Abstract
Crotoxin B (CB) is a catalytically active group IIA sPLA2 from Crotalus durissus terrificus snake venom. In contrast to most GIIA sPLA2s, CB exhibits anti-inflammatory effects, including the ability to inhibit leukocyte functions. Lipid droplets (LDs) are lipid-rich organelles associated with inflammation and recognized as a site for the synthesis of inflammatory lipid mediators. Here, the ability of CB to induce formation of LDs and the mechanisms involved in this effect were investigated in isolated macrophages. The profile of CB-induced 15-d-PGJ2 (15-Deoxy-Delta-12,14-prostaglandin J2) production and involvement of LDs in 15-d-PGJ2 biosynthesis were also investigated. Stimulation of murine macrophages with CB induced increased number of LDs and release of 15-d-PGJ2. LDs induced by CB were associated to PLIN2 recruitment and expression and required activation of PKC, PI3K, MEK1/2, JNK, iPLA2 and PLD. Both 15-d-PGJ2 and COX-1 were found in CB-induced LDs indicating that LDs contribute to the inhibitory effects of CB by acting as platform for synthesis of 15-d-PGJ2, a pro-resolving lipid mediator. Together, our data indicate that an immunomodulatory GIIA sPLA2 can directly induce LD formation and production of a pro-resolving mediator in an inflammatory cell and afford new insights into the roles of LDs in resolution of inflammatory processes.
Highlights
These organelles compartmentalize key enzymes involved in the synthesis of lipid mediators, such as COX-1, COX-2 and 5-lypoxigenase, as well as proteins related to membrane trafficking, cell signaling, such as kinases, and structural proteins, such as perilipin 2 (PLIN2)[14,15,16,17], an important protein involved in lipid droplets (LDs) assembly, adipocyte differentiation and foam cell formation[18,19,20]
We investigated the involvement of LDs in Crotoxin B (CB)-induced 15-d-PGJ2 biosynthesis as well as the involvement of COX-1, COX-2, cytosolic PLA2 (cPLA2), independent PLA2 (iPLA2) and the signaling pathway proteins PI3K, PKC, MEK1/2 and JNK in LD formation
Maximal LD numbers were observed with a concentration of 0.8 μM. This CB-induced effect was not observed in Ca2+-free, EGTA-containing medium, in a medium in which Ca2+ had been replaced by Sr2+ or when CB was inactivated by p-bromophenacyl bromide (p-BPB) (Fig. 1B), indicating that the catalytic activity of this sPLA2 is essential for induction of LD formation under the present experimental conditions
Summary
The inflammatory group IIA sPLA2s have emerged as critical regulators of LD formation, as they provide free fatty acids from membrane phospholipids that are crucial for this process, directly regulating assembly of these organelles[25] In light of this and the fact that CB, a group IIA sPLA2, is able to release arachidonic acid[7], a major fatty acid involved in both LD formation and lipid-mediator production[26, 27] during inflammatory processes, we decided to investigate the ability of CB to induce LD formation and the mechanisms involved in LD formation, as well as 15-d-PGJ2 production, in macrophages. We investigated the involvement of LDs in CB-induced 15-d-PGJ2 biosynthesis as well as the involvement of COX-1, COX-2, cPLA2, iPLA2 and the signaling pathway proteins PI3K, PKC, MEK1/2 and JNK in LD formation
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