Extracellular vesicles secreted by primary human bronchial epithelial cells reduce Pseudomonas aeruginosa burden and inflammation in cystic fibrosis mouse lung.

Cystic fibrosis (CF) results in a reduction in the volume of airway surface liquid, increased accumulation of viscous mucus, persistent antibiotic-resistant lung infections that cause chronic inflammation, and a decline in lung function. More than 50% of adults with CF are chronically colonized by Pseudomonas aeruginosa (P. aeruginosa), the primary reason for morbidity and mortality in people with CF (pwCF). Although highly effective modulator therapy (HEMT) is an important part of disease management in CF, HEMT does not eliminate P. aeruginosa or lung inflammation. Thus, new treatments are required to reduce lung infection and inflammation in CF. In a previous in vitro study, we demonstrated that primary human bronchial epithelial cells (HBECs) secrete extracellular vesicles (EVs) that block the ability of P. aeruginosa to form biofilms by reducing the abundance of several proteins necessary for biofilm formation as well as enhancing the sensitivity of P. aeruginosa to ß-lactam antibiotics. In this study, using a CF mouse model of P. aeruginosa infection, we demonstrate that intratracheal administration of EVs secreted by HBEC reduced P. aeruginosa lung burden and several proinflammatory cytokines including IFN-γ, TNF-α, and MIP-1ß in bronchoalveolar lavage fluid (BALF), even in the absence of antibiotics. Moreover, EVs decreased neutrophils in BALF. Thus, EVs secreted by HBEC reduce the lung burden of P. aeruginosa, decrease inflammation, and reduce neutrophils in a CF mouse model. These results suggest that HBEC via the secretion of EVs may play an important role in the immune response to P. aeruginosa lung infection.NEW & NOTEWORTHY Our findings show that extracellular vesicles secreted by primary human bronchial epithelial cells significantly reduce Pseudomonas aeruginosa burden, inflammation, and weight loss in a cystic fibrosis mouse model of infection.

A pilot study of cystic fibrosis exacerbation response phenotypes reveals contrasting serum and sputum iron trends.

The cystic fibrosis (CF) community seeks to explain heterogeneous outcomes of pulmonary exacerbation (PEX) treatment. Serum and sputum inflammatory mediators may identify people with CF (PwCF) at risk for suboptimal responses. However, lack of an established association between response phenotypes and these mediators limits clinical application. In this pilot study, we prospectively characterized treatment response phenotypes by assessing health-related quality-of-life (HRQoL) during PEX. We also measured lung function and iron-related biochemical parameters in serum and sputum. We classified subjects as sustained symptom-responders (SRs) or non-sustained symptom-responders (NSRs) based on the absence or presence, respectively, of worsened symptom scores after initial improvement. We used linear mixed models (LMMs) to determine whether trends in lung function, hematologic, serum, and sputum indices of inflammation differed between response cohorts. In 20 PwCF, we identified 10 SRs and 10 NSRs with no significant differences in lung function at PEX onset and treatment durations. SRs had better model-predicted trends in lung function than NSRs during PEX. Non-linear trends in serum and sputum iron levels significantly differed between SRs and NSRs. In adults with cystic fibrosis, PEX treatment response phenotypes may be correlated with distinctive trends in serum and sputum iron concentrations.

Genome-wide DNA methylation profiling shows a distinct epigenetic signature associated with lung macrophages in cystic fibrosis.

BACKGROUND: Lung macrophages are major participants in the pulmonary innate immune response. In the cystic fibrosis (CF) lung, the inability of lung macrophages to successfully regulate the exaggerated inflammatory response suggests dysfunctional innate immune cell function. In this study, we aim to gain insight into innate immune cell dysfunction in CF by investigating alterations in DNA methylation in bronchoalveolar lavage (BAL) cells, composed primarily of lung macrophages of CF subjects compared with healthy controls. All analyses were performed using primary alveolar macrophages from human subjects collected via bronchoalveolar lavage. Epigenome-wide DNA methylation was examined via Illumina MethylationEPIC (850 K) array. Targeted next-generation bisulfite sequencing was used to validate selected differentially methylated CpGs. Methylation-based sample classification was performed using the recursively partitioned mixture model (RPMM) and was tested against sample case-control status. Differentially methylated loci were identified by fitting linear models with adjustment of age, sex, estimated cell type proportions, and repeat measurement. RESULTS: RPMM class membership was significantly associated with the CF disease status (P = 0.026). One hundred nine CpG loci were differentially methylated in CF BAL cells (all FDR ≤ 0.1). The majority of differentially methylated loci in CF were hypo-methylated and found within non-promoter CpG islands as well as in putative enhancer regions and DNase hyper-sensitive regions. CONCLUSIONS: These results support a hypothesis that epigenetic changes, specifically DNA methylation at a multitude of gene loci in lung macrophages, may participate, at least in part, in driving dysfunctional innate immune cells in the CF lung.

Pulmonary microRNA profiling: implications in upper lobe predominant lung disease.

BACKGROUND: Numerous pulmonary diseases manifest with upper lobe predominance including cystic fibrosis, smoking-related chronic obstructive pulmonary disease, and tuberculosis. Zonal hypoxia, characteristic of these pulmonary maladies, and oxygen stress in general is known to exert profound effects on various important aspects of cell biology. Lung macrophages are major participants in the pulmonary innate immune response and regional differences in macrophage responsiveness to hypoxia may contribute in the development of lung disease. MicroRNAs are ubiquitous regulators of human biology and emerging evidence indicates altered microRNA expression modulates respiratory disease processes. The objective of this study is to gain insight into the epigenetic and cellular mechanisms influencing regional differences in lung disease by investigating effect of hypoxia on regional microRNA expression in the lung. All studies were performed using primary alveolar macrophages (n = 10) or bronchoalveolar lavage fluid (n = 16) isolated from human subjects. MicroRNA was assayed via the NanoString nCounter microRNA assay. RESULTS: Divergent molecular patterns of microRNA expression were observed in alternate lung lobes, specifically noted was disparate expression of miR-93 and miR-4454 in alveolar macrophages along with altered expression of miR-451a and miR-663a in bronchoalveolar lavage fluid. Gene ontology was used to identify potential downstream targets of divergent microRNAs. Targets include cytokines and matrix metalloproteinases, molecules that could have a significant impact on pulmonary inflammation and fibrosis. CONCLUSIONS: Our findings show variant regional microRNA expression associated with hypoxia in alveolar macrophages and BAL fluid in the lung-upper vs lower lobe. Future studies should address whether these specific microRNAs may act intracellularly, in a paracrine/endocrine manner to direct the innate immune response or may ultimately be involved in pulmonary host-to-pathogen trans-kingdom cross-talk.

Low levels of insulin-like growth factor-1 contribute to alveolar macrophage dysfunction in cystic fibrosis.

Alveolar macrophages are major contributors to lung innate immunity. Although alveolar macrophages from cystic fibrosis (CF) transmembrane conductance regulator(-/-) mice have impaired function, no study has investigated primary alveolar macrophages in adults with CF. CF patients have low levels of insulin-like growth factor 1 (IGF-1), and our prior studies demonstrate a relationship between IGF-1 and macrophage function. We hypothesize that reduced IGF-1 in CF leads to impaired alveolar macrophage function and chronic infections. Serum and bronchoalveolar lavage (BAL) samples were obtained from eight CF subjects and eight healthy subjects. Macrophages were isolated from BAL fluid. We measured the ability of alveolar macrophages to kill Pseudomonas aeruginosa. Subsequently, macrophages were incubated with IGF-1 prior to inoculation with bacteria to determine the effect of IGF-1 on bacterial killing. We found a significant decrease in bacterial killing by CF alveolar macrophages compared with control subjects. CF subjects had lower serum and BAL IGF-1 levels compared with healthy control subjects. Exposure to IGF-1 enhanced alveolar macrophage macrophages in both groups. Finally, exposing healthy alveolar macrophages to CF BAL fluid decreased bacterial killing, and this was reversed by the addition of IGF-1, whereas IGF-1 blockade worsened bacterial killing. Our studies demonstrate that alveolar macrophage function is impaired in patients with CF. Reductions in IGF-1 levels in CF contribute to the impaired alveolar macrophage function. Exposure to IGF-1 ex vivo results in improved function of CF alveolar macrophages. Further studies are needed to determine whether alveolar macrophage function can be enhanced in vivo with IGF-1 treatment.

Insulin-like growth factor-1 levels contribute to the development of bacterial translocation in sepsis.

RATIONALE: Many lines of evidence point toward the gastrointestinal (GI) tract in the pathophysiology of organ dysfunction in sepsis. Splanchnic hypoperfusion during sepsis leads to enterocyte apoptosis, diminished barrier function, and release of bacterial products. Sepsis lowers levels of insulin-like growth factor (IGF)-1, a known antiapoptotic factor. We recently demonstrated that treatment with IGF-1 is protective in murine sepsis. OBJECTIVES: We hypothesize that decreased IGF-1 levels in sepsis contributes to the development of bacterial translocation. METHODS: Sepsis was induced in C57BL/6 mice via intratracheal instillation of Pseudomonas aeruginosa. Human subjects with sepsis were enrolled if they had a documented positive blood culture with a nonenteric organism. Bacterial translocation was measured in serum by quantitative real-time polymerase chain reaction with primers specific for enteric bacteria. Serum IGF-1 was measured by ELISA. Apoptosis of the GI epithelium was assessed via immunohistochemistry. MEASUREMENTS AND MAIN RESULTS: We found that mice with severe sepsis had evidence of bacterial translocation by 24 hours. Enteric bacterial load correlated inversely with levels of serum IGF-1. If we treated mice with IGF-1, bacterial translocation was significantly decreased. In addition, we found increased GI epithelial cell apoptosis after sepsis, which was significantly decreased after IGF-1 treatment. Human subjects with nonenteric sepsis developed progressive enteric bacteremia over 3 days. The degree of enteric bacteremia correlated inversely with serum IGF-1 levels. CONCLUSIONS: These data support the hypothesis that sepsis-induced reductions in IGF-1 levels contribute to the development of bacterial translocation in both a murine model and human subjects.

Insulin-like growth factor-1 improves survival in sepsis via enhanced hepatic bacterial clearance.

RATIONALE: Both insulin-like growth factor (IGF)-1 and bacterial clearance by Kupffer cells are significantly reduced in severe sepsis. Kupffer cell apoptosis is triggered by tumor necrosis factor (TNF)-alpha and activation of the PI-3 kinase pathway prevents TNF-induced Kupffer cell death. OBJECTIVES: We evaluated if the marked decline in IGF-1 is related to bacterial clearance in sepsis. METHODS: Sepsis was induced in C57BL/6 mice by intratracheal inoculation with Pseudomonas aeruginosa (strain PA103). Some mice received IGF-1 24 mg/kg either before infection or 12 hours after infection. In vitro studies were performed using the clonal Kupffer cell line KC13-2. MEASUREMENTS AND MAIN RESULTS: Sepsis resulted in decreased levels of IGF-1. In vitro studies with KC13-2 cells demonstrated that IGF-1 protected Kupffer cells against TNF-alpha-induced apoptosis by activating the PI-3 kinase pathway and stabilizing the inhibitor of apoptosis protein, XIAP. In the animal model, pretreatment with IGF-1 decreased hepatic TNF-alpha and IL-6, improved hepatic bacterial clearance as demonstrated by real-time polymerase chain reaction with primers specific for P. aeruginosa, and improved survival in severe sepsis. Moreover, we rescued mice from severe sepsis by IGF-1 treatment 12 hours after infection. CONCLUSIONS: These studies show that the decline in IGF-1 levels in sepsis is related to bacterial clearance and that replacement of IGF-1 in a murine model of sepsis improves overall survival.

Pseudomonas aeruginosa delays Kupffer cell death via stabilization of the X-chromosome-linked inhibitor of apoptosis protein.

Kupffer cells are important for bacterial clearance and cytokine production during infection. We have previously shown that severe infection with Pseudomonas aeruginosa ultimately results in loss of Kupffer cells and hepatic bacterial clearance. This was associated with prolonged hepatic inflammation. However, there is a period of time during which there is both preserved hepatic bacterial clearance and increased circulating TNF-alpha. We hypothesized that early during infection, Kupffer cells are protected against TNF-alpha-induced cell death via activation of survival pathways. KC13-2 cells (a clonal Kupffer cell line) were treated with P. aeruginosa (strain PA103), TNF-alpha, or both. At early time points, TNF-alpha induced caspase-mediated cell death, but PA103 did not. When we combined the two exposures, PA103 protected KC13-2 cells from TNF-alpha-induced cell death. PA103, in the setting of TNF exposure, stabilized the X-chromosome-linked inhibitor of apoptosis protein (XIAP). Stabilization of XIAP can occur via PI3K and Akt. We found that PA103 activated Akt and that pretreatment with the PI3K inhibitor, LY294002, prevented PA103-induced protection against TNF-alpha-induced cell death. The effects of LY294002 included decreased levels of XIAP and increased amounts of cleaved caspase-3. Overexpression of Akt mimicked the effects of PA103 by protecting cells from TNF-alpha-induced cell death and XIAP cleavage. Transfection with a stable, nondegradable XIAP mutant also protected cells against TNF-alpha-induced cell death. These studies demonstrate that P. aeruginosa delays TNF-alpha-induced Kupffer cell death via stabilization of XIAP.