Faulk, Christopher

Exposure of airway epithelia to environmental allergens or nanoparticles invokes a novel genome-protective physiological response to oxidative stress. Fragments of double-stranded genomic DNA released into the airway lumen originate from repetitive, transcriptionally active, and fragile sites within the genome. Exporting DNA with high repetitive content potentially aids cell survival through the preservation of genome integrity and fidelity during the repair process.

Exposure of human bronchial epithelial cells (hBECs) and mouse airways to allergens from the fungus Alternaria alternata triggers the release of alarmin signaling molecules including ATP, IL-33 and DNA fragments. The goal of this study was to determine the mechanisms that regulate release of genomic DNA fragments and how extracellular DNA (eDNA) contributes to type 2 immunity. Treatment of hBECs with Alternaria extract induces oxidative stress, which leads to ATP release and uptake of Ca2+ following activation of P2X receptors. DNA release was dependent on a sustained increase in intracellular [Ca2+], which stimulates cleavage of caspase 3 by the proprotein convertase enzyme furin, resulting in activation of caspase 3 and ultimately nuclear DNA fragmentation as shown in comet assays. Some of the fragmented DNA was released into the media under conditions where barrier function of the epithelium was maintained, and nuclear and plasma membranes remained intact. Alternaria-induced DNA release was detected within 30 minutes following exposure, reached its peak at 4 hours and ceased after 12 hours. Pretreatment of hBECs with a calpain inhibitor resulted in continued DNA release and loss of cell viability by 24 hours. This result suggested that calpain activity contributed to termination of Alternaria-evoked DNA release. In mice, intranasal administration of Alternaria extract produced release of DNA fragments into bronchoalveolar lavage (BAL) fluid over a similar time course as observed in hBECs. In caspase 3 deficient mice however, DNA release into BAL fluid was significantly reduced. Furthermore, Alternaria-induced type 2 immune responses were suppressed following pretreatment with a DNA scavenger. Conversely, intranasal administration of mouse genomic DNA with Alternaria amplified IL-5 and IL-13 secretion into BAL fluid while DNA alone had no effect. These findings indicate that DNA release from the airway epithelium is a regulated process involving noncanonical activation of caspase 3 to produce DNA fragmentation and calpain activity, which appears to be essential for the termination of DNA release. Moreover in airways, eDNA appears to function as a secondary alarmin that acts on immune cells to enhance type 2 immunity. This work was supported by grants from the NIH (AI128729 and AI169530) to HK and SMO. This is the full abstract presented at the American Physiology Summit 2023 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.