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1.  The polymeric mucin Muc5ac is required for allergic airway hyperreactivity 
Nature communications  2015;6:6281.
In asthma, airflow obstruction is thought to result primarily from inflammation-triggered airway smooth muscle (ASM) contraction. However, anti-inflammatory and smooth muscle-relaxing treatments are often temporary or ineffective. Overproduction of the mucin MUC5AC is an additional disease feature that, while strongly associated pathologically, is poorly understood functionally. Here we show that Muc5ac is a central effector of allergic inflammation that is required for airway hyperreactivity (AHR) to methacholine (MCh). In mice bred on two well-characterized strain backgrounds (C57BL/6 and BALB/c) and exposed to two separate allergic stimuli (ovalbumin and Aspergillus extract), genetic removal of Muc5ac abolishes AHR. Residual MCh responses are identical to unchallenged controls, and although inflammation remains intact, heterogeneous mucus occlusion decreases by 74%. Thus, whereas inflammatory effects on ASM alone are insufficient for AHR, Muc5ac-mediated plugging is an essential mechanism. Inhibiting MUC5AC may be effective for treating asthma and other lung diseases where it is also overproduced.
PMCID: PMC4333679  PMID: 25687754
2.  Inducible Innate Resistance of Lung Epithelium to Infection 
Annual review of physiology  2010;72:413-435.
Most studies of innate immunity have focused on leukocytes such as neutrophils, macrophages and natural killer cells. However, epithelial cells play key roles in innate defenses that include providing a mechanical barrier to microbial entry, signaling to leukocytes, and directly killing pathogens. Importantly, all of these defenses are highly inducible in response to the sensing of microbial and host products. In healthy lungs, the level of innate immune epithelial function is low at baseline, as indicated by low levels of spontaneous microbial killing and cytokine release, reflecting low constitutive stimulation in the nearly sterile lower respiratory tract when mucociliary clearance mechanisms are functioning effectively. This contrasts with the colon, where bacteria are continuously present and epithelial cells are constitutively activated. While the surface area of the lungs presents a large target for microbial invasion, activated lung epithelial cells that are closely apposed to deposited pathogens are ideally positioned for microbial killing.
PMCID: PMC4471865  PMID: 20148683
Innate immunity: host antimicrobial defenses involving detection of conserved molecular motifs by germline-encoded pattern recognition receptors, and characterized by rapid but transient responses of both leukocytes and parenchymal cells; Adaptive immunity: host antimicrobial defenses involving pathogen detection of specific antigens by somatically recombined receptors, and characterized by clonal expansion of pathogen-specific lymphocytes and immunologic memory; Hemolymph: fluid in the body cavity of insects, homologous to vertebrate blood, with most proteins produced by the fat body, homologous to the vertebrate liver, with functions in both metabolism and immunity; Resistance: the strategy of host survival of infection that is associated with a reduction in pathogen burden; this is contrasted to tolerance, the strategy of generating a host phenotype indifferent to the pathogen burden; Non-typeable Haemophilus influenzae (NTHi): an unencapsulated (hence non-typeable) strain of a Gram-negative bacterial pathogen that is frequently cultured from the lungs of patients with chronic respiratory disease; Pathogen-associated molecular pattern (PAMP): stereotypic molecular motifs conserved across microbial species that are recognized by pattern recognition receptors, triggering innate immune responses; Damage-associated molecular pattern (DAMP): molecular motifs expressed on or released by infected or injured host cells, also known as danger signals or alarmins; Pattern recognition receptors (PRR): membrane-associated, cytosolic or secreted host products that recognize conserved molecular patterns on pathogens, initiating innate immune responses. These include Toll-like receptors (TLR), NOD-like receptors (NLR), RIG-I-like receptors (RLR), class A scavenger receptors (SR-A), and macrophage receptor with collagenous structure (MARCO); Leucine rich repeats (LRR): common molecular sequences of many pattern recognition receptors that generally occur within pathogen recognition domains; Toll/Interleukin-1 receptor adaptors (TIR adaptors): host peptides that are selectively recruited to Toll-like receptors and IL-1 receptor upon ligand binding and are required for signal propagation; Lipopolysaccharide (LPS): cell wall component of Gram-negative bacteria, the lipid A portion of which is recognized by TLR4 in association with MD2 and CD14; Interleukin (IL): a widely expressed and highly variable group of cytokine signaling molecules involved in both innate and adaptive immune responses; Complement: a system of more than thirty proteins activated by three pathways that permeabilize pathogens with the membrane attack complex, opsonize microbes and release fragments with signaling properties such as the anaphylotoxins C3a and C5a; Receptor for advanced glycation end-products (RAGE): a multifunctional member of the immunoglobulin superfamily that recognizes several host immunomodulatory proteins, including HMGB1 and S100, as well as host proteins without immunomodulatory activity that become glycated during aging or inflammation; Interferon (IFN): a subset of cytokines that inhibit viral replication within host cells and activate leukocytes. There are three classes – Type I (α, β, ω, ε, and κ), Type II (γ), and Type III (λ1-3, also known as IL-28A/B and IL-29), each with distinct receptors; Anoikis: apoptosis of epithelial cells induced by detachment from the extracellular matrix; Transcytosis: mechanism of transport across polarized epithelial cells involving endocytois of extracellular macromolecules or particles on one surface, transcellular vesicle trafficking, and exocytosis on the other surface; Secretory leukocyte proteinase inhibitor (SLPI): epithelium-derived protease inhibitor with intrinsic antibacterial activity; Transmigration: paracellular migration of leukocytes or pathogens through epithelial or endothelial barriers and associated basement membranes
3.  SNAP23 is selectively expressed in airway secretory cells and mediates baseline and stimulated mucin secretion 
Bioscience Reports  2015;35(3):e00220.
SNAP23 (23-kDa paralogue of synaptosome-associated protein of 25 kDa) is expressed in secretory but not ciliated cells of airway epithelium, suggesting that it mediates regulated but not constitutive secretion in polarized epithelia. Baseline but not stimulated mucin secretion in heterozygous mutant mice is fully compensated by increased intracellular stores.
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. However mucins are released from perfused tracheas of mutant and wild-type (WT) mice at the same rate, suggesting that increased intracellular stores balance reduced release efficiency to yield a fully compensated baseline steady state. In contrast, acute stimulated release of intracellular mucin from mutant mice is impaired whether measured by a static imaging assay 5 min after exposure to the secretagogue ATP or by kinetic analysis of mucins released from perfused tracheas during the first 10 min of ATP exposure. Together, these data indicate that increased intracellular stores cannot fully compensate for the defect in release efficiency during intense stimulation. The lungs of mutant mice develop normally and clear bacteria and instilled polystyrene beads comparable to WT mice, consistent with these functions depending on baseline secretion that is fully compensated.
PMCID: PMC4613665  PMID: 26182382
23-kDa paralogue of synaptosome-associated protein of 25 kDa (SNAP23); exocytosis; mucin; mucus; secretion
4.  Activation of Lung Toll-like Receptors Does Not Exacerbate Sickness Responses to Lipopolysaccharide in Mice 
Brain, behavior, and immunity  2014;38:211-219.
Pneumonia represents a leading cause of death. Recently, a novel treatment strategy for pneumonia has involved enhancing the host pulmonary innate immune response by pre-exposure to aerosolized toll-like receptor (TLR)9 and TLR2/6 agonists, known as O/P. O/P inhalation in mice has been demonstrated to prime the lung inflammatory response, and thus increase survival against subsequent pneumonia infection while producing barely detectable increases in systemic cytokines. Here, we examined the safety of O/P treatment when used in mice that are inflamed systemically. Swiss-Webster mice were treated with two doses of aerosolized O/P (1x or 8x) vs phosphate buffered saline (PBS) either immediately before intraperitoneal injection of 0.1 mg/kg lipopolysaccharide (LPS) or PBS (equivolume) or 2 hours after. Sickness responses (reduced body weight, food intake, activity and social interaction) were examined at 2 and 5.5 h post-treatment. Immediately following behavioral testing, mice were euthanized, perfused with PBS, and brains, spleens, livers and lungs snap frozen for assessment of pro-inflammatory cytokine mRNAs. While O/P treatment alone increased lung IL-1β, IFNγ and TNF-α, no such effects were observed in the brain, spleen or liver. Furthermore, there was no evidence that O/P treatment administered before or after LPS had any synergizing effect to potentiate the cytokine response to LPS in any compartment measured. Supportive of these findings were the measures of sickness behaviors that did not show any increased sickness response in O/P-treated mice exposed to LPS, suggestive that the cytokine signal produced in the lungs from O/P inhalation did not propagate to the brain and synergize with LPS-induced neuroinflammation. These findings support the safety of the use of O/P inhalation as a preventative measure against pneumonia and demonstrate a unique ability of the lungs to compartmentalize pulmonary inflammation and limit propagation of the cytokine signal to the brain.
PMCID: PMC4006945  PMID: 24534636
pneumonia; innate immunity; Toll-like receptor; systemic lipopolysaccharide; cytokines
5.  Muc5b Is Required for Airway Defense 
Nature  2013;505(7483):412-416.
Respiratory surfaces are exposed to billions of particulates and pathogens daily. A protective mucus barrier traps and eliminates them via mucociliary clearance (MCC)1,2. However, excessive mucus contributes to transient respiratory infections and to the pathogenesis of numerous respiratory diseases1. MUC5AC and MUC5B are evolutionarily conserved genes that encode structurally related mucin glycoproteins, the principal macromolecules in airway mucus1,3. Genetic variants are linked to diverse lung diseases4-6, but specific roles for MUC5AC and MUC5B in MCC, and the lasting effects of their inhibition, are unknown. Here we show that Muc5b (but not Muc5ac) is required for MCC, for controlling infections in the airways and middle ear, and for maintaining immune homeostasis in the lungs. Muc5b deficiency caused materials to accumulate in upper and lower airways. This defect led to chronic infection by multiple bacterial species, including Staphylococcus aureus, and to inflammation that failed to resolve normally7. Apoptotic macrophages accumulated, phagocytosis was impaired, and IL-23 production was reduced inMuc5b−/− mice. By contrast, in Muc5b transgenic (Tg) mice, macrophage functions improved. Existing dogma defines mucous phenotypes in asthma and chronic obstructive pulmonary disease (COPD) as driven by increased MUC5AC, with MUC5B levels either unaffected or increased in expectorated sputum1,8. However, in many patients, MUC5B production at airway surfaces decreases by as much as 90%9-11. By distinguishing a specific role for Muc5b in MCC, and by determining its impact on bacterial infections and inflammation in mice, our results provide a refined framework for designing targeted therapies to control mucin secretion and restore MCC.
PMCID: PMC4001806  PMID: 24317696
6.  Deletion of the Gene Encoding Calcitonin and Calcitonin Gene–Related Peptide α Does Not Affect the Outcome of Severe Infection in Mice 
Procalcitonin (PCT) is expressed in nonthryoidal tissues of humans during severe infections. Serum PCT levels are measured to diagnose and guide therapy, and there is some evidence that PCT may also contribute to the pathogenesis of sepsis. We tested whether disruption of the gene encoding PCT in mice affected the course of sepsis. Mice with exons 2–5 of the gene encoding calcitonin/calcitonin gene–related polypeptide α (Calca) knocked out and congenic C57BL/6J control mice were challenged with aerosolized Streptococcus pneumoniae or Pseudomonas aeruginosa, or injected intraperitoneally with S. pneumoniae. There were no significant differences in the survival of knockout and control mice in the two pneumonia models, and no significant differences in weight loss, splenic bacterial counts, or blood leukocyte levels in the peritoneal sepsis model. To verify disruption of the Calca gene in knockout mice, the absence of calcitonin in the serum of knockout mice and its presence and inducibility in control mice were confirmed. To evaluate PCT expression in nonthyroidal tissues of control mice, transcripts were measured in multiple organs. PCT transcripts were not significantly expressed in liver or spleen of control mice challenged with aerosolized P. aeruginosa or intraperitoneal endotoxin, and were expressed in lung only at low levels, even though serum IL-6 rose 3,548-fold. We conclude that mice are not an ideal loss-of-function model to test the role of PCT in the pathogenesis of sepsis because of low nonendocrine PCT expression during infection and inflammation. Nonetheless, our studies demonstrate that nonendocrine PCT expression is not necessary for adverse outcomes from sepsis.
PMCID: PMC3727888  PMID: 23526213
procalcitonin; calcitonin; sepsis; pneumonia
7.  Safety, tolerability, and biomarkers of the treatment of mice with aerosolized Toll-like receptor ligands 
We have previously discovered a synergistically therapeutic combination of two Toll-like receptor ligands, an oligodeoxynucleotide (ODN) and Pam2CSK4. Aerosolization of these ligands stimulates innate immunity within the lungs to prevent pneumonia from bacterial and viral pathogens. Here we examined the safety and tolerability of this treatment in mice, and characterized the expression of biomarkers of innate immune activation. We found that neutrophils appeared in lung lavage fluid 4 h after treatment, reached a peak at 48 h, and resolved by 7 days. The peak of neutrophil influx was accompanied by a small increase in lung permeability. Despite the abundance of neutrophils in lung lavage fluid, only rare neutrophils were visible histopathologically in the interstitium surrounding bronchi and veins and none were visible in alveolar airspaces. The cytokines interleukin 6 (IL-6), tumour necrosis factor, and Chemokine (C-X-C motif) ligand 2 rose several hundred-fold in lung lavage fluid 4 h after treatment in a dose-dependent and synergistic manner, providing useful biomarkers of lung activation. IL-6 rose fivefold in serum with delayed kinetics compared to its rise in lavage fluid, and might serve as a systemic biomarker of immune activation of the lungs. The dose–response relationship of lavage fluid cytokines was preserved in mice that underwent myeloablative treatment with cytosine arabinoside to model the treatment of hematologic malignancy. There were no overt signs of distress in mice treated with ODN/Pam2CSK4 in doses up to eightfold the therapeutic dose, and no changes in temperature, respiratory rate, or behavioral signs of sickness including sugar water preference, food disappearance, cage exploration or social interaction, though there was a small degree of transient weight loss. We conclude that treatment with aerosolized ODN/Pam2CSK4 is well tolerated in mice, and that innate immune activation of the lungs can be monitored by the measurement of inflammatory cytokines in lung lavage fluid and serum.
PMCID: PMC3915096  PMID: 24567720
pneumonia; innate immunity; Toll-like receptor; oligodeoxynucleotide; lipopeptide; aerosol; myeloablation
8.  β2-Adrenoceptor Agonists Are Required for Development of the Asthma Phenotype in a Murine Model 
β2-Adrenoceptor (β2AR) agonists are the most effective class of bronchodilators and a mainstay of asthma management. The first potent β2AR agonist discovered and widely used in reversing the airway constriction associated with asthma exacerbation was the endogenous activator of the β2AR, epinephrine. In this study, we demonstrate that activation of the β2AR by epinephrine is paradoxically required for development of the asthma phenotype. In an antigen-driven model, mice sensitized and challenged with ovalbumin showed marked elevations in three cardinal features of the asthma phenotype: inflammatory cells in their bronchoalveolar lavage fluid, mucin over production, and airway hyperresponsiveness. However, genetic depletion of epinephrine using mice lacking the enzyme to synthesize epinephrine, phenylethanolamine N-methyltransferase, or mice that had undergone pharmacological sympathectomy with reserpine to deplete epinephrine, had complete attenuation of these three cardinal features of the asthma phenotype. Furthermore, administration of the long-acting β2AR agonist, formoterol, a drug currently used in asthma treatment, to phenylethanolamine N-methyltransferase–null mice restored the asthma phenotype. We conclude that β2AR agonist–induced activation is needed for pathogenesis of the asthma phenotype. These findings also rule out constitutive signaling by the β2AR as sufficient to drive the asthma phenotype, and may help explain why chronic administration of β2AR agonists, such as formoterol, have been associated with adverse outcomes in asthma. These data further support the hypothesis that chronic asthma management may be better served by treatment with certain “β-blockers.”
PMCID: PMC3604060  PMID: 23204390
β2-adrenoceptor agonists; formoterol; epinephrine; murine model; asthma
9.  Lung epithelial cells are essential effectors of inducible resistance to pneumonia 
Mucosal immunology  2013;7(1):10.1038/mi.2013.26.
Infectious pneumonias are a leading cause of death worldwide, particularly among immunocompromised patients. Therapeutic stimulation of the lungs’ intrinsic defenses with a unique combination of inhaled Toll-like receptor agonists broadly protects mice against otherwise lethal pneumonias. As the survival benefit persists despite cytotoxic chemotherapy-related neutropenia, the cells required for protection were investigated. The inducibility of resistance was tested in mice with deficiencies of leukocyte lineages due to genetic deletions and in wild type mice with leukocyte populations significantly reduced by antibodies or toxins. Surprisingly, these serial reductions in leukocyte lineages did not appreciably impair inducible resistance, but targeted disruption of Toll-like receptor signaling in the lung epithelium resulted in complete abrogation of the protective effect. Isolated lung epithelial cells were also induced to kill pathogens in the absence of leukocytes. Proteomic and gene expression analyses of isolated epithelial cells and whole lungs revealed highly congruent antimicrobial responses. Taken together, these data indicate that lung epithelial cells are necessary and sufficient effectors of inducible resistance. These findings challenge conventional paradigms about the role of epithelia in antimicrobial defense and offer a novel potential intervention to protect patients with impaired leukocyte-mediated immunity from fatal pneumonias.
PMCID: PMC3735803  PMID: 23632328
10.  Mucus hypersecretion in asthma: causes and effects 
Purpose of review
Airway mucus plugging has long been recognized as a principal cause of death in asthma. However, molecular mechanisms of mucin overproduction and secretion have not been understood until recently. These mechanisms are reviewed together with ongoing investigations relating them to lung pathophysiology.
Recent findings
Of the five secreted gel-forming mucins in mammals, only MUC5AC and MUC5B are produced in significant quantities in intrapulmonary airways. MUC5B is the principal gel-forming mucin at baseline in small airways of humans and mice, and therefore likely performs most homeostatic clearance functions. MUC5AC is the principal gel-forming mucin upregulated in airway inflammation and is under negative control by forkhead box a2 and positive control by hypoxia inducible factor-1. Mucin secretion is regulated separately from production, principally by extracellular triphosphate nucleotides that bind P2Y2 receptors on the lumenal surface of airway secretory cells, generating intracellular second messengers that activate the exocytic proteins, Munc13-2 and synaptotagmin-2.
Markedly upregulated production of MUC5AC together with stimulated secretion leads to airflow obstruction in asthma. As MUC5B appears to mediate homeostatic functions, it may be possible to selectively inhibit MUC5AC production without impairing airway function. The precise roles of mucin hypersecretion in asthma symptoms such as dyspnea and cough and in physiologic phenomena such as airway hyperresponsiveness remain to be defined.
PMCID: PMC2709596  PMID: 19077699
airway; asthma; mucin; mucous; mucus
11.  Synergistic interactions of TLR2/6 and TLR9 induce a high level of resistance to lung infection in mice 
Infectious pneumonias exact an unacceptable mortality burden worldwide. Efforts to protect populations from pneumonia have historically focused on antibiotic development and vaccine-enhanced adaptive immunity. However, we have recently reported that the lungs’ innate defenses can be therapeutically induced by inhalation of a bacterial lysate that protects mice against otherwise lethal pneumonia. Here, we tested in mice the hypothesis that Toll-like receptors (TLRs) are required for this antimicrobial phenomenon, and found that resistance could not be induced in the absence of the TLR signaling adaptor protein MyD88. We then attempted to recapitulate the protection afforded by the bacterial lysate by stimulating the lung epithelium with aerosolized synthetic TLR ligands. While most single or combination treatments yielded no protection, simultaneous treatment with ligands for TLR2/6 and TLR9 conferred robust, synergistic protection against virulent Gram-positive and Gram-negative pathogens. Protection was associated with rapid pathogen killing in the lungs, and pathogen killing could be induced from lung epithelial cells in isolation. Taken together, these data demonstrate the requirement for TLRs in inducible resistance against pneumonia, reveal a remarkable, unanticipated synergistic interaction of TLR2/6 and TLR9, reinforce the emerging evidence supporting the antimicrobial capacity of the lung epithelium, and may provide the basis for a novel clinical therapeutic that can protect patients against pneumonia during periods of peak vulnerability.
PMCID: PMC3654378  PMID: 21482737
12.  Regulated Mucin Secretion from Airway Epithelial Cells 
Secretory epithelial cells of the proximal airways synthesize and secrete gel-forming polymeric mucins. The secreted mucins adsorb water to form mucus that is propelled by neighboring ciliated cells, providing a mobile barrier which removes inhaled particles and pathogens from the lungs. Several features of the intracellular trafficking of mucins make the airway secretory cell an interesting comparator for the cell biology of regulated exocytosis. Polymeric mucins are exceedingly large molecules (up to 3 × 106 Da per monomer) whose folding and initial polymerization in the ER requires the protein disulfide isomerase Agr2. In the Golgi, mucins further polymerize to form chains and possibly branched networks comprising more than 20 monomers. The large size of mucin polymers imposes constraints on their packaging into transport vesicles along the secretory pathway. Sugar side chains account for >70% of the mass of mucins, and their attachment to the protein core by O-glycosylation occurs in the Golgi. Mature polymeric mucins are stored in large secretory granules ∼1 μm in diameter. These are translocated to the apical membrane to be positioned for exocytosis by cooperative interactions among myristoylated alanine-rich C kinase substrate, cysteine string protein, heat shock protein 70, and the cytoskeleton. Mucin granules undergo exocytic fusion with the plasma membrane at a low basal rate and a high stimulated rate. Both rates are mediated by a regulated exocytic mechanism as indicated by phenotypes in both basal and stimulated secretion in mice lacking Munc13-2, a sensor of the second messengers calcium and diacylglycerol (DAG). Basal secretion is induced by low levels of activation of P2Y2 purinergic and A3 adenosine receptors by extracellular ATP released in paracrine fashion and its metabolite adenosine. Stimulated secretion is induced by high levels of the same ligands, and possibly by inflammatory mediators as well. Activated receptors are coupled to phospholipase C by Gq, resulting in the generation of DAG and of IP3 that releases calcium from apical ER. Stimulated secretion requires activation of the low affinity calcium sensor Synaptotagmin-2, while a corresponding high affinity calcium sensor in basal secretion is not known. The core exocytic machinery is comprised of the SNARE proteins VAMP8, SNAP23, and an unknown Syntaxin protein, together with the scaffolding protein Munc18b. Common and distinct features of this exocytic system in comparison to neuroendocrine cells and neurons are highlighted.
PMCID: PMC3776272  PMID: 24065956
secretion; exocytosis; mucin; mucus; MARCKS; Munc18; Munc13; synaptotagmin
13.  Munc18b is an essential gene in mice whose expression is limiting for secretion by airway epithelial and mast cells 
Biochemical Journal  2012;446(Pt 3):383-394.
Airway mucin secretion and MC (mast cell) degranulation must be tightly controlled for homoeostasis of the lungs and immune system respectively. We found the exocytic protein Munc18b to be highly expressed in mouse airway epithelial cells and MCs, and localized to the apical pole of airway secretory cells. To address its functions, we created a mouse with a severely hypomorphic Munc18b allele such that protein expression in heterozygotes was reduced by ~50%. Homozygous mutant mice were not viable, but heterozygotes showed a ~50% reduction in stimulated release of mucin from epithelial cells and granule contents from MCs. The defect in MCs affected only regulated secretion and not constitutive or transporter-mediated secretion. The severity of passive cutaneous anaphylaxis was also reduced by ~50%, showing that reduction of Munc18b expression results in an attenuation of physiological responses dependent on MC degranulation. The Munc18b promoter is controlled by INR (initiator), Sp1 (specificity protein 1), Ets, CRE (cAMP-response element), GRE (glucocorticoid-response element), GATA and E-box elements in airway epithelial cells; however, protein levels did not change during mucous metaplasia induced by allergic inflammation. Taken together, the results of the present study identify Munc18b as an essential gene that is a limiting component of the exocytic machinery of epithelial cells and MCs.
PMCID: PMC3430001  PMID: 22694344
exocytosis; mast cell; mucin; mucus; Munc18; secretion; AB-PAS, Alcian Blue/periodic acid/Schiff reagent; bHLH, basic helix–loop–helix; CCSP, Clara cell secretory protein; Clca3, chloride channel, calcium-activated, family member 3; CRE, cAMP-response element; DNP, 2,4-dinitrophenol; FBS, fetal bovine serum; FcϵRIα, high-affinity IgE receptor, α subunit; FRT, flippase recognition target; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; GRE, glucocorticoid-response element; HA, haemagglutinin; HSA, human serum albumin; HRP, horseradish peroxidase; IL-3, interleukin-3; INR, initiator; ISH, in situ hybridization; MC, mast cell; mBMMC, mouse bone-marrow-derived MC; mClca3, mouse Clca3; MFI, mean fluorescent intensity; mtCC, mouse transformed Clara cell; NK, natural killer; OCT, optimal cutting temperature compound; PAFS, periodic acid/fluorescent Schiff reagent; PBST, PBS containing 0.05% Tween 20; PGD2, prostaglandin D2; PGK, phosphoglucokinase; SCF, stem cell factor; SM, Sec1/Munc18; SNAP, soluble N-ethylmaleimide-sensitive factor-attachment protein; SNARE, SNAP receptor; Stxbp2, syntaxin-binding protein 2; TK, thymidine kinase; TNFα, tumour necrosis factor α; WT, wild-type; YFP, yellow fluorescent protein
14.  Inhaled innate immune ligands to prevent pneumonia 
British Journal of Pharmacology  2011;163(1):195-206.
Epithelial surfaces throughout the body continuously sample and respond to environmental stimuli. The accessibility of lung epithelium to inhaled therapies makes it possible to stimulate local antimicrobial defences with aerosolized innate immune ligands. This strategy has been shown to be effective in preclinical models, as delivery of innate immune ligands to the lungs of laboratory animals results in protection from subsequent challenge with microbial pathogens. Survival of the animal host in this setting correlates directly with killing of pathogens within the lungs, indicating the induction of a resistance mechanism. Resistance appears to be mediated primarily by activated epithelial cells rather than recruited leucocytes. Resistance reaches a peak within hours and persists for several days. Innate immune ligands can interact synergistically under some circumstances, and synergistic combinations of innate ligands delivered by aerosol are capable of inducing a high level of broad host resistance to bacteria, fungi and viruses. The induction of innate antimicrobial resistance within the lungs could have clinical applications in the prevention of lower respiratory tract infection in subjects transiently at high risk. These include cancer patients undergoing myeloablative chemotherapy, intubated patients being mechanically ventilated, vulnerable individuals during seasonal influenza epidemics, asthmatic subjects experiencing a respiratory viral infection, and healthy subjects exposed to virulent pathogens from a bioterror attack or emergent pandemic. In summary, stimulation of the lung epithelium to induce localized resistance to infection is a novel strategy whose clinical utility will be assessed in the near future.
This article is part of a themed issue on Respiratory Pharmacology. To view the other articles in this issue visit
PMCID: PMC3085878  PMID: 21250981
innate immunity; epithelium; lung; infection; pneumonia; resistance; aerosol; Toll-like receptor; TLR; antimicrobial peptides
15.  Synergistic TLR2/6 and TLR9 Activation Protects Mice against Lethal Influenza Pneumonia 
PLoS ONE  2012;7(1):e30596.
Lower respiratory tract infections caused by influenza A continue to exact unacceptable worldwide mortality, and recent epidemics have emphasized the importance of preventative and containment strategies. We have previously reported that induction of the lungs' intrinsic defenses by aerosolized treatments can protect mice against otherwise lethal challenges with influenza A virus. More recently, we identified a combination of Toll like receptor (TLR) agonists that can be aerosolized to protect mice against bacterial pneumonia. Here, we tested whether this combination of synthetic TLR agonists could enhance the survival of mice infected with influenza A/HK/8/68 (H3N2) or A/California/04/2009 (H1N1) influenza A viruses. We report that the TLR treatment enhanced survival whether given before or after the infectious challenge, and that protection tended to correlate with reductions in viral titer 4 d after infection. Surprisingly, protection was not associated with induction of interferon gene expression. Together, these studies suggest that synergistic TLR interactions can protect against influenza virus infections by mechanisms that may provide the basis for novel therapeutics.
PMCID: PMC3267724  PMID: 22299046
16.  Stimulated Innate Resistance of Lung Epithelium Protects Mice Broadly against Bacteria and Fungi 
Pneumonia is a serious problem worldwide. We recently demonstrated that innate defense mechanisms of the lung are highly inducible against pneumococcal pneumonia. To determine the breadth of protection conferred by stimulation of lung mucosal innate immunity, and to identify cells and signaling pathways activated by this treatment, mice were treated with an aerosolized bacterial lysate, then challenged with lethal doses of bacterial and fungal pathogens. Mice were highly protected against a broad array of Gram-positive, Gram-negative, and class A bioterror bacterial pathogens, and the fungal pathogen, Aspergillus fumigatus. Protection was associated with rapid pathogen killing within the lungs, and this effect was recapitulated in vitro using a respiratory epithelial cell line. Gene expression analysis of lung tissue showed marked activation of NF-κB, type I and II IFN, and antifungal Card9-Bcl10-Malt1 pathways. Cytokines were the most strongly induced genes, but the inflammatory cytokines TNF and IL-6 were not required for protection. Lung-expressed antimicrobial peptides were also highly up-regulated. Taken together, stimulated innate resistance appears to occur through the activation of multiple host defense signaling pathways in lung epithelial cells, inducing rapid pathogen killing, and conferring broad protection against virulent bacterial and fungal pathogens. Augmentation of innate antimicrobial defenses of the lungs might have therapeutic value for protection of patients with neutropenia or impaired adaptive immunity against opportunistic pneumonia, and for defense of immunocompetent subjects against a bioterror threat or epidemic respiratory infection.
PMCID: PMC2809220  PMID: 19329554
innate immunity; pneumonia; immunocompromised host; lung epithelium
17.  Curcumin inhibits COPD-like airway inflammation and lung cancer progression in mice 
Carcinogenesis  2009;30(11):1949-1956.
Recent studies have demonstrated that K-ras mutations in lung epithelial cells elicit inflammation that promotes carcinogenesis in mice (intrinsic inflammation). The finding that patients with chronic obstructive pulmonary disease (COPD), an inflammatory disease of the lung, have an increased risk of lung cancer after controlling for smoking suggests a further link between lung cancer and extrinsic inflammation. Besides exposure to cigarette smoke, it is thought that airway inflammation in COPD is caused by bacterial colonization, particularly with non-typeable Hemophilus influenzae (NTHi). Previously, we have shown that NTHi-induced COPD-like airway inflammation promotes lung cancer in an airway conditional K-ras-induced mouse model. To further test the role of inflammation in cancer promotion, we administered the natural anti-inflammatory agent, curcumin, 1% in diet before and during weekly NTHi exposure. This significantly reduced the number of visible lung tumors in the absence of NTHi exposure by 85% and in the presence of NTHi exposures by 53%. Mechanistically, curcumin markedly suppressed NTHi-induced increased levels of the neutrophil chemoattractant keratinocyte-derived chemokine by 80% and neutrophils by 87% in bronchoalveolar lavage fluid. In vitro studies of murine K-ras-induced lung adenocarcinoma cell lines (LKR-10 and LKR-13) indicated direct anti-tumoral effects of curcumin by reducing cell viability, colony formation and inducing apoptosis. We conclude that curcumin suppresses the progression of K-ras-induced lung cancer in mice by inhibiting intrinsic and extrinsic inflammation and by direct anti-tumoral effects. These findings suggest that curcumin could be used to protract the premalignant phase and inhibit lung cancer progression in high-risk COPD patients.
PMCID: PMC2783007  PMID: 19793800
18.  Host lung gene expression patterns predict infectious etiology in a mouse model of pneumonia 
Respiratory Research  2010;11(1):101.
Lower respiratory tract infections continue to exact unacceptable worldwide mortality, often because the infecting pathogen cannot be identified. The respiratory epithelia provide protection from pneumonias through organism-specific generation of antimicrobial products, offering potential insight into the identity of infecting pathogens. This study assesses the capacity of the host gene expression response to infection to predict the presence and identity of lower respiratory pathogens without reliance on culture data.
Mice were inhalationally challenged with S. pneumoniae, P. aeruginosa, A. fumigatus or saline prior to whole genome gene expression microarray analysis of their pulmonary parenchyma. Characteristic gene expression patterns for each condition were identified, allowing the derivation of prediction rules for each pathogen. After confirming the predictive capacity of gene expression data in blinded challenges, a computerized algorithm was devised to predict the infectious conditions of subsequent subjects.
We observed robust, pathogen-specific gene expression patterns as early as 2 h after infection. Use of an algorithmic decision tree revealed 94.4% diagnostic accuracy when discerning the presence of bacterial infection. The model subsequently differentiated between bacterial pathogens with 71.4% accuracy and between non-bacterial conditions with 70.0% accuracy, both far exceeding the expected diagnostic yield of standard culture-based bronchoscopy with bronchoalveolar lavage.
These data substantiate the specificity of the pulmonary innate immune response and support the feasibility of a gene expression-based clinical tool for pneumonia diagnosis.
PMCID: PMC2914038  PMID: 20653947
19.  Allergic lung inflammation alters neither susceptibility to Streptococcus pneumoniae infection nor inducibility of innate resistance in mice 
Respiratory Research  2009;10(1):70.
Protective host responses to respiratory pathogens are typically characterized by inflammation. However, lung inflammation is not always protective and it may even become deleterious to the host. We have recently reported substantial protection against Streptococcus pneumoniae (pneumococcal) pneumonia by induction of a robust inflammatory innate immune response to an inhaled bacterial lysate. Conversely, the allergic inflammation associated with asthma has been proposed to promote susceptibility to pneumococcal disease. This study sought to determine whether preexisting allergic lung inflammation influences the progression of pneumococcal pneumonia or reduces the inducibilty of protective innate immunity against bacteria.
To compare the effect of different inflammatory and secretory stimuli on defense against pneumonia, intraperitoneally ovalbumin-sensitized mice were challenged with inhaled pneumococci following exposure to various inhaled combinations of ovalbumin, ATP, and/or a bacterial lysate. Thus, allergic inflammation, mucin degranulation and/or stimulated innate resistance were induced prior to the infectious challenge. Pathogen killing was evaluated by assessing bacterial CFUs of lung homogenates immediately after infection, the inflammatory response to the different conditions was evaluated by measurement of cell counts of bronchoalveolar lavage fluid 18 hours after challenge, and mouse survival was assessed after seven days.
We found no differences in survival of mice with and without allergic inflammation, nor did the induction of mucin degranulation alter survival. As we have found previously, mice treated with the bacterial lysate demonstrated substantially increased survival at seven days, and this was not altered by the presence of allergic inflammation or mucin degranulation. Allergic inflammation was associated with predominantly eosinophilic infiltration, whereas the lysate-induced response was primarily neutrophilic. The presence of allergic inflammation did not significantly alter the neutrophilic response to the lysate, and did not affect the induced bacterial killing within the lungs.
These results suggest that allergic airway inflammation neither promotes nor inhibits progression of pneumococcal lung infection in mice, nor does it influence the successful induction of stimulated innate resistance to bacteria.
PMCID: PMC2724420  PMID: 19635139
20.  Stimulation of Lung Innate Immunity Protects against Lethal Pneumococcal Pneumonia in Mice 
Rationale: The lungs are a common site of serious infection in both healthy and immunocompromised subjects, and the most likely route of delivery of a bioterror agent. Since the airway epithelium shows great structural plasticity in response to inflammatory stimuli, we hypothesized it might also show functional plasticity.
Objectives: To test the inducibility of lung defenses against bacterial challenge.
Methods: Mice were treated with an aerosolized lysate of ultraviolet-killed nontypeable (unencapsulated) Haemophilus influenzae (NTHi), then challenged with a lethal dose of live Streptococcus pneumoniae (Spn) delivered by aerosol.
Measurements and Main Results: Treatment with the NTHi lysate induced complete protection against challenge with a lethal dose of Spn if treatment preceded challenge by 4 to 24 hours. Lesser levels of protection occurred at shorter (83% at 2 h) and longer (83% at 48–72 h) intervals between treatment and challenge. There was also some protection when treatment was given 2 hours after challenge (survival increased from 14 to 57%), but not 24 hours after challenge. Protection did not depend on recruited neutrophils or resident mast cells and alveolar macrophages. Protection was specific to the airway route of infection, correlated in magnitude and time with rapid bacterial killing within the lungs, and was associated with increases of multiple antimicrobial polypeptides in lung lining fluid.
Conclusions: We infer that protection derives from stimulation of local innate immune mechanisms, and that activated lung epithelium is the most likely cellular effector of this response. Augmentation of innate antimicrobial defenses of the lungs might have therapeutic value.
PMCID: PMC2427056  PMID: 18388354
innate immunity; pneumonia; immunocompromised host; lung epithelium
21.  Haemophilus influenzae Lysate Induces Aspects of the Chronic Obstructive Pulmonary Disease Phenotype 
Nontypeable Haemophilus influenzae (NTHi) commonly colonizes the lower airways of patients with chronic obstructive pulmonary disease (COPD). Whether it contributes to COPD progression is unknown. Here, we determined which aspects of the COPD phenotype can be induced by repetitive exposure to NTHi products. Mice were exposed weekly to an aerosolized NTHi lysate, and inflammation was evaluated by measurement of cells and cytokines in bronchoalveolar lavage fluid (BALF) and immunohistochemical staining; structural changes were evaluated histochemically by periodic acid fluorescent Schiff's reagent, Masson's trichrome, and Picrosirius red staining; mucin gene expression was measured by quantitative RT-PCR; and the role of TNF-α was examined by transgenic airway overexpression and use of an inhibitory antibody. NTHi lysate induced rapid activation of NF-κB in airway cells and increases of inflammatory cytokines and neutrophils in BALF. Repetitive exposure induced infiltration of macrophages, CD8+ T cells, and B cells around airways and blood vessels, and collagen deposition in airway and alveolar walls, but airway mucin staining and gel-forming mucin transcripts were not increased. Transgenic overexpression of TNF-α caused BALF neutrophilia and inflammatory cell infiltration around airways, but not fibrosis, and TNF-α neutralization did not reduce BALF neutrophilia in response to NTHi lysate. In conclusion, NTHi products elicit airway inflammation in mice with a cellular and cytokine profile similar to that in COPD, and cause airway wall fibrosis but not mucous metaplasia. TNF-α is neither required for inflammatory cell recruitment nor sufficient for airway fibrosis. Colonization by NTHi may contribute to the pathogenesis of small airways disease in patients with COPD.
PMCID: PMC2396243  PMID: 18096867
pulmonary disease, chronic obstructive; Haemophilus influenzae; bronchiolitis; inflammation; fibrosis
22.  In Vivo Demonstration and Quantification of Intracellular Bacillus anthracis in Lung Epithelial Cells▿  
Infection and Immunity  2008;76(9):3975-3983.
Inhalational anthrax is initiated by the entry of Bacillus anthracis spores into the lung. A critical early event in the establishment of an infection is the dissemination of spores from the lung. Using in vitro cell culture assays, we previously demonstrated that B. anthracis spores are capable of entering into epithelial cells of the lung and crossing a barrier of lung epithelial cells without apparent disruption of the barrier integrity, suggesting a novel portal for spores to disseminate from the lung. However, in vivo evidence for spore uptake by epithelial cells has been lacking. Here, using a mouse model, we present evidence that B. anthracis spores are taken up by lung epithelial cells in vivo soon after spores are delivered into the lung. Immunofluorescence staining of thin sections of lungs from spore-challenged BALB/c mice revealed that spores were associated with the epithelial surfaces in the airway and the alveoli at 2 and 4 h postinoculation. Confocal analysis further indicated that some of the associated spores were surrounded by F-actin, demonstrating intracellular localization. These observations were further confirmed and substantiated by a quantitative method that first isolated lung cells from spore-challenged mice and then stained these cells with antibodies specific for epithelial cells and spores. The results showed that substantial amounts of spores were taken up by lung epithelial cells in vivo. These data, combined with those in our previous reports, provided powerful evidence that the lung epithelia were directly targeted by B. anthracis spores at early stages of infection.
PMCID: PMC2519418  PMID: 18625737
23.  Chronic Exposure to Beta-Blockers Attenuates Inflammation and Mucin Content in a Murine Asthma Model 
Single-dose administration of beta-adrenoceptor agonists produces bronchodilation and inhibits airway hyperresponsiveness (AHR), and is the standard treatment for the acute relief of asthma. However, chronic repetitive administration of beta-adrenoceptor agonists may increase AHR, airway inflammation, and risk of death. Based upon the paradigm shift that occurred with the use of beta-blockers in congestive heart failure, we previously determined that chronic administration of beta-blockers decreased AHR in a murine model of asthma. To elucidate the mechanisms for the beneficial effects of beta-blockers, we examined the effects of chronic administration of several beta-adrenoceptor ligands in a murine model of allergic asthma. Administration of beta-blockers resulted in a reduction in total cell counts, eosinophils, and the cytokines IL-13, IL-10, IL-5, and TGF-β1 in bronchoalveolar lavage, and attenuated epithelial mucin content and morphologic changes. The differences in mucin content also occurred if the beta-blockers were administered only during the ovalbumin challenge phase, but administration of beta-blockers for 7 days was not as effective as administration for 28 days. These results indicate that in a murine model of asthma, chronic administration of beta-blockers reduces inflammation and mucous metaplasia, cardinal features of asthma that may contribute to airflow obstruction and AHR. Similar to heart failure, our results provide a second disease model in which beta-blockers producing an acutely detrimental effect may provide a therapeutically beneficial effect with chronic administration.
PMCID: PMC2258446  PMID: 18096872
beta-blockers; beta-adrenoceptor; asthma; mucin; airway inflammation
24.  Augmented Lung Inflammation Protects against Influenza A Pneumonia 
PLoS ONE  2009;4(1):e4176.
Influenza pneumonia causes high mortality every year, and pandemic episodes kill millions of people. Influenza-related mortality has been variously ascribed to an ineffective host response that fails to limit viral replication, an excessive host inflammatory response that results in lung injury and impairment of gas exchange, or to bacterial superinfection. We sought to determine whether lung inflammation promoted or impaired host survival in influenza pneumonia.
Methods and Findings
To distinguish among these possible causes of influenza-related death, we induced robust lung inflammation by exposing mice to an aerosolized bacterial lysate prior to challenge with live virus. The treatment induced expression of the inflammatory cytokines IL-6 and TNF in bronchoalveolar lavage fluid 8- and 40-fold greater, respectively, than that caused by lethal influenza infection. Yet, this augmented inflammation was associated with striking resistance to host mortality (0% vs 90% survival, p = 0.0001) and reduced viral titers (p = 0.004). Bacterial superinfection of virus infected lungs was not observed. When mice were repeatedly exposed to the bacterial lysate, as would be clinically desirable during an influenza epidemic, there was no tachyphylaxis of the induced viral resistance. When the bacterial lysate was administered after the viral challenge, there was still some mortality benefit, and when ribavirin was added to the aerosolized bacterial lysate, host survival was synergistically improved (0% vs 93.3% survival, p<0.0001).
Together, these data indicate that innate immune resistance to influenza can be effectively stimulated, and suggest that ineffective rather than excessive inflammation is the major cause of mortality in influenza pneumonia.
PMCID: PMC2613561  PMID: 19137067
25.  Central Role of Muc5ac Expression in Mucous Metaplasia and Its Regulation by Conserved 5′ Elements 
Mucus hypersecretion contributes to morbidity and mortality in many obstructive lung diseases. Gel-forming mucins are the chief glycoprotein components of airway mucus, and elevated expression of these during mucous metaplasia precedes the hypersecretory phenotype. Five orthologous genes (MUC2, MUC5AC, MUC5B, MUC6, and MUC19) encode the mammalian gel-forming mucin family, and several have been implicated in asthma, cystic fibrosis, and chronic obstructive pulmonary disease pathologies. However, in the absence of a comprehensive analysis, their relative contributions remain unclear. Here, we assess the expression of the entire gel-forming mucin gene family in allergic mouse airways and show that Muc5ac is the predominant gel-forming mucin induced. We previously showed that the induction of mucous metaplasia in ovalbumin-sensitized and -challenged mouse lungs occurs within bronchial Clara cells. The temporal induction and localization of Muc5ac transcripts correlate with the induced expression and localization of mucin glycoproteins in bronchial airways. To better understand the tight regulation of Muc5ac expression, we analyzed all available 5′-flanking sequences of mammalian MUC5AC orthologs and identified evolutionarily conserved regions within domains proximal to the mRNA coding region. Analysis of luciferase reporter gene activity in a mouse transformed Clara cell line demonstrates that this region possesses strong promoter activity and harbors multiple conserved transcription factor–binding motifs. In particular, SMAD4 and HIF-1α bind to the promoter, and mutation of their recognition motifs abolishes promoter function. In conclusion, Muc5ac expression is the central event in antigen-induced mucous metaplasia, and phylogenetically conserved 5′ noncoding domains control its regulation.
PMCID: PMC1994232  PMID: 17463395
mucin; metaplasia; airway; lung; epithelium

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