James D. Londino

Inhibition of Lung Fluid Clearance and Epithelial Na+ Channels by Chlorine, Hypochlorous Acid, and Chloramines

We investigated the mechanisms by which chlorine (Cl2) and its reactive byproducts inhibit Na+-dependent alveolar fluid clearance (AFC) in vivo and the activity of amiloride-sensitive epithelial Na+ channels (ENaC) by measuring AFC in mice exposed to Cl2 (0–500 ppm for 30 min) and Na+ and amiloride-sensitive currents (INa and Iamil, respectively) across Xenopus oocytes expressing human α-, β-, and γ-ENaC incubated with HOCl (1–2000 μm). Both Cl2 and HOCl-derived products decreased AFC in mice and whole cell and single channel INa in a dose-dependent manner; these effects were counteracted by serine proteases. Mass spectrometry analysis of the oocyte recording medium identified organic chloramines formed by the interaction of HOCl with HEPES (used as an extracellular buffer). In addition, chloramines formed by the interaction of HOCl with taurine or glycine decreased INa in a similar fashion. Preincubation of oocytes with serine proteases prevented the decrease of INa by HOCl, whereas perfusion of oocytes with a synthetic 51-mer peptide corresponding to the putative furin and plasmin cleaving segment in the γ-ENaC subunit restored the ability of HOCl to inhibit INa. Finally, INa of oocytes expressing wild type α- and γ-ENaC and a mutant form of βENaC (S520K), known to result in ENaC channels locked in the open position, were not altered by HOCl. We concluded that HOCl and its reactive intermediates (such as organic chloramines) inhibit ENaC by affecting channel gating, which could be relieved by proteases cleavage.

Influenza matrix protein 2 alters CFTR expression and function through its ion channel activity

The human cystic fibrosis transmembrane conductance regulator (CFTR) is a cyclic AMP-activated chloride (Cl(-)) channel in the lung epithelium that helps regulate the thickness and composition of the lung epithelial lining fluid. We investigated whether influenza M2 protein, a pH-activated proton (H(+)) channel that traffics to the plasma membrane of infected cells, altered CFTR expression and function. M2 decreased CFTR activity in 1) Xenopus oocytes injected with human CFTR, 2) epithelial cells (HEK-293) stably transfected with CFTR, and 3) human bronchial epithelial cells (16HBE14o-) expressing native CFTR. This inhibition was partially reversed by an inhibitor of the ubiquitin-activating enzyme E1. Next we investigated whether the M2 inhibition of CFTR activity was due to an increase of secretory organelle pH by M2. Incubation of Xenopus oocytes expressing CFTR with ammonium chloride or concanamycin A, two agents that alkalinize the secretory pathway, inhibited CFTR activity in a dose-dependent manner. Treatment of M2- and CFTR-expressing oocytes with the M2 ion channel inhibitor amantadine prevented the loss in CFTR expression and activity; in addition, M2 mutants, lacking the ability to transport H(+), did not alter CFTR activity in Xenopus oocytes and HEK cells. Expression of an M2 mutant retained in the endoplasmic reticulum also failed to alter CFTR activity. In summary, our data show that M2 decreases CFTR activity by increasing secretory organelle pH, which targets CFTR for destruction by the ubiquitin system. Alteration of CFTR activity has important consequences for fluid regulation and may potentially modify the immune response to viral infection.

Influenza virus M2 targets cystic fibrosis transmembrane conductance regulator for lysosomal degradation during viral infection

We sought to determine the mechanisms by which influenza infection of human epithelial cells decreases cystic fibrosis transmembrane conductance regulator (CFTR) expression and function. We infected human bronchial epithelial (NHBE) cells and murine nasal epithelial (MNE) cells with various strains of influenza A virus. Influenza infection significantly reduced CFTR short circuit currents (Isc) and protein levels at 8 hours postinfection. We then infected CFTR expressing human embryonic kidney (HEK)‐293 cells (HEK‐293 CFTRwt) with influenza virus encoding a green fluorescent protein (GFP) tag and performed whole‐cell and cell‐attached patch clamp recordings. Forskolin‐stimulated, GlyH‐101‐sensitive CFTR conductances, and CFTR open probabilities were reduced by 80% in GFP‐positive cells; Western blots also showed significant reduction in total and plasma membrane CFTR levels. Knockdown of the influenza matrix protein 2 (M2) with siRNA, or inhibition of its activity by amantadine, prevented the decrease in CFTR expression and function. Lysosome inhibition (bafilomycin‐A1), but not proteasome inhibition (lactacystin), prevented the reduction in CFTR levels. Western blots of immunoprecipitated CFTR from influenza‐infected cells, treated with BafA1, and probed with antibodies against lysine 63‐linked (K‐63) or lysine 48‐linked (K‐48) polyubiquitin chains supported lysosomal targeting. These results highlight CFTR damage, leading to early degradation as an important contributing factor to influenza infection‐associated ion transport defects.—Londino, J. D., Lazrak, A., Noah, J. W., Aggarwal, S., Bali, V., Woodworth, B. A., Bebok, Z., Matalon, S. Influenza virus M2 targets cystic fibrosis transmembrane conductance regulator for lysosomal degradation during viral infection. FASEB J. 29, 2712–2725 (2015). www.fasebj.org

Cleavage of Signal Regulatory Protein α (SIRPα) Enhances Inflammatory Signaling.

Signal regulatory protein α (SIRPα) is a membrane glycoprotein immunoreceptor abundant in cells of monocyte lineage. SIRPα ligation by a broadly expressed transmembrane protein, CD47, results in phosphorylation of the cytoplasmic immunoreceptor tyrosine-based inhibitory motifs, resulting in the inhibition of NF-κB signaling in macrophages. Here we observed that proteolysis of SIRPα during inflammation is regulated by a disintegrin and metalloproteinase domain-containing protein 10 (ADAM10), resulting in the generation of a membrane-associated cleavage fragment in both THP-1 monocytes and human lung epithelia. We mapped a charge-dependent putative cleavage site near the membrane-proximal domain necessary for ADAM10-mediated cleavage. In addition, a secondary proteolytic cleavage within the membrane-associated SIRPα fragment by γ-secretase was identified. Ectopic expression of a SIRPα mutant plasmid encoding a proteolytically resistant form in HeLa cells inhibited activation of the NF-κB pathway and suppressed STAT1 phosphorylation in response to TNFα to a greater extent than expression of wild-type SIRPα. Conversely, overexpression of plasmids encoding the proteolytically cleaved SIRPα fragments in cells resulted in enhanced STAT-1 and NF-κB pathway activation. Thus, the data suggest that combinatorial actions of ADAM10 and γ-secretase on SIRPα cleavage promote inflammatory signaling.

Chloride secretion across adult alveolar epithelial cells contributes to cardiogenic edema

In utero, fetal lung epithelial cells actively secrete chloride (Cl−) ions into the lung airspaces. Cl− ions enter the basolateral membranes through Na+/K+/2Cl− (NKCC) transporters (1), down an electrochemical gradient generated by the basolateral Na+/K+/ATPase, and exit through apical anion channels, including the cAMP- activated cystic fibrosis transmembrane regulator (CFTR) (2). Na+ ions follow passively through the paracellular junctions to preserve electroneutrality. The vectorial transport of NaCl generates an oncotic pressure, which drives fetal fluid secretion into the airway lumen. Shortly before birth, Cl− secretion diminishes and active Na+ transport begins: Na+ ions enter lung epithelial cells through amiloride-sensitive Na+ channels (ENaC) and are extruded across the basolateral membranes by the Na+/K+/ATPase (3, 4). This process is critical for the reabsorption of fetal lung fluid and the establishment of gas exchange. In the adult lung active Na+ reabsorption limits the degree of alveolar edema in hyperoxic lung injury (5) and in patients with acute respiratory distress syndrome (6, 7).

Targeting the deubiquitinase STAMBP inhibits NALP7 inflammasome activity

Inflammasomes regulate innate immune responses by facilitating maturation of inflammatory cytokines, interleukin (IL)-1β and IL-18. NACHT, LRR and PYD domains-containing protein 7 (NALP7) is one inflammasome constituent, but little is known about its cellular handling. Here we show a mechanism for NALP7 protein stabilization and activation of the inflammasome by Toll-like receptor (TLR) agonism with bacterial lipopolysaccharide (LPS) and the synthetic acylated lipopeptide Pam3CSK4. NALP7 is constitutively ubiquitinated and recruited to the endolysosome for degradation. With TLR ligation, the deubiquitinase enzyme, STAM-binding protein (STAMBP) impedes NALP7 trafficking to lysosomes to increase NALP7 abundance. STAMBP deubiquitinates NALP7 and STAMBP knockdown abrogates LPS or Pam3CSK4-induced increases in NALP7 protein. A small-molecule inhibitor of STAMBP deubiquitinase activity, BC-1471, decreases NALP7 protein levels and suppresses IL-1β release after TLR agonism. These findings describe a unique pathway of inflammasome regulation with the identification of STAMBP as a potential therapeutic target to reduce pro-inflammatory stress.

IL-4 Induces IL17Rb Gene Transcription in Monocytic Cells with Coordinate Autocrine IL-25 Signaling

&NA; IL‐25 and IL‐4 signaling in the setting of infection or allergic responses can drive Type 2 inflammation. IL‐25 requires the IL‐17 receptor B (IL‐17Rb) to mediate signaling through nuclear factor &kgr; B (NF‐&kgr;B) transcriptional activation. Despite the known coexistence of these two cytokines in the Type 2 inflammatory environment, collaborative signaling between the IL‐4 and IL‐25 axes is poorly explored. Here we demonstrate IL‐4 induction of both IL‐25 and IL‐17Rb protein in human lung tissue culture, primary alveolar macrophages, and the THP‐1 monocytic cell line. IL‐4 treatment triggers gene transcription for both IL‐25 and IL‐17Rb but does not alter the receptor mRNA stability. Genetic antagonism of the IL‐4 second messenger, signal transducer and activator of transcription 6 (STAT6), with small interfering RNA (siRNA) blunts IL‐17Rb mRNA induction by IL‐4. IL‐25 induces signaling through the canonical NF‐&kgr;B pathway, and STAT6 or NF‐&kgr;B signaling inhibitors prevent IL‐17Rb expression. Blockade of IL‐25 with monoclonal antibody suppresses NF‐&kgr;B activation after IL‐4 treatment, and IL‐4‐mediated induction of IL‐17Rb is suppressed by IL‐25 siRNA. IL‐25 and IL‐17Rb promoter regions harbor putative NF‐&kgr;B and STAT6 consensus sites, and chromatin immunoprecipitation identified these transcription factors in complex with the IL‐17Rb 5′ untranslated region. In bronchoalveolar lavage RNA preparations, IL‐25 and IL‐17Rb mRNA transcripts are increased in asthmatics compared with healthy control subjects, and IL‐25 transcript abundance correlates strongly with IL‐4 mRNA levels. Thus, these results indicate that IL‐4 signaling up‐regulates the IL‐25 axis in human monocytic cells, and that IL‐25 may provide autocrine signals in monocytes and macrophages to sustain IL‐17Rb expression and predispose to alternative activation.

Regulation of Airway Lining Fluid in Health and Disease

Abstract Mucociliary clearance in the lung epithelium is a critical protective function and is essential for the clearance of respiratory pathogens. Maintenance of the periciliary layer requires the careful coordination of Na+ absorption through epithelial Na+ (ENaC) and cation channels and Cl− secretion through the cystic fibrosis transmembrane conductance regulator (CFTR). In cystic fibrosis (CF), defective CFTR expression and activity lead to chronic bacterial respiratory infections, airway obstruction, bronchiectasis, and respiratory failure. Two recent models, the Beta ENaC overexpressing mouse and the CF pig, have shed light on the underlying mechanisms that cause dysfunction in CF. Altered fluid regulation in the airway epithelium is consistently observed in many respiratory infections. Influenza infection can lead to respiratory symptoms such as pneumonia, pulmonary edema, and acute respiratory distress syndrome in severe cases. Alteration of ENaC by influenza occurs through protein kinase C activation initiated by hemagglutinin binding during virus attachment. Influenza also decreases fluid clearance in vivo by nucleotide/nucleoside signaling well before changes in lung morphology occur. Finally, the matrix protein 2, an ion channel expressed during influenza infection, inhibits both ENaC and CFTR activity.