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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.

Competence development in the human pathogen Streptococcus pneumoniae controls several features such as genetic transformation, biofilm formation and virulence. Competent bacteria produce so called ‘fratricins’ such as CbpD, that kill non-competent siblings by cleaving peptidoglycan (PGN). CbpD is a choline-binding protein (CBP) that binds to phosphorylcholine residues found on wall- and lipoteichoic acids (WTA and LTA) that together with PGN are major constituents of the pneumococcal cell wall. Competent pneumococci are protected against fratricide by producing the immunity protein ComM. How competence and fratricide contribute to virulence is unknown. Here, using a genome-wide CRISPRi-seq screen, we show that genes involved in teichoic acid biosynthesis are essential during competence. We demonstrate that LytR is the major enzyme mediating the final step in WTA formation, and that, together with ComM, is essential for immunity against CbpD. Importantly, we show that key virulence factors PspA and PspC become more surface-exposed at midcell during competence, in a CbpD-dependent manner. Together, our work supports a model in which activation of competence is crucial for host adherence by increased surface exposure of its various CBPs.

Antibiotic resistance in the important opportunistic human pathogen

Cystic fibrosis is associated with chronic Pseudomonas aeruginosa colonization and inflammation. The role of MyD88, the shared adapter protein of the proinflammatory TLR and IL-1R families, in chronic P. aeruginosa biofilm lung infection is unknown. We report that chronic lung infection with the clinical P. aeruginosa RP73 strain is associated with uncontrolled lung infection in complete MyD88-deficient mice with epithelial damage, inflammation, and rapid death. Then, we investigated whether alveolar or myeloid cells contribute to heightened sensitivity to infection. Using cell-specific, MyD88-deficient mice, we uncover that the MyD88 pathway in myeloid or alveolar epithelial cells is dispensable, suggesting that other cell types may control the high sensitivity of MyD88-deficient mice. By contrast, IL-1R1–deficient mice control chronic P. aeruginosa RP73 infection and IL-1β Ab blockade did not reduce host resistance. Therefore, the IL-1R1/MyD88 pathway is not involved, but other IL-1R or TLR family members need to be investigated. Our data strongly suggest that IL-1 targeted neutralizing therapies used to treat inflammatory diseases in patients unlikely reduce host resistance to chronic P. aeruginosa infection.

Antibiotic resistance in the important opportunistic human pathogen Streptococcus pneumoniae is on the rise. This is particularly problematic in the case of the β-lactam antibiotic amoxicillin, which is the first-line therapy. It is therefore crucial to uncover targets that would kill or resensitize amoxicillin-resistant pneumococci. To do so, we developed a genome-wide, single-cell based, gene silencing screen using CRISPR interference called sCRilecs-seq (subsets of CRISPR interference libraries extracted by fluorescence activated cell sorting coupled to next generation sequencing). Since amoxicillin affects growth and division, sCRilecs-seq was used to identify targets that are responsible for maintaining proper cell size. Our screen revealed that downregulation of the mevalonate pathway leads to extensive cell elongation. Further investigation into this phenotype indicates that it is caused by insufficient transport of cell wall precursors across the cell membrane due to a limitation in the production of undecaprenyl phosphate (Und-P), the lipid carrier responsible for this process. The data suggest that whereas peptidoglycan synthesis continues even with reduced Und-P levels, cell constriction is specifically halted. We successfully exploited this knowledge to create a combination treatment strategy where the FDA-approved drug clomiphene, an inhibitor of Und-P synthesis, is paired up with amoxicillin. Our results show that clomiphene potentiates the antimicrobial activity of amoxicillin and that combination therapy resensitizes amoxicillin-resistant S. pneumoniae. These findings could provide a starting point to develop a solution for the increasing amount of hard-to-treat amoxicillin-resistant pneumococcal infections.

International audience; Excessive lung inflammation and airway epithelium damage are hallmarks of cystic fibrosis (CF) disease. It is unclear whether lung inflammation is related to an intrinsic defect in the immune response or to chronic infection. We aimed to determine whether TLR5-mediated response is defective in the CF airway epithelium. We used a newborn CF pig model to study intrinsic alterations in CF airway epithelium innate immune response. Airway epithelial cells (AECs) were stimulated with flagellin or lipopolysaccharide to determine responses specific for TLR5 and TLR4, respectively. We observed a significant increase in cytokine secretion when CF AECs were stimulated with flagellin compared to wild type (WT) AECs. These results were recapitulated when AECs were treated with an inhibitor of CFTR channel activity. We show that TLR5-signalling is altered in CF lung epithelium at birth. Modulation of TLR5 signalling could contribute to better control the excessive inflammatory response observed in CF lungs.

Excessive lung inflammation and airway epithelial damage are hallmarks of human inflammatory lung diseases, such as cystic fibrosis (CF). Enhancement of innate immunity provides protection against pathogens while reducing lung-damaging inflammation. However, the mechanisms underlying innate immunity–mediated protection in the lung remain mysterious, in part because of the lack of appropriate animal models for these human diseases. TLR5 (Toll-like receptor 5) stimulation by its specific ligand, the bacterial protein flagellin, has been proposed to enhance protection against several respiratory infectious diseases, although other cellular events, such as calcium signaling, may also control the intensity of the innate immune response. Here, we investigated the molecular events prompted by stimulation with flagellin and its role in regulating innate immunity in the lung of the pig, which is anatomically and genetically more similar to humans than rodent models. We found that flagellin treatment modulated NF-κB signaling and intracellular calcium homeostasis in airway epithelial cells. Flagellin pretreatment reduced the NF-κB nuclear translocation and the expression of proinflammatory cytokines to a second flagellin stimulus as well as toPseudomonas aeruginosainfection. Moreover,invivoadministration of flagellin decreased the severity ofP. aeruginosa–induced pneumonia. Then we confirmed these beneficial effects of flagellin in a pathological model of CF by usingex vivoprecision-cut lung slices from a CF pigz model. These results provide evidence that flagellin treatment contributes to a better regulation of the inflammatory response in inflammatory lung diseases such as CF.

International audience; 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.

International audience; BPZE1 is a live attenuated vaccine against infection by Bordetella pertussis, the causative agent of whooping cough. It was previously shown that BPZE1 provides heterologous protection in mouse models of disease caused by unrelated pathogens, such as influenza virus and respiratory syncytial virus. Protection was also observed in mouse models of asthma and contact dermatitis. In this study, we demonstrate that BPZE1 also displays protection against an unrelated bacterial pathogen in a mouse model of invasive pneumococcal disease mediated by Streptococcus pneumoniae. While a single administration of BPZE1 provided no protection, two doses of 10 6 colony-forming units of BPZE1 given in a three-week interval protected against mortality, lung colonization and dissemination in both BALB/c and C57BL/6 mice. Unlike for the previously reported influenza challenge model, protection was short-lived, and waned within days after booster vaccination. Formaldehyde-killed BPZE1 protected only when administered following a live prime, indicating that priming requires live BPZE1 for protection. Protection against mortality was directly linked to substantially decreased bacterial dissemination in the blood and was lost in MyD88 knockout mice, demonstrating the role of the innate immune system in the mechanism of protection. This is the first report on a heterologous protective effect of the live BPZE1 vaccine candidate against an unrelated bacterial infection.

Streptococcus pneumoniae is a commensal bacterium of the human nasopharynx, but can cause harmful infections if it spreads to other parts of the body, such as pneumonia, sepsis or meningitis. To facilitate pathogenesis studies, we constructed a doxycycline-inducible pooled CRISPR interference (CRISPRi) library targeting all operons in protypical S. pneumoniae strain D39V. Our library design allows fitness within the pool to be assessed by a one-step PCR reaction directly followed by Illumina sequencing (CRISPRi-seq). The doxycycline-inducible CRISPRi system is tightly controllable and suitable for both bottleneck exploration and evaluation of gene fitness in vitro and in vivo. Here, we applied CRISPRi-seq to identify genetic factors important for causing pneumococcal pneumonia. Mice were infected intratracheally with our CRISPRi library and bacteria collected at 24 h (from lung) and 48 h (from both lung and blood) post-infection. CRISPRi-seq showed a critical bottleneck at 48 h after intratracheal infection, with only a few bacteria surviving the brunt of the innate immune response to cause systemic infection. However, earlier at 24 h post-infection, many significant differences in gene fitness cost between in vitro and in vivo conditions were identified, including genes encoding known and putative novel virulence factors, genes essential only in vivo, and genes essential only in vitro. A key advantage of CRISPRi-seq over traditional transposon-based genetic screens is that all genes, including essential genes, can be tested for their role in virulence and pathogenicity. The approaches developed here should be generally applicable to study infection bottlenecks and in vivo fitness for other important human and animal pathogens.

Cystic fibrosis is associated with chronic

International audience; Streptococcus pneumoniae can cause disease in various human tissues and organs, including the ear, the brain, the blood, and the lung, and thus in highly diverse and dynamic environments. It is challenging to study how pneumococci control virulence factor expression, because cues of natural environments and the presence of an immune system are difficult to simulate in vitro. Here, we apply synthetic biology methods to reverse-engineer gene expression control in S. pneumoniae . A selection platform is described that allows for straightforward identification of transcriptional regulatory elements out of combinatorial libraries. We present TetR- and LacI-regulated promoters that show expression ranges of four orders of magnitude. Based on these promoters, regulatory networks of higher complexity are assembled, such as logic AND gates and IMPLY gates. We demonstrate single-copy genome-integrated toggle switches that give rise to bimodal population distributions. The tools described here can be used to mimic complex expression patterns, such as the ones found for pneumococcal virulence factors. Indeed, we were able to rewire gene expression of the capsule operon, the main pneumococcal virulence factor, to be externally inducible (YES gate) or to act as an IMPLY gate (only expressed in absence of inducer). Importantly, we demonstrate that these synthetic gene-regulatory networks are functional in an influenza A virus superinfection murine model of pneumonia, paving the way for in vivo investigations of the importance of gene expression control on the pathogenicity of S. pneumoniae .

Significance Streptococcus pneumoniae is a major human pathogen responsible for enormous global morbidity and mortality. Despite this, the pneumococcus makes up part of the commensal nasopharyngeal flora. How the pneumococcus switches from this commensal to pathogenic state and causes disease is unclear and very likely involves variability in expression of its virulence factors. Here, we used synthetic biology approaches to generate complex gene-regulatory networks such as logic gates and toggle switches. We show that these networks are functional in vivo to control capsule production in an influenza-superinfection model. This opens the field of systematically testing the role of phenotypic variation in pneumococcal virulence. The approaches used here may serve as an example for synthetic biology projects in unrelated organisms., Streptococcus pneumoniae can cause disease in various human tissues and organs, including the ear, the brain, the blood, and the lung, and thus in highly diverse and dynamic environments. It is challenging to study how pneumococci control virulence factor expression, because cues of natural environments and the presence of an immune system are difficult to simulate in vitro. Here, we apply synthetic biology methods to reverse-engineer gene expression control in S. pneumoniae. A selection platform is described that allows for straightforward identification of transcriptional regulatory elements out of combinatorial libraries. We present TetR- and LacI-regulated promoters that show expression ranges of four orders of magnitude. Based on these promoters, regulatory networks of higher complexity are assembled, such as logic AND gates and IMPLY gates. We demonstrate single-copy genome-integrated toggle switches that give rise to bimodal population distributions. The tools described here can be used to mimic complex expression patterns, such as the ones found for pneumococcal virulence factors. Indeed, we were able to rewire gene expression of the capsule operon, the main pneumococcal virulence factor, to be externally inducible (YES gate) or to act as an IMPLY gate (only expressed in absence of inducer). Importantly, we demonstrate that these synthetic gene-regulatory networks are functional in an influenza A virus superinfection murine model of pneumonia, paving the way for in vivo investigations of the importance of gene expression control on the pathogenicity of S. pneumoniae.

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 TLR5 agonist flagellin is a potent adjuvant and is currently being developed for use in vaccines. The mechanisms that drive flagellin’s activity are influenced by its administration route. Previous studies showed that lung structural cells (especially epithelial cells lining the conducting airways) are pivotal for the efficacy of intranasally administered flagellin-containing vaccines. In this study, we looked at how the airway epithelial cells (AECs) regulate the flagellin-dependent stimulation of Ag-specific CD4+ T cells and the Ab response in mice. Our results demonstrate that after sensing flagellin, AECs trigger the release of GM-CSF in a TLR5-dependent fashion and the doubling of the number of activated type 2 conventional dendritic cells (cDC2s) in draining lymph nodes. Furthermore, the neutralization of GM-CSF reduced cDC2s activation. This resulted in lower of Ag-specific CD4+ T cell count and Ab titers in mice. Our data indicate that during pulmonary immunization, the GM-CSF released by AECs orchestrates the cross-talk between cDC2s and CD4+ T cells and thus drives flagellin’s adjuvant effect.

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; Interleukin-7 (IL-7) is a critical cytokine in B- and T-lymphocyte development and maturation. Recent evidence suggests that IL-7 is a preferential homeostatic and survival factor for RORγt+ innate T cells such as natural killer T (NKT) cells, γδT cells, and mucosal-associated invariant T (MAIT) cells in the periphery. Given the important contribution of these populations in antibacterial immunity at barrier sites, we questioned whether IL-7 could be instrumental in boosting the local host immune response against respiratory bacterial infection. By using a cytokine-monoclonal antibody approach, we illustrated a role for topical IL-7 delivery in increasing the pool of RORγt+ IL-17A-producing innate T cells. Prophylactic IL-7 treatment prior to Streptococcus pneumoniae infection led to better bacterial containment, a process associated with increased neutrophilia and that depended on γδT cells and IL-17A. Last, combined delivery of IL-7 and α-galactosylceramide (α-GalCer), a potent agonist for invariant NKT (iNKT) cells, conferred an almost total protection in terms of survival, an effect associated with enhanced IL-17 production by innate T cells and neutrophilia. Collectively, we provide a proof of concept that IL-7 enables fine-tuning of innate T- cell functions. This might pave the way for considering IL-7 as an innovative biotherapeutic against bacterial infection.

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.

Pertussis is a severe respiratory disease mainly caused by Bordetella pertussis. Despite wide global vaccination coverage with efficacious pertussis vaccines, it remains one of the least well-controlled vaccine-preventable diseases, illustrating the shortcomings of the current vaccines. We have developed the live attenuated nasal pertussis vaccine BPZE1, currently undergoing clinical evaluation in human phase 2 trials. We have previously shown that in mice, BPZE1 provides strong and long-lasting protection against B. pertussis challenge by inducing potent Ab and T cell responses as well as secretory IgA and IL-17–producing resident memory T lymphocytes in the nasal cavity. In this study, we show that BPZE1 induces protection in mice against B. pertussis within days after vaccination, at a time when Ab and T cell responses were not detectable. Early protection was independent of T and B cell responses, as demonstrated by the use of SCID mice. Instead, it was due to TLR4-dependent signaling through the MyD88-dependent pathway of the innate immune response, as demonstrated in experiments with TLR4-deficient and MyD88-knockout mice. TLR2-dependent signaling did not play a major role in early protection. In addition, this study also shows that even at high doses, BPZE1 is safe in the severely immunocompromised MyD88-deficient mice, whereas virulent B. pertussis caused a severe pathological condition and death in these mice, even at a low dose. Finally, coadministration of virulent B. pertussis with BPZE1 did not cause exacerbated outgrowth of the virulent strain, thereby adding to the safety profile of this live vaccine candidate.

National audience; Pseudomonas aeruginosa, an opportunistic gram-negative bacterium that rarely infects human lungs unless the host immune system has been impaired, is one of the main pathogens found in cystic fibrosis (CF) patients. P. aeruginosa infections in CF patients are difficult to treat, becoming chronic and contributing to exacerbated lung inflammation and respiratory failure. Modulation of innate immunity has been proposed as an alternative to improve defence against infections. This approach is particularly attractive in CF since exacerbated immune response is central to the pathogenesis of CF lung disease. Innate immunity in epithelial and immune cells can be stimulated through activation of Toll-like receptors (TLRs), the main family of pattern recognition receptors. Stimulation of TLR5 through flagellin-based interventions have demonstrated protective activity against several gram-negative bacteria (Salmonella sp., Burkholderia cepacia, Yersinia pseudotuberculosis), restoring immune-competence and promoting tissue repair processes. Here, we aimed to determine the effect of flagellin stimulation on the innate immune response against P. aeruginosa using an experimental pig model of lung infection. The pig model presents several advantages since swine and human lungs are similar in terms of anatomical, histological, biochemical, and physiological features. This is especially true in CF, where pigs lacking CFTR present a similar phenotype to what is typically observed in human patients. P. aeruginosa-infected pigs showed an acute neutrophilic response with an exacerbated release of neutrophil serine proteases that peaked 3-6 h post-infection (p.i.) leading to lung destruction and tissue hepatisation 24h p.i. When animals were pre-treated with flagellin 24h before the experimental infections, we observed a significant decrease in the expression of pro-inflammatory markers. In addition, a better lung status was observed on infected animals that had been pre-treated with flagellin compared to infected controls. The effect of flagellin pre-treatment on immune response to P. aeruginosa infection was confirmed using ex-vivo and in vitro models of lung epithelium from CFTR-/- pigs. In conclusion, our data point to a modulatory role of flagellin pre-treatment on the immune response to P. aeruginosa. This approach may have a therapeutic potential to improve inflammatory manifestations of CF.

International audience; The 3-O-desacyl-4'-monophosphoryl lipid A (MPL) activates immunity through Toll-like receptor 4 (TLR4) signaling. The Adjuvant System AS01 contains MPL and is used in the candidate malaria vaccine and the licensed zoster vaccine. Recent studies reported that AS01 adjuvant activity depends on a transient inflammation at the site of vaccination, but the role of stromal or structural cells in the adjuvant effect is unknown. We investigated this question in mouse models by assessing the role of TLR4 on hematopoietic versus resident structural cells during immunization with AS01-adjuvanted vaccines. We first established that TLR4-deficient animals had a reduced immune response to an AS01-adjuvanted vaccine. Using bone marrow chimera, we consistently found that Tlr4 expression in radio-sensitive cells, i.e., hematopoietic cells, was required for an optimal adjuvant effect on antibody and T-cell responses. At day 1 after injection, the pro-inflammatory reaction at the site of injection was strongly dependent on TLR4 signaling in hematopoietic cells. Similarly, activation of dendritic cells in muscle-draining lymph nodes was strictly associated with the radio-sensitive cells expressing Tlr4. Altogether, these data suggest that MPL-mediated TLR4-signaling in hematopoietic cells is critical in the mode of action of AS01.

International audience; To assess pharmacokinetics of amoxicillin (AMX) in mice, limitations such as a small sampling volume and low drug concentrations have to be addressed. Similar challenges are faced in a clinical framework, e.g. for therapeutic drug monitoring in neonates or small-scale in vitro investigations. An assay enabling quantification of small sample volumes but still at very low concentrations covering a broad concentration range is thus needed. A simple, rapid and highly sensitive liquid chromatography tandem mass spectrometry (LC-MS/MS) method was developed and successfully validated for quantification of AMX in mouse serum according to European Medicines Agency guidelines. Sample preparation enabled the use of only 10 μL of serum, which is 5-fold less than comparable assays and allows to reduce the number of mice used in pharmacokinetic studies. After protein precipitation with 40 μL chilled methanol and dilution of the supernatant with water, the sample was injected into the LC system on a Poroshell 120 Phenyl Hexyl column (2.1 × 100 mm, 2.7 μm). Chromatographic separation was achieved using a gradient method consisting of acetonitrile and ultra-pure water, both with 0.1% (V/V) formic acid. Positive electrospray ionisation in multiple reaction monitoring mode was used for detection and quantification of AMX. Application to murine study samples demonstrated the reliability of the developed method being accurate and precise with a quantification range from 0.01 to 10 μg/mL. The assay is easily transferable due to a simple sample preparation and confirmed stability of AMX under various applied conditions.

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.

Bacterial flagellin activates the innate immune system and ultimately the adaptive immune system through a Toll-like receptor 5 (TLR5)-dependent signaling mechanism. Given that TLR5 is widely distributed in epithelia, flagellin is currently being developed as a mucosal adjuvant. Flagellin FliC from Salmonella enterica has four domains: the conserved D0 and D1 domains and the hypervariable D2 and D3 domains. The deletion of D3 and partial deletion of D2 in the recombinant FliCD174-400 strongly impairs flagellin’s intrinsic antigenicity but does not affect the TLR5-dependent immunostimulation activity, i.e., the capacity to promote innate responses and adaptive responses to co-administered antigens. Here, we describe the development of novel recombinant flagellins with various deletions encompassing all of D2 and D3, and part of D1. Most of the recombinant molecules conserved an a-helical secondary structure that was as resistant to heat denaturation as the native protein. Whereas the recombinant flagellins’ ability to trigger TLR5 varied markedly in vitro, most gave equivalent in vivo TLR5- dependent innate immune responses following intranasal administration of 2 lg of flagellin to mice. Concordantly, the recombinant flagellins were also valuable respiratory adjuvants for eliciting antibody responses to the foreign antigen ovalbumin, although their intrinsic antigenicity was decreased compared to the native flagellin and not increased compared to FliCD174-400. Our results show that the additional deletions of D2 and the distal part of D1 of FliCD174-400 does not impact on antigenicity and does not significantly modify the immunostimulatory adjuvant activity. Altogether, this study generated a novel set of recombinant flagellin that constitutes a portfolio of TLR5-dependent candidate adjuvants for vaccination. Fil: Biedma, Marina Elizabeth. 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: Cayet, Delphine. Centre d’Infection et d’Immunité de Lille; Francia. Inserm; Francia Fil: Tabareau, Julien. Centre d’Infection et d’Immunité de Lille; Francia. Inserm; Francia Fil: Rossi, Andrés Hugo. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Parque Centenario. Instituto de Investigaciones Bioquímicas de Buenos Aires. Fundación Instituto Leloir. Instituto de Investigaciones Bioquímicas de Buenos Aires; Argentina Fil: Ivičak Kocjan, Karolina. University of Ljubljana; Eslovenia Fil: Moreno, Griselda Noemí. 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: Errea, Agustina Juliana. 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: Soulard, Daphnée. Centre d’Infection et d’Immunité de Lille; Francia. Inserm; Francia Fil: Parisi, Gustavo Daniel. Universidad Nacional de Quilmes; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina Fil: Jerala, Roman. University of Ljubljana; Eslovenia Fil: Berguer, Paula Mercedes. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Parque Centenario. Instituto de Investigaciones Bioquímicas de Buenos Aires. Fundación Instituto Leloir. Instituto de Investigaciones Bioquímicas de Buenos Aires; Argentina 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: Sirard, Jean Claude. Centre d’Infection et d’Immunité de Lille; Francia. Inserm; Francia

DNA methyltransferase (Dnmt)3b mediates de novo DNA methylation and modulation of Dnmt3b in respiratory epithelial cells has been shown to affect the expression of multiple genes. Respiratory epithelial cells provide a first line of defense against pulmonary pathogens and play a crucial role in the immune response during pneumonia caused by Pseudomonas (P.) aeruginosa, a gram-negative bacterium that expresses flagellin as an important virulence factor. We here sought to determine the role of Dntm3b in respiratory epithelial cells in immune responses elicited by P. aeruginosa. DNMT3B expression was reduced in human bronchial epithelial (BEAS-2B) cells as well as in primary human and mouse bronchial epithelial cells grown in air liquid interface upon exposure to P. aeruginosa (PAK). Dnmt3b deficient human bronchial epithelial (BEAS-2B) cells produced more CXCL1, CXCL8 and CCL20 than control cells when stimulated with PAK, flagellin-deficient PAK (PAKflic) or flagellin. Dnmt3b deficiency reduced DNA methylation at exon 1 of CXCL1 and enhanced NF-ĸB p65 binding to the CXCL1 promoter. Mice with bronchial epithelial Dntm3b deficiency showed increased Cxcl1 mRNA expression in bronchial epithelium and CXCL1 protein release in the airways during pneumonia caused by PAK, which was associated with enhanced neutrophil recruitment and accelerated bacterial clearance; bronchial epithelial Dnmt3b deficiency did not modify responses during pneumonia caused by PAKflic or Klebsiella pneumoniae (an un-flagellated gram-negative bacterium). Dnmt3b deficiency in type II alveolar epithelial cells did not affect mouse pulmonary defense against PAK infection. These results suggest that bronchial epithelial Dnmt3b impairs host defense during Pseudomonas induced pneumonia, at least in part, by dampening mucosal responses to flagellin., Author summary The respiratory epithelium provides a first line of defense against respiratory pathogens. Pseudomonas (P.) aeruginosa is a common causative pathogen in pneumonia and its important virulence factor flagellin is a potent activator of epithelial cells. DNA methyltransferase (Dnmt)3b is an enzyme that mediates de novo methylation of DNA. We here tested the hypothesis that Dnmt3b is involved in the immune response generated in airway epithelial cells upon infection with P. aeruginosa. Using a combination of in vitro investigations with human bronchial epithelial cells and in vivo airway infection models in mice with targeted deletions of the gene encoding Dnmt3b in specific subtypes of airway epithelial cells we demonstrate that Dnmt3b in bronchial but not type 2 alveolar epithelial cells impairs host defense during Pseudomonas induced pneumonia, at least in part by inhibiting mucosal responses to flagellin, by an effect on epithelial cell DNA methylation. We report a thus far unknown role for bronchial epithelial cell Dnmt3b in the innate mucosal immune response to a common respiratory pathogen, providing insight into the regulatory machinery involved in reprograming of epithelial cells during pneumonia.

Aim: To evaluate efficacy of sublingual flagellin to treat acute pneumonia. Materials & methods: Mice were treated sublingually with flagellin and challenged intranasally with a lethal dose of pneumococcus. Flagellins lacking TLR5 or NLRC4 activation domains were used to assess their contribution to protection. Results: Sublingual flagellin protected mice in a TLR5-dependent, NLRC4-independent fashion. Neutrophils were required for protection. Flagellin-stimulated lung epithelial cells recapitulated the lung's transcriptional profile suggesting they could be targeted by flagellin in vivo. Conclusion: Ligation of TLR5, a pathogen recognition receptor not naturally engaged by pneumococcus, protects mice from invasive pneumonia when administered via sublingual route. This can be a highly cost-effective alternative therapy against pneumonia.

International audience; The precise impact of the somatotrope axis upon the immune system is still highly debated. We have previously shown that mice with generalized ablation of growth hormone (GH) releasing hormone (GHRH) gene (Ghrh -/-) have normal thymus and T-cell development, but present a marked spleen atrophy and B-cell lymphopenia. Therefore, in this paper we have investigated vaccinal and anti-infectious responses of Ghrh -/- mice against S. pneumoniae, a pathogen carrying T-independent antigens. Ghrh -/- mice were unable to trigger production of specific IgM after vaccination with either native pneumococcal polysaccharides (PPS, PPV23) or protein-PPS conjugate (PCV13). GH supplementation of Ghrh -/- mice restored IgM response to PPV23 vaccine but not to PCV13 suggesting that GH could exert a specific impact on the spleen marginal zone that is strongly implicated in T-independent response against pneumococcal polysaccharides. As expected, after administration of low dose of S. pneumoniae, wild type (WT) completely cleared bacteria after 24 h. In marked contrast, Ghrh -/- mice exhibited a dramatic susceptibility to S. pneumoniae infection with a time-dependent increase in lung bacterial load and a lethal bacteraemia already after 24 h. Lungs of infected Ghrh -/- mice were massively infiltrated by inflammatory macrophages and neutrophils, while lung B cells were markedly decreased. The inflammatory transcripts signature was significantly elevated in Ghrh -/- mice. In this animal model, the somatotrope GHRH/GH/IGF1 axis plays a vital and unsuspected role in vaccine and immunological defense against S. pneumoniae.

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