Tonyia Eaves-Pyles

Flagellin, a Novel Mediator of Salmonella-Induced Epithelial Activation and Systemic Inflammation: IκBα Degradation, Induction of Nitric Oxide Synthase, Induction of Proinflammatory Mediators, and Cardiovascular Dysfunction

Gram-negative sepsis is mediated by the actions of proinflammatory genes induced in response to microbes and their products. We report that flagellin, the monomeric subunit of flagella, is a potent proinflammatory species released by Salmonella. Flagellin (1 μg/ml) induces IκBα degradation, NF-κB nuclear translocation, and inducible NO synthase expression in cultured intestinal epithelial cells (IEC). Aflagellic Salmonella mutants do not induce NF-κB activation or NO production by cultured IEC. Antiserum to flagellin blocks NO production in IEC induced by medium conditioned by a variety of motile Gram-negative enteric pathogens (Escherichia coli, Salmonella muenchen, Serratia marcescens, Proteus mirabilis, and Proteus vulgaris). Flagellin, when injected systemically (∼10 μg/mouse), induces systemic inflammation characterized by the systemic expression of a range of proinflammatory cytokines and chemokines and of inducible NO synthase. At higher doses (∼300 μg/mouse), flagellin induces shock, characterized by hypotension, reduced vascular contractility in mice, and death. The effects of flagellin do not diminish in C3H/HeJ LPS-resistant mice, indicating that the Toll-like receptor-4 receptor is not involved in flagellin’s actions. In LPS-resistant mice, i.p. injection of S. dublin flagellin or medium conditioned by wild-type S. dublin induces serum IFN-γ and TNF-α, whereas medium conditioned by aflagellic mutants has no effect. Flagellin can be detected in the blood of rats with septic shock induced by live bacteria at approximately 1 μg/ml. We propose that flagellin released by Gram-negative pathogens may contribute to the inflammatory response by an LPS- and Toll-like receptor-4-independent pathway.

Salmonella flagellin-dependent proinflammatory responses are localized to the conserved amino and carboxyl regions of the protein.

Flagellin, the monomeric subunit of flagella, is an inducer of proinflammatory mediators. Bacterial flagellin genes have conserved domains (D1 and D2) at the N terminus and C terminus and a middle hypervariable domain (D3). To identify which domains induced proinflammatory activity, r6-histidine (6HIS)-tagged fusion constructs were generated from the Salmonella dublin (SD) fliC flagellin gene. A full-length r6HIS SD flagellin (6HIS flag) induced IκBα loss poststimulation and NF-κB activation in Caco-2BBe cells and was as potent as native-purified SD flagellin. IFN-γ-primed DLD-1 cells stimulated with 1 μg/ml of 6HIS flag induced high levels of NO (60 ± 0.95 μM) comparable to the combination of IL-1β and IFN-γ (77 ± 1.2) or purified native SD flag (66.3 ± 0.98). Selected rSD flagellin proteins representing the D1, D2, or D3 domains alone or in combination were tested for proinflammatory properties. Fusion proteins representing the D3, amino, or carboxyl regions alone did not induce proinflammatory mediators. The results with a recombinant protein containing the amino D1 and D2 and carboxyl D1 and D2 separated by an Escherichia coli hinge (ND1-2/ECH/CD2) indicated that D1 and D2 were bioactive when coupled to an ECH element to allow protein folding. This chimera, but not the hinge alone, induced IκBα degradation, NF-κB activation, and NO and IL-8 production in two intestinal epithelial cell lines. ND1–2/ECH/CD2–1 also induced high levels of TNF-α (900 pg/ml) in human monocytes comparable to native SD flagellin (991.5 pg/ml) and 6HIS flag (987 pg/ml). The potent proinflammatory activity of flagellin, therefore, resides in the highly conserved N and C D1 and D2 regions.

Critical role for serum opsonins and complement receptors CR3 (CD11b/CD18) and CR4 (CD11c/CD18) in phagocytosis of Francisella tularensis by human dendritic cells (DC): uptake of Francisella leads to activation of immature DC and intracellular survival of the bacteria

Francisella tularensis is one of the most infectious human pathogens known. Although much has been learned about the immune response of mice using an attenuated live vaccine strain (LVS) derived from F. tularensis subspecies holarctica (Type B), little is known about the responses of human monocyte‐derived immature dendritic cells (DC). Here, we show that optimal phagocytosis of LVS by DC is dependent on serum opsonization. We demonstrate that complement factor C3‐derived opsonins and the major complement receptors expressed by DC, the integrins CR3 (CD11b/CD18) and CR4 (CD11c/CD18), play a critical role in this adhesion‐mediated phagocytosis. LVS induced proinflammatory cytokine production and up‐regulation of costimulatory surface proteins (CD40, CD86, and MHC Class II) on DC but resisted killing. Once taken up, LVS grew intracellularly, resulting in DC death. DC maturation and cytokine production were induced by direct contact/phagocytosis of LVS or interaction with soluble products of the bacteria, and enhanced activation was seen when LVS was pretreated with serum. Sonicated LVS and supernatants from LVS cultures were potent activators of DC, but LVS LPS failed to activate DC maturation or cytokine production. Serum‐treated LVS rapidly induced (within 6 h) a number of cytokines including IL‐10, a potent suppressor of macrophage functions and down‐regulator of Th1‐like responses and the Th1 response inducer IL‐12. These results suggest that the simultaneous production of an activating (IL‐12, IL‐1β, and TNF‐α) and a suppressing (IL‐10) cytokine profile could contribute to the immunopathogenesis of tularemia.

Granulocyte macrophage colony-stimulating factor improves survival in two models of gut-derived sepsis by improving gut barrier function and modulating bacterial clearance.

ObjectiveThe effect of recombinant murine granulocyte macrophage colony-stimulating factor (rmGM-CSF) on survival and host defense was studied using two clinically relevant models of infection that included transfusion-induced immunosuppression. Summary Background DataGranulocyte macrophage colony-stimulating factor improves resistance in several models of infection, but its role in transfusion-induced immunosuppression and bacterial translocation (gut-derived sepsis) has not been defined. MethodsBalb/c mice were treated with 100 ng of rmGM-CSF or placebo for 6 days in a model of transfusion, burn, and gavage, or cecal ligation and puncture (CLP). Translocation was studied in the first model. ResultsSurvival after transfusion, burn, and gavage was 90% in rmGM-CSF-treated animals versus 35% in the control group (p < 0.001). After CLP, survival was 75% in the rmGM-CSF group versus 30% in the control group (p = 0.01). Less translocation and better killing of bacteria was observed in the tissues in animals treated with rmGM-CSF. ConclusionThe ability of rmGM-CSF to improve gut barrier function and enhance killing of translocated organisms after burn injury-induced gut origin sepsis was associated with improved outcome. Granulocyte macrophage colony-stimulating factor also improved survival after CLP.

Heat shock inhibits phosphorylation of I-κBα

Previous studies demonstrated that induction of the heat shock response is associated with inhibition of the proinflammatory transcription factor NF-kappaB by a mechanism involving inhibition of I-kappaBalpha degradation. To provide further insight regarding the interactions of these fundamental cellular responses, the present experiments were designed to elucidate the mechanism(s) by which heat shock inhibits degradation of I-kappaBalpha. In an in vitro model of inflammatory cell signaling, treatment of RAW 264.7 murine macrophages with LPS (100 ng/mL) caused rapid degradation of I-kappaBalpha. Heat shock, 1 h before treatment with LPS, completely inhibited LPS-mediated degradation of I-kappaBalpha. Immunoprecipitation studies demonstrated that heat shock inhibited LPS-mediated ubiquitination of I-kappaBalpha. Western-blot analyses using a phosphorylated I-kappaBalpha-specific antibody demonstrated that heat shock inhibited LPS-mediated phosphorylation of I-kappaBalpha. In contrast, heat shock induced phosphorylation of c-jun. In murine fibroblasts having genetic ablation of the heat shock factor-1 gene, heat shock inhibited tumor necrosis factor-alpha mediated degradation of I-kappaBalpha. We conclude that the mechanism by which heat shock inhibits LPS-mediated degradation of I-kappaBalpha involves specific inhibition of I-kappaBalpha phosphorylation and subsequent I-kappaBalpha ubiquitination. In addition, this mechanism does not involve activation of heat shock factor-1 or the heat shock proteins regulated by heat shock factor-1.

Commensal bacteria modulate innate immune responses of vaginal epithelial cell multilayer cultures.

The human vaginal microbiome plays a critical but poorly defined role in reproductive health. Vaginal microbiome alterations are associated with increased susceptibility to sexually-transmitted infections (STI) possibly due to related changes in innate defense responses from epithelial cells. Study of the impact of commensal bacteria on the vaginal mucosal surface has been hindered by current vaginal epithelial cell (VEC) culture systems that lack an appropriate interface between the apical surface of stratified squamous epithelium and the air-filled vaginal lumen. Therefore we developed a reproducible multilayer VEC culture system with an apical (luminal) air-interface that supported colonization with selected commensal bacteria. Multilayer VEC developed tight-junctions and other hallmarks of the vaginal mucosa including predictable proinflammatory cytokine secretion following TLR stimulation. Colonization of multilayers by common vaginal commensals including Lactobacillus crispatus, L. jensenii, and L. rhamnosus led to intimate associations with the VEC exclusively on the apical surface. Vaginal commensals did not trigger cytokine secretion but Staphylococcus epidermidis, a skin commensal, was inflammatory. Lactobacilli reduced cytokine secretion in an isolate-specific fashion following TLR stimulation. This tempering of inflammation offers a potential explanation for increased susceptibility to STI in the absence of common commensals and has implications for testing of potential STI preventatives.

Outer Membrane Protein A of Escherichia coli O157:H7 Stimulates Dendritic Cell Activation

ABSTRACT Outer membrane protein A (OmpA) is located in the membrane of Escherichia coli and other gram-negative bacteria and plays a multifunctional role in bacterial physiology and pathogenesis. In enterohemorrhagic E. coli (EHEC), especially serotype O157:H7, OmpA interacts with cultured human intestinal cells and likely acts as an important component to stimulate the immune response during infection. To test this hypothesis, we analyzed the effect of EHEC OmpA on cytokine production by dendritic cells (DCs) and on DC migration across polarized intestinal epithelial cells. OmpA induced murine DCs to secrete interleukin-1 (IL-1), IL-10, and IL-12 in a dose-dependent manner, and this effect was independent of Toll-like receptor 4. Although DCs displayed differential responses to EHEC OmpA and OmpA-specific antibodies enhanced DC cytokine secretion, we cannot discard that other EHEC surface elements were likely to be involved. While OmpA was required for bacterial binding to polarized Caco-2 cells, it was not needed for the induction of cytokine production by Caco-2 cells or for human DC migration across polarized cells.

Effect of Different Combinations of Dietary Additives on Bacterial Translocation and Survival in Gut-Derived Sepsis

BACKGROUND Dietary arginine, glutamine, and fish oil each have been shown to improve resistance to infection. The purpose of this study was to assess the potential benefit of different combinations and amounts of these components on bacterial translocation and related mortality during gut-derived sepsis. METHODS Balb/c mice were fed for 10 days with an AIN-76A diet supplemented with different combinations and percentages of arginine, glutamine, glycine, fish oil, and medium-chain triglycerides. Controls were fed a complete AIN-76A diet or chow. After 10 days of feeding, all animals were transfused. On day 15, the animals were gavaged with 10(10) 111In-radiolabeled or unlabeled Escherichia coli and given a 30% burn injury. Animals gavaged with unlabeled bacteria were observed for survival (n = 317). Groups that showed the best survival as well as control groups were gavaged with labeled bacteria and killed 4 hours postburn (n = 60) for harvest of mesenteric lymph nodes, liver and spleen. RESULTS Mice fed diets enriched with 5% fish oil + 2% arginine, 2% arginine + 2% glutamine, or 5% fish oil + 2% glutamine had higher survival than control groups. The animals fed fish oil+glutamine had significantly reduced translocation to the liver and spleen. Animals fed arginine+glutamine had an enhanced ability to kill translocated organisms in the liver compared with other groups. Fish oil+arginine improved both barrier function and microbial killing. CONCLUSIONS Feeding with arginine+glutamine, fish oil+arginine, or fish oil+glutamine supplemented diets positively affects the outcome in a gut-derived sepsis model.

Role of Primary Human Alveolar Epithelial Cells in Host Defense against Francisella tularensis Infection

ABSTRACT Francisella tularensis, an intracellular pathogen, is highly virulent when inhaled. Alveolar epithelial type I (ATI) and type II (ATII) cells line the majority of the alveolar surface and respond to inhaled pathogenic bacteria via cytokine secretion. We hypothesized that these cells contribute to the lung innate immune response to F. tularensis. Results demonstrated that the live vaccine strain (LVS) contacted ATI and ATII cells by 2 h following intranasal inoculation of mice. In culture, primary human ATI or ATII cells, grown on transwell filters, were stimulated on the apical (AP) surface with virulent F. tularensis Schu 4 or LVS. Basolateral (BL) conditioned medium (CM), collected 6 and 24 h later, was added to the BL surfaces of transwell cultures of primary human pulmonary microvasculature endothelial cells (HPMEC) prior to the addition of polymorphonuclear leukocytes (PMNs) or dendritic cells (DCs) to the AP surface. HPMEC responded to S4- or LVS-stimulated ATII, but not ATI, CM as evidenced by PMN and DC migration. Analysis of the AP and BL ATII CM revealed that both F. tularensis strains induced various levels of a variety of cytokines via NF-κB activation. ATII cells pretreated with an NF-κB inhibitor prior to F. tularensis stimulation substantially decreased interleukin-8 secretion, which did not occur through Toll-like receptor 2, 2/6, 4, or 5 stimulation. These data indicate a crucial role for ATII cells in the innate immune response to F. tularensis.

Levofloxacin Rescues Mice from Lethal Intra-nasal Infections with Virulent Francisella tularensis and Induces Immunity and Production of Protective Antibody

The ability to protect mice against respiratory infections with virulent Francisella tularensis has been problematic and the role of antibody-versus-cell-mediated immunity controversial. In this study, we tested the hypothesis that protective immunity can develop in mice that were given antibiotic therapy following infection via the respiratory tract with F. tularensis SCHU S4. We show that mice infected with a lethal dose of SCHU S4, via an intra-nasal challenge, could be protected with levofloxacin treatment. This protection was evident even when levofloxacin treatment was delayed 72h post-infection. At early time points after levofloxacin treatment, significant numbers of bacteria could be recovered from the lungs and spleens of mice, which was followed by a dramatic disappearance of bacteria from these tissues. Mice successfully treated with levofloxacin were later shown to be almost completely resistant to re-challenge with SCHU S4 by the intra-nasal route. Serum antibody appeared to play an important role in this immunity. Normal mice, when given sera from animals protected by levofloxacin treatment, were solidly protected from a lethal intra-nasal challenge with SCHU S4. The protective antiserum contained high titers of SCHU S4-specific IgG2a, indicating that a strong Th1 response was induced following levofloxacin treatment. Thus, this study describes a potentially valuable animal model for furthering our understanding of respiratory tularemia and provides suggestive evidence that antibody can protect against respiratory infections with virulent F. tularensis.