CAMP Potentiates Caspase‐1 Activation in Pulmonary Microvascular Endothelial Cells During Pseudomonas aeruginosa Infection

Abstract

Caspase‐1 is an inflammatory protease responsible for cytokine maturation and programmed inflammatory cell death. In the lung, Pseudomonas aeruginosa infection causes robust caspase‐1 activation in immune cells, and recent evidence indicates that a similar activation occurs in pulmonary microvascular endothelial cells (PMVECs). While P. aeruginosa‐induced caspase‐1 activation in immune cells results in hyper‐inflammation and subsequent barrier disruption, activation of caspase‐1 in PMVECs is barrier protective. The mechanism of caspase‐1 activation in response to P. aeruginosa remains poorly understood. Recently, high levels of intracellular cAMP ([cAMP]i) have been suggested to inhibit caspase‐1 activation in immune cells. We therefore sought to determine whether [cAMP]i plays a role in P. aeruginosa‐induced caspase‐1 activation in PMVECs. To measure caspase‐1 activation, PMVECs were treated with FAM‐YVAD‐FLICA, a membrane permeable, fluorescently labeled, irreversible caspase‐1 inhibitor, for up to three hours. Cells were collected, washed, and fixed for analysis on BD FACSCantoII using the FITC laser at 450 volts. Under control conditions, ~1% of the PMVECs was FITC positive, indicating that ~1% of PMVECs have active caspase‐1. Infection with P. aeruginosa strain PA103 at a multiplicity of infection (MOI) of 10:1 for three hours (infection conditions that were used throughout the course of this study) caused ~20% of the PMVECs to have active caspase‐1. The percentage of PMVECs with active caspase‐1 increased when higher MOIs were used. To determine whether [cAMP]i regulates caspase‐1 activation, PMVECs were treated with adenylyl cyclase activator forskolin (100 μM) and/or phosphodiesterase 4 inhibitor rolipram (10 μM) to increase [cAMP]i. Increasing [cAMP]i alone did not activate caspase‐1. However, in contrast to previous reports, pretreatment with forskolin and/or rolipram resulted in a ~1.5‐3‐fold increase in the percentage of PMVECs with active caspase‐1 following infection with PA103. In addition, when PMVECs were treated with the cell permeable cAMP analog, 8‐bromo‐cAMP, prior to infection with PA103, the percentage of PMVECs with active caspase‐1 increased in a dose dependent manner. To determine whether decreased [cAMP]i inhibits PA103‐induced caspase‐1 activation, PMVECs were treated with adenylyl cyclase inhibitor SQ 22,536 (200 μM) prior to infection with PA103. While treatment with SQ 22,536 alone had no effect on caspase‐1 activation, a ~2‐fold decrease in the percentage of PMVECs with active caspase‐1 was observed following infection with PA103 compared to PA103 infection alone. These studies indicate that, contrary to earlier studies in immune cells, [cAMP]i potentiates caspase‐1 activation in PMVECs. cAMP‐mediated caspase‐1 activation represents a critical signaling pathway that provides an explanation for the observed protective responses in PMVECs following P. aeruginosa infection.Support or Funding InformationR01 HL118334, P01 HL066299