The demographics of the population with cystic fibrosis (CF) is continuously changing, with nowadays adults outnumbering children and a median predicted survival of over 40 years. This leads to the challenge of treating an aging CF population, while previous research has largely focused on pediatric and adolescent patients. Chronic inflammation is not only a hallmark of CF lung disease, but also of the aging process. However, very little is known about the effects of an accelerated aging pathology in CF lungs. Several chronic lung disease pathologies show signs of chronic inflammation with accelerated aging, also termed “inflammaging”; the most notable being chronic obstructive pulmonary disease (COPD) and idiopathic pulmonary fibrosis (IPF). In these disease entities, accelerated aging has been implicated in the pathogenesis via interference with tissue repair mechanisms, alterations of the immune system leading to impaired defense against pulmonary infections and induction of a chronic pro-inflammatory state. In addition, CF lungs have been shown to exhibit increased expression of senescence markers. Sustained airway inflammation also leads to the degradation and increased turnover of cystic fibrosis transmembrane regulator (CFTR). This further reduces CFTR function and may prevent the novel CFTR modulator therapies from developing their full efficacy. Therefore, novel therapies targeting aging processes in CF lungs could be promising. This review summarizes the current research on CF in an aging population focusing on accelerated aging in the context of chronic airway inflammation and therapy implications.

An elevation in serum phosphate—also called hyperphosphatemia—is associated with reduced kidney function in chronic kidney disease (CKD). Reports show CKD patients are more likely to develop lung disease and have poorer kidney function that positively correlates with pulmonary obstruction. However, the underlying mechanisms are not well understood. Here, we report that two murine models of CKD, which both exhibit increased serum levels of phosphate and fibroblast growth factor (FGF) 23, a regulator of phosphate homeostasis, develop concomitant airway inflammation. Our in vitro studies point towards a similar increase of phosphate-induced inflammatory markers in human bronchial epithelial cells. FGF23 stimulation alone does not induce a proinflammatory response in the non-COPD bronchial epithelium and phosphate does not cause endogenous FGF23 release. Upregulation of the phosphate-induced proinflammatory cytokines is accompanied by activation of the extracellular-signal regulated kinase (ERK) pathway. Moreover, the addition of cigarette smoke extract (CSE) during phosphate treatments exacerbates inflammation as well as ERK activation, whereas co-treatment with FGF23 attenuates both the phosphate as well as the combined phosphate- and CS-induced inflammatory response, independent of ERK activation. Together, these data demonstrate a novel pathway that potentially explains pathological kidney-lung crosstalk with phosphate as a key mediator.

OBJECTIVES/GOALS: Using a cell culture model, we will determine the effects of phosphate on primary lung cell cultures and use our results to delineate a pathway through which these changes are carried out. Using animal models, we will determine the effects of phosphate on inflammatory and fibrotic lung injury, both in the presence and absence of CKD. METHODS/STUDY POPULATION: For our in vitro experiments, human lung fibroblasts were treated with concentrations of 1 to 5 mM sodium phosphate and FGFR inhibitors. Expression levels of interleukin (IL)-1beta, IL-6, and IL-8 were analyzed by qPCR and secretion of these cytokines was measured by ELISA. Phosphorylation of PLCy and ERK was measured by western blot. Using an in vivo approach, we placed C57Bl/6 mice on a high phosphate (3%) diet to elevate serum phosphate levels in the absence of kidney injury and administered bleomycin via oropharyngeal aspiration to generate an acute inflammatory response. Serum FGF23 levels were measured by ELISA and serum analysis for phosphate and renal function were obtained. Furthermore, expression of FGF23 pathway and inflammatory markers were analyzed in murine lung tissue using qPCR and western blotting. RESULTS/ANTICIPATED RESULTS: Augmented phosphate concentrations led to increased cytokine expression and secretion from human lung fibroblasts as well as a concomitant increase in PLCy and ERK phosphorylation. Inhibition of FGFR1 reversed the effects of phosphate on the inflammatory cytokines and PLCy/ERK phosphorylation. Serum FGF23 levels were significantly upregulated in mice on a high phosphate diet and further increased in mice subjected to a high phosphate diet with exposure to bleomycin. Both serum phosphate and creatinine levels were significantly elevated as well. Additionally, high phosphate and bleomycin increased local FGF23 expression in murine lung tissue, when compared to controls or each stimulus alone. DISCUSSION/SIGNIFICANCE: Phosphate has a significant impact on inflammation and fibrosis in the lung, indicating that the existence of pulmo-renal crosstalk exaggerates pulmonary injury and that there are biological pathways that may be targeted therapeutically to mediate these effects. These results could have a substantial impact on the quality of life for CKD patients.

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Pseudomonas aeruginosa (PA) is known to chronically infect airways of people with cystic fibrosis (CF) by early adulthood. PA infections can lead to increased airway inflammation and lung tissue damage, ultimately contributing to decreased lung function and quality of life. Existing models of PA infection in vitro commonly utilize 1–6-hour time courses. However, these relatively early time points may not encompass downstream airway cell signaling in response to the chronic PA infections observed in people with cystic fibrosis. To fill this gap in knowledge, the aim of this study was to establish an in vitro model that allows for PA infection of CF bronchial epithelial cells, cultured at the air liquid interface, for 24 hours. Our model shows with an inoculum of 2 x 102 CFUs of PA for 24 hours pro-inflammatory markers such as interleukin 6 and interleukin 8 are upregulated with little decrease in CF bronchial epithelial cell survival or monolayer confluency. Additionally, immunoblotting for phosphorylated phospholipase C gamma, a well-known downstream protein of fibroblast growth factor receptor signaling, showed significantly elevated levels after 24 hours with PA infection that were not seen at earlier timepoints. Finally, inhibition of phospholipase C shows significant downregulation of interleukin 8. Our data suggest that this newly developed in vitro “prolonged PA infection model” recapitulates the elevated inflammatory markers observed in CF, without compromising cell survival. This extended period of PA growth on CF bronchial epithelial cells will have impact on further studies of cell signaling and microbiological studies that were not possible in previous models using shorter PA exposures.

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