Glucose Depletion in the Airway Surface Liquid Is Essential for Sterility of the Airways

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

Eicosanoid signalling blockade protects middle-aged mice from severe COVID-19

Coronavirus disease 2019 (COVID-19) is especially severe in aged populations1. Vaccines against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) are highly effective, but vaccine efficacy is partly compromised by the emergence of SARS-CoV-2 variants with enhanced transmissibility2. The emergence of these variants emphasizes the need for further development of anti-SARS-CoV-2 therapies, especially for aged populations. Here we describe the isolation of highly virulent mouse-adapted viruses and use them to test a new therapeutic drug in infected aged animals. Many of the alterations observed in SARS-CoV-2 during mouse adaptation (positions 417, 484, 493, 498 and 501 of the spike protein) also arise in humans in variants of concern2. Their appearance during mouse adaptation indicates that immune pressure is not required for selection. For murine SARS, for which severity is also age dependent, elevated levels of an eicosanoid (prostaglandin D2 (PGD2)) and a phospholipase (phospholipase A2 group 2D (PLA2G2D)) contributed to poor outcomes in aged mice3,4. mRNA expression of PLA2G2D and prostaglandin D2 receptor (PTGDR), and production of PGD2 also increase with ageing and after SARS-CoV-2 infection in dendritic cells derived from human peripheral blood mononuclear cells. Using our mouse-adapted SARS-CoV-2, we show that middle-aged mice lacking expression of PTGDR or PLA2G2D are protected from severe disease. Furthermore, treatment with a PTGDR antagonist, asapiprant, protected aged mice from lethal infection. PTGDR antagonism is one of the first interventions in SARS-CoV-2-infected animals that specifically protects aged animals, suggesting that the PLA2G2D–PGD2/PTGDR pathway is a useful target for therapeutic interventions.This work is supported in part by grants from the National Institutes of Health USA (NIH; P01 AI060699 (S.P. and P.B.M.) and R01 AI129269 (S.P.)) and BIOAGE Labs (S.P.). The Pathology Core is partially supported by the Center for Gene Therapy for Cystic Fibrosis (NIH grant P30 DK-54759) and the Cystic Fibrosis Foundation. P.B.M. is supported by the Roy J. Carver Charitable Trust. L.-Y.R.W. is supported by Mechanism of Parasitism Training Grant (T32 AI007511). We thank M. Gelb (University of Washington) for Pla2g2d−/− mice.Peer reviewe

CFA inhibits antimicrobial peptide activity.

<p><b>Panel A.</b> PA01 in the presence of FeCl₃ (25 µM) and three different types of CFA (FA 2689, FA 2690 and FA 2691) increased growth at 10 µg/mL without AMP cocktail more than control, however the growth increase is not statistically significant. <b>Panel B.</b> PA01 growth in the presence of AMP cocktail (600 µg/mL Lysozyme, 200 µg/mL Lactoferrin and 100 ng/mL β-Defensin 1&2) inhibits PA01 growth. FeCl₃ (25 µM) and FA 2690 (10 µg/mL) inhibit AMP activity *p≤0.05, **p<0.0001. <b>Panel C</b>. CFU count of PA01 after 18 hours in the presence of AMPs. N = 3 in triplicates. SEM reported.</p

CFA increases bacterial grown <i>in vivo</i>.

<p><b>Panel A.</b> Total mouse lung sterility <i>in vivo</i>. After 24 hours, CFUs were determined after homogenizing lungs and plating to determine growth. In the presence of CFA, lung sterility was decreased by 20–30% **p<0.01, ***p<0.0001. PA01 alone N = 26, PA01+ FA 2689 N = 22, PA01+ FA 2690 N = 21, and PA01+ FA 2691 N = 24. <b>Panel B.</b> Growth in non-sterile mice. Log CFU/mouse was determined. FA 2689 and FA 2690 increased growth more than control (p<0.05).</p

CFA increases PA01 growth.

<p>Subcultured PA01 was grown in M9 with FeCl₃ (25 µM), three different CFA particles (10 µg/mL) or no particles (CT). Growth was recorded over nine hours. CFA increased growth more than CT (p<0.0001 for all three CFAs)_. FA 2691 increased PA01 growth more than FA 2689 or FA 2690 ***<i>p</i><0.0001. N = 3 in triplicates.</p

Coal Fly Ash Particles.

<p>Total iron content, aluminum silicate content, dissolved iron, particle size, and specific surface area of three different coal fly ash particles (FA 2689, FA 2690 and FA 2691).</p><p>XPS was used to determine surface composition.</p><p>EDX was used to determine bulk composition.</p>a<p>XPS/EDX Ratio: High ratio of XPS/EDX indicates elemental enrichment at the surface, low ratio content (<1) indicates enrichment of element at inner core.</p

Neonates with cystic fibrosis have a reduced nasal liquid pH; A small pilot study

AbstractBackgroundDisrupted HCO3– transport and reduced airway surface liquid (ASL) pH in cystic fibrosis (CF) may initiate airway disease. We hypothesized that ASL pH is reduced in neonates with CF.MethodsIn neonates with and without CF, we measured pH of nasal ASL. We also measured nasal pH in older children and adults.ResultsIn neonates with CF, nasal ASL (pH5.2±0.3) was more acidic than in non-CF neonates (pH6.4±0.2). In contrast, nasal pH of CF children and adults was similar to values measured in people without CF.ConclusionsAt an age when infection, inflammation and airway wall remodeling are minimal, neonates with CF had an acidic nasal ASL compared to babies without CF. The CF:non-CF pH difference disappeared in older individuals, perhaps because secondary manifestations of disease increase ASL pH. These results aid understanding of CF pathogenesis and suggest opportunities for therapeutic intervention and monitoring of disease

CFA increases PA01 growth in isolated human airway epithelia.

<p><b>Panel A.</b> After 24 hours, PA01 (12 CFU) growth in the presence of CFA (10 µg/mL) on isolated human airway epithelia was measured. CFA increases PA01 growth in cell culture. FA 2689 and FA 2691 increased PA01 growth more than CT by 11% and FA 2690 increased PA01 growth 22% **p<0.0001. <b>Panel B.</b> Hydrogen peroxide (H₂O₂) production in the presence of PA01 and CFA does not significantly increase more than PA01 alone. <b>Panel C.</b> Transepithelial electrical conductance (<i>Gt</i>) across primary human airway epithelia does not significantly decrease in the presence of PA01 and CFA when compared with PA01 alone. N = 3 in triplicates from two different human donors.</p