FGF receptors mediate cellular senescence in the cystic fibrosis airway epithelium.

The number of adults living with cystic fibrosis (CF) has already increased significantly because of drastic improvements in life expectancy attributable to advances in treatment, including the development of highly effective modulator therapy. Chronic airway inflammation in CF contributes to morbidity and mortality, and aging processes like inflammaging and cell senescence influence CF pathology. Our results show that single-cell RNA sequencing data, human primary bronchial epithelial cells from non-CF and CF donors, a CF bronchial epithelial cell line, and Cftr-knockout (Cftr-/-) rats all demonstrated increased cell senescence markers in the CF bronchial epithelium. This was associated with upregulation of fibroblast growth factor receptors (FGFRs) and mitogen-activated protein kinase (MAPK) p38. Inhibition of FGFRs, specifically FGFR4 and to some extent FGFR1, attenuated cell senescence and improved mucociliary clearance, which was associated with MAPK p38 signaling. Mucociliary dysfunction could also be improved using a combination of senolytics in a CF ex vivo model. In summary, FGFR/MAPK p38 signaling contributes to cell senescence in CF airways, which is associated with impaired mucociliary clearance. Therefore, attenuation of cell senescence in the CF airways might be a future therapeutic strategy improving mucociliary dysfunction and lung disease in an aging population with CF.

O-GlcNAc transferase regulates collagen deposition and fibrosis resolution in idiopathic pulmonary fibrosis.

Background: Idiopathic pulmonary fibrosis (IPF) is a chronic pulmonary disease that is characterized by an excessive accumulation of extracellular matrix (ECM) proteins (e.g. collagens) in the parenchyma, which ultimately leads to respiratory failure and death. While current therapies exist to slow the progression, no therapies are available to resolve fibrosis. Methods: We characterized the O-linked N-Acetylglucosamine (O-GlcNAc) transferase (OGT)/O-GlcNAc axis in IPF using single-cell RNA-sequencing (scRNA-seq) data and human lung sections and isolated fibroblasts from IPF and non-IPF donors. The underlying mechanism(s) of IPF were further investigated using multiple experimental models to modulate collagen expression and accumulation by genetically and pharmacologically targeting OGT. Furthermore, we hone in on the transforming growth factor-beta (TGF-ß) effector molecule, Smad3, by co-expressing it with OGT to determine if it is modified and its subsequent effect on Smad3 activation. Results: We found that OGT and O-GlcNAc levels are upregulated in patients with IPF compared to non-IPF. We report that the OGT regulates collagen deposition and fibrosis resolution, which is an evolutionarily conserved process demonstrated across multiple species. Co-expression of OGT and Smad3 showed that Smad3 is O-GlcNAc modified. Blocking OGT activity resulted in decreased phosphorylation at Ser-423/425 of Smad3 attenuating the effects of TGF-ß1 induced collagen expression/deposition. Conclusion: OGT inhibition or knockdown successfully blocked and reversed collagen expression and accumulation, respectively. Smad3 is discovered to be a substrate of OGT and its O-GlcNAc modification(s) directly affects its phosphorylation state. These data identify OGT as a potential target in pulmonary fibrosis resolution, as well as other diseases that might have aberrant ECM/collagen accumulation.

Fibroblast Growth Factor 23 Signaling Does Not Increase Inflammation from Pseudomonas aeruginosa Infection in the Cystic Fibrosis Bronchial Epithelium.

Background and Objectives: Chronic inflammation due to Pseudomonas aeruginosa (PA) infection in people with cystic fibrosis (CF) remains a concerning issue in the wake of modulator therapy initiation. Given the perpetuating cycle of colonization, infection, chronic inflammation, and recurrent injury to the lung, there are increases in the risk for mortality in the CF population. We have previously shown that fibroblast growth factor (FGF) 23 can exaggerate transforming growth factor (TGF) beta-mediated bronchial inflammation in CF. Our study aims to shed light on whether FGF23 signaling also plays a role in PA infection of the CF bronchial epithelium. Materials and Methods: CF bronchial epithelial cells were pretreated with FGF23 or inhibitors for FGF receptors (FGFR) and then infected with different PA isolates. After infection, immunoblot analyses were performed on these samples to assess the levels of phosphorylated phospholipase C gamma (PLCγ), total PLCγ, phosphorylated extracellular signal-regulated kinase (ERK), and total ERK. Additionally, the expression of FGFRs and interleukins at the transcript level (RT-qPCR), as well as production of interleukin (IL)-6 and IL-8 at the protein level (ELISA) were determined. Results: Although there were decreases in isoform-specific FGFRs with increases in interleukins at the mRNA level as well as phosphorylated PLCγ and the production of IL-8 protein with PA infection, treatment with FGF23 or FGFR blockade did not alter downstream targets such as IL-6 and IL-8. Conclusions: FGF23 signaling does not seem to modulate the PA-mediated inflammatory response of the CF bronchial epithelium.

A novel in vitro model to study prolonged Pseudomonas aeruginosa infection in the cystic fibrosis bronchial epithelium.

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.

Phosphate induces inflammation and exacerbates injury from cigarette smoke in the bronchial epithelium.

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.

ST6GAL1 and α2-6 Sialylation Regulates IL-6 Expression and Secretion in Chronic Obstructive Pulmonary Disease.

Chronic obstructive pulmonary disease (COPD) is a systemic disease strongly associated with cigarette smoking, airway inflammation, and acute disease exacerbations. Changes in terminal sialylation and fucosylation of asparagine (N)-linked glycans have been documented in COPD, but the role that glycosyltransferases may play in the regulation of N-linked glycans in COPD has not been fully elucidated. Recent studies suggest that modulation of ST6GAL1 (ST6 beta-galactoside alpha-2,6-sialyltransferase-1), which catalyzes terminal α2-6 sialylation of cellular proteins, may regulate inflammation and contribute to COPD phenotype(s). Interestingly, it has been previously demonstrated that ST6GAL1, a Golgi resident protein, can be proteolytically processed by BACE1 (beta-site amyloid precursor protein cleaving enzyme-1) to a circulating form that retains activity. In this study, we showed that loss of ST6GAL1 expression increased interleukin (IL)-6 expression and secretion in human bronchial epithelial cells (HBECs). Furthermore, exposure to cigarette smoke medium/extract (CSE) or BACE1 inhibition resulted in decreased ST6GAL1 secretion, reduced α2-6 sialylation, and increased IL-6 production in HBECs. Analysis of plasma ST6GAL1 levels in a small COPD patient cohort demonstrated an inverse association with prospective acute exacerbations of COPD (AECOPD), while IL-6 was positively associated. Altogether, these results suggest that reduced ST6GAL1 and α2-6 sialylation augments IL-6 expression/secretion in HBECs and is associated with poor clinical outcomes in COPD.

Targeting Aging Pathways in Chronic Obstructive Pulmonary Disease.

Chronic obstructive pulmonary disease (COPD) has become a global epidemic and is the third leading cause of death worldwide. COPD is characterized by chronic airway inflammation, loss of alveolar-capillary units, and progressive decline in lung function. Major risk factors for COPD are cigarette smoking and aging. COPD-associated pathomechanisms include multiple aging pathways such as telomere attrition, epigenetic alterations, altered nutrient sensing, mitochondrial dysfunction, cell senescence, stem cell exhaustion and chronic inflammation. In this review, we will highlight the current literature that focuses on the role of age and aging-associated signaling pathways as well as their impact on current treatment strategies in the pathogenesis of COPD. Furthermore, we will discuss established and experimental COPD treatments including senolytic and anti-aging therapies and their potential use as novel treatment strategies in COPD.

Osteogenic benefits of low-intensity pulsed ultrasound and vibration in a rodent osseointegration model.

OBJECTIVES: Osseointegrated prostheses are increasingly used for amputees, however, the lengthy rehabilitation time of these prostheses remains a challenge to their implementation. The aim of this study was to investigate the ability of locally applied vibration or low-intensity pulsed ultrasound (LIPUS) to accelerate osseointegration and increase peri-implant bone volume. METHODS: A 4-week and 8-week rodent study were conducted in a femoral intramedullary implant model (control, vibration, LIPUS, and combined treatment) to determine effects on healing. Osseointegration was evaluated quantitatively through mechanical, µCT and histological evaluations. RESULTS: Maximum pushout load at 4 weeks increased with LIPUS relative to control (37.7%, P=0.002). Histologically, LIPUS and vibration separately increased peri-implant bone formation after 4 weeks relative to control. Vibration resulted in greater peri-implant bone after 8 weeks than all other groups (25.7%, P<0.001). However, no significant group differences in pushout load were noted at 8 weeks. CONCLUSIONS: Although vibration increased bone around implants, LIPUS was superior to vibration for accelerating osseointegration and increasing bone-implant failure loads at 4 weeks. However, the LIPUS benefits on osseointegration at 4 weeks were not sustained at 8 weeks.