Lactobacillus rhamnosus dampens cytokine and chemokine secretion from primary human nasal epithelial cells infected with rhinovirus.

AIMS: To investigate the effect of Lactobacillus rhamnosus on viral replication and cellular response to human rhinovirus (HRV) infection, including the secretion of antiviral and inflammatory mediators from well-differentiated nasal epithelial cells (WD-NECs). METHODS AND RESULTS: The WD-NECs from healthy adult donors (N = 6) were cultured in vitro, exposed to different strains of L. rhamnosus (D3189, D3160, or LB21), and infected with HRV (RV-A16) after 24 h. Survival and adherence capacity of L. rhamnosus in a NEC environment were confirmed using CFSE-labelled isolates, immunofluorescent staining, and confocal microscopy. Shed virus and viral replication were quantified using TCID50 assays and RT-qPCR, respectively. Cytotoxicity was measured by lactate dehydrogenase (LDH) activity. Pro-inflammatory mediators were measured by multiplex immunoassay, and interferon (IFN)-λ1/3 was measured using a standard ELISA kit. Lactobacillus rhamnosus was able to adhere to and colonize WD-NECs prior to the RV-A16 infection. Lactobacillus rhamnosus did not affect shed RV-A16, viral replication, RV-A16-induced IFN-λ1/3 production, or LDH release. Pre-exposure to L. rhamnosus, particularly D3189, reduced the secretion of RV-A16-induced pro-inflammatory mediators by WD-NECs. CONCLUSIONS: These findings demonstrate that L. rhamnosus differentially modulates RV-A16-induced innate inflammatory immune responses in primary NECs from healthy adults.

Prime-boost, double-dose influenza vaccine immunity in COPD: a pilot observational study.

Background: COPD patients are more susceptible to viral respiratory infections and their sequelae, and have intrinsically weaker immune responses to vaccinations against influenza and other pathogens. Prime-boost, double-dose immunisation has been suggested as a general strategy to overcome weak humoral response to vaccines, such as seasonal influenza vaccination, in susceptible populations with weak immunity. However, this strategy, which may also provide fundamental insights into the nature of weakened immunity, has not been formally studied in COPD. Methods: We conducted an open-label study of seasonal influenza vaccination in 33 vaccine-experienced COPD patients recruited from established cohorts (mean age 70 (95% CI 66.9-73.2) years; mean forced expiratory volume in 1 s/forced vital capacity ratio 53.4% (95% CI 48.0-58.8%)). Patients received two sequential standard doses of the 2018 quadrivalent influenza vaccine (15 µg haemagglutinin per strain) in a prime-boost schedule 28 days apart. We measured strain-specific antibody titres, an accepted surrogate of likely efficacy, and induction of strain-specific B-cell responses following the prime and boost immunisations. Results: Whereas priming immunisation induced the expected increase in strain-specific antibody titres, a second booster dose was strikingly ineffective at further increasing antibody titres. Similarly, priming immunisation induced strain-specific B-cells, but a second booster dose did not further enhance the B-cell response. Poor antibody responses were associated with male gender and cumulative cigarette exposure. Conclusions: Prime-boost, double-dose immunisation does not further improve influenza vaccine immunogenicity in previously vaccinated COPD patients. These findings underscore the need to design more effective vaccine strategies for COPD patients for influenza.

Vaccine strain affects seroconversion after influenza vaccination in COPD patients and healthy older people.

Though clinical guidelines recommend influenza vaccination for chronic obstructive pulmonary disease (COPD) patients and other high-risk populations, it is unclear whether current vaccination strategies induce optimal antibody responses. This study aimed to identify key variables associated with strain-specific antibody responses in COPD patients and healthy older people. 76 COPD and 72 healthy participants were recruited from two Australian centres and inoculated with influenza vaccine. Serum strain-specific antibody titres were measured pre- and post-inoculation. Seroconversion rate was the primary endpoint. Antibody responses varied between vaccine strains. The highest rates of seroconversion were seen with novel strains (36-55%), with lesser responses to strains included in the vaccine in more than one consecutive year (27-33%). Vaccine responses were similar in COPD patients and healthy participants. Vaccine strain, hypertension and latitude were independent predictors of seroconversion. Our findings reassure that influenza vaccination is equally immunogenic in COPD patients and healthy older people; however, there is room for improvement. There may be a need to personalise the yearly influenza vaccine, including consideration of pre-existing antibody titres, in order to target gaps in individual antibody repertoires and improve protection.

COPD Is Associated with Elevated IFN-ß Production by Bronchial Epithelial Cells Infected with RSV or hMPV.

IFN treatment may be a viable option for treating COPD exacerbations based on evidence of IFN deficiency in COPD. However, in vitro studies have used primarily influenza and rhinoviruses to investigate IFN responses. This study aims to investigate the susceptibility to infection and IFN response of primary bronchial epithelial cells (BECs) from COPD donors to infection with RSV and hMPV. BECs from five COPD and five healthy donors were used to establish both submerged monolayer and well-differentiated (WD) cultures. Two isolates of both RSV and hMPV were used to infect cells. COPD was not associated with elevated susceptibility to infection and there was no evidence of an intrinsic defect in IFN production in either cell model to either virus. Conversely, COPD was associated with significantly elevated IFN-ß production in response to both viruses in both cell models. Only in WD-BECs infected with RSV was elevated IFN-ß associated with reduced viral shedding. The role of elevated epithelial cell IFN-ß production in the pathogenesis of COPD is not clear and warrants further investigation. Viruses vary in the responses that they induce in BECs, and so conclusions regarding antiviral responses associated with disease cannot be made based on single viral infections.

The eukaryotic translation elongation factor 1A regulation of actin stress fibers is important for infectious RSV production.

Cellular protein eukaryotic translation elongation factor 1A (eEF1A) is an actin binding protein that plays a role in the formation of filamentous actin (F-actin) bundles. F-Actin regulates multiple stages of respiratory syncytial virus (RSV) replication including assembly and budding. Our previous study demonstrated that eEF1A knock-down significantly reduced RSV replication. Here we investigated if the eEF1A function in actin bundle formation was important for RSV replication and release. To investigate this, eEF1A function was impaired in HEp-2 cells by either knock-down of eEF1A with siRNA, or treatment with an eEF1A inhibitor, didemnin B (Did B). Cell staining and confocal microscopy analysis showed that both eEF1A knock-down and treatment with Did B resulted in disruption of cellular stress fiber formation and elevated accumulation of F-actin near the plasma membrane. When treated cells were then infected with RSV, there was also reduced formation of virus-induced cellular filopodia. Did B treatment, similarly to eEF1A knock-down, reduced the release of infectious RSV, but unlike eEF1A knock-down, did not significantly affect RSV genome replication. The lower infectious virus production in Did B treated cells also reduced RSV-induced cell death. In conclusion, the cellular factor eEF1A plays an important role in the regulation of F-actin stress fiber formation required for RSV assembly and release.

Chronic IL-33 expression predisposes to virus-induced asthma exacerbations by increasing type 2 inflammation and dampening antiviral immunity.

BACKGROUND: Rhinovirus infection triggers acute asthma exacerbations. IL-33 is an instructive cytokine of type 2 inflammation whose expression is associated with viral load during experimental rhinovirus infection of asthmatic patients. OBJECTIVE: We sought to determine whether anti-IL-33 therapy is effective during disease progression, established disease, or viral exacerbation using a preclinical model of chronic asthma and in vitro human primary airway epithelial cells (AECs). METHODS: Mice were exposed to pneumonia virus of mice and cockroach extract in early and later life and then challenged with rhinovirus to model disease onset, progression, and chronicity. Interventions included anti-IL-33 or dexamethasone at various stages of disease. AECs were obtained from asthmatic patients and healthy subjects and treated with anti-IL-33 after rhinovirus infection. RESULTS: Anti-IL-33 decreased type 2 inflammation in all phases of disease; however, the ability to prevent airway smooth muscle growth was lost after the progression phase. After the chronic phase, IL-33 levels were persistently high, and rhinovirus challenge exacerbated the type 2 inflammatory response. Treatment with anti-IL-33 or dexamethasone diminished exacerbation severity, and anti-IL-33, but not dexamethasone, promoted antiviral interferon expression and decreased viral load. Rhinovirus replication was higher and IFN-λ levels were lower in AECs from asthmatic patients compared with those from healthy subjects. Anti-IL-33 decreased rhinovirus replication and increased IFN-λ levels at the gene and protein levels. CONCLUSION: Anti-IL-33 or dexamethasone suppressed the magnitude of type 2 inflammation during a rhinovirus-induced acute exacerbation; however, only anti-IL-33 boosted antiviral immunity and decreased viral replication. The latter phenotype was replicated in rhinovirus-infected human AECs, suggesting that anti-IL-33 therapy has the additional benefit of enhancing host defense.

Human Metapneumovirus Impairs Apoptosis of Nasal Epithelial Cells in Asthma via HSP70.

Asthmatics are highly susceptible to respiratory viral infections, possibly due to impaired innate immunity. However, the exact mechanisms of susceptibility are likely to differ amongst viruses. Therefore, we infected primary nasal epithelial cells (NECs) from adults with mild-to-moderate asthma, with respiratory syncytial virus (RSV) or human metapneumovirus (hMPV) in vitro and investigated the antiviral response. NECs from these asthmatics supported elevated hMPV but not RSV infection, compared to non-asthmatic controls. This correlated with reduced apoptosis and reduced activation of caspase-9 and caspase-3/7 in response to hMPV, but not RSV. The expression of heat shock protein 70 (HSP70), a known inhibitor of caspase activation and subsequent apoptosis, was amplified in response to hMPV infection. Chemical inhibition of HSP70 function restored caspase activation and reduced hMPV infection in NECs from asthmatic subjects. There was no impairment in the production of IFN by NECs from asthmatics in response to either hMPV or RSV, demonstrating that increased infection of asthmatic airway cells by hMPV is IFN-independent. This study demonstrates, for the first time, a mechanism for elevated hMPV infection in airway epithelial cells from adult asthmatics and identifies HSP70 as a potential target for antiviral and asthma therapies.

The Impact of Early-Life Exposure to Air-borne Environmental Insults on the Function of the Airway Epithelium in Asthma.

The airway epithelium is both a physical barrier protecting the airways from environmental insults and a significant component of the innate immune response. There is growing evidence that exposure of the airway epithelium to environmental insults in early life may lead to permanent changes in structure and function that underlie the development of asthma. Here we review the current published evidence concerning the link between asthma and epithelial damage within the airways and identify gaps in knowledge for future studies.

The eukaryotic elongation factor 1A is critical for genome replication of the paramyxovirus respiratory syncytial virus.

The eukaryotic translation factor eEF1A assists replication of many RNA viruses by various mechanisms. Here we show that down-regulation of eEF1A restricts the expression of viral genomic RNA and the release of infectious virus, demonstrating a biological requirement for eEF1A in the respiratory syncytial virus (RSV) life cycle. The key proteins in the replicase/transcriptase complex of RSV; the nucleocapsid (N) protein, phosphoprotein (P) and matrix (M) protein, all associate with eEF1A in RSV infected cells, although N is the strongest binding partner. Using individually expressed proteins, N, but not P or M bound to eEF1A. This study demonstrates a novel interaction between eEF1A and the RSV replication complex, through binding to N protein, to facilitate genomic RNA synthesis and virus production.

Two hepcidin-like antimicrobial peptides in Barramundi Lates calcarifer exhibit differing tissue tropism and are induced in response to lipopolysaccharide.

Genes encoding two hepcidin-like antimicrobial peptides were discovered in Barramundi, Lates calcarifer (barramundi, Giant sea perch). Analysis of the coding regions indicated that genes for each hepcidin comprised 3 exons and 2 introns. The deduced amino acid sequences for each molecule resulted in a protein comprising a signal sequence of 24 aa in each case, coupled to a prepropeptide of 75 aa for hepcidin 1 and 78 aa for hepcidin 2. A cleavage site was identified in each prepropetide at amino acid 64 with the cleavage motif--QKR/QS--resulting in mature peptides of 25 and 28 amino acids respectively. Each mature peptide contained 8 conserved cysteine residues and 3 dimensional modeling predicted a ß-hairpin and ß-sheet structure characteristic of human Liver Expressed Antimicrobial Peptide (LEAP). Analysis of the deduced amino acid sequences by BLAST with phylogenetic supported indicated that hepcidin 1 was a HAMP1-type peptide closely related to hepcidins identified in other Perciformes (Micropterus and Pseudosciaena), whilst hepcidin 2 was a HAMP2-type peptide most similar to a hepcidin previously identified in black rock fish (Sebastes schlegeli). Both hepcidin genes were inducible in barramundi following intraperitoneal injection with lipopolysaccharide, with elevated expression detected in liver and head kidney 3 h post IP injection for hepcidin 1 and in liver only for hepcidin 2. The elevated expression was transient with return to normal levels within 24-48 h. No significant expression of either peptide was detected in spleen, skin or gill following IP injection with LPS.

Linear and branched beta(1-3) D-glucans activate but do not prime teleost macrophages in vitro and are inactivated by dilute acid: implications for dietary immunostimulation.

Beta(1-3) glucans are a diverse range of carbohydrate polymers of differing lengths and structures that make up the cell walls of yeast, fungi, algae and some plants and activate innate immune responses in plants, invertebrates and higher animals. Consequently glucans are often used as dietary immunostimulants in commercial feeds for aquacultured fish species. The present study investigates the capability of purified glucans of differing structures and configurations, including curdlan, paramylon, laminarin and purified yeast beta glucan to activate innate immunity in vitro using barramundi pronephros macrophages as a model, and compares them to Zymosan, a complex mixture derived from yeast cell walls, and lipopolysaccharide from Gram negative bacteria. All of the glucans were able to stimulate respiratory burst in barramundi macrophages at concentrations of 100 microg/mL and 1000 microg/mL, with curdlan eliciting the highest respiratory burst response at 1000 microg/mL. LPS and Zymosan were the only immunostimulants tested that could prime barramundi macrophages by incubating with low concentrations (0.1 and 1 microg/mL) for 24 h before triggering respiratory burst with PMA, suggesting teleost macrophages may not prime through the glucan receptor. As glucans are used as dietary immunostimulants, the pH of the barramundi stomach was assayed for 6 h following feeding and indicated that pH was as low as 2 for up to 6 h. Treating the glucans with dilute HCl at pH 2 completely neutralised their macrophage-activating capability. These results are important as they indicate that glucans do not prime barramundi macrophages but will activate them at high concentrations. However, it is debatable whether glucans will have any effect on macrophages if administered in the diet due to the combination of high concentration required and probable hydrolysis of the polymer structures as they pass through the acid environment of the stomach.

Prevalence and effect of spawner-isolated mortality virus on the hatchery phases of Penaeus monodon and P. merguiensis in Australia.

Spawner-isolated mortality virus (SMV) has been associated with mortalities in broodstock of Penaeus monodon and with mid-crop mortality syndrome on grow-out farms. Epidemiological evidence suggested an association between the SMV status of broodstock and subsequent survival of their progeny, and this paper describes investigations into that association. The faeces of 909 broodstock in 9 different groups were tested by a polymerase chain reaction (PCR) for SMV and positive results were confirmed by an internal dot-blot. Seventy-seven spawners (8.5%) were positive for SMV with prevalence ranging from 0 to 24% among groups. The prevalence in spawners of P. monodon was higher (24%) than in P. merguiensis (4%). Three longitudinal studies were undertaken to compare the survival of progeny from broodstock that were positive to SMV with those that were not. Survival in hatchery tanks of progeny from SMV-positive spawners was lower than those from SMV-negative spawners with reductions of 23% (p = 0.01), 7.3% (p = 0.214) and 18.9% (p = 0.129) in the 3 studies. The conclusions were less consistent when examined during each of the later stages of growth in hatchery pools, nursery and grow-out ponds, with progeny from SMV-postive spawners sometimes having better survival rates. However, survival was better overall in progeny from SMV-negative spawners. Simple linear regression showed survival was negatively related to the proportion of postlarvae from SMV-positive spawners, with a decrease in survival of 5.6% for each 10% increase in the proportion of postlarvae coming from SMV-positive spawners (p = 0.006). Data from 38 ponds showed 6.71% of losses were due to SMV. If these losses were consistent across the entire industry, the annual loss due to SMV would have been approximately AUD 3 million in 1999/2000.