cAMP signaling primes lung endothelial cells to activate caspase-1 during Pseudomonas aeruginosa infection

Abstract

Activation of the inflammasome-caspase-1 axis in lung endothelial cells is emerging as a novel arm of the innate immune response to pneumonia and sepsis caused by Pseudomonas aeruginosa. Increased levels of circulating autacoids are hallmarks of pneumonia and sepsis and induce physiological responses via cAMP signaling in targeted cells. However, it is unknown whether cAMP affects other functions, such as P. aeruginosa-induced caspase-1 activation. Herein, we describe the effects of cAMP signaling on caspase-1 activation using a single cell flow cytometry-based assay. P. aeruginosa infection of cultured lung endothelial cells caused caspase-1 activation in a distinct population of cells. Unexpectedly, pharmacological cAMP elevation increased the total number of lung endothelial cells with activated caspase-1. Interestingly, addition of cAMP agonists augmented P. aeruginosa infection of lung endothelial cells as a partial explanation underlying cAMP priming of caspase-1 activation. The cAMP effect(s) appeared to function as a priming signal because addition of cAMP agonists was required either before or early during the onset of infection. However, absolute cAMP levels measured by ELISA were not predictive of cAMP-priming effects. Importantly, inhibition of de novo cAMP synthesis decreased the number of lung endothelial cells with activated caspase-1 during infection. Collectively, our data suggest that lung endothelial cells rely on cAMP signaling to prime caspase-1 activation during P. aeruginosa infection.

ExoU Induces Lung Endothelial Cell Damage and Activates Pro-Inflammatory Caspase-1 during Pseudomonas aeruginosa Infection

“…The importance of ExoU in disrupting host responses directed by lung endothelial cells is an emerging field of interest [32,[36][37][38][39][40]. Our previous work has shown that P. aeruginosa infection of PMVECs induces activation of caspase-1-dependent inflammation [44,45]. Intriguingly, the present study strongly suggests ExoU elicits a specific pattern of caspase-1 activation in PMVECs.…”

“…In innate immune cells such as macrophages, caspase-1 activation typically leads to its release to the extracellular milieu where it can be measured [69]. However, we recently demonstrated that P. aeruginosa infection of PMVECs triggers caspase-1 activation that can be measured with an intracellular staining and flow cytometry technique (FLICA) [45]. The data presented herein suggest that ExoU specifically elicits intracellular caspase-1 activation in PMVECs, and the effect is dependent on ExoU PLA 2 activity.…”

“…We have previously determined that PA103 infection of PMVECs elicits activation of the inflammasome-caspase-1 axis [44,45]. Inflammasomes are intracellular pattern recognition receptors whose activation triggers inflammation via pro-caspase-1 autoproteolysis, subsequent processing of pro-IL-1β and pro-IL-18, and Gasdermin D activation to execute a rapid form of cell death termed pyroptosis [46][47][48][49][50].…”

“…Inflammasomes are intracellular pattern recognition receptors whose activation triggers inflammation via pro-caspase-1 autoproteolysis, subsequent processing of pro-IL-1β and pro-IL-18, and Gasdermin D activation to execute a rapid form of cell death termed pyroptosis [46][47][48][49][50]. We have previously validated the use of a membrane permeable, irreversible Fluorescent Labeled Inhibitor of CAspase-1 (FLICA, FAM-YVAD-FMK), and flow cytometry as a single cell assay to identify PMVECs with activated caspase-1 [45]. In uninfected PMVECs, FLICA is freely diffusible and readily washed out of the cell.…”

“…PMVEC culture and infection conditions were previously described [45]. Briefly, PMVECs were harvested one day prior to infection by treatment with Trypsin-EDTA (4 min at 37 • C, 5% CO 2 ) and washed by centrifugation (500× g, 4 min, ambient temperature) into DMEM FBS.…”