Human Rhinovirus Infection of the Respiratory Tract Affects Sphingolipid Synthesis.

The 17q21 asthma susceptibility locus includes asthma risk alleles associated with decreased sphingolipid synthesis, likely resulting from increased expression of ORMDL3. ORMDL3 inhibits serine-palmitoyl transferase (SPT), the rate-limiting enzyme of de novo sphingolipid synthesis. There is evidence that decreased sphingolipid synthesis is critical to asthma pathogenesis. Children with asthma and 17q21 asthma risk alleles display decreased sphingolipid synthesis in blood cells. Reduced SPT activity results in airway hyperreactivity, a hallmark feature of asthma. 17q21 asthma risk alleles are also linked to childhood infections with human rhinovirus (RV). This study evaluates the interaction of RV with the de novo sphingolipid synthesis pathway, and the alterative effects of concurrent SPT inhibition in SPT-deficient mice and human airway epithelial cells. In mice, RV infection shifted lung sphingolipid synthesis gene expression to a pattern that resembles genetic SPT deficiency, including decreased expression of Sptssa, a small SPT subunit. This pattern was pronounced in lung epithelial cellular adhesion molecule (EpCAM+) cells and reproduced in human bronchial epithelial cells. RV did not affect Sptssa expression in lung CD45+ immune cells. RV increased sphingolipids unique to the de novo synthesis pathway in mouse lung and human airway epithelial cells. Interestingly, these de novo sphingolipid species were reduced in the blood of RV-infected wild-type mice. RV exacerbated SPT deficiency-associated airway hyperreactivity. Airway inflammation was similar in RV-infected wild-type and SPT-deficient mice. This study reveals the effects of RV infection on the de novo sphingolipid synthesis pathway, elucidating a potential mechanistic link between 17q21 asthma risk alleles and rhinoviral infection.

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.

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

UNLABELLED: 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-1ß (IL-1ß) 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.

Glucagon Receptor Inhibition Reduces Hyperammonemia and Lethality in Male Mice with Urea Cycle Disorder.

The liver plays a critical role in maintaining ammonia homeostasis. Urea cycle defects, liver injury, or failure and glutamine synthetase (GS) deficiency result in hyperammonemia, serious clinical conditions, and lethality. In this study we used a mouse model with a defect in the urea cycle enzyme ornithine transcarbamylase (Otcspf-ash) to test the hypothesis that glucagon receptor inhibition using a monoclonal blocking antibody will reduce the hyperammonemia and associated lethality induced by a high-protein diet, which exacerbates disease. We found reduced expression of glutaminase, which degrades glutamine and increased expression of GS in livers of Otcspf-ash mice treated with the glucagon receptor blocking antibody. The gene expression changes favor ammonia consumption and were accompanied by increased circulating glutamine levels and diminished hyperammonemia. Otcspf-ash mice treated with the glucagon receptor-blocking antibody gained lean and body mass and had increased survival. These data suggest that glucagon receptor inhibition using a monoclonal antibody could reduce the risk for hyperammonemia and other clinical manifestations of patients suffering from defects in the urea cycle, liver injury, or failure and GS deficiency.

ILC3s control airway inflammation by limiting T cell responses to allergens and microbes.

Group 3 innate lymphoid cells (ILC3s) critically regulate host-microbe interactions in the gastrointestinal tract, but their role in the airway remains poorly understood. Here, we demonstrate that lymphoid-tissue-inducer (LTi)-like ILC3s are enriched in the lung-draining lymph nodes of healthy mice and humans. These ILC3s abundantly express major histocompatibility complex class II (MHC class II) and functionally restrict the expansion of allergen-specific CD4+ T cells upon experimental airway challenge. In a mouse model of house-dust-mite-induced allergic airway inflammation, MHC class II+ ILC3s limit T helper type 2 (Th2) cell responses, eosinophilia, and airway hyperresponsiveness. Furthermore, MHC class II+ ILC3s limit a concomitant Th17 cell response and airway neutrophilia. This exacerbated Th17 cell response requires exposure of the lung to microbial stimuli, which can be found associated with house dust mites. These findings demonstrate a critical role for antigen-presenting ILC3s in orchestrating immune tolerance in the airway by restricting pro-inflammatory T cell responses to both allergens and microbes.

Increased SLC38A4 Amino Acid Transporter Expression in Human Pancreatic α-Cells After Glucagon Receptor Inhibition.

Plasma amino acids and their transporters constitute an important part of the feedback loop between the liver and pancreatic α-cell function, and glucagon regulates hepatic amino acid turnover. Disruption of hepatic glucagon receptor action activates the loop and results in high plasma amino acids and hypersecretion of glucagon associated with α-cell hyperplasia. In the present study, we report a technique to rescue implanted human pancreatic islets from the mouse kidney capsule. Using this model, we have demonstrated that expression of the amino acid transporter SLC38A4 increases in α-cells after administration of a glucagon receptor blocking antibody. The increase in SLC38A4 expression and associated α-cell proliferation was dependent on mechanistic target of rapamycin pathway. We confirmed increased α-cell proliferation and expression of SLC38A4 in pancreas sections from patients with glucagon cell hyperplasia and neoplasia (GCHN) with loss-of-function mutations in the glucagon receptor. Collectively, using a technique to rescue implanted human islets from the kidney capsule in mice and pancreas sections from patients with GCHN, we found that expression of SLC38A4 was increased under conditions of disrupted glucagon receptor signaling. These data provide support for the existence of a liver-human α-cell endocrine feedback loop.

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.

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.

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.

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.

Impaired sphingolipid synthesis in the respiratory tract induces airway hyperreactivity.

Asthma is a clinically heterogeneous genetic disease, and its pathogenesis is incompletely understood. Genome-wide association studies link ORM (yeast)-Like protein isoform 3 [corrected] (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.

Very low frequency modulation in renal autoregulation.

This study aims to examine the presence of a possible third renal autoregulatory mechanism in the very low frequency (VLF) band (approximately 10 mHz) using a high-resolution time- frequency spectral method. Blood pressure and renal blood flow data were measured from conscious and anesthetized Sprague-Dawley and spontaneously hypertensive rats, at the level of the whole kidney (via ultrasound flow probe) and local cortical tissue of a kidney (via laser Doppler flow probe). In addition, N-nitro-L-arginine (LNAME) was used in order to assess the effect of nitric oxide on the third mechanism. Using a complex demodulation method with high time and frequency resolution, a VLF band was often observed, as well as amplitude modulation at the VLF of the two other autoregulation mechanisms. The presence of amplitude modulation is an indication of a particular form of nonlinear interaction between the autoregulatory mechanisms. Physically, such interactions may arise from the fact that all three mechanisms share a common effector, the afferent arteriole. In addition, the magnitude of amplitude modulation of the VLF on the other autoregulatory mechanisms was enhanced by the addition of LNAME, suggesting an important role of nitric oxide in the autoregulatory process.