Xiaopeng Li

Transepithelial migration of neutrophils into the lung requires TREM-1

Acute respiratory infections are responsible for more than 4 million deaths each year. Neutrophils play an essential role in the innate immune response to lung infection. These cells have an armamentarium of pattern recognition molecules and antimicrobial agents that identify and eliminate pathogens. In the setting of infection, neutrophil triggering receptor expressed on myeloid cells 1 (TREM-1) amplifies inflammatory signaling. Here we demonstrate for the first time that TREM-1 also plays an important role in transepithelial migration of neutrophils into the airspace. We developed a TREM-1/3-deficient mouse model of pneumonia and found that absence of TREM-1/3 markedly increased mortality following Pseudomonas aeruginosa challenge. Unexpectedly, TREM-1/3 deficiency resulted in increased local and systemic cytokine production. TREM-1/3-deficient neutrophils demonstrated intact bacterial killing, phagocytosis, and chemotaxis; however, histologic examination of TREM-1/3-deficient lungs revealed decreased neutrophil infiltration of the airways. TREM-1/3-deficient neutrophils effectively migrated across primary endothelial cell monolayers but failed to migrate across primary airway epithelia grown at the air-liquid interface. These data define a new function for TREM-1 in neutrophil migration across airway epithelial cells and suggest that it amplifies inflammation through targeted neutrophil migration into the lung.

Cystic fibrosis transmembrane conductance regulator in sarcoplasmic reticulum of airway smooth muscle: Implications for airway contractility

RATIONALEnAn asthma-like airway phenotype has been described in people with cystic fibrosis (CF). Whether these findings are directly caused by loss of CF transmembrane conductance regulator (CFTR) function or secondary to chronic airway infection and/or inflammation has been difficult to determine.nnnOBJECTIVESnAirway contractility is primarily determined by airway smooth muscle. We tested the hypothesis that CFTR is expressed in airway smooth muscle and directly affects airway smooth muscle contractility.nnnMETHODSnNewborn pigs, both wild type and with CF (before the onset of airway infection and inflammation), were used in this study. High-resolution immunofluorescence was used to identify the subcellular localization of CFTR in airway smooth muscle. Airway smooth muscle function was determined with tissue myography, intracellular calcium measurements, and regulatory myosin light chain phosphorylation status. Precision-cut lung slices were used to investigate the therapeutic potential of CFTR modulation on airway reactivity.nnnMEASUREMENTS AND MAIN RESULTSnWe found that CFTR localizes to the sarcoplasmic reticulum compartment of airway smooth muscle and regulates airway smooth muscle tone. Loss of CFTR function led to delayed calcium reuptake following cholinergic stimulation and increased myosin light chain phosphorylation. CFTR potentiation with ivacaftor decreased airway reactivity in precision-cut lung slices following cholinergic stimulation.nnnCONCLUSIONSnLoss of CFTR alters porcine airway smooth muscle function and may contribute to the airflow obstruction phenotype observed in human CF. Airway smooth muscle CFTR may represent a therapeutic target in CF and other diseases of airway narrowing.

CFTR is required for maximal transepithelial liquid transport in pig alveolar epithelia

A balance between alveolar liquid absorption and secretion is critical for maintaining optimal alveolar subphase liquid height and facilitating gas exchange in the alveolar space. However, the role of cystic fibrosis transmembrane regulator protein (CFTR) in this homeostatic process has remained elusive. Using a newly developed porcine model of cystic fibrosis, in which CFTR is absent, we investigated ion transport properties and alveolar liquid transport in isolated type II alveolar epithelial cells (T2AECs) cultured at the air-liquid interface. CFTR was distributed exclusively to the apical surface of cultured T2AECs. Alveolar epithelia from CFTR(-/-) pigs failed to increase liquid absorption in response to agents that increase cAMP, whereas cAMP-stimulated liquid absorption in CFTR(+/-) epithelia was similar to that in CFTR(+/+) epithelia. Expression of recombinant CFTR restored stimulated liquid absorption in CFTR(-/-) T2AECs but had no effect on CFTR(+/+) epithelia. In ex vivo studies of nonperfused lungs, stimulated liquid absorption was defective in CFTR(-/-) alveolar epithelia but similar between CFTR(+/+) and CFTR(+/-) epithelia. When epithelia were studied at the air-liquid interface, elevating cAMP levels increased subphase liquid height in CFTR(+/+) but not in CFTR(-/-) T2AECs. Our findings demonstrate that CFTR is required for maximal liquid absorption under cAMP stimulation, but it is not the rate-limiting factor. Furthermore, our data define a role for CFTR in liquid secretion by T2AECs. These insights may help to develop new treatment strategies for pulmonary edema and respiratory distress syndrome, diseases in which lung liquid transport is disrupted.

CFTR gene transfer with AAV improves early cystic fibrosis pig phenotypes

The physiological components that contribute to cystic fibrosis (CF) lung disease are steadily being elucidated. Gene therapy could potentially correct these defects. CFTR-null pigs provide a relevant model to test gene therapy vectors. Using an in vivo selection strategy that amplifies successful capsids by replicating their genomes with helper adenovirus coinfection, we selected an adeno-associated virus (AAV) with tropism for pig airway epithelia. The evolved capsid, termed AAV2H22, is based on AAV2 with 5 point mutations that result in a 240-fold increased infection efficiency. In contrast to AAV2, AAV2H22 binds specifically to pig airway epithelia and is less reliant on heparan sulfate for transduction. We administer AAV2H22-CFTR expressing the CF transmembrane conductance regulator (CFTR) cDNA to the airways of CF pigs. The transduced airways expressed CFTR on ciliated and nonciliated cells, induced anion transport, and improved the airway surface liquid pH and bacterial killing. Most gene therapy studies to date focus solely on Cl- transport as the primary metric of phenotypic correction. Here, we describe a gene therapy experiment where we not only correct defective anion transport, but also restore bacterial killing in CFTR-null pig airways.

Electrolyte transport properties in distal small airways from cystic fibrosis pigs with implications for host defense

While pathological and clinical data suggest that small airways are involved in early cystic fibrosis (CF) lung disease development, little is known about how the lack of cystic fibrosis transmembrane conductance regulator (CFTR) function contributes to disease pathogenesis in these small airways. Large and small airway epithelia are exposed to different airflow velocities, temperatures, humidity, and CO2 concentrations. The cellular composition of these two regions is different, and small airways lack submucosal glands. To better understand the ion transport properties and impacts of lack of CFTR function on host defense function in small airways, we adapted a novel protocol to isolate small airway epithelial cells from CF and non-CF pigs and established an organotypic culture model. Compared with non-CF large airways, non-CF small airway epithelia cultures had higher Cl(-) and bicarbonate (HCO3 (-)) short-circuit currents and higher airway surface liquid (ASL) pH under 5% CO2 conditions. CF small airway epithelia were characterized by minimal Cl(-) and HCO3 (-) transport and decreased ASL pH, and had impaired bacterial killing compared with non-CF small airways. In addition, CF small airway epithelia had a higher ASL viscosity than non-CF small airways. Thus, the activity of CFTR is higher in the small airways, where it plays a role in alkalinization of ASL, enhancement of antimicrobial activity, and lowering of mucus viscosity. These data provide insight to explain why the small airways are a susceptible site for the bacterial colonization.

Integrin α6β4 identifies human distal lung epithelial progenitor cells with potential as a cell-based therapy for cystic fibrosis lung disease

To develop stem/progenitor cell-based therapy for cystic fibrosis (CF) lung disease, it is first necessary to identify markers of human lung epithelial progenitor/stem cells and to better understand the potential for differentiation into distinct lineages. Here we investigated integrin α6β4 as an epithelial progenitor cell marker in the human distal lung. We identified a subpopulation of α6β4+ cells that localized in distal small airways and alveolar walls and were devoid of pro-surfactant protein C expression. The α6β4+ epithelial cells demonstrated key properties of stem cells ex vivo as compared to α6β4- epithelial cells, including higher colony forming efficiency, expression of stem cell-specific transcription factor Nanog, and the potential to differentiate into multiple distinct lineages including basal and Clara cells. Co-culture of α6β4+ epithelial cells with endothelial cells enhanced proliferation. We identified a subset of adeno-associated virus (AAVs) serotypes, AAV2 and AAV8, capable of transducing α6β4+ cells. In addition, reconstitution of bronchi epithelial cells from CF patients with only 5% normal α6β4+ epithelial cells significantly rescued defects in Cl- transport. Therefore, targeting the α6β4+ epithelial population via either gene delivery or progenitor cell-based reconstitution represents a potential new strategy to treat CF lung disease.

Nominal carbonic anhydrase activity minimizes airway‐surface liquid pH changes during breathing

The airway‐surface liquid pH (pHASL) is slightly acidic relative to the plasma and becomes more acidic in airway diseases, leading to impaired host defense. CO2 in the large airways decreases during inspiration (0.04% CO2) and increases during expiration (5% CO2). Thus, we hypothesized that pHASL would fluctuate during the respiratory cycle. We measured pHASL on cultures of airway epithelia while changing apical CO2 concentrations. Changing apical CO2 produced only very slow pHASL changes, occurring in minutes, inconsistent with respiratory phases that occur in a few seconds. We hypothesized that pH changes were slow because airway‐surface liquid has little carbonic anhydrase activity. To test this hypothesis, we applied the carbonic anhydrase inhibitor acetazolamide and found minimal effects on CO2‐induced pHASL changes. In contrast, adding carbonic anhydrase significantly increased the rate of change in pHASL. Using pH‐dependent rates obtained from these experiments, we modeled the pHASL during respiration to further understand how pH changes with physiologic and pathophysiologic respiratory cycles. Modeled pHASL oscillations were small and affected by the respiration rate, but not the inspiratory:expiratory ratio. Modeled equilibrium pHASL was affected by the inspiratory:expiratory ratio, but not the respiration rate. The airway epithelium is the only tissue that is exposed to large and rapid CO2 fluctuations. We speculate that the airways may have evolved minimal carbonic anhydrase activity to mitigate large changes in the pHASL during breathing that could potentially affect pH‐sensitive components of ASL.

Development of a polarized pancreatic ductular cell epithelium for physiological studies

Pancreatic ductular epithelial cells comprise the majority of duct cells in pancreas, control cystic fibrosis transmembrane conductance regulator (CFTR)-dependent bicarbonate ([Formula: see text]) secretion, but are difficult to grow as a polarized monolayer. Using NIH-3T3-J2 fibroblast feeder cells and a Rho-associated kinase inhibitor, we produced well-differentiated and polarized porcine pancreatic ductular epithelial cells. Cells grown on semipermeable filters at the air-liquid interface developed typical epithelial cell morphology and stable transepithelial resistance and expressed epithelial cell markers (zona occludens-1 and β-catenin), duct cell markers (SOX-9 and CFTR), but no acinar (amylase) or islet cell (chromogranin) markers. Polarized cells were studied in Ussing chambers bathed in Krebs-Ringer [Formula: see text] solution at 37°C gassed with 5% CO2 to measure short-circuit currents ( Isc). Ratiometric measurement of extracellular pH was performed with fluorescent SNARF-conjugated dextran at 5% CO2. Cells demonstrated a baseline Isc (12.2u2009±u20093.2 μA/cm2) that increased significantly in response to apical forskolin-IBMX (∆ Isc: 35.4u2009±u20093.8 μA/cm2, P < 0.001) or basolateral secretin (∆ Isc: 31.4u2009±u20092.5 μA/cm2, P < 0.001), both of which increase cellular levels of cAMP. Subsequent addition of apical GlyH-101, a CFTR inhibitor, decreased the current (∆ Isc: 20.4u2009±u20093.8 μA/cm2, P < 0.01). Extracellular pH and [Formula: see text] concentration increased significantly after forskolin-IBMX (pH: 7.18u2009±u20090.23 vs. 7.53u2009±u20090.19; [Formula: see text] concentration, 14.5u2009±u20095.9 vs. 31.8u2009±u200913.4 mM; P < 0.05 for both). We demonstrate the development of a polarized pancreatic ductular epithelial cell epithelium with CFTR-dependent [Formula: see text] secretion in response to secretin and cAMP. This model is highly relevant, as porcine pancreas physiology is very similar to humans and pancreatic damage in the cystic fibrosis pig model recapitulates that of humans. NEW & NOTEWORTHY Pancreas ductular epithelial cells control cystic fibrosis transmembrane conductance regulator (CFTR)-dependent bicarbonate secretion. Their function is critical because when CFTR is deficient in cystic fibrosis bicarbonate secretion is lost and the pancreas is damaged. Mechanisms that control pancreatic bicarbonate secretion are incompletely understood. We generated well-differentiated and polarized porcine pancreatic ductular epithelial cells and demonstrated feasibility of bicarbonate secretion. This novel method will advance our understanding of pancreas physiology and mechanisms of bicarbonate secretion.