The role of ASC signalling in preterm infants at risk of chronic lung disease of prematurity

Abstract

Introduction: Pulmonary inflammation is the hallmark in infants who develop chronic lung disease of prematurity (CLD). The NLRP3/ASC inflammasome contributes to the inflammatory process by activating caspase-1 resulting in release of interleukin (IL)-1β but its role in the development of chronic lung disease of prematurity (CLD) is unclear. Extracellular apoptosis-associated speck-like protein containing a caspase activation domain (ASC) is involved in caspase-dependent IL-1β production. Aims: We sought extracellular ASC in bronchoalveolar lavage (BAL) fluid from preterm infants and analysed the ASC induced cytokine production in respiratory epithelial cell lines in culture. Methods: Pooled BAL fluid from preterm infants was used to stimulate A549 and BEAS-2B respiratory cell lines to promote cytokine secretion. BAL fluid ASC was depleted by immunoprecipitation; recombinant ASC (rASC) was produced in E.Coli and purified by immunoaffinity chromatography. Cytokines in cell culture supernatants were measured using cytometric bead array. Results: A549 and BEAS-2B cells stimulated with neat BAL produced increased IL-6 and IL-8 but not IL-1β compared to unstimulated cells; the increases in IL-6 and IL-8 were prevented by ASC-depleted BAL fluid with increases in IL-6 and IL-8 restored by re-adding rASC to the ASC-depleted BAL fluid. Conclusions: Extracellular ASC is present in BAL fluid and is involved is promoting pro-inflammatory signalling via induction of IL-6 and IL-8 secretion, independently of the NLRP3/ASC inflammasome acting most likely via the NFκB pathway. Modulation of ASC can potentially provide a therapeutic avenue to prevent CLD.