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β-Lactams are the most widely prescribed antibiotics used for the control and treatment of bacterial infections. The direct effect of β-lactams on bacteria is well studied worldwide.

Chronic obstructive pulmonary disease (COPD) is a major cause of morbidity and mortality worldwide. The major bacterial cause of COPD exacerbations is non-typeable Haemophilus influenzae (NTHi). This susceptibility to infection involves a defective production of interleukin (IL)-22 which plays an important role in mucosal defense. Prophylactic administration of flagellin, a Toll-like receptor 5 (TLR5) agonist, protects healthy mice against respiratory pathogenic bacteria. We hypothesized that TLR5-mediated stimulation of lung immunity might prevent COPD exacerbations due to NTHi. Mice were chronically exposed to cigarette smoke and then infected with NTHi. According our preventive or therapeutic protocol, flagellin was administered intraperitoneally. Cigarette smoke-exposed mice treated with flagellin showed a lower bacterial load in the airways, the lungs and the blood. This protection was associated with an early neutrophilia, a lower production of pro-inflammatory cytokines and an increased IL-22 production. Flagellin treatment decreased the recruitment of inflammatory cells and the lung damages related to exacerbation. Protective effect of flagellin against NTHi was altered by treatment with anti-IL-22 blocking antibodies in cigarette smoke-exposed mice and in Il22−/− mice. Flagellin treatment also amplified the production of the β-defensin2 anti-bacterial peptides. This study shows that stimulation of innate immunity by a TLR5 ligand is a potent antibacterial treatment in cigarette smoke exposed mice, suggesting innovative therapeutic strategies against acute exacerbation in COPD.

The emergence and spread of antibiotic resistance emphasize the need for alternative treatment strategies against bacterial infections. Boosting the host innate immunity is not only readily deployable in most individuals but can also mobilize many different antibacterial defenses. This study tested the hypothesis whereby stimulation of the innate immune receptor Toll-like receptor 4 (TLR4) can be combined with antibiotics in the treatment of invasive pneumonia. In a mouse model ofStreptococcus pneumoniaeinfection, a single oral administration of low-dose amoxicillin (AMX) or the systemic delivery of monophosphoryl lipid A (MPLA, a clinically-approved TLR4 activator) decreased the bacterial load in lung and spleen, although this was not sufficient for long-term survival. In contrast, a single treatment with a combination of MPLA and AMX induced significant bacterial clearance with little to no regrowth over time, and was associated with longer survival. Upregulation of genes related to granulocyte infiltration in lung tissue and elevation of blood levels of pro-inflammatory cytokines was immediate and transient in MPLA-treated mice; this indicates activation of the innate immune system in a context of infection. Combination treatment was associated with a well-preserved lung tissue architecture and more rapid recovery from inflammation - suggesting that immune activation by MPLA does not exacerbate pneumonia-induced damage. After AMX administration, plasma AMX concentrations rapidly reached the maximum and declined, whereas the downstream effects of MPLA extended beyond AMX elimination; these findings suggested a two-step effect. Our results demonstrated that leveraging host innate immunity increases the efficacy of antibiotic therapy in bacterial pneumonia.

International audience; Influenza infections remain a burden on health care systems despite vaccination programs and marketed antiviral drugs. Immunomodulation through activation of innate sensors could represent innovative approaches to fight the flu. This study evaluated the ability of flagellin, agonist of Toll-like receptor 5 (TLR5), to control the replication of influenza A virus (IAV) in mice. First, we showed that systemic or intranasal administration of flagellin activated transcription of anti-viral genes in lung tissue. Prophylactic and therapeutic flagellin administration resulted in decreased levels of viral RNA and infectious virus in the lungs of H3N2 IAV-infected mice. The effect of the flagellin on viral replication was also observed in Ifnar-/- and Il22-/- IAV-infected mice, suggesting a mechanism independent of type I interferon and interleukin 22 signaling. In addition, a combination therapy associating the neuraminidase inhibitor oseltamivir and flagellin was more effective than standalone treatments in reducing pulmonary viral replication. Thus, this study highlights the therapeutic potential of the flagellin to control the replication of the influenza virus.

BACKGROUND During their studies, pharmacy students must acquire the specific skills in clinical virology required for their subsequent professional practice. Recent experiments on teaching and learning in higher education have shown that hybrid courses strengthen the students’ commitment to learning and enable high-quality knowledge acquisition. OBJECTIVE This study concerned the design and deployment of a hybrid course that combines face-to-face and Web-based instruction in clinical virology for fourth-year pharmacy students. The study’s objectives were to (1) measure the students’ level of involvement in the course, (2) gauge their interest in this type of learning, and (3) highlight any associated difficulties. METHODS The study included 194 fourth-year pharmacy students from the Lille Faculty of Pharmacy (University of Lille, Lille, France) between January and June 2017. The students followed a hybrid course comprising an online learning module and 5 tutorial sessions in which professional situations were simulated. The learning module and 3 online evaluation sessions were delivered via the Moodle learning management system. Each tutorial session ended with an evaluation. The number of Moodle log-ins, the number of views of learning resources, and the evaluation marks were recorded. The coefficient for the correlation between the marks in the online evaluation and those in the tutorials was calculated. The students’ opinions and level of satisfaction were evaluated via a course questionnaire. RESULTS The course’s learning resources and Web pages were viewed 21,446 and 3413 times, respectively. Of the 194 students, 188 (96.9%) passed the course (ie, marks of at least 10 out of 20). There was a satisfactory correlation between the marks obtained in the online evaluations and those obtained after the tutorials. The course met the students’ expectations in 53.2% of cases, and 57.4% of the students stated that they were able to work at their own pace. Finally, 26.6% of the students stated that they had difficulty organizing their work around this hybrid course. CONCLUSIONS Our results showed that pharmacy students were strongly in favor of a hybrid course. The levels of attendance and participation were high. However, teachers must be aware that some students will encounter organizational difficulties.

Bacterial superantigens (SAgs) are immunostimulatory toxins that induce acute diseases mainly through the massive release of inflammatory cytokines. Yersinia pseudotuberculosis is the only Gram-negative bacterium known to produce a SAg ( Y. pseudotuberculosis -derived mitogen [YPM]). This SAg binds major histocompatibility complex class II molecules on antigen-presenting cells and T cell receptors (TcR) bearing the variable region Vβ3, Vβ9, Vβ13.1, or Vβ13.2 (in humans) and Vβ7 or Vβ8 (in mice). We have previously shown that YPM exacerbates the virulence of Y. pseudotuberculosis in mice. With a view to understanding the mechanism of YPM's toxicity, we compared the immune response in BALB/c mice infected with a YPM-producing Y. pseudotuberculosis or the corresponding isogenic, SAg-deficient mutant. Five days after infection, we observed strong CD4 + Vβ7 + T cell expansion and marked interleukin-4 (IL-4) production in mice inoculated with SAg-producing Y. pseudotuberculosis . These phenomena were correlated with the activation of ypm gene transcription in liver and spleen. A transcriptomic analysis revealed that the presence of YPM also increased expression of granzyme and perforin genes in the host's liver and spleen. This expression was attributed to a CD4 + T cell subset, rather than to natural killer T (NKT) cells that display a TcR with a Vβ region that is potentially recognized by YPM. Increased production of cytotoxic molecules was correlated with hepatotoxicity, as demonstrated by an increase in plasma alanine aminotransferase activity. Our results demonstrate that YPM activates a potentially hepatotoxic CD4 + T cell population.

Motility is often a pathogenicity determinant of bacteria targeting mucosal tissues. Flagella constitute the machinery that propels bacteria into appropriate niches. Besides motility, the structural component, flagellin, which forms the flagella, targets Toll-like receptor 5 (TLR5) to activate innate immunity. The compartmentalization of flagellin-mediated immunity and the contribution of epithelial cells and dendritic cells in detecting flagellin within luminal and basal sides are highlighted here, respectively. While a direct stimulation of the epithelium mainly results in recruitment of immune cells and production of antimicrobial molecules, TLR5 engagement on parenchymal dendritic cells can contribute to the stimulation of innate lymphocytes such as type 3 innate lymphoid cells, as well as T helper cells. This review, therefore, illustrates how the innate and adaptive immunity to flagellin are differentially regulated by the epithelium and the dendritic cells in response to pathogens that either colonize or invade mucosa. Fil: Vijayan, Aneesh. Centre National de la Recherche Scientifique; Francia Fil: Rumbo, Martín. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - La Plata. Instituto de Estudios Inmunológicos y Fisiopatológicos. Universidad Nacional de La Plata. Facultad de Ciencias Exactas. Instituto de Estudios Inmunológicos y Fisiopatológicos; Argentina Fil: Carnoy, Christophe. Centre National de la Recherche Scientifique; Francia Fil: Sirard, Jean-Claude. Centre National de la Recherche Scientifique; Francia

Chronic obstructive pulmonary disease is a major cause of morbidity and mortality worldwide. It is mainly associated to acute exacerbations due to bacterial infection, by non-typeable Haemophilus influenzae (NTHi) and Streptococcus pneumoniae (SP). Prophylactic administration of flagellin, the Toll-like receptor 5 (TLR5) agonist, protects mice against respiratory pathogenic bacteria. We hypothesized that TLR5-mediated stimulation of lung immunity might prevent COPD exacerbation. Flagellin was intraperitoneally administrated to treat NTHi or SP-infected COPD animals. Compared with control, COPD mice treated with flagellin showed a lower bacterial load in the BAL and the lungs and less bacterial dissemination in the blood. This was associated with an early neutrophilia, a lower production of proinflammatory cytokines but also an increased production of anti-bacterial peptides and of IL-22. Interestingly, the treatment with flagellin decreased the inflammatory cell recruitment and limited lung damage related to exacerbation. Lastly, administration of neutralizing anti-IL-22 antibody attenuated the beneficial effect of Flagellin against NTHI infection. This study shows for the first time that innate immunity stimulation with flagellin is a potent antibacterial treatment against exacerbation during experimental COPD through an IL-22 dependent mechanism. This opens the way to innovative therapeutic strategies. This work is sponsored by INSERM-Transfert, Paris, France.

Signaling through Toll-like receptors (TLRs), the main receptors in innate immunity, is essential for the defense of mucosal surfaces. It was previously shown that systemic TLR5 stimulation by bacterial flagellin induces an immediate, transient interleukin-22 (IL-22)-dependent antimicrobial response to bacterial or viral infections of the mucosa. This process was dependent on the activation of type 3 innate lymphoid cells (ILCs). The objective of the present study was to analyze the effects of flagellin treatment in a murine model of oral infection with Yersinia pseudotuberculosis (an invasive, Gram-negative, enteropathogenic bacterium that targets the small intestine). We found that systemic administration of flagellin significantly increased the survival rate after intestinal infection (but not systemic infection) by Y. pseudotuberculosis . This protection was associated with a low bacterial count in the gut and the spleen. In contrast, no protection was afforded by administration of the TLR4 agonist lipopolysaccharide, suggesting the presence of a flagellin-specific effect. Lastly, we found that TLR5- and MyD88-mediated signaling was required for the protective effects of flagellin, whereas neither lymphoid cells nor IL-22 was involved.

Flagellins are the main constituents of bacterial flagella and belong to the group of microbe-associated molecular patterns. Their ability to stimulate signaling through Toll-like receptor 5 (TLR5) is the main activity that was used to develop flagellin-based vaccines. This report will summarize the current knowledge on flagellin's mode of action and will focus on the compartmentalization of TLR5 signaling in various sites of the body host. The impact of human TLR5 genetic polymorphism on the susceptibility to mucosal infections and on vaccination will also be discussed.

Mucosal sites are continuously exposed to pathogenic microorganisms and are therefore equipped to control respiratory infections. Type 3 innate lymphoid cells (ILC3) are key players in antimicrobial defense in intestinal mucosa, through interleukin 17 and interleukin 22 (IL-22) production. The present study aimed at analyzing the distribution and function of ILC3 in the respiratory tract. We first observed that lung mucosa harbors a discrete population of ILC3 expressing CD127, CD90, CCR6, and the transcriptional factor RORγt. In addition, lung ILC3 were identified as a major source of IL-22 in response to interleukin 23 stimulation. During Streptococcus pneumoniae infection, ILC3 rapidly accumulated in the lung tissue to produce IL-22. In response to S. pneumoniae, dendritic cells and MyD88, an important adaptor of innate immunity, play critical functions in IL-22 production by ILC3. Finally, administration of the Toll-like receptor 5 agonist flagellin during S. pneumoniae challenge exacerbated IL-22 production by ILC3, a process that protects against lethal infection. In conclusion, boosting lung ILC3 might represent an interesting strategy to fight respiratory bacterial infections. © The Author 2014.

Prophylactic intranasal administration of the Toll-like receptor 5 (TLR5) agonist flagellin protects mice against respiratory pathogenic bacteria. We hypothesized that TLR5-mediated stimulation of lung immunity might improve the therapeutic index of antibiotics for the treatment of Streptococcus pneumoniae respiratory infections in mice. Intranasal administration of flagellin was combined with either oral administration of amoxicillin or intraperitoneal injection of trimethoprim-sulfamethoxazole to treat S. pneumoniae -infected animals. Compared with standalone treatments, the combination of antibiotic and flagellin resulted in a lower bacterial load in the lungs and greater protection against S. pneumoniae dissemination and was associated with an early increase in neutrophil infiltration in the airways. The antibiotic-flagellin combination treatment was, however, not associated with any exacerbation of inflammation. Moreover, combination treatment was more efficacious than standalone antibiotic treatments in the context of post-influenza virus pneumococcal infection. Lastly, TLR5 signaling was shown to be mandatory for the efficacy of the combined antibacterial therapy. This report is the first to show that combining antibiotic treatment with the stimulation of mucosal innate immunity is a potent antibacterial strategy against pneumonia.

Among Yersinia spp., Y. enterocolitica is the species most frequently isolated from infected aneurysms. This report describes the first case of postaneurysmal prosthetic vascular infection due to a superantigen-negative Yersinia pseudotuberculosis strain, showing a potential affinity of this species for endovascular tissue.

Diffusely adheringEscherichia coli(DAEC) strains expressing F1845 fimbrial adhesin or Dr hemagglutinin belonging to the Afa/Dr family of adhesins infect cultured polarized human intestinal cells through recognition of the brush border-associated decay-accelerating factor (DAF; CD55) as a receptor. The wild-type Afa/Dr DAEC strain C1845 has been shown to induce brush border lesions by an adhesin-dependent mechanism triggering apical F-actin rearrangements. In the present study, we undertook to further characterize cell injuries following the interaction of wild-type Afa/Dr DAEC strains C1845 and IH11128 expressing fimbrial F1845 adhesin and Dr hemagglutinin, respectively, with polarized, fully differentiated Caco-2/TC7 cells. In both cases, bacterium-cell interaction was followed by rearrangement of the major brush border-associated cytoskeletal proteins F-actin, villin, and fimbrin, proteins which play a pivotal role in brush border assembly. In contrast, distribution of G-actin, actin-depolymerizing factor, and tubulin was not modified. UsingdraEmutants, we found that a mutant in which cysteine replaces aspartic acid at position 54 conserved binding capacity but failed to induce F-actin disassembly. Accompanying the cytoskeleton injuries, we found that the distribution of brush border-associated functional proteins sucrase-isomaltase (SI), dipeptidylpeptidase IV (DPPIV), glucose transporter SGLT1, and fructose transporter GLUT5 was dramatically altered. In parallel, SI and DPPIV enzyme activity decreased.

The Afa/Dr family of diffusely adheringEscherichia coli(Afa/Dr DAEC) includes bacteria expressing afimbrial adhesins (AFA), Dr hemagglutinin, and fimbrial F1845 adhesin. We show that infection of human intestinal Caco-2/TC7 cells by the Afa/Dr DAEC strains C1845 and IH11128 is followed by clustering of CD55 around adhering bacteria. Mapping of CD55 epitopes involved in CD55 clustering by Afa/Dr DAEC was conducted using CD55 deletion mutants expressed by stable transfection in CHO cells. Deletion in the short consensus repeat 1 (SCR1) domain abolished Afa/Dr DAEC-induced CD55 clustering. In contrast, deletion in the SCR4 domain does not modify Afa/Dr DAEC-induced CD55 clustering. We show that the brush border-associated glycosylphosphatidylinositol (GPI)-anchored protein CD66e (carcinoembryonic antigen) is recruited by the Afa/Dr DAEC strains C1845 and IH11128. This conclusion is based on the observations that (i) infection of Caco-2/TC7 cells by Afa/Dr DAEC strains is followed by clustering of CD66e around adhering bacteria and (ii) Afa/Dr DAEC strains bound efficiently to stably transfected HeLa cells expressing CD66e, accompanied by CD66e clustering around adhering bacteria. Inhibition assay using monoclonal antibodies directed against CD55 SCR domains, and polyclonal anti-CD55 and anti-CD66e antibodies demonstrate that CD55 and CD66e function as a receptors for the C1845 and IH11128 bacteria. Moreover, using structuraldraEgene mutants, we found that a mutant in which cysteine replaced aspartic acid at position 54 displayed conserved binding capacity but failed to induce CD55 and CD66e clustering. Taken together, these data give new insights into the mechanisms by which Afa/Dr DAEC induces adhesin-dependent cross talk in the human polarized intestinal epithelial cells by mobilizing brush border-associated GPI-anchored proteins known to function as transducing molecules.

The gram-negative bacterium Yersinia pseudotuberculosis is widely spread in nature and is responsible for sporadic infection in many animal species (10). Humans are commonly infected after ingestion of food or water contaminated with excreta of infected animals. Y. pseudotuberculosis causes acute ileitis and mesenteric lymphadenitis, sometimes complicated by septicemia (10, 26), but is also responsible for the occurrence of postinfection complications such as reactive arthritis and erythema nodosum (10, 35, 45, 46). This microorganism has been suggested as one of the causative agents of Kawasaki syndrome, an acute, self-limited vasculitis affecting predominantly infants and young children (4, 25, 39). A few years ago, Y. pseudotuberculosis strains producing a mitogen activity were isolated from a mass outbreak in Japan (48) and from a patient with Kawasaki-like symptoms (1, 51). The substance, purified from bacterial lysates and exhibiting a mitogenic activity on human peripheral blood mononuclear cells (PBMC), was first designated YPM, for Y. pseudotuberculosis-derived mitogen (30), and then YPMa after the discovery of a variant (36). YPMa was characterized as a 14.5-kDa superantigenic toxin activating human T lymphocytes bearing T-cell receptors exhibiting the Vβ3, Vβ9, Vβ13.1, and Vβ13.2 variable regions (1, 48). The geographic distribution of the 456-bp superantigen-encoding gene ypmA is rather heterogeneous, being present in most Y. pseudotuberculosis strains from the Far East but only in about 20% of European clinical isolates (13, 52). A recent study showed that 61% of patients acutely infected with Y. pseudotuberculosis, especially those with systemic complications, had elevated anti-YPM immunoglobulin G level in blood, thereby demonstrating the production of YPMa in vivo (2). Furthermore, Vβ3-bearing T cells were increased in patients during the acute phase of the disease (2). Additionally, Miyoshi-Akiyama et al. (32) found that purified YPMa was able to induce lethal shock in a murine experimental model, demonstrating the toxicity of the Y. pseudotuberculosis superantigenic toxin in vivo. The toxin was also found to alter in vitro epithelial function by reducing active ion transport and increasing epithelial permeability (16). Altogether, these data suggest a role of YPMa in the pathogenesis of Y. pseudotuberculosis infections, but experimental evidence necessary to confirm this hypothesis is lacking. In this study, we constructed a superantigen-deficient mutant of Y. pseudotuberculosis and tested its virulence in a mouse experimental model of infection. Rodents have been extensively used as the animal model to study Yersinia infections since they develop a disease resembling yersiniosis in humans (22). We found that inactivation of ypmA reduced the virulence of Y. pseudotuberculosis after intravenous (i.v.) challenge but not after intragastric (i.g.) inoculation, demonstrating an exacerbated toxicity of YPMa-producing Y. pseudotuberculosis in systemic infection.

The fimbrial and afimbrial adhesins of the Dr family mediate the adherence of uropathogenic and diarrhoea-associated Escherichia coli to decay-accelerating factor (DAF) present on erythrocytes and other cell types. The Dr haemagglutinin binds type IV collagen and, unlike other members of the Dr family, mediates an adherence inhibited in the presence of chloramphenicol. We examined the ability of other members of the Dr family-AFAI, AFAIII, and F1845-to bind to type IV collagen, and demonstrated that the collagen-binding phenotype was unique to the Dr haemagglutinin. We employed site-directed mutagenesis to demonstrate the requirement of a negatively charged amino-acid at position 54 of the Dr haemagglutinin subunit for chloramphenicol sensitivity of binding. Mutations at position 32, 40, 54, 90, and 113 differently affected type IV collagen binding and chloramphenicol sensitivity of binding, while retaining DAF-binding capability. These results suggest the existence of a conformational receptor-binding domain in the major structural subunit of Dr family adhesins and demonstrate that chloramphenicol sensitivity of binding and adherence to type IV collagen were independent and separable phenotypes. Finally, we showed that the two conserved cysteine residues of Dr family structural subunits form a disulphide bond and that mutations of these residues abolish haemagglutination and binding to type IV collagen.

International audience; Chronic ingestion of environmental heavy metals such as lead (Pb) and cadmium (Cd) causes various well-documented pathologies in specific target organs following their intestinal absorption and subsequent accumulation. However, little is known about the direct impact of the non-absorbed heavy metals on the small intestine and the colon homeostasis. The aim of our study was to compare the specific bioaccumulation and retention of Cd and Pb and their effect on the essential metal balance in primary organs, with those occurring specifically in the gastrointestinal tract of mice. Various doses of Cd (5, 20 and 100 mg l−1) and Pb (100 and 500 mg l−1) chloride salts were provided in drinking water for subchronic to chronic exposures (4, 8 and 12 weeks). In contrast to a clear dose- and time-dependent accumulation in target organs, results showed that intestines are poor accumulators for Cd and Pb. Notwithstanding, changes in gene expression of representative intestinal markers revealed that the transport-, oxidative- and inflammatory status of the gut epithelium of the duodenum, ileum and colon were specifically affected by both heavy metal species. Additionally, in vivo comet assay used to evaluate the impact of heavy metals on DNA damage showed clear genotoxic activities of Cd, on both the upper and distal parts of the gastrointestinal tract. Altogether, these results outline the resilience of the gut which balances the various effects of chronic Cd and Pb in the intestinal mucosa. Collectively, it provides useful information for the risk assessment of heavy metals in gut homeostasis and further disease’s susceptibility.

There was an error in affiliation 1 for authors Aleksandra W. Debowski, Phebe Verbrugghe, Hans-Olof Nilsson, Jonathan C. Gauntlett, Alma Fulurija, Tania Camilleri, Barry J. Marshall, and Mohammed Benghezal. Affiliation 1 should be: Ondek Pty Ltd and Helicobacter pylori Research Laboratory, School of Pathology & Laboratory Medicine, M504, Marshall Centre for Infectious Disease Research and Training, University of Western Australia, Nedlands, Western Australia

In the model organism E. coli, recombination mediated by the related XerC and XerD recombinases complexed with the FtsK translocase at specialized dif sites, resolves dimeric chromosomes into free monomers to allow efficient chromosome segregation at cell division. Computational genome analysis of Helicobacter pylori, a slow growing gastric pathogen, identified just one chromosomal xer gene (xerH) and its cognate dif site (difH). Here we show that recombination between directly repeated difH sites requires XerH, FtsK but not XerT, the TnPZ transposon associated recombinase. xerH inactivation was not lethal, but resulted in increased DNA per cell, suggesting defective chromosome segregation. The xerH mutant also failed to colonize mice, and was more susceptible to UV and ciprofloxacin, which induce DNA breakage, and thereby recombination and chromosome dimer formation. xerH inactivation and overexpression each led to a DNA segregation defect, suggesting a role for Xer recombination in regulation of replication. In addition to chromosome dimer resolution and based on the absence of genes for topoisomerase IV (parC, parE) in H. pylori, we speculate that XerH may contribute to chromosome decatenation, although possible involvement of H. pylori's DNA gyrase and topoisomerase III homologue are also considered. Further analyses of this system should contribute to general understanding of and possibly therapy development for H. pylori, which causes peptic ulcers and gastric cancer; for the closely related, diarrheagenic Campylobacter species; and for unrelated slow growing pathogens that lack topoisomerase IV, such as Mycobacterium tuberculosis.

In adaptive immunity, Th17 lymphocytes produce the IL-17 and IL-22 cytokines that stimulate mucosal antimicrobial defenses and tissue repair. In this study, we observed that the TLR5 agonist flagellin induced swift and transient transcription of genes encoding IL-17 and IL-22 in lymphoid, gut, and lung tissues. This innate response also temporarily enhanced the expression of genes associated with the antimicrobial Th17 signature. The source of the Th17-related cytokines was identified as novel populations of CD3negCD127+ immune cells among which CD4-expressing cells resembling lymphoid tissue inducer cells. We also demonstrated that dendritic cells are essential for expression of Th17-related cytokines and so for stimulation of innate cells. These data define that TLR-induced activation of CD3negCD127+ cells and production of Th17-related cytokines may be crucial for the early defenses against pathogen invasion of host tissues.

Mucin glycopeptides were prepared from the salivary mucins of 20 healthy donors with blood group O. The carbohydrate chains of the high-molecular-weight mucins were released by alkaline borohydride treatment. Neutral and monosialylated oligosaccharide-alditols were purified by ion-exchange chromatography, gel filtration, and HPLC. The structures of the oligosaccharide-alditols were determined by high-resolution 1H-NMR spectroscopy in combination with fast atom bombardment mass spectrometry and methylation analysis. Thirty-seven oligosaccharide-alditols were characterized and illustrate the extreme diversity of the salivary mucins carbohydrate chains. This diversity might represent a mosaic of bacterial adhesion sites and be involved in the early events of the nonimmune defense of the oral cavity. Among these 37 oligosaccharide-alditols, 31 have not been previously described in human saliva and five of these are novel structures: [formula: see text]

The carbohydrate chains of the respiratory-mucus glycoproteins of a patient (blood group O) suffering from bronchiectasis due to Kartagener's syndrome, were released by alkaline borohydride treatment of a pronase digest. Neutral oligosaccharides were obtained after ion-exchange chromatography and were subsequently separated utilizing gel filtration, HPLC on normal-phase alkylamine-bonded silica and reverse-phase HPLC, into 46 fractions

Oligosaccharide-alditols derived from the respiratory mucins of a patient suffering from bronchiectasis were separated by HPLC into 46 fractions. The structures of 11 oligosaccharides with GlcNAcβ(1→3)Galβ(1→4)-GlcNAcβ(1→6)GalNAc-ol in common have been established (preceding paper in this journal). In this second part the structures of 12 oligosaccharides were established 8 of which have not been described before

In 1993, a mitogen with superantigenic features was characterized in the Gram-negative bacillus Yersinia pseudotuberculosis. It was described as the causative agent of tuberculosis-like lesions (pseudotuberculosis) in guinea pigs. Human infection is most probably established by ingestion of food or water contaminated by the excreta of infected animals. In immunocompetent hosts, infection is self-limited and causes relatively mild illness. The most frequent clinical symptoms are fever and abdominal pain rather than the diarrhea and vomiting usually associated with gastrointestinal infections. A typical feature of Y. pseudotuberculosis infection is the occurrence of post-infection complications, such as reactive arthritis and erythema nodosum. The typical post-infection immunopathological complications that may occur after infection with Y pseudotuberculosi suggested the involvement of a superantigen-like molecule. Although structurally and genetically unrelatedto streptococcal, staphylococcal, and mycoplasmal superantigens, YPM exhibits all the classical features of a superantigenic toxin. The YPM structural and genetic originality raises the central question of what makes a molecule a superantigen. YPM interaction with its ligands is still unknown but deserves serious attention. The recent characterization of the toxin 3D structure will undoubtedly unveil the molecular mechanism responsible for YPM-mediated mitogenicity.

An internal control of amplification was constructed by recombinant PCR to detect PCR inhibitors. This exogenous DNA was included in the reaction mixture and coamplified with the target gene. This detection was successfully applied to the diagnosis of whooping cough by amplification of a fragment of Bordetella pertussis IS 481 .

The superantigen-encoding ypm gene and the pil gene cluster governing type IV pilus biogenesis have been laterally acquired by Yersinia pseudotuberculosis. PCR assays on 270 unrelated strains from various environmental and animal sources revealed a significant association of ypm and pil in isolates.

The pathogenic mechanism of recurrent or chronic urinary tract infection is poorly understood. Escherichia coli cells bearing Dr fimbriae display unique tropism to the basement membrane (BM)-renal interstitium that enables the bacteria to cause chronic pyelonephritis in experimental mice. The renal receptors for Dr-fimbriated E. coli are type IV collagen and decay-accelerating factor (DAF). We hypothesized that type IV collagen receptor-mediated BM-interstitial tropism is essential for E. coli to cause chronic pyelonephritis. To test the role of the type IV collagen tropism of Dr-fimbriated E. coli in renal persistence, we constructed an isogenic mutant in the DraE adhesin subunit that was unable to bind type IV collagen but retained binding to DAF and examined its virulence in the mouse model. The collagen-binding mutant DrI113T was eliminated from the mouse renal tissues in 6 to 8 weeks, while the parent strain caused persistent renal infection that lasted at least 14 weeks ( P ≤ 0.02). Transcomplementation with the intact Dr operon restored collagen-binding activity, BM-interstitial tropism, and the ability to cause persistent renal infection. We conclude that type IV collagen binding mediated by DraE adhesin is a critical step for the development of persistent renal infection in a murine model of E. coli pyelonephritis.

Yersinia pseudotuberculosisproduces YPM (Y. pseudotuberculosis-derived mitogen), a superantigenic toxin that exacerbates the virulence of the bacterium in vivo. To date, three alleles of the superantigen gene (ypmA,ypmB, andypmC) have been described. These genes are not found in allY. pseudotuberculosisstrains and have a low GC content, suggesting their location on mobile genetic elements. To elucidate this question, the genetic environment of the superantigen-encoding genes was characterized and 11 open reading frames (ORFs) were defined. Sequence analysis revealed that theypmgenes were not associated with plasmids, phages, transposons, or pathogenicity islands and that the superantigen genes were always located in the chromosome between ORF3 and ORF4. Nonsuperantigenic strains exhibited the same genetic organization of the locus but lacked theypmgene between ORF3 and ORF4. A new insertion sequence, designated IS1398, which displays features of the Tn3family, was characterized downstream of theypmAandypmCgenes. A 13.3-kb region containing theypmgenes was not found in the genome ofY. pestis(CO92 and KIM 5 strains). We experimentally induced deletion of theypmgene from a superantigen-expressingY. pseudotuberculosis: using the association ofaph(3′)-IIIaandsacBgenes, we demonstrated that when these reporter genes were present in theypmlocus, deletion of these genes was about 250 times more frequent than when they were located in another region of theY. pseudotuberculosischromosome. These results indicate that unlike other superantigenic toxin genes, theYersinia ypmgenes are not associated with mobile genetic elements but are inserted in an unstable locus of the genome.

Superantigens (SAgs) are viral and bacterial proteins exhibiting a highly potent polyclonal lymphocyte-proliferating activity for CD4(+), CD8(+) and sometimes gammadelta(+) T cells of human and (or) various animal species. Unlike conventional antigens, SAgs bind as unprocessed proteins to invariant regions of major histocompatibility complex (MHC) class II molecules on the surface of antigen-presenting cells (APCs) and to particular motifs of the variable region of the beta chain (Vbeta) of T-cell receptor (TcR) outside the antigen-binding groove. As a consequence, SAgs stimulate at nano-to picogram concentrations up to 10 to 30% of host T-cell repertoire while only one in 10(5)-10(6) T cells (0.01-0.0001%) are activated upon conventional antigenic peptide binding to TcR. SAg activation of an unusually high percentage of T lymphocytes initiates massive release of pro-inflammatory and other cytokines which play a pivotal role in the pathogenesis of the diseases provoked by SAg-producing microorganisms. We briefly describe in this review the molecular and biological properties of the bacterial superantigen toxins and mitogens identified in the past decade.

The culture supernatant from a single Pseudomonas aeruginosa strain has been reported to show neuraminidase activity, leading to the speculation that this bacterium may use this enzyme as a virulence factor to act on host macromolecules. In order to extend this finding, we have examined the activity of concentrated P. aeruginosa culture supernatants and cells on synthetic and natural substrates containing sialic acid, such as human respiratory mucins. Four P. aeruginosa strains showed some activity on the synthetic substrate 4-methylumbelliferyl-alpha-D-N-acetylneuraminic acid but failed to liberate N-acetylneuraminic acid from six different natural substrates. Attempts to induce enzyme production by use of human respiratory mucins in the culture medium were also unsuccessful. The supernatants also showed N-acetyl-beta-D-glucosaminidase-like activity on a synthetic substrate but did not liberate N-acetylhexosamines from natural substrates. We conclude that the neuraminidase-like activity observed in P. aeruginosa can be defined as an arylneuraminidase and that the possession of a neuraminidase active on natural substrates is not a common attribute of P. aeruginosa strains.

We compared the chemical composition of salivary mucin glycopeptides from cystic fibrosis (CF) and from non-CF subjects and the adhesion of Pseudomonas aeruginosa to these different salivary glycopeptides. Three pools of CF saliva, four pools of non-CF saliva, one individual CF saliva, and one individual non-CF saliva were studied. The soluble fraction of the saliva was treated with pronase, and gel filtration was performed to obtain high and low molecular mass salivary mucin glycopeptides. The yield of total glycopeptides was significantly higher from CF than from non-CF saliva. Furthermore, the chemical composition revealed a significantly higher sialic acid content in CF than in non-CF mucin glycopeptides, and higher sulfate and fucose content in CF than in non-CF high molecular mass glycopeptides. We studied the adhesion of a nonmucoid strain of P. aeruginosa (1244), its nonpiliated isogenic derivative, and a mucoid strain (M35) to salivary mucin glycopeptides from patients with CF and from non-CF subjects. The three strains bound significantly more to the CF salivary glycopeptides than to the corresponding non-CF salivary glycopeptides. The nonpiliated isogenic mutant of P. aeruginosa 1244 also bound to CF salivary glycopeptides, suggesting that the adhesion of P. aeruginosa could involve nonpilus adhesions. Furthermore, neuraminidase treatment of CF glycopeptides decreased the adhesion of P. aeruginosa 1244. Altogether these results suggested that differences in mucins may in part explain the specificity of P. aeruginosa for CF.

The carbohydrate chains of the respiratory-mucus glycoproteins of a patient (blood group O) suffering from bronchiectasis due to Kartagener's syndrome, were released by alkaline borohydride treatment of a pronase digest. The structures of 82 neutral and low-molecular-mass sialylated oligosaccharides have been described previously [van Kuik A., de Waard P., Vliegenthart J. F. G., Klein A., Carnoy C., Lamblin G. Roussel P. (1991) Eur. J. Biochem. 198, 169-182]. In the present work, medium-size sialylated oligosaccharides were obtained after ion-exchange chromatography and were subsequently separated into 36 fractions utilizing gel filtration, HPLC on normal-phase alkylamine-bonded silica and reverse-phase HPLC. From these fractions, the following six sialylated hepta- and octa-saccharide-alditols have been characterized by employing 500-MHz 1H-NMR spectroscopy, in conjunction with fast-atom-bombardment mass spectroscopy and methylation analysis. [formula: see text]

Forty five oligosaccharide-alditols, purified after reductive β-elimination of human bronchial mucins, were analyzed by HPLC on alkylamine-bonded silica column (Lichrosorb-NH 2 ). The comparison of their structural features and retention times permitted the extension of some previous findings. The chromatographic behavior of the oligosaccharide depends more on the accessibility of oligosaccharide hydroxyl groups than on the sugar composition. The use of this type of fractionation is very efficient for low-molecular-mass oligosaccharide-alditols but needs to be completed by a second chromatographic step for higher molecular-mass oligosaccharide-alditols.

The attachment of Pseudomonas aeruginosa to human respiratory mucus represents an important step in the development of lung infection, especially in cystic fibrosis. Local factors in the respiratory tract, such as osmolarity or iron concentration, might influence this colonization. In the present work, we have observed that overall levels of adhesion of two nonmucoid, nonpiliated strains of P. aeruginosa, 1244-NP and PAK-NP, to human airway mucins were higher when these strains were grown in a minimal medium of low osmolarity (M9) than when they were grown in a rich medium of higher osmolarity (tryptic soy broth [TSB]). However, increasing the NaCl concentration of M9 to increase the osmolarity did not modify the level of binding. In order to find out whether these differences were due to variations in nutrients, the influence of iron concentration was investigated: the levels of binding of the two strains increased after TSB was depleted of iron and decreased after iron was added to M9. Since the outer membranes from the two strains have been shown to contain proteins reacting with human bronchial mucins, we compared the mucin-binding proteins expressed by the two strains grown in different media. When the nonpiliated strains 1244-NP and PAK-NP were grown in the different media, previously observed mucin-binding bands were detected in the 42- to 48-kDa range but additional mucin-binding bands in the 77- to 85-kDa range were detected when these strains were grown in M9 or iron-deprived TSB. These results demonstrate that the adhesion of P. aeruginosa and the expression of mucin-binding proteins in the outer membranes of nonpiliated P. aeruginosa are affected by the iron content of the medium in which the bacteria are grown and not by the osmolarity.

The attachment of Pseudomonas aeruginosa to human respiratory mucus represents an important step in the development of lung infection, especially in cases of cystic fibrosis. For this purpose, microtiter plate adhesion assays have been developed and have suggested that nonpilus adhesins of P. aeruginosa are the most important ones for binding to human respiratory mucins. In order to characterize these mucin-binding adhesins, outer membrane proteins (OMP) from two adhesive strains, 1244-NP and PAK-NP, and their poorly adhesive rpoN mutants, 1244-N3 and PAK-N1, were prepared by a mild extraction with Zwittergent 3-14. Mucin-binding adhesins were detected after polyacrylamide gel electrophoresis and blotting of the OMP on nitrocellulose replicas, using human bronchial mucins labeled with 125I. The binding properties of these OMP with lactotransferrin, another glycoprotein abundant in respiratory mucus, were also studied. Radiolabeled mucins detected four bands at 48, 46, 28, and 25 kDa with strain PAK-NP. With the nonmucoid strain 1244-NP, five bands were observed at 48, 46, 42, 28, and 25 kDa. The bands at 48 and 25 kDa were also visualized by radiolabeled lactotransferrin. These bands were partially or completely displaced by nonradiolabeled respiratory mucin glycopeptides but not by tetramethylurea, suggesting that they recognized carbohydrate sites. In contrast, the poorly adhesive strains showed weakly binding bands. These results demonstrate that outer membranes from two different nonpiliated P. aeruginosa strains express multiple adhesins with an affinity for human respiratory mucins and/or lactotransferrin.

Theadhesion ofPseudomonas aeruginosa totype1 (Gal,11-3GlcNAc) andtype2 (Galpll-4GlcNAc) disaccharide determinants wasstudied ina microtiter adhesion assayanda thin-layer chromatography bacterial overlay assay. Theoligosaccharides wereprepared fromhumanbreast milkandhumanurine and wereconjugated tohexadecylaniline toformneoglycolipids thatwereusedintheassays. Boththemucoidand thenonmucoid strains thatwerestudied recognized thedisaccharide determinants. Sialylation ofthe oligosaccharides didnotsuppress binding inthethin-layer chromatography assay, butt2-6-linked sialic acid blocked binding inthemicrotiter assay. Theuseofbovine serumalbumin instead ofgelatin asablocking agent against nonspecific binding completely suppressed binding inthethin-layer chromatography assay. Isogenic nonpiliated mutants ofnonmucoid strains constructed byinterrupting thepilin generetained their adhesive capacity forthedisaccharide units, indicating thatbinding tothedisaccharides wasmediated byanonpilus adhesin(s). Furthermore, twomonoclonal antibodies thatrecognize thetype2 disaccharide determinant (GaI01-4GlcNAc) partially inhibited adhesion ofapair ofpiliated andnonpiliated isogenic strains tomucin. Thisstudy suggests thatP.aeruginosa utilizes anonpilus adhesin(s) tobindtodisaccharide units commonly found inmucins, inaddition topili andalginate, twopreviously described adhesins. Receptors forPseudomonas aeruginosa havebeenfound onatleast twotypes ofglycoconjugates, namely, glycolipids andmucins. Theminimal structure recognized onglycolipids isclaimed tobeGalNAc1-4Gal sequence commonly found intheglycosphingolipid

Bacterial infections of the respiratory tract constitute a major cause of death worldwide. Given the constant rise in bacterial resistance to antibiotics, treatment failure is increasingly frequent. In this context, innovative therapeutic strategies are urgently needed. Stimulation of innate immune cells in the respiratory tract (via activation of Toll-like receptors (TLRs)) is an attractive approach for rapidly activating the body’s immune defenses against a broad spectrum of microorganisms. Previous studies of the TLR5 agonist flagellin in animal models showed that standalone TLR stimulation does not result in the effective treatment of pneumococcal respiratory infection but does significantly improve the therapeutic outcome of concomitant antibiotic treatment.The objectives of my thesis are, firstly, to define the type of therapeutic interaction between antibiotics and flagellin (additive or synergic) and to study its effectiveness against respiratory infection caused by antibiotic-resistant bacteria. Secondly, to investigate the molecular and cellular mechanisms implicated in flagellin's ant-infectious efficacy, particularly the role of neutrophils, wich are massively recruted in the lung upon intranasale administration of flagellin., Les pneumonies bactériennes, notamment à Streptococcus pneumoniae,représentent la troisième cause de mortalité au niveau mondiale et la progression constantede la résistance aux antibiotiques complique la prise en charge des patients. La stimulationde l’immunité innée, de par sa rapidité d’activation et la grande variété des mécanismes dedéfense mis en jeu, est une approche intéressante pour le développement d’alternativesthérapeutiques anti-infectieuses. Notre laboratoire a montré précédemment quel’administration intra-nasale de flagelline, agoniste du récepteur de l’immunité innée Toll-likereceptor 5, en association avec l’antibiothérapie, était plus efficace que l’antibiothérapieseule contre l’infection pulmonaire à S. pneumoniae chez la souris.Au cours de ce travail de thèse, nous avons montré que l’association antibiotique – flagellineétait synergique dans ce modèle, avec deux antibiotiques différents, l’amoxicilline et lecotrimoxazole. Nous avons également montré la synergie du traitement combiné amoxicilline– flagelline dans un modèle murin de surinfection bactérienne post-grippale par une souchede S. pneumoniae résistante à l’amoxicilline. En parallèle, nous nous sommes intéressésaux mécanismes de défense immunitaires cellulaires et moléculaires responsables del’efficacité anti-infectieuse de la flagelline en association avec les antibiotiques contrel’infection à S. pneumoniae. Des études de transcriptomique et protéomique sur le poumonet le liquide de lavage broncho-alvéolaire nous ont permit de mettre en évidence uneactivation par la flagelline des voies de signalisation associées au recrutement, à l’activationet aux capacités antimicrobiennes des cellules immunitaires innées, notamment desneutrophiles. Un recrutement significatif, rapide et transitoire de neutrophiles dans leparenchyme pulmonaire et le LBA a été observé par cytométrie en flux chez les sourisinfectées par S. pneumoniae et traitées par l’association amoxicilline – flagelline par rapportaux souris traitées par amoxicilline seule. L’étude de l’effet protecteur du traitement combinéamoxicilline – flagelline dans un modèle murin de déplétion sélective des neutrophiles etchez des souris chimères Cxcr2-/- présentant un défaut de recrutement des neutrophilesnous a permit de confirmer le rôle essentiel de ces cellules pour l’efficacité de la flagellinecontre l’infection pneumococcique. Cependant, nous n’avons pas mis en évidence dedifférence phénotypique ou fonctionnelle des neutrophiles pulmonaires recrutés dans lecontexte de l’infection pneumococcique en présence ou en l’absence de flagelline, suggérantque le rôle essentiel des neutrophiles dans l’efficacité de la flagelline contre l’infection à S.pneumoniae serait lié à une augmentation de leur nombre, mais pas de leurs capacitésfonctionnelles.Ces résultats démontrent l’intérêt de la stimulation de l’immunité innée par la flagelline entant que stratégie thérapeutique anti-infectieuse innovante pour le traitement despneumonies, permettant de renforcer l’efficacité de l’antibiothérapie contre des souchesrésistantes, de diminuer les doses d’antibiotiques utilisées et de limiter l’apparition derésistances.

Les pneumonies bactériennes, notamment à Streptococcus pneumoniae,représentent la troisième cause de mortalité au niveau mondiale et la progression constantede la résistance aux antibiotiques complique la prise en charge des patients. La stimulationde l’immunité innée, de par sa rapidité d’activation et la grande variété des mécanismes dedéfense mis en jeu, est une approche intéressante pour le développement d’alternativesthérapeutiques anti-infectieuses. Notre laboratoire a montré précédemment quel’administration intra-nasale de flagelline, agoniste du récepteur de l’immunité innée Toll-likereceptor 5, en association avec l’antibiothérapie, était plus efficace que l’antibiothérapieseule contre l’infection pulmonaire à S. pneumoniae chez la souris.Au cours de ce travail de thèse, nous avons montré que l’association antibiotique – flagellineétait synergique dans ce modèle, avec deux antibiotiques différents, l’amoxicilline et lecotrimoxazole. Nous avons également montré la synergie du traitement combiné amoxicilline– flagelline dans un modèle murin de surinfection bactérienne post-grippale par une souchede S. pneumoniae résistante à l’amoxicilline. En parallèle, nous nous sommes intéressésaux mécanismes de défense immunitaires cellulaires et moléculaires responsables del’efficacité anti-infectieuse de la flagelline en association avec les antibiotiques contrel’infection à S. pneumoniae. Des études de transcriptomique et protéomique sur le poumonet le liquide de lavage broncho-alvéolaire nous ont permit de mettre en évidence uneactivation par la flagelline des voies de signalisation associées au recrutement, à l’activationet aux capacités antimicrobiennes des cellules immunitaires innées, notamment desneutrophiles. Un recrutement significatif, rapide et transitoire de neutrophiles dans leparenchyme pulmonaire et le LBA a été observé par cytométrie en flux chez les sourisinfectées par S. pneumoniae et traitées par l’association amoxicilline – flagelline par rapportaux souris traitées par amoxicilline seule. L’étude de l’effet protecteur du traitement combinéamoxicilline – flagelline dans un modèle murin de déplétion sélective des neutrophiles etchez des souris chimères Cxcr2-/- présentant un défaut de recrutement des neutrophilesnous a permit de confirmer le rôle essentiel de ces cellules pour l’efficacité de la flagellinecontre l’infection pneumococcique. Cependant, nous n’avons pas mis en évidence dedifférence phénotypique ou fonctionnelle des neutrophiles pulmonaires recrutés dans lecontexte de l’infection pneumococcique en présence ou en l’absence de flagelline, suggérantque le rôle essentiel des neutrophiles dans l’efficacité de la flagelline contre l’infection à S.pneumoniae serait lié à une augmentation de leur nombre, mais pas de leurs capacitésfonctionnelles.Ces résultats démontrent l’intérêt de la stimulation de l’immunité innée par la flagelline entant que stratégie thérapeutique anti-infectieuse innovante pour le traitement despneumonies, permettant de renforcer l’efficacité de l’antibiothérapie contre des souchesrésistantes, de diminuer les doses d’antibiotiques utilisées et de limiter l’apparition derésistances. Bacterial infections of the respiratory tract constitute a major cause of death worldwide. Given the constant rise in bacterial resistance to antibiotics, treatment failure is increasingly frequent. In this context, innovative therapeutic strategies are urgently needed. Stimulation of innate immune cells in the respiratory tract (via activation of Toll-like receptors (TLRs)) is an attractive approach for rapidly activating the body’s immune defenses against a broad spectrum of microorganisms. Previous studies of the TLR5 agonist flagellin in animal models showed that standalone TLR stimulation does not result in the effective treatment of pneumococcal respiratory infection but does significantly improve the therapeutic outcome of concomitant antibiotic treatment.The objectives of my thesis are, firstly, to define the type of therapeutic interaction between antibiotics and flagellin (additive or synergic) and to study its effectiveness against respiratory infection caused by antibiotic-resistant bacteria. Secondly, to investigate the molecular and cellular mechanisms implicated in flagellin's ant-infectious efficacy, particularly the role of neutrophils, wich are massively recruted in the lung upon intranasale administration of flagellin.

Viral and bacterial infections remain a burden for health care systems. The current therapeutic approaches by antibiotic have shown their effectiveness but also their limit especially in cases of multidrug-resistant microorganisms or in the treatment of patients with aggravating pathologies. Stimulation of innate immunity could be a complementary therapeutic approach to current treatments. Indeed, the activation of the innate immune system induces local production of antimicrobial molecules and the recruitment of effector cells involved in controlling infection.The Toll-like Receptors (TLRs) are innate sensors that trigger signaling cascades and promotes the archetypal pro-inflammatory responses involved in antimicrobial defense. TLR5 recognizes flagellin, the structural protein of bacterial flagella. TLR5 is expressed on the surface of macrophages, dendritic cells and epithelial cells.In the first part of the work, we assessed the ability of flagellin to control the replication of influenza A virus H3N2. We first showed that intranasal or systemic administration of flagellin activated transcription of anti-viral genes in lung tissue. Administration of flagellin reduced viral replication in the lung of mice infected with H3N2 IAV. This effect is also observed in Il22-/- and Ifnar-/- mice suggesting a mechanism independent of type I interferon and interleukin 22 signaling. Finally, we also showed that the combination of flagellin with oseltamivir (neuraminidase inhibitor) was more efficacious than standalone treatments.The second part of this work is the ASTRAL clinical study, which assesses the ability of blood mononuclear cells to respond to different TLR agonists in patients with acute lobar pneumonia. This study started in October 2017 and plans to include 38 patients. The objective is to observe the response after TLR agonist stimulation by transcriptional analysis and cytokine assays. The first results (analysis of 7 patients) show that blood mononuclear cells can be activated by TLR agonists in the context of pneumonia., Les infections virales et bactériennes pulmonaires représentent un problème de santé publique majeur. Les approches thérapeutiques actuelles par antibiotique ont montré leur efficacité mais également leur limite notamment dans des cas de micro-organismes multirésistants ou dans le traitement de patients atteints de pathologies aggravantes. La stimulation de l’immunité innée pourrait représenter une voie thérapeutique complémentaire aux traitements actuels. En effet, l’activation du système immunitaire inné induit la production locale de molécules antimicrobiennes et le recrutement de cellules effectrices qui interviennent dans le contrôle de l’infection.Les « Toll-like receptors » (TLR) sont des senseurs de l’immunité innée. Leur activation entraîne une cascade de signalisation qui module la transcription de gènes de l’immunité comme ceux codant certaines cytokines. TLR5 reconnaît la flagelline, protéine structurale des flagelles bactériens. TLR5 est exprimé à la surface des macrophages, des cellules dendritiques et des cellules épithéliales.Dans la première partie de travail, nous avons évalué la capacité de la flagelline à contrôler la réplication du virus de la grippe A H3N2. Nous avons tout d’abord montré que l’administration intranasale ou systémique de flagelline était capable d’activer des gènes antiviraux au niveau pulmonaire. L’administration de la flagelline conduit à une diminution de la réplication virale chez les souris infectées par le virus de la grippe A H3N2. Cet effet est également observé chez les souris Il22-/- et Ifnar-/- suggérant un mécanisme indépendant de l’interféron de type I et de l’IL-22. Enfin, nous avons également montré que l’association de la flagelline à l’oseltamivir (inhibiteur de neuraminidase) était plus efficace que les traitements administrés séparément.La deuxième partie de ce travail correspond à l’étude clinique ASTRAL qui évalue la capacité des cellules mononucléées sanguines à répondre à différents agonistes de TLR chez des patients atteints de pneumonie franche lobaire aiguë. Cette étude a démarré en octobre 2017 et prévoit d’inclure 38 patients. L’objectif est d’observer la réponse induite par des agonistes de TLR à travers des analyses transcriptionnelles et des dosages de cytokines. Les premiers résultats (analyse chez 7 patients) montrent que les cellules mononucléées sanguines peuvent être activées par des agonistes de TLR dans le contexte de pneumonie.