Effect of Broccoli Sprouts and Live Attenuated Influenza Virus on Peripheral Blood Natural Killer Cells: A Randomized, Double-Blind Study

Enhancing antiviral host defense responses through nutritional supplementation would be an attractive strategy in the fight against influenza. Using inoculation with live attenuated influenza virus (LAIV) as an infection model, we have recently shown that ingestion of sulforaphane-containing broccoli sprout homogenates (BSH) reduces markers of viral load in the nose. To investigate the systemic effects of short-term BSH supplementation in the context of LAIV-inoculation, we examined peripheral blood immune cell populations in non-smoking subjects from this study, with a particular focus on NK cells. We carried out a randomized, double-blinded, placebo-controlled study measuring the effects of BSH (N = 13) or placebo (alfalfa sprout homogenate, ASH; N = 16) on peripheral blood mononuclear cell responses to a standard nasal vaccine dose of LAIV in healthy volunteers. Blood was drawn prior to (day-1) and post (day2, day21) LAIV inoculation and analyzed for neutrophils, monocytes, macrophages, T cells, NKT cells, and NK cells. In addition, NK cells were enriched, stimulated, and assessed for surface markers, intracellular markers, and cytotoxic potential by flow cytometry. Overall, LAIV significantly reduced NKT (day2 and day21) and T cell (day2) populations. LAIV decreased NK cell CD56 and CD158b expression, while significantly increasing CD16 expression and cytotoxic potential (on day2). BSH supplementation further increased LAIV-induced granzyme B production (day2) in NK cells compared to ASH and in the BSH group granzyme B levels appeared to be negatively associated with influenza RNA levels in nasal lavage fluid cells. We conclude that nasal influenza infection may induce complex changes in peripheral blood NK cell activation, and that BSH increases virus-induced peripheral blood NK cell granzyme B production, an effect that may be important for enhanced antiviral defense responses

Nasal lavage natural killer cell function is suppressed in smokers after live attenuated influenza virus

<p>Abstract</p> <p>Background</p> <p>Modified function of immune cells in nasal secretions may play a role in the enhanced susceptibility to respiratory viruses that is seen in smokers. Innate immune cells in nasal secretions have largely been characterized by cellular differentials using morphologic criteria alone, which have successfully identified neutrophils as a significant cell population within nasal lavage fluid (NLF) cells. However, flow cytometry may be a superior method to fully characterize NLF immune cells. We therefore characterized immune cells in NLF by flow cytometry, determined the effects of live attenuated influenza virus (LAIV) on NLF and peripheral blood immune cells, and compared responses in samples obtained from smokers and nonsmokers.</p> <p>Methods</p> <p>In a prospective observational study, we characterized immune cells in NLF of nonsmokers at baseline using flow cytometry and immunohistochemistry. Nonsmokers and smokers were inoculated with LAIV on day 0 and serial nasal lavages were collected on days 1-4 and day 9 post-LAIV. LAIV-induced changes of NLF cells were characterized using flow cytometry. Cell-free NLF was analyzed for immune mediators by bioassay. Peripheral blood natural killer (NK) cells from nonsmokers and smokers at baseline were stimulated <it>in vitro </it>with LAIV followed by flow cytometric and mediator analyses.</p> <p>Results</p> <p>CD45(+)CD56(-)CD16(+) neutrophils and CD45(+)CD56(+) NK cells comprised median 4.62% (range 0.33-14.52) and 23.27% (18.29-33.97), respectively, of non-squamous NLF cells in nonsmokers at baseline. LAIV did not induce changes in total NK cell or neutrophil percentages in either nonsmokers or smokers. Following LAIV inoculation, CD16(+) NK cell percentages and granzyme B levels increased in nonsmokers, and these effects were suppressed in smokers. LAIV inoculation enhanced expression of activating receptor NKG2D and chemokine receptor CXCR3 on peripheral blood NK cells from both nonsmokers and smokers <it>in vitro </it>but did not induce changes in CD16(+) NK cells or granzyme B activity in either group.</p> <p>Conclusions</p> <p>These data are the first to identify NK cells as a major immune cell type in the NLF cell population and demonstrate that mucosal NK cell cytotoxic function is suppressed in smokers following LAIV. Altered NK cell function in smokers suggests a potential mechanism that may enhance susceptibility to respiratory viruses.</p

Sequential analysis of surfactant, lung function and inflammation in cystic fibrosis patients

BACKGROUND: In a cross-sectional analysis of cystic fibrosis (CF) patients with mild lung disease, reduced surfactant activity was correlated to increased neutrophilic airway inflammation, but not to lung function. So far, longitudinal measurements of surfactant function in CF patients are lacking and it remains unclear how these alterations relate to the progression of airway inflammation as well as decline in pulmonary function over time. METHODS: As part of the BEAT trial, a longitudinal study to assess the course of airway inflammation in CF, we studied lung function, surfactant function and endobronchial inflammation using bronchoalveolar lavage fluid from 20 CF patients with normal pulmonary function (median FEV(1 )94% of predicted) at three times over a three year period. RESULTS: There was a progressive loss of surfactant function, assessed as minimal surface tension. The decline in surfactant function was negatively correlated to an increase in neutrophilic inflammation and a decrease in lung function, assessed by FEV(1), MEF(75/25%VC), and MEF(25%VC). The concentrations of the surfactant specific proteins A, C and D did not change, whereas SP-B increased during this time period. CONCLUSION: Our findings suggest a link between loss of surfactant function driven by progressive airway inflammation and loss of small airway function in CF patients with limited lung disease

Azithromycin reduces spontaneous and induced inflammation in ΔF508 cystic fibrosis mice

BACKGROUND: Inflammation plays a critical role in lung disease development and progression in cystic fibrosis. Azithromycin is used for the treatment of cystic fibrosis lung disease, although its mechanisms of action are poorly understood. We tested the hypothesis that azithromycin modulates lung inflammation in cystic fibrosis mice. METHODS: We monitored cellular and molecular inflammatory markers in lungs of cystic fibrosis mutant mice homozygous for the ΔF508 mutation and their littermate controls, either in baseline conditions or after induction of acute inflammation by intratracheal instillation of lipopolysaccharide from Pseudomonas aeruginosa, which would be independent of interactions of bacteria with epithelial cells. The effect of azithromycin pretreatment (10 mg/kg/day) given by oral administration for 4 weeks was evaluated. RESULTS: In naive cystic fibrosis mice, a spontaneous lung inflammation was observed, characterized by macrophage and neutrophil infiltration, and increased intra-luminal content of the pro-inflammatory cytokine macrophage inflammatory protein-2. After induced inflammation, cystic fibrosis mice combined exaggerated cellular infiltration and lower anti-inflammatory interleukin-10 production. In cystic fibrosis mice, azithromycin attenuated cellular infiltration in both baseline and induced inflammatory condition, and inhibited cytokine (tumor necrosis factor-α and macrophage inflammatory protein-2) release in lipopolysaccharide-induced inflammation. CONCLUSION: Our findings further support the concept that inflammatory responses are upregulated in cystic fibrosis. Azithromycin reduces some lung inflammation outcome measures in cystic fibrosis mice. We postulate that some of the benefits of azithromycin treatment in cystic fibrosis patients are due to modulation of lung inflammation

Potential therapeutic implications of new insights into respiratory syncytial virus disease

Viral bronchiolitis is the most common cause of hospitalization in infants under 6 months of age, and 70% of all cases of bronchiolitis are caused by respiratory syncytial virus (RSV). Early RSV infection is associated with respiratory problems such as asthma and wheezing later in life. RSV infection is usually spread by contaminated secretions and infects the upper then lower respiratory tracts. Infected cells release proinflammatory cytokines and chemokines, including IL-1, tumor necrosis factor-α, IL-6, and IL-8. These activate other cells and recruit inflammatory cells, including macrophages, neutrophils, eosinophils, and T lymphocytes, into the airway wall and surrounding tissues. The pattern of cytokine production by T lymphocytes can be biased toward 'T-helper-1' or 'T-helper-2' cytokines, depending on the local immunologic environment, infection history, and host genetics. T-helper-1 responses are generally efficient in antiviral defense, but young infants have an inherent bias toward T-helper-2 responses. The ideal intervention for RSV infection would be preventive, but the options are currently limited. Vaccines based on protein subunits, live attenuated strains of RSV, DNA vaccines, and synthetic peptides are being developed; passive antibody therapy is at present impractical in otherwise healthy children. Effective vaccines for use in neonates continue to be elusive but simply delaying infection beyond the first 6 months of life might reduce the delayed morbidity associated with infantile disease

Phosphoinositide-3 kinase inhibition modulates responses to rhinovirus by mechanisms that are predominantly independent of autophagy

Human rhinoviruses (HRV) are a major cause of exacerbations of airways disease. Aspects of cell signalling responses to HRV infection remain unclear, particularly with regard to signalling via PI3K, and the PI3K-dependent pathway, autophagy. We investigated the roles of PI3K and autophagy in the responses of epithelial cells to major and minor group HRV infection. The PI3K inhibitor 3-MA, commonly used to inhibit autophagy, markedly reduced HRV-induced cytokine induction. Further investigation of potential targets of 3-MA and comparison of results using this inhibitor to a panel of general and class I-selective PI3K inhibitors showed that several PI3Ks cooperatively regulate responses to HRV. Targeting by siRNA of the autophagy proteins Beclin-1, Atg7, LC3, alone or in combination, or targeting of the autophagy-specific class III PI3K had at most only modest effects on HRV-induced cell signalling as judged by induction of proinflammatory cytokine production. Our data indicate that PI3K and mTOR are involved in induction of proinflammatory cytokines after HRV infection, and that autophagy has little role in the cytokine response to HRV or control of HRV replication

NGF Is an Essential Survival Factor for Bronchial Epithelial Cells during Respiratory Syncytial Virus Infection

Background: Overall expression of neurotrophins in the respiratory tract is upregulated in infants infected by the respiratory syncytial virus (RSV), but it is unclear where (structural vs. inflammatory cells, upper vs. lower airways) and why, these changes occur. We analyzed systematically the expression of neurotrophic factors and receptors following RSV infection of human nasal, tracheal, and bronchial epithelial cells, and tested the hypothesis that neurotrophins work as innate survival factors for infected respiratory epithelia. Methodology: Expression of neurotrophic factors (nerve growth factor, NGF; brain-derived neurotrophic factor, BDNF) and receptors (trkA, trkB, p75) was analyzed at the protein level by immunofluorescence and flow cytometry and at the mRNA level by real-time PCR. Targeted siRNA was utilized to blunt NGF expression, and its effect on virus-induced apoptosis/ necrosis was evaluated by flow cytometry following annexin V/7-AAD staining. Principal Findings: RSV infection was more efficient in cells from more distal (bronchial) vs. more proximal origin. In bronchial cells, RSV infection induced transcript and protein overexpression of NGF and its high-affinity receptor trkA, with concomitant downregulation of the low-affinity p75 NTR. In contrast, tracheal cells exhibited an increase in BDNF, trkA and trkB, and nasal cells increased only trkA. RSV-infected bronchial cells transfected with NGF-specific siRNA exhibited decreased trkA and increased p75 NTR expression. Furthermore, the survival of bronchial epithelial cells was dramaticall

Respiratory syncytial virus infection induces higher Toll-like receptor-3 expression and TNF-α production than human metapneumovirus infection

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SNPs Associated with Cerebrospinal Fluid Phospho-Tau Levels Influence Rate of Decline in Alzheimer's Disease

Alzheimer's Disease (AD) is a complex and multifactorial disease. While large genome-wide association studies have had some success in identifying novel genetic risk factors for AD, case-control studies are less likely to uncover genetic factors that influence progression of disease. An alternative approach to identifying genetic risk for AD is the use of quantitative traits or endophenotypes. The use of endophenotypes has proven to be an effective strategy, implicating genetic risk factors in several diseases, including anemia, osteoporosis and heart disease. In this study we identify a genetic factor associated with the rate of decline in AD patients and present a methodology for identification of other such factors. We have used an established biomarker for AD, cerebrospinal fluid (CSF) tau phosphorylated at threonine 181 (ptau181) levels as an endophenotype for AD, identifying a SNP, rs1868402, in the gene encoding the regulatory sub-unit of protein phosphatase B, associated with CSF ptau181 levels in two independent CSF series . We show no association of rs1868402 with risk for AD or age at onset, but detected a very significant association with rate of progression of disease that is consistent in two independent series . Our analyses suggest that genetic variants associated with CSF ptau181 levels may have a greater impact on rate of progression, while genetic variants such as APOE4, that are associated with CSF Aβ42 levels influence risk and onset but not the rate of progression. Our results also suggest that drugs that inhibit or decrease tau phosphorylation may slow cognitive decline in individuals with very mild dementia or delay the appearance of memory problems in elderly individuals with low CSF Aβ42 levels. Finally, we believe genome-wide association studies of CSF tau/ptau181 levels should identify novel genetic variants which will likely influence rate of progression of AD