.

INTRODUCTION The past decade has seen dramatic progress in our understanding of immune mechanisms and host defense. Along with the ability that vertebrates have to acquire immunity to pathogenic antigens by expanding specific populations of T and B cells and making cytokines and antibodies, scientists have discovered that like invertebrates, we have multiple innate pathways to activate more generic host responses through a whole new family of receptors. Over 80 years ago, Ramon demonstrated that it was possible to artificially increase antigen-specific levels of diphtheria or tetanus antitoxin by the addition of bread crumbs, agar, tapioca, starch oil, lecithin, or saponin to the vaccines (1). Since then, aluminum salts have been the dominant substance used and are still the only adjuvant currently used in licensed vaccines in the United States. The field has become much more sophisticated recently with the introduction of numerous new adjuvants and new concepts regarding the mechanisms of action. In this brief chapter, we review the modern adjuvants used in a variety of current and experimental human vaccines. After a more general discussion of adjuvants, including their definition, mechanisms of action, and safety, we will discuss recent clinical trials of investigational adjuvants. For additional study of this complex subject, including a historical perspective, the reader is referred to published reviews of vaccine adjuvants (see Refs. 2-4).

Neuraminidase-1 (NEU1) is the predominant sialidase expressed in human airway epithelia and lung microvascular endothelia where it mediates multiple biological processes. We tested whether the NEU1-selective sialidase inhibitor, C9-butyl-amide-2-deoxy-2,3-dehydro-N-acetylneuraminic acid (C9-BA-DANA), inhibits one or more established NEU1-mediated bioactivities in human lung cells. We established the IC50 values of C9-BA-DANA for total sialidase activity in human airway epithelia, lung microvascular endothelia and lung fibroblasts to be 3.74 µM, 13.0 µM and 4.82 µM, respectively. In human airway epithelia, C9-BA-DANA dose-dependently inhibited flagellin-induced, NEU1-mediated mucin-1 ectodomain desialylation, adhesiveness for Pseudomonas aeruginosa and shedding. In lung microvascular endothelia, C9-BA-DANA reversed NEU1-driven restraint of cell migration into a wound and disruption of capillary-like tube formation. NEU1 and its chaperone/transport protein, protective protein/cathepsin A (PPCA), were differentially expressed in these same cells. Normalized NEU1 protein expression correlated with total sialidase activity whereas PPCA expression did not. In contrast to eukaryotic sialidases, C9-BA-DANA exerted far less inhibitory activity for three selected bacterial neuraminidases (IC50 > 800 µM). Structural modeling of the four human sialidases and three bacterial neuraminidases revealed a loop between the seventh and eighth strands of the β-propeller fold, that in NEU1, was substantially shorter than that seen in the six other enzymes. Predicted steric hindrance between this loop and C9-BA-DANA could explain its selectivity for NEU1. Finally, pretreatment of mice with C9-BA-DANA completely protected against flagellin-induced increases in lung sialidase activity. Our combined data indicate that C9-BA-DANA inhibits endogenous and ectopically expressed sialidase activity and established NEU1-mediated bioactivities in human airway epithelia, lung microvascular endothelia, and fibroblasts in vitro and murine lungs in vivo.

The IFN family of cytokines operates a frontline defense against pathogens and neoplastic cells in vivo by controlling the expression of several genes. The death-associated protein kinase 1 (DAPK1), an IFN-γ–induced enzyme, controls cell cycle, apoptosis, autophagy, and tumor metastasis, and its expression is frequently down-regulated in a number of human tumors. Although the biochemical action of DAPK1 is well understood, mechanisms that regulate its expression are unclear. Previously, we have shown that transcription factor C/EBP-β is required for the basal and IFN-γ–induced expression of DAPK1 . Here, we show that ATF6, an ER stress-induced transcription factor, interacts with C/EBP-β in an IFN-stimulated manner and is obligatory for Dapk1 expression. IFN-stimulated proteolytic processing of ATF6 and ERK1/2-mediated phosphorylation of C/EBP-β are necessary for these interactions. More importantly, IFN-γ failed to activate autophagic response in cells lacking either ATF6 or C/EBP-β. Consistent with these observations, the Atf6 −/− mice were highly susceptible to lethal bacterial infections compared with the wild-type mice. These studies not only unravel an IFN signaling pathway that controls cell growth and antibacterial defense, but also expand the role of ATF6 beyond ER stress.

The microvascular endothelial surface expresses multiple molecules whose sialylation state regulates multiple aspects of endothelial function. To better regulate these sialoproteins, we asked whether endothelial cells (ECs) might express one or more catalytically active sialidases. Human lung microvascular EC lysates contained heat-labile sialidase activity for a fluorogenic substrate, 2′-(4-methylumbelliferyl)-α-d-N-acetylneuraminic acid (4-MU-NANA), that was dose-dependently inhibited by the competitive sialidase inhibitor, 2,3-dehydro-2-deoxy-N-acetylneuraminic acid but not its negative control. The EC lysates also contained sialidase activity for a ganglioside mixture. Using real time RT-PCR to detect mRNAs for the four known mammalian sialidases, NEU1, -2, -3, and -4, NEU1 mRNA was expressed at levels 2700-fold higher that those found for NEU2, -3, or -4. Western analyses indicated NEU1 and -3 protein expression. Using confocal microscopy and flow cytometry, NEU1 was immunolocalized to both the plasma membrane and the perinuclear region. NEU3 was detected both in the cytosol and nucleus. Prior siRNA-mediated knockdown of NEU1 and NEU3 each decreased EC sialidase activity for 4-MU-NANA by >65 and >17%, respectively, and for the ganglioside mixture by 0 and 40%, respectively. NEU1 overexpression in ECs reduced their migration into a wound by >40%, whereas NEU3 overexpression did not. Immunohistochemical studies of normal human tissues immunolocalized NEU1 and NEU3 proteins to both pulmonary and extrapulmonary vascular endothelia. These combined data indicate that human lung microvascular ECs as well as other endothelia express catalytically active NEU1 and NEU3. NEU1 restrains EC migration, whereas NEU3 does not.

Epithelial cells (ECs) lining the airways provide a protective barrier between the external environment and the internal host milieu. These same airway epithelia express receptors that respond to danger signals and initiate repair programs. Because the sialylation state of a receptor can influence its function and is dictated in part by sialidase activity, we asked whether airway epithelia express catalytically active sialidase(s). Human primary small airway and A549 ECs expressed NEU1 sialidase at the mRNA and protein levels, and NEU1 accounted for >70% of EC sialidase activity. Blotting with Maackia amurensis and peanut agglutinin lectins established epidermal growth factor receptor (EGFR) and MUC1 as in vivo substrates for NEU1. NEU1 associated with EGFR and MUC1, and NEU1-EGFR association was regulated by EGF stimulation. NEU1 overexpression diminished EGF-stimulated EGFR Tyr-1068 autophosphorylation by up to 44% but enhanced MUC1-dependent Pseudomonas aeruginosa adhesion by 1.6–1.7-fold and flagellin-stimulated ERK1/2 activation by 1.7–1.9-fold. In contrast, NEU1 depletion increased EGFR activation (1.5-fold) and diminished MUC1-mediated bacterial adhesion (38–56%) and signaling (73%). These data indicate for the first time that human airway epithelia express catalytically active NEU1 sialidase that regulates EGFR- and MUC1-dependent signaling and bacterial adhesion. NEU1 catalytic activity may offer an additional level of regulation over the airway epithelial response to ligands, pathogens, and injurious stimuli.

Macrophages, upon phagocytosing endospores of Bacillus anthracis, up-regulate the expression of the immunological isoform of nitric oxide synthase, NOS 2, concomitant with production of nitric oxide (NO•) from metabolism of l-arginine. We have previously demonstrated that macrophages that secrete NO• kill the bacilli of B. anthracis. To circumvent this microbicidal activity of NO•, B. anthracis has evolved pathways that include the enzyme arginase, which metabolizes l-arginine to ornithine and urea. Compounds that inhibit arginase might, therefore, offer a therapeutic approach to controlling B. anthracis infection. 2(S)-Amino-6-boronohexanoic acid (ABH) has been reported to be an inhibitor of mammalian arginase. In this study, we explore the inhibitory effect of ABH against B. anthracis arginase and its potential for future development, as an effective therapeutic agent against microbial infection. We found that ABH is an inhibitor of bacterial arginase in several different endospore strains of B. anthracis. Further, ABH inhibits neither the phagocytosis of these endospores nor the up-regulation of NOS 2 concomitant with secretion of NO•. These findings set the stage to determine how efficacious ABH will be in promoting NO•-mediating killing of B. anthracis.

Diapedesis is a dynamic, highly regulated process by which leukocytes are recruited to inflammatory sites. We reported previously that removal of sialyl residues from PMNs enables these cells to become more adherent to EC monolayers and that sialidase activity within intracellular compartments of resting PMNs translocates to the plasma membrane following activation. We did not identify which surface adhesion molecules were targeted by endogenous sialidase. Upon activation, β2 integrin (CD11b/CD18) on the PMN surface undergoes conformational change, which allows it to bind more tightly to the ICAM-1 and ICAM-2 on the EC surface. Removal of sialyl residues from CD18 and CD11b, by exogenous neuraminidase or mobilization of PMN sialidase, unmasked activation epitopes, as detected by flow cytometry and enhanced binding to ICAM-1. One sialidase isoform, Neu1, colocalized with CD18 on confocal microscopy. Using an autoperfused microflow chamber, desialylation of immobilized ICAM-1 enhanced leukocyte arrest in vivo. Further, treatment with a sialidase inhibitor in vivo reversed endotoxin-induced binding of leukocytes to ICAM-1, thereby suggesting a role for leukocyte sialidase in the cellular arrest. These data suggest that PMN sialidase could be a physiologic source of the enzymatic activity that removes sialyl residues on β2 integrin and ICAM-1, resulting in their enhanced interaction. Thus, PMN sialidase may be an important regulator of the recruitment of these cells to inflamed sites.

A comparative analysis of antibody and cell-mediated immune responses was performed in ambulatory medically stable elderly and young adults who received the standard-dose of trivalent inactivated influenza vaccine, containing 15 μg of hemagglutinin (HA) per virus strain, or a high-dose vaccine containing 60 μg HA per virus strain. Among the elderly, the high dose vaccine induced greater HAI (hemagglutination inhibition) and virus neutralization antibody titers than the standard dose vaccine. These responses, however, did not achieve the magnitude of those induced by the standard dose vaccine in young adults. Vaccine-specific circulating T cells producing IFN-γ were detected in the elderly and young adults following immunization. However, there were no significant differences in the IFN-γ responses among groups. On the other hand, the standard dose vaccine in the elderly resulted in the highest proportion of complete non-responders who failed to elicit either an HAI or an IFN-γ response. This study provides further evidence that a higher dose vaccine for the elderly may result in enhanced immune responses which are predicted to improve protection although still of lower magnitude than those induced in younger healthier individuals.

Neu1 and Neu3 are up-regulated as monocytes differentiate into DCs; and desialylation of cell surface glycoconjugates by one or both sialidase promotes cytokine production. Removal of sialic acid from glycoconjugates on the surface of monocytes enhances their response to bacterial LPS. We tested the hypothesis that endogenous sialidase activity creates a permissive state for LPS-induced cytokine production in human monocyte-derived DCs. Of the four genetically distinct sialidases (Neu1–4), Neu1, Neu3, and Neu4 are expressed in human monocytes, but only Neu1 and Neu3 are up-regulated as cells differentiate into DCs. Neu1 and Neu3 are present on the surface of monocytes and DCs and are also present intracellularly. DCs contain a greater amount of sialic acid than monocytes, but the amount of sialic acid/mg total protein declines during differentiation to DCs. This relative hyposialylation of cells does not occur in mature DCs grown in the presence of zanamivir, a pharmacologic inhibitor of Neu3 but not Neu1, or DANA, an inhibitor of Neu1 and Neu3. Inhibition of sialidase activity during differentiation to DCs causes no detectable change in cell viability or expression of DC surface markers. Differentiation of monocytes into DCs in the presence of zanamivir results in reduced LPS- induced expression of IL-6, IL-12p40, and TNF-α by mature DCs, demonstrating a role for Neu3 in cytokine production. A role for Neu3 is supported by inhibition of cytokine production by DANA in DCs from Neu1–/– and WT mice. We conclude that sialidase-mediated change in sialic acid content of specific cell surface glycoconjugates in DCs regulates LPS-induced cytokine production, thereby contributing to development of adaptive immune responses.

Recent advances in lipopolysaccharide (LPS) biology have led to its use in drug discovery pipelines, including vaccine and vaccine adjuvant discovery. Desirable characteristics for LPS vaccine candidates include both the ability to produce a specific antibody titer in patients and a minimal host inflammatory response directed by the innate immune system. However, in-depth chemical characterization of most LPS extracts has not been performed; hence, biological activities of these extracts are unpredictable. Additionally, the most widely adopted workflow for LPS structure elucidation includes nonspecific chemical decomposition steps before analyses, making structures inferred and not necessarily biologically relevant. In this work, several different mass spectrometry workflows that have not been previously explored were employed to show proof-of-principle for top down LPS primary structure elucidation, specifically for a rough-type mutant (J5) E. coli-derived LPS component of a vaccine candidate. First, ion mobility filtered precursor ions were subjected to collision induced dissociation (CID) to define differences in native J5 LPS v. chemically detoxified J5 LPS (dLPS). Next, ultra-high mass resolving power, accurate mass spectrometry was employed for unequivocal precursor and product ion empirical formulae generation. Finally, MS(3) analyses in an ion trap instrument showed that previous knowledge about dissociation of LPS components can be used to reconstruct and sequence LPS in a top down fashion. A structural rationale is also explained for differential inflammatory dose-response curves, in vitro, when HEK-Blue hTLR4 cells were administered increasing concentrations of native J5 LPS v. dLPS, which will be useful in future drug discovery efforts.

Francisella tularensis is a category A select agent. J5dLPS/OMP is a novel vaccine construct consisting of detoxified, O-polysaccharide side chain-deficient, lipopolysaccharide non-covalently complexed with the outer membrane protein of N. meningitidis group B. Immunization elicits high-titer polyclonal antibodies specific for the highly-conserved epitopes expressed within the glycolipid core that constitutes gram-negative bacteria (e.g., F. tularensis). Mice immunized intranasally with J5dLPS/OMP exhibited protective immunity to intratracheal challenge with the live vaccine strain, as well as the highly-virulent SchuS4 strain, of F. tularensis. The efficacy of J5dLPS/OMP vaccine suggests its potential utility in immunizing the general population against several different gram-negative select agents concurrently.

When given passively or elicited actively, antibodies induced by a detoxified Escherichia coli Rc chemotype (J5) mutant lipopolysaccharide (J5dLPS)—group B meningococcal outer membrane protein (OMP) complex vaccine protected animals from lethal sepsis. The protection from sepsis is believed to be dependent on high levels of antibodies against the core glycolipid (CGL), a region of LPS that is rather conserved among Enterobacteriaceae. The addition of unmethylated deoxycytidyl-deoxyguanosine dinucleotide (CpG)-containing oligodeoxynucleotides (ODN) was used as an immuno-adjuvant to improve antibody responses. In preparation for a Phase I human trial, we elucidated potential contributions by which the sepsis vaccine (J5dLPS—OMP) and CpG ODN might enhance the antibody response and provide evidence that the generation of immune responses is Toll-like receptor (TLR) dependent. Toll-like receptor 2, TLR4, and TLR9 were each essential for generating robust cytokine and antibody responses. The signature cytokine of dendritic cells, interleukin-12, was one of the cytokines that demonstrated synergy with the optimal TLR ligand/ engagement combination. We conclude that the involvement of multiple TLRs upon immunization was critical for the generation of optimal antibody responses. These observations provide further evidence for the inclusion of innate immune-based adjuvants during the development of next-generation vaccines.

Type I IFNs were discovered as the primary antiviral cytokines and are now known to serve critical functions in host defense against bacterial pathogens. Accordingly, established mediators of IFN antiviral activity may mediate previously unrecognized antibacterial functions. RNase-L is the terminal component of an RNA decay pathway that is an important mediator of IFN-induced antiviral activity. Here, we identify a role for RNase-L in the host antibacterial response. RNase-L −/− mice exhibited a dramatic increase in mortality after challenge with Bacillus anthracis and Escherichia coli ; this increased susceptibility was due to a compromised immune response resulting in increased bacterial load. Investigation of the mechanisms of RNase-L antibacterial activity indicated that RNase-L is required for the optimal induction of proinflammatory cytokines that play essential roles in host defense from bacterial pathogens. RNase-L also regulated the expression of the endolysosomal protease, cathepsin-E, and endosome-associated activities, that function to eliminate internalized bacteria and may contribute to RNase-L antimicrobial action. Our results reveal a unique role for RNase-L in the antibacterial response that is mediated through multiple mechanisms. As a regulator of fundamental components of the innate immune response, RNase-L represents a viable therapeutic target to augment host defense against diverse microbial pathogens.

Previous reports suggested that lethal toxin (LT)-induced caspase-1 activity and/or IL-1β accounted for Bacillus anthracis (BA) infection lethality. In contrast, we now report that caspase-1-mediated IL-1β expression in response to BA spores is required for anti-BA host defenses. Caspase-1–/– and IL-1β–/– mice are more susceptible than wild-type (WT) mice to lethal BA infection, are less able to kill BA both in vivo and in vitro, and addition of rIL-1β to macrophages from these mice restored killing in vitro. Non-germinating BA spores induced caspase-1 activity, IL-1β and nitric oxide, by which BA are killed in WT but not in caspase-1–/– mice, suggesting that the spore itself stimulated inflammatory responses. While spores induced IL-1β in LT-susceptible and -resistant macrophages, LT induced IL-1β only in LT-susceptible macrophages. Cooperation between MyD88-dependent and -independent signaling pathways was required for spore-induced, but not LT-induced, IL-1β. While both spores and LT induced caspase-1 activity and IL-1β, LT did not induce IL-1β mRNA, and spores did not induce cell death. Thus different components of the same bacterium each induce IL-1β by distinct signaling pathways. Whereas the spore-induced IL-1β limits BA infection, LT-induced IL-1β enables BA to escape host defenses. Supporting Information for this article is available at www.wiley-vch.de/contents/jc_2040/2008/38141_s.pdf