Diabetes mellitus predisposes the host to bacterial infections. Moreover, hyperglycemia has been shown to be an independent risk factor for respiratory infections. The luminal surface of airway ...epithelia is covered by a thin layer of airway surface liquid (ASL) and is normally sterile despite constant exposure to bacteria. The balance between bacterial growth and killing in the airway determines the outcome of exposure to inhaled or aspirated bacteria: infection or sterility. We hypothesized that restriction of carbon sources--including glucose--in the ASL is required for sterility of the lungs. We found that airway epithelia deplete glucose from the ASL via a novel mechanism involving polarized expression of GLUT-1 and GLUT-10, intracellular glucose phosphorylation, and low relative paracellular glucose permeability in well-differentiated cultures of human airway epithelia and in segments of airway epithelia excised from human tracheas. Moreover, we found that increased glucose concentration in the ASL augments growth of P. aeruginosa in vitro and in the lungs of hyperglycemic ob/ob and db/db mice in vivo. In contrast, hyperglycemia had no effect on intrapulmonary bacterial growth of a P. aeruginosa mutant that is unable to utilize glucose as a carbon source. Our data suggest that depletion of glucose in the airway epithelial surface is a novel mechanism for innate immunity. This mechanism is important for sterility of the airways and has implications in hyperglycemia and conditions that result in disruption of the epithelial barrier in the lung.
Smoking is a leading cause of respiratory infections worldwide. Tobacco particulate matter disrupts iron homeostasis in the lungs and increases the iron content in the airways of smokers. The airway ...epithelia secrete lactoferrin to quench iron required for bacteria to proliferate and cause lung infections. We hypothesized that smokers would have increased bacterial growth and biofilm formation via iron lactoferrin imbalance.
We collected bronchoalveolar lavage (BAL) samples from non-smokers and smokers. We challenged these samples using a standard inoculum of Staphylococcus aureus and Pseudomonas aeruginosa and quantified bacterial growth and biofilm formation. We measured both iron and lactoferrin in the samples. We investigated the effect of supplementing non-smoker BAL with cigarette smoke extract (CSE) or ferric chloride and the effect of supplementing smoker BAL with lactoferrin on bacterial growth and biofilm formation.
BAL from smokers had increased bacterial growth and biofilm formation compared to non-smokers after both S. aureus and P. aeruginosa challenge. In addition, we found that samples from smokers had a higher iron to lactoferrin ratio. Supplementing the BAL of non-smokers with cigarette smoke extract and ferric chloride increased bacterial growth. Conversely, supplementing the BAL of smokers with lactoferrin had a concentration-dependent decrease in bacterial growth and biofilm formation.
Cigarette smoking produces factors which increase bacterial growth and biofilm formation in the BAL. We propose that smoking disrupts the iron-to-lactoferrin in the airways. This finding offers a new avenue for potential therapeutic interventions to prevent respiratory infections in smokers.
The pathogenic bacterium
Pseudomonas aeruginosa uses acyl-HSL quorum-sensing signals to regulate genes controlling virulence and biofilm formation. We found that paraoxonase 1 (PON1), a mammalian ...lactonase with an unknown natural substrate, hydrolyzed the
P. aeruginosa acyl-HSL 3OC12-HSL. In in vitro assays, mouse serum-PON1 was required and sufficient to degrade 3OC12-HSL. Furthermore, PON2 and PON3 also degraded 3OC12-HSL effectively. Serum-PON1 prevented
P. aeruginosa quorum-sensing and biofilm formation in vitro by inactivating the quorum-sensing signal. Although 3OC12-HSL production by
P. aeruginosa was important for virulence in a mouse sepsis model,
Pon1-knock-out mice were paradoxically protected. These mice showed increased levels of PON2 and PON3 mRNA in epithelial tissues suggesting a possible compensatory mechanism. Thus, paraoxonase interruption of bacterial communication represents a novel mechanism to modulate quorum-sensing by bacteria. The consequences for host immunity are yet to be determined.
People with cystic fibrosis (CF) exhibit growth defects. That observation has been attributed, in part, to decreased insulin-like growth factor 1 (IGF1) levels, and the reduction has been blamed on ...malnutrition and pulmonary inflammation. However, patients with CF already have a reduced weight at birth, a manifestation not likely secondary to poor nutrition or inflammation. We found that, like humans, CF pigs were smaller than non-CF littermates and had lower IGF1 levels. To better understand the basis of IGF1 reduction, we studied newborn pigs and found low IGF1 levels within 12 h of birth. Moreover, humerus length and bone mineral content were decreased, consistent with less IGF1 activity in utero. These findings led us to test newborn humans with CF, and we found that they also had reduced IGF1 levels. Discovering lower IGF1 levels in newborn pigs and humans indicates that the decrease is not solely a consequence of malnutrition or pulmonary inflammation and that loss of cystic fibrosis transmembrane conductance regulator function has a more direct effect. Consistent with this hypothesis, we discovered reduced growth hormone release in organotypic pituitary slice cultures of newborn CF pigs. These findings may explain the long-standing observation that CF newborns are smaller than non-CF babies and why some patients with good clinical status fail to reach their growth potential. The results also suggest that measuring IGF1 levels might be of value as a biomarker to predict disease severity or the response to therapeutics. Finally, they raise the possibility that IGF1 supplementation beginning in infancy might be beneficial in CF.
Interactions between ligands and receptors are central to communication between cells and tissues. Human airway epithelia constitutively produce both a ligand, the growth factor heregulin, and its ...receptors-erbB2, erbB3 and erbB4 (refs 1-3). Although heregulin binding initiates cellular proliferation and differentiation, airway epithelia have a low rate of cell division. This raises the question of how ligand-receptor interactions are controlled in epithelia. Here we show that in differentiated human airway epithelia, heregulin-α is present exclusively in the apical membrane and the overlying airway surface liquid, physically separated from erbB2-4, which segregate to the basolateral membrane. This physical arrangement creates a ligand-receptor pair poised for activation whenever epithelial integrity is disrupted. Indeed, immediately following a mechanical injury, heregulin-α activates erbB2 in cells at the edge of the wound, and this process hastens restoration of epithelial integrity. Likewise, when epithelial cells are not separated into apical and basolateral membranes ('polarized'), or when tight junctions between adjacent cells are opened, heregulin-α activates its receptor. This mechanism of ligand-receptor segregation on either side of epithelial tight junctions may be vital for rapid restoration of integrity following injury, and hence critical for survival. This model also suggests a mechanism for abnormal receptor activation in diseases with increased epithelial permeability.
Recombinant adeno-associated virus vectors based on serotype 2 (rAAV2) can direct transgene expression in the central nervous system (CNS), but it is not known how other rAAV serotypes perform as CNS ...gene transfer vectors. Serotypes 4 and 5 are distinct from rAAV2 and from each other in their capsid regions, suggesting that they may direct binding and entry into different cell types. In this study, we examined the tropisms and transduction efficiencies of β -galactosidase-encoding vectors made from rAAV4 and rAAV5 compared with similarly designed rAAV2-based vectors. Injection of rAAV5 β -galactosidase (β gal) or rAAV4β gal into the lateral ventricle resulted in stable transduction of ependymal cells, with approximately 10-fold more positive cells than in mice injected with rAAV2β gal. Major differences between the three vectors were revealed upon striatal injections. Intrastriatal injection of rAAV4β gal resulted again in striking ependyma-specific expression of transgene, with a notable absence of transduced cells in the parenchyma. rAAV2β gal and rAAV5β gal intrastriatal injections led to β -gal-positive parenchymal cells, but, unlike rAAV2β gal, rAAV5β gal transduced both neurons and astrocytes. The number of transgene-positive cells in rAAV5β gal-injected brains was 130 and 5,000 times higher than in rAAV2β gal-injected brains at 3 and 15 wk, respectively. Moreover, transgene-positive cells were widely dispersed throughout the injected hemisphere in rAAV5β gal-transduced animals. Together, our data provide in vivo support for earlier in vitro work, suggesting that rAAV4 and rAAV5 gain cell entry by means of receptors distinct from rAAV2. These differences could be exploited to improve gene therapy for CNS disorders.
A recently discovered enzyme system produces antibacterial hypothiocyanite (OSCN−) in the airway lumen by oxidizing the secreted precursor thiocyanate (SCN−). Airway epithelial cultures have been ...shown to secrete SCN− in a CFTR-dependent manner. Thus, reduced SCN− availability in the airway might contribute to the pathogenesis of cystic fibrosis (CF), a disease caused by mutations in the CFTR gene and characterized by an airway host defense defect. We tested this hypothesis by analyzing the SCN− concentration in the nasal airway surface liquid (ASL) of CF patients and non-CF subjects and in the tracheobronchial ASL of CFTR-ΔF508 homozygous pigs and control littermates. In the nasal ASL, the SCN− concentration was ~30-fold higher than in serum independent of the CFTR mutation status of the human subject. In the tracheobronchial ASL of CF pigs, the SCN− concentration was somewhat reduced. Among human subjects, SCN− concentrations in the ASL varied from person to person independent of CFTR expression, and CF patients with high SCN− levels had better lung function than those with low SCN− levels. Thus, although CFTR can contribute to SCN− transport, it is not indispensable for the high SCN− concentration in ASL. The correlation between lung function and SCN− concentration in CF patients may reflect a beneficial role for SCN−.
It is widely unknown why respiratory infections follow a seasonal pattern. Variations in ultraviolet B (UVB) light during seasons affects cutaneous synthesis of vitamin D
. Serum vitamin D ...concentration influences the expression of airway surface liquid (ASL) antimicrobial peptides such as LL-37.
We sought to determine the effect of seasons on serum vitamin D levels and ASL antimicrobial activity.
Forty participants, 18-60 years old, were randomized 1:1 to receive 90 days of 1000 IU vitamin D
or placebo. We collected ASL via bronchoscopy and measured serum 25(OH) vitamin D from participants before and after intervention across seasons. We measured ASL antimicrobial activity by challenging samples with bioluminescent
and measured relative light units (RLUs) after four minutes. We also investigated the role of LL-37 using a monoclonal neutralizing antibody.
We found that participants, prior to any intervention, during summer-fall (
= 20) compared to winter-spring (
= 20) had (1) decreased live bacteria after challenge (5542 ± 175.2 vs. 6585 ± 279 RLU,
= 0.003) and (2) higher serum vitamin D (88.25 ± 24.25 vs. 67.5 ± 45.25 nmol/L,
= 0.026). Supplementation with vitamin D
increased vitamin D levels and restored ASL antimicrobial activity only during the winter-spring. The increased ASL antimicrobial activity seen during the summer-fall was abrogated by adding the LL-37 neutralizing antibody.
ASL kills bacteria more effectively during the summer-fall compared to the winter-spring. Supplementation of vitamin D during winter-spring restores ASL antimicrobial activity by increasing the expression of antimicrobial peptides including LL-37.
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