Antimicrobial mitochondrial reactive oxygen species induction by lung epithelial metabolic reprogramming.

Unlabelled: Pneumonia is a worldwide threat, making discovery of novel means to combat lower respiratory tract infections an urgent need. We have previously shown that manipulating the lungs' intrinsic host defenses by therapeutic delivery of a unique dyad of pathogen-associated molecular patterns protects mice against pneumonia in a reactive oxygen species (ROS)-dependent manner. Here we show that antimicrobial ROS are induced from lung epithelial cells by interactions of CpG oligodeoxynucleotides (ODNs) with mitochondrial voltage-dependent anion channel 1 (VDAC1) without dependence on Toll-like receptor 9 (TLR9).

Epithelial immunomodulation by aerosolized Toll-like receptor agonists prevents allergic inflammation in airway mucosa in mice.

Allergic asthma is a chronic inflammatory respiratory disease associated with eosinophilic infiltration, increased mucus production, airway hyperresponsiveness, and airway remodeling. Epidemiologic data reveal that the prevalence of allergic sensitization and associated diseases has increased in the twentieth century. This has been hypothesized to be partly due to reduced contact with microbial organisms (the hygiene hypothesis) in industrialized society.

Screening of Hydrocarbon-Stapled Peptides for Inhibition of Calcium-Triggered Exocytosis.

The so-called primary interface between the SNARE complex and synaptotagmin-1 (Syt1) is essential for Ca-triggered neurotransmitter release in neuronal synapses. The interacting residues of the primary interface are conserved across different species for synaptotagmins (Syt1, Syt2, Syt9), SNAP-25, and syntaxin-1A homologs involved in fast synchronous release. This Ca-independent interface forms prior to Ca-triggering and plays a role in synaptic vesicle priming.

Mucins MUC5AC and MUC5B Are Variably Packaged in the Same and in Separate Secretory Granules.

MUC5AC (mucin 5AC, oligomeric gel-forming) and MUC5B (mucin 5B, oligomeric gel-forming) are the predominant secreted polymeric mucins in mammalian airways. They contribute differently to the pathogenesis of various muco-obstructive and interstitial lung diseases, and their genes are separately regulated, but whether they are packaged together or in separate secretory granules is not known. To determine the packaging of MUC5AC and MUC5B within individual secretory granules in mouse and human airways under varying conditions of inflammation and along the proximal-distal axis.

Intermediary Role of Lung Alveolar Type 1 Cells in Epithelial Repair upon Sendai Virus Infection.

The lung epithelium forms the first barrier against respiratory pathogens and noxious chemicals; however, little is known about how more than 90% of this barrier, made of AT1 (alveolar type 1) cells, responds to injury. Using the Sendai virus to model natural infection in mice, we find evidence that AT1 cells have an intermediary role by persisting in areas depleted of AT2 cells, upregulating IFN responsive genes, and receding from invading airway cells. Sendai virus infection mobilizes airway cells to form alveolar SOX2 (Sry-box 2) clusters without differentiating into AT1 or AT2 cells.

Inhibition of calcium-triggered secretion by hydrocarbon-stapled peptides.

Membrane fusion triggered by Ca is orchestrated by a conserved set of proteins to mediate synaptic neurotransmitter release, mucin secretion and other regulated exocytic processes. For neurotransmitter release, the Ca sensitivity is introduced by interactions between the Ca sensor synaptotagmin and the SNARE complex, and sequence conservation and functional studies suggest that this mechanism is also conserved for mucin secretion. Disruption of Ca-triggered membrane fusion by a pharmacological agent would have therapeutic value for mucus hypersecretion as it is the major cause of airway obstruction in the pathophysiology of respiratory viral infection, asthma, chronic obstructive pulmonary disease and cystic fibrosis.

Airway Epithelial Innate Immunity.

Besides providing an essential protective barrier, airway epithelial cells directly sense pathogens and respond defensively. This is a frontline component of the innate immune system with specificity for different pathogen classes. It occurs in the context of numerous interactions with leukocytes, but here we focus on intrinsic epithelial mechanisms.

Immune Modulation to Improve Survival of Viral Pneumonia in Mice.

Viral pneumonias remain global health threats, as exemplified in the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) pandemic, requiring novel treatment strategies both early and late in the disease process. We have reported that mice treated before or soon after infection with a combination of inhaled Toll-like receptor (TLR) 2/6 and 9 agonists (Pam2-ODN) are broadly protected against microbial pathogens including respiratory viruses, but the mechanisms remain incompletely understood. The objective of this study was to validate strategies for immune modulation in a preclinical model of viral pneumonia and determine their mechanisms.

Potentiating TMEM16A does not stimulate airway mucus secretion or bronchial and pulmonary arterial smooth muscle contraction.

The calcium-activated chloride channel (CaCC) TMEM16A enables chloride secretion across several transporting epithelia, including in the airways. Additional roles for TMEM16A have been proposed, which include regulating mucus production and secretion and stimulating smooth muscle contraction. The aim of the present study was to test whether the pharmacological regulation of TMEM16A channel function, could affect any of these proposed biological roles in the airways.

Inducible epithelial resistance against acute Sendai virus infection prevents chronic asthma-like lung disease in mice.

Background And Purpose: Respiratory viral infections play central roles in the initiation, exacerbation and progression of asthma in humans. An acute paramyxoviral infection in mice can cause a chronic lung disease that resembles human asthma. We sought to determine whether reduction of Sendai virus lung burden in mice by stimulating innate immunity with aerosolized Toll-like receptor (TLR) agonists could attenuate the severity of chronic asthma-like lung disease.

Lipocalin-2 is dispensable in inflammation-induced sickness and depression-like behavior.

Rationale: While the relationship between inflammation and depression is well-established, the molecular mechanisms mediating this relationship remain unclear. RNA sequencing analysis comparing brains of vehicle- and lipopolysaccharide-treated mice revealed LCN2 among the most dysregulated genes. As LCN2 is known to be an important regulator of the immune response to bacterial infection, we investigated its role in the behavioral response to lipopolysaccharide.

Different Munc18 proteins mediate baseline and stimulated airway mucin secretion.

Airway mucin secretion is necessary for ciliary clearance of inhaled particles and pathogens but can be detrimental in pathologies such as asthma and cystic fibrosis. Exocytosis in mammals requires a Munc18 scaffolding protein, and airway secretory cells express all 3 Munc18 isoforms. Using conditional airway epithelial cell-deletant mice, we found that Munc18a has the major role in baseline mucin secretion, Munc18b has the major role in stimulated mucin secretion, and Munc18c does not function in mucin secretion.

Airway Mucin Secretion.

Exocytosis of secreted mucins is the final step in their intracellular processing, resulting in their release into the airway lumen to interact with water and ions to form mucus. Mucins are secreted at a low baseline rate and a high stimulated rate, and both rates are regulated by second messengers acting on components of the exocytic machinery. The principal physiologic function of the low baseline rate is to support steady-state mucociliary clearance of inhaled particles and pathogens that enter the airways during normal breathing.

Inflammation-induced upregulation of P2X expression augments mucin secretion in airway epithelia.

Mucus clearance provides an essential innate defense mechanism to keep the airways and lungs free of particles and pathogens. Baseline and stimulated mucin secretion from secretory airway epithelial cells need to be tightly regulated to prevent mucus hypersecretion and mucus plugging of the airways. It is well established that extracellular ATP is a potent stimulus for regulated mucus secretion.

COPD-Type lung inflammation promotes K-ras mutant lung cancer through epithelial HIF-1α mediated tumor angiogenesis and proliferation.

Chronic obstructive pulmonary disease (COPD), an inflammatory disease of the lung, is an independent risk factor for lung cancer. Lung tissues obtained from human smokers with COPD and lung cancer demonstrate hypoxia and up-regulated hypoxia inducible factor-1 (HIF-1). HIF-1 activation is the central mechanism for controlling the cellular response to hypoxia during inflammation and tumor development.

Inducible Lung Epithelial Resistance Requires Multisource Reactive Oxygen Species Generation To Protect against Viral Infections.

Viral pneumonias cause profound worldwide morbidity, necessitating novel strategies to prevent and treat these potentially lethal infections. Stimulation of intrinsic lung defenses via inhalation of synergistically acting Toll-like receptor (TLR) agonists protects mice broadly against pneumonia, including otherwise-lethal viral infections, providing a potential opportunity to mitigate infectious threats. As intact lung epithelial TLR signaling is required for the inducible resistance and as these cells are the principal targets of many respiratory viruses, the capacity of lung epithelial cells to be therapeutically manipulated to function as autonomous antiviral effectors was investigated.

Platelet Munc13-4 regulates hemostasis, thrombosis and airway inflammation.

Platelet degranulation is crucial for hemostasis and may participate in inflammation. Exocytosis in platelets is mediated by SNARE proteins and should be controlled by Munc13 proteins. We found that platelets express Munc13-2 and -4.

Munc18-2, but not Munc18-1 or Munc18-3, controls compound and single-vesicle-regulated exocytosis in mast cells.

Mast cells (MCs) play pivotal roles in many inflammatory conditions including infections, anaphylaxis, and asthma. MCs store immunoregulatory compounds in their large cytoplasmic granules and, upon stimulation, secrete them via regulated exocytosis. Exocytosis in many cells requires the participation of Munc18 proteins (also known as syntaxin-binding proteins), and we found that mature MCs express all three mammalian isoforms: Munc18-1, -2, and -3.

Munc13 proteins control regulated exocytosis in mast cells.

Mast cells (MCs) are involved in host defenses against pathogens and inflammation. Stimulated MCs release substances stored in their granules via regulated exocytosis. In other cell types, Munc13 (mammalian homolog of uncoordinated gene 13) proteins play essential roles in regulated exocytosis.

β-Adrenoceptor signaling in airway epithelial cells promotes eosinophilic inflammation, mucous metaplasia, and airway contractility.

The mostly widely used bronchodilators in asthma therapy are β-adrenoreceptor (βAR) agonists, but their chronic use causes paradoxical adverse effects. We have previously determined that βAR activation is required for expression of the asthma phenotype in mice, but the cell types involved are unknown. We now demonstrate that βAR signaling in the airway epithelium is sufficient to mediate key features of the asthmatic responses to IL-13 in murine models.

Combined aerosolized Toll-like receptor ligands are an effective therapeutic agent against influenza pneumonia when co-administered with oseltamivir.

Influenza pneumonia remains a common and debilitating viral infection despite vaccination programs and antiviral agents developed for prophylaxis and treatment. The neuraminidase inhibitor oseltamivir is frequently prescribed for established influenza A virus infections, but the emergence of neuraminidase inhibitor resistant viruses, a brief therapeutic window and competing diagnoses complicate its use. PUL-042 is a clinical stage, aerosol drug comprised of synthetic ligands for Toll-like receptor (TLR) 2/6 and TLR 9.

Inducible epithelial resistance protects mice against leukemia-associated pneumonia.

Despite widespread infection prevention efforts, pneumonia remains the leading cause of death among patients with acute leukemia, due to complex disease- and treatment-dependent immune defects. We have reported that a single inhaled treatment with a synergistic combination of Toll-like receptor 2/6 (TLR 2/6) and TLR9 agonists (Pam2-ODN) induces protective mucosal defenses in mice against a broad range of pathogens. As Pam2-ODN-induced protection persists despite depletion of several leukocyte populations, we tested whether it could prevent pneumonia in a mouse model of acute myeloid leukemia (AML) remission induction therapy.

SNAP23 is selectively expressed in airway secretory cells and mediates baseline and stimulated mucin secretion.

Airway mucin secretion is important pathophysiologically and as a model of polarized epithelial regulated exocytosis. We find the trafficking protein, SNAP23 (23-kDa paralogue of synaptosome-associated protein of 25 kDa), selectively expressed in secretory cells compared with ciliated and basal cells of airway epithelium by immunohistochemistry and FACS, suggesting that SNAP23 functions in regulated but not constitutive epithelial secretion. Heterozygous SNAP23 deletant mutant mice show spontaneous accumulation of intracellular mucin, indicating a defect in baseline secretion.