Neutrophils cast neutrophil extracellular traps (NETs) to ensnare microbial pathogens. Nevertheless, the molecular rheostats that regulate NETosis in response to bacteria are not clearly established. We hypothesized that stress-activated protein kinase or c-Jun N-terminal Kinase (SAPK/JNK) is a molecular switch that turns on NETosis in response to increasing concentrations of lipopolysaccharide (LPS)- and Gram-negative bacteria. Here we show that Escherichia coli LPS (0111:B4; 10–25 μg/ml), but not phorbol myristate acetate (PMA), activates JNK in human neutrophils in a dose-dependent manner. JNK inhibitors SP600125 and TCSJNK6o, and a TLR4 inhibitor TAK242 suppress reactive oxygen species production and NETosis in LPS-, but not PMA-treated neutrophils. Diphenyleneiodonium suppresses LPS-induced NETosis, confirming that endotoxin induces NADPH oxidase-dependent NETosis. Immunoblots, Sytox Green assays, and confocal microscopy of cleaved caspase-3 and nuclear morphology show that JNK inhibition does not induce apoptosis in LPS-stimulated neutrophils. JNK inhibition also suppresses NETosis induced by two typical Gram-negative bacteria, E. coli and Pseudomonas aeruginosa. Therefore, we propose that neutrophils use a TLR4-dependent, JNK-mediated molecular sensing mechanism to initiate NADPH oxidase-dependent suicidal NETosis in response to increasing concentrations of LPS, and Gram-negative bacteria. The LPS-TLR4-JNK activation axis determines the fate of these cells: to be or not to be NETotic neutrophils.

The lungs of newborn rats exposed to 60% oxygen for 14 days develop an injury that shares morphologic similarities to human bronchopulmonary dysplasia (BPD). Neutrophil influx into the lung, as part of an inflammatory response, may play a pivotal role in the development of BPD. A neutrophil chemokine, cytokine-induced neutrophil chemoattractant-1, which signals through the neutrophil CXC chemokine receptor-2, is increased in the lung tissue of newborn rats exposed to 60% oxygen. The purpose of this study was to explore the role of neutrophils in the rat model of BPD by inhibiting neutrophil influx using SB265610, a selective CXC chemokine receptor-2 antagonist. SB265610, administered to 60% oxygen-exposed newborn rats from birth to 14 days, completely inhibited neutrophil influx. It also attenuated increased production of reactive oxygen species in newborn rat lung tissue after exposure to 60% oxygen for 4 days. Lung morphometric analysis revealed that 60% oxygen for 14 days, when accompanied by treatment with SB265610 to prevent neutrophil accumulation, increased alveolar formation over that seen in newborn rats exposed to air. These data suggest that exposure of the neonatal lung to moderate hyperoxia may enhance postnatal lung growth, provided postnatal pulmonary inflammation is suppressed.

Cystic fibrosis (CF) lung disease is characterized by chronic neutrophilic inflammation and infection. Effective management of airway inflammation could complement other therapies for the treatment of CF. Recent progress has been made in understanding the signaling pathways regulating inflammatory cytokines in the lung. Here we examine the mechanisms responsible for inflammation in the CF lung, and discuss potential therapeutic strategies targeting inflammation.

BackgroundAmong patients with cystic fibrosis (CF), females have worse pulmonary function and survival than males, primarily due to chronic lung inflammation and infection with Pseudomonas aeruginosa (P. aeruginosa). A role for gender hormones in the causation of the CF "gender gap" has been proposed. The female gender hormone 17β-estradiol (E2) plays a complex immunomodulatory role in humans and in animal models of disease, suppressing inflammation in some situations while enhancing it in others. Helper T-cells were long thought to belong exclusively to either T helper type 1 (Th1) or type 2 (Th2) lineages. However, a distinct lineage named Th17 is now recognized that is induced by interleukin (IL)-23 to produce IL-17 and other pro-inflammatory Th17 effector molecules. Recent evidence suggests a central role for the IL-23/IL-17 pathway in the pathogenesis of CF lung inflammation. We used a mouse model to test the hypothesis that E2 aggravates the CF lung inflammation that occurs in response to airway infection with P. aeruginosa by a Th17-mediated mechanism.ResultsExogenous E2 caused adult male CF mice with pneumonia due to a mucoid CF clinical isolate, the P. aeruginosa strain PA508 (PA508), to develop more severe manifestations of inflammation in both lung tissue and in bronchial alveolar lavage (BAL) fluid, with increased total white blood cell counts and differential and absolute cell counts of polymorphonuclear leukocytes (neutrophils). Inflammatory infiltrates and mucin production were increased on histology. Increased lung tissue mRNA levels for IL-23 and IL-17 were accompanied by elevated protein levels of Th17-associated pro-inflammatory mediators in BAL fluid. The burden of PA508 bacteria was increased in lung tissue homogenate and in BAL fluid, and there was a virtual elimination in lung tissue of mRNA for lactoferrin, an antimicrobial peptide active against P. aeruginosa in vitro.ConclusionsOur data show that E2 increases the severity of PA508 pneumonia in adult CF male mice, and suggest two potential mechanisms: enhancement of Th17-regulated inflammation and suppression of innate antibacterial defences. Although this animal model does not recapitulate all aspects of human CF lung disease, our present findings argue for further investigation of the effects of E2 on inflammation and infection with P. aeruginosa in the CF lung.

Genetic defects in cystic fibrosis (CF) transmembrane conductance regulator (CFTR) gene cause CF. Infants with CFTR mutations show a peribronchial neutrophil infiltration prior to the establishment of infection in their lung. The inflammatory response progressively increases in children that include both upper and lower airways. Infectious and inflammatory response leads to an increase in mucus viscosity and mucus plugging of small and medium-size bronchioles. Eventually, neutrophils chronically infiltrate the airways with biofilm or chronic bacterial infection. Perpetual infection and airway inflammation destroy the lungs, which leads to increased morbidity and eventual mortality in most of the patients with CF. Studies have now established that neutrophil cytotoxins, extracellular DNA, and neutrophil extracellular traps (NETs) are associated with increased mucus clogging and lung injury in CF. In addition to opportunistic pathogens, various aspects of the CF airway milieux (e.g., airway pH, salt concentration, and neutrophil phenotypes) influence the NETotic capacity of neutrophils. CF airway milieu may promote the survival of neutrophils and eventual pro-inflammatory aberrant NETosis, rather than the anti-inflammatory apoptotic death in these cells. Degrading NETs helps to manage CF airway disease; since DNAse treatment release cytotoxins from the NETs, further improvements are needed to degrade NETs with maximal positive effects. Neutrophil-T cell interactions may be important in regulating viral infection-mediated pulmonary exacerbations in patients with bacterial infections. Therefore, clarifying the role of neutrophils and NETs in CF lung disease and identifying therapies that preserve the positive effects of neutrophils, while reducing the detrimental effects of NETs and cytotoxic components, are essential in achieving innovative therapeutic advances.

pH is highly variable in different tissues and affects many enzymatic reactions in neutrophils. In response to calcium ionophores such as A23187 and ionomycin, neutrophils undergo nicotinamide adenine dinucleotide phosphate oxidase (NOX)-independent neutrophil extracellular trap (NET) formation (NETosis). However, how pH influences calcium-dependent Nox-independent NET formation is not well understood. We hypothesized that increasing pH promotes Nox-independent NET formation by promoting calcium influx, mitochondrial reactive oxygen species (mROS) generation, histone citrullination, and histone cleavage. Here, we show that stimulating human neutrophils isolated from peripheral blood with calcium ionophore A23187 or ionomycin in the media with increasing extracellular pH (6.6, 6.8, 7.0, 7.2, 7.4, 7.8) drastically increases intracellular pH within in 10–20 min. These intracellular pH values are much higher compared to unstimulated cells placed in the media with corresponding pH values. Raising pH slightly drastically increases intracellular calcium concentration in resting and stimulated neutrophils, respectively. Like calcium, mROS generation also increases with increasing pH. An mROS scavenger, MitoTempo, significantly suppresses calcium ionophore-mediated NET formation with a greater effect at higher pH, indicating that mROS production is at least partly responsible for pH-dependent suppression of Nox-independent NETosis. In addition, raising pH increases PAD4 activity as determined by the citrullination of histone (CitH3) and histone cleavage determined by Western blots. The pH-dependent histone cleavage is reproducibly very high during ionomycin-induced NETosis compared to A23187-induced NETosis. Little or no histone cleavage was noted in unstimulated cells, at any pH. Both CitH3 and cleavage of histones facilitate DNA decondensation. Therefore, alkaline pH promotes intracellular calcium influx, mROS generation, PAD4-mediated CitH3 formation, histone 4 cleavage and eventually NET formation. Calcium-mediated NET formation and CitH3 formation are often related to sterile inflammation. Hence, understanding these important mechanistic steps helps to explain how pH regulates NOX-independent NET formation, and modifying pH may help to regulate NET formation during sterile inflammation or potential damage caused by compounds such as ionomycin, secreted by Streptomyces, a group of Gram-positive bacteria well known for producing antibiotics.

We used differential display-PCR (DD-PCR) to identify glucocorticoid-inducible genes that regulate lung development in late gestation. DD-PCR, a method to screen for differentially expressed genes, is based on a comparison of mRNAs isolated from a subset of two or more cell populations by analysis of RT-PCR products on DNA-sequencing gels. We isolated cDNA probes representing mRNAs expressed in primary cultures of rat lung fibroblasts, but not in epithelial cells, on fetal day 20. A day 20 glucocorticoid-treated fibroblast cDNA library was screened with a single probe to isolate the 3.1-kb cDNA late-gestation lung 1( LGL1; GenBank accession no. AF109674 ) encoding a deduced polypeptide of 188 amino acids. Northern analysis confirmed that LGL1is expressed in human, rat, and mouse fetal lungs, induced by glucocorticoid, developmentally regulated in fibroblasts but not detectable in epithelium. In situ hybridization confirmed LGL1 expression in the mesenchyme, but not in the epithelium, of fetal rat lung, kidney, and gut. The predicted LGL1 gene product (lgl1) showed 81% homology to P25TI, a polypeptide trypsin inhibitor recently identified in human glioblastoma and neuroblastoma cells but not detected in normal human tissues. Both lgl1 and P25TI belong to the CRISP family of cysteine-rich extracellular proteins. Trypsin is produced by both normal bronchial epithelial and lung adenocarcinoma cells. Although additional studies will be necessary to clearly establish a functional role for lgl1, we propose that lgl1 has a role in normal lung development that is likely to be via regulation of extracellular matrix degradation.

Cystic fibrosis (CF) is a complex, multi-system, autosomal recessive disease predominantly affecting Caucasians that leads to vigorous airway inflammation and chronic respiratory infection, commonly with Pseudomonas aeruginosa. A variety of factors significantly modify the progression and severity of CF lung disease and the timing of the resulting mortality. We summarize here data indicating that there is in CF a female disadvantage in survival and morbidity, called the "CF gender gap". Although controversy exists regarding the nature and relative importance of the various contributing mechanisms involved, gender affects the progression of CF disease with respect to lung infection, decline in pulmonary function and nutritional status. These interrelated factors in turn have a negative impact on survival. This review will emphasize the increasing evidence that suggest a role for the effects of gender, and particularly the female sex hormone estrogen, on infection, inflammation and transepithelial ion transport, all major determinants of CF lung disease. Future elucidation of the pathophysiology of hormonal aggravation of CF lung disease may pave the way for novel therapeutic interventions. This, combined with the magnitude of the gender gap in CF mortality, strongly suggests that further work in this field is well justified.