Airway inflammation plays a major role in the pathogenesis of influenza viruses and can lead to a fatal outcome. One of the challenging objectives in the field of influenza research is the identification of the molecular bases associated to the immunopathological disorders developed during infection. While its precise function in the virus cycle is still unclear, the viral protein PB1-F2 is proposed to exert a deleterious activity within the infected host. Using an engineered recombinant virus unable to express PB1-F2 and its wild-type homolog, we analyzed and compared the pathogenicity and host response developed by the two viruses in a mouse model. We confirmed that the deletion of PB1-F2 renders the virus less virulent. The global transcriptomic analyses of the infected lungs revealed a potent impact of PB1-F2 on the response developed by the host. Thus, after two days post-infection, PB1-F2 invalidation severely decreased the number of genes activated by the host. PB1-F2 expression induced an increase in the number and level of expression of activated genes linked to cell death, inflammatory response and neutrophil chemotaxis. When generating interactive gene networks specific to PB1-F2, we identified IFN-γ as a central regulator of PB1-F2-regulated genes. The enhanced cell death of airway-recruited leukocytes was evidenced using an apoptosis assay, confirming the pro-apoptotic properties of PB1-F2. Using a NF-kB luciferase adenoviral vector, we were able to quantify in vivo the implication of NF-kB in the inflammation mediated by the influenza virus infection; we found that PB1-F2 expression intensifies the NF-kB activity. Finally, we quantified the neutrophil recruitment within the airways, and showed that this type of leukocyte is more abundant during the infection of the wild-type virus. Collectively, these data demonstrate that PB1-F2 strongly influences the early host response during IAV infection and provides new insights into the mechanisms by which PB1-F2 mediates virulence.

Improvements in our knowledge of the gut microbiota have broadened our vision of the microbes associated with the intestine. These microbes are essential actors and protectors of digestive and extra-digestive health and, by extension, crucial for human physiology. Similar reconsiderations are currently underway concerning the endogenous microbes of the lungs, with a shift in focus away from their involvement in infections toward a role in physiology. The discovery of the lung microbiota was delayed by the long-held view that the lungs of healthy individuals were sterile and by sampling difficulties. The lung microbiota has a low density, and the maintenance of small numbers of bacteria seems to be a critical determinant of good health. This review aims to highlight how knowledge about the lung microbiota can change our conception of lung physiology and respiratory health. We provide support for this point of view with knowledge acquired about the gut microbiota and intestinal physiology. We describe the main characteristics of the lung microbiota and its functional impact on lung physiology, particularly in healthy individuals, after birth, but also in asthma. We describe some of the physiological features of the respiratory tract potentially favoring the installation of a dysbiotic microbiota. The gut microbiota feeds and matures the intestinal epithelium and is involved in immunity, when the principal role of the lung microbiota seems to be the orientation and balance of aspects of immune and epithelial responsiveness. This implies that the local and remote effects of bacterial communities are likely to be determinant in many respiratory diseases caused by viruses, allergens or genetic deficiency. Finally, we discuss the reciprocal connections between the gut and lungs that render these two compartments inseparable.

De novo protein design has been successful in expanding the natural protein repertoire. However, most de novo proteins lack biological function, presenting a major methodological challenge. In vaccinology, the induction of precise antibody responses remains a cornerstone for next-generation vaccines. Here, we present a protein design algorithm called TopoBuilder, with which we engineered epitope-focused immunogens displaying complex structural motifs. In both mice and nonhuman primates, cocktails of three de novo–designed immunogens induced robust neutralizing responses against the respiratory syncytial virus. Furthermore, the immunogens refocused preexisting antibody responses toward defined neutralization epitopes. Overall, our design approach opens the possibility of targeting specific epitopes for the development of vaccines and therapeutic antibodies and, more generally, will be applicable to the design of de novo proteins displaying complex functional motifs.

The protease inhibitor Elafin prevents intestinal inflammation in mouse models of colitis and might be developed as a therapeutic agent for inflammatory bowel disease.

Respiratory syncytial virus (RSV) is the major cause of lower respiratory tract infections in infants and is characterized by pulmonary infiltration of B cells in fatal cases. We analyzed the B cell compartment in human newborns and identified a population of neonatal regulatory B lymphocytes (nBreg cells) that produced interleukin 10 (IL-10) in response to RSV infection. The polyreactive B cell receptor of nBreg cells interacted with RSV protein F and induced upregulation of chemokine receptor CX3CR1. CX3CR1 interacted with RSV glycoprotein G, leading to nBreg cell infection and IL-10 production that dampened T helper 1 (Th1) cytokine production. In the respiratory tract of neonates with severe RSV-induced acute bronchiolitis, RSV-infected nBreg cell frequencies correlated with increased viral load and decreased blood memory Th1 cell frequencies. Thus, the frequency of nBreg cells is predictive of the severity of acute bronchiolitis disease and nBreg cell activity may constitute an early-life host response that favors microbial pathogenesis.

A deficit in early clearance of Pseudomonas aeruginosa (P. aeruginosa) is crucial in nosocomial pneumonia and in chronic lung infections. Few studies have addressed the role of Toll-like receptors (TLRs), which are early pathogen associated molecular pattern receptors, in pathogen uptake and clearance by alveolar macrophages (AMs). Here, we report that TLR5 engagement is crucial for bacterial clearance by AMs in vitro and in vivo because unflagellated P. aeruginosa or different mutants defective in TLR5 activation were resistant to AM phagocytosis and killing. In addition, the clearance of PAK (a wild-type P. aeruginosa strain) by primary AMs was causally associated with increased IL-1β release, which was dramatically reduced with PAK mutants or in WT PAK-infected primary TLR5−/− AMs, demonstrating the dependence of IL-1β production on TLR5. We showed that this IL-1β production was important in endosomal pH acidification and in inducing the killing of bacteria by AMs through asparagine endopeptidase (AEP), a key endosomal cysteine protease. In agreement, AMs from IL-1R1 −/− and AEP −/− mice were unable to kill P. aeruginosa. Altogether, these findings demonstrate that TLR5 engagement plays a major role in P. aeruginosa internalization and in triggering IL-1β formation.flagellin | interleukin-1 | lysosomal protease T he opportunist Gram-negative bacterium, Pseudomonas aeruginosa, is particularly important in nosocomial pneumonia and in chronic lung diseases such as cystic fibrosis (1). Alveolar macrophages (AMs) lie at the forefront of lung defense against pathogens such as P. aeruginosa. The main function of AMs is to clear pathogens (2), and a deficiency in early recognition of P. aeruginosa by AMs has been suspected in these pathologies (3,4). Research has shown that pathogen-associated molecular patterns (PAMPs) are recognized by specific Toll-like receptors (TLRs) at the surface of phagocytes and mucosal epithelial cells. Surprisingly, although numerous studies have associated the ligand-induced TLR engagement to cytokine and chemokine production from phagocytes (5, 6), comparatively fewer studies have investigated the importance of TLRs in pathogen phagocytosis and killing. Furthermore, these studies have mostly used macrophages from bone marrow-differentiated cells (BMDMs), few have used live bacteria, and even fewer have used flagellated bacteria such as P. aeruginosa. Moreover most studies have used primed phagocytes (with LPS, zymosan) to boost pathogen uptake. Despite these caveats, the recruitment of membrane TLRs to phagosomes upon phagocytosis has been demonstrated (7-10), except for TLR5. TLR2, TLR4, and the adaptor molecule MyD88 have been shown to be important molecules in processing of heat-killed Escherichia coli and Staphylococcus aureus by BMDMs in late endosomes and lysosomes (9-11), suggesting that a blockade in phagosome maturation was occurring in phagocytes deleted for these molecules.TLR5 is thought to be one of the key receptors implicated in the recognition of P. aeruginosa (5, 8), bu...

Liver tropism potentially leading to massive hepatocyte transduction and hepatotoxicity still represents a major drawback to adenovirus (Ad)-based gene therapy. We previously demonstrated that substitution of the hexon hypervariable region 5 (HVR5), the most abundant capsid protein, constituted a valuable platform for efficient Ad retargeting. The use of different mouse strains revealed that HVR5 substitution also led to dramatically less adenovirus liver transduction and associated toxicity, whereas HVR5-modified Ad were still able to transduce different cell lines efficiently, including primary hepatocytes. We showed that HVR5 modification did not significantly change Ad blood clearance or liver uptake at early times. However, we were able to link the lower liver transduction to enhanced HVR5-modified Ad liver clearance and impaired use of blood factors. Most importantly, HVR5-modified vectors continued to transduce tumors in vivo as efficiently as their wild-type counterparts. Taken together, our data provide a rationale for future design of retargeted vectors with a safer profile.

Asthma is a chronic, non-curable, multifactorial disease with increasing incidence in industrial countries. This study evaluates the direct contribution of lung microbial components in allergic asthma in mice. Germ-Free and Specific-Pathogen-Free mice display similar susceptibilities to House Dust Mice-induced allergic asthma, indicating that the absence of bacteria confers no protection or increased risk to aeroallergens. In early life, allergic asthma changes the pattern of lung microbiota, and lung bacteria reciprocally modulate aeroallergen responsiveness. Primo-colonizing cultivable strains were screened for their immunoregulatory properties following their isolation from neonatal lungs. Intranasal inoculation of lung bacteria influenced the outcome of allergic asthma development: the strain CNCM I 4970 exacerbated some asthma features whereas the pro-Th1 strain CNCM I 4969 had protective effects. Thus, we confirm that appropriate bacterial lung stimuli during early life are critical for susceptibility to allergic asthma in young adults.