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1.  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.
doi:10.1146/annurev-physiol-021909-135909
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
2.  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.
doi:10.1165/rcmb.2012-0489OC
PMCID: PMC3727888  PMID: 23526213
procalcitonin; calcitonin; sepsis; pneumonia
3.  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.
doi:10.3389/fphar.2014.00008
PMCID: PMC3915096  PMID: 24567720
pneumonia; innate immunity; Toll-like receptor; oligodeoxynucleotide; lipopeptide; aerosol; myeloablation
4.  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.
doi:10.3389/fendo.2013.00129
PMCID: PMC3776272  PMID: 24065956
secretion; exocytosis; mucin; mucus; MARCKS; Munc18; Munc13; synaptotagmin
5.  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.
LINKED ARTICLES
This article is part of a themed issue on Respiratory Pharmacology. To view the other articles in this issue visit http://dx.doi.org/10.1111/bph.2011.163.issue-1
doi:10.1111/j.1476-5381.2011.01237.x
PMCID: PMC3085878  PMID: 21250981
innate immunity; epithelium; lung; infection; pneumonia; resistance; aerosol; Toll-like receptor; TLR; antimicrobial peptides
6.  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.
Summary
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.
doi:10.1097/MCP.0b013e32831da8d3
PMCID: PMC2709596  PMID: 19077699
airway; asthma; mucin; mucous; mucus
7.  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.
doi:10.1164/rccm.200607-1038OC
PMCID: PMC2427056  PMID: 18388354
innate immunity; pneumonia; immunocompromised host; lung epithelium
8.  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.
doi:10.1165/rcmb.2007-0366OC
PMCID: PMC2396243  PMID: 18096867
pulmonary disease, chronic obstructive; Haemophilus influenzae; bronchiolitis; inflammation; fibrosis
9.  Baseline Goblet Cell Mucin Secretion in the Airways Exceeds Stimulated Secretion over Extended Time Periods, and Is Sensitive to Shear Stress and Intracellular Mucin Stores 
PLoS ONE  2015;10(5):e0127267.
Airway mucin secretion studies have focused on goblet cell responses to exogenous agonists almost to the exclusion of baseline mucin secretion (BLMS). In human bronchial epithelial cell cultures (HBECCs), maximal agonist-stimulated secretion exceeds baseline by ~3-fold as measured over hour-long periods, but mucin stores are discharged completely and require 24 h for full restoration. Hence, over 24 h, total baseline exceeds agonist-induced secretion by several-fold. Studies with HBECCs and mouse tracheas showed that BLMS is highly sensitive to mechanical stresses. Harvesting three consecutive 1 h baseline luminal incubations with HBECCs yielded equal rates of BLMS; however, lengthening the middle period to 72 h decreased the respective rate significantly, suggesting a stimulation of BLMS by the gentle washes of HBECC luminal surfaces. BLMS declined exponentially after washing HBECCs (t1/2 = 2.75 h), to rates approaching zero. HBECCs exposed to low perfusion rates exhibited spike-like increases in BLMS when flow was jumped 5-fold: BLMS increased >4 fold, then decreased within 5 min to a stable plateau at 1.5–2-fold over control. Higher flow jumps induced proportionally higher BLMS increases. Inducing mucous hyperplasia in HBECCs increased mucin production, BLMS and agonist-induced secretion. Mouse tracheal BLMS was ~6-fold higher during perfusion, than when flow was stopped. Munc13-2 null mouse tracheas, with their defect of accumulated cellular mucins, exhibited similar BLMS as WT, contrary to predictions of lower values. Graded mucous metaplasia induced in WT and Munc13-2 null tracheas with IL-13, caused proportional increases in BLMS, suggesting that naïve Munc13-2 mouse BLMS is elevated by increased mucin stores. We conclude that BLMS is, [i] a major component of mucin secretion in the lung, [ii] sustained by the mechanical activity of a dynamic lung, [iii] proportional to levels of mucin stores, and [iv] regulated differentially from agonist-induced mucin secretion.
doi:10.1371/journal.pone.0127267
PMCID: PMC4449158  PMID: 26024524
10.  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.
doi:10.1016/j.bbi.2014.02.004
PMCID: PMC4006945  PMID: 24534636
pneumonia; innate immunity; Toll-like receptor; systemic lipopolysaccharide; cytokines
11.  Pneumonia during Remission Induction Chemotherapy in Patients with Acute Leukemia 
Background: Pneumonia is a major cause of death during induction chemotherapy for acute leukemia. The purpose of this study was to quantify the incidence, risk factors, and outcomes of pneumonia in patients with acute leukemia.
Methods: We conducted a retrospective cohort study of 801 patients with acute myeloid leukemia (AML), myelodysplastic syndrome (MDS), or acute lymphocytic leukemia (ALL) who underwent induction chemotherapy.
Measurements and Main Results: Pneumonia was present at induction start in 85 patients (11%). Of the 716 remaining patients, 148 (21%) developed pneumonia. The incidence rate of pneumonia was higher in MDS and AML than in ALL (0.013 vs. 0.008 vs. 0.003 pneumonias per day, respectively; P < 0.001). In multivariate analysis, age greater than or equal to 60 years, AML, low platelet count, low albumin level, neutropenia, and neutrophil count greater than 7,300 were risk factors. The case fatality rate of pneumonia was 17% (40 of 233). Competing risk analysis demonstrated that in the absence of pneumonia, death was rare: 28-day mortality was 6.2% for all patients but only 1.26% in those without pneumonia. Compared with patients without pneumonia, patients with pneumonia had more intensive care unit days, longer hospital stays, and 49% higher costs (P < 0.001).
Conclusions: Pneumonia after induction chemotherapy for acute leukemia continues to be common, and it is the most important determinant of early mortality after induction chemotherapy. Given the high incidence, morbidity, mortality, and cost of pneumonia, interventions aimed at prevention are warranted in patients with acute leukemia.
doi:10.1513/AnnalsATS.201304-097OC
PMCID: PMC3960911  PMID: 23987587
pneumonia; opportunistic infections; leukemia; fungal pneumonia; epidemiology
12.  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.
doi:10.1038/nature12807
PMCID: PMC4001806  PMID: 24317696
13.  Airway Mucus Function and Dysfunction 
The New England journal of medicine  2010;363(23):2233-2247.
doi:10.1056/NEJMra0910061
PMCID: PMC4048736  PMID: 21121836
14.  Toll-Like Receptor–2/6 and Toll-Like Receptor–9 Agonists Suppress Viral Replication but Not Airway Hyperreactivity in Guinea Pigs 
Respiratory virus infections cause airway hyperreactivity (AHR). Preventative strategies for virus-induced AHR remain limited. Toll-like receptors (TLRs) have been suggested as a therapeutic target because of their central role in triggering antiviral immune responses. Previous studies showed that concurrent treatment with TLR2/6 and TLR9 agonists reduced lethality and the microbial burden in murine models of bacterial and viral pneumonia. This study investigated the effects of TLR2/6 and TLR9 agonist pretreatment on parainfluenza virus pneumonia and virus-induced AHR in guinea pigs in vivo. Synthetic TLR2/6 lipopeptide agonist Pam2CSK4 and Class C oligodeoxynucleotide TLR9 agonist ODN2395, administered in combination 24 hours before virus infection, significantly reduced viral replication in the lung. Despite a fivefold reduction in viral titers, concurrent TLR2/6 and TLR9 agonist pretreatment did not prevent virus-induced AHR or virus-induced inhibitory M2 muscarinic receptor dysfunction. Interestingly, the TLR agonists independently caused non–M2-dependent AHR. These data confirm the therapeutic antiviral potential of TLR agonists, while suggesting that virus inhibition may be insufficient to prevent virus-induced airway pathophysiology. Furthermore, TLR agonists independently cause AHR, albeit through a distinctly different mechanism from that of parainfluenza virus.
doi:10.1165/rcmb.2012-0498OC
PMCID: PMC3727870  PMID: 23449736
Toll-like receptor; airway hyperreactivity; muscarinic receptor; parainfluenza virus
15.  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.”
doi:10.1165/rcmb.2012-0364OC
PMCID: PMC3604060  PMID: 23204390
β2-adrenoceptor agonists; formoterol; epinephrine; murine model; asthma
16.  Promoting effect of neutrophils on lung tumorigenesis is mediated by CXCR2 and neutrophil elastase 
Molecular Cancer  2013;12:154.
Background
Tumor cells produce various cytokines and chemokines that attract leukocytes. Leukocytes can amplify parenchymal innate immune responses, and have been shown to contribute to tumor promotion. Neutrophils are among the first cells to arrive at sites of inflammation, and the increased number of tumor-associated neutrophils is linked to poorer outcome in patients with lung cancer.
Results
We have previously shown that COPD-like airway inflammation promotes lung cancer in a K-ras mutant mouse model of lung cancer (CC-LR). This was associated with severe lung neutrophilic influx due to the increased level of neutrophil chemoattractant, KC. To further study the role of neutrophils in lung tumorigenesis, we depleted neutrophils in CC-LR mice using an anti-neutrophil antibody. This resulted in a significant reduction in lung tumor number. We further selectively inhibited the main receptor for neutrophil chemo-attractant KC, CXCR2. Similarly, this resulted in suppression of neutrophil recruitment into the lung of CC-LR mice followed by significant tumor reduction. Neutrophil elastase (NE) is a potent elastolytic enzyme produced by neutrophils at the site of inflammation. We crossed the CC-LR mice with NE knock-out mice, and found that lack of NE significantly inhibits lung cancer development. These were associated with significant reduction in tumor cell proliferation and angiogenesis.
Conclusion
We conclude that lung cancer promotion by inflammation is partly mediated by activation of the IL-8/CXCR2 pathway and subsequent recruitment of neutrophils and release of neutrophil elastase. This provides a baseline for future clinical trials using the IL-8/CXCR2 pathway or NE inhibitors in patients with lung cancer.
doi:10.1186/1476-4598-12-154
PMCID: PMC3923587  PMID: 24321240
Neutrophil; Elastase; Lung cancer; Inflammation; CXCR2; K-ras
17.  Assessment of Intracellular Mucin Content In Vivo 
Airway mucus presents a first line of defense against inhaled materials. It also, however, is a significant pathological contributor to chronic lung diseases such as asthma, cystic fibrosis, and chronic obstructive pulmonary disease. Thus, gaining a better understanding of the mechanisms of mucus production and secretion is an important goal for improving respiratory health. Mucins, the chief glycoprotein components of airway mucus, are very large polymeric glycoproteins, and measuring their production and secretion in experimental animals present significant technical challenges. Over the past several years, we have developed assays for accurately quantifying mucin production and secretion using histological and biochemical assays. These methods are described here.
doi:10.1007/978-1-61779-513-8_17
PMCID: PMC3751172  PMID: 22259143
airways; asthma; cystic fibrosis; chronic obstructive pulmonary disease; goblet cell; lungs; mouse; mucin; mucous; mucus
18.  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.
doi:10.4049/jimmunol.1002122
PMCID: PMC3654378  PMID: 21482737
19.  Pleuropericarditis, obliterative bronchiolitis and lymphocytic interstitial pneumonitis after allogeneic haematopoietic stem cell transplantation 
BMJ Case Reports  2011;2011:bcr1120103488.
Chronic graft-versus-host disease (GVHD) is a common complication of allogeneic haematopoietic cell transplantation, with pulmonary involvement occurring in 5–10% of cases. Obliterative bronchiolitis (OB) is recognised as a diagnostic manifestation of chronic GVHD, whereas lymphocytic interstitial pneumonitis (LIP) has been reported but is not considered diagnostic, and pleuritis is not clearly associated. The authors describe a transplant patient who simultaneously manifested three distinct pulmonary processes: OB, patchy LIP and pleuropericarditis. The onset of these entities along with other manifestations of chronic GVHD, their resolution with increased immunosuppression and their recurrence upon tapering support all three entities as manifestations of GVHD in the lungs.
doi:10.1136/bcr.11.2010.3488
PMCID: PMC3062890  PMID: 22707568
20.  Performance of a Standardized Bronchoalveolar Lavage Protocol in a Comprehensive Cancer Center 
Cancer  2011;117(15):3424-3433.
BACKGROUND
Flexible bronchoscopy with bronchoalveolar lavage (BAL) is performed widely for the diagnosis of pulmonary infections in patients with cancer, but there is no consensus regarding the technical parameters of the lavage procedure in this setting.
METHODS
The authors evaluated the mechanics (instilled and recovered volumes), diagnostic yield, and safety of a standardized BAL protocol in 284 patients with cancer who underwent bronchoscopy for the evaluation of new radiologic infiltrates.
RESULTS
Physician adherence to the BAL protocol was > 90%. The most common protocol deviations were reductions in the saline volume instilled because of actual or anticipated oxyhemoglobin desaturation during the procedure. The mean volume instilled was 121.5 ± 13.9 mL, the mean volume recovered was 68.7 ± 18.1 mL, and the mean ratio of volume instilled to that recovered was 56.7% ± 14.5%. The overall diagnostic yield of BAL was 33.8% and was higher in the nonhematologic malignancy group (42.3% vs 29.4%; P = .021). The diagnostic yield in neutropenic patients was significantly higher than in nonneutropenic patients (41.5% vs 24.6%; P = .019). No major complications were encountered.
CONCLUSIONS
In summary, the diagnostic performance of a standardized BAL protocol was comparable to that of nonprotocolized BAL reported in the literature with few complications. Adherence to a standardized BAL protocol may improve clinical and laboratory comparisons between studies, potentially facilitating research into the diagnosis and management of pneumonia in patients with cancer.
doi:10.1002/cncr.25905
PMCID: PMC3457647  PMID: 21246537
pneumonia; bronchoalveolar lavage; immunocompromise; cancer; diagnosis
21.  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.
doi:10.1042/BJ20120057
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
22.  A Common MUC5B Promoter Polymorphism and Pulmonary Fibrosis 
The New England Journal of Medicine  2011;364(16):1503-1512.
BACKGROUND
The mutations that have been implicated in pulmonary fibrosis account for only a small proportion of the population risk.
METHODS
Using a genomewide linkage scan, we detected linkage between idiopathic interstitial pneumonia and a 3.4-Mb region of chromosome 11p15 in 82 families. We then evaluated genetic variation in this region in gel-forming mucin genes expressed in the lung among 83 subjects with familial interstitial pneumonia, 492 subjects with idiopathic pulmonary fibrosis, and 322 controls. MUC5B expression was assessed in lung tissue.
RESULTS
Linkage and fine mapping were used to identify a region of interest on the p-terminus of chromosome 11 that included gel-forming mucin genes. The minor-allele of the single-nucleotide polymorphism (SNP) rs35705950, located 3 kb upstream of the MUC5B transcription start site, was present at a frequency of 34% among subjects with familial interstitial pneumonia, 38% among subjects with idiopathic pulmonary fibrosis, and 9% among controls (allelic association with familial interstitial pneumonia, P = 1.2×10−15; allelic association with idiopathic pulmonary fibrosis, P = 2.5×10−37). The odds ratios for disease among subjects who were heterozygous and those who were homozygous for the minor allele of this SNP were 6.8 (95% confidence interval [CI], 3.9 to 12.0) and 20.8 (95% CI, 3.8 to 113.7), respectively, for familial interstitial pneumonia and 9.0 (95% CI, 6.2 to 13.1) and 21.8 (95% CI, 5.1 to 93.5), respectively, for idiopathic pulmonary fibrosis. MUC5B expression in the lung was 14.1 times as high in subjects who had idiopathic pulmonary fibrosis as in those who did not (P<0.001). The variant allele of rs35705950 was associated with up-regulation in MUC5B expression in the lung in unaffected subjects (expression was 37.4 times as high as in unaffected subjects homozygous for the wild-type allele, P<0.001). MUC5B protein was expressed in lesions of idiopathic pulmonary fibrosis.
CONCLUSIONS
A common polymorphism in the promoter of MUC5B is associated with familial interstitial pneumonia and idiopathic pulmonary fibrosis. Our findings suggest that dys-regulated MUC5B expression in the lung may be involved in the pathogenesis of pulmonary fibrosis. (Funded by the National Heart, Lung, and Blood Institute and others.)
doi:10.1056/NEJMoa1013660
PMCID: PMC3379886  PMID: 21506741
23.  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.
doi:10.1371/journal.pone.0030596
PMCID: PMC3267724  PMID: 22299046
24.  Enhancement of lung tumorigenesis in a Gprc5a Knockout mouse by chronic extrinsic airway inflammation 
Molecular Cancer  2012;11:4.
Background
Although cigarette smoking is the principal cause of lung carcinogenesis, chronic obstructive pulmonary disease (COPD), an inflammatory disease of the lung, has been identified as an independent risk factor for lung cancer. Bacterial colonization, particularly with non-typeable Haemophilus influenzae (NTHi), has been implicated as a cause of airway inflammation in COPD besides cigarette smoke. Accordingly, we hypothesized that lung cancer promotion may occur in a chronic inflammatory environment in the absence of concurrent carcinogen exposure.
Results
Herein, we investigated the effects of bacterial-induced COPD-like inflammation and tobacco carcinogen-enhanced tumorigenesis/inflammation in the retinoic acid inducible G protein coupled receptor knock out mouse model (Gprc5a-/- mouse) characterized by late-onset, low multiplicity tumor formation. Three-month-old Gprc5a-/- mice received 4 intraperitoneal injections of the tobacco-specific carcinogen, NNK, followed by weekly exposure to aerosolized NTHi lysate for 6 months. The numbers of inflammatory cells in the lungs and levels of several inflammatory mediators were increased in Gprc5a-/- mice treated with NTHi alone, and even more so in mice pretreated with NNK followed by NTHi. The incidence of spontaneous lung lesions in the Gprc5a-/- mice was low, but NTHi exposure led to enhanced development of hyperplastic lesions. Gprc5a-/- mice exposed to NNK alone developed multiple lung tumors, while NTHi exposure increased the number of hyperplastic foci 6-fold and the tumor multiplicity 2-fold. This was associated with increased microvessel density and HIF-1α expression.
Conclusion
We conclude that chronic extrinsic lung inflammation induced by bacteria alone or in combination with NNK enhances lung tumorigenesis in Gprc5a-/- mice.
doi:10.1186/1476-4598-11-4
PMCID: PMC3281775  PMID: 22239913
lung cancer; inflammation; COPD; Gpcr5a; NTHi
25.  Interleukin 6 But Not T Helper 2 Cytokines Promotes Lung Carcinogenesis 
Several epidemiologic studies have found that smokers with chronic obstructive pulmonary disease (COPD), an inflammatory disease of the lung, have an increased risk of lung cancer compared to smokers without COPD. We have shown a causal role for COPD-like airway inflammation in lung cancer promotion in the CCSPCre/LSL–K-rasG12D mouse model (CC-LR). In contrast, existing epidemiologic data do not suggest any definite association between allergic airway inflammation and lung cancer. To test this, CC-LR mice were sensitized to ovalbumin (OVA) then challenged with an OVA aerosol weekly for eight weeks. This resulted in eosinophilic lung inflammation associated with increased levels of T helper 2 cytokines and mucous metaplasia of airway epithelium, similar to what is seen in asthma patients. However, this type of inflammation did not result in a significant difference in lung surface tumor number (49 ± 9 in OVA vs 52 ± 5 in control), in contrast to a 3.2-fold increase with COPD-like inflammation. Gene expression analysis of NTHi-treated lungs showed up-regulation of a different profile of inflammatory genes, including interleukin 6 (IL-6), compared to OVA-treated lungs. Therefore, to determine the causal role of cytokines that mediate COPD-like inflammation in lung carcinogenesis, we genetically ablated IL-6 in CC-LR mice. This not only inhibited intrinsic lung cancer development (1.7-fold), but also inhibited the promoting effect of extrinsic COPD-like airway inflammation (2.6-fold). We conclude that there is a clear specificity for the nature of inflammation in lung cancer promotion, and IL-6 has an essential role in lung cancer promotion.
doi:10.1158/1940-6207.CAPR-10-0180
PMCID: PMC3058282  PMID: 21098042
lung cancer; inflammation; COPD; asthma; IL-6

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