mTOR-driven glycolysis governs induction of innate immune responses by bronchial epithelial cells exposed to the bacterial component flagellin.

Human bronchial epithelial (HBE) cells play an essential role during bacterial infections of the airways by sensing pathogens and orchestrating protective immune responses. We here sought to determine which metabolic pathways are utilized by HBE cells to mount innate immune responses upon exposure to a relevant bacterial agonist. Stimulation of HBE cells by the bacterial component flagellin triggered activation of the mTOR pathway resulting in an increased glycolytic flux that sustained the secretory activity of immune mediators by HBE cells. The mTOR inhibitor rapamycin impeded glycolysis and limited flagellin-induced secretion of immune mediators. The role of the mTOR pathway was recapitulated in vivo in a mouse model of flagellin-triggered lung innate immune responses. These data demonstrate that metabolic reprogramming via the mTOR pathway modulates activation of the respiratory epithelium, identifying mTOR as a potential therapeutic target to modulate mucosal immunity in the context of bacterial infections.

Boosting the IL-22 response using flagellin prevents bacterial infection in cigarette smoke-exposed mice.

The progression of chronic obstructive pulmonary disease (COPD), a lung inflammatory disease being the fourth cause of death worldwide, is marked by acute exacerbations. These episodes are mainly caused by bacterial infections, frequently due to Streptococcus pneumoniae. This susceptibility to infection involves a defect in interleukin (IL)-22, which plays a pivotal role in mucosal defense mechanism. Administration of flagellin, a Toll-like receptor 5 (TLR-5) agonist, can protect mice and primates against respiratory infections in a non-pathological background. We hypothesized that TLR-5-mediated stimulation of innate immunity might improve the development of bacteria-induced exacerbations in a COPD context. Mice chronically exposed to cigarette smoke (CS), mimicking COPD symptoms, are infected with S. pneumoniae, and treated in a preventive and a delayed manner with flagellin. Both treatments induced a lower bacterial load in the lungs and blood, and strongly reduced the inflammation and lung lesions associated with the infection. This protection implicated an enhanced production of IL-22 and involved the recirculation of soluble factors secreted by spleen cells. This is also associated with higher levels of the S100A8 anti-microbial peptide in the lung. Furthermore, human mononuclear cells from non-smokers were able to respond to recombinant flagellin by increasing IL-22 production while active smoker cells do not, a defect associated with an altered IL-23 production. This study shows that stimulation of innate immunity by a TLR-5 ligand reduces CS-induced susceptibility to bacterial infection in mice, and should be considered in therapeutic strategies against COPD exacerbations.

Characterization of structural and immunological properties of a fusion protein between flagellin from Salmonella and lumazine synthase from Brucella.

Aiming to combine the flexibility of Brucella lumazine synthase (BLS) to adapt different protein domains in a decameric structure and the capacity of BLS and flagellin to enhance the immunogenicity of peptides that are linked to their structure, we generated a chimeric protein (BLS-FliC131) by fusing flagellin from Salmonella in the N-termini of BLS. The obtained protein was recognized by anti-flagellin and anti-BLS antibodies, keeping the oligomerization capacity of BLS, without affecting the folding of the monomeric protein components determined by circular dichroism. Furthermore, the thermal stability of each fusion partner is conserved, indicating that the interactions that participate in its folding are not affected by the genetic fusion. Besides, either in vitro or in vivo using TLR5-deficient animals we could determine that BLS-FliC131 retains the capacity of triggering TLR5. The humoral response against BLS elicited by BLS-FliC131 was stronger than the one elicited by equimolar amounts of BLS + FliC. Since BLS scaffold allows the generation of hetero-decameric structures, we expect that flagellin oligomerization on this protein scaffold will generate a new vaccine platform with enhanced capacity to activate immune responses.

Neutrophilic NLRP3 inflammasome-dependent IL-1ß secretion regulates the γδT17 cell response in respiratory bacterial infections.

Traditionally regarded as simple foot soldiers of the innate immune response limited to the eradication of pathogens, neutrophils recently emerged as more complex cells endowed with a set of immunoregulatory functions. Using a model of invasive pneumococcal disease, we highlighted an unexpected key role for neutrophils as accessory cells in innate interleukin (IL)-17A production by lung resident Vγ6Vδ1+ T cells via nucleotide-binding oligomerization domain receptor, pyrin-containing 3 (NLRP3) inflammasome-dependent IL-1ß secretion. In vivo activation of the NLRP3 inflammasome in neutrophils required both host-derived and bacterial-derived signals. Elaborately, it relies on (i) alveolar macrophage-secreted TNF-α for priming and (ii) subsequent exposure to bacterial pneumolysin for activation. Interestingly, this mechanism can be translated to human neutrophils. Our work revealed the cellular and molecular dynamic events leading to γδT17 cell activation, and highlighted for the first time the existence of a fully functional NLRP3 inflammasome in lung neutrophils. This immune axis thus regulates the development of a protective host response to respiratory bacterial infections.

Airway structural cells regulate TLR5-mediated mucosal adjuvant activity.

Antigen-presenting cell (APC) activation is enhanced by vaccine adjuvants. Most vaccines are based on the assumption that adjuvant activity of Toll-like receptor (TLR) agonists depends on direct, functional activation of APCs. Here, we sought to establish whether TLR stimulation in non-hematopoietic cells contributes to flagellin's mucosal adjuvant activity. Nasal administration of flagellin enhanced T-cell-mediated immunity, and systemic and secretory antibody responses to coadministered antigens in a TLR5-dependent manner. Mucosal adjuvant activity was not affected by either abrogation of TLR5 signaling in hematopoietic cells or the presence of flagellin-specific, circulating neutralizing antibodies. We found that flagellin is rapidly degraded in conducting airways, does not translocate into lung parenchyma and stimulates an early immune response, suggesting that TLR5 signaling is regionalized. The flagellin-specific early response of lung was regulated by radioresistant cells expressing TLR5 (particularly the airway epithelial cells). Flagellin stimulated the epithelial production of a small set of mediators that included the chemokine CCL20, which is known to promote APC recruitment in mucosal tissues. Our data suggest that (i) the adjuvant activity of TLR agonists in mucosal vaccination may require TLR stimulation of structural cells and (ii) harnessing the effect of adjuvants on epithelial cells can improve mucosal vaccines.

Toll-like receptor 2 is critical for induction of Reg3 beta expression and intestinal clearance of Yersinia pseudotuberculosis.

OBJECTIVE: Yersinia pseudotuberculosis causes ileitis and mesenteric lymphadenitis by mainly invading the Peyer's patches that are positioned in the terminal ileum. Whereas toll-like-receptor 2 (TLR2) controls mucosal inflammation by detecting certain microbiota-derived signals, its exact role in protecting Peyer's patches against bacterial invasion has not been defined. DESIGN: Wild-type, Tlr2-, Nod2- and MyD88-deficient animals were challenged by Y pseudotuberculosis via the oral or systemic route. The role of microbiota in conditioning Peyer's patches against Yersinia through TLR2 was assessed by delivering, ad libitum, exogenous TLR2 agonists in drinking water to germ-free and streptomycin-treated animals. Bacterial eradication from Peyer's patches was measured by using a colony-forming unit assay. Expression of cryptdins and the c-type lectin Reg3 beta was quantified by quantitative reverse transcriptase polymerase chain reaction analysis. RESULTS: Our data demonstrated that Tlr2-deficient mice failed to limit Yersinia dissemination from the Peyer's patches and succumbed to sepsis independently of nucleotide-binding and oligomerisation domain 2 (NOD2). Recognition of both microbiota-derived and myeloid differentiation factor 88 (MyD88)-mediated elicitors was found to be critically involved in gut protection against Yersinia-induced lethality, while TLR2 was dispensable to systemic Yersinia infection. Gene expression analyses revealed that optimal epithelial transcript level of the anti-infective Reg3 beta requires TLR2 activation. Consistently, Yersinia infection triggered TLR2-dependent Reg3 beta expression in Peyer's patches. Importantly, oral treatment with exogenous TLR2 agonists in germ-free animals was able to further enhance Yersinia-induced expression of Reg3 beta and to restore intestinal resistance to Yersinia. Lastly, genetic ablation of Reg3 beta resulted in impaired clearance of the bacterial load in Peyer's patches. CONCLUSIONS: TLR2/REG3 beta is thus an essential component in conditioning epithelial defence signalling pathways against bacterial invasion.

Physical activity in overweight and nonoverweight preschool children.

OBJECTIVE: To compare the physical activity levels of overweight and non overweight 3- to 5-y-old children while attending preschool. A secondary aim was to evaluate weight-related differences in hypothesized parental determinants of child physical activity behavior. DESIGN: Cross-sectional study. SUBJECTS: A total of 245, 3- to 5-y-olds (127 girls, 118 boys) and their parent(s) (242 mothers, 173 fathers) recruited from nine preschools. Overweight status determined using the age- and sex-specific 85th percentile for body mass index (BMI) from CDC Growth Charts. MEASUREMENTS: Physical activity during the preschool day was assessed on multiple days via two independent objective measures-direct observation using the observation system for recording activity in preschools (OSRAP) and real-time accelerometry using the MTI/CSA 7164 accelerometer. Parents completed a take-home survey assessing sociodemographic information, parental height and weight, modeling of physical activity, support for physical activity, active toys and sporting equipment at home, child's television watching, frequency of park visitation, and perceptions of child competence. RESULTS: Overweight boys were significantly less active than their nonoverweight peers during the preschool day. No significant differences were observed in girls. Despite a strong association between childhood overweight status and parental obesity, no significant differences were observed for the hypothesized parental influences on physical activity behavior. CONCLUSIONS: Our results suggest that a significant proportion of overweight children may be at increased risk for further gains in adiposity because of low levels of physical activity during the preschool day.

Flagellin stimulation of intestinal epithelial cells triggers CCL20-mediated migration of dendritic cells.

Enteropathogenic bacteria elicit mucosal innate and adaptive immune responses. We investigated whether gut epithelial cells played a role in triggering an adaptive immune response by recruiting dendritic cells (DCs). Immature DCs are selectively attracted by the CCL20 chemokine. The expression of the CCL20 gene in human intestinal epithelial cell lines was up-regulated by pathogenic bacteria, including Salmonella species, but not by indigenous bacteria of the intestinal flora. The Salmonella machinery for epithelial cell invasion was not required for CCL20 gene activation. Flagellin but not the lipopolysaccharide was found to be the Salmonella factor responsible for stimulation of epithelial CCL20 production. CCL20 in turn triggered a specific migration of immature DCs. Our data show that crosstalk between bacterial flagellin and epithelial cells is essential for the recruitment of DCs, a mechanism that could be instrumental to initiate adaptive immune responses in the gut.

Nasal immunisation with Salmonella typhimurium producing rotavirus VP2 and VP6 antigens stimulates specific antibody response in serum and milk but fails to protect offspring.

Rotavirus specifically infects the small intestine of young infants resulting in severe diarrhoea. Mucosal antibody responses are required to cure the infection, and mucosal administration of rotavirus-like particles induces protective immunity without requiring a mucosal adjuvant such as cholera toxin. In addition, the rotavirus protein VP6 has been defined as a protective antigen in an adult mouse rotavirus infection model. Salmonella typhimurium is an epithelium-invasive bacterium that induces specific immune responses in mucosal tissues against itself and carried antigens. In this work, we investigated the capacity of a live recombinant S. typhimurium vaccine to stimulate antibody responses against rotavirus. We constructed an attenuated S. typhimurium strain simultaneously producing VP6 and VP2 rotavirus proteins in the cytoplasm. In contrast to expression in eukaryotic cells, VP6 and VP2 did not form virus-like particles in our bacterial system. After nasal administration of female mice, the live recombinant Salmonella were able to elicit an antibody response specific to both VP2 and VP6 in serum and milk. However, these antibodies failed to passively protect the offspring against rotavirus-induced diarrhoea.

Physical activity assessment in children and adolescents.

Chronic disease risk factors, including a sedentary lifestyle, may be present even in young children, suggesting that early prevention programmes may be critical to reducing the rates of chronic disease. Accurate assessment of physical activity in children is necessary to identify current levels of activity and to assess the effectiveness of intervention programmes designed to increase physical activity. This article summarises the strengths and limitations of the methods used to evaluate physical activity in children and adolescents. MEDLINE searches and journal article citations were used to locate 59 articles that validated physical activity measurement methods in children and adolescents. Only those methods that were validated against a more stringent measure were included in the review. Based on the definition of physical activity as any bodily movement resulting in energy expenditure (EE), direct observation of the individual's movement should be used as the gold standard for physical activity research. The doubly labelled water technique and indirect calorimetry can also be considered criterion measures for physical activity research, because they measure EE, a physiologic consequence closely associated with physical activity. Devices such as heart rate monitors, pedometers and accelerometers have become increasingly popular as measurement tools for physical activity. These devices reduce the subjectivity inherent in survey methods and can be used with large groups of individuals. Heart rate monitoring is sufficiently valid to use in creating broad physical activity categories (e.g. highly active, somewhat active, sedentary) but lacks the specificity needed to estimate physical activity in individuals. Laboratory and field validations of pedometers and accelerometers yield relatively high correlations using oxygen consumption (r = 0.62 to 0.93) or direct observation (r = 0.80 to 0.97) as criterion measures, although, they may not be able to capture all physical activity. Physical activity has traditionally been measured with surveys and recall instruments. These techniques must be used cautiously in a paediatric population that has difficulty recalling such information. Still, some studies have reported 73.4% to 86.3% agreement between these instruments and direct observation. Future investigations of physical activity instruments should validate the novel instrument against a higher standard. Additional studies are needed to investigate the possibility of improving the accuracy of measurement by combining 2 or more techniques. The accurate measurement of physical activity is critical for determining current levels of physical activity, monitoring compliance with physical activity guidelines, understanding the dose-response relationship between physical activity and health and determining the effectiveness of intervention programmes designed to improve physical activity.

Characterization of a plasmid region involved in Bacillus anthracis toxin production and pathogenesis.

The germination of spores within the host is the initial step of anthrax infection. We have shown, using immunofluorescence staining, confocal scanning laser microscopy and image cytometry analysis, that the alveolar macrophage is the primary site of B. anthracis germination in a murine inhalation infection model. B. anthracis germinated inside macrophages, in vesicles derived from the phagosomal compartment. We have demonstrated that the toxin genes and their trans-activator, AtxA, are expressed within the macrophages after germination. It was also shown that the pXO1 plasmid strongly enhanced capsule formation and that this influence is mediated by AtxA. This indicates the existence of a regulon where AtxA is the regulatory protein acting on genes located on different plasmids. We identified a tricistronic germination operon gerX located between the pag and atxA genes on the 40-kb toxin-encoding fragment of pXO1 . Analysis of a gerX null mutant indicated that gerX-encoded proteins are involved in the virulence of B. anthracis.

Entry and survival of Salmonella typhimurium in dendritic cells and presentation of recombinant antigens do not require macrophage-specific virulence factors.

Macrophages have long been regarded as the main target encountered by Salmonella typhimurium, a Gram-negative facultative intracellular pathogen that invades the intestinal mucosa. S. typhimurium, however, are first internalized by dendritic cells. To gain new insights into the interactions between Salmonella and the dendritic cells, we compared the fate of wild-type S. typhimurium and the virulence-attenuated PhoP constitutive (PhoP(c)) strain. The PhoP(c) strain is impaired for entry and survival in mammalian cells and is poorly processed by macrophages for antigen presentation on MHC class II molecules. Here, we show that bone marrow-derived dendritic cells can similarly process and present a foreign antigen expressed by the invasive wild-type and the attenuated PhoP(c) S. typhimurium. This property correlates with equivalent entry and survival efficiencies of both strains in dendritic cells. In addition, Salmonella strains mutated in mgtCB, sseC, and orfL genes required for macrophage survival showed no defect in survival in dendritic cells. Together, these results indicate that uptake of Salmonella by dendritic cells and subsequent antigen processing and presentation do not depend on virulence factors important in macrophages.

Protective antigen-mediated antibody response against a heterologous protein produced in vivo by Bacillus anthracis.

Bacillus anthracis secretes a lethal toxin composed of two proteins, the lethal factor (LF) and the protective antigen (PA), which interact within the host or in vitro at the surfaces of eukaryotic cells. Immunization with attenuated B. anthracis strains induces an antibody response against PA and LF. The LF-specific response is potentiated by the binding of LF to PA. In this study, we investigated the capacity of PA to increase the antibody response against a foreign antigen. We constructed a chimeric gene encoding the PA-binding part of LF (LF254) fused to the C fragment of tetanus toxin (ToxC). The construct was introduced by allelic exchange into the locus encoding LF. Two recombinant B. anthracis strains secreting the hybrid protein LF254-ToxC were generated, one in a PA-producing background and the other in a PA-deficient background. Mice were immunized with spores of the strains, and the humoral response and protection against tetanus toxin were assessed. The B. anthracis strain producing both PA and LF254-ToxC induced significantly higher antibody titers and provided better protection against a lethal challenge with tetanus toxin than did its PA-deficient counterpart. Thus, PA is able to potentiate protective immunity against a heterologous antigen, demonstrating the potential of B. anthracis recombinant strains for use as live vaccine vehicles.

Nasal immunization of mice with virus-like particles protects offspring against rotavirus diarrhea.

Rotavirus is the major cause of diarrhea among young infants in both humans and animals. Immune protection of newborns by vaccination is difficult to achieve since there is not enough time to mount an immune response before exposure to the virus. We have designed a vaccination strategy mediating transfer of neutralizing antibodies from the mother to the offspring during pregnancy and/or lactation. Adult female mice were nasally immunized with virus-like particles (VLPs) made of viral proteins VP2 and 6 (VLP2/6) or VP 2, 6, and 7 (VLP2/6/7) derived from the RF rotavirus strain in the presence or absence of cholera toxin. Both vaccines elicited serum and milk antibodies against the respective VPs. Four days after parturition, suckling pups were challenged orally with RF rotavirus. Pups from mothers immunized with VLP2/6/7 but not VLP2/6 were protected against rotavirus diarrhea, indicating that VP7 plays a key role in protection. Protection was mediated by milk rather than serum antibodies, and mucosal adjuvants were not required. In conclusion, VLPs containing VP7 administered nasally to mothers represent a promising vaccine candidate for the protection of suckling newborns against rotavirus-induced diarrhea, even in the absence of a mucosal adjuvant.

Field evaluation of the Computer Science and Applications, Inc. physical activity monitor.

PURPOSE: The purpose of this study was to evaluate the Computer Science and Applications, Inc. (CSA) activity monitor to quantify physical activity in free living subjects using an activity diary as the criterion measure. METHODS: Subjects also completed a 7-d physical activity recall at the end of the monitoring period. Nine male and 10 female subjects (mean, SD) (25.0, 3.6 yr) wore the CSA monitor for 7 consecutive days. On 3 of those days, subjects completed an activity diary (2 weekdays and 1 weekend day). Total kcal per day (Dkcal(tot)) was calculated from the self-reported diary classifications of the subject's activities. For the 3 days that coincided with the diary, total number of counts accumulated per day (cnt(tot)) was obtained from the monitor. RESULTS: The amount of activity per day recorded by the CSA monitor followed the same pattern of change as the activity diary. The cnt(tot) and Dkcal(tot) were significantly (P < or = 0.05) correlated on day 1 (r = 0.65), day 2 (r = 0.49), day 3 (r = 0.55), and for the 3 days pooled (r = 0.51). Subjects were classified as low, moderate, or highly active based on tertiles of kcal from the diary and counts from the CSA monitor. The percentage agreement between the CSA and the activity diary was 68.4% (Kappa = 0.53, P < 0.01). The number of minutes spent in the various activity categories were compared between instruments using an ANOVA model. The results of these analyses suggest that the CSA overestimated light activity and underestimated vigorous activity compared with the diary. CONCLUSION: In conclusion, the CSA monitor may be useful in a field situation where total physical activity and patterns of physical activity are the desired outcomes.

Role of toxin functional domains in anthrax pathogenesis.

We investigated the role of the functional domains of anthrax toxins during infection. Three proteins produced by Bacillus anthracis, the protective antigen (PA), the lethal factor (LF), and the edema factor (EF), combine in pairs to produce the lethal (PA+LF) and edema (PA+EF) toxins. A genetic strategy was developed to introduce by allelic exchange specific point mutations or in-frame deletions into B. anthracis toxin genes, thereby impairing either LF metalloprotease or EF adenylate cyclase activity or PA functional domains. In vivo effects of toxin mutations were analyzed in an experimental infection of mice. A tight correlation was observed between the properties of anthrax toxins delivered in vivo and their in vitro activities. The synergic effects of the lethal and edema toxins resulted purely from their enzymatic activities, suggesting that in vivo these toxins may act together. The PA-dependent antibody response to LF induced by immunization with live B. anthracis was used to follow the in vivo interaction of LF and PA. We found that the binding of LF to PA in vivo was necessary and sufficient for a strong antibody response against LF, whereas neither LF activity nor binding of lethal toxin complex to the cell surface was required. Mutant PA proteins were cleaved in mice sera. Thus, our data provide evidence that, during anthrax infection, PA may interact with LF before binding to the cell receptor. Immunoprotection studies indicated that the strain producing detoxified LF and EF, isogenic to the current live vaccine Sterne strain, is a safe candidate for use as a vaccine against anthrax.

Live attenuated Salmonella: a paradigm of mucosal vaccines.

Two key steps control immune responses in mucosal tissues: the sampling and transepithelial transport of antigens, and their targeting into professional antigen-presenting cells in mucosa-associated lymphoid tissue. Live Salmonella bacteria use strategies that allow them to cross the epithelial barrier of the gut, to survive in antigen-presenting cells where bacterial antigens are processed and presented to the immune cells, and to express adjuvant activity that prevents induction of oral tolerance. Two Salmonella serovars have been used as vaccines or vectors, S. typhimurium in mice and S. typhi in humans. S. typhimurium causes gastroenteritis in a broad host range, including humans, while S. typhi infection is restricted to humans. Attenuated S. typhimurium has been used successfully in mice to induce systemic and mucosal responses against more than 60 heterologous antigens. This review aims to revisit S. typhimurium-based vaccination, as an alternative to S. typhi, with special emphasis on the molecular pathogenesis of S. typhimurium and the host response. We then discuss how such knowledge constitutes the basis for the rational design of novel live mucosal vaccines.

Antigen delivery by attenuated Bacillus anthracis: new prospects in veterinary vaccines.

This report summarizes the recent investigations on the use of Bacillus anthracis as a live vector for delivery of antigens. Recombinant strains were constructed by engineering the current live Sterne vaccine. This vaccine, used to prevent anthrax in cattle, causes side-effects due to anthrax toxin activities. Bacteria producing a genetically detoxified toxin factor were devoid of lethal effects and were as protective as the Sterne strain against experimental anthrax. Moreover, B. anthracis expressing a foreign antigen controlled by an in vivo inducible promoter were able to generate either antibody or cellular protective responses against heterologous diseases.

Identification and characterization of a germination operon on the virulence plasmid pXO1 of Bacillus anthracis.

The spores of Bacillus anthracis, the agent of anthrax disease, germinate within professional phagocytes, such as murine macrophage-like RAW264.7 cells and alveolar macrophages. We identified a cluster of germination genes extending for 3608 nucleotides between the pag and atxA genes on the B. anthracis virulence plasmid pXO1. The three predicted proteins (40, 55 and 37 kDa in size) have significant sequence similarities to B. subtilis, B. cereus and B. megaterium germination proteins. Northern blot analysis of total RNA from sporulating cells indicated that the gerX locus was organized as a tricistronic operon (gerXB, gerXA and gerXC). Primer extension analysis identified a major potential transcriptional start site 31 bp upstream from the translation initiation codon of gerXB. Expression of the gerX operon was studied using a gerXB-lacZ transcriptional fusion. Expression began 2.5-3 h after the initiation of sporulation and was detected exclusively in the forespore compartment. A gerX null mutant was constructed. It was less virulent than the parental strain and did not germinate efficiently in vivo or in vitro within phagocytic cells. These data strongly suggest that gerX-encoded proteins are involved in the virulence of B. anthracis.