Biin Sung

Impaired Sphingolipid Synthesis in the Respiratory Tract Induces Airway Hyperreactivity

Decreased activity of serine palmitoyl-CoA transferase is associated with asthma. Breathing Easy The explosion in genome-wide association studies (GWAS) has implicated countless genes in the pathology of diverse diseases. Yet, when it comes to developing new therapies, associations aren’t enough. Studies must functionally connect GWAS-implicated targets to disease pathology. Now, Worgall et al. demonstrate that orosomucoid-like 3 (ORMDL3) and downstream sphingolipids may play a pathogenic role in asthma. ORM proteins negatively regulate sphingolipid synthesis through their effects on serine palmitoyl-CoA transferase (SPT), which is required for sphingolipid generation. ORMDL3 has been associated with asthma in several GWAS; the authors therefore hypothesized that SPT and sphingolipids may contribute to asthma pathogenesis. They found that either decreasing sphingolipid synthesis or inhibiting SPT with myriocin increased bronchioreactivity in the absence of inflammation in part by altered bronchial sensitivity to magnesium. These data functionally link sphingolipid synthesis and airway hyperactivity, and suggest that this pathway may be a new target for asthma therapy. Asthma is a clinically heterogeneous genetic disease, and its pathogenesis is incompletely understood. Genome-wide association studies link ORM (yeast)-Like protein isoform3 (ORMDL3), a member of the ORM gene family, to nonallergic childhood-onset asthma. Orm proteins negatively regulate sphingolipid (SL) synthesis by acting as homeostatic regulators of serine palmitoyl-CoA transferase (SPT), the rate-limiting enzyme of de novo SL synthesis, but it is not known how SPT activity or SL synthesis is related to asthma. The present study analyzes the effect of decreased de novo SL synthesis in the lung on airway reactivity after administration of myriocin, an inhibitor of SPT, and in SPT heterozygous knockout mice. We show that, in both models, decreased de novo SL synthesis increases bronchial reactivity in the absence of inflammation. Decreased SPT activity affected intracellular magnesium homeostasis and altered the bronchial sensitivity to magnesium. This functionally links decreased de novo SL synthesis to asthma and so identifies this metabolic pathway as a potential target for therapeutic interventions.

Adenovirus-Based Vaccine with Epitopes Incorporated in Novel Fiber Sites to Induce Protective Immunity against Pseudomonas aeruginosa

Adenovirus (Ad) vector-based vaccines displaying pathogen-derived epitopes on Ad capsid proteins can elicit anti-pathogen immunity. This approach seems to be particularly efficient with epitopes incorporated into the Ad fiber protein. Here, we explore epitope insertion into various sites of the Ad fiber to elicit epitope-specific immunity. Ad vectors expressing the 14-mer Pseudomonas aeruginosa immune-dominant outer membrane protein F (OprF) epitope 8 (Epi8) in five distinct sites of the Ad5 fiber, loops CD (AdZ.F(CD)Epi8), DE (AdZ.F(DE)Epi8), FG (AdZ.F(FG)Epi8), HI (AdZ.F(HI)Epi8) and C terminus (AdZ.F(CT)Epi8), or the hexon HVR5 loop (AdZ.HxEpi8) were compared in their capacity to elicit anti-P. aeruginosa immunity to AdOprF, an Ad expressing the entire OprF protein. Intramuscular immunization of BALB/c mice with AdZ.F(FG)Epi8 or AdZ.F(HI)Epi8 elicited higher anti-OprF humoral and cellular CD4 and CD8 responses as well as enhanced protection against respiratory infection with P. aeruginosa compared to immunization with AdZ.F(CD)Epi8, AdZ.F(DE)Epi8, AdZ.F(CT)Epi8 or AdZ.HxEpi8. Importantly, repeat administration of the fiber- and hexon-modified Ad vectors boosted the OprF-specific humoral immune response in contrast to immunization with AdOprF. Strikingly, following three doses of AdZ.F(FG)Epi8 or AdZ.F(HI)Epi8 anti-OprF immunity surpassed that induced by AdOprF. Furthermore, in the presence of anti-Ad5 immunity, immunization with AdZ.F(FG)Epi8 or AdZ.F(HI)Epi8, but not with AdOprF, induced protective immunity against P. aeruginosa. This suggests that incorporation of epitopes into distinct sites of the Ad fiber is a promising vaccine strategy.

Maternal Immunization with Chimpanzee Adenovirus Expressing RSV Fusion Protein Protects Against Neonatal RSV Pulmonary Infection

Respiratory syncytial virus (RSV) is a leading cause of lower respiratory tract disease with high morbidity and mortality in young infants and children. Despite numerous efforts, a licensed vaccine against RSV remains elusive. Since young infants form the primary target group of RSV disease, maternal immunization to boost the protection in neonates is an attractive strategy. In this study we tested the efficacy of maternal immunization with a chimpanzee adenovirus expressing codon-optimized RSV fusion protein (AdC7-Fsyn) to protect infants against RSV infection. Single intranasal immunization of mice by AdC7-Fsyn induced robust anti-RSV systemic and mucosal immunity that protected against RSV without causing vaccine-enhanced RSV disease. RSV humoral immunity was transferred to pups born to immunized mothers that provided protection against RSV. Immunization with AdC7-Fsyn was effective even in the presence of Ad5 preimmunity. The maternally derived immunity was durable with the half-life of 14.63 days that reduced the viral replication up to 15 weeks of age. Notably, the passively immunized mice could be actively re-immunized with AdC7-Fsyn to boost and extend the protection. This substantiates maternal immunization with an AdC7-based vaccine expressing RSV F as feasible approach to protect against RSV early in life.

Respiratory Syncytial Virus (RSV) Pulmonary Infection in Humanized Mice Induces Human Anti-RSV Immune Responses and Pathology

ABSTRACT Respiratory syncytial virus (RSV) is a leading cause of lower respiratory tract disease, which causes high rates of morbidity and mortality in infants and the elderly. Models of human RSV pulmonary disease are needed to better understand RSV pathogenesis and to assess the efficacy of RSV vaccines. We assessed the RSV-specific human innate, humoral, and cellular immune responses in humanized mice (mice with a human immune system [HIS mice]) with functional human CD4+ T and B cells. These mice were generated by introduction of HLA class II genes, various human cytokines, and human B cell activation factor into immunodeficient NOD scid gamma (NSG) mice by the use of an adeno-associated virus vector, followed by engraftment of human hematopoietic stem cells. During the first 3 days of infection, HIS mice lost more weight and cleared RSV faster than NSG mice. Human chemokine (C-C motif) ligand 3 (CCL3) and human interleukin-1β (IL-1β) expression was detected in the RSV-infected HIS mice. The pathological features induced by RSV infection in HIS mice included peribronchiolar inflammation, neutrophil predominance in the bronchioalveolar lavage fluid, and enhanced airway mucus production. Human anti-RSV IgG and RSV-neutralizing antibodies were detected in serum and human anti-RSV mucosal IgA was detected in bronchioalveolar lavage fluid for up to 6 weeks. RSV infection induced an RSV-specific human gamma interferon response in HIS mouse splenocytes. These results indicate that human immune cells can induce features of RSV lung disease, including mucus hyperplasia, in murine lungs and that HIS mice can be used to elicit human anti-RSV humoral and cellular immunity. IMPORTANCE Infections with respiratory syncytial virus (RSV) are common and can cause severe lung disease in infants and the elderly. The lack of a suitable animal model with disease features similar to those in humans has hampered efforts to predict the efficacy of novel anti-RSV therapies and vaccines for use in humans. A murine model consisting of mice with a human immune system (HIS mice) could be useful for assessment of RSV disease and anti-RSV responses specific to humans. This study investigates an HIS mouse model to imitate human RSV disease and immune responses. We found that RSV lung infection in HIS mice results in an RSV-specific pathology that mimics RSV disease in humans and induces human anti-RSV immune responses. This model could be useful for better understanding of human RSV disease and for the development of RSV therapies.

Mast cells and exosomes in hyperoxia-induced neonatal lung disease

Chronic lung disease of prematurity (CLD) is a frequent sequela of premature birth and oxygen toxicity is a major associated risk factor. Impaired alveolarization, scarring, and inflammation are hallmarks of CLD. Mast cell hyperplasia is a feature of CLD but the role of mast cells in its pathogenesis is unknown. We hypothesized that mast cell hyperplasia is a consequence of neonatal hyperoxia and contributes to CLD. Additionally, mast cell products may have diagnostic and prognostic value in preterm infants predisposed to CLD. To model CLD, neonatal wild-type and mast cell-deficient mice were placed in an O2 chamber delivering hyperoxic gas mixture [inspired O2 fraction (FiO2 ) of 0.8] (HO) for 2 wk and then returned to room air (RA) for an additional 3 wk. Age-matched controls were kept in RA (FiO2 of 0.21). Lungs from HO mice had increased numbers of mast cells, alveolar simplification and enlargement, and increased lung compliance. Mast cell deficiency proved protective by preserving air space integrity and lung compliance. The mast cell mediators β-hexosaminidase (β-hex), histamine, and elastase increased in the bronchoalveolar lavage fluid of HO wild-type mice. Tracheal aspirate fluids (TAs) from oxygenated and mechanically ventilated preterm infants were analyzed for mast cell products. In TAs from infants with confirmed cases of CLD, β-hex was elevated over time and correlated with FiO2 Mast cell exosomes were also present in the TAs. Collectively, these data show that mast cells play a significant role in hyperoxia-induced lung injury and their products could serve as potential biomarkers in evolving CLD.

The fermentation product 2,3-butanediol alters P. aeruginosa clearance, cytokine response and the lung microbiome.

Diseases that favor colonization of the respiratory tract with Pseudomonas aeruginosa are characterized by an altered airway microbiome. Virulence of P. aeruginosa respiratory tract infection is likely influenced by interactions with other lung microbiota or their products. The bacterial fermentation product 2,3-butanediol enhances virulence and biofilm formation of P. aeruginosa in vitro. This study assessed the effects of 2,3-butanediol on P. aeruginosa persistence, inflammatory response, and the lung microbiome in vivo. Here, P. aeruginosa grown in the presence of 2,3-butanediol and encapsulated in agar beads persisted longer in the murine respiratory tract, induced enhanced TNF-α and IL-6 responses and resulted in increased colonization in the lung tissue by environmental microbes. These results led to the following hypothesis that now needs to be tested with a larger study: fermentation products from the lung microbiota not only have a role in P. aeruginosa virulence and abundance, but also on the increased colonization of the respiratory tract with environmental microbes, resulting in dynamic shifts in microbiota diversity and disease susceptibility.

Post-exposure immunization by capsid-modified AdC7 vector expressing Pseudomonas aeruginosa OprF clears P. aeruginosa respiratory infection

Respiratory infections with Pseudomonas aeruginosa are major health problems, particularly in patients with cystic fibrosis (CF). No vaccine against P. aeruginosa is yet available. A vaccine that controls colonization of the respiratory tract with P. aeruginosa could be useful to prevent chronic infection and exacerbations. Replication-deficient adenoviral (Ad) vectors based on non-human serotypes are attractive vaccine platforms as they can circumvent the problem of pre-existing anti-Ad immunity in humans. The primate-based AdC7 vector AdC7OprF.RGD that expresses the outer membrane protein F (OprF) of P. aeruginosa (AdC7OprF) and that displays an integrin-binding arginine-glycine-aspartic acid (RGD) sequence is a potent inducer of lung mucosal and protective immunity. Here, we investigated the efficacy of immunization with AdC7OprF.RGD to clear an already established P. aeruginosa respiratory infection in mice (wild-type and CF) and rats. Intratracheal administration of the clinical P. aeruginosa strain RP73 embedded in agar beads was used to establish persistent infection. Subsequent intranasal immunization with AdC7OprF.RGD induced robust P. aeruginosa-specific systemic and mucosal, humoral and cellular immune responses. Importantly, the AdC7OprF.RGD immunized mice effectively cleared P. aeruginosa from the lungs. Likewise, immunization with AdC7OprF.RGD of CF mice and Sprague Dawley rats with established P. aeruginosa respiratory infection showed enhanced anti-Pseudomonas immune responses and increased clearance of P. aeruginosa from the lungs. These data suggest that AdC7OprF.RGD can be effective as a post-exposure vaccine and may be useful in clinical settings in particular for patients with CF who frequently harbor the bacteria over prolonged periods.

Regulation of the Coxsackie and adenovirus receptor expression is dependent on cystic fibrosis transmembrane regulator in airway epithelial cells

The coxsackievirus and adenovirus receptor (CAR), in addition to serving as viral receptor, is a component of tight junctions and plays an important role in tissue homeostasis. Defects in the cystic fibrosis transmembrane regulator (CFTR) in lung epithelial cells are linked to inflammation and susceptibility for respiratory tract infections. Here, we demonstrate that CAR expression and infectivity with adenovirus (Ad) are increased in cystic fibrosis airway epithelial cells. Inhibition of CFTR or histone deacetylase (HDAC) enhanced CAR expression while CFTR overexpression or restoration of the diminished HDAC activity in cystic fibrosis cells reduced CAR expression. This connects the CFTR to CAR expression and infectivity with adenovirus through HDAC.

Regulation of the Coxsackie and adenovirus receptor expression is dependent on cystic fibrosis transmembrane regulator in airway epithelial cells: CFTR regulates CAR expression

The coxsackievirus and adenovirus receptor (CAR), in addition to serving as viral receptor, is a component of tight junctions and plays an important role in tissue homeostasis. Defects in the cystic fibrosis transmembrane regulator (CFTR) in lung epithelial cells are linked to inflammation and susceptibility for respiratory tract infections. Here, we demonstrate that CAR expression and infectivity with adenovirus (Ad) are increased in cystic fibrosis airway epithelial cells. Inhibition of CFTR or histone deacetylase (HDAC) enhanced CAR expression while CFTR overexpression or restoration of the diminished HDAC activity in cystic fibrosis cells reduced CAR expression. This connects the CFTR to CAR expression and infectivity with adenovirus through HDAC.

22. Post-Exposure Vaccination by Capsid-Modified AdC7 Vector Expressing Pseudomonas aeruginosa OprF in Chronic P. aeruginosa Lung Infection

Chronic infections by Pseudomonas aeruginosa are a common problem in cystic fibrosis and other chronic lung diseases associated with bronchiectasis. With antimicrobial resistance of P. aeruginosa becoming more common, alternate prophylactic and therapeutic approaches are needed. Despite extensive efforts, a vaccine against P. aeruginosa is not yet available. A post-exposure vaccine that eliminates already established P. aeruginosa from the respiratory tract, could be useful in the management of chronic P. aeruginosa colonization. Replication-deficient adenoviral (Ad) vectors are an attractive platform for vaccines against respiratory pathogens. We have previously found that, in addition to circumvent pre-existing anti-human Ad immunity, a non-human primate-based AdC7 vector expressing outer membrane protein F (OprF) of P. aeruginosa (AdC7OprF) was more potent in inducing lung mucosal and protective immunity compared to a human Ad5-based vector. In addition, genetic modification of the AdC7 fiber to display an integrin-binding arginine-glycine-aspartic acid (RGD) sequence can further enhance mucosal protective immunogenicity of AdC7OprF. In this study we investigated if post-exposure vaccination by AdC7OprF. RGD can clear the already established P. aeruginosa in a mouse model. Intratracheal inoculation of P. aeruginosa (clinical strain RP73) encapsulated in agar beads (10^6 cfu/mouse) was used to establish persistent infection. Intranasal immunization of P. aeruginosa infected mice with AdC7OprF. RGD (10^10 pu/mouse) induced significantly high serum anti-OprF IgG antibodies as early as 1 week of immunization that further increased to higher levels after 2 week of immunization compared to AdC7Null or PBS inoculated mice (p<0.05; all comparisons). In addition to robust humoral response, immunization with AdC7OprF. RGD induced OprF-specific T-cell responses, as indicated by the higher secretion of IFN-γ or IL-4 from the OprF-stimulated cultured splenocytes compared to AdNull or PBS inoculated mice (p<0.05; all comparisons). Importantly, the AdC7OprF. RGD immunized mice showed significantly higher clearance of P. aeruginosa from the infected lungs after 1 week or 2 weeks of immunization (p<0.05; all comparisons). In fact, after 2 weeks of immunization, 50% of AdC7OprF. RGD immunized mice (3 out of 6) completely cleared the P. aeruginosa from the infected lungs. These data suggest that immunization with AdC7OprF. RGD induced robust humoral and cellular anti-P. aeruginosa immunity that could clear established pulmonary P. aeruginosa infections.