Role of cytosolic phospholipase A2 in Pseudomonas aeruginosa-induced inflammation

Abstract

Phospholipase A2 (PLA2) belongs to a superfamily of enzymes that hydrolyse phospholipids. This family can be classified into secretory PLA2 (sPLA2), cytosolic PLA2 (cPLA2) and Ca 2+ -independent PLA2 (iPLA2). cPLA2 selectively release arachidonic acid (AA) from membrane phospholipids. AA metabolites play a role in inflammation and have cytotoxic effects. Pseudomonas aeruginosa , a Gram-negative bacteria, is an opportunistic pathogen that infects immuno-compromised and cystic fibroblast patients. This results in high mortality due to the notorious resistance to antibiotics of this bacterium. But, the down-stream enzymes of the host involved in the pathogenesis of lung inflammation induced by P aeruginosa have not been identified. We postulated that cPLA2 plays an important role in P aeruginosa -induced inflammation. Alveolar epithelial cells A459 were pre-incubated with the indicated drugs 1 h before infection with laboratory P aeruginosa strains PAK or PAO1 or stimulation with bacterial toxins, PMA or TNFα A release was measured using [ 3 H]-AA labeled cells. IL-8 and PGE2 levels were determined using ELISA methods. Activation of MAPKs and expression of COX enzymes were analyzed by western blot. results showed that incubation of A549 epithelial cells with a wild-type strain of P aeruginosa , PAK, led to an increased cPLA2 phosphorylation which reflects its activation. This suggests that cPLA2 may be involved in P aeruginosa -induced inflammation. We found that either PAK or another P aeruginosa strain, PAO1, induced a time-dependent AA release from A549 cells. The production of prostaglandin E2 (PGE2), a downstream metabolite of AA, was increased significantly after stimulation with PAK PAK also induced IL-8 synthesis. In addition, our results showed that PAK induced p38MAPK phosphorylation in a time- and concentration- dependent manner. SB2035803, a specific inhibitor of p38 MAPK, abolished the effect of PAK on both PGE2 and IL-8 synthesis. Neither Erk nor Junk MAPK was involved in this process. The expression of cyclooxygenase (COX), which converts AA into PGE2, was increased after PAK stimulation. Finally, the effects of different virulence factors isolated from P aeruginosa , including LPS, flagellin and pili were investigated. LPS had no effect on PGE2 release and IL-8 synthesis. Flagellin stimulation increased IL- 8 expression but had no effect on PGE2 release. The δflic strain, in which the flagellin gene was deleted, had less effect on IL-8 synthesis but had similar effect on PGE2, as compared to the PAK strain. Collectively, our results show that cPLA2 is involved in P aeruginosa -induced inflammation through a process probably involving p38MAPK. However, the virulence factors involved in cPLA2 activation remains to be identified.